Alan J. Taege, MD

Lyme disease is a complex multisystem bacterial infection affecting the skin, joints, heart, and nervous system. The full spectrum of disease was first recognized and the disease was named in the 1970s during an outbreak of arthritis in children in the town of Lyme, Connecticut.¹

This review describes the epidemiology and pathogenesis of Lyme disease, the advantages and disadvantages of current diagnostic methods, and diagnostic algorithms.

THE MOST COMMON TICK-BORNE INFECTION IN NORTH AMERICA

Lyme disease is the most common tick-borne infection in North America.^2,3 In the United States, more than 30,000 cases are reported annually. In fact, in 2017, the number of cases was about 42,000, a 16% increase from the previous year, according to the US Centers for Disease Control and Prevention (CDC).

From Sigal LH. Myths and facts about Lyme disease. Cleve Clin J Med 1997; 64(4):203–209.

The infection is caused by Borrelia burgdorferi, a particularly arthritogenic spirochete transmitted by Ixodes scapularis (the black-legged deer tick, (Figure 1) and Ixodes pacificus (the Western black-legged tick). Although the infection can occur at any time of the year, its peak incidence is in May to late September, coinciding with increased outdoor recreational activity in areas where ticks live.^3,4 The typical tick habitat consists of deciduous woodland with sufficient humidity provided by a good layer of decaying vegetation. However, people can contract Lyme disease in their own backyard.³

Most cases of Lyme disease are seen in the northeastern United States, mainly in suburban and rural areas.^2,3 Other areas affected include the midwestern states of Minnesota, Wisconsin, and Michigan, as well as northern California.⁴ Fourteen states and the District of Columbia report a high average incidence (> 10 cases per 100,000 persons) (Table 1).²

FIRST COMES IgM, THEN IgG

The pathogenesis and the different stages of infection should inform laboratory testing in Lyme disease.

It is estimated that only 5% of infected ticks that bite people actually transmit their spirochetes to the human host.⁵ However, once infected, the patient’s innate immune system mounts a response that results in the classic erythema migrans rash at the bite site. A rash develops in only about 85% of patients who are infected and can appear at any time between 3 and 30 days, but most commonly after 7 days. Hence, a rash occurring within the first few hours of tick contact is not erythema migrans and does not indicate infection, but rather an early reaction to tick salivary antigens.⁵

Antibody levels remain below the detection limits of currently available serologic tests in the first 7 days after exposure. Immunoglobulin M (IgM) antibody titers peak between 8 and 14 days after tick contact, but IgM antibodies may never develop if the patient is started on early appropriate antimicrobial therapy.⁵

If the infection is not treated, the spirochete may disseminate through the blood from the bite site to different tissues.³ Both cell-mediated and antibody-mediated immunity swing into action to kill the spirochetes at this stage. The IgM antibody response occurs in 1 to 2 weeks, followed by a robust IgG response in 2 to 4 weeks.⁶

Because IgM can also cross-react with antigens other than those associated with B burgdorferi, the IgM test is less specific than the IgG test for Lyme disease.

Once a patient is exposed and mounts an antibody-mediated response to the spirochete, the antibody profile may persist for months to years, even after successful antibiotic treatment and cure of the disease.⁵

Despite the immune system’s robust series of defenses, untreated B burgdorferi infection can persist, as the organism has a bag of tricks to evade destruction. It can decrease its expression of specific immunogenic surface-exposed proteins, change its antigenic properties through recombination, and bind to the patient’s extracellular matrix proteins to facilitate further dissemination.³

Certain host-genetic factors also play a role in the pathogenesis of Lyme disease, such as the HLA-DR4 allele, which has been associated with antibiotic-refractory Lyme-related arthritis.³

LYME DISEASE EVOLVES THROUGH STAGES

Lyme disease evolves through stages broadly classified as early and late infection, with significant variability in its presentation.⁷

Early infection

Early disease is further subdivided into “localized” infection (stage 1), characterized by a single erythema migrans lesion and local lymphadenopathy, and “disseminated” infection (stage 2), associated with multiple erythema migrans lesions distant from the bite site, facial nerve palsy, radiculoneuritis, meningitis, carditis, or migratory arthritis or arthralgia.⁸

Highly specific physical findings include erythema migrans, cranial nerve palsy, high-grade or progressive conduction block, and recurrent migratory polyarthritis. Less specific symptoms and signs of Lyme disease include arthralgia, myalgia, neck stiffness, palpitations, and myocarditis.⁵

Erythema migrans lesions are evident in at least 85% of patients with early disease.⁹ If they are not apparent on physical examination, they may be located at hidden sites and may be atypical in appearance or transient.⁵

If treatment is not started in the initial stage of the disease, 60% of infected patients may develop disseminated infection.⁵ Progressive, untreated infection can manifest with Lyme arthritis and neuroborreliosis.⁷

Noncutaneous manifestations are less common now than in the past due to increased awareness of the disease and early initiation of treatment.¹⁰

Late infection

Manifestations of late (stage 3) infection include oligoarthritis (affecting any joint but often the knee) and neuroborreliosis. Clinical signs and symptoms of Lyme disease may take months to resolve even after appropriate antimicrobial therapy is completed. This should not be interpreted as ongoing, persistent infection, but as related to host immune-mediated activity.⁵

The usefulness of a laboratory test depends on the individual patient’s pretest probability of infection, which in turn depends on the patient’s epidemiologic risk of exposure and clinical features of Lyme disease. Patients with a high pretest probability—eg, a history of a tick bite followed by the classic erythema migrans rash—do not need testing and can start antimicrobial therapy right away.¹¹

Serologic tests are the gold standard

Prompt diagnosis is important, as early Lyme disease is easily treatable without any future sequelae.¹¹

Tests for Lyme disease can be divided into direct methods, which detect the spirochete itself by culture or by polymerase chain reaction (PCR), and indirect methods, which detect antibodies (Table 2). Direct tests lack sensitivity for Lyme disease; hence, serologic tests remain the gold standard. Currently recommended is a standard 2-tier testing strategy using an enzyme-linked immunosorbent assay (ELISA) followed by Western blot for confirmation.

DIRECT METHODS

Culture lacks sensitivity

A number of factors limit the sensitivity of direct culture for diagnosing Lyme disease. B burgdorferi does not grow easily in culture, requiring special media, low temperatures, and long periods of incubation. Only a relatively few spirochetes are present in human tissues and body fluids to begin with, and bacterial counts are further reduced with duration and dissemination of infection.⁵ All of these limit the possibility of detecting this organism.

Polymerase chain reaction may help in some situations

Molecular assays are not part of the standard evaluation and should be used only in conjunction with serologic testing.⁷ These tests have high specificity but lack consistent sensitivity.

That said, PCR testing may be useful:

• In early infection, before antibody responses develop
• In reinfection, when serologic tests are not reliable because the antibodies persist for many years after an infection in many patients
• In endemic areas where serologic testing has high false-positive rates due to high baseline population seropositivity for anti-Borrelia antibodies caused by subclinical infection.³

PCR assays that target plasmid-borne genes encoding outer surface proteins A and C (OspA and OspC) and VisE (variable major protein-like sequence, expressed) are more sensitive than those that detect chromosomal 16s ribosomal ribonucleic acid (rRNA) genes, as plasmid-rich “blebs” are shed in larger concentrations than chromosomal DNA during active infection.⁷ However, these plasmid-contained genes persist in body tissues and fluids even after the infection is cleared, and their detection may not necessarily correlate with ongoing disease.⁸ Detection of chromosomal 16s rRNA genes is a better predictor of true organism viability.

The sensitivity of PCR for borrelial DNA depends on the type of sample. If a skin biopsy sample is taken of the leading edge of an erythema migrans lesion, the sensitivity is 69% and the specificity is 100%. In patients with Lyme arthritis, PCR of the synovial fluid has a sensitivity of up to 80%. However, the sensitivity of PCR of the cerebrospinal fluid of patients with neurologic manifestations of Lyme disease is only 19%.⁷ PCR of other clinical samples, including blood and urine, is not recommended, as spirochetes are primarily confined to tissues, and very few are present in these body fluids.^3,12

The disadvantage of PCR is that a positive result does not always mean active infection, as the DNA of the dead microbe persists for several months even after successful treatment.⁸

INDIRECT METHODS

Enzyme-linked immunosorbent assay

ELISAs detect anti-Borrelia antibodies. Early-generation ELISAs, still used in many laboratories, use whole-cell extracts of B burgdorferi. Examples are the Vidas Lyme screen (Biomérieux, biomerieux-usa.com) and the Wampole B burgdorferi IgG/M EIA II assay (Alere, www.alere.com). Newer ELISAs use recombinant proteins.¹³

Three major targets for ELISA antibodies are flagellin (Fla), outer surface protein C (OspC), and VisE, especially the invariable region 6 (IR6). Among these, VisE-IR6 is the most conserved region in B burgdorferi.

Early-generation assays have a sensitivity of 89% and specificity of 72%.¹¹ However, the patient’s serum may have antibodies that cross-react with unrelated bacterial antigens, leading to false-positive results (Table 3). Whole-cell sonicate assays are not recommended as an independent test and must be confirmed with Western blot testing when assay results are indeterminate or positive.¹¹

Newer-generation ELISAs detect antibodies targeting recombinant proteins of VisE, especially a synthetic peptide C6, within IR6.¹³ VisE-IR6 is the most conserved region of the B burgdorferi complex, and its detection is a highly specific finding, supporting the diagnosis of Lyme disease. Antibodies against VisE-IR6 antigen are the earliest to develop.⁵ An example of a newer-generation serologic test is the VisE C6 Lyme EIA kit, approved as a first-tier test by the US Food and Drug Administration in 2001. This test has a specificity of 99%,^14,15 and its specificity is further increased when used in conjunction with Western blot (99.5%).¹⁵ The advantage of the C6 antibody test is that it is more sensitive than 2-tier testing during early infection (sensitivity 29%–74% vs 17%–40% in early localized infection, and 56%–90% vs 27%–78% in early disseminated infection).⁶

During early infection, older and newer ELISAs are less sensitive because of the limited number of antigens expressed at this stage.¹³ All patients suspected of having early Lyme disease who are seronegative at initial testing should have follow-up testing to look for seroconversion.¹³

Western blot

Western blot (immunoblot) testing identifies IgM and IgG antibodies against specific B burgdorferi antigens. It is considered positive if it detects at least 2 of a possible 3 specific IgM bands in the first 4 weeks of disease or at least 5 of 10 specific IgG bands after 4 weeks of disease (Table 4 and Figure 2).¹⁶

The nature of the bands indicates the duration of infection: Western blot bands against 23-kD OspC and 41-kD FlaB are seen in early localized infection, whereas bands against all 3 B burgdorferi proteins will be seen after several weeks of disease.¹⁷ The IgM result should be interpreted carefully, as only 2 bands are required for the test to be positive, and IgM binds to antigen less specifically than IgG.¹²

If clinical symptoms and signs of Lyme disease have been present for more than 1 month, IgM reactivity alone should not be used to support the diagnosis, in view of the likelihood of a false-positive test result in this situation.¹⁸ This is called the “1-month rule” in the diagnosis of Lyme disease.¹³

In early localized infection, Western blot is only half as sensitive as ELISA testing. Since the overall sensitivity of a 2-step algorithm is equal to that of its least sensitive component, 2-tiered testing is not useful in early disease.¹³

Although currently considered the most specific test for confirmation of Lyme disease, Western blot has limitations. It is technically and interpretively complex and is thus not universally available.¹³ The blots are scored by visual examination, compromising the reproducibility of the test, although densitometric blot analysis techniques and automated scanning and scoring attempt to address some of these limitations.¹³ Like the ELISA, Western blot can have false-positive results in healthy individuals without tick exposure, as nonspecific IgM immunoblots develop faint bands. This is because of cross-reaction between B burgdorferi antigens and antigens from other microorganisms. Around 50% of healthy adults show low-level serum IgG reactivity against the FlaB antigen, leading to false-positive results as well. In cases in which the Western blot result is indeterminate, other etiologies must be considered.

False-positive IgM Western blots are a significant problem. In a 5-year retrospective study done at 63 US Air Force healthcare facilities, 113 (53.3%) of 212 IgM Western blots were falsely positive.¹⁹ A false-positive test was defined as one that failed to meet seropositivity (a first-tier test omitted or negative, > 30 days of symptoms with negative IgG blot), lack of exposure including residing in areas without documented tick habitats, patients having atypical or no symptoms, and negative serology within 30 days of a positive test.

In a similar study done in a highly endemic area, 50 (27.5%) of 182 patients had a false-positive test.²⁰ Physicians need to be careful when interpreting IgM Western blots. It is always important to consider locale, epidemiology, and symptoms when interpreting the test.

Limitations of serologic tests for Lyme disease

Currently available serologic tests have inherent limitations:

• Antibodies against B burgdorferi take at least 1 week to develop
• The background rate of seropositivity in endemic areas can be up to 4%, affecting the utility of a positive test result
• Serologic tests cannot be used as tests of cure because antibodies can persist for months to years even after appropriate antimicrobial therapy and cure of disease; thus, a positive serologic result could represent active infection or remote exposure²¹
• Antibodies can cross-react with related bacteria, including other borrelial or treponemal spirochetes
• False-positive serologic test results can also occur in association with other medical conditions such as polyclonal gammopathies and systemic lupus erythematosus.¹²

RECOMMENDATIONS FOR TESTING

Standard 2-tier testing

The CDC released recommendations for diagnosing Lyme disease after a second national conference of serologic diagnosis of Lyme disease in October 1994.¹⁸ The 2-tiered testing method, involving a sensitive ELISA followed by the Western blot to confirm positive and indeterminate ELISA results, was suggested as the gold standard for diagnosis (Figure 3). Of note, negative ELISA results do not require further testing.¹¹

The sensitivity of 2-tiered testing depends on the stage of the disease. Unfortunately, this method has a wide range of sensitivity (17% to 78%) in stage 1 disease. In the same stage, the sensitivity increases from 14.1% in patients with a single erythema migrans lesion and early localized infection to 65.4% in those with multiple lesions. The algorithm has excellent sensitivity in late stage 3 infection (96% to 100%).⁵

A 2-step ELISA algorithm

A 2-step ELISA algorithm (without the Western blot) that includes the whole-cell sonicate assay followed by the VisE C6 peptide assay actually showed higher sensitivity and comparable specificity compared with 2-tiered testing in early localized disease (sensitivity 61%–74% vs 29%–48%, respectively; specificity 99.5% for both methods).²² This higher sensitivity was even more pronounced in early disseminated infection (sensitivity 100% vs 40%, respectively). By late infection, the sensitivities of both testing strategies reached 100%. Compared with the Western blot, the 2-step ELISA algorithm was simpler to execute in a reproducible fashion.⁵

The Infectious Diseases Society of America is revising its current guidelines, with an update expected late this year, which may shift the recommendation from 2-tiered testing to the 2-step ELISA algorithm.

Multiplex testing

To overcome the intrinsic problems of protein-based assays, a multiplexed, array-based assay for the diagnosis of tick-borne infections called Tick-Borne Disease Serochip (TBD-Serochip) was established using recombinant antigens that identify key immunodominant epitopes.⁸ More studies are needed to establish the validity and usefulness of these tests in clinical practice.

Who should not be tested?

The American College of Physicians⁶ recommends against testing in patients:

• Presenting with nonspecific symptoms (eg, headache, myalgia, fatigue, arthralgia) without objective signs of Lyme disease
• With low pretest probability of infection based on epidemiologic exposures and clinical features
• Living in Lyme-endemic areas with no history of tick exposure⁶
• Presenting less than 1 week after tick exposure⁵
• Seeking a test of cure for treated Lyme disease.

DIAGNOSIS IN SPECIAL SITUATIONS

Early Lyme disease

The classic erythema migrans lesion on physical examination of a patient with suspected Lyme disease is diagnostic and does not require laboratory confirmation.¹⁰

In ambiguous cases, 2-tiered testing of a serum sample during the acute presentation and again 4 to 6 weeks later can be useful. In patients who remain seronegative on paired serum samples despite symptoms lasting longer than 6 weeks and no antibiotic treatment in the interim, the diagnosis of Lyme disease is unlikely, and another diagnosis should be sought.³

Antimicrobial therapy may block the serologic response; hence, negative serologic testing in patients started on empiric antibiotics should not rule out Lyme disease.⁶

PCR or bacterial culture testing is not recommended in the evaluation of suspected early Lyme disease.

Central nervous system Lyme disease

Central nervous system Lyme disease is diagnosed by 2-tiered testing using peripheral blood samples because all patients with this infectious manifestation should have mounted an adequate IgG response in the blood.¹¹

B cells migrate to and proliferate inside the central nervous system, leading to intrathecal production of anti-Borrelia antibodies. An index of cerebrospinal fluid to serum antibody greater than 1 is thus also indicative of neuroborreliosis.¹² Thus, performing lumbar puncture to detect intrathecal production of antibodies may support the diagnosis of central nervous system Lyme disease; however, it is not necessary.¹¹

Antibodies persist in the central nervous system for many years after appropriate antimicrobial treatment.

Lyme arthritis

Articular involvement in Lyme disease is characterized by a robust humoral response such that a negative IgG serologic test virtually rules out Lyme arthritis.²³ PCR testing of synovial fluid for borrelial DNA has a sensitivity of 80% but may become falsely negative after 1 to 2 months of antibiotic treatment.^24,25 In an algorithm suggested by Puius et al,²³ PCR testing of synovial fluid should be done in patients who have minimal to no response after 2 months of appropriate oral antimicrobial therapy to determine whether intravenous antibiotics are merited.

Table 5 summarizes the tests of choice in different clinical stages of infection.

Acknowledgment: The authors would like to acknowledge Anita Modi, MD, and Ceena N. Jacob, MD, for reviewing the manuscript and providing valuable suggestions, and Belinda Yen-Lieberman, PhD, for contributing pictures of the Western blot test results.

Lyme disease is a complex multisystem bacterial infection affecting the skin, joints, heart, and nervous system. The full spectrum of disease was first recognized and the disease was named in the 1970s during an outbreak of arthritis in children in the town of Lyme, Connecticut.¹

This review describes the epidemiology and pathogenesis of Lyme disease, the advantages and disadvantages of current diagnostic methods, and diagnostic algorithms.

THE MOST COMMON TICK-BORNE INFECTION IN NORTH AMERICA

Lyme disease is the most common tick-borne infection in North America.^2,3 In the United States, more than 30,000 cases are reported annually. In fact, in 2017, the number of cases was about 42,000, a 16% increase from the previous year, according to the US Centers for Disease Control and Prevention (CDC).

From Sigal LH. Myths and facts about Lyme disease. Cleve Clin J Med 1997; 64(4):203–209.

The infection is caused by Borrelia burgdorferi, a particularly arthritogenic spirochete transmitted by Ixodes scapularis (the black-legged deer tick, (Figure 1) and Ixodes pacificus (the Western black-legged tick). Although the infection can occur at any time of the year, its peak incidence is in May to late September, coinciding with increased outdoor recreational activity in areas where ticks live.^3,4 The typical tick habitat consists of deciduous woodland with sufficient humidity provided by a good layer of decaying vegetation. However, people can contract Lyme disease in their own backyard.³

Most cases of Lyme disease are seen in the northeastern United States, mainly in suburban and rural areas.^2,3 Other areas affected include the midwestern states of Minnesota, Wisconsin, and Michigan, as well as northern California.⁴ Fourteen states and the District of Columbia report a high average incidence (> 10 cases per 100,000 persons) (Table 1).²

FIRST COMES IgM, THEN IgG

The pathogenesis and the different stages of infection should inform laboratory testing in Lyme disease.

It is estimated that only 5% of infected ticks that bite people actually transmit their spirochetes to the human host.⁵ However, once infected, the patient’s innate immune system mounts a response that results in the classic erythema migrans rash at the bite site. A rash develops in only about 85% of patients who are infected and can appear at any time between 3 and 30 days, but most commonly after 7 days. Hence, a rash occurring within the first few hours of tick contact is not erythema migrans and does not indicate infection, but rather an early reaction to tick salivary antigens.⁵

Antibody levels remain below the detection limits of currently available serologic tests in the first 7 days after exposure. Immunoglobulin M (IgM) antibody titers peak between 8 and 14 days after tick contact, but IgM antibodies may never develop if the patient is started on early appropriate antimicrobial therapy.⁵

If the infection is not treated, the spirochete may disseminate through the blood from the bite site to different tissues.³ Both cell-mediated and antibody-mediated immunity swing into action to kill the spirochetes at this stage. The IgM antibody response occurs in 1 to 2 weeks, followed by a robust IgG response in 2 to 4 weeks.⁶

Because IgM can also cross-react with antigens other than those associated with B burgdorferi, the IgM test is less specific than the IgG test for Lyme disease.

Once a patient is exposed and mounts an antibody-mediated response to the spirochete, the antibody profile may persist for months to years, even after successful antibiotic treatment and cure of the disease.⁵

Despite the immune system’s robust series of defenses, untreated B burgdorferi infection can persist, as the organism has a bag of tricks to evade destruction. It can decrease its expression of specific immunogenic surface-exposed proteins, change its antigenic properties through recombination, and bind to the patient’s extracellular matrix proteins to facilitate further dissemination.³

Certain host-genetic factors also play a role in the pathogenesis of Lyme disease, such as the HLA-DR4 allele, which has been associated with antibiotic-refractory Lyme-related arthritis.³

LYME DISEASE EVOLVES THROUGH STAGES

Lyme disease evolves through stages broadly classified as early and late infection, with significant variability in its presentation.⁷

Early infection

Early disease is further subdivided into “localized” infection (stage 1), characterized by a single erythema migrans lesion and local lymphadenopathy, and “disseminated” infection (stage 2), associated with multiple erythema migrans lesions distant from the bite site, facial nerve palsy, radiculoneuritis, meningitis, carditis, or migratory arthritis or arthralgia.⁸

Highly specific physical findings include erythema migrans, cranial nerve palsy, high-grade or progressive conduction block, and recurrent migratory polyarthritis. Less specific symptoms and signs of Lyme disease include arthralgia, myalgia, neck stiffness, palpitations, and myocarditis.⁵

Erythema migrans lesions are evident in at least 85% of patients with early disease.⁹ If they are not apparent on physical examination, they may be located at hidden sites and may be atypical in appearance or transient.⁵

If treatment is not started in the initial stage of the disease, 60% of infected patients may develop disseminated infection.⁵ Progressive, untreated infection can manifest with Lyme arthritis and neuroborreliosis.⁷

Noncutaneous manifestations are less common now than in the past due to increased awareness of the disease and early initiation of treatment.¹⁰

Late infection

Manifestations of late (stage 3) infection include oligoarthritis (affecting any joint but often the knee) and neuroborreliosis. Clinical signs and symptoms of Lyme disease may take months to resolve even after appropriate antimicrobial therapy is completed. This should not be interpreted as ongoing, persistent infection, but as related to host immune-mediated activity.⁵

The usefulness of a laboratory test depends on the individual patient’s pretest probability of infection, which in turn depends on the patient’s epidemiologic risk of exposure and clinical features of Lyme disease. Patients with a high pretest probability—eg, a history of a tick bite followed by the classic erythema migrans rash—do not need testing and can start antimicrobial therapy right away.¹¹

Serologic tests are the gold standard

Prompt diagnosis is important, as early Lyme disease is easily treatable without any future sequelae.¹¹

Tests for Lyme disease can be divided into direct methods, which detect the spirochete itself by culture or by polymerase chain reaction (PCR), and indirect methods, which detect antibodies (Table 2). Direct tests lack sensitivity for Lyme disease; hence, serologic tests remain the gold standard. Currently recommended is a standard 2-tier testing strategy using an enzyme-linked immunosorbent assay (ELISA) followed by Western blot for confirmation.

DIRECT METHODS

Culture lacks sensitivity

A number of factors limit the sensitivity of direct culture for diagnosing Lyme disease. B burgdorferi does not grow easily in culture, requiring special media, low temperatures, and long periods of incubation. Only a relatively few spirochetes are present in human tissues and body fluids to begin with, and bacterial counts are further reduced with duration and dissemination of infection.⁵ All of these limit the possibility of detecting this organism.

Polymerase chain reaction may help in some situations

Molecular assays are not part of the standard evaluation and should be used only in conjunction with serologic testing.⁷ These tests have high specificity but lack consistent sensitivity.

That said, PCR testing may be useful:

• In early infection, before antibody responses develop
• In reinfection, when serologic tests are not reliable because the antibodies persist for many years after an infection in many patients
• In endemic areas where serologic testing has high false-positive rates due to high baseline population seropositivity for anti-Borrelia antibodies caused by subclinical infection.³

PCR assays that target plasmid-borne genes encoding outer surface proteins A and C (OspA and OspC) and VisE (variable major protein-like sequence, expressed) are more sensitive than those that detect chromosomal 16s ribosomal ribonucleic acid (rRNA) genes, as plasmid-rich “blebs” are shed in larger concentrations than chromosomal DNA during active infection.⁷ However, these plasmid-contained genes persist in body tissues and fluids even after the infection is cleared, and their detection may not necessarily correlate with ongoing disease.⁸ Detection of chromosomal 16s rRNA genes is a better predictor of true organism viability.

The sensitivity of PCR for borrelial DNA depends on the type of sample. If a skin biopsy sample is taken of the leading edge of an erythema migrans lesion, the sensitivity is 69% and the specificity is 100%. In patients with Lyme arthritis, PCR of the synovial fluid has a sensitivity of up to 80%. However, the sensitivity of PCR of the cerebrospinal fluid of patients with neurologic manifestations of Lyme disease is only 19%.⁷ PCR of other clinical samples, including blood and urine, is not recommended, as spirochetes are primarily confined to tissues, and very few are present in these body fluids.^3,12

The disadvantage of PCR is that a positive result does not always mean active infection, as the DNA of the dead microbe persists for several months even after successful treatment.⁸

INDIRECT METHODS

Enzyme-linked immunosorbent assay

ELISAs detect anti-Borrelia antibodies. Early-generation ELISAs, still used in many laboratories, use whole-cell extracts of B burgdorferi. Examples are the Vidas Lyme screen (Biomérieux, biomerieux-usa.com) and the Wampole B burgdorferi IgG/M EIA II assay (Alere, www.alere.com). Newer ELISAs use recombinant proteins.¹³

Three major targets for ELISA antibodies are flagellin (Fla), outer surface protein C (OspC), and VisE, especially the invariable region 6 (IR6). Among these, VisE-IR6 is the most conserved region in B burgdorferi.

Early-generation assays have a sensitivity of 89% and specificity of 72%.¹¹ However, the patient’s serum may have antibodies that cross-react with unrelated bacterial antigens, leading to false-positive results (Table 3). Whole-cell sonicate assays are not recommended as an independent test and must be confirmed with Western blot testing when assay results are indeterminate or positive.¹¹

Newer-generation ELISAs detect antibodies targeting recombinant proteins of VisE, especially a synthetic peptide C6, within IR6.¹³ VisE-IR6 is the most conserved region of the B burgdorferi complex, and its detection is a highly specific finding, supporting the diagnosis of Lyme disease. Antibodies against VisE-IR6 antigen are the earliest to develop.⁵ An example of a newer-generation serologic test is the VisE C6 Lyme EIA kit, approved as a first-tier test by the US Food and Drug Administration in 2001. This test has a specificity of 99%,^14,15 and its specificity is further increased when used in conjunction with Western blot (99.5%).¹⁵ The advantage of the C6 antibody test is that it is more sensitive than 2-tier testing during early infection (sensitivity 29%–74% vs 17%–40% in early localized infection, and 56%–90% vs 27%–78% in early disseminated infection).⁶

During early infection, older and newer ELISAs are less sensitive because of the limited number of antigens expressed at this stage.¹³ All patients suspected of having early Lyme disease who are seronegative at initial testing should have follow-up testing to look for seroconversion.¹³

Western blot

Western blot (immunoblot) testing identifies IgM and IgG antibodies against specific B burgdorferi antigens. It is considered positive if it detects at least 2 of a possible 3 specific IgM bands in the first 4 weeks of disease or at least 5 of 10 specific IgG bands after 4 weeks of disease (Table 4 and Figure 2).¹⁶

The nature of the bands indicates the duration of infection: Western blot bands against 23-kD OspC and 41-kD FlaB are seen in early localized infection, whereas bands against all 3 B burgdorferi proteins will be seen after several weeks of disease.¹⁷ The IgM result should be interpreted carefully, as only 2 bands are required for the test to be positive, and IgM binds to antigen less specifically than IgG.¹²

If clinical symptoms and signs of Lyme disease have been present for more than 1 month, IgM reactivity alone should not be used to support the diagnosis, in view of the likelihood of a false-positive test result in this situation.¹⁸ This is called the “1-month rule” in the diagnosis of Lyme disease.¹³

In early localized infection, Western blot is only half as sensitive as ELISA testing. Since the overall sensitivity of a 2-step algorithm is equal to that of its least sensitive component, 2-tiered testing is not useful in early disease.¹³

Although currently considered the most specific test for confirmation of Lyme disease, Western blot has limitations. It is technically and interpretively complex and is thus not universally available.¹³ The blots are scored by visual examination, compromising the reproducibility of the test, although densitometric blot analysis techniques and automated scanning and scoring attempt to address some of these limitations.¹³ Like the ELISA, Western blot can have false-positive results in healthy individuals without tick exposure, as nonspecific IgM immunoblots develop faint bands. This is because of cross-reaction between B burgdorferi antigens and antigens from other microorganisms. Around 50% of healthy adults show low-level serum IgG reactivity against the FlaB antigen, leading to false-positive results as well. In cases in which the Western blot result is indeterminate, other etiologies must be considered.

False-positive IgM Western blots are a significant problem. In a 5-year retrospective study done at 63 US Air Force healthcare facilities, 113 (53.3%) of 212 IgM Western blots were falsely positive.¹⁹ A false-positive test was defined as one that failed to meet seropositivity (a first-tier test omitted or negative, > 30 days of symptoms with negative IgG blot), lack of exposure including residing in areas without documented tick habitats, patients having atypical or no symptoms, and negative serology within 30 days of a positive test.

In a similar study done in a highly endemic area, 50 (27.5%) of 182 patients had a false-positive test.²⁰ Physicians need to be careful when interpreting IgM Western blots. It is always important to consider locale, epidemiology, and symptoms when interpreting the test.

Limitations of serologic tests for Lyme disease

Currently available serologic tests have inherent limitations:

• Antibodies against B burgdorferi take at least 1 week to develop
• The background rate of seropositivity in endemic areas can be up to 4%, affecting the utility of a positive test result
• Serologic tests cannot be used as tests of cure because antibodies can persist for months to years even after appropriate antimicrobial therapy and cure of disease; thus, a positive serologic result could represent active infection or remote exposure²¹
• Antibodies can cross-react with related bacteria, including other borrelial or treponemal spirochetes
• False-positive serologic test results can also occur in association with other medical conditions such as polyclonal gammopathies and systemic lupus erythematosus.¹²

RECOMMENDATIONS FOR TESTING

Standard 2-tier testing

The CDC released recommendations for diagnosing Lyme disease after a second national conference of serologic diagnosis of Lyme disease in October 1994.¹⁸ The 2-tiered testing method, involving a sensitive ELISA followed by the Western blot to confirm positive and indeterminate ELISA results, was suggested as the gold standard for diagnosis (Figure 3). Of note, negative ELISA results do not require further testing.¹¹

The sensitivity of 2-tiered testing depends on the stage of the disease. Unfortunately, this method has a wide range of sensitivity (17% to 78%) in stage 1 disease. In the same stage, the sensitivity increases from 14.1% in patients with a single erythema migrans lesion and early localized infection to 65.4% in those with multiple lesions. The algorithm has excellent sensitivity in late stage 3 infection (96% to 100%).⁵

A 2-step ELISA algorithm

A 2-step ELISA algorithm (without the Western blot) that includes the whole-cell sonicate assay followed by the VisE C6 peptide assay actually showed higher sensitivity and comparable specificity compared with 2-tiered testing in early localized disease (sensitivity 61%–74% vs 29%–48%, respectively; specificity 99.5% for both methods).²² This higher sensitivity was even more pronounced in early disseminated infection (sensitivity 100% vs 40%, respectively). By late infection, the sensitivities of both testing strategies reached 100%. Compared with the Western blot, the 2-step ELISA algorithm was simpler to execute in a reproducible fashion.⁵

The Infectious Diseases Society of America is revising its current guidelines, with an update expected late this year, which may shift the recommendation from 2-tiered testing to the 2-step ELISA algorithm.

Multiplex testing

To overcome the intrinsic problems of protein-based assays, a multiplexed, array-based assay for the diagnosis of tick-borne infections called Tick-Borne Disease Serochip (TBD-Serochip) was established using recombinant antigens that identify key immunodominant epitopes.⁸ More studies are needed to establish the validity and usefulness of these tests in clinical practice.

Who should not be tested?

The American College of Physicians⁶ recommends against testing in patients:

• Presenting with nonspecific symptoms (eg, headache, myalgia, fatigue, arthralgia) without objective signs of Lyme disease
• With low pretest probability of infection based on epidemiologic exposures and clinical features
• Living in Lyme-endemic areas with no history of tick exposure⁶
• Presenting less than 1 week after tick exposure⁵
• Seeking a test of cure for treated Lyme disease.

DIAGNOSIS IN SPECIAL SITUATIONS

Early Lyme disease

The classic erythema migrans lesion on physical examination of a patient with suspected Lyme disease is diagnostic and does not require laboratory confirmation.¹⁰

In ambiguous cases, 2-tiered testing of a serum sample during the acute presentation and again 4 to 6 weeks later can be useful. In patients who remain seronegative on paired serum samples despite symptoms lasting longer than 6 weeks and no antibiotic treatment in the interim, the diagnosis of Lyme disease is unlikely, and another diagnosis should be sought.³

Antimicrobial therapy may block the serologic response; hence, negative serologic testing in patients started on empiric antibiotics should not rule out Lyme disease.⁶

PCR or bacterial culture testing is not recommended in the evaluation of suspected early Lyme disease.

Central nervous system Lyme disease

Central nervous system Lyme disease is diagnosed by 2-tiered testing using peripheral blood samples because all patients with this infectious manifestation should have mounted an adequate IgG response in the blood.¹¹

B cells migrate to and proliferate inside the central nervous system, leading to intrathecal production of anti-Borrelia antibodies. An index of cerebrospinal fluid to serum antibody greater than 1 is thus also indicative of neuroborreliosis.¹² Thus, performing lumbar puncture to detect intrathecal production of antibodies may support the diagnosis of central nervous system Lyme disease; however, it is not necessary.¹¹

Antibodies persist in the central nervous system for many years after appropriate antimicrobial treatment.

Lyme arthritis

Articular involvement in Lyme disease is characterized by a robust humoral response such that a negative IgG serologic test virtually rules out Lyme arthritis.²³ PCR testing of synovial fluid for borrelial DNA has a sensitivity of 80% but may become falsely negative after 1 to 2 months of antibiotic treatment.^24,25 In an algorithm suggested by Puius et al,²³ PCR testing of synovial fluid should be done in patients who have minimal to no response after 2 months of appropriate oral antimicrobial therapy to determine whether intravenous antibiotics are merited.

Table 5 summarizes the tests of choice in different clinical stages of infection.

Acknowledgment: The authors would like to acknowledge Anita Modi, MD, and Ceena N. Jacob, MD, for reviewing the manuscript and providing valuable suggestions, and Belinda Yen-Lieberman, PhD, for contributing pictures of the Western blot test results.

Lyme disease is a complex multisystem bacterial infection affecting the skin, joints, heart, and nervous system. The full spectrum of disease was first recognized and the disease was named in the 1970s during an outbreak of arthritis in children in the town of Lyme, Connecticut.¹

This review describes the epidemiology and pathogenesis of Lyme disease, the advantages and disadvantages of current diagnostic methods, and diagnostic algorithms.

THE MOST COMMON TICK-BORNE INFECTION IN NORTH AMERICA

Lyme disease is the most common tick-borne infection in North America.^2,3 In the United States, more than 30,000 cases are reported annually. In fact, in 2017, the number of cases was about 42,000, a 16% increase from the previous year, according to the US Centers for Disease Control and Prevention (CDC).

From Sigal LH. Myths and facts about Lyme disease. Cleve Clin J Med 1997; 64(4):203–209.

The infection is caused by Borrelia burgdorferi, a particularly arthritogenic spirochete transmitted by Ixodes scapularis (the black-legged deer tick, (Figure 1) and Ixodes pacificus (the Western black-legged tick). Although the infection can occur at any time of the year, its peak incidence is in May to late September, coinciding with increased outdoor recreational activity in areas where ticks live.^3,4 The typical tick habitat consists of deciduous woodland with sufficient humidity provided by a good layer of decaying vegetation. However, people can contract Lyme disease in their own backyard.³

Most cases of Lyme disease are seen in the northeastern United States, mainly in suburban and rural areas.^2,3 Other areas affected include the midwestern states of Minnesota, Wisconsin, and Michigan, as well as northern California.⁴ Fourteen states and the District of Columbia report a high average incidence (> 10 cases per 100,000 persons) (Table 1).²

FIRST COMES IgM, THEN IgG

The pathogenesis and the different stages of infection should inform laboratory testing in Lyme disease.

It is estimated that only 5% of infected ticks that bite people actually transmit their spirochetes to the human host.⁵ However, once infected, the patient’s innate immune system mounts a response that results in the classic erythema migrans rash at the bite site. A rash develops in only about 85% of patients who are infected and can appear at any time between 3 and 30 days, but most commonly after 7 days. Hence, a rash occurring within the first few hours of tick contact is not erythema migrans and does not indicate infection, but rather an early reaction to tick salivary antigens.⁵

Antibody levels remain below the detection limits of currently available serologic tests in the first 7 days after exposure. Immunoglobulin M (IgM) antibody titers peak between 8 and 14 days after tick contact, but IgM antibodies may never develop if the patient is started on early appropriate antimicrobial therapy.⁵

If the infection is not treated, the spirochete may disseminate through the blood from the bite site to different tissues.³ Both cell-mediated and antibody-mediated immunity swing into action to kill the spirochetes at this stage. The IgM antibody response occurs in 1 to 2 weeks, followed by a robust IgG response in 2 to 4 weeks.⁶

Because IgM can also cross-react with antigens other than those associated with B burgdorferi, the IgM test is less specific than the IgG test for Lyme disease.

Once a patient is exposed and mounts an antibody-mediated response to the spirochete, the antibody profile may persist for months to years, even after successful antibiotic treatment and cure of the disease.⁵

Despite the immune system’s robust series of defenses, untreated B burgdorferi infection can persist, as the organism has a bag of tricks to evade destruction. It can decrease its expression of specific immunogenic surface-exposed proteins, change its antigenic properties through recombination, and bind to the patient’s extracellular matrix proteins to facilitate further dissemination.³

Certain host-genetic factors also play a role in the pathogenesis of Lyme disease, such as the HLA-DR4 allele, which has been associated with antibiotic-refractory Lyme-related arthritis.³

LYME DISEASE EVOLVES THROUGH STAGES

Lyme disease evolves through stages broadly classified as early and late infection, with significant variability in its presentation.⁷

Early infection

Early disease is further subdivided into “localized” infection (stage 1), characterized by a single erythema migrans lesion and local lymphadenopathy, and “disseminated” infection (stage 2), associated with multiple erythema migrans lesions distant from the bite site, facial nerve palsy, radiculoneuritis, meningitis, carditis, or migratory arthritis or arthralgia.⁸

Highly specific physical findings include erythema migrans, cranial nerve palsy, high-grade or progressive conduction block, and recurrent migratory polyarthritis. Less specific symptoms and signs of Lyme disease include arthralgia, myalgia, neck stiffness, palpitations, and myocarditis.⁵

Erythema migrans lesions are evident in at least 85% of patients with early disease.⁹ If they are not apparent on physical examination, they may be located at hidden sites and may be atypical in appearance or transient.⁵

If treatment is not started in the initial stage of the disease, 60% of infected patients may develop disseminated infection.⁵ Progressive, untreated infection can manifest with Lyme arthritis and neuroborreliosis.⁷

Noncutaneous manifestations are less common now than in the past due to increased awareness of the disease and early initiation of treatment.¹⁰

Late infection

Manifestations of late (stage 3) infection include oligoarthritis (affecting any joint but often the knee) and neuroborreliosis. Clinical signs and symptoms of Lyme disease may take months to resolve even after appropriate antimicrobial therapy is completed. This should not be interpreted as ongoing, persistent infection, but as related to host immune-mediated activity.⁵

The usefulness of a laboratory test depends on the individual patient’s pretest probability of infection, which in turn depends on the patient’s epidemiologic risk of exposure and clinical features of Lyme disease. Patients with a high pretest probability—eg, a history of a tick bite followed by the classic erythema migrans rash—do not need testing and can start antimicrobial therapy right away.¹¹

Serologic tests are the gold standard

Prompt diagnosis is important, as early Lyme disease is easily treatable without any future sequelae.¹¹

Tests for Lyme disease can be divided into direct methods, which detect the spirochete itself by culture or by polymerase chain reaction (PCR), and indirect methods, which detect antibodies (Table 2). Direct tests lack sensitivity for Lyme disease; hence, serologic tests remain the gold standard. Currently recommended is a standard 2-tier testing strategy using an enzyme-linked immunosorbent assay (ELISA) followed by Western blot for confirmation.

DIRECT METHODS

Culture lacks sensitivity

A number of factors limit the sensitivity of direct culture for diagnosing Lyme disease. B burgdorferi does not grow easily in culture, requiring special media, low temperatures, and long periods of incubation. Only a relatively few spirochetes are present in human tissues and body fluids to begin with, and bacterial counts are further reduced with duration and dissemination of infection.⁵ All of these limit the possibility of detecting this organism.

Polymerase chain reaction may help in some situations

Molecular assays are not part of the standard evaluation and should be used only in conjunction with serologic testing.⁷ These tests have high specificity but lack consistent sensitivity.

That said, PCR testing may be useful:

• In early infection, before antibody responses develop
• In reinfection, when serologic tests are not reliable because the antibodies persist for many years after an infection in many patients
• In endemic areas where serologic testing has high false-positive rates due to high baseline population seropositivity for anti-Borrelia antibodies caused by subclinical infection.³

PCR assays that target plasmid-borne genes encoding outer surface proteins A and C (OspA and OspC) and VisE (variable major protein-like sequence, expressed) are more sensitive than those that detect chromosomal 16s ribosomal ribonucleic acid (rRNA) genes, as plasmid-rich “blebs” are shed in larger concentrations than chromosomal DNA during active infection.⁷ However, these plasmid-contained genes persist in body tissues and fluids even after the infection is cleared, and their detection may not necessarily correlate with ongoing disease.⁸ Detection of chromosomal 16s rRNA genes is a better predictor of true organism viability.

The sensitivity of PCR for borrelial DNA depends on the type of sample. If a skin biopsy sample is taken of the leading edge of an erythema migrans lesion, the sensitivity is 69% and the specificity is 100%. In patients with Lyme arthritis, PCR of the synovial fluid has a sensitivity of up to 80%. However, the sensitivity of PCR of the cerebrospinal fluid of patients with neurologic manifestations of Lyme disease is only 19%.⁷ PCR of other clinical samples, including blood and urine, is not recommended, as spirochetes are primarily confined to tissues, and very few are present in these body fluids.^3,12

The disadvantage of PCR is that a positive result does not always mean active infection, as the DNA of the dead microbe persists for several months even after successful treatment.⁸

INDIRECT METHODS

Enzyme-linked immunosorbent assay

ELISAs detect anti-Borrelia antibodies. Early-generation ELISAs, still used in many laboratories, use whole-cell extracts of B burgdorferi. Examples are the Vidas Lyme screen (Biomérieux, biomerieux-usa.com) and the Wampole B burgdorferi IgG/M EIA II assay (Alere, www.alere.com). Newer ELISAs use recombinant proteins.¹³

Three major targets for ELISA antibodies are flagellin (Fla), outer surface protein C (OspC), and VisE, especially the invariable region 6 (IR6). Among these, VisE-IR6 is the most conserved region in B burgdorferi.

Early-generation assays have a sensitivity of 89% and specificity of 72%.¹¹ However, the patient’s serum may have antibodies that cross-react with unrelated bacterial antigens, leading to false-positive results (Table 3). Whole-cell sonicate assays are not recommended as an independent test and must be confirmed with Western blot testing when assay results are indeterminate or positive.¹¹

Newer-generation ELISAs detect antibodies targeting recombinant proteins of VisE, especially a synthetic peptide C6, within IR6.¹³ VisE-IR6 is the most conserved region of the B burgdorferi complex, and its detection is a highly specific finding, supporting the diagnosis of Lyme disease. Antibodies against VisE-IR6 antigen are the earliest to develop.⁵ An example of a newer-generation serologic test is the VisE C6 Lyme EIA kit, approved as a first-tier test by the US Food and Drug Administration in 2001. This test has a specificity of 99%,^14,15 and its specificity is further increased when used in conjunction with Western blot (99.5%).¹⁵ The advantage of the C6 antibody test is that it is more sensitive than 2-tier testing during early infection (sensitivity 29%–74% vs 17%–40% in early localized infection, and 56%–90% vs 27%–78% in early disseminated infection).⁶

During early infection, older and newer ELISAs are less sensitive because of the limited number of antigens expressed at this stage.¹³ All patients suspected of having early Lyme disease who are seronegative at initial testing should have follow-up testing to look for seroconversion.¹³

Western blot

Western blot (immunoblot) testing identifies IgM and IgG antibodies against specific B burgdorferi antigens. It is considered positive if it detects at least 2 of a possible 3 specific IgM bands in the first 4 weeks of disease or at least 5 of 10 specific IgG bands after 4 weeks of disease (Table 4 and Figure 2).¹⁶

The nature of the bands indicates the duration of infection: Western blot bands against 23-kD OspC and 41-kD FlaB are seen in early localized infection, whereas bands against all 3 B burgdorferi proteins will be seen after several weeks of disease.¹⁷ The IgM result should be interpreted carefully, as only 2 bands are required for the test to be positive, and IgM binds to antigen less specifically than IgG.¹²

If clinical symptoms and signs of Lyme disease have been present for more than 1 month, IgM reactivity alone should not be used to support the diagnosis, in view of the likelihood of a false-positive test result in this situation.¹⁸ This is called the “1-month rule” in the diagnosis of Lyme disease.¹³

In early localized infection, Western blot is only half as sensitive as ELISA testing. Since the overall sensitivity of a 2-step algorithm is equal to that of its least sensitive component, 2-tiered testing is not useful in early disease.¹³

Although currently considered the most specific test for confirmation of Lyme disease, Western blot has limitations. It is technically and interpretively complex and is thus not universally available.¹³ The blots are scored by visual examination, compromising the reproducibility of the test, although densitometric blot analysis techniques and automated scanning and scoring attempt to address some of these limitations.¹³ Like the ELISA, Western blot can have false-positive results in healthy individuals without tick exposure, as nonspecific IgM immunoblots develop faint bands. This is because of cross-reaction between B burgdorferi antigens and antigens from other microorganisms. Around 50% of healthy adults show low-level serum IgG reactivity against the FlaB antigen, leading to false-positive results as well. In cases in which the Western blot result is indeterminate, other etiologies must be considered.

False-positive IgM Western blots are a significant problem. In a 5-year retrospective study done at 63 US Air Force healthcare facilities, 113 (53.3%) of 212 IgM Western blots were falsely positive.¹⁹ A false-positive test was defined as one that failed to meet seropositivity (a first-tier test omitted or negative, > 30 days of symptoms with negative IgG blot), lack of exposure including residing in areas without documented tick habitats, patients having atypical or no symptoms, and negative serology within 30 days of a positive test.

In a similar study done in a highly endemic area, 50 (27.5%) of 182 patients had a false-positive test.²⁰ Physicians need to be careful when interpreting IgM Western blots. It is always important to consider locale, epidemiology, and symptoms when interpreting the test.

Limitations of serologic tests for Lyme disease

Currently available serologic tests have inherent limitations:

• Antibodies against B burgdorferi take at least 1 week to develop
• The background rate of seropositivity in endemic areas can be up to 4%, affecting the utility of a positive test result
• Serologic tests cannot be used as tests of cure because antibodies can persist for months to years even after appropriate antimicrobial therapy and cure of disease; thus, a positive serologic result could represent active infection or remote exposure²¹
• Antibodies can cross-react with related bacteria, including other borrelial or treponemal spirochetes
• False-positive serologic test results can also occur in association with other medical conditions such as polyclonal gammopathies and systemic lupus erythematosus.¹²

RECOMMENDATIONS FOR TESTING

Standard 2-tier testing

The CDC released recommendations for diagnosing Lyme disease after a second national conference of serologic diagnosis of Lyme disease in October 1994.¹⁸ The 2-tiered testing method, involving a sensitive ELISA followed by the Western blot to confirm positive and indeterminate ELISA results, was suggested as the gold standard for diagnosis (Figure 3). Of note, negative ELISA results do not require further testing.¹¹

The sensitivity of 2-tiered testing depends on the stage of the disease. Unfortunately, this method has a wide range of sensitivity (17% to 78%) in stage 1 disease. In the same stage, the sensitivity increases from 14.1% in patients with a single erythema migrans lesion and early localized infection to 65.4% in those with multiple lesions. The algorithm has excellent sensitivity in late stage 3 infection (96% to 100%).⁵

A 2-step ELISA algorithm

A 2-step ELISA algorithm (without the Western blot) that includes the whole-cell sonicate assay followed by the VisE C6 peptide assay actually showed higher sensitivity and comparable specificity compared with 2-tiered testing in early localized disease (sensitivity 61%–74% vs 29%–48%, respectively; specificity 99.5% for both methods).²² This higher sensitivity was even more pronounced in early disseminated infection (sensitivity 100% vs 40%, respectively). By late infection, the sensitivities of both testing strategies reached 100%. Compared with the Western blot, the 2-step ELISA algorithm was simpler to execute in a reproducible fashion.⁵

The Infectious Diseases Society of America is revising its current guidelines, with an update expected late this year, which may shift the recommendation from 2-tiered testing to the 2-step ELISA algorithm.

Multiplex testing

To overcome the intrinsic problems of protein-based assays, a multiplexed, array-based assay for the diagnosis of tick-borne infections called Tick-Borne Disease Serochip (TBD-Serochip) was established using recombinant antigens that identify key immunodominant epitopes.⁸ More studies are needed to establish the validity and usefulness of these tests in clinical practice.

Who should not be tested?

The American College of Physicians⁶ recommends against testing in patients:

• Presenting with nonspecific symptoms (eg, headache, myalgia, fatigue, arthralgia) without objective signs of Lyme disease
• With low pretest probability of infection based on epidemiologic exposures and clinical features
• Living in Lyme-endemic areas with no history of tick exposure⁶
• Presenting less than 1 week after tick exposure⁵
• Seeking a test of cure for treated Lyme disease.

DIAGNOSIS IN SPECIAL SITUATIONS

Early Lyme disease

The classic erythema migrans lesion on physical examination of a patient with suspected Lyme disease is diagnostic and does not require laboratory confirmation.¹⁰

In ambiguous cases, 2-tiered testing of a serum sample during the acute presentation and again 4 to 6 weeks later can be useful. In patients who remain seronegative on paired serum samples despite symptoms lasting longer than 6 weeks and no antibiotic treatment in the interim, the diagnosis of Lyme disease is unlikely, and another diagnosis should be sought.³

Antimicrobial therapy may block the serologic response; hence, negative serologic testing in patients started on empiric antibiotics should not rule out Lyme disease.⁶

PCR or bacterial culture testing is not recommended in the evaluation of suspected early Lyme disease.

Central nervous system Lyme disease

Central nervous system Lyme disease is diagnosed by 2-tiered testing using peripheral blood samples because all patients with this infectious manifestation should have mounted an adequate IgG response in the blood.¹¹

B cells migrate to and proliferate inside the central nervous system, leading to intrathecal production of anti-Borrelia antibodies. An index of cerebrospinal fluid to serum antibody greater than 1 is thus also indicative of neuroborreliosis.¹² Thus, performing lumbar puncture to detect intrathecal production of antibodies may support the diagnosis of central nervous system Lyme disease; however, it is not necessary.¹¹

Antibodies persist in the central nervous system for many years after appropriate antimicrobial treatment.

Lyme arthritis

Articular involvement in Lyme disease is characterized by a robust humoral response such that a negative IgG serologic test virtually rules out Lyme arthritis.²³ PCR testing of synovial fluid for borrelial DNA has a sensitivity of 80% but may become falsely negative after 1 to 2 months of antibiotic treatment.^24,25 In an algorithm suggested by Puius et al,²³ PCR testing of synovial fluid should be done in patients who have minimal to no response after 2 months of appropriate oral antimicrobial therapy to determine whether intravenous antibiotics are merited.

Table 5 summarizes the tests of choice in different clinical stages of infection.

Acknowledgment: The authors would like to acknowledge Anita Modi, MD, and Ceena N. Jacob, MD, for reviewing the manuscript and providing valuable suggestions, and Belinda Yen-Lieberman, PhD, for contributing pictures of the Western blot test results.

The immunosuppressed state of liver transplant recipients makes them vulnerable to infections after surgery.¹ These infections are directly correlated with the net state of immunosuppression. Higher levels of immunosuppression mean a higher risk of infection, with rates of infection typically highest in the early posttransplant period.

Common infections during this period include operative and perioperative nosocomial bacterial and fungal infections, reactivation of latent infections, and invasive fungal infections such as candidiasis, aspergillosis, and pneumocystosis. Donor-derived infections also must be considered. As time passes and the level of immunosuppression is reduced, liver recipients are less prone to infection.¹

The risk of infection can be minimized by appropriate antimicrobial prophylaxis, strategies for safe living after transplant,² vaccination,³ careful balancing of immunosuppressive therapy,⁴ and thoughtful donor selection.⁵ Drug-drug interactions are common and must be carefully considered to minimize the risk.

This review highlights common infectious complications encountered after liver transplant.

INTRA-ABDOMINAL INFECTIONS

Intra-abdominal infections are common in the early postoperative period.^6,7

Risk factors include:

• Pretransplant ascites
• Posttransplant dialysis
• Wound infection
• Reoperation⁸
• Hepatic artery thrombosis
• Roux-en-Y choledochojejunostomy anastomosis.⁹

Signs that may indicate intra-abdominal infection include fever, abdominal pain, leukocytosis, and elevated liver enzymes. But because of their immunosuppressed state, transplant recipients may not manifest fever as readily as the general population. They should be evaluated for cholangitis, peritonitis, biloma, and intra-abdominal abscess.

Organisms. Intra-abdominal infections are often polymicrobial. Enterococci, Staphylococcus aureus, gram-negative species including Pseudomonas, Klebsiella, and Acinetobacter, and Candida species are the most common pathogens. Strains are often resistant to multiple drugs, especially in patients who received antibiotics in the weeks before transplant.^8,10

Liver transplant recipients are also particularly susceptible to Clostridium difficile-associated colitis as a result of immunosuppression and frequent use of antibiotics perioperatively and postoperatively.¹¹ The spectrum of C difficile infection ranges from mild diarrhea to life-threatening colitis, and the course in liver transplant patients tends to be more complicated than in immunocompetent patients.¹²

Diagnosis. Intra-abdominal infections should be looked for and treated promptly, as they are associated with a higher mortality rate, a greater risk of graft loss, and a higher incidence of retransplant.^6,10 Abdominal ultrasonography or computed tomography (CT) can confirm the presence of fluid collections.

Treatment. Infected collections can be treated with percutaneous or surgical drainage and antimicrobial therapy. In the case of biliary tract complications, retransplant or surgical correction of biliary leakage or stenosis decreases the risk of death.⁶

Suspicion should be high for C difficile-associated colitis in cases of posttransplant diarrhea. C difficile toxin stool assays help confirm the diagnosis.¹² Oral metronidazole is recommended in mild to moderate C difficile infection, with oral vancomycin and intravenous metronidazole reserved for severe cases. Colectomy may be necessary in patients with toxic megacolon.

CYTOMEGALOVIRUS INFECTION

Cytomegalovirus is an important opportunistic pathogen in liver transplant recipients.¹³ It causes a range of manifestations, from infection (viremia with or without symptoms) to cytomegalovirus syndrome (fever, malaise, and cell-line cytopenias) to tissue-invasive disease with end-organ disease.¹⁴ Without preventive measures and treatment, cytomegalovirus disease can increase the risk of morbidity, allograft loss and death.^15,16

Risk factors for cytomegalovirus infection (Table 1) include:

• Discordant serostatus of the donor and recipient (the risk is highest in seronegative recipients of organs from seropositive donors)
• Higher levels of immunosuppression, especially when antilymphocyte antibodies are used
• Treatment of graft rejection
• Coinfection with other human herpesviruses, such as Epstein-Barr virus.^4,17

Preventing cytomegalovirus infection

The strategy to prevent cytomegalovirus infection depends on the serologic status of the donor and recipient and may include antiviral prophylaxis or preemptive treatment (Table 2).¹⁸

Prophylaxis involves giving antiviral drugs during the early high-risk period, with the goal of preventing the development of cytomegalovirus viremia. The alternative preemptive strategy emphasizes serial testing for cytomegalovirus viremia, with the goal of intervening with antiviral medications while viremia is at a low level, thus avoiding potential progression to cytomegalovirus disease. Both strategies have pros and cons that should be considered by each transplant center when setting institutional policy.

A prophylactic approach seems very effective at preventing both infection and disease from cytomegalovirus and has been shown to reduce graft rejection and the risk of death.¹⁸ It is preferred in cytomegalovirus-negative recipients when the donor was cytomegalovirus-positive—a high-risk situation.¹⁹ However, these patients are also at higher risk of late-onset cytomegalovirus disease. Higher cost and potential drug toxicity, mainly neutropenia from ganciclovir-based regimens, are additional considerations.

Preemptive treatment, in contrast, reserves drug treatment for patients who are actually infected with cytomegalovirus, thus resulting in fewer adverse drug events and lower cost; but it requires regular monitoring. Preemptive methods, by definition, cannot prevent infection, and with this strategy tissue-invasive disease not associated with viremia does occasionally occur.²⁰ As such, patients with a clinical presentation that suggests cytomegalovirus but have negative results on blood testing should be considered for tissue biopsy with culture and immunohistochemical stain.

The most commonly used regimens for antiviral prophylaxis and treatment in liver transplant recipients are intravenous ganciclovir and oral valganciclovir.²¹ Although valganciclovir is the most commonly used agent in this setting because of ease of administration, it has not been approved by the US Food and Drug Administration in liver transplant patients, as it was associated with higher rates of cytomegalovirus tissue-invasive disease.^22–24 Additionally, drug-resistant cytomegalovirus strains have been associated with valganciclovir prophylaxis in cytomegalovirus-negative recipients of solid organs from cytomegalovirus-positive donors.²⁵

Prophylaxis typically consists of therapy for 3 months from the time of transplant. In higher-risk patients (donor-positive, recipient-negative), longer courses of prophylaxis have been extrapolated from data in kidney transplant recipients.²⁶ Extension or reinstitution of prophylaxis should also be considered in liver transplant patients receiving treatment for rejection with antilymphocyte therapy.

Routine screening for cytomegalovirus is not recommended while patients are receiving prophylaxis. High-risk patients who are not receiving prophylaxis should be monitored with nucleic acid or pp65 antigenemia testing as part of the preemptive strategy protocol.

Treatment of cytomegalovirus disease

Although no specific threshold has been established, treatment is generally indicated if a patient has a consistent clinical syndrome, evidence of tissue injury, and persistent or increasing viremia.

Treatment involves giving antiviral drugs and also reducing the level of immunosuppression, if possible, until symptoms and viremia have resolved.

The choice of antiviral therapy depends on the severity of disease. Intravenous ganciclovir (5 mg/kg twice daily adjusted for renal impairment) or oral valganciclovir (900 mg twice daily, also renally dose-adjusted when necessary) can be used for mild to moderate disease if no significant gastrointestinal involvement is reported. Intravenous ganciclovir is preferred for patients with more severe disease or gastrointestinal involvement. The minimum duration of treatment is 2 weeks and may need to be prolonged until both symptoms and viremia completely resolve.¹⁸

Drug resistance can occur and should be considered in patients who have a history of prolonged ganciclovir or valganciclovir exposure who do not clinically improve or have persistent or rising viremia. In such cases, genotype assays are helpful, and initiation of alternative therapy should be considered. Mutations conferring resistance to ganciclovir are often associated with cross-resistance to cidofovir. Cidofovir can therefore be considered only when genotype assays demonstrate specific mutations conferring an isolated resistance to ganciclovir.²⁷ The addition of foscarnet to the ganciclovir regimen or substitution of foscarnet for ganciclovir are accepted approaches.

Although cytomegalovirus hyperimmunoglobulin has been used in prophylaxis and invasive disease treatment, its role in the management of ganciclovir-resistant cytomegalovirus infections remains controversial.²⁸

EPSTEIN-BARR VIRUS POSTTRANSPLANT LYMPHOPROLIFERATIVE DISEASE

Epstein-Barr virus-associated posttransplant lymphoproliferative disease is a spectrum of disorders ranging from an infectious mononucleosis syndrome to aggressive malignancy with the potential for death and significant morbidity after liver transplant.²⁹ The timeline of risk varies, but the disease is most common in the first year after transplant.

Risk factors for this disease (Table 1) are:

• Primary Epstein-Barr virus infection
• Cytomegalovirus donor-recipient mismatch
• Cytomegalovirus disease
• Higher levels of immunosuppression, especially with antilymphocyte antibodies.³⁰

The likelihood of Epstein-Barr virus playing a contributing role is lower in later-onset posttransplant lymphoproliferative disease. Patients who are older at the time of transplant, who receive highly immunogenic allografts including a liver as a component of a multivisceral transplant, and who receive increased immunosuppression to treat rejection are at even greater risk of late posttransplant lymphoproliferative disease.³¹ This is in contrast to early posttransplant lymphoproliferative disease, which is seen more commonly in children as a result of primary Epstein-Barr virus infection.

Recognition and diagnosis. Heightened suspicion is required when considering posttransplant lymphoproliferative disease, and careful evaluation of consistent symptoms and allograft dysfunction are required.

Clinically, posttransplant lymphoproliferative disease should be suspected if a liver transplant recipient develops unexplained fever, weight loss, lymphadenopathy, or cell-line cytopenias.^30,32 Other signs and symptoms may be related to the organ involved and may include evidence of hepatitis, pneumonitis, and gastrointestinal disease.³¹

Adjunctive diagnostic testing includes donor and recipient serology to characterize overall risk before transplantation and quantification of Epstein-Barr viral load, but confirmation relies on tissue histopathology.

Treatment focuses on reducing immunosuppression.^30,32 Adding antiviral agents does not seem to improve outcome in all cases.³³ Depending on clinical response and histologic classification, additional therapies such as anti-CD20 humanized chimeric monoclonal antibodies, surgery, radiation, and conventional chemotherapy may be required.³⁴

Preventive approaches remain controversial. Chemoprophylaxis with an antiviral such as ganciclovir is occasionally used but has not been shown to consistently decrease rates of posttransplant lymphoproliferative disease. These agents may act in an indirect manner, leading to decreased rates of cytomegalovirus infection, a major cofactor for posttransplant lymphoproliferative disease.²⁴

Although oral valganciclovir is used more than intravenous ganciclovir, it is not approved for liver transplant patients

Passive immunoprophylaxis with immunoglobulin targeting cytomegalovirus has shown to decrease rates of non-Hodgkin lymphoma from posttransplant lymphoproliferative disease in renal transplant recipients in the first year after transplant,³⁵ but data are lacking regarding its use in liver transplant recipients. Monitoring of the viral load and subsequent reduction of immunosuppression remain the most efficient measures to date.³⁶

FUNGAL INFECTIONS

Candida species account for more than half of fungal infections in liver transplant recipients.³⁷ However, a change has been noted in the past 20 years, with a decrease in Candida infections accompanied by an increase in Aspergillus infections.³⁸ Endemic mycoses such as coccidioidomycosis, blastomycosis, and histoplasmosis should be considered with the appropriate epidemiologic history or if disease develops early after transplant and the donor came from a highly endemic region.³⁹Cryptococcus may also be encountered.

Diagnosis. One of the most challenging aspects of fungal infection in liver transplant recipients is timely diagnosis. Heightened suspicion and early biopsy for pathological and microbiological confirmation are necessary. Although available noninvasive diagnostic tools often lack specificity, early detection of fungal markers may be of great use in guiding further diagnostic workup or empiric treatment in the critically ill.

Noninvasive tests include galactomannan, cryptococcal antigen, histoplasma antigen, (1-3)-beta-D-glucan assay and various antibody tests. Galactomannan testing has been widely used to aid in the diagnosis of invasive aspergillosis. Similarly, the (1-3)-beta-D-glucan assay is a non–culture-based tool for diagnosing and monitoring the treatment of invasive fungal infections. However, a definite diagnosis cannot be made on the basis of a positive test alone.⁴⁰ The complementary diagnostic characteristics of combining noninvasive assays have yet to be fully elucidated.⁴¹ Cultures and tissue histopathology are also used when possible.

Treatment is based on targeted specific antifungal drug therapy and reduction of immunosuppressive therapy, when possible. The choice of antifungal agent varies with the pathogen, the site of involvement, and the severity of the disease. A focus on potential drug interactions, their management, and therapeutic drug monitoring when using antifungal medications is essential in the posttransplant period. Combination therapy can be considered in some situations to enhance synergy. The following sections discuss in greater detail Candida species, Aspergillus species, and Pneumocystis jirovecii infections.

Candida infections

Candidiasis after liver transplant is typically nosocomial, especially when diagnosed during the first 3 months (Table 3).³⁷

Risk factors for invasive candidiasis include perioperative colonization, prolonged operative time, retransplant, greater transfusion requirements, and postoperative renal failure.^37,42,43 Invasive candidiasis is of concern for its effects on morbidity, mortality, and cost of care.^43–46

Organisms. The frequency of implicated species, in particular those with a natural resistance to fluconazole, differs in various reports.^37,45,46Candida albicans remains the most commonly isolated pathogen; however, non-albicans species including those resistant to fluconazole have been reported more frequently and include Candida glabrata and Candida krusei.^47,48

Signs and diagnosis. Invasive candidiasis in liver transplant recipients generally manifests itself in catheter-related blood stream infections, urinary tract infections, or intra-abdominal infections. Diagnosis can be made by isolating Candida from blood cultures, recovering organisms in culture of a normally sterile site, or finding direct microscopic evidence of the fungus on tissue specimens.⁴⁹

Disseminated candidiasis refers to the involvement of distant anatomic sites. Clinical manifestations may cause vision changes, abdominal pain or skin nodules with findings of candidemia, hepatosplenic abscesses, or retinal exudates on funduscopy.⁴⁹

Treatment of invasive candidiasis in liver recipients often involves antifungal therapy and reduction of immunosuppression. Broad-spectrum antifungals are initially advocated in an empirical approach to cover fluconazole-resistant strains of the non-albicans subgroups.⁵⁰ Depending on antifungal susceptibility, treatment can later be adjusted.

Fluconazole remains the agent of choice in most C albicans infections.⁴⁷ However, attention should be paid to the possibility of resistance in patients who have received fluconazole prophylaxis within the past 30 days. Additional agents used in treatment may include echinocandins, amphotericin, and additional azoles.

Antifungal prophylaxis is recommended in high-risk liver transplant patients, although its optimal duration remains undetermined.⁴⁴ Antifungal prophylaxis has been associated with decreased incidence of both superficial and invasive candidiasis.⁵¹

Aspergillus infection

Aspergillus, the second most common fungal pathogen, has become a more common concern in liver transplant recipients. Aspergillus fumigatus is the most frequently encountered species.^38,52

Risk factors. These infections typically occur in the first year, during intense immunosuppression. Retransplant, renal failure, and fulminant hepatic failure are major risk factors.⁵² In the presence of risk factors and a suggestive clinical setting, invasive aspergillosis should be considered and the diagnosis pursued.

Diagnosis is suggested by positive findings on CT accompanied by lower respiratory tract symptoms, focal lesions on neuroimaging, or demonstration of the fungus on cultures.⁴⁹ However, Aspergillus is rarely grown in blood culture. The galactomannan antigen is a noninvasive test that can provide supporting evidence for the diagnosis.^41,52 False-positive results do occur in the setting of certain antibiotics and cross-reacting fungi.⁵³

Treatment consists of antifungal therapy and immunosuppression reduction.⁵²

Candida accounts for more than half of fungal infections in liver transplant recipients, but Aspergillus is gaining

Voriconazole is the first-line agent for invasive aspergillosis. Monitoring for potential drug-drug interactions and side effects is required.^54,55 Amphotericin B is considered a second-line choice due to toxicity and lack of an oral formulation. In refractory cases, combined antifungal therapy could be considered.⁵² The duration of treatment is generally a minimum of 12 weeks.

Prophylaxis. Specific prophylaxis against invasive aspergillosis is not currently recommended; however, some authors suggest a prophylactic approach using echinocandins or liposomal amphotericin B in high-risk patients.^51,52 Aspergillosis is associated with a considerable increase in mortality in liver transplant recipients, which highlights the importance of timely management.^52,56

Pneumocystis jirovecii

P jirovecii remains a common opportunistic pathogen in people with impaired immunity, including transplant and human immunodeficiency virus patients.

Prophylaxis. Widespread adoption of antimicrobial prophylaxis by transplant centers has decreased the rates of P jirovecii infection in liver transplant recipients.^57,58 Commonly used prophylactic regimens after liver transplantation include a single-strength trimeth­oprim-sulfamethoxazole tablet daily or a double-strength tablet three times per week for a minimum of 6 to 12 months after transplant. Atovaquone and dapsone can be used as alternatives in cases of intolerance to tri­methoprim-sulfamethoxazole (Table 2).

Inhaled pentamidine is clearly inferior and should be used only when the other medications are contraindicated.⁵⁹

Signs and diagnosis. P jirovecii pneumonia is characterized by fever, cough, dyspnea, and chest pain. Insidious hypoxemia, abnormal chest examination, and bilateral interstitial pneumonia on chest radiography are common.

CT may be more sensitive than chest radiography.⁵⁷ Findings suggestive of P jirovecii pneumonia on chest CT are extensive bilateral and symmetrical ground-glass attenuations. Other less-characteristic findings include upper lobar parenchymal opacities and spontaneous pneumothorax.^57,60

The serum (1,3)-beta-D-glucan assay derived from major cell-wall components of P jiro­vecii might be helpful. Studies report a sensitivity for P jirovecii pneumonia as high as 96% and a negative predictive value of 99.8%.^61,62

Definitive diagnosis requires identification of the pathogen. Routine expectorated sputum sampling is generally associated with a poor diagnostic yield. Bronchoscopy and bronchoalveolar lavage with silver or fluorescent antibody staining of samples, polymerase chain reaction testing, or both significantly improves diagnosis. Transbronchial or open lung biopsy are often unnecessary.⁵⁷

Treatment. Trimethoprim-sulfamethoxazole is the first-line agent for treating P jirovecii pneumonia.⁵⁷ The minimum duration of treatment is 14 days, with extended courses for severe infection.

Intravenous pentamidine or clindamycin plus primaquine are alternatives for patients who cannot tolerate trimethoprim-sulfamethoxazole. The major concern with intravenous pentamidine is renal dysfunction. Hypoglycemia or hyperglycemia, neutropenia, thrombocytopenia, nausea, dysgeusia, and pancreatitis may also occur.⁶³

Atovaquone might also be beneficial in mild to moderate P jirovecii pneumonia. The main side effects include skin rashes, gastrointestinal intolerance, and elevation of transaminases.⁶⁴

A corticosteroid (40–60 mg of prednisone or its equivalent) may be beneficial in conjunction with antimicrobial therapy in patients with significant hypoxia (partial pressure of arterial oxygen < 70 mm Hg on room air) in decreasing the risk of respiratory failure and need for intubation.

With appropriate and timely antimicrobial prophylaxis, cases of P jirovecii pneumonia should continue to decrease.

TUBERCULOSIS

Development of tuberculosis after transplantation is a catastrophic complication, with mortality rates of up to 30%.⁶⁵ Most cases of posttransplant tuberculosis represent reactivation of latent disease.⁶⁶ Screening with tuberculin skin tests or interferon-gamma-release assays is recommended in all liver transplant candidates. Chest radiography before transplant is necessary when assessing a positive screening test.⁶⁷

The optimal management of latent tuberculosis in these cases remains controversial. Patients at high risk or those with positive screening results on chest radiography warrant treatment for latent tuberculosis infection with isoniazid unless contraindicated.^67,68

The ideal time to initiate prophylactic isoniazid therapy is unclear. Some authors suggest delaying it, as it might be associated with poor tolerance and hepatotoxicity.⁶⁹ Others have found that early isoniazid use was not associated with negative outcomes.⁷⁰

Risk factors for symptomatic tuberculosis after liver transplant include previous infection with tuberculosis, intensified immunosuppression (especially anti-T-lymphocyte therapies), diabetes mellitus, and other co-infections (Table 1).⁷¹

The increased incidence of atypical presentations in recent years makes the diagnosis of active tuberculosis among liver transplant recipients challenging. Sputum smears can be negative due to low mycobacterial burdens, and tuberculin skin testing and interferon-gamma-release assays may be falsely negative due to immunosuppression.⁶⁷

Treatment of active tuberculosis consists initially of a four-drug regimen using isoniazid, rifampin, pyrazinamide, and ethambutol for 2 months. Adjustments are made in accordance with culture and sensitivity results. Treatment can then be tapered to two drugs (isoniazid and rifampin) for a minimum of 4 additional months. Prolonged treatment may be required in instances of extrapulmonary or disseminated disease.^65,72

Tuberculosis treatment can be complicated by hepatotoxicity in liver transplant recipients because of direct drug effects and drug-drug interactions with immunosuppressive agents. Close monitoring for rejection and hepatotoxicity is therefore imperative while liver transplant recipients are receiving antituberculosis therapy. Drug-drug interactions may also be responsible for marked reductions in immunosuppression levels, especially with regimens containing rifampin.⁷¹ Substitution of rifabutin for rifampin reduces the effect of drug interactions.⁶⁶

VIRAL HEPATITIS

Hepatitis B virus

Hepatitis B virus-related end-stage liver disease and hepatocellular carcinoma are common indications for liver transplant in Asia. It is less common in the United States and Europe, accounting for less than 10% of all liver transplant cases. Prognosis is favorable in recipients undergoing liver transplant for hepatitis B virus, with excellent survival rates. Prevention of reinfection is crucial in these patients.

Treatment with combination antiviral agents and hepatitis B immunoglobulin (HBIG) is effective.⁷³ Lamivudine was the first nucleoside analogue found to be effective against hepatitis B virus. Its low cost and relative safety are strong arguments in favor of its continued use in liver transplant recipients.⁷⁴ In patients without evidence of hepatitis B viral replication at the time of transplant, monotherapy with lamivudine has led to low recurrence rates, and adefovir can be added to control resistant viral strains.⁷⁵

Widespread adoption of prophylaxis has decreased the rate of P jirovecii infection in liver transplant recipients

The frequent emergence of resistance with lamivudine favors newer agents such as entecavir or tenofovir. These nucleoside and nucleotide analogues have a higher barrier to resistance, and thus resistance to them is rare. They are also more efficient, potentially allowing use of an HBIG-sparing protocol.⁷⁶ However, they are associated with a higher risk of nephrotoxicity and require dose adjustments in renal insufficiency. Data directly comparing entecavir and tenofovir are scarce.

Prophylaxis. Most studies support an individualized approach for prevention of hepatitis B virus reinfection. High-risk patients, ie, those positive for HBe antigen or with high viral loads (> 100,000 copies/mL) are generally treated with both HBIG and antiviral agents.⁷⁷ Low-risk patients are those with a negative HBe antigen, low hepatitis B virus DNA levels, hepatitis B virus-related acute liver failure, and cirrhosis resulting from coinfection with both hepatitis B and hepatitis D virus.⁷⁵ In low-risk patients, discontinuation of HBIG after 1 to 2 years of treatment is appropriate, and long-term prophylaxis with antiviral agents alone is an option. However, levels of hepatitis B DNA should be monitored closely.^78,79

Hepatitis C virus

Recurrence of hepatitis C virus infection is the rule among patients who are viremic at the time of liver transplant.^80,81 Most of these patients will show histologic evidence of recurrent hepatitis within the first year after liver transplant. It is often difficult to distinguish between the histopathological appearance of a recurrent hepatitis C virus infection and acute cellular rejection.

Progression to fibrosis and subsequently cirrhosis and decompensation is highly variable in hepatitis C virus-infected liver transplant recipients. Diabetes, insulin resistance, and possibly hepatitis steatosis have been associated with a rapid progression to advanced fibrosis. The contribution of immunosuppression to the progression of hepatitis C virus remains an area of active study. Some studies point to antilymphocyte immunosuppressive agents as a potential cause.⁸² Liver biopsy is a useful tool in this situation. It allows monitoring of disease severity and progression and may distinguish recurrent hepatitis C virus disease from other causes of liver enzyme elevation.

The major concern with the recurrence of hepatitis C virus infection after liver transplant is allograft loss. Rates of patient and graft survival are reduced in infected patients compared with hepatitis C virus-negative patients.^83,84 Prophylactic antiviral therapy has no current role in the management of hepatitis C virus disease. Those manifesting moderate to severe necroinflammation or mild to moderate fibrosis indicative of progressive disease should be treated.^81,85

Sustained viral clearance with antiviral agents confers a graft survival benefit.

The combination of peg-interferon and weight-based ribavirin has been the standard of treatment but may be associated with increased rates of rejection.^86,87 The sustained virologic response rates for hepatitis C virus range from 60% in genotypes 4, 5, and 6 after 48 weeks of treatment to 60% to 80% in genotypes 2 and 3 after 24 weeks, but only about 30% in genotype 1.⁸⁸

The major concern with hepatitis C recurrence after liver transplant is allograft loss

Treatment with the newer agents, especially protease inhibitors, in genotype 1 (peg-interferon, ribavirin, and either telaprevir or boceprevir) has been evaluated. Success rates reaching 70% have been achieved.⁸⁹ Adverse effects can be a major setback. Serious complications include severe anemia, renal dysfunction, increased risk of infection, and death.

Triple therapy should be carefully considered in liver transplant patients with genotype 1 hepatitis C virus.⁹⁰ Significant drug-drug interactions are reported between hepatitis C virus protease inhibitors and immunosuppression regimens. Additional new oral direct- acting antivirals have been investigated. They bring promising advances in hepatitis C virus treatment and pave the way for interferon-free regimens with pangenotypic activity.

IMMUNIZATION

Immunization can decrease the risk of infectious complications in liver transplant recipients, as well as in close contacts and healthcare professionals.³

Influenza. Pretransplant influenza vaccine and posttransplant annual influenza vaccines are necessary.

Pneumococcal immunization should additionally be provided prior to transplant and repeated every 3 to 5 years thereafter.^3,91

A number of other vaccinations should also be completed before transplant, including the hepatitis A and B vaccines and the tetanus/diphtheria/acellular pertussis vaccines. However, these vaccinations have not been shown to be detrimental to patients after transplant.⁹¹

Varicella and zoster vaccines should be given before liver transplant—zoster in patients over age 60, and varicella in patients with no immunity. Live vaccines, including varicella and zoster vaccines, are contraindicated after liver transplant.³

Human papillomavirus. The bivalent human papillomavirus vaccine can be given before transplant in females ages 9 to 26; the quadrivalent vaccine is beneficial in those ages 9 to 26 and in women under age 45.^3,91

IMMUNOSUPPRESSION CARRIES RISK OF INFECTION

Most liver transplant patients require prolonged immunosuppressive therapy. This comes with an increased risk of new or recurrent infections, potentially causing death and significant morbidity.

Evaluation of existing risk factors, appropriate prophylaxis and immunization, timely diagnosis, and treatment of such infections are therefore essential steps for the successful management of liver transplant recipients.

The immunosuppressed state of liver transplant recipients makes them vulnerable to infections after surgery.¹ These infections are directly correlated with the net state of immunosuppression. Higher levels of immunosuppression mean a higher risk of infection, with rates of infection typically highest in the early posttransplant period.

Common infections during this period include operative and perioperative nosocomial bacterial and fungal infections, reactivation of latent infections, and invasive fungal infections such as candidiasis, aspergillosis, and pneumocystosis. Donor-derived infections also must be considered. As time passes and the level of immunosuppression is reduced, liver recipients are less prone to infection.¹

The risk of infection can be minimized by appropriate antimicrobial prophylaxis, strategies for safe living after transplant,² vaccination,³ careful balancing of immunosuppressive therapy,⁴ and thoughtful donor selection.⁵ Drug-drug interactions are common and must be carefully considered to minimize the risk.

This review highlights common infectious complications encountered after liver transplant.

INTRA-ABDOMINAL INFECTIONS

Intra-abdominal infections are common in the early postoperative period.^6,7

Risk factors include:

• Pretransplant ascites
• Posttransplant dialysis
• Wound infection
• Reoperation⁸
• Hepatic artery thrombosis
• Roux-en-Y choledochojejunostomy anastomosis.⁹

Signs that may indicate intra-abdominal infection include fever, abdominal pain, leukocytosis, and elevated liver enzymes. But because of their immunosuppressed state, transplant recipients may not manifest fever as readily as the general population. They should be evaluated for cholangitis, peritonitis, biloma, and intra-abdominal abscess.

Organisms. Intra-abdominal infections are often polymicrobial. Enterococci, Staphylococcus aureus, gram-negative species including Pseudomonas, Klebsiella, and Acinetobacter, and Candida species are the most common pathogens. Strains are often resistant to multiple drugs, especially in patients who received antibiotics in the weeks before transplant.^8,10

Liver transplant recipients are also particularly susceptible to Clostridium difficile-associated colitis as a result of immunosuppression and frequent use of antibiotics perioperatively and postoperatively.¹¹ The spectrum of C difficile infection ranges from mild diarrhea to life-threatening colitis, and the course in liver transplant patients tends to be more complicated than in immunocompetent patients.¹²

Diagnosis. Intra-abdominal infections should be looked for and treated promptly, as they are associated with a higher mortality rate, a greater risk of graft loss, and a higher incidence of retransplant.^6,10 Abdominal ultrasonography or computed tomography (CT) can confirm the presence of fluid collections.

Treatment. Infected collections can be treated with percutaneous or surgical drainage and antimicrobial therapy. In the case of biliary tract complications, retransplant or surgical correction of biliary leakage or stenosis decreases the risk of death.⁶

Suspicion should be high for C difficile-associated colitis in cases of posttransplant diarrhea. C difficile toxin stool assays help confirm the diagnosis.¹² Oral metronidazole is recommended in mild to moderate C difficile infection, with oral vancomycin and intravenous metronidazole reserved for severe cases. Colectomy may be necessary in patients with toxic megacolon.

CYTOMEGALOVIRUS INFECTION

Cytomegalovirus is an important opportunistic pathogen in liver transplant recipients.¹³ It causes a range of manifestations, from infection (viremia with or without symptoms) to cytomegalovirus syndrome (fever, malaise, and cell-line cytopenias) to tissue-invasive disease with end-organ disease.¹⁴ Without preventive measures and treatment, cytomegalovirus disease can increase the risk of morbidity, allograft loss and death.^15,16

Risk factors for cytomegalovirus infection (Table 1) include:

• Discordant serostatus of the donor and recipient (the risk is highest in seronegative recipients of organs from seropositive donors)
• Higher levels of immunosuppression, especially when antilymphocyte antibodies are used
• Treatment of graft rejection
• Coinfection with other human herpesviruses, such as Epstein-Barr virus.^4,17

Preventing cytomegalovirus infection

The strategy to prevent cytomegalovirus infection depends on the serologic status of the donor and recipient and may include antiviral prophylaxis or preemptive treatment (Table 2).¹⁸

Prophylaxis involves giving antiviral drugs during the early high-risk period, with the goal of preventing the development of cytomegalovirus viremia. The alternative preemptive strategy emphasizes serial testing for cytomegalovirus viremia, with the goal of intervening with antiviral medications while viremia is at a low level, thus avoiding potential progression to cytomegalovirus disease. Both strategies have pros and cons that should be considered by each transplant center when setting institutional policy.

A prophylactic approach seems very effective at preventing both infection and disease from cytomegalovirus and has been shown to reduce graft rejection and the risk of death.¹⁸ It is preferred in cytomegalovirus-negative recipients when the donor was cytomegalovirus-positive—a high-risk situation.¹⁹ However, these patients are also at higher risk of late-onset cytomegalovirus disease. Higher cost and potential drug toxicity, mainly neutropenia from ganciclovir-based regimens, are additional considerations.

Preemptive treatment, in contrast, reserves drug treatment for patients who are actually infected with cytomegalovirus, thus resulting in fewer adverse drug events and lower cost; but it requires regular monitoring. Preemptive methods, by definition, cannot prevent infection, and with this strategy tissue-invasive disease not associated with viremia does occasionally occur.²⁰ As such, patients with a clinical presentation that suggests cytomegalovirus but have negative results on blood testing should be considered for tissue biopsy with culture and immunohistochemical stain.

The most commonly used regimens for antiviral prophylaxis and treatment in liver transplant recipients are intravenous ganciclovir and oral valganciclovir.²¹ Although valganciclovir is the most commonly used agent in this setting because of ease of administration, it has not been approved by the US Food and Drug Administration in liver transplant patients, as it was associated with higher rates of cytomegalovirus tissue-invasive disease.^22–24 Additionally, drug-resistant cytomegalovirus strains have been associated with valganciclovir prophylaxis in cytomegalovirus-negative recipients of solid organs from cytomegalovirus-positive donors.²⁵

Prophylaxis typically consists of therapy for 3 months from the time of transplant. In higher-risk patients (donor-positive, recipient-negative), longer courses of prophylaxis have been extrapolated from data in kidney transplant recipients.²⁶ Extension or reinstitution of prophylaxis should also be considered in liver transplant patients receiving treatment for rejection with antilymphocyte therapy.

Routine screening for cytomegalovirus is not recommended while patients are receiving prophylaxis. High-risk patients who are not receiving prophylaxis should be monitored with nucleic acid or pp65 antigenemia testing as part of the preemptive strategy protocol.

Treatment of cytomegalovirus disease

Although no specific threshold has been established, treatment is generally indicated if a patient has a consistent clinical syndrome, evidence of tissue injury, and persistent or increasing viremia.

Treatment involves giving antiviral drugs and also reducing the level of immunosuppression, if possible, until symptoms and viremia have resolved.

The choice of antiviral therapy depends on the severity of disease. Intravenous ganciclovir (5 mg/kg twice daily adjusted for renal impairment) or oral valganciclovir (900 mg twice daily, also renally dose-adjusted when necessary) can be used for mild to moderate disease if no significant gastrointestinal involvement is reported. Intravenous ganciclovir is preferred for patients with more severe disease or gastrointestinal involvement. The minimum duration of treatment is 2 weeks and may need to be prolonged until both symptoms and viremia completely resolve.¹⁸

Drug resistance can occur and should be considered in patients who have a history of prolonged ganciclovir or valganciclovir exposure who do not clinically improve or have persistent or rising viremia. In such cases, genotype assays are helpful, and initiation of alternative therapy should be considered. Mutations conferring resistance to ganciclovir are often associated with cross-resistance to cidofovir. Cidofovir can therefore be considered only when genotype assays demonstrate specific mutations conferring an isolated resistance to ganciclovir.²⁷ The addition of foscarnet to the ganciclovir regimen or substitution of foscarnet for ganciclovir are accepted approaches.

Although cytomegalovirus hyperimmunoglobulin has been used in prophylaxis and invasive disease treatment, its role in the management of ganciclovir-resistant cytomegalovirus infections remains controversial.²⁸

EPSTEIN-BARR VIRUS POSTTRANSPLANT LYMPHOPROLIFERATIVE DISEASE

Epstein-Barr virus-associated posttransplant lymphoproliferative disease is a spectrum of disorders ranging from an infectious mononucleosis syndrome to aggressive malignancy with the potential for death and significant morbidity after liver transplant.²⁹ The timeline of risk varies, but the disease is most common in the first year after transplant.

Risk factors for this disease (Table 1) are:

• Primary Epstein-Barr virus infection
• Cytomegalovirus donor-recipient mismatch
• Cytomegalovirus disease
• Higher levels of immunosuppression, especially with antilymphocyte antibodies.³⁰

The likelihood of Epstein-Barr virus playing a contributing role is lower in later-onset posttransplant lymphoproliferative disease. Patients who are older at the time of transplant, who receive highly immunogenic allografts including a liver as a component of a multivisceral transplant, and who receive increased immunosuppression to treat rejection are at even greater risk of late posttransplant lymphoproliferative disease.³¹ This is in contrast to early posttransplant lymphoproliferative disease, which is seen more commonly in children as a result of primary Epstein-Barr virus infection.

Recognition and diagnosis. Heightened suspicion is required when considering posttransplant lymphoproliferative disease, and careful evaluation of consistent symptoms and allograft dysfunction are required.

Clinically, posttransplant lymphoproliferative disease should be suspected if a liver transplant recipient develops unexplained fever, weight loss, lymphadenopathy, or cell-line cytopenias.^30,32 Other signs and symptoms may be related to the organ involved and may include evidence of hepatitis, pneumonitis, and gastrointestinal disease.³¹

Adjunctive diagnostic testing includes donor and recipient serology to characterize overall risk before transplantation and quantification of Epstein-Barr viral load, but confirmation relies on tissue histopathology.

Treatment focuses on reducing immunosuppression.^30,32 Adding antiviral agents does not seem to improve outcome in all cases.³³ Depending on clinical response and histologic classification, additional therapies such as anti-CD20 humanized chimeric monoclonal antibodies, surgery, radiation, and conventional chemotherapy may be required.³⁴

Preventive approaches remain controversial. Chemoprophylaxis with an antiviral such as ganciclovir is occasionally used but has not been shown to consistently decrease rates of posttransplant lymphoproliferative disease. These agents may act in an indirect manner, leading to decreased rates of cytomegalovirus infection, a major cofactor for posttransplant lymphoproliferative disease.²⁴

Although oral valganciclovir is used more than intravenous ganciclovir, it is not approved for liver transplant patients

Passive immunoprophylaxis with immunoglobulin targeting cytomegalovirus has shown to decrease rates of non-Hodgkin lymphoma from posttransplant lymphoproliferative disease in renal transplant recipients in the first year after transplant,³⁵ but data are lacking regarding its use in liver transplant recipients. Monitoring of the viral load and subsequent reduction of immunosuppression remain the most efficient measures to date.³⁶

FUNGAL INFECTIONS

Candida species account for more than half of fungal infections in liver transplant recipients.³⁷ However, a change has been noted in the past 20 years, with a decrease in Candida infections accompanied by an increase in Aspergillus infections.³⁸ Endemic mycoses such as coccidioidomycosis, blastomycosis, and histoplasmosis should be considered with the appropriate epidemiologic history or if disease develops early after transplant and the donor came from a highly endemic region.³⁹Cryptococcus may also be encountered.

Diagnosis. One of the most challenging aspects of fungal infection in liver transplant recipients is timely diagnosis. Heightened suspicion and early biopsy for pathological and microbiological confirmation are necessary. Although available noninvasive diagnostic tools often lack specificity, early detection of fungal markers may be of great use in guiding further diagnostic workup or empiric treatment in the critically ill.

Noninvasive tests include galactomannan, cryptococcal antigen, histoplasma antigen, (1-3)-beta-D-glucan assay and various antibody tests. Galactomannan testing has been widely used to aid in the diagnosis of invasive aspergillosis. Similarly, the (1-3)-beta-D-glucan assay is a non–culture-based tool for diagnosing and monitoring the treatment of invasive fungal infections. However, a definite diagnosis cannot be made on the basis of a positive test alone.⁴⁰ The complementary diagnostic characteristics of combining noninvasive assays have yet to be fully elucidated.⁴¹ Cultures and tissue histopathology are also used when possible.

Treatment is based on targeted specific antifungal drug therapy and reduction of immunosuppressive therapy, when possible. The choice of antifungal agent varies with the pathogen, the site of involvement, and the severity of the disease. A focus on potential drug interactions, their management, and therapeutic drug monitoring when using antifungal medications is essential in the posttransplant period. Combination therapy can be considered in some situations to enhance synergy. The following sections discuss in greater detail Candida species, Aspergillus species, and Pneumocystis jirovecii infections.

Candida infections

Candidiasis after liver transplant is typically nosocomial, especially when diagnosed during the first 3 months (Table 3).³⁷

Risk factors for invasive candidiasis include perioperative colonization, prolonged operative time, retransplant, greater transfusion requirements, and postoperative renal failure.^37,42,43 Invasive candidiasis is of concern for its effects on morbidity, mortality, and cost of care.^43–46

Organisms. The frequency of implicated species, in particular those with a natural resistance to fluconazole, differs in various reports.^37,45,46Candida albicans remains the most commonly isolated pathogen; however, non-albicans species including those resistant to fluconazole have been reported more frequently and include Candida glabrata and Candida krusei.^47,48

Signs and diagnosis. Invasive candidiasis in liver transplant recipients generally manifests itself in catheter-related blood stream infections, urinary tract infections, or intra-abdominal infections. Diagnosis can be made by isolating Candida from blood cultures, recovering organisms in culture of a normally sterile site, or finding direct microscopic evidence of the fungus on tissue specimens.⁴⁹

Disseminated candidiasis refers to the involvement of distant anatomic sites. Clinical manifestations may cause vision changes, abdominal pain or skin nodules with findings of candidemia, hepatosplenic abscesses, or retinal exudates on funduscopy.⁴⁹

Treatment of invasive candidiasis in liver recipients often involves antifungal therapy and reduction of immunosuppression. Broad-spectrum antifungals are initially advocated in an empirical approach to cover fluconazole-resistant strains of the non-albicans subgroups.⁵⁰ Depending on antifungal susceptibility, treatment can later be adjusted.

Fluconazole remains the agent of choice in most C albicans infections.⁴⁷ However, attention should be paid to the possibility of resistance in patients who have received fluconazole prophylaxis within the past 30 days. Additional agents used in treatment may include echinocandins, amphotericin, and additional azoles.

Antifungal prophylaxis is recommended in high-risk liver transplant patients, although its optimal duration remains undetermined.⁴⁴ Antifungal prophylaxis has been associated with decreased incidence of both superficial and invasive candidiasis.⁵¹

Aspergillus infection

Aspergillus, the second most common fungal pathogen, has become a more common concern in liver transplant recipients. Aspergillus fumigatus is the most frequently encountered species.^38,52

Risk factors. These infections typically occur in the first year, during intense immunosuppression. Retransplant, renal failure, and fulminant hepatic failure are major risk factors.⁵² In the presence of risk factors and a suggestive clinical setting, invasive aspergillosis should be considered and the diagnosis pursued.

Diagnosis is suggested by positive findings on CT accompanied by lower respiratory tract symptoms, focal lesions on neuroimaging, or demonstration of the fungus on cultures.⁴⁹ However, Aspergillus is rarely grown in blood culture. The galactomannan antigen is a noninvasive test that can provide supporting evidence for the diagnosis.^41,52 False-positive results do occur in the setting of certain antibiotics and cross-reacting fungi.⁵³

Treatment consists of antifungal therapy and immunosuppression reduction.⁵²

Candida accounts for more than half of fungal infections in liver transplant recipients, but Aspergillus is gaining

Voriconazole is the first-line agent for invasive aspergillosis. Monitoring for potential drug-drug interactions and side effects is required.^54,55 Amphotericin B is considered a second-line choice due to toxicity and lack of an oral formulation. In refractory cases, combined antifungal therapy could be considered.⁵² The duration of treatment is generally a minimum of 12 weeks.

Prophylaxis. Specific prophylaxis against invasive aspergillosis is not currently recommended; however, some authors suggest a prophylactic approach using echinocandins or liposomal amphotericin B in high-risk patients.^51,52 Aspergillosis is associated with a considerable increase in mortality in liver transplant recipients, which highlights the importance of timely management.^52,56

Pneumocystis jirovecii

P jirovecii remains a common opportunistic pathogen in people with impaired immunity, including transplant and human immunodeficiency virus patients.

Prophylaxis. Widespread adoption of antimicrobial prophylaxis by transplant centers has decreased the rates of P jirovecii infection in liver transplant recipients.^57,58 Commonly used prophylactic regimens after liver transplantation include a single-strength trimeth­oprim-sulfamethoxazole tablet daily or a double-strength tablet three times per week for a minimum of 6 to 12 months after transplant. Atovaquone and dapsone can be used as alternatives in cases of intolerance to tri­methoprim-sulfamethoxazole (Table 2).

Inhaled pentamidine is clearly inferior and should be used only when the other medications are contraindicated.⁵⁹

Signs and diagnosis. P jirovecii pneumonia is characterized by fever, cough, dyspnea, and chest pain. Insidious hypoxemia, abnormal chest examination, and bilateral interstitial pneumonia on chest radiography are common.

CT may be more sensitive than chest radiography.⁵⁷ Findings suggestive of P jirovecii pneumonia on chest CT are extensive bilateral and symmetrical ground-glass attenuations. Other less-characteristic findings include upper lobar parenchymal opacities and spontaneous pneumothorax.^57,60

The serum (1,3)-beta-D-glucan assay derived from major cell-wall components of P jiro­vecii might be helpful. Studies report a sensitivity for P jirovecii pneumonia as high as 96% and a negative predictive value of 99.8%.^61,62

Definitive diagnosis requires identification of the pathogen. Routine expectorated sputum sampling is generally associated with a poor diagnostic yield. Bronchoscopy and bronchoalveolar lavage with silver or fluorescent antibody staining of samples, polymerase chain reaction testing, or both significantly improves diagnosis. Transbronchial or open lung biopsy are often unnecessary.⁵⁷

Treatment. Trimethoprim-sulfamethoxazole is the first-line agent for treating P jirovecii pneumonia.⁵⁷ The minimum duration of treatment is 14 days, with extended courses for severe infection.

Intravenous pentamidine or clindamycin plus primaquine are alternatives for patients who cannot tolerate trimethoprim-sulfamethoxazole. The major concern with intravenous pentamidine is renal dysfunction. Hypoglycemia or hyperglycemia, neutropenia, thrombocytopenia, nausea, dysgeusia, and pancreatitis may also occur.⁶³

Atovaquone might also be beneficial in mild to moderate P jirovecii pneumonia. The main side effects include skin rashes, gastrointestinal intolerance, and elevation of transaminases.⁶⁴

A corticosteroid (40–60 mg of prednisone or its equivalent) may be beneficial in conjunction with antimicrobial therapy in patients with significant hypoxia (partial pressure of arterial oxygen < 70 mm Hg on room air) in decreasing the risk of respiratory failure and need for intubation.

With appropriate and timely antimicrobial prophylaxis, cases of P jirovecii pneumonia should continue to decrease.

TUBERCULOSIS

Development of tuberculosis after transplantation is a catastrophic complication, with mortality rates of up to 30%.⁶⁵ Most cases of posttransplant tuberculosis represent reactivation of latent disease.⁶⁶ Screening with tuberculin skin tests or interferon-gamma-release assays is recommended in all liver transplant candidates. Chest radiography before transplant is necessary when assessing a positive screening test.⁶⁷

The optimal management of latent tuberculosis in these cases remains controversial. Patients at high risk or those with positive screening results on chest radiography warrant treatment for latent tuberculosis infection with isoniazid unless contraindicated.^67,68

The ideal time to initiate prophylactic isoniazid therapy is unclear. Some authors suggest delaying it, as it might be associated with poor tolerance and hepatotoxicity.⁶⁹ Others have found that early isoniazid use was not associated with negative outcomes.⁷⁰

Risk factors for symptomatic tuberculosis after liver transplant include previous infection with tuberculosis, intensified immunosuppression (especially anti-T-lymphocyte therapies), diabetes mellitus, and other co-infections (Table 1).⁷¹

The increased incidence of atypical presentations in recent years makes the diagnosis of active tuberculosis among liver transplant recipients challenging. Sputum smears can be negative due to low mycobacterial burdens, and tuberculin skin testing and interferon-gamma-release assays may be falsely negative due to immunosuppression.⁶⁷

Treatment of active tuberculosis consists initially of a four-drug regimen using isoniazid, rifampin, pyrazinamide, and ethambutol for 2 months. Adjustments are made in accordance with culture and sensitivity results. Treatment can then be tapered to two drugs (isoniazid and rifampin) for a minimum of 4 additional months. Prolonged treatment may be required in instances of extrapulmonary or disseminated disease.^65,72

Tuberculosis treatment can be complicated by hepatotoxicity in liver transplant recipients because of direct drug effects and drug-drug interactions with immunosuppressive agents. Close monitoring for rejection and hepatotoxicity is therefore imperative while liver transplant recipients are receiving antituberculosis therapy. Drug-drug interactions may also be responsible for marked reductions in immunosuppression levels, especially with regimens containing rifampin.⁷¹ Substitution of rifabutin for rifampin reduces the effect of drug interactions.⁶⁶

VIRAL HEPATITIS

Hepatitis B virus

Hepatitis B virus-related end-stage liver disease and hepatocellular carcinoma are common indications for liver transplant in Asia. It is less common in the United States and Europe, accounting for less than 10% of all liver transplant cases. Prognosis is favorable in recipients undergoing liver transplant for hepatitis B virus, with excellent survival rates. Prevention of reinfection is crucial in these patients.

Treatment with combination antiviral agents and hepatitis B immunoglobulin (HBIG) is effective.⁷³ Lamivudine was the first nucleoside analogue found to be effective against hepatitis B virus. Its low cost and relative safety are strong arguments in favor of its continued use in liver transplant recipients.⁷⁴ In patients without evidence of hepatitis B viral replication at the time of transplant, monotherapy with lamivudine has led to low recurrence rates, and adefovir can be added to control resistant viral strains.⁷⁵

Widespread adoption of prophylaxis has decreased the rate of P jirovecii infection in liver transplant recipients

The frequent emergence of resistance with lamivudine favors newer agents such as entecavir or tenofovir. These nucleoside and nucleotide analogues have a higher barrier to resistance, and thus resistance to them is rare. They are also more efficient, potentially allowing use of an HBIG-sparing protocol.⁷⁶ However, they are associated with a higher risk of nephrotoxicity and require dose adjustments in renal insufficiency. Data directly comparing entecavir and tenofovir are scarce.

Prophylaxis. Most studies support an individualized approach for prevention of hepatitis B virus reinfection. High-risk patients, ie, those positive for HBe antigen or with high viral loads (> 100,000 copies/mL) are generally treated with both HBIG and antiviral agents.⁷⁷ Low-risk patients are those with a negative HBe antigen, low hepatitis B virus DNA levels, hepatitis B virus-related acute liver failure, and cirrhosis resulting from coinfection with both hepatitis B and hepatitis D virus.⁷⁵ In low-risk patients, discontinuation of HBIG after 1 to 2 years of treatment is appropriate, and long-term prophylaxis with antiviral agents alone is an option. However, levels of hepatitis B DNA should be monitored closely.^78,79

Hepatitis C virus

Recurrence of hepatitis C virus infection is the rule among patients who are viremic at the time of liver transplant.^80,81 Most of these patients will show histologic evidence of recurrent hepatitis within the first year after liver transplant. It is often difficult to distinguish between the histopathological appearance of a recurrent hepatitis C virus infection and acute cellular rejection.

Progression to fibrosis and subsequently cirrhosis and decompensation is highly variable in hepatitis C virus-infected liver transplant recipients. Diabetes, insulin resistance, and possibly hepatitis steatosis have been associated with a rapid progression to advanced fibrosis. The contribution of immunosuppression to the progression of hepatitis C virus remains an area of active study. Some studies point to antilymphocyte immunosuppressive agents as a potential cause.⁸² Liver biopsy is a useful tool in this situation. It allows monitoring of disease severity and progression and may distinguish recurrent hepatitis C virus disease from other causes of liver enzyme elevation.

The major concern with the recurrence of hepatitis C virus infection after liver transplant is allograft loss. Rates of patient and graft survival are reduced in infected patients compared with hepatitis C virus-negative patients.^83,84 Prophylactic antiviral therapy has no current role in the management of hepatitis C virus disease. Those manifesting moderate to severe necroinflammation or mild to moderate fibrosis indicative of progressive disease should be treated.^81,85

Sustained viral clearance with antiviral agents confers a graft survival benefit.

The combination of peg-interferon and weight-based ribavirin has been the standard of treatment but may be associated with increased rates of rejection.^86,87 The sustained virologic response rates for hepatitis C virus range from 60% in genotypes 4, 5, and 6 after 48 weeks of treatment to 60% to 80% in genotypes 2 and 3 after 24 weeks, but only about 30% in genotype 1.⁸⁸

The major concern with hepatitis C recurrence after liver transplant is allograft loss

Treatment with the newer agents, especially protease inhibitors, in genotype 1 (peg-interferon, ribavirin, and either telaprevir or boceprevir) has been evaluated. Success rates reaching 70% have been achieved.⁸⁹ Adverse effects can be a major setback. Serious complications include severe anemia, renal dysfunction, increased risk of infection, and death.

Triple therapy should be carefully considered in liver transplant patients with genotype 1 hepatitis C virus.⁹⁰ Significant drug-drug interactions are reported between hepatitis C virus protease inhibitors and immunosuppression regimens. Additional new oral direct- acting antivirals have been investigated. They bring promising advances in hepatitis C virus treatment and pave the way for interferon-free regimens with pangenotypic activity.

IMMUNIZATION

Immunization can decrease the risk of infectious complications in liver transplant recipients, as well as in close contacts and healthcare professionals.³

Influenza. Pretransplant influenza vaccine and posttransplant annual influenza vaccines are necessary.

Pneumococcal immunization should additionally be provided prior to transplant and repeated every 3 to 5 years thereafter.^3,91

A number of other vaccinations should also be completed before transplant, including the hepatitis A and B vaccines and the tetanus/diphtheria/acellular pertussis vaccines. However, these vaccinations have not been shown to be detrimental to patients after transplant.⁹¹

Varicella and zoster vaccines should be given before liver transplant—zoster in patients over age 60, and varicella in patients with no immunity. Live vaccines, including varicella and zoster vaccines, are contraindicated after liver transplant.³

Human papillomavirus. The bivalent human papillomavirus vaccine can be given before transplant in females ages 9 to 26; the quadrivalent vaccine is beneficial in those ages 9 to 26 and in women under age 45.^3,91

IMMUNOSUPPRESSION CARRIES RISK OF INFECTION

Most liver transplant patients require prolonged immunosuppressive therapy. This comes with an increased risk of new or recurrent infections, potentially causing death and significant morbidity.

Evaluation of existing risk factors, appropriate prophylaxis and immunization, timely diagnosis, and treatment of such infections are therefore essential steps for the successful management of liver transplant recipients.

The immunosuppressed state of liver transplant recipients makes them vulnerable to infections after surgery.¹ These infections are directly correlated with the net state of immunosuppression. Higher levels of immunosuppression mean a higher risk of infection, with rates of infection typically highest in the early posttransplant period.

Common infections during this period include operative and perioperative nosocomial bacterial and fungal infections, reactivation of latent infections, and invasive fungal infections such as candidiasis, aspergillosis, and pneumocystosis. Donor-derived infections also must be considered. As time passes and the level of immunosuppression is reduced, liver recipients are less prone to infection.¹

The risk of infection can be minimized by appropriate antimicrobial prophylaxis, strategies for safe living after transplant,² vaccination,³ careful balancing of immunosuppressive therapy,⁴ and thoughtful donor selection.⁵ Drug-drug interactions are common and must be carefully considered to minimize the risk.

This review highlights common infectious complications encountered after liver transplant.

INTRA-ABDOMINAL INFECTIONS

Intra-abdominal infections are common in the early postoperative period.^6,7

Risk factors include:

• Pretransplant ascites
• Posttransplant dialysis
• Wound infection
• Reoperation⁸
• Hepatic artery thrombosis
• Roux-en-Y choledochojejunostomy anastomosis.⁹

Signs that may indicate intra-abdominal infection include fever, abdominal pain, leukocytosis, and elevated liver enzymes. But because of their immunosuppressed state, transplant recipients may not manifest fever as readily as the general population. They should be evaluated for cholangitis, peritonitis, biloma, and intra-abdominal abscess.

Organisms. Intra-abdominal infections are often polymicrobial. Enterococci, Staphylococcus aureus, gram-negative species including Pseudomonas, Klebsiella, and Acinetobacter, and Candida species are the most common pathogens. Strains are often resistant to multiple drugs, especially in patients who received antibiotics in the weeks before transplant.^8,10

Liver transplant recipients are also particularly susceptible to Clostridium difficile-associated colitis as a result of immunosuppression and frequent use of antibiotics perioperatively and postoperatively.¹¹ The spectrum of C difficile infection ranges from mild diarrhea to life-threatening colitis, and the course in liver transplant patients tends to be more complicated than in immunocompetent patients.¹²

Diagnosis. Intra-abdominal infections should be looked for and treated promptly, as they are associated with a higher mortality rate, a greater risk of graft loss, and a higher incidence of retransplant.^6,10 Abdominal ultrasonography or computed tomography (CT) can confirm the presence of fluid collections.

Treatment. Infected collections can be treated with percutaneous or surgical drainage and antimicrobial therapy. In the case of biliary tract complications, retransplant or surgical correction of biliary leakage or stenosis decreases the risk of death.⁶

Suspicion should be high for C difficile-associated colitis in cases of posttransplant diarrhea. C difficile toxin stool assays help confirm the diagnosis.¹² Oral metronidazole is recommended in mild to moderate C difficile infection, with oral vancomycin and intravenous metronidazole reserved for severe cases. Colectomy may be necessary in patients with toxic megacolon.

CYTOMEGALOVIRUS INFECTION

Cytomegalovirus is an important opportunistic pathogen in liver transplant recipients.¹³ It causes a range of manifestations, from infection (viremia with or without symptoms) to cytomegalovirus syndrome (fever, malaise, and cell-line cytopenias) to tissue-invasive disease with end-organ disease.¹⁴ Without preventive measures and treatment, cytomegalovirus disease can increase the risk of morbidity, allograft loss and death.^15,16

Risk factors for cytomegalovirus infection (Table 1) include:

• Discordant serostatus of the donor and recipient (the risk is highest in seronegative recipients of organs from seropositive donors)
• Higher levels of immunosuppression, especially when antilymphocyte antibodies are used
• Treatment of graft rejection
• Coinfection with other human herpesviruses, such as Epstein-Barr virus.^4,17

Preventing cytomegalovirus infection

The strategy to prevent cytomegalovirus infection depends on the serologic status of the donor and recipient and may include antiviral prophylaxis or preemptive treatment (Table 2).¹⁸

Prophylaxis involves giving antiviral drugs during the early high-risk period, with the goal of preventing the development of cytomegalovirus viremia. The alternative preemptive strategy emphasizes serial testing for cytomegalovirus viremia, with the goal of intervening with antiviral medications while viremia is at a low level, thus avoiding potential progression to cytomegalovirus disease. Both strategies have pros and cons that should be considered by each transplant center when setting institutional policy.

A prophylactic approach seems very effective at preventing both infection and disease from cytomegalovirus and has been shown to reduce graft rejection and the risk of death.¹⁸ It is preferred in cytomegalovirus-negative recipients when the donor was cytomegalovirus-positive—a high-risk situation.¹⁹ However, these patients are also at higher risk of late-onset cytomegalovirus disease. Higher cost and potential drug toxicity, mainly neutropenia from ganciclovir-based regimens, are additional considerations.

Preemptive treatment, in contrast, reserves drug treatment for patients who are actually infected with cytomegalovirus, thus resulting in fewer adverse drug events and lower cost; but it requires regular monitoring. Preemptive methods, by definition, cannot prevent infection, and with this strategy tissue-invasive disease not associated with viremia does occasionally occur.²⁰ As such, patients with a clinical presentation that suggests cytomegalovirus but have negative results on blood testing should be considered for tissue biopsy with culture and immunohistochemical stain.

The most commonly used regimens for antiviral prophylaxis and treatment in liver transplant recipients are intravenous ganciclovir and oral valganciclovir.²¹ Although valganciclovir is the most commonly used agent in this setting because of ease of administration, it has not been approved by the US Food and Drug Administration in liver transplant patients, as it was associated with higher rates of cytomegalovirus tissue-invasive disease.^22–24 Additionally, drug-resistant cytomegalovirus strains have been associated with valganciclovir prophylaxis in cytomegalovirus-negative recipients of solid organs from cytomegalovirus-positive donors.²⁵

Prophylaxis typically consists of therapy for 3 months from the time of transplant. In higher-risk patients (donor-positive, recipient-negative), longer courses of prophylaxis have been extrapolated from data in kidney transplant recipients.²⁶ Extension or reinstitution of prophylaxis should also be considered in liver transplant patients receiving treatment for rejection with antilymphocyte therapy.

Routine screening for cytomegalovirus is not recommended while patients are receiving prophylaxis. High-risk patients who are not receiving prophylaxis should be monitored with nucleic acid or pp65 antigenemia testing as part of the preemptive strategy protocol.

Treatment of cytomegalovirus disease

Although no specific threshold has been established, treatment is generally indicated if a patient has a consistent clinical syndrome, evidence of tissue injury, and persistent or increasing viremia.

Treatment involves giving antiviral drugs and also reducing the level of immunosuppression, if possible, until symptoms and viremia have resolved.

The choice of antiviral therapy depends on the severity of disease. Intravenous ganciclovir (5 mg/kg twice daily adjusted for renal impairment) or oral valganciclovir (900 mg twice daily, also renally dose-adjusted when necessary) can be used for mild to moderate disease if no significant gastrointestinal involvement is reported. Intravenous ganciclovir is preferred for patients with more severe disease or gastrointestinal involvement. The minimum duration of treatment is 2 weeks and may need to be prolonged until both symptoms and viremia completely resolve.¹⁸

Drug resistance can occur and should be considered in patients who have a history of prolonged ganciclovir or valganciclovir exposure who do not clinically improve or have persistent or rising viremia. In such cases, genotype assays are helpful, and initiation of alternative therapy should be considered. Mutations conferring resistance to ganciclovir are often associated with cross-resistance to cidofovir. Cidofovir can therefore be considered only when genotype assays demonstrate specific mutations conferring an isolated resistance to ganciclovir.²⁷ The addition of foscarnet to the ganciclovir regimen or substitution of foscarnet for ganciclovir are accepted approaches.

Although cytomegalovirus hyperimmunoglobulin has been used in prophylaxis and invasive disease treatment, its role in the management of ganciclovir-resistant cytomegalovirus infections remains controversial.²⁸

EPSTEIN-BARR VIRUS POSTTRANSPLANT LYMPHOPROLIFERATIVE DISEASE

Epstein-Barr virus-associated posttransplant lymphoproliferative disease is a spectrum of disorders ranging from an infectious mononucleosis syndrome to aggressive malignancy with the potential for death and significant morbidity after liver transplant.²⁹ The timeline of risk varies, but the disease is most common in the first year after transplant.

Risk factors for this disease (Table 1) are:

• Primary Epstein-Barr virus infection
• Cytomegalovirus donor-recipient mismatch
• Cytomegalovirus disease
• Higher levels of immunosuppression, especially with antilymphocyte antibodies.³⁰

The likelihood of Epstein-Barr virus playing a contributing role is lower in later-onset posttransplant lymphoproliferative disease. Patients who are older at the time of transplant, who receive highly immunogenic allografts including a liver as a component of a multivisceral transplant, and who receive increased immunosuppression to treat rejection are at even greater risk of late posttransplant lymphoproliferative disease.³¹ This is in contrast to early posttransplant lymphoproliferative disease, which is seen more commonly in children as a result of primary Epstein-Barr virus infection.

Recognition and diagnosis. Heightened suspicion is required when considering posttransplant lymphoproliferative disease, and careful evaluation of consistent symptoms and allograft dysfunction are required.

Clinically, posttransplant lymphoproliferative disease should be suspected if a liver transplant recipient develops unexplained fever, weight loss, lymphadenopathy, or cell-line cytopenias.^30,32 Other signs and symptoms may be related to the organ involved and may include evidence of hepatitis, pneumonitis, and gastrointestinal disease.³¹

Adjunctive diagnostic testing includes donor and recipient serology to characterize overall risk before transplantation and quantification of Epstein-Barr viral load, but confirmation relies on tissue histopathology.

Treatment focuses on reducing immunosuppression.^30,32 Adding antiviral agents does not seem to improve outcome in all cases.³³ Depending on clinical response and histologic classification, additional therapies such as anti-CD20 humanized chimeric monoclonal antibodies, surgery, radiation, and conventional chemotherapy may be required.³⁴

Preventive approaches remain controversial. Chemoprophylaxis with an antiviral such as ganciclovir is occasionally used but has not been shown to consistently decrease rates of posttransplant lymphoproliferative disease. These agents may act in an indirect manner, leading to decreased rates of cytomegalovirus infection, a major cofactor for posttransplant lymphoproliferative disease.²⁴

Although oral valganciclovir is used more than intravenous ganciclovir, it is not approved for liver transplant patients

Passive immunoprophylaxis with immunoglobulin targeting cytomegalovirus has shown to decrease rates of non-Hodgkin lymphoma from posttransplant lymphoproliferative disease in renal transplant recipients in the first year after transplant,³⁵ but data are lacking regarding its use in liver transplant recipients. Monitoring of the viral load and subsequent reduction of immunosuppression remain the most efficient measures to date.³⁶

FUNGAL INFECTIONS

Candida species account for more than half of fungal infections in liver transplant recipients.³⁷ However, a change has been noted in the past 20 years, with a decrease in Candida infections accompanied by an increase in Aspergillus infections.³⁸ Endemic mycoses such as coccidioidomycosis, blastomycosis, and histoplasmosis should be considered with the appropriate epidemiologic history or if disease develops early after transplant and the donor came from a highly endemic region.³⁹Cryptococcus may also be encountered.

Diagnosis. One of the most challenging aspects of fungal infection in liver transplant recipients is timely diagnosis. Heightened suspicion and early biopsy for pathological and microbiological confirmation are necessary. Although available noninvasive diagnostic tools often lack specificity, early detection of fungal markers may be of great use in guiding further diagnostic workup or empiric treatment in the critically ill.

Noninvasive tests include galactomannan, cryptococcal antigen, histoplasma antigen, (1-3)-beta-D-glucan assay and various antibody tests. Galactomannan testing has been widely used to aid in the diagnosis of invasive aspergillosis. Similarly, the (1-3)-beta-D-glucan assay is a non–culture-based tool for diagnosing and monitoring the treatment of invasive fungal infections. However, a definite diagnosis cannot be made on the basis of a positive test alone.⁴⁰ The complementary diagnostic characteristics of combining noninvasive assays have yet to be fully elucidated.⁴¹ Cultures and tissue histopathology are also used when possible.

Treatment is based on targeted specific antifungal drug therapy and reduction of immunosuppressive therapy, when possible. The choice of antifungal agent varies with the pathogen, the site of involvement, and the severity of the disease. A focus on potential drug interactions, their management, and therapeutic drug monitoring when using antifungal medications is essential in the posttransplant period. Combination therapy can be considered in some situations to enhance synergy. The following sections discuss in greater detail Candida species, Aspergillus species, and Pneumocystis jirovecii infections.

Candida infections

Candidiasis after liver transplant is typically nosocomial, especially when diagnosed during the first 3 months (Table 3).³⁷

Risk factors for invasive candidiasis include perioperative colonization, prolonged operative time, retransplant, greater transfusion requirements, and postoperative renal failure.^37,42,43 Invasive candidiasis is of concern for its effects on morbidity, mortality, and cost of care.^43–46

Organisms. The frequency of implicated species, in particular those with a natural resistance to fluconazole, differs in various reports.^37,45,46Candida albicans remains the most commonly isolated pathogen; however, non-albicans species including those resistant to fluconazole have been reported more frequently and include Candida glabrata and Candida krusei.^47,48

Signs and diagnosis. Invasive candidiasis in liver transplant recipients generally manifests itself in catheter-related blood stream infections, urinary tract infections, or intra-abdominal infections. Diagnosis can be made by isolating Candida from blood cultures, recovering organisms in culture of a normally sterile site, or finding direct microscopic evidence of the fungus on tissue specimens.⁴⁹

Disseminated candidiasis refers to the involvement of distant anatomic sites. Clinical manifestations may cause vision changes, abdominal pain or skin nodules with findings of candidemia, hepatosplenic abscesses, or retinal exudates on funduscopy.⁴⁹

Treatment of invasive candidiasis in liver recipients often involves antifungal therapy and reduction of immunosuppression. Broad-spectrum antifungals are initially advocated in an empirical approach to cover fluconazole-resistant strains of the non-albicans subgroups.⁵⁰ Depending on antifungal susceptibility, treatment can later be adjusted.

Fluconazole remains the agent of choice in most C albicans infections.⁴⁷ However, attention should be paid to the possibility of resistance in patients who have received fluconazole prophylaxis within the past 30 days. Additional agents used in treatment may include echinocandins, amphotericin, and additional azoles.

Antifungal prophylaxis is recommended in high-risk liver transplant patients, although its optimal duration remains undetermined.⁴⁴ Antifungal prophylaxis has been associated with decreased incidence of both superficial and invasive candidiasis.⁵¹

Aspergillus infection

Aspergillus, the second most common fungal pathogen, has become a more common concern in liver transplant recipients. Aspergillus fumigatus is the most frequently encountered species.^38,52

Risk factors. These infections typically occur in the first year, during intense immunosuppression. Retransplant, renal failure, and fulminant hepatic failure are major risk factors.⁵² In the presence of risk factors and a suggestive clinical setting, invasive aspergillosis should be considered and the diagnosis pursued.

Diagnosis is suggested by positive findings on CT accompanied by lower respiratory tract symptoms, focal lesions on neuroimaging, or demonstration of the fungus on cultures.⁴⁹ However, Aspergillus is rarely grown in blood culture. The galactomannan antigen is a noninvasive test that can provide supporting evidence for the diagnosis.^41,52 False-positive results do occur in the setting of certain antibiotics and cross-reacting fungi.⁵³

Treatment consists of antifungal therapy and immunosuppression reduction.⁵²

Candida accounts for more than half of fungal infections in liver transplant recipients, but Aspergillus is gaining

Voriconazole is the first-line agent for invasive aspergillosis. Monitoring for potential drug-drug interactions and side effects is required.^54,55 Amphotericin B is considered a second-line choice due to toxicity and lack of an oral formulation. In refractory cases, combined antifungal therapy could be considered.⁵² The duration of treatment is generally a minimum of 12 weeks.

Prophylaxis. Specific prophylaxis against invasive aspergillosis is not currently recommended; however, some authors suggest a prophylactic approach using echinocandins or liposomal amphotericin B in high-risk patients.^51,52 Aspergillosis is associated with a considerable increase in mortality in liver transplant recipients, which highlights the importance of timely management.^52,56

Pneumocystis jirovecii

P jirovecii remains a common opportunistic pathogen in people with impaired immunity, including transplant and human immunodeficiency virus patients.

Prophylaxis. Widespread adoption of antimicrobial prophylaxis by transplant centers has decreased the rates of P jirovecii infection in liver transplant recipients.^57,58 Commonly used prophylactic regimens after liver transplantation include a single-strength trimeth­oprim-sulfamethoxazole tablet daily or a double-strength tablet three times per week for a minimum of 6 to 12 months after transplant. Atovaquone and dapsone can be used as alternatives in cases of intolerance to tri­methoprim-sulfamethoxazole (Table 2).

Inhaled pentamidine is clearly inferior and should be used only when the other medications are contraindicated.⁵⁹

Signs and diagnosis. P jirovecii pneumonia is characterized by fever, cough, dyspnea, and chest pain. Insidious hypoxemia, abnormal chest examination, and bilateral interstitial pneumonia on chest radiography are common.

CT may be more sensitive than chest radiography.⁵⁷ Findings suggestive of P jirovecii pneumonia on chest CT are extensive bilateral and symmetrical ground-glass attenuations. Other less-characteristic findings include upper lobar parenchymal opacities and spontaneous pneumothorax.^57,60

The serum (1,3)-beta-D-glucan assay derived from major cell-wall components of P jiro­vecii might be helpful. Studies report a sensitivity for P jirovecii pneumonia as high as 96% and a negative predictive value of 99.8%.^61,62

Definitive diagnosis requires identification of the pathogen. Routine expectorated sputum sampling is generally associated with a poor diagnostic yield. Bronchoscopy and bronchoalveolar lavage with silver or fluorescent antibody staining of samples, polymerase chain reaction testing, or both significantly improves diagnosis. Transbronchial or open lung biopsy are often unnecessary.⁵⁷

Treatment. Trimethoprim-sulfamethoxazole is the first-line agent for treating P jirovecii pneumonia.⁵⁷ The minimum duration of treatment is 14 days, with extended courses for severe infection.

Intravenous pentamidine or clindamycin plus primaquine are alternatives for patients who cannot tolerate trimethoprim-sulfamethoxazole. The major concern with intravenous pentamidine is renal dysfunction. Hypoglycemia or hyperglycemia, neutropenia, thrombocytopenia, nausea, dysgeusia, and pancreatitis may also occur.⁶³

Atovaquone might also be beneficial in mild to moderate P jirovecii pneumonia. The main side effects include skin rashes, gastrointestinal intolerance, and elevation of transaminases.⁶⁴

A corticosteroid (40–60 mg of prednisone or its equivalent) may be beneficial in conjunction with antimicrobial therapy in patients with significant hypoxia (partial pressure of arterial oxygen < 70 mm Hg on room air) in decreasing the risk of respiratory failure and need for intubation.

With appropriate and timely antimicrobial prophylaxis, cases of P jirovecii pneumonia should continue to decrease.

TUBERCULOSIS

Development of tuberculosis after transplantation is a catastrophic complication, with mortality rates of up to 30%.⁶⁵ Most cases of posttransplant tuberculosis represent reactivation of latent disease.⁶⁶ Screening with tuberculin skin tests or interferon-gamma-release assays is recommended in all liver transplant candidates. Chest radiography before transplant is necessary when assessing a positive screening test.⁶⁷

The optimal management of latent tuberculosis in these cases remains controversial. Patients at high risk or those with positive screening results on chest radiography warrant treatment for latent tuberculosis infection with isoniazid unless contraindicated.^67,68

The ideal time to initiate prophylactic isoniazid therapy is unclear. Some authors suggest delaying it, as it might be associated with poor tolerance and hepatotoxicity.⁶⁹ Others have found that early isoniazid use was not associated with negative outcomes.⁷⁰

Risk factors for symptomatic tuberculosis after liver transplant include previous infection with tuberculosis, intensified immunosuppression (especially anti-T-lymphocyte therapies), diabetes mellitus, and other co-infections (Table 1).⁷¹

The increased incidence of atypical presentations in recent years makes the diagnosis of active tuberculosis among liver transplant recipients challenging. Sputum smears can be negative due to low mycobacterial burdens, and tuberculin skin testing and interferon-gamma-release assays may be falsely negative due to immunosuppression.⁶⁷

Treatment of active tuberculosis consists initially of a four-drug regimen using isoniazid, rifampin, pyrazinamide, and ethambutol for 2 months. Adjustments are made in accordance with culture and sensitivity results. Treatment can then be tapered to two drugs (isoniazid and rifampin) for a minimum of 4 additional months. Prolonged treatment may be required in instances of extrapulmonary or disseminated disease.^65,72

Tuberculosis treatment can be complicated by hepatotoxicity in liver transplant recipients because of direct drug effects and drug-drug interactions with immunosuppressive agents. Close monitoring for rejection and hepatotoxicity is therefore imperative while liver transplant recipients are receiving antituberculosis therapy. Drug-drug interactions may also be responsible for marked reductions in immunosuppression levels, especially with regimens containing rifampin.⁷¹ Substitution of rifabutin for rifampin reduces the effect of drug interactions.⁶⁶

VIRAL HEPATITIS

Hepatitis B virus

Hepatitis B virus-related end-stage liver disease and hepatocellular carcinoma are common indications for liver transplant in Asia. It is less common in the United States and Europe, accounting for less than 10% of all liver transplant cases. Prognosis is favorable in recipients undergoing liver transplant for hepatitis B virus, with excellent survival rates. Prevention of reinfection is crucial in these patients.

Treatment with combination antiviral agents and hepatitis B immunoglobulin (HBIG) is effective.⁷³ Lamivudine was the first nucleoside analogue found to be effective against hepatitis B virus. Its low cost and relative safety are strong arguments in favor of its continued use in liver transplant recipients.⁷⁴ In patients without evidence of hepatitis B viral replication at the time of transplant, monotherapy with lamivudine has led to low recurrence rates, and adefovir can be added to control resistant viral strains.⁷⁵

Widespread adoption of prophylaxis has decreased the rate of P jirovecii infection in liver transplant recipients

The frequent emergence of resistance with lamivudine favors newer agents such as entecavir or tenofovir. These nucleoside and nucleotide analogues have a higher barrier to resistance, and thus resistance to them is rare. They are also more efficient, potentially allowing use of an HBIG-sparing protocol.⁷⁶ However, they are associated with a higher risk of nephrotoxicity and require dose adjustments in renal insufficiency. Data directly comparing entecavir and tenofovir are scarce.

Prophylaxis. Most studies support an individualized approach for prevention of hepatitis B virus reinfection. High-risk patients, ie, those positive for HBe antigen or with high viral loads (> 100,000 copies/mL) are generally treated with both HBIG and antiviral agents.⁷⁷ Low-risk patients are those with a negative HBe antigen, low hepatitis B virus DNA levels, hepatitis B virus-related acute liver failure, and cirrhosis resulting from coinfection with both hepatitis B and hepatitis D virus.⁷⁵ In low-risk patients, discontinuation of HBIG after 1 to 2 years of treatment is appropriate, and long-term prophylaxis with antiviral agents alone is an option. However, levels of hepatitis B DNA should be monitored closely.^78,79

Hepatitis C virus

Recurrence of hepatitis C virus infection is the rule among patients who are viremic at the time of liver transplant.^80,81 Most of these patients will show histologic evidence of recurrent hepatitis within the first year after liver transplant. It is often difficult to distinguish between the histopathological appearance of a recurrent hepatitis C virus infection and acute cellular rejection.

Progression to fibrosis and subsequently cirrhosis and decompensation is highly variable in hepatitis C virus-infected liver transplant recipients. Diabetes, insulin resistance, and possibly hepatitis steatosis have been associated with a rapid progression to advanced fibrosis. The contribution of immunosuppression to the progression of hepatitis C virus remains an area of active study. Some studies point to antilymphocyte immunosuppressive agents as a potential cause.⁸² Liver biopsy is a useful tool in this situation. It allows monitoring of disease severity and progression and may distinguish recurrent hepatitis C virus disease from other causes of liver enzyme elevation.

The major concern with the recurrence of hepatitis C virus infection after liver transplant is allograft loss. Rates of patient and graft survival are reduced in infected patients compared with hepatitis C virus-negative patients.^83,84 Prophylactic antiviral therapy has no current role in the management of hepatitis C virus disease. Those manifesting moderate to severe necroinflammation or mild to moderate fibrosis indicative of progressive disease should be treated.^81,85

Sustained viral clearance with antiviral agents confers a graft survival benefit.

The combination of peg-interferon and weight-based ribavirin has been the standard of treatment but may be associated with increased rates of rejection.^86,87 The sustained virologic response rates for hepatitis C virus range from 60% in genotypes 4, 5, and 6 after 48 weeks of treatment to 60% to 80% in genotypes 2 and 3 after 24 weeks, but only about 30% in genotype 1.⁸⁸

The major concern with hepatitis C recurrence after liver transplant is allograft loss

Treatment with the newer agents, especially protease inhibitors, in genotype 1 (peg-interferon, ribavirin, and either telaprevir or boceprevir) has been evaluated. Success rates reaching 70% have been achieved.⁸⁹ Adverse effects can be a major setback. Serious complications include severe anemia, renal dysfunction, increased risk of infection, and death.

Triple therapy should be carefully considered in liver transplant patients with genotype 1 hepatitis C virus.⁹⁰ Significant drug-drug interactions are reported between hepatitis C virus protease inhibitors and immunosuppression regimens. Additional new oral direct- acting antivirals have been investigated. They bring promising advances in hepatitis C virus treatment and pave the way for interferon-free regimens with pangenotypic activity.

IMMUNIZATION

Immunization can decrease the risk of infectious complications in liver transplant recipients, as well as in close contacts and healthcare professionals.³

Influenza. Pretransplant influenza vaccine and posttransplant annual influenza vaccines are necessary.

Pneumococcal immunization should additionally be provided prior to transplant and repeated every 3 to 5 years thereafter.^3,91

A number of other vaccinations should also be completed before transplant, including the hepatitis A and B vaccines and the tetanus/diphtheria/acellular pertussis vaccines. However, these vaccinations have not been shown to be detrimental to patients after transplant.⁹¹

Varicella and zoster vaccines should be given before liver transplant—zoster in patients over age 60, and varicella in patients with no immunity. Live vaccines, including varicella and zoster vaccines, are contraindicated after liver transplant.³

Human papillomavirus. The bivalent human papillomavirus vaccine can be given before transplant in females ages 9 to 26; the quadrivalent vaccine is beneficial in those ages 9 to 26 and in women under age 45.^3,91

IMMUNOSUPPRESSION CARRIES RISK OF INFECTION

Most liver transplant patients require prolonged immunosuppressive therapy. This comes with an increased risk of new or recurrent infections, potentially causing death and significant morbidity.

Evaluation of existing risk factors, appropriate prophylaxis and immunization, timely diagnosis, and treatment of such infections are therefore essential steps for the successful management of liver transplant recipients.

In this issue of the Cleveland Clinic Journal of Medicine, Drs. Short and Anderson give an overview of the epidemic of human immunodeficiency virus (HIV) infection in US women and the various aspects of health care of this group, including pregnancy.¹ They introduce a much broader topic and bring to light a number of additional concerns.

HIV PREYS ON THE VULNERABLE

The authors review epidemiologic trends and the evolving demographics of HIV, which deserve specific discussion.

See related article

In the early years of the epidemic, ie, the early 1980s, HIV infection in women was overshadowed by the epidemic in men, particularly men who have sex with men. The epidemic in men who have sex with men remains the larger component of the HIV picture in the United States. But worldwide, HIV is an evenly balanced problem, with nearly half of all infections occurring in women.² Women have received much more attention recently.

In the United States, about 300,000 women are living with HIV, and 10% of them are unaware of it. Between 1985 and 2013, the number of HIV cases in US women tripled.

The epidemic continues to disproportionately affect women of color. Two-thirds of all women with HIV are African American,² and estimates suggest that 1 of every 32 African American women will acquire HIV during her lifetime. On a positive note, there was a 20% reduction in new infections among African American women between 2008 and 2010.³

The epidemic preys on the vulnerable and is fueled by poverty, lack of education (general and health literacy), substance abuse, and restricted access to health care. Major metropolitan areas such as New York, Washington, DC, Miami, and Los Angeles are “hot spots,” where high concentrations of infected people reside.⁴

Many women underestimate or do not perceive their susceptibility. They unknowingly acquire HIV infection from their male partners, many of whom are unaware of their infection. Some of their partners may lead a dual life of bisexuality. In some areas, an estimated 20% of men who have sex with men also engage in sex with women.⁵ If these women contract the disease, they may be diagnosed at a late stage and when they are symptomatic, or coincidentally during pregnancy and childbirth.

Negotiating safe sex practices can be difficult for a woman. She may perceive or lack empowerment to do so, fearing rejection, isolation, or violence. Sexual violence may have been initiated in childhood, through intimate partners, rape, sex trafficking, or prostitution. Patterns vary throughout the world, but sexual violence is more common than perceived.⁶ Because of shame, embarrassment, and isolation, many victims do not seek medical care and so may carry undiagnosed infections. Even when they access care, they are less likely to remain in the HIV care system.⁷ Greater efforts are needed to reach these women, make them feel supported in care, and keep them in the system.

TESTING IS CRUCIAL

Diagnosis remains a weak link in the chain of care for both men and women. Success has been noted in the form of a marked reduction in cases of mother-to-child transmission, thanks to near-universal opt-out screening during pregnancy or at delivery.

If appropriate routine testing were done for all people, as advocated by the US Centers for Disease Control and Prevention guidelines,⁸ more cases could be diagnosed, behaviors changed, and treatment offered. Control of HIV through treatment can lead to a 96% reduction in transmission between serodiscordant partners, as demonstrated in HPTN 052, an ongoing phase 3 trial.⁹ Early diagnosis and treatment offer the potential for improved immune regeneration and healthier lives.

PRE-EXPOSURE PROPHYLAXIS

Pre-exposure prophylaxis (PrEP) is one approach to empowering women and preventing HIV infection. Studies have demonstrated the efficacy of this approach, although some studies have not.^10,11

An important finding in the failed studies appeared to be a lack of adherence to the regimen.¹¹ Unless taken faithfully, PrEP will not succeed. Additionally, there may be inherent differences in outcomes for unknown reasons. Lack of access to the necessary two-drug combination regimen is another barrier.

PrEP is expensive, requires regular monitoring, and requires patients to remain engaged in medical care. Currently, not all medical programs offer PrEP, and not all insurance policies cover it. Further insight into long-term side effects and complications is needed.

Although PrEP is an attractive concept and a reality for some, it is an incomplete solution to prevention at this time.

MEN AND WOMEN ARE DIFFERENT

Men and women are different physiologically and psychologically. Women typically have a lower body mass, lower bone mass, and higher content of body fat. As a result, women may differ from men in their ability to tolerate medications, and long-term side effects may be more pronounced.

Women are also more likely to place family responsibilities above self-preservation and personal health concerns. As a result, providing for and taking care of their children takes precedence over care of their own health.

Providing care to women presents many challenges and opportunities to improve their health. Health care access, transportation, assistance with child care during medical visits, the availability of counseling to deal with shame, guilt, and depression, and maintaining women within the care system are but a few examples.

AGING WITH HIV: STUDY NEEDED

Antiretroviral therapy has enabled patients to survive and often to reach a normal life expectancy if the infection is diagnosed and treated early. As a result, HIV-associated causes of death have been replaced by non-HIV comorbidities typical of aging, such as cardiovascular disease, organ failure (heart, lung, kidney, liver), non-HIV cancers, and bone disease.

Women face unique aspects of aging with menopause, including an accelerated rate of bone loss resulting in osteoporosis. HIV itself and some antiretroviral drugs may increase the loss of bone mineral density. Alcohol abuse, sedentary lifestyle, smoking, hepatitis C co-infection, and poor nutrition also contribute to this problem. Bone disease and many other aspects of aging and HIV in women require more research and intervention.

Other areas that need to be studied are the unique mucosal immune system of the female genital tract, the interplay of sex hormones and the immune system, the role of genital tract inflammation in increasing the risk of HIV acquisition, sexual violence and HIV acquisition, and the safety and efficacy of PrEP for women. This will require prioritization and ongoing funding, which is becoming scarcer. If there is to be hope of containing this disease, our efforts to understand it must not diminish.

In this issue of the Cleveland Clinic Journal of Medicine, Drs. Short and Anderson give an overview of the epidemic of human immunodeficiency virus (HIV) infection in US women and the various aspects of health care of this group, including pregnancy.¹ They introduce a much broader topic and bring to light a number of additional concerns.

HIV PREYS ON THE VULNERABLE

The authors review epidemiologic trends and the evolving demographics of HIV, which deserve specific discussion.

See related article

In the early years of the epidemic, ie, the early 1980s, HIV infection in women was overshadowed by the epidemic in men, particularly men who have sex with men. The epidemic in men who have sex with men remains the larger component of the HIV picture in the United States. But worldwide, HIV is an evenly balanced problem, with nearly half of all infections occurring in women.² Women have received much more attention recently.

In the United States, about 300,000 women are living with HIV, and 10% of them are unaware of it. Between 1985 and 2013, the number of HIV cases in US women tripled.

The epidemic continues to disproportionately affect women of color. Two-thirds of all women with HIV are African American,² and estimates suggest that 1 of every 32 African American women will acquire HIV during her lifetime. On a positive note, there was a 20% reduction in new infections among African American women between 2008 and 2010.³

The epidemic preys on the vulnerable and is fueled by poverty, lack of education (general and health literacy), substance abuse, and restricted access to health care. Major metropolitan areas such as New York, Washington, DC, Miami, and Los Angeles are “hot spots,” where high concentrations of infected people reside.⁴

Many women underestimate or do not perceive their susceptibility. They unknowingly acquire HIV infection from their male partners, many of whom are unaware of their infection. Some of their partners may lead a dual life of bisexuality. In some areas, an estimated 20% of men who have sex with men also engage in sex with women.⁵ If these women contract the disease, they may be diagnosed at a late stage and when they are symptomatic, or coincidentally during pregnancy and childbirth.

Negotiating safe sex practices can be difficult for a woman. She may perceive or lack empowerment to do so, fearing rejection, isolation, or violence. Sexual violence may have been initiated in childhood, through intimate partners, rape, sex trafficking, or prostitution. Patterns vary throughout the world, but sexual violence is more common than perceived.⁶ Because of shame, embarrassment, and isolation, many victims do not seek medical care and so may carry undiagnosed infections. Even when they access care, they are less likely to remain in the HIV care system.⁷ Greater efforts are needed to reach these women, make them feel supported in care, and keep them in the system.

TESTING IS CRUCIAL

Diagnosis remains a weak link in the chain of care for both men and women. Success has been noted in the form of a marked reduction in cases of mother-to-child transmission, thanks to near-universal opt-out screening during pregnancy or at delivery.

If appropriate routine testing were done for all people, as advocated by the US Centers for Disease Control and Prevention guidelines,⁸ more cases could be diagnosed, behaviors changed, and treatment offered. Control of HIV through treatment can lead to a 96% reduction in transmission between serodiscordant partners, as demonstrated in HPTN 052, an ongoing phase 3 trial.⁹ Early diagnosis and treatment offer the potential for improved immune regeneration and healthier lives.

PRE-EXPOSURE PROPHYLAXIS

Pre-exposure prophylaxis (PrEP) is one approach to empowering women and preventing HIV infection. Studies have demonstrated the efficacy of this approach, although some studies have not.^10,11

An important finding in the failed studies appeared to be a lack of adherence to the regimen.¹¹ Unless taken faithfully, PrEP will not succeed. Additionally, there may be inherent differences in outcomes for unknown reasons. Lack of access to the necessary two-drug combination regimen is another barrier.

PrEP is expensive, requires regular monitoring, and requires patients to remain engaged in medical care. Currently, not all medical programs offer PrEP, and not all insurance policies cover it. Further insight into long-term side effects and complications is needed.

Although PrEP is an attractive concept and a reality for some, it is an incomplete solution to prevention at this time.

MEN AND WOMEN ARE DIFFERENT

Men and women are different physiologically and psychologically. Women typically have a lower body mass, lower bone mass, and higher content of body fat. As a result, women may differ from men in their ability to tolerate medications, and long-term side effects may be more pronounced.

Women are also more likely to place family responsibilities above self-preservation and personal health concerns. As a result, providing for and taking care of their children takes precedence over care of their own health.

Providing care to women presents many challenges and opportunities to improve their health. Health care access, transportation, assistance with child care during medical visits, the availability of counseling to deal with shame, guilt, and depression, and maintaining women within the care system are but a few examples.

AGING WITH HIV: STUDY NEEDED

Antiretroviral therapy has enabled patients to survive and often to reach a normal life expectancy if the infection is diagnosed and treated early. As a result, HIV-associated causes of death have been replaced by non-HIV comorbidities typical of aging, such as cardiovascular disease, organ failure (heart, lung, kidney, liver), non-HIV cancers, and bone disease.

Women face unique aspects of aging with menopause, including an accelerated rate of bone loss resulting in osteoporosis. HIV itself and some antiretroviral drugs may increase the loss of bone mineral density. Alcohol abuse, sedentary lifestyle, smoking, hepatitis C co-infection, and poor nutrition also contribute to this problem. Bone disease and many other aspects of aging and HIV in women require more research and intervention.

Other areas that need to be studied are the unique mucosal immune system of the female genital tract, the interplay of sex hormones and the immune system, the role of genital tract inflammation in increasing the risk of HIV acquisition, sexual violence and HIV acquisition, and the safety and efficacy of PrEP for women. This will require prioritization and ongoing funding, which is becoming scarcer. If there is to be hope of containing this disease, our efforts to understand it must not diminish.

In this issue of the Cleveland Clinic Journal of Medicine, Drs. Short and Anderson give an overview of the epidemic of human immunodeficiency virus (HIV) infection in US women and the various aspects of health care of this group, including pregnancy.¹ They introduce a much broader topic and bring to light a number of additional concerns.

HIV PREYS ON THE VULNERABLE

The authors review epidemiologic trends and the evolving demographics of HIV, which deserve specific discussion.

See related article

In the early years of the epidemic, ie, the early 1980s, HIV infection in women was overshadowed by the epidemic in men, particularly men who have sex with men. The epidemic in men who have sex with men remains the larger component of the HIV picture in the United States. But worldwide, HIV is an evenly balanced problem, with nearly half of all infections occurring in women.² Women have received much more attention recently.

In the United States, about 300,000 women are living with HIV, and 10% of them are unaware of it. Between 1985 and 2013, the number of HIV cases in US women tripled.

The epidemic continues to disproportionately affect women of color. Two-thirds of all women with HIV are African American,² and estimates suggest that 1 of every 32 African American women will acquire HIV during her lifetime. On a positive note, there was a 20% reduction in new infections among African American women between 2008 and 2010.³

The epidemic preys on the vulnerable and is fueled by poverty, lack of education (general and health literacy), substance abuse, and restricted access to health care. Major metropolitan areas such as New York, Washington, DC, Miami, and Los Angeles are “hot spots,” where high concentrations of infected people reside.⁴

Many women underestimate or do not perceive their susceptibility. They unknowingly acquire HIV infection from their male partners, many of whom are unaware of their infection. Some of their partners may lead a dual life of bisexuality. In some areas, an estimated 20% of men who have sex with men also engage in sex with women.⁵ If these women contract the disease, they may be diagnosed at a late stage and when they are symptomatic, or coincidentally during pregnancy and childbirth.

Negotiating safe sex practices can be difficult for a woman. She may perceive or lack empowerment to do so, fearing rejection, isolation, or violence. Sexual violence may have been initiated in childhood, through intimate partners, rape, sex trafficking, or prostitution. Patterns vary throughout the world, but sexual violence is more common than perceived.⁶ Because of shame, embarrassment, and isolation, many victims do not seek medical care and so may carry undiagnosed infections. Even when they access care, they are less likely to remain in the HIV care system.⁷ Greater efforts are needed to reach these women, make them feel supported in care, and keep them in the system.

TESTING IS CRUCIAL

Diagnosis remains a weak link in the chain of care for both men and women. Success has been noted in the form of a marked reduction in cases of mother-to-child transmission, thanks to near-universal opt-out screening during pregnancy or at delivery.

If appropriate routine testing were done for all people, as advocated by the US Centers for Disease Control and Prevention guidelines,⁸ more cases could be diagnosed, behaviors changed, and treatment offered. Control of HIV through treatment can lead to a 96% reduction in transmission between serodiscordant partners, as demonstrated in HPTN 052, an ongoing phase 3 trial.⁹ Early diagnosis and treatment offer the potential for improved immune regeneration and healthier lives.

PRE-EXPOSURE PROPHYLAXIS

Pre-exposure prophylaxis (PrEP) is one approach to empowering women and preventing HIV infection. Studies have demonstrated the efficacy of this approach, although some studies have not.^10,11

An important finding in the failed studies appeared to be a lack of adherence to the regimen.¹¹ Unless taken faithfully, PrEP will not succeed. Additionally, there may be inherent differences in outcomes for unknown reasons. Lack of access to the necessary two-drug combination regimen is another barrier.

PrEP is expensive, requires regular monitoring, and requires patients to remain engaged in medical care. Currently, not all medical programs offer PrEP, and not all insurance policies cover it. Further insight into long-term side effects and complications is needed.

Although PrEP is an attractive concept and a reality for some, it is an incomplete solution to prevention at this time.

MEN AND WOMEN ARE DIFFERENT

Men and women are different physiologically and psychologically. Women typically have a lower body mass, lower bone mass, and higher content of body fat. As a result, women may differ from men in their ability to tolerate medications, and long-term side effects may be more pronounced.

Women are also more likely to place family responsibilities above self-preservation and personal health concerns. As a result, providing for and taking care of their children takes precedence over care of their own health.

Providing care to women presents many challenges and opportunities to improve their health. Health care access, transportation, assistance with child care during medical visits, the availability of counseling to deal with shame, guilt, and depression, and maintaining women within the care system are but a few examples.

AGING WITH HIV: STUDY NEEDED

Antiretroviral therapy has enabled patients to survive and often to reach a normal life expectancy if the infection is diagnosed and treated early. As a result, HIV-associated causes of death have been replaced by non-HIV comorbidities typical of aging, such as cardiovascular disease, organ failure (heart, lung, kidney, liver), non-HIV cancers, and bone disease.

Women face unique aspects of aging with menopause, including an accelerated rate of bone loss resulting in osteoporosis. HIV itself and some antiretroviral drugs may increase the loss of bone mineral density. Alcohol abuse, sedentary lifestyle, smoking, hepatitis C co-infection, and poor nutrition also contribute to this problem. Bone disease and many other aspects of aging and HIV in women require more research and intervention.

Other areas that need to be studied are the unique mucosal immune system of the female genital tract, the interplay of sex hormones and the immune system, the role of genital tract inflammation in increasing the risk of HIV acquisition, sexual violence and HIV acquisition, and the safety and efficacy of PrEP for women. This will require prioritization and ongoing funding, which is becoming scarcer. If there is to be hope of containing this disease, our efforts to understand it must not diminish.