Leishmania Species : Visceral (Kala-Azar), Cutaneous, and Mucosal Leishmaniasis

Life Cycle and Morphology

Leishmania organisms are dimorphic protozoa existing in two distinct morphologies within sand fly vectors and mammalian hosts, respectively. Although no clear sexual form has been identified, there is evidence that sexual recombination does occur. Additionally, whereas generally diploid, Leishmania organisms use aneuploidy at various points in their life cycle, likely as a mechanism of gene regulation. Mammalian infection begins when a female sand fly, taking a blood meal, regurgitates promastigotes into the bite site. Promastigotes (1.5–3.5 µm × 15–200 µm) are elongate, flagellated, and motile forms found in the digestive tract and proboscis of the sand fly. Promastigotes avoid the innate immune response and are phagocytized by neutrophils, macrophages, and dendritic cells. Within 24 to 48 hours after infection, intracellular promastigotes transform into oval or round amastigotes (1.5–3 µm × 3–5 µm) that lack a visible flagellum on light microscopy. Amastigotes have a distinct, rod-shaped structure called a kinetoplast, which is a specialized mitochondrial structure. Visualization of a kinetoplast, as seen in Fig. 275.1 , confirms the diagnosis of leishmaniasis.

Amastigotes multiply by simple binary division, eventually rupturing the cell and invading other mononuclear phagocytes. Depending on the species and host factors, amastigotes may spread regionally or systemically from the bite site through lymphatics and/or the vascular system to infect mononuclear phagocytes elsewhere. This variable tropism is important in the clinical manifestations characteristic for a given species. The cycle is completed when female sand flies ingest parasitized cells. When in the digestive tract of the sand flies, Leishmania parasites develop through a series of flagellated intermediate stages to become infectious metacyclic promastigotes over the course of approximately 1 week.

Taxonomy and Classification

The taxonomy of Leishmania parasites can be confusing and is evolving. Traditional classification schemes using geographic, clinical, isozyme, or vectorial criteria are useful and widespread in the literature. However, molecular techniques such as sequencing and multilocus sequence typing are influencing taxonomy, as exemplified by the recognition of the synonymous nature of Leishmania donovani and Leishmania archibaldi . The division of the genus Leishmania into the subgenera Leishmania and Viannia was originally based on the location of promastigote development within sand flies, and this has generally been confirmed by molecular techniques. The advancement of molecular techniques, including rapid whole-genome sequencing, transcriptomics, and proteomics, will continue to refine the taxonomy of Leishmania .

From the clinician's perspective, a useful classification accurately predicts the natural history of infection and response to treatment. However, different definitions, standards, and technical methods have been used over the past several decades to determine the species-level characterization of Leishmania parasites, making the correlation of species with treatment outcomes and prognosis challenging. The Leishmania species that infect humans and the clinical syndromes they produce are summarized in Table 275.1 .

Transmission Principles and Sand Fly Biology

Transmission depends on the sand fly vector, the presence of a suitable reservoir, and susceptible humans. Female sand flies of the genera Lutzomyia and Phlebotomus transmit Leishmania species in the Americas and elsewhere, respectively. As seen in Fig. 275.2 , phlebotomine sand flies are small, delicate insects ranging from about 1.5 to 3.5 mm in length. Sand flies breed in the cracks of dwelling walls, rubbish, rubble, or rodent burrows. They are weak fliers and tend to remain close to the ground near their breeding sites and feed on nearby hosts. Sand flies make small cuts in host skin and feed on pooled blood. Saliva from the sand fly enhances the infectivity of promastigotes through its antihemostatic and immunomodulatory properties. Factors in the saliva such as adenosine or maxadilan influence the host immune response, favoring a Th2-type response along with decreased macrophage activation and diminished nitric oxide (NO) production.

Transmission back to the sand fly occurs when a susceptible sand fly feeds on an infected reservoir host. It is unclear whether host parasitemia or parasites within the skin are responsible for this transmission. Sand fly saliva has been shown to increase host parasitemia after repeated feedings, but the variability of sand fly infection seen in xenodiagnostic studies may be better accounted for by patchy skin infection.

Depending on the Leishmania species, the sand fly genus, and the geographic location, the major reservoirs for leishmaniasis may be canines, rodents, or humans.

Pathogenesis and Immunology

Leishmaniasis can be thought of as a polar disorder similar to other intracellular infections such as leprosy ( Fig. 275.3 ). On one end of the spectrum are polyparasitic infections (e.g., diffuse CL or VL) characterized by a predominantly Th2-type immune response with relative anergy, akin to lepromatous leprosy. Heavily parasitized macrophages are abundant, and diagnosis is readily made by smear. The oligoparasitic end of the spectrum includes ML and latent VL infection. Amastigotes are sparse, and a mononuclear cell infiltrate predominates in a Th1-type immune response, analogous to tuberculoid leprosy. In reality, the interaction between Th1 and Th2 is much more complex and includes variable modulation by Th17-type CD4 ⁺ T cells among others.

Why some species spread systemically causing visceral disease and others remain relatively confined to the skin is not fully understood. The A2 gene family, found as a functional gene among species that cause VL, is likely important in visceralization, protecting the parasite from heat shock and oxidative stress. This is further supported by the observation that subspecies of L . donovani isolated from patients with CL have much less A2 production, but when A2 production is increased, the capacity to visceralize is restored. Comparing the sequences of different CL and VL species shows that only 19 of >8000 total genes are L . donovani –specific genes. Insertion of some of these L . donovani –specific genes into Leishmania major promotes the survival of L . major in the viscera of mice in murine models.

Regardless of tropism, intracellular survival is essential to the pathogenesis of all forms of leishmaniasis. Multiple virulence factors enable Leishmania organisms to survive intracellularly, including lipophosphoglycan and leishmanolysin. The former aids in the transformation of phagosomes into parasitophorous vacuoles by promastigotes. Leishmanolysin, or GP63, is expressed on the parasite cell surface and secreted in exosomes; it facilitates complement and natural killer cell inactivation, prevents antigen cross-presentation to T cells, and modulates macrophage signaling and transcription pathways.

Diagnostic Principles

There are three ways to approach the diagnosis of all suspected Leishmania infections: clinical, parasitologic, and immunologic. Clinical diagnosis combines epidemiology with clinical manifestations and serves as a useful guide but is rarely adequate in and of itself due to the cost and toxicity of available therapies as well as the prognostic importance of species identification, particularly in CL.

A parasitologic diagnosis is confirmed by visualizing amastigotes in a tissue biopsy specimen or smear, visualizing promastigotes in culture, or amplifying Leishmania -specific nucleic acids by polymerase chain reaction (PCR). Optimally, parasites should be speciated using either isoenzyme techniques from cultured parasites or molecular methods such as multilocus sequence typing or PCR from a clinical sample, although molecular methods lack standardization. Specimens for culture can be inoculated into one of several media (Schneider's modified medium, Novy-MacNeal-Nicolle medium, and others) and maintained at 22°C to 26°C. In vitro, amastigotes transform into motile promastigotes and multiply, taking a few days to several weeks to reach detectable levels depending on the inoculum size. Parasite culture and Leishmania molecular assays are generally relegated to reference laboratories, and it is helpful to contact the laboratories in advance to ensure appropriate collection methods. In the United States, culture media and expert assistance are available from the Centers for Disease Control and Prevention ( http://www.cdc.gov/parasites/leishmaniasis/health_professionals/index.html#dx ). Protocols using matrix-assisted laser desorption/ionization may allow more widespread future availability of species identification.

Immunologic diagnosis is an adjunct in most cases, with various antibody tests, cytokine release assays, and the leishmanin (Montenegro) skin test. The leishmanin skin test and cytokine release assays such as the interferon (IFN)-γ release assay (IGRA), evaluate for cell-mediated immune responses, but neither the skin test nor cytokine release assays are standardized or commercially available.

The choice of the optimal diagnostic test or procedure depends on the parasite burden of the leishmaniasis syndrome (see Fig. 275.3 ). The guidelines from the Infectious Disease Society of America (IDSA) and the American Society for Tropical Medicine and Hygiene (ASTMH) recommend a multipronged approach in most cases, with the use of molecular methods whenever possible.

Treatment Principles

The diversity of Leishmania infections makes standard treatment recommendations impossible. Each region has different species complexes with a greater or lesser degree of genetic heterogeneity within the complex. These genetic differences may be reflected in a variable natural history of infection and response to treatment. In addition, each geographic region has its own unique combination of sand fly vectors, mammalian reservoirs, and human hosts with different genetic backgrounds in varying zoonotic or anthroponotic cycles, leading to different outcomes and treatment responses. Optimal drug treatment regimens for each geographic region and major syndrome are best defined in consideration of demonstrated regional efficacy, available resources, and risk-benefit assessments. In resource-rich countries, the efficacy, safety, availability, and tolerability of drug regimens can be the primary factors regarding choice, whereas in low-resource endemic areas, cost and availability are crucial.

In addition to regional differences, variations in trial design, particularly treatment end points, make definitive recommendations difficult. The end points used in clinical trials include clinical, parasitologic, and immunologic cure. These refer to the resolution of clinical symptoms and signs within a defined time period; the absence of parasites by smear, culture, or PCR; and a falling antibody titer or conversion of a skin test from negative to positive. Although current chemotherapy options result in a clinical cure, they may seldom lead to true parasitologic cure, with persistence of Leishmania parasites in host tissue being the rule, not the exception.

The IDSA-ASTMH guidelines summarize the treatment recommendations along with approved or recommended doses and side-effect profiles for providers practicing in North America.

Epidemiology

VL is endemic in 75 countries across the tropics and subtropics, predominantly in low-income or low-middle–income countries ( Fig. 275.5 ). More than 90% of reported cases from the last 10 years were in Bangladesh, Brazil, Ethiopia, India, South Sudan, and Sudan. The World Health Organization (WHO) estimated the incidence of VL to be 200,000 to 400,000 clinical cases/year in 2010 ; as of 2015, the number of reported cases had fallen by nearly 60% corresponding to an estimated 82,000 to 164,000 cases/year globally (assuming similar rates of underreporting). The global decline has largely been driven by declines seen in the Indian subcontinent. VL is often clustered locally and regionally and may undergo epidemic cycles in some regions. VL is generally either an anthroponosis (sand fly–human–sand fly) or zoonosis depending on the region and parasite species. In anthroponotic disease, clinically ill patients with VL have the greatest potential of transmitting the parasite, although persons with post–kala-azar dermal leishmaniasis (PKDL) or latent infection also play a role in maintaining the disease.

There is an increasing recognition of asymptomatic or latent infection occurring in endemic areas. Depending on the region, testing modality, and definition of asymptomatic infection, the prevalence is 0.3% to 37%, with the ratio of latent to active VL in the range of 4 : 1 to 50 : 1.

Both L . donovani and L . infantum (syn L . chagasi ) cause VL in the Old World (see Fig. 275.5A ). In the Indian subcontinent, humans serve as the reservoir for L . donovani, and transmission is by Phlebotomus argentipes and other anthropophilic species. As a result of a multinational elimination effort in South Asia, reported cases and deaths in India have decreased by greater than 70% and 95%, respectively, from 2010 to 2015.

In East Africa, VL caused by L . donovani is endemic in Eritrea, Ethiopia, Kenya, Somalia, South Sudan, Sudan, and Uganda and sporadic in other countries of the region. Conflict-related instability and resulting displaced people have produced multiple waves of epidemic VL. The exact reservoir in the region is unclear, but putative reservoirs include humans (especially during epidemics), rats, gerbils, other rodents, small carnivores, and potentially dogs. Also, VL caused by L . donovani exists in southwestern Saudi Arabia, western Yemen, northeast Afghanistan, Pakistan, and western China, with humans and black rats serving as potential reservoirs, depending on the region.

In Europe and the Mediterranean littoral region, VL caused by L . infantum (syn L . chagasi ) is a zoonosis with dogs being the principal reservoir. Clinically apparent cases are typically encountered among infants, young children, and immunocompromised persons. In the 1990s VL emerged as an important opportunistic disease among patients with acquired immunodeficiency syndrome (AIDS) in southern Europe (Spain, France, and Italy). The rates of coinfection with human immunodeficiency virus (HIV) and VL have declined significantly with the advent of antiretroviral therapy (ART). Highlighting the clustered, cyclic nature of VL, a large outbreak occurred near several parks in southwestern Madrid, Spain, in the years 2009–12. VL caused by L . infantum (syn L . chagasi ) also extends eastward out of the Mediterranean into Southwest and Central Asia as well as northwestern and central China, where dogs, other canines, cats, and hares are reservoirs.

In Latin America, L . infantum (syn L . chagasi ) is endemic and broadly distributed (see Fig. 275.5B ). Most areas have focal disease risk with a background of latent or subclinical infection with sporadic clinical cases in rural areas. The clustering of cases in households suggests that humans may also be reservoirs in these settings. Since the 1950s disease epidemiology has shifted from rural to predominantly urban, and major periurban outbreaks of VL have been reported from cities in northeastern Brazil. Children are most frequently affected. Lutzomyia longipalpis is the major vector. Domestic dogs and wild foxes are reservoirs of infection.

Although sand fly bites account for most VL transmission, the parasite can be transmitted by blood transfusion, needle sharing, occupational exposure, and congenital exposure. Clear evidence of sexual transmission exists in canines, but only a single case of human sexual transmission has been reported.

Leishmania species that are typically associated with cutaneous syndromes—such as Leishmania amazonensis in Latin America and Leishmania tropica in Kenya, India, Iran, and Saudi Arabia—are isolated in rare instances from patients with oligosymptomatic visceral syndromes. For example, a small group of American military personnel who served in the Persian Gulf War acquired a viscerotropic form of L . tropica infection. Additionally, Leishmania martiniquensis in the Caribbean and related species in the Leishmania enrietti complex in Thailand can cause visceral disease.

Pathogenesis and Immunology

After infection the parasites of the L . donovani complex are phagocytized and spread systemically. From a pathologic perspective, VL is characterized by inadequately controlled parasite replication leading to increasing numbers of amastigote-infected mononuclear phagocytes in the liver and spleen with progressive hypertrophy and clinically apparent hepatosplenomegaly. The splenic lymphoid follicles are replaced by parasitized mononuclear cells. There is a marked increase in the number and size of Kupffer cells in the liver, many of which contain amastigotes. Autopsy studies reveal wide dissemination of the parasite with infected mononuclear phagocytes observed in the bone marrow, lymph nodes, skin, and other organs.

The immune response is complex and modulated by multiple factors. A protective immune response involves the production and interaction of interleukin (IL)-12, IFN-γ, and tumor necrosis factor (TNF)-α, among others. IL-12 is produced by macrophages and dendritic cells in response to intracellular parasites and costimulation by T cells. IL-12 not only promotes differentiation of Th1 CD4 ⁺ T cells but also stimulates the production of IFN-γ by multiple cell types. IFN-γ stimulates the secretion of TNF-α, and together these cytokines activate macrophages to kill intracellular amastigotes through the production of NO and reactive oxygen species. In the liver, granuloma formation seems to be important to the control of VL, although perhaps not essential, and is influenced by TNF-α, various chemokines, and T-cell granzyme production.

An ineffectual immune response is characterized by the production of IL-10 and transforming growth factor (TGF)-β. IL-10 inhibits IFN-γ–mediated macrophage activation, suppresses NO production, and inhibits the production of IL-12 and TNF-α. TGF-β interferes with macrophage activation and suppresses expression of major histocompatibility complex type II on monocytes. Although IFN-γ, TNF-α, and other Th1-type cytokines are often elevated in active VL, the influence of IL-10 and the Th2 environment results in a pathologic response.

Multiple other cell types contribute to an ineffectual immune response including macrophages, neutrophils, and B cells. B-cell proliferation and antibody production is generally regarded as nonspecific and ineffectual, but N -glycosylation patterns may be involved in the pathophysiology.

The reason some people develop a protective response when others develop disease is not fully understood. The sequence of early cytokine responses and the manner in which Leishmania antigens are presented by macrophages and dendritic cells—influenced by vector, parasite, and host factors—may be important variables determining the response to an individual infection.

Vector determinants associated with progressive disease include the vasodilatory effects of sand fly saliva, a smaller size of the parasite inoculum, and an absence of preexisting immune responses to sand fly salivary proteins. Parasite factors associated with visceralization were discussed previously. It is likely that there are subspecies differences among VL-causing Leishmania species that influence immune response and parasite virulence, but further genomic, transcriptomic, and proteomic comparisons are needed.

Host determinants associated with the outcome of infection and the spectrum of clinical manifestations include genetically determined human immune responses, host nutritional status, and immunocompetence. Genetic risk factors include loci on chromosomes 22q12 and 2q22-23 in Sudan, the HLA-CDR-HLA-DQA1 locus in Brazil and India, and various cytokine pathways (including IL-12, IL-10, TNF-α, and mannose-binding lectin). Additionally, risk for disease relapse has been seen in families from Sudan with mutations involving the alkylglycerol monooxygenase gene. Malnutrition is a risk factor for progression of infection to disease and results in lower concentrations of leptin, which influences IFN-γ and IL-12 production. As discussed subsequently, various forms of immunosuppression predispose infected individuals to develop symptomatic VL. Additionally, the extremes of age and in utero exposure to the parasite are associated with disease.

Natural History and Latent Visceral Leishmaniasis Infection

The natural history of leishmaniasis generally progresses along two pathways: latent VL infection or progression to disease. There is not a consensus in the literature regarding the definition of asymptomatic VL, with patients with latent infection being identified variably by skin test, IGRA, serology, or PCR. When multiple tests are performed on the same asymptomatic population, they may not show agreement. Latent infection progresses to active VL in 2% to 29% of individuals at up to 3 years. Most individuals with latent infection remain asymptomatic, but such individuals may develop subclinical symptoms or splenomegaly before progressing to frank disease or resolving spontaneously. Activation of latent infection is more common in patients who are antibody positive or immunosuppressed and can occur many years after exposure.

For patients progressing more directly to symptomatic disease, the incubation time has generally been regarded as 2 to 8 months but can range from 10 days to >1 year. However, incubation periods are often extrapolated from travelers with known exposure periods, and the latent infection paradigm adds further uncertainty. Individuals may have a subclinical course with spontaneous resolution or progression to overt disease, or they may immediately develop classic kala-azar. Although most patients with fully symptomatic kala-azar will die without intervention, there are reports of spontaneous disease resolution. Various studies have shown that risk of death is increased with delayed diagnosis, incomplete treatment, lower social status, age <2 years or >40 to 45 years, HIV or tuberculosis coinfection, low body mass index, jaundice, ascites or edema, bleeding, severe anemia, thrombocytopenia, hypoalbuminemia, or hyponatremia.

Persons with self-resolving infection and persons who have undergone successful chemotherapy develop protective immune responses. However, the parasite is not eradicated, and disease can redevelop years later if the infected person becomes immunocompromised.

Clinical Manifestations

The fully developed clinical manifestations of VL are similar in all endemic areas. Skin lesions at the site of inoculation are usually not apparent in persons with VL. In cases with a subacute or chronic course, there is an insidious onset of fever, weakness, loss of appetite, weight loss, failure to thrive, and abdominal enlargement caused by hepatosplenomegaly. In endemic areas, low-grade symptoms may persist for weeks to months but may not be sufficiently severe to warrant medical attention in low-resource areas, and the condition of such patients may be called subclinical when oligosymptomatic would be more appropriate. Fever may be intermittent, remittent with twice-daily temperature spikes (double quotidian), or, less commonly, continuous. Fever is relatively well tolerated, and older clinical references routinely noted that patients were not acutely ill or toxic in appearance.

Acute presentation in nonimmune persons comprises abrupt onset with high fever and chills, sometimes with a periodicity that suggests malaria. Chills, but seldom rigors, accompany the temperature spikes. As time passes, the spleen can become massively enlarged. It is usually soft to firm and nontender. The presence of a hard spleen suggests a hematologic disorder or another diagnosis such as schistosomiasis. The liver also enlarges; it usually has a sharp edge, soft consistency, and a smooth surface. Lymphadenopathy is common in patients in Sudan but uncommon in other geographic areas. Elevated liver enzymes and bilirubin may be observed. Peripheral edema may be seen late in disease, particularly in malnourished children. Hemorrhage can occur from one or more sites; epistaxis and gingival bleeding may be noted as well as petechiae and ecchymoses on the extremities in late-stage disease. Many patients with VL become cachectic, mediated in part by TNF-α and other cytokines known to have catabolic and anorectic effects.

Secondary bacterial infections are common in persons with advanced VL. Patients can present with coinfection or acquire secondary bacterial infections during hospitalizations. It is important to recognize and treat clinically significant bacterial coinfections. Death may result from pneumonia, septicemia, tuberculosis, dysentery, or measles, or it may be the consequence of malnutrition, severe anemia, or hemorrhage.

The laboratory findings include anemia, leukopenia, thrombocytopenia, and hypergammaglobulinemia. Anemia is almost always present and may be severe. It is usually normocytic and normochromic and appears to be due to a combination of factors including hemolysis, marrow replacement with Leishmania -infected macrophages, hemorrhage, splenic sequestration of erythrocytes, hemodilution, and marrow suppressive effects of cytokines such as TNF-α. Secondary hemophagocytic lymphohistiocytosis is increasingly reported, frequently in children. Leukopenia is also prominent, with white blood cell counts occasionally as low as 1000/mm ³ . Severe eosinopenia is frequently observed. Of note, anemia and neutropenia have not been prominent in patients with VL who have undergone splenectomy. Hypergammaglobulinemia, circulating immune complexes, and rheumatoid factors are present in the sera of most patients with VL. The globulin level may be as high as 9 g/dL; the ratio of globulin to albumin is typically high. The erythrocyte sedimentation rate is usually elevated.

Renal involvement in VL is common and includes proteinuria, acute renal failure, nephrotic syndrome, glomerulonephritis, acute interstitial nephritis, tubular necrosis, and tubulitis. Elevated serum creatinine has been observed both retrospectively and prospectively in 26% to 28% of patients with VL in Brazil, but abnormalities in urinary concentration and acidification were seen in >60%.

Viscerotropic disease caused by atypical species is characterized by chronic low-grade fever, malaise, fatigue, and in some cases diarrhea. Mild splenomegaly can occur, but generally patients do not develop classic kala-azar or progressive VL.

Diagnosis

The positive predictive value of a clinical diagnosis of VL in patients with the pentad of prolonged fever, progressive weight loss, pronounced hepatosplenomegaly (especially splenomegaly), pancytopenia, and hypergammaglobulinemia from a known endemic area is very high. However, clinical diagnosis can be challenging in patients with atypical presentations such as oligosymptomatic or immunosuppressed patients or in returned travelers presenting to physicians in nonendemic regions months to years after an exposure.

Samples for parasitologic diagnosis can be obtained by splenic aspiration, bone marrow aspiration, liver biopsy, and lymph node aspiration. Splenic aspiration to obtain a few drops of fluid for Wright-Giemsa–stained smears, culture, or PCR assay is the most sensitive method for parasite identification. Splenic aspirations have sensitivities of 93% to 99%, and the parasite burden can be quantified to stage patients and monitor response to treatment. However, splenic aspiration carries the risk of life-threatening hemorrhage, particularly in advanced disease, and is not recommended in North America. Bone marrow aspiration is safer and preferred in nonendemic settings but is less sensitive, ranging from 60% to 85%, but improving to as high as 95% when microscopists review smears for an hour. Liver biopsy is even less likely to yield the diagnosis but occasionally is positive when bone marrow is negative. Lymph node aspiration or biopsy may be diagnostic when enlarged nodes are present, as is often the case in East Africa. Rarely, amastigotes may also be seen on blood smears or cultured from the buffy coat or blood. When the diagnosis of VL is suspected, parasitologic confirmation may require more than one technique and repeated procedures. Whenever possible, aspirates should also be cultured.

Molecular approaches have the potential to replace traditional parasitologic methods due to their diagnostic accuracy even when using blood samples rather than more invasive splenic or bone marrow aspirates. A meta-analysis found a pooled sensitivity and specificity from whole blood of 93% and 96%, respectively. Certain molecular assays are more sensitive than microscopic methods for the detection of parasites and can detect parasites in asymptomatic individuals with exposure risks. Whereas various techniques exist, quantitative PCR assay has the advantage of being able to assess parasite load in patients with VL, which is highly correlated to splenic score. Also, quantitative PCR may provide a threshold for distinguishing latent infection from active disease and is useful in monitoring response to treatment. There is a lack of standardization in methodology and target sequence, and larger studies are needed to compare different methods, both within and between VL regions. Assays targeting kinetoplast DNA minicircles have been shown to be the most sensitive target, likely as a result of each parasite containing approximately 10 ⁴ minicircles, although this number is variable, making quantification less reliable. Replacing traditional parasitologic methods with PCR has also been shown to be potentially cost saving. However, most molecular methods require well-equipped laboratory facilities, minimizing their applicability to resource-limited settings. Techniques such as loop-mediated isothermal amplification or recombinase polymerase amplification may offer reliable alternatives for field application but require further standardization and development.

Immunologic tests are widely used in the diagnosis of VL worldwide. High-titer antileishmanial antibodies are typically present in immunocompetent persons with symptomatic VL and are variably present in individuals with latent infection. A number of serologic tests using different antigens and assays are available; the most widely used include the recombinant K39 (rK39) (a kinesin-related protein) test and direct agglutination test (DAT). Both tests perform well with pooled sensitivities and specificities >90%, but sensitivity is diminished in some regions, particularly East Africa—potentially related to host differences or, for rK39, the extensive diversity of kinesin-related proteins found in East African isolates. Performance of DAT may be improved by using locally derived antigens instead of commercial preparations. Both rK39 and DAT are suited for use in resource-limited settings, with the former using rapid immunochromatographic test strips. Antibody tests are limited by remaining detectable for months to years after successful treatment and by the potential to cross-react with antibodies from past infections such as leprosy, Chagas disease, CL, and others. Although antigen-based tests have the potential for overcoming many of these shortcomings, the commercially available KAtex antigen test has poor sensitivity in clinical specimens, and other potential alternatives are still early in development.

Other immunologic tests measuring cell-mediated immunity such as the IGRA or leishmanin skin test are often negative in patients with active VL, potentially becoming positive after spontaneous or treatment-related disease resolution. Individuals with latent infection can be positive as well, making these tests epidemiologically useful. In transplant patients, cytokine release assays can be positive in asymptomatic patients but cannot distinguish between latent infection and successfully treated cases.

A reasonable approach to the diagnosis of VL in immunocompetent hosts in North America consists of screening clinically suspect cases with serologic tests and confirming the diagnosis via bone marrow aspirate/biopsy for histopathology, parasite culture, and PCR.

The differential diagnosis of late-stage VL with the full complement of symptoms and signs is limited to hematologic and lymphatic malignancies and, occasionally, disseminated histoplasmosis and tropical splenomegaly syndrome. Acute VL has a much broader differential diagnosis including malaria, miliary tuberculosis, hemophagocytic syndromes, enteric fevers, HIV, bacterial endocarditis, sarcoidosis, acute Chagas disease, acute schistosomiasis, typhus, and amebic liver abscess. Subacute or chronic VL may be confused with brucellosis, prolonged Salmonella bacteremia, infectious mononucleosis, myeloproliferative disease, hepatosplenic schistosomiasis, and chronic malaria.

Visceral Leishmaniasis in Immunocompromised Patients

Immunocompromised patients represent a unique spectrum of VL with multiple differences across the clinical picture from presentation to management. Immunosuppressing conditions that have been shown to influence VL include HIV/AIDS, transplant-related immunosuppression, exposure to immunosuppressive drugs such as TNF-α inhibitors or steroids, malignancies or related chemotherapy, and other rare conditions such as genetic disorders in the IL-12 pathway. Most data in this context are from patients with HIV/AIDS, but there is a growing body of evidence from other etiologies of immunosuppression.

The prevalence of VL in immunosuppression is not fully known. In the Mediterranean, HIV previously accounted for most VL cases, but with the use of ART it is much less common, and other etiologies of immunosuppression have become increasingly recognized. In an outbreak of VL in Spain, HIV coinfection was seen in 10% of patients, whereas 13% had another form of immunosuppression. Elsewhere in the world, HIV clearly predominates, with estimated coinfection rates highest in East Africa (20%–40%), followed by Brazil (4%) and India (<1%). VL is seen in <1% of transplant patients globally, but increasing rates of transplantation in VL endemic regions such as Brazil and India may result in more cases. Immunosuppressed individuals are often older at diagnosis than immunocompetent patients.

Immunosuppressed patients with VL often have a condition impairing cell-medicated immunity making it harder, if not impossible, for the immune system to control parasite replication, resulting in a wider dissemination and greater burden of mononuclear cells infected with amastigotes. VL also impacts the pathophysiology of HIV infection, causing increased immune activation, which leads to immunosenescence, progression of HIV disease, and poorer CD4 ⁺ T-cell recovery with ART.

The natural history of VL in immunosuppressed patients differs in several aspects. All immunosuppressed persons are at higher risk of progressing to clinical VL (4-fold in transplant patients and 100- to 2300-fold in HIV-infected patients). In endemic regions, it is often impossible to distinguish activation of latent infection versus an incident case in immunocompromised patients, although patients have been observed to experience reactivation on becoming immunosuppressed years after their last potential VL exposure. Anyone with a history of birth, residence, or travel in a Leishmania -endemic area is at risk of late reactivation if he or she becomes immunocompromised. The role of primary preventive chemotherapy among latently infected immunocompromised persons has not been studied. Regardless of etiology, spontaneous resolution is unlikely to occur in immunosuppressed individuals.

Immunosuppressed individuals often present similarly to patients with classic VL, although atypical presentations are more common, correlating with the degree of immunosuppression. In general, HIV-infected patients have more weight loss, weakness, bleeding, and secondary infections but relatively less fever, pallor, and organomegaly. When CD4 ⁺ counts fall below 50 cells/mm ³ , atypical presentations are much more common and include atypical skin lesions similar to PKDL; extensive gastrointestinal tract involvement, which may manifest with chronic diarrhea and malabsorption; intraabdominal lymphadenopathy; airway, pleural, or pericardial involvement; aplastic anemia; and anterior uveitis. Compared with patients with HIV coinfection, patients with other types of immunosuppression tend to have more fever, lower leukocyte counts, and less hepatomegaly.

Immunosuppression, particularly due to HIV, influences the diagnosis of VL by increasing the burden of parasites but simultaneously impairing specific antibody production. Parasitologic diagnosis is easier with parasites being found in a wide variety of samples including bronchoalveolar lavage fluid, pleural effusions, or biopsy specimens of the oropharynx, stomach, or intestine. Patients with HIV infection are also more likely to have circulating parasites found in their blood compared with immunocompetent patients. PCR remains highly sensitive in a wide variety of samples. Serologic tests are variable in immunosuppressed individuals, with less sensitivity overall. In immunocompromised patients, the optimal testing regimen is not clear and should involve a multipronged approach, ideally including quantitative PCR of the blood for its utility in monitoring response to therapy.

Treatment and secondary prophylaxis are discussed later in this chapter. Posttreatment relapse is problematic, especially in patients with HIV infection. Rates of relapse vary significantly in heterogeneously designed studies, but more recent rates range from 26% to 37%. Relapse is more common in HIV-coinfected patients not on ART or with a history of prior relapse, high parasite loads at diagnosis, baseline CD4 ⁺ counts <100 cells/mm ³ , or persistence of low CD4 ⁺ counts. Although relapses can occur at higher CD4 ⁺ counts, consideration can be given to stopping secondary prophylaxis in patients on ART if the CD4 ⁺ count is >200 cells/mm ³ for at least 6 months, especially in the setting of negative blood PCR. Continued monitoring by PCR could be useful to predict relapse in this context as well. Although ART is essential for sustained prevention of relapse, timing of ART initiation is not clear. Whereas relapse is less common in patients without HIV/AIDS, transplant patients still have a rate of approximately 25%.

Finally, death is more common in all immunosuppressed patients, even with treatment. Mortality rates of 25% to 46% are reported in patients with HIV (the higher end of range in patients not on secondary prophylaxis) and 22% in transplant patients.

Post–Kala-Azar Dermal Leishmaniasis

PKDL follows after active VL caused by L . donovani in 5% to 10% of patients within 2 to 4 years after treatment in India and approximately 50% of patients within 0 to 6 months after treatment in Sudan ( Fig. 275.6 ). In Bangladesh, active surveillance resulted in higher than previously observed rates, with >25% developing PKDL after therapy. PKDL is rarely seen in areas where L . infantum (syn L . chagasi ) predominates and, when reported, has been seen in patients with concurrent AIDS. Additionally, studies in India have shown that 4% to 29% of patients with PKDL have no history of clinical VL.

The pathophysiology of PKDL is not conclusively known, but suboptimal therapy, host genetic predisposition, parasite strain difference, and environmental factors such as ultraviolet light exposure or water arsenic levels likely combine to create a Th2-type response in the skin with alternative macrophage activation despite a Th1 response predominating in the viscera. This is even more pronounced in nodular lesions, resulting in a higher parasite burden relative to macular lesions.

Skin lesions of PKDL are macular, papular, nodular, or verrucous. The lesions are more often macular and chronic in India, whereas in Sudan, they are more frequently papulonodular but generally resolve within 12 months without therapy. Lesions are more typical on sun-exposed skin. Patients generally feel well. The diagnosis is mainly clinical, but amastigotes can be detected in the skin microscopically or with PCR; the latter has high sensitivity even in macular lesions. PKDL must be differentiated from syphilis, leprosy, and yaws.

Patients with PKDL have been shown to be infectious to sand flies with no difference in infectiveness seen between nodular and macular lesions. Because of this, patients with PKDL are thought to be reservoirs for anthroponotic infection and may become increasingly important to elimination programs. However, further study is needed regarding the true potential of PKDL in disease transmission, as no VL seroconversion was seen in households with patients with PKDL over a 2-year period. Treatment, discussed next, speeds resolution and is thought to decrease the risk of transmission, but this also remains to be demonstrated.