What is the deal with Visceral Leishmaniasis?

Visceral leishmaniasis is a protozoan disease caused by the Leishmania donovani complex, which is transmitted by sand flies in the genus phlebotomus in the old world and Lutzomyia in the new world (1). Leishmaniasis tends to be an intracellular organism, infecting macrophages and the reticuloendothelial system, leading to severe bone marrow suppression and splenomegaly. The life cycle is actually quite simple when compared to other protozoans (2). The promastigote in the sand fly digestive tract is transmitted into the skin of the host when the fly takes a blood meal. The promastigotes are phagocytized by macrophages in the dermis and transform into intracellular amastigotes. These are typically found in phagolysosomes, where they multiply by binary division, eventually rupturing the cell and invading other reticuloendothelial cells. 

To know the epidemiology of VL, know about the “old world” aka not North America. Of course there is some crossover but in general VL tends to be in Africa, Middle East, while cutaneous tends to be in Latin America:

Again, this is incredibly simplistic, but gets the basic point across. Leishmania can be both zoonotic, where animals serve as the reservoir, or anthroponotic, where humans serve as the reservoir. This plays a role mostly in epidemiology and infection control, where zoonotic transmission plays a role in rural areas while anthroponotic transmission plays a larger role in urban one. Though L. donovani tends to be the main anthroponotic species of Leishmaniasis that causes VL. 

Pathophysiology

As shown above, visceral leishmaniasis exists on a spectrum depending on the immune response. At one end, delayed-type hypersensitivity reactions predominate (2) and these depend on Th1, IL-2 and IFN-gamma to control infection with visceral leishmaniasis. Indeed, CD4 Th1 cell response in a host are required to control initial infection (3), with Th2 responses with IL-4 and IL-10 production result in susceptibility to infection and development of severe disease due to inhibition of macrophage activation and consequent intracellular replication of the parasite. At the cellular level, IL-1 and TNF-alpha prime macrophages for activation by IFN-gamma, which in turn activates macrophages to kill amastigotes through NO production (2). 

The role that T-cells play in disease regulation was  demonstrated in an animal study, where mice without a thymus were infected with L. donovani (4). Parasite liver burden was higher at any dose of Pentostam in mice without thymus compared to those with one:

The parasite killing effect of Pentostam was restored in athymic mice after injection with spleen cells obtained from normal mice:

Nutrition also plays a role, with malnutrition being cited as a common risk factor for VL. In mice fed a diet rich in protein, zinc, and iron (5), parasite burden was significantly lower than those who were fed a nutrient poor diet. Further, cytokines were significantly higher in the nutrient rich group, reflecting their role in infection control. Moreover, NO production was significantly higher in the nutrient rich diet (diet A) in comparison with the other diets in the presence of IFN-gamma:

This reflects the role that cellular immunity plays in the pathophysiology of VL, and how its derangement may play a role in its clinical picture (read: co-infection with HIV, see later).

Clinical Characteristics

Asymptomatic infections tend to be the rule rather than the exception. In general, asymptomatic infections tend to be 5-10 times greater than the number of apparent VL cases (3). The incubation period ranges from 10 days up to one year, with many patients presenting roughly around 3 months into the disease course. The holy trinity of symptoms that suggest visceral leishmaniasis seems to be:

• Fever
• Splenomegaly
• Fatigue

However, these are not very specific. Indeed, one of the issues that makes the diagnosis of VL difficult is that it mimics others. For instance, a case series of 241 patients found that weakness, weight loss, splenomegaly, fever, and body weakness were found in >90 percent of patients (6):

Another case control studies (7) found similar cluster of symptoms, with the added bonus of darkening of the skin:

These patients also tend to present in a subacute fashion. For instance, one study found the mean number of months prior to therapy was roughly 3-4 (8). In terms of lab values, these also tend to be very vague. In general, normocytic anemia, leukopenia, and thrombocytopenia is found, with the anemia seen in these patients being a combination of hemolysis, bone marrow replacement by parasite-infected cells, marrow suppression from TNF-alpha, and splenic sequestration (9). A high globulin is also seen, so elevated globulin gap (globulin – albumin >4) in a person coming in from an endemic area should be a tip off to VL. 

Risk factors for mortality tend to be pretty straight forward as well. An analysis of 2543 leishmaniasis patients (8) found that age >45, edema, low Hbg, positive HIV, and presence of TB were associated with increased mortality: 

And in another case series (6), the presence of TB, sepsis, and HIV positivity were associated with poor treatment outcomes: 

What happens in those who recover from VL? In areas where L. donovani is the main parasite, many patients will develop a skin condition known as post-kala azar dermal leishmaniasis (PKDL) as a complication (2, 22). This is typically seen in India (where it follows anywhere from 5-10% of cases of VL) as well as east Africa, particularly Kenya and Sudan (follows up to 50% of VL cases). The presentation ranges from papular or nodular rash predominantly in the face, to a maculopapular or macular rash:

Sudanese lesions appear in the area around the mouth and face prior to spreading downwards towards the chest and arms. Differences between the PKDL seen in India and Sudan is as follows (22):

Patients with PKDL are usually immune, and its development depends on a capacity to mount an immune response, with increased CD4 count, restored IFN-gamma production, and lower IL-10 concentrations seen in PKDL compared to VL (22). Further, it seems to follow treatment with pentavalent antimony rather than amphotericin B compounds, though the clinical significance of this is unknown (2). It is thought these lesions may be a reservoir for parasites, especially in areas such as India where humans tend to be the main reservoir, as parasites are seen in 90% of skin biopsies. As a result, it may play an important role in the transmission of disease in interepidemic periods of VL. Sudanese lesions have a tendency to spontaneously heal and it is generally the rule, while Indian PKDL rarely heals and needs to be treated. In certain scenarios, especially if lesions are long-lasting (>1yr) or severe, Sudanese lesions may require treatment.

Diagnosis

The gold standard for the diagnosis of VL is parasitological diagnosis (i.e. looking at the parasites yourself) due to its high specificity. This is accomplished by seeing amastigote forms (aka LD bodies) in tissue smears from lymph nodes, bone marrow, or spleen (10). In preparations stained with Giemsa or Leishman stain, cytoplasm appears pale blue, with a relatively large nucleus that stains red. In the same plane as the nucleus, but at a right angle to it, is a deep red or violet rod-like body called a kinetoplast (11. You have no clue how many times I read this exact same sentence in other reviews; I assume these people all work for the same center since they quote they’ve only had 2 fatal bleeds in >9600 splenic aspirate procedures over 10 years). 

From here, parasite density can be scored, ranging from 0 (no parasite per 1000 oil immersion fields) to 6+ (>100 parasites per field). The sensitivity for microscopy varies depending on where the sample is obtained. For instance, splenic smears have a sensitivity of 93-98.7%, compared to bone marrow which ranges from 52-85% and lymph nodes, ranging from 52-58%. Splenic aspiration is not without its risk, however, so outside of experienced centers (i.e in endemic areas where they do this all the time), it is likely not a modality that you will see often. Microscopy of peripheral blood has also been attempted, but this method does not have high sensitivity to be useful. In an analysis of 208 patients with confirmed VL (12), microscopy of isolated mononuclear cells barely achieved a sensitivity over 30%, though better than the abysmal microscopy of the buffy coat component of serum:

As a result, ELISA and other molecular testing such as PCR have been used for the diagnosis of leishmaniasis. One of the caveats here is the fact these tests require significant infrastructure which is not feasible in many of the areas where VL is endemic. The other factor is the fact that specificity of the tests depends on the target sequence (in the case of PCR) or the target antigen (for ELISA) used. Depending on the species of Leishmania and where the test is implemented, the sensitivity and specificity will vary. For instance, an 8 year prospective study (13) compared microscopy, serology, and PCR-based diagnostic tools. 1678 samples were obtained from 594 patients, with 68 patients receiving a diagnosis of VL. Overall sensitivity for PCR off bone marrow was 95.7% and 98.5% off peripheral blood. This was much higher than blood culture or bone marrow culture as well as the standard, microscopic examination of bone marrow sample:

A literature review in the same paper found the sensitivity of PCR from blood ranged from 70-100%, and 67-100% in bone marrow, with specificity from 95-100%. This has also been used to track treatment progress, with anywhere from 90-100 percent of patients without HIV being negative by PCR after about 2 months of therapy. Another review noted that PCR was more sensitive than microscopy in lymph node and bone marrow aspirations but the sensitivity in blood was only 70% (11). Despite its relatively high sensitivity, these cannot be used on the field. 

ELISA is a more viable option for field use. This, of course, requires the use of an antigen. One of the most commonly used antigens for VL is the crude soluble antigen (CSA), which is a suspension of promastigotes in phosphate-buffered saline. The sensitivity ranges from 80-100%, but the specificity is hampered by cross-reactivity with trypanosomiasis, tuberculosis, and toxoplasmosis (11). On the flip side, using more specific antigens drops the sensitivity substantially, in once case down to 37%. The recombinant antigen, rK39 has been used as it has been shown to be specific for antibodies in patients with VL caused by members of the L. donovani complex. A meta-analysis found that the rK39 dipstick test had a sensitivity of 94.8% and specificity of 85%, while the agglutination test had a sensitivity 93.9% of and specificity of 90% (14) . Despite these high numbers, the sensitivity of data from East Africa suggested a sensitivity of 70%. A study from Ethiopia demonstrated a sensitivity of 72% (15) while another one from Sudan found a sensitivity of 81% (16). As such, while this assay may work in certain areas, it is likely not a universal method for non-invasive diagnosis. 

Despite these shortcomings, one of the advantages of antigen testing is its ability to be used to trend progression as you treat. rK39 tends to have higher titers when compared to CSA (17). There was an overall trend towards decreasing titers as time progressed, with relapses being better reflected with rK39:

The other advantage is its utility in HIV-positive patients. Compared to IFA, rK39 detected 82% of patients with confirmed VL compared to 53.1% (18) in a study of 59 seropositive patients. In this group, titers also decreased with treatment, although rK39 antigen was significantly higher at all points:

Other methods for diagnosis have been proposed, including detection of polypeptide fractions of 72-75kDa and 123 kDa antigens in the urine (11). While sensitivity was high, it is not broadly used, though it is a viable option as a test of cure.

You may have heard about the Montenegro test, which is similar to the tuberculin skin test where 0.5ml of phenol-killed whole parasites are injected into the forearm of a patient (11). The size of induration is then measured and compared to the induration size of a control in the opposite forearm. Given this test depends on delayed-type hypersensitivity reaction, this is typically negative in VL and positive in cases where it was cured. Further, it seems it has no clinical utility anyways. 

HIV and Visceral Leishmaniasis

Going back to the issue of cell-mediated immunity and VL, HIV presents an issue in terms of diagnostics and clinical characteristics. I mentioned previously how antigen based testing may circumvent this issue so I’ll focus on the clinical presentation. While it may be similar to the presentation of immunocompetent individuals, there are some differences to be aware of. A retrospective study (19) analyzed 120 cases of VL in both HIV and non-HIV patients. While both had similar symptoms, including fever and splenomegaly, though HIV patients were less likely to present with these symptoms compared to non-HIV ones:

HIV patients were also more likely to be diagnosed via skin biopsy rather than bone marrow biopsy:

In another study of 91 HIV positive patients co-infected with VL (20), the triad of fever, splenomegaly, and hepatomegaly was found in 43% of patients, and was more frequent in those whose CD4 count was >50 (58% vs 33%, p-value 0.021). In this case series, 31 of these patients were found to have VL in atypical locations including digestive tract, skin, lungs, and tonsils, with this being the sole method of diagnosis in 15 of these. These were more frequent in patients whose CD4 count was <50 than in those whose CD4 >50 (41 vs 21%, p-value 0.05).

As mentioned previously, HIV has an impact on mortality. Indeed, HIV infected patients are less likely to achieve a clinical cure a month after cessation of therapy (19):

Though with the advent of ART, this does not seem to be the issue. Patients on ART treated for VL had excellent survival in one retrospective analysis (19), with median survival in patients with no ART being 16mo vs 66mo in those who were on ART. The timing of initiation of ART in context of co-infection in naive patients is unclear, but given what we know about immune reconstitution in other diseases such as TB, I assume that initiating ART and then doing VL treatment is reasonable. A literature review (21) identified 31 cases of Leishmania-associated IRIS, of which 19 constituted visceral leishmaniasis. Moreover, mortality was not addressed in this review, so I assume this may be a relatively rare occurrence of unmasking rather than paradoxical IRIS.

References:

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