Causes and consequences of higher Leishmania infantum burden in patients with kala-azar: a study of 625 patients.

BACKGROUND: An infected host's Leishmania infantum load in blood is considered to be an estimate of his or her total parasite burden. Therefore, the measurement of blood parasite burden is important in the identification of factors involved in parasite control. METHODS: Quantitative polymerase chain reaction was performed on blood samples from 625 patients with kala-azar consecutively admitted to a reference hospital in Teresina, Brazil. Primers were used to amplify a segment of kDNA using the TaqMan system. Non-parametric statistical tests were applied. RESULTS: The median blood parasite burden was 499.2 amastigote equivalents (AE)/ml. Children <1 year old (yo) had a high parasite burden, which dropped sharply after the first year of life (192.8, AE/ml at 1 < 2 yo) and remained lower until adolescence. Following adolescence, the parasite burden increased with age, peaking among elderly individuals. Men had a higher parasite burden than women. HIV-infected patients had a much higher parasite burden than non-infected patients. The parasite burden of children under 5 years with acute moderate to severe malnourishment (weight-for-age and body mass index z-scores <-2) was almost three times greater than that of better-nourished children. The parasite burden identified in deceased patients was more than twice that of surviving patients; those with a higher risk of death, sepsis, pneumonia and jaundice also had increased parasite burdens. All of these differences were statistically significant at P-values <0.05. CONCLUSIONS: These data indicate that the parasite burden in patients with kala-azar was associated with age- and gender-associated factors and with HIV infection status. Acute malnutrition could be either a cause or a consequence of a higher parasite burden. An individual's parasite burden influences his or her clinical profile, disease severity and mortality risk. The best explanation for the presence of a higher parasite burden in individuals with these immunoregulatory conditions and severe disease is the occurrence of acquired immunosuppression followed by heightened innate immunity.

Comparison of optical microscopy and quantitative polymerase chain reaction for estimating parasitaemia in patients with kala-azar and modelling infectiousness to the vector Lutzomyia longipalpis

Currently, the only method for identifying infective hosts with Leishmania infantum to the vector Lutzomyia longipalpis is xenodiagnosis. More recently, quantitative polymerase chain reaction (qPCR) has been used to model human reservoir competence by assuming that detection of parasite DNA indicates the presence of viable parasites for infecting vectors. Since this assumption has not been proven, this study aimed to verify this hypothesis. The concentration of amastigotes in the peripheral blood of 30 patients with kala-azar was microscopically verified by leukoconcentration and was compared to qPCR estimates. Parasites were identified in 4.8 mL of peripheral blood from 67% of the patients, at a very low concentration (average 0.3 parasites/mL). However, qPCR showed 93% sensitivity and the estimated parasitaemia was over a thousand times greater, both in blood and plasma, with higher levels in plasma than in blood. Furthermore, the microscopic count of circulating parasites and the qPCR parasitaemia estimates were not mathematically compatible with the published proportions of infected sandflies in xenodiagnostic studies. These findings suggest that qPCR does not measure the concentration of circulating parasites, but rather measures DNA from other sites, and that blood might not be the main source of infection for vectors.

Comparison of optical microscopy and quantitative polymerase chain reaction for estimating parasitaemia in patients with kala-azar and modelling infectiousness to the vector Lutzomyia longipalpis.

Currently, the only method for identifying infective hosts with Leishmania infantum to the vector Lutzomyia longipalpis is xenodiagnosis. More recently, quantitative polymerase chain reaction (qPCR) has been used to model human reservoir competence by assuming that detection of parasite DNA indicates the presence of viable parasites for infecting vectors. Since this assumption has not been proven, this study aimed to verify this hypothesis. The concentration of amastigotes in the peripheral blood of 30 patients with kala-azar was microscopically verified by leukoconcentration and was compared to qPCR estimates. Parasites were identified in 4.8 mL of peripheral blood from 67% of the patients, at a very low concentration (average 0.3 parasites/mL). However, qPCR showed 93% sensitivity and the estimated parasitaemia was over a thousand times greater, both in blood and plasma, with higher levels in plasma than in blood. Furthermore, the microscopic count of circulating parasites and the qPCR parasitaemia estimates were not mathematically compatible with the published proportions of infected sandflies in xenodiagnostic studies. These findings suggest that qPCR does not measure the concentration of circulating parasites, but rather measures DNA from other sites, and that blood might not be the main source of infection for vectors.

Bone marrow parasite burden among patients with New World kala-azar is associated with disease severity.

Kala-azar or visceral leishmaniasis, found mostly throughout the Indian Subcontinent, East Africa, and Brazil, kills 20,000-40,000 persons annually. The agents, Leishmania donovani and Leishmania infantum, are obligatory intracellular protozoa of mononuclear phagocytes found principally in the spleen and bone marrow. Protracted fever, anemia, wasting, hepatosplenomegaly, hemorrhages, and bacterial co-infections are typical features. One hundred and twenty-two (122) in-hospital patients were studied to verify if higher bone marrow parasite load estimated by quantitative polymerase chain reaction is associated with severe disease. The estimated median parasite load was 5.0 parasites/10(6) human nucleated cells. It is much higher in deceased than among survivors (median 75.0 versus 4.2). Patients who lost more weight had a higher parasite burden, as well as patients with epistaxis, abdominal pain, edema, and jaundice. This study suggests that higher parasite load is influenced by wasting, which may lead to more severe disease.