We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.

During an epidemiological study of visceral leishmaniasis in an endemic region of Brazil, new perspectives emerged on a subclinical form of the disease. A group of 86 children with antibody to Leishmania were identified. None of these children had a history of leishmaniasis. The children were segregated into four groups: One group remained asymptomatic (n = 20), whereas another developed classic kala-azar within weeks of the index serology (n = 15). The remaining 51 patients initially had subclinical disease; 13 (25%) of these patients progressed to classic kala-azar (mean, five months). The others (75%) resolved their illness after a prolonged period (mean, 35 months). The initial illness in the subclinical group was characterized by hepatomegaly, frequent splenomegaly, intermittent cough, diarrhea, and low-grade fever. Malaise and poor weight gain were common. Giemsa-stained smears and cultures of bone marrow aspirates were usually negative for Leishmania in the absence of symptoms of classic kala-azar.

The epidemiology, clinical patterns, and risk factors for visceral leishmaniasis were prospectively studied in an endemic area of Brazil. The prevalence of disease was 3.1% for children less than 15 years of age, and the annual incidence was 4.3 cases per 1,000 children. The number of children with disease fluctuated yearly and seasonally, and distribution of the disease varied within the endemic area. Risk factors included young age (median, three years) and malnutrition before the onset of disease. Intestinal parasitism, recent migration into the area, and house location within the area did not influence the progression of infection to disease. Serological testing indicated that 7.5% of children were infected with Leishmania each year and that the ratio of disease to infection was 1:18.5 for the whole area and 1:6.5 for the section with the highest prevalence of disease. Early diagnosis and therapy altered clinical patterns of the disease.

No abstract

The diagnosis of visceral leishmaniasis (VL), a serious and often fatal parasitic disease caused by members of the Leishmania donovani complex, remains problematic. Current methods rely on clinical criteria, parasite identification in aspirate material, and serology. The latter methods use crude antigen preparations lacking in specificity. A previously described cloned antigen, rK39, of Leishmania specific for all members of the L. donovani complex (L. chagasi, L. donovani, L. infantum) was very useful in the serodiagnosis by ELISA of both human and canine VL. The present study demonstrated that rK39 seroreactivity correlated with active disease. The sera from early or self-healing infected subjects reacted with leishmanial lysate and were generally nonreactive with rK39. These data demonstrate the utility of rK39 in the serodiagnosis of VL and as an indicator of active disease.

MethodsThe lymphocytes from eight patients with active visceral leishmaniasis (VL), a disease associated with marked immunologic dysfunction, were examined for ability to produce interleukin 2 (IL-2) and gamma interferon during in vitro cultivation. It was found that both IL-2 and gamma interferon production, in response to leishmania antigen, was absent during the active disease, but was restored after successful chemotherapy. Untreated VL patients produced IL-2 and gamma interferon when stimulated with phytohemagglutinin (PHA). Six patients with either active cutaneous or mucosal leishmaniasis, a disease not associated with immunosuppression, showed high levels of gamma interferon in response to leishmania antigen and PHA. Since IL-2 and gamma interferon have been shown to have important roles in the immune response and in the killing of leishmania, their absence may represent a key defect in the immune response in VL.

A five-year prospective study of cutaneous leishmaniasis in an endemic area of Brazil revealed an annual incidence of disease of 8.1 per 1000 inhabitants and a prevalence of 14.9%. The disease fluctuated as a series of mini-epidemics. Most disease occurred in individuals who were 10-30 years of age. Mucosal disease occurred in 2.7% of patients with primary lesions and occurred a median of six years after this lesion. Disease was more common in males, in those with either large or multiple antecedent skin lesions, and in those with incomplete antimony therapy for the primary lesions. An ELISA was positive in 85% of those tested during the first two years after the primary lesion and remained positive for five to 40 years in 27% of patients. Skin testing was positive in 96% of patients with recent lesions and remained positive in 70% of patients. All patients with mucosal disease had positive serological and skin tests.

To assess the effect of removing leishmania-infected dogs on the incidence of visceral leishmaniasis, a controlled intervention study was performed in northeast Brazil. The attempted elimination of seropositive dogs resulted in an initial significant decrease in the annual incidence of seroconversion among dogs from 36% to 6% over the first two years. In the following two years, the incidence increased to 11% and 14%, respectively. In a control area in which dogs were surveyed but seropositive dogs were not removed, the cumulative incidence did not vary significantly from year to year, ranging from 16% to 27%. In the intervention area, the prevalence of dog seropositivity decreased from 36% before the intervention to 10% and remained stable. These findings suggest that attempting to remove seropositive dogs is insufficient as a measure for eradicating visceral leishmaniasis in dogs. However, the force of transmission of infection among dogs can be reduced by such programs. Also, when the number of human cases before and after the start of the intervention was calculated, a significant decrease in incidence of disease in the intervention area was observed among children less than 15 years of age (P Ͻ 0.01). The results of this intervention study suggest that the elimination of the majority of seropositive dogs may affect the cumulative incidence of seroconversion in dogs temporarily and may also diminish the incidence of human cases of visceral leishmaniasis.