A. Nandy

Genetic diversity of Plasmodium vivax in Kolkata, India

BackgroundPlasmodium vivax malaria accounts for approximately 60% of malaria cases in Kolkata, India. There has been limited information on the genotypic polymorphism of P. vivax in this malaria endemic area. Three highly polymorphic and single copy genes were selected for a study of genetic diversity in Kolkata strains.MethodsBlood from 151 patients with P. vivax infection diagnosed in Kolkata between April 2003 and September 2004 was genotyped at three polymorphic loci: the P. vivax circumsporozoite protein (pvcs), the merozoite surface protein 1 (pvmsp 1) and the merozoite surface protein 3-alpha (pvmsp 3-alpha).ResultsAnalysis of these three genetic markers revealed that P. vivax populations in Kolkata are highly diverse. A large number of distinguishable alleles were found from three genetic markers: 11 for pvcs, 35 for pvmsp 1 and 37 for pvmsp 3-alpha. These were, in general, randomly distributed amongst the isolates. Among the 151 isolates, 142 unique genotypes were detected the commonest genotype at a frequency of less than 2% (3/151). The overall rate of mixed genotype infections was 10.6%.ConclusionThese results indicate that the P. vivax parasite population is highly diverse in Kolkata, despite the low level of transmission. The genotyping protocols used in this study may be useful for differentiating re-infection from relapse and recrudescence in studies assessing of malarial drug efficacy in vivax malaria.

Genotyping of Plasmodium vivax Reveals Both Short and Long Latency Relapse Patterns in Kolkata

Background The Plasmodium vivax that was once prevalent in temperate climatic zones typically had an interval between primary infection and first relapse of 7–10 months, whereas in tropical areas P.vivax infections relapse frequently at intervals of 3–6 weeks. Defining the epidemiology of these two phenotypes from temporal patterns of illness in endemic areas is difficult or impossible, particularly if they overlap. Methods A prospective open label comparison of chloroquine (CQ) alone versus CQ plus unobserved primaquine for either 5 days or 14 days was conducted in patients presenting with acute vivax malaria in Kolkata. Patients were followed for 15 months and primary and recurrent infections were genotyped using three polymorphic antigen and up to 8 microsatellite markers. Results 151 patients were enrolled of whom 47 (31%) had subsequent recurrent infections. Recurrence proportions were similar in the three treatment groups. Parasite genotyping revealed discrete temporal patterns of recurrence allowing differentiation of probable relapse from newly acquired infections. This suggested that 32 of the 47 recurrences were probable relapses of which 22 (69%) were genetically homologous. The majority (81%) of probable relapses occurred within three months (16 homologous, 10 heterologous) and six genetically homologous relapses (19%) were of the long latency (8–10 month interval) phenotype. Conclusions With long follow-up to assess temporal patterns of vivax malaria recurrence, genotyping of P.vivax can be used to assess relapse rates. A 14 day unobserved course of primaquine did not prevent relapse. Genotyping indicates that long latency P.vivax is prevalent in West Bengal, and that the first relapses after long latent periods are genetically homologous. Trial Registration Controlled-Trials.com ISRCTN14027467

Subpopulations of T lymphocytes in the peripheral blood and lymph nodes of Indian kala-azar patients

Abstract Distribution of T cells in the peripheral blood and lymph nodes of Indian kala-azar (KA) patients was studied by using appropriate phenotypic markers for CD2+, CD4+ and CD8+ cells. Significant reduction in the CD2+, CD4+ cell numbers as well as CD4+/CD8+ cell ratio was noted in the peripheral blood of active KA cases. Such alteration in the T cell population appeared to be a manifestation of the disease process as it showed a tendency to return close to normalcy several months after successful chemotherapy. Histopathological studies of KA patients with lymphadenopathy demonstrated gradual destruction of lymph node follicular architecture which correlated well with the severity and duration of illness. Massive infiltration of CD2+ cells in the cortical region of lymph node was evident. The observed preponderance of CD4+ cells over CD8+ ones in these infiltrates was in sharp contrast to the distribution pattern of these cells in the periphery. Significance of these findings is discussed in relation to the current concepts on the immunology of leishmaniasis and related diseases.

Laryngeal involvement during post kala-azar dermal leishmaniasis in India

Summary Post kala‐azar dermal leishmaniasis (PKDL) involving the mucus membranes is relatively rare on the Indian sub‐continent. We describe 3 cases of PKDL presenting with hoarseness of voice. In one case the skin, nasal, oral, oropharyngeal and laryngeal mucosa had nodular and nodulo‐ulcerative lesions; in the 2 other cases, genitalia and anorectal mucosa were also affected. Laryngoscopic examination revealed nodular lesions on the vocal cords. Biopsy smear and culture confirmed their leishmanial origin.

Recurrence of kala-azar after PKDL: role of co-factors.

Recurrence of kala‐azar after post kala‐azar dermal leishmaniasis (PKDL) has remained uncommon. We report here two patients with recurrence of kala‐azar (KA) after development of PKDL. In one case the second attack of KA was preceded by repeated attacks of malaria and tuberculosis, and in the other the recurrence of KA followed an attack of measles. While measles has earlier been suggested as co‐factor in inducing transformation from sub‐clinical to clinical kala‐azar, malaria was demonstrated to enhance the virulence and invasiveness of Leishmania in an experimental model as well as under natural condition. We propose that in our cases, measles and repeated attacks of malaria or tuberculosis led to immunosuppression and recurrence of visceral leishmaniasis (VL).

Dynamics of the antibodies in cohorts of cured cases of visceral leishmaniasis: its implication on the validity of serological test, value in prognosis and in post therapeutic assessment.

The major disadvantage of a Serological test like Direct Agglutination Test (DAT) for Visceral Leishmaniasis (also called Kala-azar) is its inability to distinguish between recent and past infection. The objective of our study was to look at rate of decline of antibodies in fully cured cases of Kala-azar and length of time it takes for DAT to become negative. Cohort Study involving completely treated Kala-azar cases from Government Hospital during one calendar year of study. Cases were selected on the basis of treatment cohorts 0,3,6,9 &12 mo after completion of treatment.. Phase I- The cases were traced and after obtaining the informed consent they were subjected to Direct Agglutination Test (DAT). Phase II-The five treatment cohorts, constituting 82 cured cases (average of 15 cured cases per each treatment cohort) were tested again with DAT three months after the first test. The titers of Phase-I and phase-II tests were analyzed for the dynamics of the antibodies for the period. Cutoff- Values of DAT below 1:800 are considered negative. Values of 1:800, 1:1200, 1:1600 and so on are considered positive. The mean titer [Geometric Mean Titer (GMT)] at the start of treatment was 1:1120, which showed steady decline up to six months, plummeting below the cutoff titer for the DAT (1:800) at the ninth month. Antibodies continue to linger for about one year in cured Kala-azar cases even after correct and complete treatment. Single DAT results may be misleading due to high false positivity up to one year after the cure. Paired test defined as two tests 3 mo apart on the same subject. Paired test is highly recommended for diagnosis and prognosis. DAT is still a very useful tool for diagnosis if used along with clinical correlation