Diane Jacquet

Putative Leishmania hybrids in the Eastern Andean valley of Huanuco, Peru

During an outbreak of tegumentary leishmaniasis that developed in the 1990s in the Eastern Andean valley of Huanuco, Peru, the coexistence of Andean (uta) and sylvatic leishmaniases was suspected for ecological and geographical reasons, and sympatric sampling was carried out. Seven human isolates of Leishmania were characterized by multilocus enzyme electrophoresis, random amplification of polymorphic DNA and molecular karyotyping. The three methods identified 3 isolates as L. braziliensis, and 4 isolates as putative hybrids with characters of L. braziliensis and L. peruviana. Data from Huanuco are compared to previous results from other areas endemic for uta. Biological and epidemiological implications are discussed.

Evaluation of a urinary antigen‐based latex agglutination test in the diagnosis of kala‐azar in eastern Nepal

Background  We evaluated the diagnostic accuracy as well as the reproducibility of the urine latex agglutination test ‘KAtex’ in the diagnosis of kala‐azar in patients recruited at a tertiary care centre in Dharan, Nepal, between November 2000 and January 2002.

Molecular karyotype variation in Leishmania (Viannia) peruviana: indication of geographical populations in Peru distributed along a north-south cline.

Forty-one Leishmania peruviana isolates were selected along a north-south transect which crossed areas endemic for uta in three different biogeographical regions in the Peruvian Andes. The isolates were analysed by molecular karyotyping and hybridization with three chromosome-derived DNA probes. All the isolates could be distinguished from L. braziliensis by their pLb-134 hybridization patterns. However, the patterns with the other probes (pLb-168 and -22) could be used to cluster the Peruvian isolates in discrete groups (karyodemes) which varied in their level of similarity with L. braziliensis. The geographical distribution of these karyodemes supports the hypothesis that eco-graphical isolation has contributed to the heterogeneity of L. peruviana.

Is Leishmania (Viannia) peruviana a distinct species? A MLEE/RAPD evolutionary genetics answer

Abstract A set of 38 Leishmania stocks from the Andean valleys of Peru was characterized by both Multilocus Enzyme Electrophoresis (MLEE) and Random Amplified Polymorphic DNA (RAPD). Data were analyzed in terms of taxonomy and evolutionary genetics. Synapomorphic MLEE and RAPD characters, clear-cut clustering, and strong agreement between the phylogenies inferred from either MLEE or RAPD supported the view that Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis correspond to two closely related, but distinct monophyletic lines (clades) and can therefore be considered as “discrete typing units” (DTUs). The question whether the L. (V.) peruviana DTU deserves species status is dependent upon the desirability of it, in terms of epidemiological and medical relevance. A previous Orthogonal Field Alternating Gel Electrophoresis (OFAGE) analysis of the same L. (V.) peruviana isolates was published by Dujardin et al. (1995b). The data from the different markers (i.e. MLEE, RAPD and OFAGE) were compared by population genetics analysis. RAPD and OFAGE provided divergent results, since RAPD showed a strong linkage disequilibrium whereas OFAGE revealed no apparent departure from panmictic expectation. MLEE showed no linkage disequilibrium. Nevertheless, contrary to OFAGE, this is most probably explainable by the limited variability revealed by this marker in L. (V.) peruviana (statistical type II error). RAPD data were consistent with the hypothesis that the present L. (V.) peruviana sample displays a basically clonal population structure with limited or no genetic exchange. Disagreement between RAPD and OFAGE can be explained either by accumulation of chromosomal rearrangements due to amplification/deletion of repeated sequences, or by pseudo-recombinational events.

Multi-centre evaluation of repeatability and reproducibility of the direct agglutination test for visceral leishmaniasis

Summary objective  To evaluate the repeatability and reproducibility of the serological direct agglutination test (DAT) for visceral leishmaniasis (VL) with aqueous antigen in a multi‐centre study in VL‐endemic areas in Sudan, Kenya and Nepal.

Comparative evaluation of freeze-dried and liquid antigens in the direct agglutination test for serodiagnosis of visceral leishmaniasis (ITMA-DAT/VL).

Objective  The direct agglutination test (DAT) for visceral leishmaniasis (VL) with liquid (LQ) antigen is known to be only moderately reproducible because of inter‐observer and batch‐to‐batch variability as well as its sensitivity to temperature and shaking during transport. We evaluated a DAT with freeze‐dried (FD) antigen and compared it with the LQ antigen version.

Karyotype plasticity in Neotropical Leishmania : an index for measuring genomic distance among L. (V.) peruviana and L. (V.) braziliensis populations

A method for phenetic analysis of karyotype data has been developed for Leishmania populations. Measurement of size difference between chromosomes recognized by a given DNA probe in different isolates led to the formulation of a Chromosome Size Difference Index (CSDI). The method was applied to phenetic analysis of 4 sets of chromosomes--each set being recognized by a different probe--in 37 L. (Viannia) peruviana isolates sampled along a North-South transect through the Peruvian Andes and, in 11 L. (V.) braziliensis isolates from the Amazonian forest (Peru, Bolivia and Brazil). Karyotype variability was better accounted for by CSDI than by a method based on disjunctive encoding of karyotype data. CSDI evidenced the nature of relationships between L. braziliensis and L. peruviana and it provided a coherent picture of geographical and genomic differentiation among parasite populations. The latter did cluster according to their geographical origin. L. braziliensis was found karyotypically more homogeneous than L. peruviana. Within L. peruviana, Northern populations were closer to L. braziliensis than to Southern L. peruviana populations. The validity of karyotypic populations, or karyodemes, was sustained.

Sensitivity and specificity of HAT Sero-K-SeT, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by Trypanosoma brucei gambiense: a case-control study

BACKGROUND Human African trypanosomiasis (HAT) is a life-threatening infection affecting rural populations in sub-Saharan Africa. Large-scale population screening by antibody detection with the Card Agglutination Test for Trypanosomiasis (CATT)/Trypanosoma brucei (T b) gambiense helped reduce the number of reported cases of gambiense HAT to fewer than 10 000 in 2011. Because low case numbers lead to decreased cost-effectiveness of such active screening, we aimed to assess diagnostic accuracy of a rapid serodiagnostic test (HAT Sero-K-SeT) applicable in primary health-care centres. METHODS In our case-control study, we assessed participants older than 11 years who presented for HAT Sero-K-SeT and CATT/T b gambiense at primary care centres or to mobile teams (and existing patients with confirmed disease status at these centres) in Bandundu Province, DR Congo. We defined cases as patients with trypanosomes that had been identified in lymph node aspirate, blood, or cerebrospinal fluid. During screening, we recruited controls without previous history of HAT or detectable trypanosomes in blood or lymph who resided in the same area as the cases. We assessed diagnostic accuracy of three antibody detection tests for gambiense HAT: HAT Sero-K-SeT and CATT/T b gambiense (done with venous blood at the primary care centres) and immune trypanolysis (done with plasma at the Institute of Tropical Medicine, Antwerp, Belgium). FINDINGS Between June 6, 2012, and Feb 25, 2013, we included 134 cases and 356 controls. HAT Sero-K-SeT had a sensitivity of 0·985 (132 true positives, 95% CI 0·947-0·996) and a specificity of 0·986 (351 true negatives, 0·968-0·994), which did not differ significantly from CATT/T b gambiense (sensitivity 95% CI 0·955, 95% CI 0·906-0·979 [128 true positives] and specificity 0·972, 0·949-0·985 [346 true negatives]) or immune trypanolysis (sensitivity 0·985, 0·947-0·996 [132 true positives] and specificity 0·980, 0·960-0·990 [349 true negatives]). INTERPRETATION The diagnostic accuracy of HAT Sero-K-SeT is adequate for T b gambiense antibody detection in local health centres and could be used for active screening whenever a cold chain and electricity supply are unavailable and CATT/T b gambiense cannot be done.

Development of a slide ELISA for canine leishmaniasis and comparison with four serological tests

A slide ELISA for canine leishmaniasis was developed by using promastigotes of Leishmania infantum, and compared with microimmunodiffusion, immunoelectrophoresis, direct agglutination and indirect immunofluorescence assays. The sensitivity of all the tests was 100 per cent. The specificity of the direct agglutination test was 95 per cent but it was 100 per cent for the three other tests. There was also a positive correlation and a high level of concordance between the titres measured by the different tests.

Chromosome rearrangement in Leishmania mexicana M379

Circular extrachromosomal elements were observed in a variety of Leishmania species. We show here that two lines originating from the same isolate have been found to contain a circular DNA molecule of 26.6 kb and a linear chromosome of about 250 kb, respectively, which share a homology of more than 20 kb. The circular DNA molecule and its related region on the linear chromosome were cloned and their restriction maps compared. This investigation reveals information about chromosome rearrangement in L. mexicana M379. Further examination will enable us to understand the nature of chromosome rearrangement such as circularization or linearization.