Yemane B. Mebrahtu

Cutaneous leishmaniasis caused by Leishmania tropica in Kenya

Cutaneous leishmaniasis is endemic in Algeria, but clinical and parasitological data from this area are scarce. In order to document the transmission of this disease in a peri-urban setting, cutaneous lesions from patients living in Constantine City and surrounding areas were spotted on filter paper for diagnosis and species identification using real-time PCR. Surprisingly, Leishmania tropica was detected in 6/69 patients, and confirmation was obtained by sequencing. This observation suggests a modification of the epidemiology of cutaneous leishmaniasis in Algeria and should alert physicians and policy-makers to the risk of antimony treatment failure with this species.9 leishmanial strains, isolated from cutaneous papulonodular lesions on 3 patients, were characterized by cellulose acetate electrophoresis using 7 enzymes. The patterns obtained were indistinguishable from those of a Leishmania tropica reference strain and these 9 strains were similar to L. tropica in failing to infect mice. Although these 3 patients were Americans, their only potential exposure to sandflies was in Kenya, and thus they are believed to be the first cases of cutaneous leishmaniasis due to L. tropica in Kenya.

Isolation of Leishmania donovani from Phlebotomus martini in Baringo District, Kenya

An 18-month sandfly survey was conducted at 4 locations in Baringo District, Rift Valley Province, Kenya. 3 collection techniques were used: aspiration, sticky paper trap, and light trap in sites selected because of their proximity to homes of visceral leishmaniasis patients diagnosed and treated within 6 months before the survey. Over 2000 female Phlebotomus martini were collected of which 6 females were found to have flagellate protozoan infections. 3 of these infections were cultured successfully and cryopreserved. 2 isolates were identified as Leishmania donovani by cellulose acetate electrophoresis. The zymogram of the third isolate was different from all Old World Leishmania reference strains examined, and it is still unidentified. The finding of 2 P. martini naturally infected with L. donovani strongly supports the hypothesis that this species is a vector of visceral leishmaniasis in this area.

Human cutaneous leishmaniasis caused by Leishmania donovani s.l. in Kenya

Our laboratory is characterizing Leishmania stabilates and isolates from active leishmaniasis cases. Smears and cultures from aspirates made on different dates from a single lesion on the bridge of the nose of an 18 years old Kenyan male from Nyandarua District contained Leishmania. The isolates, NLB-271 and NLB-271-IA, were characterized by cellulose acetate electrophoresis (CAE) using 20 enzyme systems and by Southern analysis using 2 deoxyribonucleic acid (DNA) probes (pDK10 and pDK20) from a Dakar strain of L. major (MHOM/SN/00/DK1) and a third probe, p7-059 from L. infantum strain ITMAP-263. Digestion of the two Leishmania DNAs with endonucleases HindIII and PstI, followed by hybridization with the 3 probes, revealed DNA fragment banding patterns indistinguishable from those of the L. donovani species complex. The CAE isoenzyme profiles of these 2 Kenyan isolates were indistinguishable from those of Kenyan L. donovani strains we designated as zymodeme Z6. Excluding post-kala-azar dermal leishmaniasis, this constitutes the first human case of cutaneous leishmaniasis caused by L. donovani s.l. in Kenya. Previously, cutaneous leishmaniasis cases in Kenya have been due to L. aethiopica, L. major and L. tropica only.

Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes

In situ hybridization on cultured promastigotes and sandfly smears were performed with nonradioactively labeled total DNA and recombinant DNA probes containing minicircle kinetoplast DNA (kDNA) or nuclear DNA inserts. Total DNA probes lack specificity whereas recombinant nuclear DNA probes work only if they contain repetitive sequences. Minicircle kDNAs of five Leishmania isolates, representative of five Leishmania taxa found in Kenya, were sequenced. Comparison of the sequences showed a 150-bp region with around 80% homology, whereas the rest of the minicircles had about 50% homology. Nevertheless, application of these probes in in situ hybridization assays as tested on Leishmania promastigotes in the vector gave good specificity and hybridization signal. Two types of labeling were tested: incorporation of biotin-labeled dUTP or directly horseradish peroxidase (HRP)-labeled nucleotides. Both techniques provided good sensitivity and signal-to-noise ratio on cultured promastigotes. Hybridization with HRP-labeled kDNA probes gave a superior signal-to-noise ratio if tested on sandfly preparations. This method provided a reliable and fast identification and facilitated the detection of promastigotes in sandflies. The technique presented here may be helpful in rapid identification of Leishmania promastigotes, and thus make epidemiological studies easier and less time consuming.

Mouse foot-pad inoculation as an aid to the isolation of Leishmania spp. from patients

Portions of splenic or subcutaneous saline aspirates from suspected visceral or cutaneous leishmaniasis patients were inoculated into NNN media with an overlay of Schneiders medium or Schneiders medium alone for routine parasitological diagnosis. The remaining portions of the aspirates were used for preparing Giemsa-stained smears and for subcutaneous inoculation into hind foot-pads of Balb/c mice. Saline aspirates obtained from the foot-pads 2-14 d after inoculation were inoculated into Schneiders medium and examined for promastigotes. Parasite isolation was achieved from 90% of confirmed leishmaniasis patients by either culture method alone. Mouse foot-pad aspiration demonstrated parasites in 95% of all patients, and in over 80% of the confirmed cases of leishmaniasis. Combined culturing and aspirate smear examination was more efficient than foot-pad inoculation alone for the demonstration of leishmanial infection. Foot-pad aspiration does not entail killing animals and was sensitive for parasite isolation; it may be a useful short-term adjunct to existing parasite isolation methods, especially under field conditions where the risks of culture contamination may be high.

Leishmaniasis Research in Kenya: Parasite-Vector-Host Associations

Clinically speaking, there are 2 types of leishmaniasis in Kenya, visceral leishmaniasis, or kala-azar, caused by Leishmania donovani and cutaneous leishmaniasis caused by L. aethiopica, L. major, L. tropica (a recent discovery), and L. donovani (post kala-azar dermal leishmaniasis). These will each be discussed from a historical perspective, then from a perspective of current research on Leishmania parasite-vector-host associations.

Characterization of Simulium (Edwardsellum) damnosum s. l. populations from six river systems in Kenya by cellulose acetate electrophoresis

Isoenzyme characterization of the Simulium damnosum Theobald sibling species complex from two widely separated geographical areas in Kenya is presented based on 10 enzymatic loci. Four river systems in Western and Nyanza Provinces, namely, the Yala, Lusumu, Isiukhu and the Nzoia harbouring S. damnosum s.l. were compared among themselves and with S. damnosum s.l. collected from the Thiba and the Nyamindi river systems in the Mt. Kenya area. The two populations were easily separable using PGM, HK and, more than 73% of the time, with PGI. Using the first two enzymatic loci, PGM and HK, all the western Kenya S. damnosum s.l. belong to the same population while those from Mt. Kenya areas belong to a different population. In both geographical zones there was less than 20% qualitative and quantitative polymorphism within infraspecific forms in any given breeding area of S. damnosum s.l. Three enzymes, ME, XDH, and G-6-PDH had isomorphic mobilities for both the Mt. Kenya and western Kenya populations. Four other enzyme/substrate systems tested had no satisfactory resolution as a diagnostic value.

Rapid enzymological identification of leishmanial isolates in Kenya

Leishmanial isolates from Kenya were identified as being either Leishmania major, L. donovani or L. aethiopica by using cellulose acetate electrophoresis. Species determination is based on the electrophoretic pattern for glucose phosphate isomerase alone. This report describes the technique and presents data on its reliability as a rapid identification test for Leishmania isolates from the same geographical region.