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A two-year study was conducted of phlebotomine sand fly fauna in a defined focus of Leishmania tropica. A total of 17,947 sand flies representing 10 species were collected from the location. Phlebotomus guggisbergi, a vector of L. tropica in Kenya, was the most prevalent species through the entire period, representing about 80% of the total catch. There was marked seasonal fluctuation in the populations of the three most common species, with highest population levels reached in December and lowest levels reached in July and August. Leishmania-like infections were encountered in 489 P. guggisbergi. No flagellate infections were observed in any other species of sand fly. Although infected P. guggisbergi were collected during each month of the year, the percent parous infected flies was highest (27.5%) during the November through January time period. These data show that the greatest risk of transmission to humans at this focus occurs during December, when the vector is prevalent and infections are common.

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The genetic mechanisms that confer larval permethrin resistance were investigated in two strains of Aedes aegypti, vectors of yellow fever and dengue hemorrhagic fever. Larval resistance to permethrin in an Ae. aegypti field-collected resistant Couva (R) strain was associated with the sex-determining locus by analysis of backcrosses to the susceptible Rockefeller (S) strain. The median lethal concentrations (LC50s) of these strains were 23.1 (95% confidence interval = 22.0-24.3) and 2.2 (2.0-2.3) parts/billion of permethrin, respectively. The estimated resistance ratio (RR) for the R strain was 10.8 (10.3-11.4) compared with the S strain. Resistance was inherited as partly recessive (dominance [D] = -0.31) with an estimated RR of 2.3 (2.1-2.4) in the F1 hybrids when the R parent was male. There were also significantly male-biased sex ratios for this cross. In contrast, inheritance was slightly dominant (D = 0.19) with an estimated RR of 4.1 (3.8-4.4) when the R parent was female, and no significant sex ratio bias of progeny was observed. Analysis revealed a strong paternal-strain effect in bioassay mortality, sex ratio, egg hatch, and fecundity. A maternal-strain effect was also evident for bioassay mortality. Similarly, a strong maternal by paternal strain interaction was also evident for sex ratio. Progeny of single-family backcrosses of F1 hybrids to R were statistically homogeneous for sex ratio, duration of oviposition, fecundity, and hatch rate. A significant increase in male bias was found for only one backcross to R, after treatment with permethrin. In contrast, complex patterns of inheritance of life histories were observed among backcrosses to S. Backcrosses to S had greater mean fecundities, shorter mean times to the start of oviposition, and shorter mean oviposition periods than did backcrosses to R. Hatch rates were statistically homogeneous among backcrosses, but all strikingly reduced relative to the parental generation. Times of start and duration of oviposition were highly negatively correlated with fecundity (first gonotropic cycle only) and rate of egg hatch. Females with lower fecundities had lower hatch rates, and there was a threshold of approximately 80 eggs per female, below which no eggs hatched. Generally all backcrosses had higher LC50s than expected from single-locus inheritance. Association between sex bias and inheritance of resistance was apparent, but no single genetic linkage model based on current understanding of sex chromosome genetics was consistent with these observations. These results may have epidemiologic importance considering that permethrin-soaked bed nets are being used in many countries to control the biting activity of disease vectors.

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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.

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In the early 1930s, investigators of visceral leishmaniasis stated that Leishman-Donovan bodies are found in body fluids of kala-azar patients, for example, in urine, feces, semen, and nasal and pharyngeal secretions. Based on this finding, we investigated the diagnostic potential of nasal secretions, tonsillopharyngeal mucosal swabs, and urine centrifugates inoculated into Schneider's Drosophila Medium (containing antibiotics and antifungal agents) as well as with Giemsa-stained smears. Consequently, 64 randomly selected patients with visceral leishmaniasis from Kenya (59 who were splenic culture or Giemsa stain positive and five who were culture negative but Giemsa stain positive) were tested by three noninvasive methods. These tests were all performed before the patients were treated with Pentostam. Cultures of nasal and tonsillopharyngeal swabs and urine centrifugates produced 28 positive samples representing 24 patients (37.5%). Moreover, a set of 25 Giemsa-stained slide smears made from the nasal and tonsillopharyngeal mucosa of 25 patients with visceral leishmaniasis who had not tested positive in cultures produced nine positives. Therefore, the overall total of patients who tested positive by all of the above methods was 33 or 51.6%. The cryopreserved Leishmania isolates were characterized by cellulose acetate electrophoresis using 20 enzyme systems. The isoenzyme profiles produced by the parasites were represented in five different L. donovani s.l. zymodemes. Representatives of these isolates were also characterized by DNA Southern blotting analysis, which corroborated the isoenzyme results.

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A total of 407 Leishmania and other Leishmania-like isolates obtained from patients, other vertebrates, sand fly vectors, and other arthropods from Kenya and other countries were characterized and compared with several World Health Organization and other well-characterized reference strains of Leishmania, Trypanosoma, Crithidia, Herpetomonas, and Leptomonas by cellulose acetate electrophoresis (CAE), using 20 enzyme systems. Analysis of the isoenzyme banding patterns (IBP) of the isolates generated isoenzyme profiles that were resolved as zymodemes and tabulated. Isolates that produced similar isoenzyme profiles in all 20 enzyme systems were placed into a particular Leishmania isoenzyme taxon, with the zymodeme designated numerically as Zn. A total of 66 zymodemes were recorded for the 407 isolates studied. To obviate the need to draw all 66 representative IBP for each of the 20 enzyme systems, the 66 zymodemes (Z1-Z66) were again placed into similarity groups represented by pattern number or Pn. This resulted in 23-50 IBP (Pn) per enzyme system. The highest number of IBP scored was for malate dehydrogenase (MDH) (P1-50) and the lowest score was for glucose-6-phosphate isomerase (GPI) (P1-23). From these different isoenzyme profiles or zymodemes, IBP of 14 (MDH, GPI, nucleoside hydrolase, phosphoglucomutase, malic enzyme, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, mannose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, glutamate oxaloacetate transferase/aspartate aminotransferase, glutathione reductase, superoxide dismutase, fumarase, and glyceraldehyde-3-phosphate dehydrogenase) of the 20 enzyme systems were selected for computer-calculated numerical taxonomy. Consistent individual isoenzyme bands with similar relative mobilities of the 14 enzyme systems were scored into groups (allelomorphs, allozymes, or electromorphs) and used in cluster analysis. For each pattern in every profile, the presence of a consistent band was entered as 1 and its absence as 0. A total of 419 allozyme characters (variables) were scored for the 14 enzyme systems. Lastly, all different zymodemes sharing a particular IBP (Pn) within an enzyme system were counted and the total number was shown as a zymodeme frequency (Zf). Final analysis of the CAE isoenzyme profiles and cluster-dendrograms resulted in the identification of several potentially new species and subspecies of Leishmania and other Leishmania-like isolates from patients, sand flies, and animal reservoir hosts collected from Kenya and other locations in Africa. Zymodeme analysis of the Kenyan visceral and cutaneous leishmaniasis isolates resulted in the identification of 11 subpopulations of the L. donovani species complex and six subpopulations of the L. tropica species complex endemic to different geographic areas of Kenya.

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We have identified a new rural focus of cutaneous leishmaniasis caused by Leishmania tropica in Muruku sublocation, Salama location, Laikipia district, Rift Valley province, Kenya. Based on a few available case histories, previous reports of L. tropica in Kenya indicated a tentative geographical distribution. Recently 6 indigenous Kenyans from the new focus, who had never travelled outside Kenya, developed cutaneous lesions on the face and/or extremities found to contain Leishmania by culture and smear. Most of the patients manifested the typical 'urban' dry sore which grew slowly into a nodule measuring 2 x 1 cm to 9.5 x 3 cm, and after some months formed a central crust surrounded by small satellite papules. After treatment with Pentostam (sodium stibogluconate), about 40% of the sores failed to heal completely, either scarring centrally with fulminating papules at the edges and spreading peripherally, or healing but then recrudescing at the edge of the scar. Stationary-phase promastigotes from culture isolates were analysed by cellulose acetate electrophoresis. Isoenzyme profiles of 6 isolates were compared with those of World Health Organization reference strains using 12 enzyme loci; they were indistinguishable from those of 2 L. tropica reference strains. All 6 case sites lay within a radius of 4 km. Several other suspected cases from the same area are being investigated.

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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.

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Leishmania isolates aspirated a few months apart from the spleen of an indigenous adult male kala-azar patient from Baringo District, Kenya, were biochemically characterized and compared. The patient lived within a dual focus of L. donovani kalazar and L. major cutaneous leishmaniasis. A primary Leishmania isolate from splenic aspirates was cryopreserved (NLB-294). The patient was treated with sodium stibogluconate for kala-azar and discharged. Three months later, he had clinical relapse and returned for retreatment. During his second visit, the patient participated in a diagnostic study in which urine and nasopharyngeal samples were cultured for leishmaniasis. Urine, nasopharyngeal, and splenic samples were positive for Leishmania. Secondary isolates from splenic (NLB-294-I) and urine (NLB-318) cultures were cryopreserved and characterized by cellulose acetate electrophoresis (CAE) using 20 enzymes. Whereas the urine isolate was typed as L. donovani, the splenic aspirate culture revealed a mixed infection with L. donovani and L. major. The primary isolate (NLB-294) was then characterized and also showed a mixed infection. To exclude the possibility of protein post-translational modifications in electrophoretic assays, the primary and secondary isolates were grown and processed under identical cultural and lysis conditions, and compared using CAE. The results were identical to the first electrophoretic assays showing mixed promastigote banding patterns. Stationary-phase promastigotes of the secondary splenic isolate (NLB-294-I) inoculated subcutaneously, intraperitoneally, and intracardially into Syrian hamsters and BALB/c mice produced both kala-azar and cutaneous leishmaniasis within 6.5 months.(ABSTRACT TRUNCATED AT 250 WORDS)

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Sand flies were collected in light traps and on oiled papers at four active case sites of human cutaneous leishmaniasis due to Leishmania tropica at Muruku Sublocation, Laikipia District, Kenya. Nearly 5,200 females of five species, including Phlebotomus guggisbergi, were dissected and examined for flagellates. Of 3,867 P. guggisbergi females collected at a multiple case site, 168 (4.3%) harbored mature infections (to include metacyclic promastigotes) of flagellates morphologically identical to Leishmania, while all other flies were negative. Of the infected flies, 164 were collected in a cave near the patients' home, three from crevices on an escarpment immediately behind the house, and one from the bedroom of one of the patients. One hundred sixty-four of the isolates were successfully grown in Schneider's Drosophila medium and harvested for typing by cellulose-acetate electrophoresis. Isoenzyme profiles of the first 22 of these were compared with those of WHO reference strains and well characterized local strains using 12 enzyme loci. The isolates yielded isoenzyme migration patterns that were indistinguishable from those of two L. tropica reference strains and of six L. tropica patient isolates from the same locality. This is the first reported isolation of L. tropica from a sand fly in Kenya, the first reported isolation of Leishmania parasites from P. guggisbergi, and the first confirmed isolation of this Leishmania from a sand fly other than P. sergenti. The finding of such a large number of P. guggisbergi naturally harboring mature infections of L. tropica at an active case site of cutaneous leishmaniasis due to this agent strongly implicates this fly as a vector.

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Quantitative in vitro drug sensitivity of 32 Leishmania isolates (16 from patients failing pentavalent antimony [SbV] therapy) was determined using a radiorespirometric microtechnique (RAM). Of 30 isolates with histories, 22 (73%) RAM tests agreed with patient history; the remaining 8 (27%) did not. There was no difference in RAM drug sensitivity: clinical correlation between 15 isolates tested blindly and 15 with known clinical history (4 did not agree with clinical history in both). Test sensitivity appeared to be limited only by the sensitivity of the Leishmania to SbV and could be detected at 2 micrograms/ml Sb (about 10% of serum drug level). An isolate from a patient with untreated self-healing cutaneous disease was drug resistant. Using RAM, parasite drug sensitivity can be quantified apart from patient physiologic and immunologic variables intrinsic to clinical data. Potency differed a maximum of 100% (weight% Sb:weight% Sb) among drug lots and also between Glucantime and Pentostam. Potency changes between drug lots were not explainable based on Sb content or test-to-test variability. This microtest offers a rapid method for evaluating the drug sensitivity of patient isolates and for determining of the activity of pentavalent antimonials and other candidate anti-leishmanials prior to the initiation of therapy.

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The extrinsic development of Leishmania major was observed in 2 man-biting sand flies, Phlebotomus duboscqi, a known vector, and Sergentomyia schwetzi, an assumed non-vector. Flies fed on a leishmanial lesion on the nose of a hamster were examined for infection at 0, 6, 12, 18, 24, 36, 48, and 60 hr and at approximately 24 hr intervals from day 3 to day 14 post-feeding. Infection rates, determined by light microscopy, were 47% (n = 258) in P. duboscqi and 5% (n = 162) in S. schwetzi. Transformation from amastigotes to "procyclic" promastigotes occurred in both species at 6-18 hr post-feeding. In P. duboscqi, the parasites multiplied rapidly and developed through as many as 10 forms, including at least 3 dividing-promastigote forms. Metacyclic promastigotes, the "infective" form, appeared at 6 days post-feeding, first in the region of the stomodeal valve, then in the pharynx, cibarium, and proboscis. In a single attempt 14 days post-feeding, a P. duboscqi transmitted L. major to a mouse by bite. In contrast, the parasites multiplied slowly in S. schwetzi, and did not develop beyond "procyclic" promastigotes. The parasites did not migrate anteriorly nor survive beyond 90 hr post-feeding, indicating that S. schwetzi is not a vector of L. major. Classical strategies for vector incrimination may be confounded by the isolation of non-infective early developmental forms of Leishmania from wild-caught non-vectors.

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We report the characterization of 6 Leishmania tropica isolates from 2 patients with visceral leishmaniasis who were unresponsive to treatment with sodium stibogluconate. The Leishmania isolates, MHOM/KE/81/NLB-029A, -029XIB, and -029XIC and MHOM/KE/81/NLB-030I, -030B, and -030XXA, all from splenic aspirates, were characterized by cellulose acetate electrophoresis using 11 enzymes: malate dehydrogenase, malic enzyme, phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, superoxide dismutase, glutamate-oxaloacetate transaminase, adenylate kinase, nucleoside hydrolase, mannose phosphate isomerase, glucose phosphate isomerase, and phosphoglucomutase. Isozyme migration patterns were indistinguishable from those of 2 WHO reference strains of Leishmania tropica (MHOM/SU/60/LRC-L39, NLB-305 and MHOM/IQ/OO/LRC-L36, NLB-067). These are the first reported cases of visceral leishmaniasis (kala-azar) caused by L. tropica in Africa; these cases were refractory to sodium stibogluconate.

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Six Leishmania isolates from 3 indigenous Kenyans (2 isolates from one patient) and 2 Canadian visitors in Kenya were characterized by cellulose acetate electrophoresis. The isolates were compared among themselves and with reference strains of Leishmania donovani, L. aethiopica, L. major, L. tropica, and L. arabica using 9 enzymes: malate dehydrogenase (MDH), malic enzyme (ME), phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), aspartate aminotransferase (ASAT), adenylate kinase (AK), mannose phosphate isomerase (MPI), glucose phosphate isomerase (GPI), and phosphoglucomutase (PGM). Enzyme migration patterns of isolates from the 3 indigenous Kenyans were indistinguishable from those of 2 L. tropica reference strains. The isolates from the 2 Canadians yielded migration patterns of 7 enzymes that were indistinguishable from those of 2 L. tropica reference strains. However, migration patterns of 2 enzymes, PGM and ME, differed from all migration patterns of the 10 reference strains. Balb/c mice were inoculated with stationary phase promastigotes cultured from 3 stabilates from the lesions of 2 of the Kenyan patients. The mice developed no gross pathological lesions in 6 months time. All of the study patients developed cutaneous leishmaniasis while living in or visiting districts in Central and Rift Valley Provinces, Kenya. This is the first report of human cutaneous leishmaniasis caused by L. tropica indigenous to Africa south of the Sahara.

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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.

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Portions of splenic or subcutaneous saline aspirates from suspected visceral or cutaneous leishmaniasis patients were inoculated into NNN media with an overlay of Schneider's medium or Schneider's 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 Schneider's 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.

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Isozyme variation of the Simulium damnosum sibling species complex was studied by cellulose acetate electrophoresis (CAE) from four Kenyan river systems. Two enzymes, PGM and HK, were diagnostic and differentiated the larvae collected in Western and Nyanza provinces from the larvae collected at Mt. Kenya. Allele frequency differences of the enzyme PGI allowed about 75% separation of the geographically distinct populations.

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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.

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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.

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