Phlebotomine sand fly (Diptera: Psychodidae) seasonal distribution and infection rates in a defined focus of Leishmania tropica.

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.

Inheritance of larval resistance to permethrin in Aedes aegypti and association with sex ratio distortion and life history variation.

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.

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.

Leishmania donovani parasites in the nasal secretions, tonsillopharyngeal mucosa, and urine centrifugates of visceral leishmaniasis patients in Kenya.

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.

Biochemical characterization and zymodeme classification of Leishmania isolates from patients, vectors, and reservoir hosts in Kenya.

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.

A new rural focus of cutaneous leishmaniasis caused by Leishmania tropica in Kenya.

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.

Concurrent infection with Leishmania donovani and Leishmania major in a Kenyan patient: clinical description and parasite characterization.

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)

Phlebotomus guggisbergi (Diptera: Psychodidae), a vector of Leishmania tropica in Kenya.

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.

Quantitative in vitro drug potency and drug susceptibility evaluation of Leishmania ssp. from patients unresponsive to pentavalent antimony therapy.

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.

Development of Leishmania major in Phlebotomus duboscqi and Sergentomyia schwetzi (Diptera: Psychodidae).

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.

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 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.