Random amplified polymorphic DNA (RAPD) analysis of Lutzomyia longipalpis laboratory populations.

The phlebotomine sand fly Lutzomyia longipalpis has been incriminated as a vector of American visceral leishmaniasis, caused by Leishmania chagasi. However, some evidence has been accumulated suggesting that it may exist in nature not as a single but as a species complex. Our goal was to compare four laboratory reference populations of L. longipalpis from distinct geographic regions at the molecular level by RAPD-PCR. We screened genomic DNA for polymorphic sites by PCR amplification with decamer single primers of arbitrary nucleotide sequences. One primer distinguished one population (Marajó Island, Pará State, Brazil) from the other three (Lapinha Cave, Minas Gerais State, Brazil; Melgar, Tolima Department, Colombia and Liberia, Guanacaste Province, Costa Rica). The population-specific and the conserved RAPD-PCR amplified fragments were cloned and shown to differ only in number of internal repeats.

Plasmodium falciparum incidence relative to entomologic inoculation rates at a site proposed for testing malaria vaccines in western Kenya.

Relationships between Plasmodium falciparum incidence and entomologic inoculation rates (EIRs) were determined for a 21-month period in Saradidi, western Kenya, in preparation for malaria vaccine field trials. Children, ranging in age from six months to six years and treated to clear malaria parasites, were monitored daily for up to 12 weeks to detect new malaria infections. Overall, new P. falciparum infections were detected in 77% of 809 children. The percentage of children that developed infections per two-week period averaged 34.7%, ranging from 7.3% to 90.9%. Transmission by vector populations was detected in 86.4% (38 of 44) of the two-week periods, with daily EIRs averaging 0.75 infective bites per person. Periods of intense transmission during April to August, and from November to January, coincided with seasonal rains. Relationships between daily malaria attack rates and EIRs indicated that an average of only 7.5% (1 in 13) of the sporozoite inoculations produced new infections in children. Regression analysis demonstrated that EIRs accounted for 74% of the variation in attack rates. One of the components of the EIR, the human-biting rate, alone accounted for 68% of the variation in attack rates. Thus, measurements of either the EIR or the human-biting rate can be used to predict corresponding attack rates in children. These baseline epidemiologic studies indicate that the intense transmission patterns of P. falciparum in Saradidi will provide excellent conditions for evaluating malaria vaccine efficacy.

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.

Laboratory evaluation of repellents against four anopheline mosquitoes (Diptera: Culicidae) and two phlebotomine sand flies (Diptera: Psychodidae).

Deet, the lactone CIC-4, and the piperidine compounds A13-37220 and A13-35765 were evaluated for initial repellency against laboratory-reared Anopheles albimanus Wiedemann, An. freeborni Aitken, An. gambiae Giles, An. stephensi Liston, and Phlebotomus papatasi (Scopoli) using a dose-response testing procedure on human volunteers. In addition, deet and CIC-4 were tested against Lutzomyia longipalpis (Lutz & Neiva). In general, the repellency of A13-37220, A13-35765, and CIC-4 was not markedly different from that of deet against each species tested; however, the different species varied greatly in response to the repellents. Overall, An. stephensi, L. longipalpis, and P. papatasi were the most sensitive, and An. albimanus the most tolerant species. The four repellents subsequently were tested against An. stephensi and An. albimanus to determine the duration of repellency. AI3-37220 provided effective (> 90%) protection against An. stephensi bites for 7 h, whereas deet, AI3-35765, and CIC-4 provided 6, 5, and 3 h of protection, respectively. Each of the four compounds provided < 1 h of protection against An. albimanus bites.

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.

Stage-specific adhesion of Leishmania promastigotes to the sandfly midgut.

Although leishmaniasis is transmitted to humans almost exclusively by the bite of infected phlebotomine sandflies, little is known about the molecules controlling the survival and development of Leishmania parasites in their insect vectors. Adhesion of Leishmania promastigotes to the midgut epithelial cells of the sandfly was found to be an inherent property of noninfective-stage promastigotes, which was lost during their transformation to metacyclic forms, thus permitting the selective release of infective-stage parasites for subsequent transmission by bite. Midgut attachment and release was found to be controlled by specific developmental modifications in terminally exposed saccharides on lipophosphoglycan, the major surface molecule on Leishmania promastigotes.

Recent advances in laboratory mass rearing of phlebotomine sand flies.

Recent technical and procedural advances in mass rearing of sand flies have resulted in larger, healthier, and less labor-intensive colonies. We now maintain closed colonies of Phlebotomus papatasi, P. duboscqi, P. argentipes, and Lutzomyia longipalpis which produce up to 1,000 females per week, in excess of colony-maintenance requirements, for use in research. Advances include larval food preparation in acrylic-plastic incubator cabinets, strict regulation of food quantity and moisture in 500-ml plaster-lined rearing jars, use of large plaster-lined adult holding/mating cages and vacuum-powered aspirators for trauma-free handling of adults.

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)

Characterization of malaria transmission by Anopheles (Diptera: Culicidae) in western Kenya in preparation for malaria vaccine trials.

Malaria transmission was studied for 33 mo in the villages of Kisian and Saradidi in western Kenya in preparation for field trials of malaria vaccines. Abundance estimates of Anopheles gambiae Giles sensu lato and Anopheles funestus Giles, which constituted over 99% of 26,645 anophelines collected, were compared for all-night biting collections inside houses, outdoors, and in tents. The overall numbers of Anopheles per man-night were 2.3 times greater in Kisian than in Saradidi. For the three types of collections, mean sporozoite rates by dissection ranged from 2.2 to 5.4% for 13,072 Anopheles in Kisian and from 9.9 to 13.6% for 7,058 Anopheles in Saradidi; greater than 90% of the infections were Plasmodium falciparum, either alone or mixed with P. malariae or P. ovale. Heaviest transmission from April to July coincided with the end of the long rainy season. Entomological inoculation rates (EIR) averaged 0.82 infective bites per man per night inside houses in Kisian and 0.65 in Saradidi. Outdoors, EIRs averaged 0.09 in Kisian and 0.52 in Saradidi. In tents, which were evaluated to identify methods for exposing nonindigenous volunteers during vaccine efficacy trials, EIRs were 3.3 and 2.5 times less than inside houses for Kisian (EIR = 0.25) and Saradidi (EIR = 0.26), respectively. Exposure in tents averaged one infective bite every 4.0 d in Kisian and every 3.8 d in Saradidi. The use of tents in vaccine efficacy trials should provide adequate exposure for nonindigenous volunteers. Malaria vaccine trials could be conducted efficiently in western Kenya, with timing dependent upon the intensity of transmission required by vaccine trial objectives.

Malaria sporozoite detection by dissection and ELISA to assess infectivity of afrotropical Anopheles (Diptera: Culicidae).

Malaria infection rates determined by dissection and Plasmodium falciparum enzyme-linked immunosorbent assay (ELISA) were compared for 26,935 Anopheles gambiae Giles sensu lato and 17,739 Anopheles funestus Giles collected during 20 mo in western Kenya. ELISA infection rates were about 43% higher than dissection sporozoite rates. In dissection-negative Anopheles, circumsporozoite (CS) protein was detected by ELISA in 5.2% of 10,017 salivary gland samples and in 12.2% of 237 thorax samples. The accuracy of dissection and ELISA techniques was compared by the following tests on a group of 352 field-collected Anopheles (held 10 d to ensure sporogonic development): salivary gland dissection, examination of Giemsa-stained dissection slides, ELISA tests on salivary gland and thorax body parts, and microscopic techniques for determining sporozoite loads. Respective infection rates were 9.9%, 10.8%, and 15.6% for dissection, stained slides, and ELISA. Sporozoite loads were associated significantly with ELISA absorbance values (r = 0.76). Compared with Giemsa-stained dissection slide results, the sensitivity of sporozoite detection was 92.1% for dissection compared with 78.9% for ELISA; specificity was 100.0% for dissection versus 92.0% for ELISA. Immunological detection of CS protein in head-thorax samples of Afrotropical vectors overestimated the proportion of infective Anopheles because the comparison of techniques indicated that 45.4% of the ELISA positive Anopheles did not contain salivary gland sporozoites.

Transmission of Rift Valley fever virus by the sand fly, Phlebotomus duboscqi (Diptera: Psychodidae).

Studies were conducted to determine if the sand fly Phlebotomus duboscqi could serve as a vector of Rift Valley fever (RVF) virus. When 145 P. duboscqi were fed on a hamster with RVF viremia (approximately 10(9) PFU/ml of blood), 72 (50%) became infected. Of 5 with disseminated infections (i.e., virus recovered from their legs) 4 transmitted virus to hamsters by bite. Sand flies were uniformly infected when RVF virus was inoculated by the intrathoracic route, and each of 31 sand flies so inoculated that fed on a hamster transmitted virus. None of 331 progeny of inoculated sand flies or 230 progeny of orally exposed sand flies contained virus. Sand flies could serve as vectors of RVF virus.

Identification of malaria species by ELISA in sporozoite and oocyst infected Anopheles from western Kenya.

Enzyme-linked immunosorbent assays (ELISAs) for the circumsporozoite (CS) antigens of Plasmodium falciparum, P. malariae, and P. ovale were used to identify species of sporozoite and oocyst infections detected by dissection in Anopheles gambiae s.1. and An. funestus collected in western Kenya. ELISAs identified 92.5% of 1,113 salivary gland infections; Plasmodium species infections included 79.4% P. falciparum, 3.2% P. malariae, 1.7% P. ovale, and 2 or more Plasmodium species were detected in 15.7% of the Anopheles in which the species of parasite was identified. Identification was more likely with greater numbers of sporozoites observed in dissections, increasing from 65% ELISA positivity in mosquitoes with 1-10 sporozoites in their salivary glands to 96% in mosquitoes with over 1,000 sporozoites. ELISAs detected CS antigen in 66% of 294 Anopheles that by dissection had oocysts but uninfected salivary glands. Of 112 Anopheles with a single species of Plasmodium detected in the salivary glands, 29 (25.9%) had 1 or more additional species detected in the midgut, indicating a high potential for multiple infections. Similar proportions of Plasmodium species were found in An. gambiae s.1. and An. funestus.

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.

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.

Detection and enumeration of Leishmania in sand flies using agar-based media.

An agar plating technique was used to determine the number of amastigotes ingested by Lutzomyia longipalpis fed on papules on Mesocricetus auratus caused by Leishmania mexicana amazonensis and on lesions on Mystromys albicaudatus caused by Leishmania braziliensis panamensis. The technique involved homogenizing sand flies after bloodfeeding on the infected animals and spreading the homogenate over the surface of agar plates. A great variation in the number of amastigotes ingested by individual sand flies was demonstrated. Not all amastigotes ingested developed anterior stomodeal infections.

Field evaluation of an enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum sporozoite detection in anopheline mosquitoes from Kenya.

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody that recognizes a repetitive epitope on the circumsporozoite protein of Plasmodium falciparum was used in Kenya to assess malaria infections in Anopheles gambiae s.l. and An. funestus. The ELISA confirmed that 88% of 44 sporozoite-positive gland dissections were P. falciparum. The ELISA infection rate of 18.6% (n = 736) for individually tested mosquitoes for both species was significantly higher than the 10.4% (n = 537) salivary gland sporozoite rate determined by dissection. This difference was due to ELISA detection of medium and large sized oocysts on the midguts of infected mosquitoes which did not contain salivary gland sporozoites. From a series of 379 Anopheles that were cut at the thorax, ELISA tests on "head" and "body" portions showed that 29.5% of 95 positive mosquitoes contained circumsporozoite antigen in the body portion in the absence of salivary gland infections. This field evaluation demonstrates that the ELISA can most accurately be used to estimate sporozoite rates by cutting mosquitoes at the thorax and testing anterior portions.