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.

Lutzomyia longipalpis is a species complex: genetic divergence and interspecific hybrid sterility among three populations.

The sand fly Lutzomyia longipalpis is the vector of Leishmania donovani chagasi in Latin America. An analysis of genetic variability at 27 enzyme coding loci among three laboratory populations of Lu. longipalpis revealed substantial genetic polymorphism. Levels of genetic distance between all pairwise comparisons of colonies were very high, and consistent with those previously reported among separate species in the genus Lutzomyia. Between 7% and 22% of the loci studied were diagnostic for any two of the colony populations. Experimental hybridization between colonies resulted in the production of sexually sterile male progeny. Our results provide strong evidence that Lu. longipalpis exists in nature as a complex of at least three distinct species. The possible effects of colonization on the genetic makeup of laboratory populations is considered in extending our results to natural populations.

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.

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)

Human cutaneous leishmaniasis acquired in Texas.

Four cases of autochthonous human cutaneous leishmaniasis have been identified in south-central Texas since 1980. The patients presented with chronic ulcerating papules on the face, earlobe, and lateral thigh. In two patients, the infections healed without treatment. In the other two patients, the lesions healed following treatment with intramuscular sodium stibogluconate or topical antimony potassium tartrate. Serologic testing of family members, using four different techniques, indicates that asymptomatic infections may occur. These are the first reported cases of cutaneous leishmaniasis acquired in Texas since 1974. Organisms isolated from patients in 1974 and 1980 belonged to the Leishmania mexicana complex when tested by the isoenzyme technique. Although no animal reservoir or insect vector has been identified, six species of sand flies belonging to the genus Lutzomyia do inhibit this part of Texas. Accumulated evidence strongly suggests that cutaneous leishmaniasis is endemic in south-central Texas.

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.

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.

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.

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.

Epidemiologic investigation of an outbreak of cutaneous leishmaniasis in a defined geographic focus of transmission.

An outbreak of cutaneous leishmaniasis occurred in a unit of 608 Puerto Rican national guardsmen conducting jungle warfare training in the Panama Canal Area in July 1984. An epidemiologic investigation of reported nonhealing, ulcerating skin lesions was conducted among 540 (89%) unit members in November and December 1984. Fifteen (88%) of 17 individuals with chronic, ulcerating skin lesions were confirmed as cases of cutaneous leishmaniasis by culture or histopathology. Twelve cases yielded positive Leishmania cultures, identified as L. braziliensis panamensis by cellulose acetate electrophoresis. Evaluation of different diagnostic techniques revealed that direct examination of tissues by Giemsa-stained histological examination was the most sensitive test (87% sensitivity), with an indirect immunofluorescent antibody test being rather insensitive (67%). All but one of the confirmed cases operated in small units that trained and slept overnight at a mortar firing site for a period of three days, yielding a site-specific attack rate of 22% (14 of 64). This contrasted with a much lower attack rate of 0.2% (1 of 476), experienced by unit members who trained at other locations during the same time frame (P less than 0.001). The median incubation period calculated from day of arrival at the mortar firing site was 17 days (range 2-78) for the 15 confirmed cases. Available personal protection methods, such as the use of insect repellents, were not appropriately implemented by unit personnel and thus, were not found to effectively protect against Leishmania infection. This is the largest reported outbreak of cutaneous leishmaniasis in military personnel associated with a single geographic focus of infection and contrasts with the usual sporadic disease experience in Panama.

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.

Experimental transmission of Leishmania major to vervet monkeys (Cercopithecus aethiops) by bites of Phlebotomus duboscqi (Diptera: Psychodidae).

Experimental transmission of Leishmania major to vervet monkeys (Cercopithecus aethiops) was accomplished by bites of Phlebotomus duboscqi sandflies. Three-day-old, laboratory-reared P. duboscqi were fed on leishmanial lesions on hamsters infected with L. major. The flies were re-fed on monkeys 10 d after infection. Five adult male vervet monkeys were used in concurrent transmission trials. Two of the monkeys received subcutaneous inoculations with stationary-phase promastigotes (2 x 10(6) promastigotes in 0.1 ml of medium) on the base of the tail. Putatively infected P. duboscqi were allowed to feed on the remaining 3 monkeys at sites on the base of the tail and on the right eyebrow. Challenges by sandfly bites resulted in multiple leishmanial lesions at all bite sites and, consequently, more lesion area than was produced by needle challenges. Post-feeding dissection of sandflies indicated that multiple lesions could be caused by bites of a single fly, and that probing alone, without imbibing blood, was sufficient for transmission. These first experimental transmissions of L. major to vervets by bites of P. duboscqi demonstrate that sandfly challenge is an efficient alternative to needle challenge, making available a unique Leishmania-sandfly-non-human primate model for use in vaccine development.

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.

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 transmission of Rift Valley fever virus by Phlebotomus duboscqi, Phlebotomus papatasi, Phlebotomus sergenti, and Sergentomyia schwetzi (Diptera: Psychodidae).

We examined the potential for Phlebotomus papatasi (Scopoli), Phlebotomus duboscqi (Neveu-Lemarie), Phlebotomus sergenti (Parrot), and Sergentomyia schwetzi (Adler, Theodor, & Parrot) to transmit Rift Valley fever (RVF) virus. After feeding on hamsters that had been inoculated with RVF virus, P. papatasi, P. sergenti, and S. schwetzi became infected and developed disseminated infections. All P. papatasi and P. duboscqi inoculated with RVF virus developed high-titer infections. In contrast, only 41% of the inoculated S. schwetzi contained detectable virus, and infected individuals contained significantly less virus than the two Phlebotomus species. Although 50% of the inoculated P. duboscqi transmitted RVF virus to hamsters, only 14% of P. papatasi and none of the S. schwetzi transmitted this virus. Additional studies are needed to determine the role of sand flies as vectors of RVF virus.

Field evaluation of four repellents against Leptoconops americanus (Diptera: Ceratopogonidae) biting midges.

Four repellents, deet, AI3-37220, AI3-35765, and CIC-4, prepared as 12.5% ethanol solutions, were evaluated against biting midges on Stansbury Islands, UT. Leptoconops americanus Carter was the only species that was biting human volunteers during the study. This species bit primarily on the ears at rates up to 840 bites per hour. All four repellents significantly reduced the number of bites on treated volunteers. AI3-37220 consistently provided the longest period of protection, giving 97 and 74% protection at 4 and 8 h, respectively. In a direct statistical comparison, AI3-37220 significantly outperformed deet. CIC-4 and AI3-35765 were the least effective repellents, providing 45-47% protection 8 h after application.

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.

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.

Phlebotomine sand fly control using bait-fed adults to carry the larvicide Bacillus sphaericus to the larval habitat.

Sugar meals of plant origin are an important component of the sand fly diet. We show that sugar solution baits have potential as vehicles for phlebotomine sand fly control. In the laboratory, adult Phlebotomus duboscqi Neveu-Lemaire and Sergentomyia schwetzi (Adler, Theodor, and Parrot) that have consumed an aqueous sucrose solution containing Bacillus sphaericus Neide toxins and are subsequently eaten by larvae produce significant larval death (P < 0.01). In the field, when vegetation near animal burrows and eroded termite mounds was sprayed with sucrose solution with or without incorporation of the larval toxicant B. sphaericus, 40% of female sand flies fed in situ. Dispersing B. sphaericus-carrier sand flies caused significant larval mortality (P < 0.01) in resting and breeding sites in animal burrows 10-30 m from the sprayed vegetation for 2-12 wk posttreatment. Also, adult sand fly populations breeding and resting inside animal burrows were significantly reduced (P < 0.01) following direct application of the sucrose/B. sphaericus solution to the burrow entrances. This control effect lasted 4-10 wk post-treatment. The effect was not seen for sand fly populations breeding and resting inside eroded termite mounds. This approach may be useful for the application of biological control agents against phlebotomine sand flies in biotypes where larvae and adults use the same habitats.

Development and application of the polymerase chain reaction for the detection and identification of Leishmania parasites in clinical material.

Detection, diagnosis and identification of Leishmaniasis may be difficult owing to low numbers of parasites present in clinical samples. The PCR has improved the sensitivity and specificity of diagnosis of several infectious diseases. A leishmania specific PCR assay was developed based on the SSUrRNA genes which amplifies DNA of all Leishmania species. Point mutations occurring within the rRNA genes allow differentiation of the Leishmania complexes using primers constructed with the 3/ ends complementary to the specific point mutations present in the SSU rRNA genes of the Leishmania species. Biopsy material, blood, lesion impressions and blood spots on filter paper can be used in the assay. In a longitudinal study on the incidence rates of VL, subclinical cases and PKDL in an endemic region of Sudan, filter paper blood spots from proven and suspected VL patients, PKDL and control samples from an endemic region in Sudan are being taken. The blood spots were analyzed in the DAT and by PCR and results compared with clinical and parasitological data. The first results indicate that the PCR on blood spots is a simple and sensitive means of detecting active VL; in PKDL patients parasites are detectable in the skin.