The effect of 5 years of annual treatment with ivermectin (Mectizan) on the prevalence and morbidity of onchocerciasis in the village of Gami in the Central African Republic.

To assess the impact of 5 years of annual community treatment with ivermectin (Mectizan) on the prevalence of onchocerciasis and onchocerciasis-associated morbidity, data collected, before and after such treatment, in the village of Gami, in a hyper-endemic area of the Central African Republic, were analysed. Skin snips from all the villagers treated in 1990 and/or 1995 were used to assess the prevalence and intensity of infection with Onchocerca volvulus. Ocular and dermatological morbidity was assessed by ophthalmological and clinical examinations of the same subjects. Following the five annual treatments, there was a reduction in the prevalence of infection and a dramatic decrease in the microfilarial load of the community. The prevalences of pruritus, onchocercal nodules and impaired vision were all significantly reduced. The results emphasise the long-term benefits of the mass-treatment programmes, particularly for children aged <10 years.

DNA in situ hybridization on polytene chromosomes of Simulium sanctipauli at loci relevant to insecticide resistance.

A DNA technique for in situ hybridization developed by Kumar & Collins (1994) for use on polytene chromosomes of adult Anopheles mosquitoes (Diptera: Culicidae) was modified for use with Simulium larval salivary gland chromosomes (Diptera: Simuliidae). Cloned fragments of several Simulium genes (coding for aspartate amino transferase, cytochrome P450 and DNA polymerase) were successfully mapped physically by assigning specific band locations in Simulim sanctipauli V. & D. This represents the first attempt at locating genes beyond the resolution of linkage to inversions in any blackfly species.

The Mectizan Donation Programme--a 10-year report.

In the 10 years since 1987 when Merck & Co. announced a plan to donate its safe and effective microfilaricide, Mectizan (ivermectin, MSD), to treat onchocerciasis wherever and for as long as needed, more than 96 million treatments have been enabled in community-based treatment programmes in all 34 countries, in Africa, the Middle East, and Latin America, where the disease is endemic. It is expected that donations enabling some 33 million treatments will be approved in 1997. Since the beginning of the donation programme, it is estimated that some 19-20 million people have received at least one dose of the drug and that many have received their sixth, seventh, eighth, or even ninth annual dose.

Biochemical and molecular characterization of Leishmania parasites isolated from an endemic focus in eastern Sudan.

Twelve Leishmania isolates from visceral leishmaniasis patients in eastern Sudan were characterized using isoenzyme analysis, Southern blotting and polymerase chain reaction (PCR) 'fingerprinting'. Isoenzyme analysis revealed the presence of 3 zymodemes: MON-18, MON-30 and MON-82, corresponding to Leishmania donovani sensu stricto, L. infantum and L. archibaldi (still of uncertain taxonomic status), respectively. Southern blotting and PCR 'fingerprinting' revealed identical patterns for all 3 zymodemes, which were indistinguishable from those of L. donovani s.s.

The direct agglutination test for diagnosis of visceral leishmaniasis under field conditions in Sudan: comparison of aqueous and freeze-dried antigens.

The performance of the direct agglutination test (DAT) was evaluated under field conditions in an endemic area of visceral leishmaniasis in eastern Sudan, using aqueous (Aq) antigen which has to be kept refrigerated and a newly developed freeze-dried (FD) antigen which is stable at ambient temperature. Both antigens compared well, with 92-98% of readings being identical or only with one dilution difference in titre. FD antigen gave titres that were identical with Aq antigen in 73% of samples, higher in 19%, and lower in 8%. Owing to high ambient temperatures and low humidity, microtitre plate wells dried out during the standard procedures for elution and incubation. However, shortening the elution time from 12 to 4 h proved possible for both antigens; incubation could be reduced from 24 to 10 h for Aq antigen, after which the plates could still be read. Incubation with FD antigen required 18 h and the plates needed to be kept cool because of evaporation. Despite the longer procedure with the FD antigen, the DAT can be completed in 24 h and the use of this stable antigen, that does not require refrigeration, is a major improvement in performing the DAT under unfavourable field conditions.

Sensitivity of a polymerase chain reaction-based assay to detect Onchocerca volvulus DNA in skin biopsies.

A polymerase chain reaction (PCR) of a 150-bp tandem repeat of Onchocerca volvulus (O-150) combined with Southern-blot hybridization to species-specific DNA probes was employed for DNA detection. O-150 was amplified from parasites originating from Uganda, Benin, Cameroon, Liberia, Ghana, Burkina Faso, Mali, and Zaire and was successfully hybridized to digoxigenin-labeled oligonucleotides. To investigate the sensitivity of the PCR, 2 skin biopsies were taken from each of 227 persons from Uganda with proven O. volvulus infections but with low microfilaria (mf) densities due to ivermectin treatment. One biopsy was tested by PCR and the other was digested using collagenase to assess the total number of mf. The PCR revealed 76.2% of the samples to be positive, and the collagenase method showed that 78.9% were positive, indicating similar sensitivity for the two methods. It is probable that for both techniques the biopsy must contain at least one live mf or fragments of a dead mf. In this study, no free or circulating O. volvulus DNA could be detected in skin biopsies by PCR.

Sudanese mucosal leishmaniasis: epidemiology, clinical features, diagnosis, immune responses and treatment.

The epidemiology, clinical features, pathology, immune responses, diagnosis and treatment of 14 patients with mucosal leishmaniasis in the Sudan are described. The condition occurred mainly in adult males, particularly in certain closely related tribes from the western Sudan. It affected the mucosa of the upper respiratory tract and/or the oral mucosa and sometimes followed treated kala azar. The parasites were sometimes confined to the mucosa, sometimes spread to the lymph nodes, and rarely infected the bone marrow and spleen. One of the 2 patients with both visceral and mucosal leishmaniasis differed from classical kala azar cases; his infection was longer lasting, he was leishmanin positive, and his peripheral mononuclear cells proliferated in response to leishmanial antigens. Mucosal leishmaniasis following treated kala azar is a similar phenomenon to post-kala azar dermal leishmaniasis and post-kala azar uveitis. Post-kala azar mucosal leishmaniasis can therefore be added to the other post-kala azar leishmanial infections. Using the polymerase chain reaction, Southern blot analysis with specific probes, and isoenzyme characterization, the causative parasite was identified as Leishmania donovani in 4 patients and as L. major in one. Unlike American mucocutaneous leishmaniasis, mucosal leishmaniasis in the Sudan was not preceded or accompanied by cutaneous lesions and the response to pentavalent antimony or ketoconazole was good.

Visceral leishmaniasis: use of the polymerase chain reaction in an epidemiological study in Baringo District, Kenya.

The polymerase chain reaction was applied to capillary blood spots dried on filter paper from 20 parasitologically proved cases of visceral leishmaniasis (VL), 21 subclinical cases, and 11 healthy controls in a longitudinal study of anthroponotic VL in Baringo District, Kenya. Leishmania deoxyribonucleic acid (DNA) was detected 10.5 months before diagnosis and up to 3 years after diagnosis and apparently successful treatment. Subclinical cases can have detectable circulating parasite DNA in their blood. These findings may indicate that subclinical cases can be a reservoir and formerly treated VL patients can remain a reservoir for a long time. Xenodiagnosis should be performed on subclinical cases and former VL patients to establish their role in transmission of VL in Kenya.

Leish-KIT, a stable direct agglutination test based on freeze-dried antigen for serodiagnosis of visceral leishmaniasis.

In order to increase the application potential of the direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in human serum samples, we developed an antigen based on stained and freeze-dried Leishmania donovani promastigotes. We describe here the evaluation of the performance of the DAT based on this freeze-dried antigen. It was shown that the freeze-dried antigen remains fully active, even after storage at 56 degrees C for 18 months. With a cutoff value of 1:1,600, the sensitivity of the DAT was shown to be 92% and the specificity of the test was 99.7%, which were comparable with the results found for the DAT based on liquid antigen. The major advantages of the freeze-dried antigen are that the production of a large batch of this antigen allows reproducible results in the DAT over a long period of time and that the freeze-dried antigen can be stored at ambient temperature, which, as was shown, makes the test a valuable diagnostic tool for use in the field.

Genomic fingerprinting of Onchocerca species using random amplified polymorphic DNA.

A method based on amplification of genomic DNA by decamer primers of random nucleotide sequence was used to obtain DNA fingerprints from different species of the genus Onchocerca. Each of the 20 primers tested allowed a clear distinction between the different species of the genus on an agarose gel. The technique offers the potential to construct species or strain specific probes and oligonucleotides PCR primers from the species specific fragments. A combination of these primers or others could be useful as population markers.

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.

Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite.

Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop O. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologically most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difficulty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate oligonucleotides. These oligonucleotides have been shown to support the amplification of the 150-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microfilariae. In addition, the method is capable of detecting O. volvulus infective larvae directly in extracts of blackfly vectors.

The onchocerciasis focus at Kinsuka/Kinshasa (Republic of Zaire) in 1985. I. Entomological aspect.

The entomological aspect of the onchocerciasis focus at Kinsuka/Kinshasa has not been studied since vector control was carried out in 1948. As in 1940, larvae of Simulium damnosum s.l. are located in the arm of the Zaire River flowing between Mimosa Island and the Zairian bank. They are, however, scarce. Cytotaxonomic studies showed that the S. squamosum group was the only member of the S. damnosum complex present in the region of Kinshasa. Physico-chemical analysis of the water at breeding sites determined that S. squamosum larvae develop in neutral or acid water with a conductivity of less than 50 micronhos cm-1 and a low concentration of dissolved salts. Longitudinal study showed that the 'annual biting rate' (ABR) and the 'annual transmission potential' (ATP) fluctuate from 2406-5999 bites man-1 year-1 and from 120-236 infective larvae man-1 year-1 respectively. The low transmission rate at Kinsuka is mainly due to the low level of the biting population occurring during the main river rising. The increase in Kinsuka's population has reinforced this situation by 'diluting' the parasite in the human reservoir.