Fc gamma receptor IIa (CD32) polymorphism and antibody responses to asexual blood-stage antigens of Plasmodium falciparum malaria in Sudanese patients.

In a prospective clinical study in New Halfa Teaching Hospital, the possible association between FcgammaRIIa-R/H131 polymorphism and anti-malarial antibody responses with clinical outcome of Plasmodium falciparum malaria among Sudanese patients was investigated. A total of 256 individuals were consecutively enrolled, comprising 115 patients with severe malaria, 85 with mild malaria and 56 malaria-free controls. Genotyping of FcgammaRIIa-R/H131 dimorphism was performed using gene-specific polymerase chain reaction (PCR) amplification with allele-specific restriction enzyme digestion of the PCR product. The antibody responses to asexual blood-stage antigens were assessed by an enzyme-linked immunosorbent assay. The frequency of the FcgammaRIIa-R/R131 genotype was significantly higher in those with severe malaria when compared with patients with mild malaria, while the FcgammaRIIa-H/H131 genotype showed a significant association with mild malaria. A reduced risk of severe malaria with IgG3 antibodies in combination with the H/H131 genotype was observed. Furthermore, low levels of IgG2 antibodies reactive with the Pf332-C231 antigen were also associated with lower risk of severe malaria in individuals carrying the H131 allele. The levels of IgG1 and IgG3 antibodies were statistically significantly higher in the mild malaria patients when compared with the severe malaria patients. Taken together, our study revealed that the FcgammaRIIa-R/R131 genotype is associated with the development of severe malaria, while the H/H131 genotype is more likely to be associated with mild malaria. Our results also revealed that the natural acquisition of immunity against clinical malaria appeared to be more associated with IgG1 and IgG3 antibodies, signifying their roles in parasite-neutralizing immune mechanisms.

Factors affecting variations in exposure to infections by Leishmania donovani in eastern Sudan.

A cross-sectional survey was carried out in Gedaref state, eastern Sudan to investigate the prevalence of positive leishmanin skin tests and environmental factors related to Leishmania donovani infection. A total of 3835 people living in 11 villages in 3 regions were screened. Soil types and tree densities were determined in 33 villages inhabited by 44 different tribes. The highest rates of positive skin tests were in Rahad region (33.9%), Atbara (21.6%) and Gedaref (10.6%), with an average of 21.1% for the state. Risk of infection by L. donovani varied significantly between different tribes. Higher densities of Acacia and Balanites spp. trees were in Masaleet villages, suggesting that the relatively high risk of L. donovani exposure in this tribe is due to environmental factors.

Factors affecting variations in exposure to infections by Leishmania donovani in eastern Sudan

A cross-sectional survey was carried out in Gedaref state, eastern Sudan to investigate the prevalence of positive leishmanin skin tests and environmental factors related to Leishmania donovani infection. A total of 3835 people living in 11 villages in 3 regions were screened. Soil types and tree densities were determined in 33 villages inhabited by 44 different tribes. The highest rates of positive skin tests were in Rahad region [33.9%], Atbara [21.6%] and Gedaref [10.6%], with an average of 21.1% for the state. Risk of infection by L. donovani varied significantly between different tribes. Higher densities of Acacia and Balanites spp. trees were in Masaleet villages, suggesting that the relatively high risk of L. donovani exposure in this tribe is due to environmental factors

Leishmaniasis in the Sudan: a literature review with emphasis on clinical aspects.

The literature on the leishmaniases in the Sudan is reviewed with an emphasis on clinical aspects and on literature related to the recent outbreaks in the south and east of the country. The numbers of cases of subclinical infection and post-kala azar dermal leishmaniasis in the recent outbreaks are remarkable. New diagnostic techniques have been introduced and evaluated, notably the direct agglutination test and polymerase chain reaction technology. The latter gives very promising results and further research into application of the technique is warranted. Treatment with pentavalent antimony is still satisfactory. The reservoir host has not been identified definitely.

Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing.

A polymerase chain reaction and single-strand conformation polymorphism determination (PCR-SSCP) was used to detect deoxyribonucleic acid sequence polymorphisms in the transcribed non-coding regions between the small and large sub-unit ribosomal ribonucleic acid (rRNA) genes in Leishmania donovani from 63 clinical samples collected in eastern Sudan, between April 1997 and October 1998. Specific Leishmania primers were used to amplify the internal transcribed spacer (ITS) regions of L. donovani isolates directly from clinical samples spotted on filter papers. Amplification products were subsequently analysed by SSCP. Eleven polymorphic patterns were detected in the first part of the spacer, the ITS1 region, and were sequenced. Most of the changes were due to deletions of adenine bases and AT pairs within the first 192 nucleotides of the ITS region. This is the first application of PCR-linked SSCP analysis for the detection of population variation with direct display of sequence variation in parasitologically positive clinical samples spotted on filter paper. Culturing the parasite is thus not required, which is beneficial particularly in epidemiological studies based on field work where obtaining cultures can be extremely difficult.

Safety and immunogenicity of an autoclaved Leishmania major vaccine.

OBJECTIVE: To test the safety and immunogenicity of two doses of autoclaved L. major (ALM) vaccine mixed with BCG. SETTING: Kala-azar endemic area of eastern Sudan. DESIGN: This was a randomised, double blind and BCG controlled phase I/II study. SUBJECTS: Eighty healthy volunteers (forty children and forty adults) with no past history of kala-azar, no reactivity to leishmanin antigen and with a reciprocal direct agglutination test (DAT) titre of <200 were recruited. Informed consents were obtained from volunteers or their guardians in case of children. MAIN OUTCOME MEASURES: Conversion in the leishmanin skin and the DAT tests. INTERVENTION: Two intra-dermal injections of either ALM+BCG or BCG alone. The injections were three weeks apart. RESULTS: Side effects were minimal and confined to the injection site, with no significant difference between the ALM+BCG and the BCG alone groups. The leishmanin skin conversion was significantly higher in the ALM+BCG group compared to the BCG alone group (p<0.0005). Furthermore, the Leishmanin skin test conversion was significantly higher in children than adults (p<0.0005). One adult volunteer in the ALM+BCG group converted in both the Leishmanin skin and the DAT tests. CONCLUSION: We conclude that two doses of ALM+BCG are safe and immunogenic, especially in children.

Use of the polymerase chain reaction to assess the success of visceral leishmaniasis treatment.

We investigated whether the polymerase chain reaction (PCR) performed with aspirates of bone marrow or lymph node can be used as a test of cure of visceral leishmaniasis (VL). Sixty-one VL patients who had received supervised treatment with sodium stibogluconate in the health centre of Médecins sans Frontières (MSF) Holland in Um-Kuraa, eastern Sudan, were studied. Immediately after treatment, no parasite could be demonstrated by microscopy in aspirates of bone marrow or lymph node. In contrast, PCR detected Leishmania deoxyribonucleic acid in 50 of the 61 lymph node aspirates (82%). Forty-nine patients were examined 3 and 6 months later; the other 12 were reported to be alive but had left the area. With 10 of these 49 patients, the PCR was negative and the patients remained free from signs and symptoms of VL; they were apparently cured. Of the 39 patients with a positive PCR after treatment, 14 (36%) developed post kala-azar dermal leishmaniasis and 9 (23%) had a recurrence of VL symptoms with reappearance of parasites in the aspirates. Four relapsed patients subsequently died of VL. We concluded that the PCR on lymph node aspirates can be used to assess treatment and cure of VL. The fact that 23 of 49 patients who received standard supervised treatment were not completely cured indicated that there is a need to investigate extended or alternative treatments.

Use of PCR for diagnosis of post-kala-azar dermal leishmaniasis.

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.

Evaluation of PCR for diagnosis of visceral leishmaniasis.

An evaluation of Leishmania PCR was performed with bone marrow, lymph node, and blood samples from 492 patients, 60 positive controls, and 90 negative controls. Results were compared with microscopy results for Giemsa-stained smears. PCR and microscopy of lymph node and bone marrow aspirates from patients with microscopically confirmed visceral leishmaniasis (VL) were equally sensitive. However, in patients clinically suspected of having VL and in whom parasites could not be demonstrated by microscopy, PCR was positive for 12 of 23 (52.2%) lymph node aspirates and 8 of 12 (66.7%) bone marrow aspirates, thus confirming the clinical diagnosis of VL. With PCR on filter paper, Leishmania DNA was detected in the blood of 33 of 47 (70%) patients with confirmed VL and in 2 of 11 (19%) patients suspected of having VL. Positive PCR results were more frequently found for blood samples on filter paper than for samples stored in EDTA. In conclusion, PCR is a more sensitive method than microscopy for the detection of Leishmania in lymph node and bone marrow aspirates, being especially useful for the confirmation of cases of suspected VL. Blood from a finger prick may be used for the initial PCR screening of people suspected of having VL. If the PCR of blood is negative, one should perform PCR with lymph node and/or bone marrow material, because PCR with these materials is more often positive.

Use of PCR on lymph-node sample as test of cure of visceral leishmaniasis.

When the polymerase chain reaction (PCR) was used to test lymph-node aspirates from 35 patients from eastern Sudan, who had had visceral leishmaniasis but were believed cured, leishmanial DNA was detected in samples from 14 of the patients. There were no significant differences between the PCR-positives and -negatives in terms of age, sex, spleen size, malaria status or presence of anti-Leishmania antibodies. However, PCR was more often positive in the patients who tested negative by the leishmanin skin test (LST) than in those who gave positive skin tests. Moreover, patients with a positive PCR and a negative LST converted more often to LST positivity than those with a negative PCR and a negative LST. The most important finding was that, during follow-up, eight (57%) of the PCR-positives, but none of the 21 negatives, developed post-kala-azar dermal leishmaniasis (PKDL) In conclusion, PCR-based testing of lymph-node aspirates after treatment may be used as a prognostic marker for the future development of PKDL and may be useful in the follow-up of patients.

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.

Extended use of scrotal septal island skin flap for the repair of penile hypospadias.

In this article, one-stage repair of complicated cases of penile hypospadias when there is no penile skin available for safe reconstruction is described. An island of the most median hairless part of the scrotal skin around the median raphe was used as a skin flap based on its scrotal septal pedicle with an extended and free arc of rotation for repair of distant urethral defects. The bilaterally stretched, most median area of scrotal skin is almost hairless. This well-vascularized flap was used in reconstruction of 18 patients with recurrent, complicated, proximal, and distal penile hypospadias from January 1989 through July 1993. Of these, 17 patients were post pubertal and 1 was prepubertal. A maximum of 5 years and a minimum of 8 months follow-up showed no hair growth problems. Complications were sacculation of the reconstructed urethra in 1 patient, meatal stenosis in 1 patient, and urethral fistula in 1 patient. Technique refinements and encouraging results as well as complications are discussed.