M.E. Ibrahim

Autoclaved Leishmania major vaccine for prevention of visceral leishmaniasis: a randomised, double-blind, BCG-controlled trial in Sudan

BACKGROUND Visceral leishmaniasis is a major cause of morbidity and mortality in the Sudan. Drug treatment is expensive, and drug resistance is becoming increasingly common. Safe, effective, and cheap vaccines are needed. We report the results of a vaccine trial against human visceral leishmaniasis. METHODS We undertook a double-blind randomised trial to test the safety and efficacy of an autoclaved Leishmania major (ALM) promastigote vaccine (1 mg per dose). Of 5093 volunteers screened, 2306 had negative leishmanin skin tests and reciprocal titres of less than 6400 in the direct agglutination test. They were randomly assigned two doses of ALM mixed with BCG or BCG alone. Volunteers were followed up for 2 years. The primary endpoint was clinical visceral leishmaniasis or post-kala-azar dermal leishmaniasis. Analyses were by intention to treat. FINDINGS Side-effects were confined to the injection site. The cumulative frequency of visceral leishmaniasis at 2 years did not differ significantly between the group assigned ALM plus BCG and that assigned BCG alone (133/1155 [11.5%] vs 141/1151 [12.3%], p=0.6). The vaccine efficacy was 6% (95% CI -18 to 25). The proportion of individuals showing leishmanin skin conversion was significantly higher in the ALM plus BCG group than in the BCG alone group throughout follow-up (303 [30%] vs 72 [7%] at 42 days). Individuals whose leishmanin test converted after vaccination (induration > or =5 mm) had a significantly lower frequency of visceral leishmaniasis than non-responders (27/375 [7.2%] vs 210/1660 [12.7%], p=0.003). INTERPRETATION We found no evidence that two doses of ALM plus BCG offered significant protective immunity against visceral leishmaniasis compared with BCG alone. Leishmanin skin conversion with an induration of 5 mm or more in either group was associated with protection from the disease.

Genetic susceptibility to visceral leishmaniasis in The Sudan: linkage and association with IL4 and IFNGR1

Longitudinal studies in Sudan show ethnic differences in incidence and clinical phenotypes associated with Leishmania donovani. Immunologically, bias in type 1 vs type 2 cytokine responses is important. To determine whether polymorphisms at IL4/IL9 or IFNGR1 contribute to susceptibility, we examined 59 multicase families of visceral leishmaniasis (VL) with/without post Kala-azar dermal leishmaniasis (PKDL). Multipoint nonparametric analysis (Allegro) linked IL4/IL9 to VL per se (P=0.002). Transmission disequilibrium testing with robust variance estimates confirmed association in the presence of linkage between VL per se and IL4 (P=0.008) but not IL9. Stepwise logistic regression analysis showed both IL4RP2 and IL4RP1 markers contributed significantly to the association, suggesting a common disease-associated haplotype. In contrast, IFNGR1 was linked (P=0.031) and associated (P=0.007) to PKDL but not VL or VL per se. Hence, polymorphism in a type 2 cytokine gene influences underlying susceptibility to VL, whereas IFNGR1 is specifically related to susceptibility to PKDL.

Treatment of visceral leishmaniasis with sodium stibogluconate in Sudan: management of those who do not respond

Almost all (98%) of 1593 visceral leishmaniasis (VL) patients treated with sodium stibogluconate (Pentostam; Wellcome) in Sudan between 1989 and 1995 and follow-up responded well to treatment. However, the other 33 patients, all of whom were seronegative for HIV, showed partial or no response. The two main causes of unresponsiveness were primary drug resistance (39.3%) and low drug dosages given at peripheral dispensaries (30.3%). All of those who had been sub-optimal doses were cured when adequate doses of the drug were given. A third cause was concurrent disease, particularly pulmonary tuberculosis (18%). With treatment of the concurrent disease, patients responded well to Pentostam. Eight patients who failed to respond to repeated courses of Pentostam did not benefit from pentamidine or sterol inhibitors. Three of these patients responded to liposomal amphotericin B, two responded to splenectomy in association with Pentostam therapy, and three died. Pentostam, given in adequate doses, still appears to be the drug of choice for the treatment of VL in the Sudan Liposomal amphotericin B is a suitable second-line drug.

Post kala-azar ocular leishmaniasis

The clinical features, diagnosis and treatment of 6 patients with post kala-azar ocular leishmaniasis are described. The eye lesions were associated with past or concomitant post kala-azar dermal leishmaniasis (PKDL). Four patients had post kala-azar leishmanial conjunctivitis and blepharitis. Using the polymerase chain reaction, the causative parasite was characterized as Leishmania donovani in 2 of these 4 patients. Two patients had post kala-azar anterior uveitis. The diagnosis of uveitis was based on the clinical manifestations, temporal relation to treated visceral leishmaniasis, the association with PKDL and positive anti-Leishmania serology. All patients were treated with systemic sodium stibogluconate. Patients with anterior uveitis were also treated with steroid and atropine eyedrops. The response to treatment was good. The importance of early diagnosis and treatment of ocular leishmaniasis is stressed.

Minor Role for BRCA2 (Exon11) and p53 (Exon 5–9) Among Sudanese Breast Cancer Patients

A cohort of 20 breast cancer patients from the Sudan was tested for germ line and somatic mutation in their BRCA2 exon 11 as well as the main conserved area of the p53 tumor suppressor gene. The results indicate that both regions may play a limited role in the pathogenesis of breast cancer in those patients. The fact that there are no somatic mutations detected in p53 was particularly surprising as the expected rate for mutations in breast cancer is 30–50%.

Interleukin 10 gene polymorphisms and development of post kala-azar dermal leishmaniasis in a selected sudanese population.

Background: Post kala-azar dermal leishmaniasis (PKDL) is a cutaneous form of disease that develops at variable times after individuals have received treatment for clinical visceral leishmaniasis (VL). The study aimed to investigate the possible role of interleukin 10 (IL-10) and development of PKDL. Methods: 77 families composed of 41 complete case-parent trios and 36 case-parent pairs from the Masalit ethnic group were genotyped for 3 IL10 promoter polymorphisms: -1082A/G, -819C/T and -592C/A. Results: Single point analysis using the transmission disequilibrium test showed no evidence of association between any of these IL10 promoter single nucleotide polymorphisms (SNPs) and development of PKDL. Haplotype analysis performed using TRANSMIT showed borderline significance between PKDL and the haplotype AA across -592C/A and -1082A/G (p = 0.053). Haplotypes GCC (0.33) and ATA (0.30) were the common haplotypes in this Sudanese population. Allele frequencies for the 3 SNPs differed significantly in Sudan compared to other African (Gambian, Malawian, YRI) populations. Conclusion: There is no evidence for an association between 3 SNPs in the IL10 gene promoter and susceptibility to PKDL in the Masalit ethnic group in Sudan, although some evidence for haplotype association was observed.

Allele frequency and genotype distribution of polymorphisms within disease-related genes is influenced by ethnic population sub-structuring in Sudan

Four single nucleotide polymorphisms (SNPs) and a variable number of tandem repeats (VNTR) polymorphism located within disease associated/causing genes were typed in four populations of different tribal and ethnic affiliation from the Sudan. The genotype and allele frequencies were compared with those of other groups from published and unpublished data of world populations. The combined Sudanese sample conformed with Hardy–Weinberg equilibrium (HWE) expectation. However, population sub-structuring according to ethnic/linguistic group indicated at least two SNPs in departure from HWE. Differences in allele frequencies and genotype distribution between groups was also noted in three of the four SNPs. The other loci were distributed homogeneously within the populations studied with genotype frequencies in agreement with HWE expectation. These results highlight the importance of inter-population stratification for polymorphic markers, as well as the potential influence of evolutionary history and ethnic variation of loci, in the general distribution of SNPs and other polymorphisms.

Frequencies of BCR-ABL1 fusion transcripts among Sudanese chronic myeloid leukaemia patients

The incidence of one or other rearrangement in chronic myeloid leukemia (CML) patients varies in different reported series. In this study we report the frequencies of BCR-ABL1 fusion transcript variants studied in 43 CML patients from Sudan. The study includes 46 Sudanese patients, three of which negative for the BCR-ABL1 fusion transcript. More than half of 43 positive patients showed b2a2 fusion transcript (53.5%), while (41.9%) showed b3a2 transcript and the remaining (4.6%) coexpression of b3a2/ b2a2 and b3a2/b2a2/e19a2. We detected neither coexpression of p210/p190 nor e1a2 alone. Male patients showed a tendency to express b2a2, while female tende to express b3a2 (p = 0.017). Moreover, a single nucleotide polymorphism was detected in BCR exon 13 in one out of four patients and this patient showed only b2a2 expression. In conclusion, we observed a significant correlation between sex and type of BCR-ABL1 transcript, an observation that deserves further investigation.

Nasopharyngeal Cancer in Sudan: Epidemiology, Clinical and Histological Characteristics

Objectives To study the epidemiology, clinical features, staging, etiology and pathology of nasopharyngeal cancer in Sudan. Study design This is a retrospective study. Setting Ear, Nose and Throat Department Khartoum Teaching Hospital, Khartoum City, Sudan. Subjects and methods Patients suspected to have nasopharyngeal cancer were assessed during the period March 2004 to May 2010. Data from confirmed cases was obtained; it included clinical and epidemiological information. Results Three hundred and eighty five cases were studied. Bimodal age distribution of the disease was noted with two peaks, one at 15–19 years and one at 50–54 years. The male to female ratio was 2.6:1 and a distinct geographical distribution of the disease was noted, with clustering of cases in the towns of Dilling, Kadogli and the surrounding rural area of the Nuba Mountains. These areas in the Western States were reported to be of high background radiation due to naturally produced radioactive uranium. The Nuba tribe headed the list among other tribes, demonstrating a clear ethnic predilection. Sixty-eight cases presented at stage IV. There was a predominance of Type II (15.58%) and Type III (65.97%). Patients were treated by neoadjuvant chemoradiotherapy. Conclusions NPC is an important form of cancer in Sudan. Some tribes are significantly more affected than others. Patients present with advanced disease. Environmental and genetic factors need further studies. Screening at risk populations that aim at early diagnosis and management of patients is recommended.

Leishmania Donovani: Genetic Diversity Of Isolates From Sudan Characterized By PCR-Based RAPD

Drug unresponsiveness in patients with visceral leishmaniasis (VL) is a problem in many endemic areas. This study aimed to determine genetic diversity of Leishmania donovani isolates from a VL endemic area in Sudan as a possible explanation for drug unresponsiveness in some patients. Thirty clinically stibogluconate (SSG)-sensitive isolates were made SSG-unresponsive in vitro by gradually increasing SSG concentrations. The sensitive isolates and their SSG-unresponsive counterparts were typed using mini-circle kDNA and categorized using PCR-RAPD. All the isolates were typed as L. donovani, the resulting PCR-RAPD characterization of the SSG-sensitive isolates gave three distinct primary genotypes while, the SSG-unresponsive isolates showed only a single band. L. donovani isolates from eastern Sudan are diverse; this probably resulted from emergence of new L. donovani strains during epidemics due to the pressure of widespread use of antimonials. In this communication the possible role of isolates diversity in antimonial unresponsiveness and the in vitro changing PCR-RAPD band pattern in SSG-unresponsive strains were discussed.