Expression profiling of Sudanese visceral leishmaniasis patients pre- and post-treatment with sodium stibogluconate.

Visceral leishmaniasis (VL) in Sudan caused by Leishmania donovani is fatal in susceptible individuals if untreated. Treatment with sodium stibogluconate (SSG) leads to post-kala-azar dermal leishmaniasis (PKDL) in 58% of patients. Here, Affymetrix microarrays were used to identify genes differentially expressed in lymph nodes (N=9 paired samples) pre- and post-treatment with SSG. Using the Bioconductor package limma, 438 genes from 28 869 post-quality-control probe sets were differentially expressed (Pnominal ≤.02) post- vs pretreatment. Canonical pathway analysis using Ingenuity Pathway Analysis™ identified "role of nuclear factor of activated T-cell in regulation of immune response" (Pnominal =1.35×10-5 ; PBH-adjusted =4.79×10-3 ), "B-cell development" (Pnominal =2.04×10-4 ; PBH-adjusted =.024), "Fcγ receptor-mediated phagocytosis in macrophages and monocytes" (Pnominal =2.04×10-4 ; PBH-adjusted =.024) and "OX40 signalling" (Pnominal =2.82×10-4 ; PBH-adjusted =.025) as pathways differentially regulated post- vs pretreatment. Major network hub genes included TP53, FN1, MYC, BCL2, JUN, SYK, RUNX2, MMP1 and ACTA2. Top endogenous upstream regulators included IL-7 (P=2.28×10-6 ), TNF (P=4.26×10-6 ), Amyloid Precursor Protein (P=4.23×10-5 ) and SPI1/PI.1 (P=1.17×10-7 ). Top predicted chemical drug regulators included the flavonoid genistein (P=4.56×10-7 ) and the quinoline alkaloid camptothecin (P=5.14×10-5 ). These results contribute to our understanding of immunopathology associated with VL and response to SSG treatment. Further replication could identify novel therapeutic strategies that improve on SSG treatment and reduce the likelihood of progression to PKDL.

The 5q31 region in two African populations as a facet of natural selection by infectious diseases.

Cases of extreme natural selection could lead either to rapid fixation or extinction of alleles depending on the population structure and size. It may also manifest in excess of heterozygosity and the locus concerned will be displaying such drastic features of allele change. We suspect the 5q31 in chromosome 5 to mirror situation of such extreme natural selection particularly that the region encompasses genes of type 2 cytokine known to associate with a number of infectious and non-infectious diseases. We typed two sets of single nucleotide polymorphisms (SNPS) in two populations: an initial limited set of only 4 SNP within the genes of IL-4, IL-13, IL-5 and IL-9 in 108 unrelated individuals and a replicating set of 14 SN P in 924 individuals from the same populations with disregard to relatedness. The results suggest the 5q31 area to be under intense selective pressure as indicated by marked heterozygosity independent of Linkage Disequilibrium (LD); difference in heterozygosity, allele, and haplotype frequencies between generations and departure from Hardy-Weinberg expectations (DHWE). The study area is endemic for several infectious diseases including malaria and visceral leishmaniasis (VL). Malaria caused by Plasmodiumfalciparum, however, occurs mostly with mild clinical symptoms in all ages, which makes it unlikely to account for these indices. The strong selection signals seems to emanate from recent outbreaks of VL which affected both populations to varying extent.

Ultrasound-guided fascia iliaca block versus quadratus lumborum block for perioperative analgesia in patients undergoing hip surgery. A randomised controlled trial.

INTRODUCTION: Femoral neck fractures are common orthopaedic fractures, especially in old age, and they represent a life-threatening condition requiring surgical intervention. In this study, we aimed to compare 2 regional techniques used to decrease perioperative pain. MATERIAL AND METHODS: In this parallel group randomized controlled clinical trial we enrolled 68 patients from both sexes scheduled for hip surgery after femoral neck fractures. The patients were randomly allocated to 2 equal groups with one receiving ultrasound- guided supra-inguinal fascia iliaca block (FIB) and the other receiving ultrasound- guided anterior quadratus lumborum block (QLB). Our primary outcome was the duration of postoperative analgesia. The secondary outcome was measuring the Visual Analog Scale (VAS) during patient positioning while applying the neuraxial block, the total analgesic requirement in the postoperative period, patient satisfaction in the postoperative period, and the frequency of adverse effects. RESULTS: The group receiving supra-inguinal FIB had a significantly longer time of postoperative analgesia 18 (4-24), compared to the group receiving anterior QLB 2 (1-24), P = 0.005. They consumed less morphine throughout 24 hours postoperatively, 5.3 ± 0.9 mg compared to 6.9 ± 1.87 mg (95% CI: 6.45-3.92, P = 0.008), and they showed less pain during positioning for spinal anaesthesia. CONCLUSIONS: Supra-inguinal FIB provides prolonged postoperative analgesia compared to anterior QLB in patients undergoing hip surgery. It was associated with less pain during positioning in spinal anaesthesia and decreased total morphine consumption.

Noninvasive high frequency oscillatory ventilation versus noninvasive positive pressure ventilation in preterm neonates after extubation: A randomized controlled trial.

BACKGROUND: Weaning from mechanical ventilation is a challenging phase of neonatal respiratory support [1]. Choosing efficient and safe noninvasive modality to prevent re-intubation and choosing the optimal time for weaning are key points for weaning success. The aim of the study is to compare the efficiency and safety of noninvasive high frequency oscillatory ventilation (NHFOV) versus noninvasive positive pressure ventilation (NIPPV) as respiratory support after extubation in preterms with respiratory distress syndrome (RDS). Also, the study compared the lung ultrasound findings between these 2 modalities and assessed the use of lung ultrasound score (LUS) as predictor for extubation outcome. METHODS: This study is a randomized controlled trial conducted on 60 preterm neonates with RDS. Patients were allocated into one of 2 groups: NIPPV or NHFOV as post-extubation noninvasive respiratory support. The 2 groups were compared regarding the incidence of extubation failure within 72 hours from extubation, oxygen needs, duration of application of the noninvasive modality, duration of admission, safety and mortality rate. LUS was assessed pre-extubation and 2 hours post-extubation. RESULTS: The study did not show a statistically significant difference in re-ventilation rate in NHFOV group (23.3%) compared to NIPPV group (30.0%), p = 0.56. Oxygen needs were significantly lower in NHFOV group compared to NIPPV groups (mean FiO2 31.8±6.09 vs 38±0.55, p = 0.007). The duration of the used noninvasive modality, CO2 concentration, LUS, and mortality rate showed statistically insignificant difference between both groups. There was a significant correlation between LUS and extubation outcome. CONCLUSION: NHFOV is a feasible noninvasive modality for respiratory support post-extubation in premature infants. LUS is a good predictor of extubation outcome in neonates.

Leishmania mexicana: expression; characterization and activity assessment of E. coli-expressed recombinant CRK3.

OBJECTIVES AND METHODS: Previous studies have shown that CRK3 protein kinase of Leishmania mexicana is a potential drug target. Therefore, the aim of this study was to provide an active protein kinase for chemical inhibitors testing. A system was developed to express and affinity-purify recombinant L. mexicana CRK3 protein from Escherichia coli. RESULTS: Biochemical analysis has confirmed the expression of the pure kinase. The bacterial-expressed kinase was found to be inactive as a monomer. The mutated CRK3-E178 protein kinase was also found to be inactive. CONCLUSION: This study suggests that cyclin binding and phosphorylation status are both important for reconstituting protein kinase activity. Work presented by this paper has confirmed the usefulness of the prokaryotic system for production of pure homogenous recombinant protein kinase of Leishmania parasite, though this system is unable to produce active CRK3 protein kinase  

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.

Serum LINC00305 expression and its genetic variant rs2850711 are associated with clinical and laboratory features of rheumatoid arthritis.

BACKGROUND: Long intergenic non-protein coding (lnc) RNA 00305 (LINC00305) is a pro-inflammatory atherosclerosis-associated lncRNA. We hypothesised that LINC00305 expression and its variant rs2850711 (A/T) are implicated in rheumatoid arthritis (RA) and linked with clinical and routine laboratory markers. METHODS: 100 RA patients and 100 healthy controls were recruited. LINC00305 genotyping and expression were performed using allelic-discrimination PCR and quantitative real-time PCR. LINC00305 diagnostic power was evaluated using area under the receiver operating characteristic curve (AUC). Serum nuclear factor- κB (NF-κB) and matrix metalloproteinase-3 (MMP-3) levels were determined by ELISA, standard laboratory markers by routine methods. RESULTS: LINC00305 expression was significantly increased in RA patients and positively correlated with DAS28, C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor and anti-cyclic citrullinated peptide antibody. In multivariate analysis, NF-κB, MMP-3 and LINC00305 were significant predictors of RA (P< 0.0001). Individuals carrying AT and TT genotypes of rs2850711 polymorphism had significantly more likely to have RA than AA genotype carriers (P< 0.05). LINC00305 expression, DAS28 score and serum levels of NF-κB and MMP-3 were significantly increased in the patients carrying LINC00305 AT and TT genotypes as compared with AA genotype patients (P< 0.01). CONCLUSION: Increased expression level of LINC00305 and its rs2850711 genetic variant may play a role in the diagnosis and management of RA, and its severity and activity.

Genetics and visceral leishmaniasis: of mice and man.

Ninety per cent of the 500,000 annual new cases of visceral leishmaniasis (VL) occur in India/Bangladesh/Nepal, Sudan and Brazil. Importantly, 80-90% of human infections are sub-clinical or asymptomatic, usually associated with strong cell-mediated immunity. Understanding the environmental and genetic risk factors that determine why two people with the same exposure to infection differ in susceptibility could provide important leads for improved therapies. Recent research using candidate gene association analysis and genome-wide linkage studies (GWLS) in collections of families from Sudan, Brazil and India have identified a number of genes/regions related both to environmental risk factors (e.g. iron), as well as genes that determine type 1 vs. type 2 cellular immune responses. However, until now all of the allelic association studies carried out have been underpowered to find genes of small effect sizes (odds ratios or OR < 2), and GWLS using multicase pedigrees have only been powered to find single major genes, or at best oligogenic control. The accumulation of large DNA banks from India and Brazil now makes it possible to undertake genome-wide association studies (GWAS), which are ongoing as part of phase 2 of the Wellcome Trust Case Control Consortium. Data from this analysis should seed research into novel genes and mechanisms that influence susceptibility to VL.

Naltrexone treatment in clomiphene resistant women with polycystic ovary syndrome.

BACKGROUND: Endogenous opiates may affect various aspects of reproductive and metabolic function in patients with polycystic ovary syndrome (PCOS). This study evaluated long-term inhibition of the opioid system using naltrexone in clomiphene citrate (CC)-resistant women with PCOS. METHODS: A group of 30 infertile females with PCOS were evaluated; all subjects were obese, hyperandrogenic and hyperinsulinemic; 16 patients were amenorrhic and 14 were oligomenorrhic. All subjects received natrexone (50 mg p.o. daily) for 6 months. Patients who did not ovulate after 12 weeks of naltrexone monotherapy, also received CC (starting at 50 mg/day for 5 days and, for non-responders, increasing it up to 150 mg/day). RESULTS: Of the 30 women, 3 ovulated during naltrexone monotherapy and 19 of the remaining 27 ovulated during naltrexone + CC therapy. There were no conceptions during naltrexone monotherapy, but 9 of 27 women (33.3%) conceived during naltrexone + CC; there was one missed abortion at 9 weeks, one preterm delivery at 34 weeks and seven term live births. Naltrexone therapy was also followed by significant reductions in BMI, fasting serum insulin, luteinizing hormone (LH), LH/follicle-stimulating hormone ratio and testosterone. CONCLUSIONS: In this preliminary trial, naltrexone improved endocrine and metabolic function in women with CC-resistant PCOS. Furthermore, naltrexone restored CC sensitivity in the majority of subjects, resulting in a significant number of pregnancies.

Acute intestinal schistosomiasis among school-aged children presented to King Abdullah Hospital, Bisha province, Saudi Arabia: A Case Series.

Acute intestinal schistosomiasis is one of the clinical manifestations of infection with S. mansoni fluke. School aged-children are most at risk for this infection. To present cases of acute intestinal schistosomiasis among school-aged children attending the pediatric unit at King Abdullah Hospital, Bisha province, southwest of Saudi Arabia. This was a retrospective case study of nine school aged-children who were diagnosed with intestinal schistosomiasis in 2015. Data regarding clinical presentation, development of infections, diagnosis and management were recorded. Direct microscopical examination of stool sample for detection of S. mansoni egg's had been applied as a diagnostic tool. Laboratory findings were obtained to assess the severity of the infection. Nine children (7 boys and 2 girls) having acute intestinal schistosomiasis were reviewed. The age of the children were between six to 13 years old [mean 8.8 ± 2.17 years (SD)]. The duration of signs and symptoms prior to admission ranged from three to 21 days [mean 9.0 ± 5.8 days (SD)]. Most of the patients (n=7) presented with fever associated with abdominal pain followed by vomiting and cough. Four patients have a family history of intestinal schistosomiasis. Children had history of water contact for playing and swimming purposes. Infected children were treated with praziquantel (PZQ) oral dose of 20 mg/kg every eight hours for a day. None of the children presented late complications of schistosomiasis after three months follow up. The existence of intestinal schistosomiasis among school aged-children in Bisha suburb is alarming. The severity of the clinical manifestations of acute intestinal schistosomiasis were non-specific and varied that need of high expectation of physicians to diagnosis such disease. Obtaining of patients travelling history to endemic areas and visiting of infested water resources are necessary for detection of schistosomiasis cases.

Putative periodontal pathogens in the subgingival plaque of Sudanese subjects with aggressive periodontitis.

BACKGROUND AND OBJECTIVES: There has been limited study of the bacterial species associated with aggressive periodontitis (AgP) in high-risk populations in Africa. The aim of this study was to investigate and quantify the presence of four putative periodontal pathogens in the subgingival plaque of Sudanese subjects with AgP. A secondary aim was to investigate the effect of varying the detection threshold on the reported prevalence of the bacterial species investigated. MATERIALS AND METHODS: Subgingival plaque samples were collected from AgP cases (n=73) and healthy controls (n=71). Bacterial DNA was extracted and analyzed by quantitative polymerase chain reaction for the detection and quantification of four putative periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola and Tannerella forsythia. RESULTS: At the lowest detection threshold (>101 cells), P. gingivalis (p<0.0001) was more prevalent in AgP cases than controls. T. forsythia and T. denticola had a high prevalence (>70%) in AgP cases at all detection levels. While T. forsythia was significantly more frequently identified in AgP than in controls at all detection thresholds, this was only the case for T. denticola at the intermediate threshold (>102 cells). A. actinomycetemcomitans was identified less frequently than the other bacterial species with no difference in its prevalence between AgP cases and controls. CONCLUSION: The prevalence of the putative periodontal pathogens investigated varied considerably in Sudanese subjects with AgP and in periodontally healthy controls depending on the detection thresholds applied. T. forsythia was identified as having the strongest association with AgP.

A del(X)(p11) carrying SRY sequences in an infant with ambiguous genitalia.

BACKGROUND: SRY (sex-determining region, Y) is the gene responsible of gonadal differentiation in the male and it is essential for the regular development of male genitalia. Translocations involving the human sex chromosomes are rarely reported, however here we are reporting a very rare translocation of SRY gene to the q -arm of a deleted X chromosome. This finding was confirmed by cytogenetic, fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). CASE PRESENTATION: A 7-month infant was clinically diagnosed as an intersex case, with a phallus, labia majora and minora, a blind vagina and a male urethra. Neither uterus nor testes was detected by Ultrasonography. G-banding of his chromosomes showed 46,X,del(X)(p11) and fluorescent in situ hybridization (FISH) analysis showed a very small piece from the Y chromosome translocated to the q-arm of the del(X). Polymerase chain reaction (PCR) analysis revealed the presence of material from the sex-determining region Y (SRY) gene. CONCLUSION: It is suggested that the phenotype of the patient was caused by activation of the deleted X chromosome with SRY translocation, which is responsible for gonadal differentiation.

The pathogenesis of post kala-azar dermal leishmaniasis from the field to the molecule: does ultraviolet light (UVB) radiation play a role?

Post kala-azar dermal leishmaniasis (PKDL) is a dermatosis caused by persistence of Leishmania donovani parasites in the skin following apparently successful treatment of visceral leishmaniasis. The distribution of PKDL lesions in Sudanese patients often mirrors the clothing habits of those affected. It is most severe in or confined to the sun-exposed parts of the skin. It is well established that elimination of Leishmania parasites requires activation of parasitised macrophages by a Th1 immune response and that the latter is depressed by ultraviolet light (UVB). In this paper, we hypothesized that UVB light might be a key player in the pathogenesis of PKDL. This paper links observations made in the field with immunological data that are compatible with this hypothesis. We therefore investigated patients with PKDL immunologically for a possible role of UVB exposure in the pathogenesis of this condition. We marshal evidence that the changes in the tissues are compatible with the effects of UVB light and it is probable that UVB appears to be a key factor in the pathogenesis of PKDL. Immunopathologically the lesions were characterized by an influx of various inflammatory cells. The number of CD1a (Langerhans' cells) was decreased, they lost their dendrites, their HLA-DR and B7-1 expression was down regulated while B7-2 was expressed. Others have shown that Langerhans' cells with these features result from UVB exposure and that such cells are unable to present antigen to Th1 cells while retaining the capacity to present antigen to Th2 cells. Various cytokines known to be induced by UVB radiation could be demonstrated in PKDL lesions. Of these IL-10, TGF-beta, IL-12, IL-4 and TNF-alpha were found in different quantities. The Th-1 cytokine IFN-gamma was constantly present. The tissue origin of the Th-1 cells in PKDL is unknown. We believe that the antagonistic action of the different cytokines is the cause of the inflammation and chronicity of PKDL.

Uncommon clinical presentations of cutaneous leishmaniasis in Sudan.

Cutaneous leishmaniasis in Sudan is caused by Leishmania major zymodeme LON1. Self-healing usually occurs within 1 year but occasionally its duration is prolonged and treatment is required. The clinical forms are ulcers, nodules and noduloulcerative lesions. Here we describe seven patients with uncommon lesions that were difficult to recognize as Leishmania infections. These included mycetoma-like lesions, lesions that resembled L. tropica infection and others. One HIV/AIDS patient had Kaposi's sarcoma with Leishmania parasites in the Kaposi lesions. Most of these uncommon clinical forms were difficult to treat. The diagnosis depended on a high degree of suspicion and the demonstration of parasites in smears or culture. PCR was used to characterize parasites from the patients described here. Leishmania major was found by kDNA PCR in all patients, except one, who had a leishmanioma due to L. donovani. In three patients, including one with a L. tropica like-lesion, the parasites were confirmed as L. major by gp63 PCR-RFLP.

The origin and evolution of the Leishmania donovani complex as inferred from a mitochondrial cytochrome oxidase II gene sequence.

Members of the Leishmania donovani complex are parasites of the reticulo-endothelial system that are often associated with serious epidemics of a life threatening disease known as visceral leishmaniasis or kala-azar. Twenty-two Leishmania isolates representative of the geographical range of the parasite were analysed for sequence variations in their cytochrome oxidase II gene. In performing phylogenetic analysis, the maximum parsimonious, neighbour joining and maximum likelihood trees were congruent and produced a tree that differentiated between two clades conforming to the current classification of the species complex into two species: Leishmania donovani and Leishmania infantum. Furthermore, the molecular haplotypes were concordant, in general, with the isoenzyme data of the complex. The donovani isolates from the Sudan that possessed the most ancestral sequence were of a single haplotype that significantly resembled the sequence of Leishmania major. Our sequence data tallied with a general neutral model of sequence evolution with manifestations of weak selection. The data allowed an approximate dating of the origin of the complex to a period contemporary to or predating the spread of modern humans out of Africa.

Seroepidemiology of viral and toxoplasmal infections during pregnancy among Arab women of child-bearing age in Kuwait.

Five hundred and seventy three of 606 (94.6%), 583 of 604 (96.5%) and 575 of 595 (96.6%) women were found to have antibody to rubella virus, cytomegalovirus and herpes simplex virus, respectively. The Geometric Mean Titre (GMT) of haemagglutination-inhibition antibody to rubella virus was 60.97, while the GMT of complement-fixation antibody to cytomegalovirus and herpes simplex virus were 28.49 and 25.01, respectively. Furthermore, 301 of 517 (58.2%) women were also found to possess antibody to toxoplasma (GMT = 10.12) as detected by the passive-haemagglutination test. No significant differences in antibody prevalence or GMT could be found among the various nationalities studied except among Bedouins who showed a significantly higher rate and GMT of antibody to rubella virus and for the Mediterranean Arabs who showed significantly higher rate of immunity to and GMT of toxoplasma antibody when compared to other nationalities. Finally, no significant differences in antibody prevalence or GMT could be found among the various age groups.

Kala-azar in a high transmission focus: an ethnic and geographic dimension.

In 1994-1996, we studied a group of 58 game wardens stationed in an area known to be highly endemic for visceral leishmaniasis (kala-azar) for evidence of infection with Leishmania donovani. Leishmania DNA was detected by the polymerase chain reaction in the peripheral blood of cases of active kala-azar, former patients with visceral leishmaniasis, patients, and asymptomatic subjects. Using the cloned antigen rk39, antibodies were detected in 44.2% of the game wardens while leishmanin skin test result was positive in 77% of our sample. It was shown that certain tribes from northern Sudan were more likely to develop subclinical infections, while those of the Baria tribe from southern Sudan and those of the Nuba tribe from western Sudan were more likely to develop visceral leishmaniasis. Whether this is due to genetic factors or previous exposure to Leishmania parasites remains to be elucidated.

Diversity among Leishmania isolates from the Sudan: isoenzyme homogeneity of L. donovani versus heterogeneity of L. major.

Leishmania isolates from patients in the Sudan suffering from either visceral or cutaneous leishmaniasis were characterized using a battery of 12 enzymes. Aspartate aminotransferase separated the L. donovani isolates into 2 distinct zymodemes, but the overall results showed no significant geographical variation among L. donovani isolates. In contrast, the isolates of L. major were polymorphic, exhibiting differences in nucleoside hydrolase, 6-phosphogluconate dehydrogenase, superoxide dismutase, esterase, mannose phosphate isomerase, and aspartate aminotransferase, resulting in the description of 4 new enzymatic variants.

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.

Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Guérrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers.

In a previous efficacy study, autoclaved Leishmania major (ALM) + bacille Calmette-Guérrin (BCG) vaccine was shown to be safe, but not superior to BCG alone, in protecting against visceral leishmaniasis. From June 1999 to June 2000, we studied the safety and immunogenicity of different doses of alum-precipitated ALM + BCG vaccine mixture administered intradermally to evaluate whether the addition of alum improved the immunogenicity of ALM. Twenty-four healthy adult volunteers were recruited and sequentially allocated to receive either 10 microg, 100 microg, 200 microg, or 400 microg of leishmanial protein in the alum-precipitated ALM + BCG vaccine mixture. Side effects were minimal for all doses and confined to the site of injection. All volunteers in the 10 microg, 100 microg, and 400 microg groups had a leishmanin skin test (LST) reaction of > or = 5 mm by day 42 and this response was maintained when tested after 90 d. Only 1 volunteer out of 5 in the 200 microg group had a LST reaction of > or = 5 mm by day 42 and the reasons for the different LST responses in this group are unclear. This is the first time that an alum adjuvant with ALM has been in used in humans and the vaccine mixture was safe and induced a strong delayed type hypersensitivity (DTH) reaction in the study volunteers. On the basis of this study we suggest that 100 1 microg of leishmanial protein in the vaccine mixture is a suitable dose for future efficacy studies, as it induced the strongest DTH reaction following vaccination.