Diagnostic predictive values of the Hain genotype MTBDRsl assay in mycobacterial strains isolated from Sudan.

INTRODUCTION: hain GenoType MTBDRsl is nucleic acid amplification assay based on reverse hybridization with specific oligonucleotide probes on nitrocellulose strips. MTBDRsl identifies M. tuberculosis complex and detects resistance to fluoroquinolone, second line injectable drugs and ethambutol evident as mutations of gyrA, rrs and embB genes respectively. This study aimed to evaluate the diagnostic performance of the Hain GenoType MTBDRsl Assay using 1% proportion method on LJ medium as gold standard. METHODS: a total of 52 rifampicin resistant (RR) isolates were tested for second line drug sensitivity by 1% proportion method and by MTBDRsl assay. RESULTS: two strains were identified as mycobacteria other than tuberculosis MOTT and the rest were Mycobacterium tuberculosis complex MTBC. Five of the MTBC isolates (5/50; 10%) showed resistance to at least one second line drug and one isolate (1/50; 2%) was XDR. XDR strain was concordantly detected by the two methods. One of two Kanamycin-resistant isolates showed discordant results. Ofloxacin showed one false positive and one false negative result. Most discrepancies were detected with Ethambutol. The sensitivity, specificity, positive and negative predictive values were respectively as follows: Ethambutol (63.3.4%, 85.7%, 94.4% and 62%), for Kanamycin (67%, 100%, 100% and 97.9%), for Amikacin and Capreomycin (100%, 100%, 100% and 100%), for Ofloxacin (75%, 97.5%, 75% and 97.8%). For XDR isolate the values were 100%, 100%, 100% and 100% respectively. CONCLUSION: MTBDRsl showed high specificity and negative predictive values making it acceptable and time-saving for early presumptive detection of resistance to second-line drugs in Sudan.

Initial second-line drug resistance of Mycobacterium tuberculosis isolates from Sudanese retreatment-patients.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are a major public health threat. OBJECTIVE: This study aimed to determine resistance patterns to second line anti-TB drugs (SLDs), and to determine the frequency of extensively drug resistant Mycobacterium tuberculosis (XDR-TB). DESIGN: During the period from July 2009 to July 2010; sputum specimens were collected from TB retreatment patients; isolates were tested for sensitivity to first line anti-TB drugs by the 1% proportion method; MDR strains were tested for second line anti-TB drugs sensitivity by 1% proportion method and by version 1. Hain GenoType MTBDRsl Assay. RESULTS: One hundred and forty three mycobacterial isolates were successfully recovered from a total of 239 specimens (143/239; 59.8%). Fifty six strains were rifampicin resistant (RR); of these 54 were multi-drug resistant (MDR); two were RIF/INH-resistant mycobacterium other than tuberculosis (MOTT). Five of MDR (5/50; 10%) showed resistance to at least one second line drug and one isolate (1/50; 2%) was XDR. The XDR strain was concordantly detected by the two methods. CONCLUSION: Initial resistance to second line anti-TB drugs among MDR-TB patients is at 10% levels and XDR-TB is prevalent at low levels (2%). Nevertheless; without great efforts from national tuberculosis control program (NTP) this figure can fuel the TB epidemics in Sudan.

Efficacy and Safety of AmBisome in Combination with Sodium Stibogluconate or Miltefosine and Miltefosine Monotherapy for African Visceral Leishmaniasis: Phase II Randomized Trial.

BACKGROUND: SSG&PM over 17 days is recommended as first line treatment for visceral leishmaniasis in eastern Africa, but is painful and requires hospitalization. Combination regimens including AmBisome and miltefosine are safe and effective in India, but there are no published data from trials of combination therapies including these drugs from Africa. METHODS: A phase II open-label, non-comparative randomized trial was conducted in Sudan and Kenya to evaluate the efficacy and safety of three treatment regimens: 10 mg/kg single dose AmBisome plus 10 days of SSG (20 mg/kg/day), 10 mg/kg single dose AmBisome plus 10 days of miltefosine (2.5mg/kg/day) and miltefosine alone (2.5 mg/kg/day for 28 days). The primary endpoint was initial parasitological cure at Day 28, and secondary endpoints included definitive cure at Day 210, and pharmacokinetic (miltefosine) and pharmacodynamic assessments. RESULTS: In sequential analyses with 49-51 patients per arm, initial cure was 85% (95% CI: 73-92) in all arms. At D210, definitive cure was 87% (95% CI: 77-97) for AmBisome + SSG, 77% (95% CI 64-90) for AmBisome + miltefosine and 72% (95% CI 60-85) for miltefosine alone, with lower efficacy in younger patients, who weigh less. Miltefosine pharmacokinetic data indicated under-exposure in children compared to adults. CONCLUSION: No major safety concerns were identified, but point estimates of definitive cure were less than 90% for each regimen so none will be evaluated in Phase III trials in their current form. Allometric dosing of miltefosine in children needs to be evaluated. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov, number NCT01067443.

Prevalence of latent tuberculosis infection in Sudan: a case-control study comparing interferon-γ release assay and tuberculin skin test.

BACKGROUND: Most people exposed to M. tuberculosis show no evidence of clinical disease. Five to 10% of individuals with latent infection progress to develop overt disease during their life time. Identification of people with latent TB infection will increase case detection rates and may dictate new treatment policies to control tuberculosis. This study aimed to determine LTBI point prevalence in a population from Sudan using two different diagnostic methods: the tuberculin skin test (TST) and the IFN-γ release assay (IGRA). METHODS: This was a prospective, community-based and case-controlled study. Following informed consent, household contacts (HHCs; n = 98) of smear-positive index cases and Community controls (CCs; 186), were enrolled. Tuberculin skin test (TST), whole blood stimulation with ESAT-6/CFP-10 ± TB7.7 antigens or purified protein derivative (PPD) and IFN-γ levels determination with ELISA were performed. The levels of IFN-γ and TST induration between the CCs and the HHCs were compared using student t-test, Chi-square and Kappa coefficient. Pearson correlation test was used to compare TST and IFN-γ. P levels of <0.05 were considered significant. RESULTS: TST induration of ≥ 10 mm gave an LTBI point prevalence of 327 cases/1000 individuals among HHCs compared to 126 cases/1000 individuals among CCs (p = 0.000). PPD-induced IFN-γ release assay gave an LTBI point prevalence of 418 cases/1000 individuals among HHCs compared to 301 cases/1000 individuals among CCs (p =0.06). On the other hand ESAT-6/CFP-10 ± TB7.7-induced IFN-γ gave an LTBI point prevalence of 429 cases/1000 individuals among HHCs compared to 268 cases/1000 individuals among CCs (p = 0.01). IFN-γ productions levels induced by ESAT-6/CPF-10 ± TB7.7 antigens in HHCS and CCs were not significantly different from those induced by PPD (p = 0.7). CONCLUSION: IFN-γ release assay (IGRA) gave higher LTBI point prevalence compared to TST in HHCs and CCs. PPD gave comparable results to ESAT-6/CFP-10 ± TB7.7 antigens in whole blood IFN-γ release, making it a cheap alternative to the recombinant antigens.

Post-Kala-Azar Dermal Leishmaniasis: A Paradigm of Paradoxical Immune Reconstitution Syndrome in Non-HIV/AIDS Patients.

Visceral leishmaniasis (VL) is a parasitic disease characterized by immune suppression. Successful treatment is usually followed by immune reconstitution and a dermatosis called post-Kala-azar dermal leishmaniasis (PKDL). Recently, PKDL was described as one of the immune reconstitution syndromes (IRISs) in HIV/VL patients on HAART. This study aimed to present PKDL as a typical example of paradoxical IRIS in non-HIV/AIDS individuals. Published and new data on the pathogenesis and healing of PKDL was reviewed and presented. The data suggested that PKDL is a typical example of paradoxical IRIS, being a new disease entity that follows VL successful treatment and immune recovery. PKDL lesions are immune inflammatory in nature with granuloma, adequate response to immunochemotherapy, and an ensuing hypersensitivity reaction, the leishmanin skin test (LST). The data also suggested that the cytokine patterns of PKDL pathogenesis and healing are probably as follows: an active disease state dominated by IL-10 followed by spontaneous/treatment-induced IL-12 priming, IL-2 stimulation, and INF- γ production. INF- γ -activated macrophages eliminate the Leishmania parasites/antigen to be followed by LST conversion and healing. In conclusion, PKDL is a typical example of paradoxical IRIS in non-HIV/AIDS individuals with anti-inflammatory cytokine patterns that are superseded by treatment-induced proinflammatory cytokines and lesions healing.

Misdiagnosis and mistreatment of post-kala-azar dermal leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL) is a known complication of visceral leishmaniasis (VL) caused by L. donovani. It is rare in VL caused by L. infantum and L. chagasi. In Sudan, it occurs with a frequency of 58% among successfully treated VL patients. In the majority of cases, PKDL can be diagnosed on the basis of clinical appearance, distribution of the lesions, and past history of treated VL. The ideal diagnostic method is to demonstrate the parasite in smears, by culture or PCR. Diagnosis is particularly difficult in patients who develop PKDL in the absence of previous history of visceral leishmaniasis. We describe a case of cutaneous leishmaniasis misdiagnosed as PKDL and 3 cases of PKDL who were either misdiagnosed or mistreated as other dermatoses. This caused exacerbation of their disease leading to high parasite loads in the lesions and dissemination to internal organs in one of the patients, who was also diabetic. The latter patient had L. major infection. A fourth patient with papulonodular lesions on the face and arms of 17-year duration and who was misdiagnosed as having PKDL is also described. He turned out to have cutaneous leishmaniasis due to L. major. Fortunately, he was not treated with steroids. He was cured with intravenous sodium stibogluconate.

Immunochemotherapy of persistent post-kala-azar dermal leishmaniasis: a novel approach to treatment.

Post-kala-azar dermal leishmaniasis (PKDL) is a recognized dermatosis that follows successful treatment of visceral leishmaniasis in the Sudan. This randomized and double-blind study aimed to assess safety, immunogenicity and curative potentials of a novel immunochemotherapy regimen in patients with persistent PKDL. Following informed consent, 30 patients were randomized to receive alum-precipitated autoclaved Leishmania major (Alum/ALM) vaccine+Bacille Calmette-Guérin (BCG) and sodium stibogluconate (SSG) or vaccine diluent and SSG. The SSG+Alum/ALM+BCG proved safe with minimal local adverse events. In the SSG+vaccine group, 87% of the patients were cured by day 60 compared with 53% in the SSG alone group (SSG+vaccine efficacy=71%, 95% CI for risk ratio 0.7-1.16). On day 90 of follow-up there were two relapses in the SSG alone arm and none in the SSG+vaccine arm. Pre-treatment cytokines showed high IFN-gamma or high IFN-gamma/IL-10 levels and leishmanin skin test (LST) non-reactivity, while healing/clinical improvement were associated with LST reactivity and low IFN-gamma levels in both study groups (P=0.004). In conclusion, SSG+Alum/ALM+BCG is safe and immunogenic with significant healing potentials in persistent PKDL lesions. Immunochemotherapy probably augmented IFN-gamma production, which induced healing. Leishmanin skin reactivity is a good surrogate marker of cure in persistent PKDL lesions.

Innovative serum-free medium for in vitro cultivation of promastigote forms of Leishmania species.

We described a comparatively simple medium formula (CML) using common, available and reasonably priced ingredients that could be used in place of medium that requires calf serum enhancement for cultivation of Leishmania promastigote forms. This medium equivalently supported the growth of parasites at rates comparable with those obtained with serum supplemented RPMI-1640 medium. Leishmania promastigotes reproduced in CML exhibited moderate to high infectivity capacities when tested against J774 macrophage cell line. No significant difference was noted between Leishmania strains cultivated in the newly modified medium and those grown in RPMI-1640 medium in their cells infectivity and replication potentials. The use of new CML can easily take the place of other biphasic or liquid media because of its easy preparation and instantaneous use, reasonable price, availability of ingredients, and its long shelf life, which is 30-45 days. The fact that this medium is similar to other culture media as far as durability and quantity of produced parasites might give it an advantage over the other currently used media.

Safety and immunogenicity of a candidate vaccine for visceral leishmaniasis (Alum-precipitated autoclaved Leishmania major + BCG) in children: an extended phase II study.

BACKGROUND: Untreated visceral leishmaniasis (VL) is an inevitably fatal childhood disease. First-generation candidate vaccines for VL [autoclaved Leishmania major (ALM) + BCG] have been found to be safe and immunogenic but not superior to BCG alone. Modulation of ALM by adsorption to Alum significantly increases the immunogenicity. The Alum-adsorbed ALM vaccine was found to be safe and strongly immunogenic in healthy adult volunteers in a non-VL-endemic area. This study aimed at establishing the safety and immunogenicity of Alum-precipitated autoclaved L. major + BCG vaccine in children under field conditions. METHODS: A total of 544 healthy, leishmanin non-reactive children (<15 y) were randomly allocated to receive either a single intradermal injection of Alum/ALM + BCG or vaccine diluent (placebo). Volunteers were closely followed for 2 years at 6-month intervals for vaccine safety and immunogenicity. RESULTS: The vaccine was well tolerated with minimal side-effects. Leishmanin skin test conversion (>or=5 mm) was seen in 56%, 50%, 25% and 31% at 6, 12, 18 and 24 months post-vaccination, respectively; conversion in the placebo group was 4%, 12%, 3% and 13% at the same follow-up visits. There was no significant increase in anti-leishmanial antibodies in either study arm at any of the follow-up visits. During the study, four patients in the placebo arm developed parasitologically confirmed VL. CONCLUSION: Alum/ALM + BCG vaccine is safe and immunogenic in children under field conditions. Multiple injections might be needed to obtain results similar to those obtained in healthy volunteers.

SLC11A1 (formerly NRAMP1) and susceptibility to visceral leishmaniasis in The Sudan.

Genetic susceptibility to visceral leishmaniasis (VL) is indicated by differences in incidence and clinical phenotypes between ethnic groups in Sudan. In mice, innate susceptibility to Leishmania donovani, the etiological agent of VL, is controlled by Slc11a1 (formerly Nramp1). We therefore examined polymorphisms at SLC11A1 in 59 multicase families of VL from the high-incidence Masalit tribe in Sudan. Multipoint nonparametric analysis in ALLEGRO shows a significant linkage across SLC11A1 (Zlr scores 2.38-2.55; 0.008< or =P< or =0.012; information content 0.88). The extended transmission disequilibrium test shows biased transmission of alleles at 5' polymorphisms in the promoter (P=0.0145), exon 3 (P=0.0037) and intron 4 (P=0.0049), and haplotypes formed by them (P=0.0089), but not for 3' polymorphisms at exon 15 or the 3'UTR. Stepwise logistic regression analysis using a case/pseudo-control data set derived from the 59 families was consistent with main effects contributed by the intron 4 469+14G/C polymorphism. Although the two alleles for 469+14G/C lie on haplotypes carrying different alleles for the functional promoter GTn polymorphism, the latter did not itself contribute separate main effects. Sequence analysis of 36 individuals failed to identify new putative functional polymorphisms in the coding region, intron 1, intron/exon boundaries, intron 4/exon 4a, or in the 3'UTR. One novel promoter polymorphism (-86G/A) was located within a putative nuclear factor kappa B binding site that could be functional. Further work will determine whether additional polymorphisms occur upstream in the promoter, which could be in linkage disequilibrium with the intron 4 polymorphism. These studies contribute to knowledge of the role of SLC11A1 in infectious disease.