Dissou Affolabi

Quantification of Hepatitis Delta Virus RNA in Serum by Consensus Real-Time PCR Indicates Different Patterns of Virological Response to Interferon Therapy in Chronically Infected Patients

ABSTRACT Hepatitis delta virus (HDV), in association with hepatitis B virus, is responsible for severe acute and chronic hepatitis. Treatment of the infection relies on the long-term administration of high doses of alpha interferon (IFN), and the treatment efficiency is monitored by the detection of anti-HDV immunoglobulin M and HDV genome in serum. Like the case for other chronic viral infections, HDV genome quantification in serum should be useful for the follow-up of infected patients. The aims of this study were to develop a quantitative assay for the detection of any type of HDV in serum and to evaluate the benefit of HDV RNA quantification for the follow-up of chronically infected patients receiving IFN. A real-time reverse transcription-PCR assay was developed to quantify the HDV RNA load in serum. Its efficacy was evaluated with 160 serum samples, 76 of which were collected from 11 chronically infected patients who were treated with pegylated IFN. The assay was sensitive (100 copies/ml of serum) and efficient for all HDV types, including type 3 and the recently described types 5, 6, and 7. The viral load determinations for treated patients allowed us to identify different profiles of virological responses to IFN therapy with more accuracy than that attainable with the qualitative approach. In conclusion, we have developed a quantitative HDV RNA assay for serum which is adapted to the follow-up of antiviral treatment for patients infected with any HDV type. The assay will help us to understand the natural history of HDV infection and to define guidelines for the management of chronic delta hepatitis.

Buruli ulcer disease: prospects for a vaccine

Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, is a neglected bacterial infection of the poor in remote rural areas, mostly affecting children. BUD is a mutilating disease leading to severe disability; it is the third most common mycobacterial infection in immunocompetent people after tuberculosis and leprosy. It is most endemic in West Africa, but cases have been reported from more than 30 countries. Treatment with antibiotics is possible, long-lasting and requires injections; there are cases of treatment failures, and the disease is prone to resistance. A vaccine against M. ulcerans would protect persons at risk in highly endemic areas, and could be used as a therapeutic vaccine to shorten the duration of treatment and prevent relapses. There is considerable evidence supporting the notion that generation of a vaccine is feasible. This article reviews the present state of the art with special emphasis on the immunology of the infection and the prospects for development of a vaccine.

Evaluation of direct detection of Mycobacterium tuberculosis rifampin resistance by a nitrate reductase assay applied to sputum samples in Cotonou, Benin

ABSTRACT The aim of this study was to evaluate a nitrate reductase assay (NRA) performed on smear-positive sputa for the direct detection of rifampin resistance in Mycobacterium tuberculosis. A total of 213 smear-positive sputa with a positivity score of 1+ or more (>1 acid-fast bacillus per field by fluorescence microscopy) were used in the study. The samples were decontaminated using the modified Petroff method, and portions of the resulting suspension were used to perform the NRA. The NRA results were compared with the reference indirect proportion method for 177 specimens for which comparable results were available. NRA results were obtained at day 10 for 15 specimens (9%), results for 88 specimens (50%) were obtained at day 14, results for 66 specimens (37%) were obtained at day 18, and results for the remaining 8 specimens (4%) were obtained at day 28. Thus, 96% of NRA results were obtained in 18 days. Of the 177 specimens, there was only one discrepancy (susceptible according to the NRA and resistant according to the indirect proportion method). NRA is simple to perform and provides a rapid, accurate, and cost-effective means for the detection of rifampin resistance in M. tuberculosis isolates.

Rapid and Inexpensive Detection of Multidrug-Resistant Mycobacterium tuberculosis with the Nitrate Reductase Assay Using Liquid Medium and Direct Application to Sputum Samples

ABSTRACT For rapid and low-cost detection of multidrug-resistant (MDR) Mycobacterium tuberculosis, we applied the nitrate reductase assay (NRA) using a liquid medium directly to sputum samples. A total of 179 sputum samples were analyzed by the NRA, and results were compared to those obtained by the indirect proportion method (IPM) as a standard reference. Out of 144 specimens for which comparable results were available, only one discrepant result was obtained: MDR by NRA but susceptible by the IPM. In total 56% of the results were obtained in 10 days by the NRA. NRA performed in liquid medium is rapid and inexpensive and can be easily implemented in low-income countries.

Possible Outbreak of Streptomycin-Resistant Mycobacterium tuberculosis Beijing in Benin

Using geographic information system and molecular tools, we characterized a possible outbreak of tuberculosis caused by Mycobacterium tuberculosis Beijing strain in 17 patients in Cotonou, Benin, during July 2005–October 2006. Most patients lived or worked in the same area and frequented the same local drinking bar. The isolates were streptomycin resistant.

Buruli ulcer : a review of in vitro tests to screen natural products for activity against mycobacterium ulcerans

Buruli ulcer (BU), caused by Mycobacterium ulcerans, has recently been recognized by the World Health Organization (WHO) as an important emerging disease. It is largely a problem of the poor in remote rural areas and has emerged as an important cause of human suffering. While antimycobacterial therapy is often effective for the earliest nodular or ulcerative lesions, for advanced ulcerated lesions, surgery is sometimes necessary. Antimycobacterial drugs may also prevent relapses or disseminated infections. Efficient alternatives different from surgery are presently explored because this treatment deals with huge restrictive factors such as the necessity of prolonged hospitalization, its high cost, and the scars after surgery. Traditional treatment remains the first option for poor populations of remote areas who may have problems of accessibility to synthetic products because of their high cost. The search for efficient natural products active on M. ulcerans should then be encouraged because they are part of the natural heritage of these populations; they are affordable financially and can be used at the earliest stage. This review provides a number of tests that will help to evaluate the antimycobacterial activity of natural products against M. ulcerans, which are adapted to its slow growing rate, and lists active extracts published up to now in Medline.

Case Report: Severe Multifocal Form of Buruli Ulcer after Streptomycin and Rifampin Treatment: Comments on Possible Dissemination Mechanisms

Buruli ulcer (BU), a disease caused by Mycobacterium ulcerans, leads to the destruction of skin and sometimes bone. Here, we report a case of severe multifocal BU with osteomyelitis in a 6-year-old human immunodeficiency virus (HIV)-negative boy. Such disseminated forms are poorly documented and generally occur in patients with HIV co-infection. The advent of antibiotic treatment with streptomycin (S) and rifampin (R) raised hope that these multifocal BU cases could be reduced. The present case raises two relevant points about multifocal BU: the mechanism of dissemination that leads to the development of multiple foci and the difficulties of treatment of multifocal forms of BU. Biochemical (hypoproteinemia), hematological (anemia), clinical (traditional treatment), and genetic factors are discussed as possible risk factors for dissemination.

The First Phylogeographic Population Structure and Analysis of Transmission Dynamics of M. africanum West African 1— Combining Molecular Data from Benin, Nigeria and Sierra Leone

Mycobacterium africanum is an important cause of tuberculosis (TB) in West Africa. So far, two lineages called M. africanum West African 1 (MAF1) and M. africanum West African 2 (MAF2) have been defined. Although several molecular studies on MAF2 have been conducted to date, little is known about MAF1. As MAF1 is mainly present in countries around the Gulf of Guinea we aimed to estimate its prevalence in Cotonou, the biggest city in Benin. Between 2005–06 we collected strains in Cotonou/Benin and genotyped them using spoligo- and 12-loci-MIRU-VNTR-typing. Analyzing 194 isolates, we found that 31% and 6% were MAF1 and MAF2, respectively. Therefore Benin is one of the countries with the highest prevalence (37%) of M. africanum in general and MAF1 in particular. Moreover, we combined our data from Benin with publicly available genotyping information from Nigeria and Sierra Leone, and determined the phylogeographic population structure and genotypic clustering of MAF1. Within the MAF1 lineage, we identified an unexpected great genetic variability with the presence of at least 10 sub-lineages. Interestingly, 8 out of 10 of the discovered sub-lineages not only clustered genetically but also geographically. Besides showing a remarkable local restriction to certain regions in Benin and Nigeria, the sub-lineages differed dramatically in their capacity to transmit within the human host population. While identifying Benin as one of the countries with the highest overall prevalence of M. africanum, this study also contains the first detailed description of the transmission dynamics and phylogenetic composition of the MAF1 lineage.

Insertion Sequence Element Single Nucleotide Polymorphism Typing Provides Insights into the Population Structure and Evolution of Mycobacterium ulcerans across Africa

ABSTRACT Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the “pan-African clade” were found to be widespread throughout Africa, while the ISE-SNP types of the “Gabonese/Cameroonian clade” were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.

Prospective multicentre evaluation of the direct nitrate reductase assay for the rapid detection of extensively drug-resistant tuberculosis

OBJECTIVES To perform a multicentre study evaluating the performance of the direct nitrate reductase assay (NRA) for the detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in sputum samples. METHODS The study was conducted in six laboratories performing tuberculosis diagnosis that were located in six different countries. The NRA was performed directly on sputum samples in parallel with the reference method used at each site. Detection of resistance was performed for rifampicin, isoniazid, ofloxacin and kanamycin. RESULTS Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 93.7%-100% for rifampicin, 88.2%-100% for isoniazid, 94.6%-100% for ofloxacin and 100% for kanamycin. The majority of NRA results were available at day 21 for sites 1, 2 and 5. Site 3 had a turnaround time of 13.9 days, at site 4 it was 18.4 days and at site 6 it was 16.2 days. The contamination rate ranged between 2.5% and 12%. CONCLUSIONS Rapid detection of drug resistance by the direct NRA on sputum smear-positive samples was accurate and easy to implement in clinical diagnostic laboratories, making it a good alternative for rapid screening for MDR and XDR tuberculosis.