A multiplex PCR assay for the differentiation of Mycobacterium tuberculosis complex reveals high rates of mixed-lineage tuberculosis infections among patients in Ghana.

In low-resource settings with high tuberculosis (TB) burdens, lack of rapid diagnostic methods for detection and differentiation of Mycobacterium tuberculosis complex (MTBC) is a major challenge affecting TB management. This study utilized comparative genomic analyses of MTBC lineages; M. tuberculosis, M. africanum Lineages 5/6 and M. bovis to identify lineage-specific genes. Primers were designed for the development of a Multiplex PCR assay which was successful in differentiating the MTBC lineages. There was no cross-reaction with other respiratory pathogens tested. Validation of the assay using clinical samples was performed with sputum DNA extracts from 341 clinically confirmed active TB patients. It was observed that 24.9% of cases were caused by M. tuberculosis, while M. africanum L5 & L6 reported 9.0% and 14.4%, respectively. M. bovis infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. This multiplex PCR assay will allow speciation of MTBC lineages in low-resource regions, providing rapid differentiation of TB infections to select appropriate medication at the earliest possible time point. It will also be useful in epidemiological surveillance studies providing reliable information on the prevalence of TB lineages as well as identifying difficult to treat cases of mixed-lineage tuberculosis infections.

The Efficacy of Doxycycline Treatment on Mansonella perstans Infection: An Open-Label, Randomized Trial in Ghana.

Treating Mansonella perstans is challenged by the low efficacy of registered antihelminthics. Wolbachia endobacteria provide an alternative treatment target because depletion results in amicrofilaremia in filarial infections with Wuchereria bancrofti and Onchocerca volvulus infections. This open-label, randomized study sought to confirm that i) Wolbachia are present in M. perstans in Ghana and ii) doxycycline treatment will deplete Wolbachia and cause a slow, sustained decline in microfilariae (MF). Two hundred and two Ghanaians with M. perstans infection were randomized into early (immediate) and delayed (6 months deferred) treatment groups, given doxycycline 200 mg/day for 6 weeks, and monitored for MF and Wolbachia levels at baseline, 4, 12, and 24 months after the study onset (= time of randomization and start of treatment for the early group). Per protocol analysis revealed that the median MF/mL in the early group declined from 138 at baseline to 64 at month 4 and further to 0 at month 12. In the delayed group, MF load did not change from a baseline median of 97 to 102 at month 4 but declined to 42 at month 12, that is, 6 months after receiving treatment, trailing the early group as expected. By month 24, both treatment groups had reached a median MF level of 0. After treatment, Wolbachia were depleted from MF by ≥ 1-log drop compared with baseline levels. We conclude that M. perstans in Ghana harbor Wolbachia that are effectively depleted by doxycycline with subsequent reduction in MF loads, most likely because of interruption of fertility of adult worms.

Analysis of Mycobacterium ulcerans-specific T-cell cytokines for diagnosis of Buruli ulcer disease and as potential indicator for disease progression.

BACKGROUND: Buruli ulcer disease (BUD), caused by Mycobacterium (M.) ulcerans, is the third most common mycobacterial disease after tuberculosis and leprosy. BUD causes necrotic skin lesions and is a significant problem for health care in the affected countries. As for other mycobacterial infections, T cell mediated immune responses are important for protection and recovery during treatment, but detailed studies investigating these immune responses in BUD patients are scarce. In this study, we aimed to characterise M. ulcerans-specific CD4+ T cell responses in BUD patients and to analyse specific cytokine-producing T cells in the context of disease severity and progression. METHODOLOGY/PRINCIPAL FINDINGS: For this case-control study, whole blood samples of BUD patients (N = 36, 1.5-17 years of age) and healthy contacts (N = 22, 3-15 years of age) were stimulated with antigen prepared from M. ulcerans and CD4+ T cells were analysed for the expression of TNFα, IFNγ and CD40L by flow cytometry. The proportions and profile of cytokine producing CD4+ T cells was compared between the two study groups and correlated with disease progression and severity. Proportions of cytokine double-positive IFNγ+TNFα+, TNFα+CD40L+, IFNγ+CD40L+ (p = 0.014, p = 0.010, p = 0.002, respectively) and triple positive IFNγ+TNFα+CD40L+ (p = 0.010) producing CD4+ T cell subsets were increased in BUD patients. In addition, TNFα+CD40L-IFNγ- CD4+ T cells differed between patients and controls (p = 0.034). TNFα+CD40L-IFNγ- CD4+ T cells were correlated with lesion size (p = 0.010) and proportion were higher in 'slow' healers compared to 'fast healers' (p = 0.030). CONCLUSIONS: We were able to identify M. ulcerans-specific CD4+ T cell subsets with specific cytokine profiles. In particular a CD4+ T cell subset, producing TNFα but not IFNγ and CD40L, showed association with lesion size and healing progress. Further studies are required to investigate, if the identified CD4+ T cell subset has the potential to be used as biomarker for diagnosis, severity and/or progression of disease.

Epidemiology of Mansonella perstans in the middle belt of Ghana.

BACKGROUND: Mansonellosis was first reported in Ghana by Awadzi in the 1990s. Co-infections of Mansonella perstans have also been reported in a small cohort of patients with Buruli ulcer and their contacts. However, no study has assessed the exact prevalence of the disease in a larger study population. This study therefore aimed to find out the prevalence of M. perstans infection in some districts in Ghana and to determine the diversity of Culicoides that could be potential vectors for transmission. METHODS: From each participant screened in the Asante Akim North (Ashanti Region), Sene West and Atebubu Amantin (Brong Ahafo Region) districts, a total of 70 µl of finger prick blood was collected for assessment of M. perstans microfilariae. Centre for Disease Control (CDC) light traps as well as the Human Landing Catch (HLC) method were used to assess the species diversity of Culicoides present in the study communities. RESULTS: From 2,247 participants, an overall prevalence of 32% was recorded although up to 75% prevalence was demonstrated in some of the communities. Culicoides inornatipennis was the only species of Culicoides caught with the HLC method. By contrast, C. imicola (47%), C. neavei (25%) and C. schultzei (15%) were caught by the CDC light trap method. A wide diversity of other Culicoides spp. was also identified but correlation was only found between the prevalence of C. inornatipennis and M. perstans during the dry season. CONCLUSIONS: Here we demonstrate for the first time that M. perstans is highly prevalent in three districts in Ghana. We found a wide spectrum of Culicoides spp. Culicoides inornatipennis was the most anthropophilic and is therefore likely to be the species responsible for transmission of infection but formal proof has yet to be obtained. TRIAL REGISTRATION: NCT02281643 . Registered October 26, 2014. 'Retrospectively registered'. TRIAL REGISTRY: ClinicalTrials.gov.

Hygienic Practices among Food Vendors in Educational Institutions in Ghana: The Case of Konongo.

With the booming street food industry in the developing world there is an urgent need to ensure food vendors adhere to hygienic practices to protect public health. This study assessed the adherence to food hygiene practices by food vendors in educational institutions in Konongo, Ghana. Structured questionnaires, extensive observation and interviews were used for the study involving 60 food vendors from 20 basic schools. Attributable to the influence of school authorities and the level of in-training of food vendors, the study points out that food vendors in educational institutions generally adhered to good food hygiene practices, namely, regular medical examination (93%), protection of food from flies and dust (55%); proper serving of food (100%); good hand hygiene (63%); and the use of personal protective clothing (52%). The training of food vendors on food hygiene, instead of the level of education had a significant association (p < 0.05) with crucial food hygiene practices such as medical examination, hand hygiene and protection of food from flies and dust. Further, regulatory bodies legally mandated to efficiently monitor the activities of food vendors lacked the adequate capacity to do so. The study proposes that efforts should be geared towards developing training programmes for food vendors as well as capacity building of the stakeholders.