Browsing by Author "Bratschi, M.W."
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Item Late Onset of the Serological Response against the 18 kDa Small Heat Shock Protein of Mycobacterium ulcerans in Children(Public Library of Science, 2014) Röltgen, K.; Bratschi, M.W.; Aboagye, S.Y.; Ampah, K.A.; Bolz, M.; Andreoli, A.; Pritchard, J.; Minyem, J.C.; Noumen, D.; Koka, E.; Um Boock, A.; Yeboah-Manu, D.; Pluschke, G.A previous survey for clinical cases of Buruli ulcer (BU) in the Mapé Basin of Cameroon suggested that, compared to older age groups, very young children may be less exposed to Mycobacterium ulcerans. Here we determined serum IgG titres against the 18 kDa small heat shock protein (shsp) of M. ulcerans in 875 individuals living in the BU endemic river basins of the Mapé in Cameroon and the Densu in Ghana. While none of the sera collected from children below the age of four contained significant amounts of 18 kDa shsp specific antibodies, the majority of sera had high IgG titres against the Plasmodium falciparum merozoite surface protein 1 (MSP-1). These data suggest that exposure to M. ulcerans increases at an age which coincides with the children moving further away from their homes and having more intense environmental contact, including exposure to water bodies at the periphery of their villages. © 2014 Röltgen et al.Item Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens(2014-11-30) Bratschi, M.W.; Bolz, M.; Grize, L.; Kerber, S.; Minyem, J.C.; Um Boock, A.; Yeboah-Manu, D.; Ruf, M.; Pluschke, G.Abstract Background While cultivation of pathogens represents a foundational diagnostic approach in the study of infectious diseases, its value for the confirmation of clinical diagnosis of Buruli ulcer is limited by the fact that colonies of Mycobacterium ulcerans appear only after about eight weeks of incubation at 30°C. However, for molecular epidemiological and drug sensitivity studies, primary isolation of M. ulcerans remains an essential tool. Since for most of the remote Buruli ulcer endemic regions of Africa cultivation laboratories are not easily accessible, samples from lesions often have to be stored for extended periods of time prior to processing. The objective of the current study therefore was to determine which transport medium, decontamination method or other factors decrease the contamination rate and increase the chance of primary isolation of M. ulcerans bacilli after long turnover time. Methods Swab and fine needle aspirate (FNA) samples for the primary cultivation were collected from clinically confirmed Buruli ulcer patients in the Mapé Basin of Cameroon. The samples were either stored in the semi-solid transport media 7H9 or Amies or dry for extended period of time prior to processing. In the laboratory, four decontamination methods and two inoculation media were evaluated and statistical methods applied to identify factors that decrease culture contamination and factors that increase the probability of M. ulcerans recovery. Results The analysis showed: i) that the use of moist transport media significantly increased the recovery rate of M. ulcerans compared to samples kept dry; ii) that the choice of the decontamination method had no significant effect on the chance of M. ulcerans isolation; and iii) that Löwenstein-Jensen supplemented with antibiotics as inoculation medium yielded the best results. We further found that, ten extra days between sampling and inoculation lead to a relative decrease in the isolation rate of M. ulcerans by nearly 20%. Finally, collection and processing of multiple samples per patient was found to significantly increase the M. ulcerans isolation rate. Conclusions Based on our analysis we suggest a procedure suitable for the primary isolation of M. ulcerans strains from patients following long delay between sample collection and processing to establish a M. ulcerans strain collection for research purposes.Item Primary cultivation: Factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens(BMC Infectious Diseases, 2014-11) Bratschi, M.W.; Bolz, M.; Grize, L.; Kerber, S.; Minyem, J.C.; Um Boock, A.; Yeboah-Manu, D.; Ruf, M.-T.; Pluschke, G.Background: While cultivation of pathogens represents a foundational diagnostic approach in the study of infectious diseases, its value for the confirmation of clinical diagnosis of Buruli ulcer is limited by the fact that colonies of Mycobacterium ulcerans appear only after about eight weeks of incubation at 30°C. However, for molecular epidemiological and drug sensitivity studies, primary isolation of M. ulcerans remains an essential tool. Since for most of the remote Buruli ulcer endemic regions of Africa cultivation laboratories are not easily accessible, samples from lesions often have to be stored for extended periods of time prior to processing. The objective of the current study therefore was to determine which transport medium, decontamination method or other factors decrease the contamination rate and increase the chance of primary isolation of M. ulcerans bacilli after long turnover time. Methods: Swab and fine needle aspirate (FNA) samples for the primary cultivation were collected from clinically confirmed Buruli ulcer patients in the Mapé Basin of Cameroon. The samples were either stored in the semi-solid transport media 7H9 or Amies or dry for extended period of time prior to processing. In the laboratory, four decontamination methods and two inoculation media were evaluated and statistical methods applied to identify factors that decrease culture contamination and factors that increase the probability of M. ulcerans recovery. Results: The analysis showed: i) that the use of moist transport media significantly increased the recovery rate of M. ulcerans compared to samples kept dry; ii) that the choice of the decontamination method had no significant effect on the chance of M. ulcerans isolation; and iii) that Löwenstein-Jensen supplemented with antibiotics as inoculation medium yielded the best results. We further found that, ten extra days between sampling and inoculation lead to a relative decrease in the isolation rate of M. ulcerans by nearly 20%. Finally, collection and processing of multiple samples per patient was found to significantly increase the M. ulcerans isolation rate. Conclusions: Based on our analysis we suggest a procedure suitable for the primary isolation of M. ulcerans strains from patients following long delay between sample collection and processing to establish a M. ulcerans strain collection for research purposes. © 2014 Bratschi et al.Item Spatial Distribution of Mycobacterium ulcerans in Buruli Ulcer Lesions: Implications for Laboratory Diagnosis(Public Library of Science, 2016) Ruf, M.T.; Bolz, M.; Vogel, M.; Bayi, B.F.; Bratschi, M.W.; Sopho, G.E.; Yeboah-Manu, D.; Um Boock, A.; Junghanss, T.; Pluschke, G.Current laboratory diagnosis of Buruli ulcer (BU) is based on microscopic detection of acid fast bacilli, quantitative real-time PCR (qPCR), histopathology or cultivation. Insertion sequence (IS) 2404 qPCR, the most sensitive method, is usually only available at reference laboratories. The only currently available point-of-care test, microscopic detection of acid fast bacilli (AFB), has limited sensitivity and specificity. Methodology/ Principal Findings: Here we analyzed AFB positive tissue samples (n = 83) for the presence, distribution and amount of AFB. AFB were nearly exclusively present in the subcutis with large extracellular clusters being most frequently (67%) found in plaque lesions. In ulcerative lesions small clusters and dispersed AFB were more common. Beside this, 151 swab samples from 37 BU patients were analyzed by IS2404 qPCR and ZN staining in parallel. The amount of M. ulcerans DNA in extracts from swabs correlated well with the probability of finding AFB in direct smear microscopy, with 56.1% of the samples being positive in both methods and 43.9% being positive only in qPCR. By analyzing three swabs per patient instead of one, the probability to have at least one positive swab increased from 80.2% to 97.1% for qPCR and from 45% to 66.1% for AFB smear examination. Conclusion / Significance: Our data show that M. ulcerans bacteria are primarily located in the subcutis of BU lesions, making the retrieval of the deep subcutis mandatory for examination of tissue samples for AFB. When laboratory diagnosis is based on the recommended less invasive collection of swab samples, analysis of three swabs from different areas of ulcerative lesions instead of one increases the sensitivity of both qPCR and of smear microscopy substantially. © 2016 Ruf et al.