Dataset Information

Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens.

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.

SUBMITTER: Bratschi MW

PROVIDER: S-EPMC4264541 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Publications

Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens.

Bratschi Martin W MW Bolz Miriam M Grize Leticia L Kerber Sarah S Minyem Jacques C JC Um Boock Alphonse A Yeboah-Manu Dorothy D Ruf Marie-Thérèse MT Pluschke Gerd G

BMC infectious diseases 20141130

<h4>Background</h4>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 endem ...[more]

PMID: 25433390

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Project description:BACKGROUND: Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin. METHODOLOGY/PRINCIPAL FINDINGS: One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5' end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3' end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone. CONCLUSION: Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.

Dataset Information

Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens.

Background

Methods

Results

Conclusions

Publications

Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens.

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