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Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis.

Rabodoarivelo MS, Imperiale B, Andrianiavomikotroka R, Brandao A, Kumar P, Singh S, Ferrazoli L, Morcillo N, Rasolofo V, Palomino JC, Vandamme P, Martin A - PLoS ONE (2015)

Bottom Line: Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing.For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%.These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur de Madagascar, Antananarivo, Madagascar; Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.

ABSTRACT

Background: Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus.

Methods: Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing.

Results: For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%.

Conclusion: The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.

No MeSH data available.


Related in: MedlinePlus

16SrRNA PCR results on 2% agarose gel.The appearance of 921 bp PCR amplicon indicates the positive results for MTB. M: marker, lane 1: from ethanol; lane 2: from FTA card; lane 4; from GenoCard; lane 4: from slide.
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pone.0139382.g001: 16SrRNA PCR results on 2% agarose gel.The appearance of 921 bp PCR amplicon indicates the positive results for MTB. M: marker, lane 1: from ethanol; lane 2: from FTA card; lane 4; from GenoCard; lane 4: from slide.

Mentions: We first standardized and optimized the DNA extraction protocol from the different storage supports using NTM. For the DNA extraction from suspensions spotted on FTA cards, we initially found that 1 punched disc was not enough for PCR amplification. The 16SrRNA amplification product was negative in 2% agarose gel electrophoresis. However, DNA extraction from 4 discs by using the 10% Chelex-100 method showed a very good and an optimal PCR result (Fig 1). Similarly, DNA extraction from the mycobacterial cells spotted on GenoCard was standardized with 8 punched discs by using the 10% Chelex-100 method (Fig 1). Equally satisfactory results with or without 10% Chelex-100 were obtained for both cards. Nevertheless, the DNA extraction protocol using 10% Chelex-100 was selected for the field evaluation because it has the advantage of allowing having a sufficient amount of DNA (75μl) for subsequent additional tests, which is not possible using directly the punched discs in the PCR mix. The Chelex DNA extraction methods from ZN smear and from the strain kept in ethanol gave also satisfactory results (Fig 1). All NTM were correctly identified by GenoType Mycobacterium CM using the 4 storage and transport systems.


Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis.

Rabodoarivelo MS, Imperiale B, Andrianiavomikotroka R, Brandao A, Kumar P, Singh S, Ferrazoli L, Morcillo N, Rasolofo V, Palomino JC, Vandamme P, Martin A - PLoS ONE (2015)

16SrRNA PCR results on 2% agarose gel.The appearance of 921 bp PCR amplicon indicates the positive results for MTB. M: marker, lane 1: from ethanol; lane 2: from FTA card; lane 4; from GenoCard; lane 4: from slide.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591989&req=5

pone.0139382.g001: 16SrRNA PCR results on 2% agarose gel.The appearance of 921 bp PCR amplicon indicates the positive results for MTB. M: marker, lane 1: from ethanol; lane 2: from FTA card; lane 4; from GenoCard; lane 4: from slide.
Mentions: We first standardized and optimized the DNA extraction protocol from the different storage supports using NTM. For the DNA extraction from suspensions spotted on FTA cards, we initially found that 1 punched disc was not enough for PCR amplification. The 16SrRNA amplification product was negative in 2% agarose gel electrophoresis. However, DNA extraction from 4 discs by using the 10% Chelex-100 method showed a very good and an optimal PCR result (Fig 1). Similarly, DNA extraction from the mycobacterial cells spotted on GenoCard was standardized with 8 punched discs by using the 10% Chelex-100 method (Fig 1). Equally satisfactory results with or without 10% Chelex-100 were obtained for both cards. Nevertheless, the DNA extraction protocol using 10% Chelex-100 was selected for the field evaluation because it has the advantage of allowing having a sufficient amount of DNA (75μl) for subsequent additional tests, which is not possible using directly the punched discs in the PCR mix. The Chelex DNA extraction methods from ZN smear and from the strain kept in ethanol gave also satisfactory results (Fig 1). All NTM were correctly identified by GenoType Mycobacterium CM using the 4 storage and transport systems.

Bottom Line: Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing.For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%.These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur de Madagascar, Antananarivo, Madagascar; Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.

ABSTRACT

Background: Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus.

Methods: Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing.

Results: For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%.

Conclusion: The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.

No MeSH data available.


Related in: MedlinePlus