Browsing by Author "Bosch, Corne"

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    Molecular detection of Mycobacterium tuberculosis in stool of children with suspected intrathoracic tuberculosis
    (Stellenbosch : Stellenbosch University, 2018-03) Bosch, Corne; Hoek, Kim Gilberte Pauline; Walters, Elisabetta; Warren, Robin; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
    ENGLISH SUMMARY: The bacteriological confirmation of tuberculosis in children is challenging. The current diagnostic gold standard, liquid culture of respiratory specimens, has low sensitivity in paucibacillary paediatric tuberculosis, and sputum collection in young children is relatively invasive and resource-intensive. Stool is easy to collect and may contain mycobacterial deoxyribonucleic acid (DNA) from swallowed sputum. However, the performance of polymerase chain reaction (PCR) assays, including Xpert MTB/RIF and HAIN FluoroType may be affected by PCR inhibition from stool enzymes and by instrument failure due to particulate matter blocking filters. In this study, we aimed to evaluate the diagnostic performance of stool specimens using a variety of stool pre-processing steps, including decontamination and lyophilisation; as well as various DNA extraction and molecular detection protocols. This study formed part of a larger prospective study involving children with suspected intrathoracic tuberculosis where up to 6 respiratory specimens were collected. Stool specimens were collected at enrolment where one portion was tested by a direct Xpert MTB/RIF protocol; the second portion was frozen for lyophilisation and/or DNA extraction protocols followed by PCR-based molecular detection. DNA was extracted from stools using either a manual commercial stool or soil kit. Extracted DNA was tested for the presence of mycobacterial DNA using the Xpert MTB/RIF cartridge according to standard manufacturer’s protocol and/or a modified “Tube Fill” protocol; and/or the HAIN FluoroType® MTB assay. The results were compared to a composite reference standard and a secondary reference standard (first respiratory culture), which was a better reflection of true performance in our setting. Our results indicate that the standard and Tube Fill Xpert MTB/RIF protocols, as well as the FluoroType MTB detection platforms are able to detect mycobacterial DNA from stool specimens. The Xpert MTB/RIF performed directly on decontaminated stool specimens was found to have the best diagnostic accuracy with sensitivities of 45.8% - 47.1% and specificities of 97.8% - 98.2%. This method was also found to have the lowest indeterminate rate of 3.4% - 10.3%. The other protocols investigated displayed unacceptable sensitivity and specificity combinations with high rates of indeterminate results. The high indeterminate rates were concerning and further optimisation and method simplification are required to propose stool as a non-invasive specimen type for the rapid confirmation of TB in children.
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    Stool culture for diagnosis of pulmonary tuberculosis in children
    (American Society for Microbiology, 2017-12) Walters, Elisabetta; Demers, Anne-Marie; Van der Zalm, Marieke M.; Whitelaw, Andrew; Palmer, Megan; Bosch, Corne; Draper, Heather R.; Gie, Robert P.; Hesseling, Anneke C.
    Bacteriological confirmation of Mycobacterium tuberculosis is achieved in the minority of young children with tuberculosis (TB), since specimen collection is resource intensive and respiratory secretions are mostly paucibacillary, leading to limited sensitivity of available diagnostic tests. Although molecular tests are increasingly available globally, mycobacterial culture remains the gold standard for diagnosis and determination of drug susceptibility and is more sensitive than molecular methods for paucibacillary TB. We evaluated stool culture as an alternative to respiratory specimens for the diagnosis of suspected intrathoracic TB in a subgroup of 188 children (median age, 14.4 months; 15.4% HIV infected) enrolled in a TB diagnostic study at two local hospitals in Cape Town, South Africa. One stool culture was compared to overall bacteriological confirmation by stool Xpert and by Xpert and culture of multiple respiratory specimens. After decontamination/digestion with NALC (N-acetyl-l-cysteine)-NaOH (1.25%), concentrated fluorescent smear microscopy, Xpert MTB/RIF, and liquid culture were completed for all specimens. Culture contamination of stool specimens was high at 41.5%. Seven of 90 (7.8%) children initiating TB treatment were stool culture positive for M. tuberculosis. Excluding contaminated cultures, the sensitivity of stool culture versus confirmed TB was 6/25 (24.0%; 95% confidence interval [CI] = 9.4 to 45.1%). In addition, stool culture detected TB in 1/93 (1.1%) children with “unconfirmed TB.” Testing the same stool by Xpert increased sensitivity to 33.3% (95% CI = 18.0 to 51.8%). In conclusion, stool culture had low sensitivity for M. tuberculosis detection in children with intrathoracic TB. Reducing culture contamination through improved laboratory protocols may enable more reliable estimates of its diagnostic utility.