Paediatric pulmonary disease-are we diagnosing it right?

Background: It has been reported that differential diagnosis of bacterial or viral pneumonia and tuberculosis (TB) in infants and young children is complex. This could be due to the difficulty in microbiological confirmation in this age group. In this study, we aimed to assess the utility of a real-time multiplex PCR for diagnosis of respiratory pathogens in children with pulmonary TB. Methods: A total of 185 respiratory samples [bronchoalveolar lavage (15), gastric aspirates (98), induced sputum (21), and sputum (51)] from children aged 3-12 years, attending tertiary care hospitals, Chennai, India, were included in the study. The samples were processed by N acetyl L cysteine (NALC) NAOH treatment and subjected to microbiological investigations for Mycobacterium tuberculosis (MTB) diagnosis that involved smear microscopy, Xpert® MTB/RIF testing, and liquid culture. In addition, DNA extraction from the processed sputum was carried out and was subjected to a multiplex real-time PCR comprising a panel of bacterial and fungal pathogens. Results: Out of the 185 samples tested, a total of 20 samples were positive for MTB by either one or more identification methods (smear, culture, and GeneXpert). Out of these 20 MTB-positive samples, 15 were positive for one or more bacterial or fungal pathogens, with different cycle threshold values. Among patients with negative MTB test results (n = 165), 145 (87%) tested positive for one or more than one bacterial or fungal pathogens. Conclusion: The results suggest that tuberculosis could coexist with other respiratory pathogens causing pneumonia. However, a large-scale prospective study from different geographical settings that uses such simultaneous detection methods for diagnosis of childhood tuberculosis and pneumonia will help in assessing the utility of these tests in rapid diagnosis of respiratory infections.

Standardization of a stool concentration method for Mycobacterium tuberculosis detection in the pediatric population.

Background: The inability of young children to expectorate sputum and paucibacillary status of Mycobacterium tuberculosis (MTB) increases its diagnostic complexity. In this study, we aimed to standardize a stool concentration method for the detection of MTB and its drug resistance by line probe assay (LPA). Methods: The stool from 10 healthy children spiked with H37Rv in five different dilutions (1:1, 1:10, 1:100, 1:1000, and 1:10,000), and stool from 10 confirmed TB and 54 clinically diagnosed TB children were subjected to an in-house stool concentration protocol. All the processed filtrates were subjected to smear microscopy, solid culture, Xpert ultra testing, and LPA. Results: Of 10 control samples, growth was seen in four samples (neat 1:1). In smear microscopy, bacilli could be seen in eight samples (1:1 and 1:10). Xpert ultra testing could detect MTB in eight samples in all dilutions with different loads. LPA could detect MTB in all samples and dilutions. In microbiologically confirmed children, seven out of 10 stool samples tested were positive. Out of 54 children with clinically diagnosed TB, 4 (7.4%) could be confirmed by microbiological diagnosis. Conclusion: The protocol standardized in this study proves to be better working in the molecular detection of MTB.

Characterization of probes associated with rifampicin resistance in M.tuberculosis detected by GenXpert from a national reference laboratory at Chennai.

With increasing use of Xpert MTB/RIF a point of care molecular test for simultaneous detection of TB and resistance to rifampicin, a growing number of rifampicin resistant cases are being detected and notified. Insights into the variation and frequencies in the probe mutations obtained through Xpert testing in the RRTB case will form the baseline information for further investigation on drug resistance. In this study we did a retrospective analysis of the GeneXpert data obtained from patient samples received at a National reference laboratory in Chennai between the years 2014 and 2020 to look at the probe distribution, the variation in the mutation and explore its significance. Probe E mutation was most commonly identified followed by Probe D, Probe A, Probe B and Probe C. Coexistence of multiple probe mutations in low bacillary load samples could be related to prolonged amplification cycle leading to delayed hybridization of probes. In such instances reporting false RR in xpert testing is possible. The probe mutations of RR should be monitored in depth with inclusion of codon specific targets for management of drug sensitive TB. In addition, heteroresistance needs to be further tested by alternative genotypic methods to avoid false resistance.