Comparison of methods and samples used in the diagnosis of childhood PTB and characterization of mycobacterium tuberculosis isolates
Ogunbayo, Ayodeji Emmanuel
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The diagnosis of childhood pulmonary tuberculosis (PTB) remains an ongoing challenge due to the atypical clinical presentations of the disease. Bacteriological confirmation of PTB and drug susceptibility testing (DST) is imperative in an era of increasing drug resistance, but is seldom achieved in children. This is due to the challenges in obtaining adequate specimens and the low sensitivity of currently available microbiological tests owing to the paucibacillary nature of TB in children as well co-infection with human immunodeficiency virus. The difficulty in obtaining spontaneously expectorated sputum has necessitated the use of induced sputum or gastric aspirate (GA), which both requires infrastructure and technical expertise. To promote decentralisation and enhance the acceptance of routine specimen collection in children, feasible alternatives (such as stool, urine, and nasopharyngeal specimens) have been proposed. However, operational data on the performance and diagnostic yield of these specimens requires further study. Furthermore, the occurrence and transmission of Mycobacterium tuberculosis (M. tuberculosis) strain families varies by regions and has not yet been documented in children from the Free State. Moreover, since disease progression in children after primary infection mostly occurs within 12 months, genotypic analysis of isolates from children could indicate current transmission patterns of M. tuberculosis in a community. This study aimed to determine and compare the diagnostic yield of various samples [Nasopharyngeal aspirate (NPA), Nasopharyngeal swab (NPS), GA, urine and stool] and methods [smear microscopy, culture and GeneXpert® MTB/RIF (Xpert®)] used in the diagnosis of childhood PTB. Our study further characterise the TB positive isolates with regard to drug resistance using the BACTEC™ MGIT™ 960 System and Genotype® MTBDRplus, and strain diversity using spoligotyping and a 24 loci Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats typing. A total of 126 children with suspected PTB in two hospitals in Mangaung Free State, South Africa, were enrolled in the study. GA, stool, urine and NPA/NPS were collected from each patient. Four children were bacteriologically diagnosed of TB. Two children(1 and 3) were diagnosed only on urine and NPS culture respectively, child 2 on smear microscopy of urine and stool, Xpert® (stool, urine, GA) and culture (stool and urine), and child 4 on Xpert® and culture (GA, urine and stool). Of the remaining children, 18/126 (14.2%) were classified as “unconfirmed TB”, whilst 104/126 (82.5%) were classified as ‘TB unlikely”. DST revealed all the children had a susceptible strain of M. tuberculosis. Genotyping showed that child 1 had an X3 strain, child 2 and 4 had a Beijing strain, while child 3 had a T1 strain. Collectively, our results showed that culture remains the gold standard of diagnosis.. While Xpert® was more sensitive (33%) than smear microscopy (14%), its sensitivity remains suboptimal to culture detecting only 2/4 cases. The inclusion of alternative specimens was valuable as urine enabled the bacteriological confirmation of TB in 3/4 children compared to GA (2/4). While urine and NPS solely, respectively, allowed the detection of TB in children not detected by routine specimen, stool confirmed the diagnosis obtained by GA. DST result concurred across samples and patients in both assays employed. While the Beijing genotype was a predominant lineage, it was not associated with drug resistance in our study. Alternative samples outperformed the routine specimen in this study. Although a limitation of this study was the small number of bacteriologically confirmed TB cases, we would suggest at least, the inclusion of urine for routine TB diagnosis in children. However, further studies are required to validate the use of NPS specimen and evaluate other decontamination procedures that can adeqautely prevent the over growth of normal microflora without inhibiting mycobacteria in stool samples. More so, the presence of Beijing strain in 2/4 of the TB positive children raises concern, as Beijing was previously not reported as a predominant strain in the FS population.