Bacterial pathogens in Xpert MTB/RIF Ultra-negative sputum samples of patients with presumptive tuberculosis in a high TB burden setting: a 16S rRNA analysis

Abstract

In patients with presumptive tuberculosis (TB) in whom the diagnosis of TB was excluded, understanding the bacterial etiology of lower respiratory tract infections (LRTIs) is important for optimal patient management. A secondary analysis was performed on a cohort of 250 hospitalized patients with symptoms of TB. Bacterial DNA was extracted from sputum samples for Illumina 16S rRNA sequencing to identify bacterial species based on amplicon sequence variant level. The bacterial pathogen most likely to be responsible for the patients' LRTI could only be identified in a minority (6.0%, 13/215) of cases based on 16S rRNA amplicon sequencing: Mycoplasma pneumoniae (n = 7), Bordetella pertussis (n = 2), Acinetobacter baumanii (n = 2), and Pseudomonas aeruginosa (n = 2). Other putative pathogens were present in similar proportions of Xpert Ultra-positive and Xpert Ultra-negative sputum samples. The presence of Streptococcus (pseudo)pneumoniae appeared to increase the odds of radiological abnormalities (aOR 2.5, 95% CI 1.12-6.16) and the presence of S. (pseudo)pneumoniae (aOR 5.31, 95% CI 1.29-26.6) and Moraxella catarrhalis/nonliquefaciens (aOR 12.1, 95% CI 2.67-72.8) increased the odds of 6-month mortality, suggesting that these pathogens might have clinical relevance. M. pneumoniae, B. pertussis, and A. baumanii appeared to be the possible causes of TB-like symptoms. S. (pseudo)pneumoniae and M. catarrhalis/nonliquefaciens also appeared of clinical relevance based on 16S rRNA amplicon sequencing. Further research using tools with higher discriminatory power than 16S rRNA sequencing is required to develop optimal diagnostic and treatment strategies for this population.IMPORTANCEThe objective of this study was to identify possible bacterial lower respiratory tract infection (LRTI) pathogens in hospitalized patients who were initially suspected to have TB but later tested negative using the Xpert Ultra test. Although 16S rRNA was able to identify some less common or difficult-to-culture pathogens such as Mycoplasma pneumoniae and Bordetella pertussis, one of the main findings of the study is that, in contrast to what we had hypothesized, 16S rRNA is not a method that can be used to assist in the management of patients with presumptive TB having a negative Xpert Ultra test. Even though this could be considered a negative finding, we believe it is an important finding to report as it highlights the need for further research using different approaches.

Keywords: Ethiopia; LRTIs; M. tuberculosis; bacterial etiology; diagnostics; presumptive TB cases; sequencing.

Fig 1

Distribution of the bacterial genera in sputum samples identified by 16S rRNA gene amplicon sequencing in 250 Xpert MTB/RIF-negative presumptive tuberculosis cases.