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Tuberculosis is a major public health issue in Yemen, a country located at the southwestern tip of the Arabian Peninsula, while the situation of tuberculosis had been further exacerbated since the war started in 2015. The objective of this study is to investigate the incidence of tuberculosis in Yemen before the outbreak of COVID-19, from 2006 to 2018. During the 13-year period, 92 482 patients were enrolled in the TB program records from the 22 governorates. Almost equal number of cases were diagnosed between males and females (a male to female ratio, 1.03: 1). A notable rising incidence was observed in all age groups starting from 2011. The sharpest increase occurred in children under age 15, rising by 8.0 folds from 0.5 in the period 2006-2010 to 4.1 in the period 2011-2018. Pediatric TB accounted for 9.6% of all reported cases. In terms of the patient residence, incidence has more than doubled in Sana'a city, Sana'a Gov., Hajjah and Saadah. Concomitant diseases with tuberculosis included diabetes mellitus (14.0%), brucellosis (6.1%), hepatitis (6.0%), rheumatoid arthritis (4.3%), renal disorders (2.5%), and HIV infection (2.5%). Development of interventions to reduce tuberculosis incidence in children and concomitant communicable diseases are urgently needed.

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Background Normalization of cell-free RNA (cf-RNA) concentration can be affected by variable experimental conditions and thus impact the performance of their diagnostic potential. Our study aimed to identify appropriate endogenous reference genes for cf-RNA biomarker evaluation in the diagnosis of tuberculosis (TB). Methods Subjects consisting of patients with TB with and without malignancy, patients with pneumonia, and healthy controls were recruited. Candidate reference genes were screened and identified by literature reviewing and RNA-Seq analysis. Expression levels of the candidate genes were determined by reverse-transcription real-time quantitative polymerase chain reaction in plasma from patients with TB and healthy controls. The stability of gene expression was assessed by geNorm, NormFinder, BestKeeper, the Comparative Delta Ct method, and RefFinder. Differential expression of 2 small RNAs (sRNAs) encoding by genome of Mycobacterium tuberculosis in plasma of patients with TB were determined by both absolute quantification and relative quantification with candidate reference genes. Results According to the stability ranking analyzed with the 5 computational programs, the top 4 candidates—miR-93, RNU44, miR-16, and glyceraldehyde 3-phosphate dehydrogenase—were used to normalize the transcript levels of 2 mycobacterial sRNAs, MTS2823 and MTS1338, which were observed to have higher copy numbers in the plasma of patients with TB. Normalization with RNU44 displayed significantly higher levels of the 2 M tuberculosis sRNAs in the patients’ plasma than those of healthy controls. Conclusions RNU44 was demonstrated as a proper endogenous gene for cf-RNA normalization in TB diagnosis.