Molecular detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary samples in a hospital-based study

Objective Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a deadly infectious disease. India contributes to one-third of the global TB burden. However, no studies have been carried out in the Telangana (Hyderabad) population using real-time polymerase chain reaction (RT-PCR). Therefore, the current study evaluated the role of RT-PCR as a rapid and non-invasive test to diagnose TB by testing for pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB). Materials and methods This hospital-based study examined 1670 samples (900 EPTB; 770 PTB) comprising tissue (n = 537), peritoneal fluid (n = 420), sputum (n = 166), bronchial fluid (n = 126), cerebrospinal fluid (n = 145), ascetic fluid (n = 76), sputum pus (n = 78), urine (n = 79), and bronchoalveolar fluid (n = 43) samples. DNA from samples was separated using specific isolation kits and subjected to RT-PCR. Results In this study, we enrolled 1670 subjects and categorized 54.4% as females and 45.6% as males. The collected samples were categorized as 48.5% of fluid samples, followed by tissue (32.2%), sputum (9.9%), urine (4.7%), and pus-swab (4.6%). RT-PCR analysis revealed that 4.7% patients were positive for Mtb. Our results revealed that 61% of the affected patients were male and 39% were female. Among the specimen types, tissue samples gave the highest proportion of positive results (36.3%). Conclusion The results showed that RT-PCR should be implemented and given top priority in TB diagnosis to save time and facilitate a definitive diagnosis. Tissue samples are highly recommended to screen the Mtb through the technique RTPCR. Future studies should extend the technique to the global population and exome sequencing analysis should be performed to identify TB risk markers.


Introduction
Tuberculosis (TB), caused by acid-fast aerobic bacteria comprising the Mycobacterium tuberculosis (Mtb) complex, is a contagious infection that generally affects the lungs. TB is a communicable infectious disease that is transmitted through cough aerosols, and is characterized pathologically by necrotizing granulomatous inflammation, usually in the lungs 1 . In 2015,10.4 million new active TB cases were registered according to the World Health Organization (WHO) and 1.8 million deaths were documented 2 . TB has remained one of the major causes of morbidity and mortality in lowto-middle income countries 3 . The risk factors for TB are: patients infected with HIV, diabetes, alcoholism, leukemia and patients who receive immunosuppressive drugs 4,5 . TB is usually diagnosed through clinical findings of chest radiography and microbiological tests. Smear microscopy of either sputum or tissue, and microbial cultures are used to confirm TB 6 . Ongoing treatment for drug-susceptible TB comprises a fourdrug regimen of isoniazid, rifampin, pyrazinamide, and ethambutol or streptomycin, which are administered for 6 months. Currently, new pharmaceutical agents are urgently required to control TB because of the increasing incidence of drug-resistant TB, multidrug-resistant TB (MDR-TB), and extensively drug-resistant TB (XDR-TB), which pose a major public health problem worldwide 7 . Pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) are the two clinical manifestations of TB. The involvement of TB other than lungs is termed as EPTB. Any patient affeted with the combination of pulmonary and EPTB is defined as PTB 8 . In cases of suspected EPTB, rapid and accurate laboratory diagnosis is important, because traditional techniques of detecting acid-fast bacilli have limitations. During the last decade, remarkable progress has been made in the diagnostics of PTB. However, diagnostic challenges in EPTB remain to be addressed. A more accurate test to diagnose various forms of EPTB, which can easily be incorporated into the routine TB control program, would contribute significantly towards improving EPTB case-detection 9 . EPTB constitutes about 15-20% of TB cases 10 . The common cause of tuberculosis between human and animals is a group of mycobacterial species that form the Mtb complex 11 . TB is a chronic disease and has long-lasting effects on the human body, with complications that are less common but life threatening 12 . Molecular techniques have proven very successful as diagnostic tools. However, these molecular typing techniques target particularly polymorphic genetic sequences, but interrogate less than 1% of the genome; therefore, they have an intrinsically restricted discriminatory power. This limitation could be overcome by the application of next-generation whole-genome sequencing (WGS) for genome-based epidemiology. Various platforms for WGS have been developed in the last decade 13 . Therefore, the current study aimed to evaluate the role of real-time polymerase chain reaction (RT-PCR) to test PTB and EPTB specimens as a rapid and non-invasive test for the fast diagnosis of TB.

Subjects
This was a hospital-based study carried out in Hyderabad, the capital city of Telangana, India. In this study we selected 1670 cases, of whom 770 were PTB and 900 were EPTB. The patients were recruited after an ethical grant (AHJ-114/10-18) from the Apollo Research committee to the hospital. The samples included tissue (n = 537), peritoneal fluid (n = 420), sputum (n = 166), bronchial fluid (n = 126), cerebrospinal fluid (n = 145), ascetic fluid (n = 76), sputum pus (n = 78), urine (n = 79), and bronchoalveolar fluid (n = 43) samples. The patients were recruited from January 2012 to March 2017. All subjects gave their consent to participate in the study. Sample processing differed according to the specimen type. Non-sterile samples were subjected to standard N-acetyl cysteine sodium hydroxide decontamination. The average duration of symptoms was 113 days (range, 35-320 days). Symptoms like uncontrolled cough for a minimum of 2 weeks with or without chronic fever or weight loss were evaluated for TB. The inclusion criteria of this study were: (i) suspected active TB; (ii) an age range of 16-60 years of age; (iii) no previous history of anti-TB treatment; and (iv) a negative status for HIV. The exclusion criteria were: (i) noinalized diagnosis after examination and treatment; (ii) previous identification of TB; and (iii) previous treatment for TB 14 . Based on the typical TB clinical symptoms, all the patients were screened by bacterial culture and imaging examination. The acid-fast bacilli (AFB) culture results of these cases were positive, but some of the patients showed the negative TB clinical features. The patients had clinical and radiological features suggestive of TB. The AFB staining procedure was performed using N-acetyl-l-cysteine-NaOH, as described in an earlier publication 15 . The formalin-fixed paraffin-embedded (FFPE) human specimens were examined after Ziehl-Neelsen (ZN) staining, which was performed according to standard protocols 16 . All the slides stained with AFB were reconfirmed microscopically by two independent pathologists who were unaware of each patient's clinical details, as per the National Accreditation Board for Testing and Calibration Laboratories (NABL) protocol.

DNA extraction
DNA was isolated from all the different types of samples using a QIAamp DNA mini kit (QIAGEN, Germany) according to the manufacturer's recommendations. For the FFPE specimens, 3-μm thick sections were cut from each paraffin block; from the resected specimens, three sections were taken; and from the biopsy specimen, five or more sections were taken. For the FFPE specimens, DNA was extracted using a QIAamp FFPE tissue kit (QIAGEN, Germany) and subsequently analyzed by PCR. The quantification of Mtb DNA was performed using an RG PCR kit, based on the manufacturer's instructions (QIAGEN, Germany). This kit detects specific variants appearing in the Mtb complex, including M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, M. microti, and M. pinnipedii, using a single primer. Using Rotor-Gene Q with 5-plex HRM (Qiagen), RT-PCR was performed in a total volume of 25 µl, comprising 15 µl of master-mix, 7 µl of double distilled water, and 3 µl of template DNA. The PCR conditions were as follows: 2 min at 95 °C; followed by 45 cycles of 15 sec at 95 °C, 30 sec at 64 °C, and 20 sec at 72 °C. Standard curves from four quantification standards were used to calculate the amount of Mtb DNA (copies/μl).

Results
In this hospital-based study, 1670 patients were recruited, of whom 45.6% were male and 54.4% were female. The mean age of the 1670 subjects was 41.36 ± 17.76 years; amongst, males were 47.87±18.39 years old and females were 36.28 ± 15.46 years' old ( Table  1). The total samples were categorized as 53.2% were fluid samples, followed by tissue (32.2%), sputum (9.9%), and pus-swab (4.6%) ( Table 1).The total number of fluids used in this study are 48.5% (n=810) and amongst them fluids are sub-categorized as follows; peripheral fluid (n=420; 25.2%), bronchial fluid (n=126; 7.5%), cerebrospinal fluid (n=145; 8.7%), ascetic fluid (n=76; 4.6%), bronchoalveolar fluid (n=43; 2.6%) and urine fluid (n=79; 4.7%). In this study, only 4.6% of the samples were found to be Mtb-positive by RT-PCR.   WGS is the process of reading the complete DNA sequence of an organism (i.e., screening of genetic material). In Mtb, the WGS means decoding the exact nucleotide sequence from bacterial genome 36 . Next-generation whole genome sequencing (NG-WGS) of TB has been implemented earlier but its application in the clinical practice is still very limited. The WHO has been recognized NG-WGS has great potentiality for diagnosing drug-resistant TB in diverse clinical settings. NG-WGS allows the researchers to generate millions of genome-sequences in a short-time. This technique in the diagnosis of Tb identifies the accurate type of Mtb affecting the patient which involves the drug resistance profile which allows clinicians to recognize the suitable treatment regimen to combat the disease 37 .
The strength of the current study was that it analyzed a relatively large number (1670) of cases with symptoms of TB, and diagnosis was carried out using RT-PCR. A limitation of our study was that we did not take into account the microbiological results, TB culture result, and patients' clinical details.

Conclusion
The study results showed RT-PCR was used to screen the PTB and EPTB patients and our study has showed 4.7% were positive for Mtb. Our study also confirms RT-PCR has an ability and latent to increase the rapid diagnosis of PTB and EPTB. Future studies with RT-PCR should be implemented and given top priority for a faster initial diagnosis of TB so that patients requiring further diagnostic tests may be identified. Tissue samples are highly recommended to screen the Mtb through the technique RT-PCR. We recommend RT-PCR, molecular techniques should be implemented in the global population, and future studies should perform second generation sequencing analysis to identify risk markers for TB.