This observational prospective cross-sectional study was carried out in the Paediatric Department of UPUMS Saifai, Etawah from January 2018 to June 2019. Children of age group 3 months to 12 years who were admitted in Department of Paediatrics with clinical diagnosis of TB and fulfilling the inclusion criteria were enrolled in the study. Ethical clearance was obtained from Ethical Committee of the University (Letter no. 795/UPUMS/Dean/2019/EC No. 27/2018). Written and informed consent was taken from guardian of each patient.

Inclusion criteria: A clinically diagnosed TB case refers to a pulmonary or extra-pulmonary TB patient who has been diagnosed with active TB by a clinician on the basis of clinical findings or having clinical features suggestive of TB anywhere in the body with one or more below mentioned findings:

Exclusion criteria: Patient already diagnosed to have TB or already taking Anti-Tubercular Drugs (ATT) patient having Human Immunodeficiency Virus (HIV) infection or whose guardian did not give consent for enrolling in study were excluded from the study.

During the study period, 156 children were presented to the Department with clinical TB out of which 49 were excluded from the study and finally 107 children were studied and analysed. All the children who were included in the study were evaluated for relevant clinical history, family history and history of contact with tubercular patient, dietary history and immunisation status. The socio-demographic data obtained and clinical examination was performed on each of participants with emphasis on anthropometry, general physical examination and systemic examination.

Complete blood count with Erythrocyte Sedimentation Rate (ESR) and relevant investigations were sent to diagnose TB. ESR >15 mm in 1 hour considered as raised or high [6].

The chest radiograph was done in all patients suspected to have TB. Patients having radiological signs like hilar lymphadenopathy, effusion, miliary pattern, cavity, consolidation, calcification, fibrosis, collapse were considered suggestive of TB [7]. Mantoux test was done in all patients with 5 TU PPD RT23 with standard method. The result was interpreted after 72 hours of injection. The induration was measured in horizontal direction. Induration of 10 mm or more is considered positive and less than 10 mm considered negative [8]. Sputum/gastric aspirate specimens were collected for detection of MTB by CBNAAT from all subjects suffering from TB, diagnosed by one of the conventional methods. A single morning sputum specimen was collected. Those who could not produce sputum, 10 mL gastric aspirate sample were collected by standard procedure after at least eight hours, of fasting. CSF study and neuroimaging in the form of Contrast-Enhanced Computed Tomography (CECT) was performed only in selected subjects under clinical suspicion of TBM.

On CSF analysis, cell count of 10-500 predominantly lymphocytes in CSF with sugar <50 mg/dL and protein 100-3000 mg/dL was considered positive for TBM [9]. A 2 mL CSF was properly stored for CBNAAT analysis.

Patients having CECT head findings like basal exudates, Lepto-meningeal enhancement, hydrocephalus, cerebral infarction, tuberculoma, vasculitis, diffuse brain oedema were considered to have TBM [10].

USG abdomen was done in suspected gastrointestinal TB patients and findings like mesenteric lymph nodes >15 mm, fluid in abdomen, hepatosplenomegaly, ileocecal TB, were considered to have gastrointestinal TB [11].

CBNAAT was performed as per the manufacturer’s instructions. In brief, 1-2 mL of sample (CSF/sputum/gastric aspirate) was collected in the conical tube and sample reagent (sodium hydroxide and isopropanolol) was added in 1:2 proportion and shaken 10-20 times vigorously. A 2 mL of this mixture was transferred into cartridge. This loaded cartridge was inserted into MTB-RiF platform where sample automatically filtered and washed. Ultrasonic lysis of filter captured organism occurs to release DNA molecules mixed with dry Polymerase Chain Reaction (PCR) reagents than semi-nested real time amplification and detection in integrated reaction tube with printed result in 2 hours [12].

In the present study, the cases were enrolled on the basis of clinical suspicion of TB and further subjected to other conventional methods for the diagnosis of TB. These patients were tested for CBNAAT positivity and Rifampicin resistance. ZN staining was not performed in this study as it is time consuming, provide lesser yield and not advocated by RNTCP in children.

All the data was collected, compiled, analysed and interpreted statistically through relevant statistical methods. Software used was SPSS (version 22) for Windows. A p-value of <0.05 considered statistically significant.

One hundred and seven children, between 3 months to 12 years, with clinical diagnosis of TB were investigated, out of which 59 (55.1%) were male. The mean age was 6.55±3.93 years. Out of 107 enrolled patients, 29 (27.1%) had pulmonary TB and 78 (72.9%) had extra-pulmonary TB. Only 35 (32.71%) patients had history of contact with TB patient. Various conventional investigations done to diagnose TB and their results are given in [Table/Fig-1].

Total 107 sputum/gastric aspirate and 69 CSF samples were collected for CBNAAT analysis. CBNAAT was able to detect MTB in 15 (14%) of the gastric aspirate/ sputum sample and in 9 (13.04%) cases of CSF sample. In 5 cases, MTB was detected in both CSF as well as in gastric aspirate/sputum sample. Among the 19 CBNAAT positive cases none found rifampicin resistant.

Various conventional diagnostic methods done for the diagnosis of TB were compared with CBNAAT positivity and observations are given in [Table/Fig-2]. CBNAAT positivity showed a significant association with positive Mantoux test and CSF analysis suggestive of TBM.

In this study, the performance of CBNAAT in sputum/gastric aspirate and CSF of clinically diagnosed TB cases was evaluated. The MTB was detected in 19 (17.8%) patients.

Das PK et al., reported, 3% to 5% quarter-to-quarter period CBNAAT positivity in the paediatric age group and prevalence of MTB detection was reported to be 3.88% [13]. One study from Institute of Medical Sciences, India, reported 11.1% CBNAAT positivity in children [14]. This might be due to selection of patients on the clinical basis only. Kasat S et al., found 15% positivity of CBNAAT in adult extra pulmonary TB cases, which is comparable to this study [15]. Khorgade RR and Bhise PR found significantly high (42.56%) CBNAAT positivity in pulmonary TB adult cases [16]. The high positivity of CBNAAT can be explained by the selection of adult pulmonary TB cases.

CXR was suggestive of TB in 47.7% of all cases while it was suggestive in 52.6% of CBNAAT positive cases. There was no association between CBNAAT positivity and chest CXR finding. Swaminathan S et al., found normal chest radiographs in more than half of the children with confirmed TB [17]. Pepper T et al., also reported high rate of normal CXR among persons with culture-confirmed pulmonary TB [18]. Chest radiography is a relatively insensitive tool apart from its known limitations of poor specificity and high inter-observer variability [17].

In this study, Mantoux test was suggestive of TB in 69.2% patients. A significant association was found between positive Mantoux test and CBNAAT positivity (p=0.02). Mantoux test serves as an indirect evidence for diagnosing paediatric TB, its positivity in 69.2% cases highlighting the relevance of this cheap, easily available and simple test in diagnosis of TB. Positivity was very high when compared to a study done in the Institute of child health, Chennai which reported Mantoux positivity of 34.7% in various forms of paediatric TB [19]. Vijayasekaran D et al., found that the positivity of Mantoux was highest in lymph node TB (53%) and lowest in CNS TB (21.2%) [20].

ESR was raised in 76.6% of patients but there was no association between raised ESR and CBNAAT. An elevated ESR was observed by Al-Marri MR and Kirkpatrick MB in children with TB, but they found that one-third of children with TB had a normal ESR at the time of diagnosis, and consequently there would be little value in using ESR as a diagnostic test for childhood TB [21].

CECT was advised in 34 cases and was suggestive of TBM in 29 (85.3%) patients, which is comparable to Ozates M et al., who reported abnormal CT head findings in 88% cases of TBM [10]. The advent of CT has provided insight into disease progression, and gives prognostic and diagnostic information. As CECT findings are considerably high in cases of TBM in children, few authors recommend CECT should be part of the initial work-up in every child suspected of having central nervous system TB [22].

CSF cytological and biochemical analysis was suggestive of TBM in 29 (42.0%) cases of the 69 clinically diagnosed TBM patients. Out of these 29 patient, 10 (34.5%) were confirmed by CBNAAT. Similar to a previous study done by Mittal M et al., found 34% of the TBM patient had positive CBNAAT in CSF [23]. This study shows significant association between CSF analysis suggestive of TBM and CBNAAT positivity.

In the present study, Rifampicin resistance was not detected in any case by CBNAAT. Das PK et al., reported point and periodic prevalence of Rifampicin resistant among the children vary from 0.2% to 0.5% [13]. The possible reason may be less incidence of drug resistance in children or the small sample size.

The limitation of the study was small sample size. If the study is extended to a wider sample size or is conducted as a multi-centric study, it may give better results. This study was done in rural area of central UP. There can be difference in prevalence of TB in urban population. So, results of this study cannot be projected in urban population.

In the present study, CBNAAT positivity showed significant association with positive Mantoux test and CSF analysis suggestive of TBM. Drug resistance for the Rifampicin was also evaluated. Use of CBNAAT like newer diagnostic modalities, will help in early diagnosis and management of paediatric TB.