Diagnostic accuracy of CBNAAT (Gene-Xpert) Vs liquid culture in clinically diagnosed presumptive childhood tuberculosis admitted in Pt. J.N.M. Medical College &

Background: Paediatric tuberculosis comprises 10% of all tuberculosis in developing countries. Smear microscopy is diagnostic for Pulmonary TB whereas for extra-pulmonary TB, CBNAAT (Gene-Xpert) and liquid-culture are available choices. Objectives: To establish the comparative diagnostic efficacy of CBNAAT and Liquid-culture in clinically presumptive childhood Tuberculosis. Outcome: Assess sensitivity and specificity of CBNAAT Vs Liquid-culture in clinically diagnosed presumptive childhood tuberculosis. Material & Method: After approval from institutional ethical committee this Cross-sectional study was performed in department of paediatrics Pt. J. N. M. Medical College, Raipur C.G. Total 97 paediatric presumptive TB cases were enrolled for systematic screening from June-2017 to July-2018. Samples were collected and analysed by CBNAAT and liquid-culture. Data for Diagnostic efficacy of both techniques were analysed with appropriate statistical method. Results: Among study subject group 41.2% belongs to 11-15 age-group and 53.6% were female. Majority of them belongs to joint family (81.4%) and low-socioeconomic (85.6%) status. Among 97 presumptive cases CBNAAT was positive in 24.7% whereas liquid-culture was positive in 22.7%. CBNNAT has sensitivity (100%) and specificity (98.61%) when compared with liquid-culture as gold-standard. It has 95.65% positive and 100% negative predictive values. CBNAAT had 100% of sensitivity and specificity in diagnosis of abdominal-tuberculosis and TB-meningitis. In Pulmonary-TB diagnosis its specificity reduced to 98%. Conclusion: CBNAAT is equally efficacious diagnostic tool to diagnose clinically presumptive TB cases and it should be used in public health system for early diagnosis of paediatric tuberculosis. Chest X- ray, CT scan and Ultrasonography observations : The subjects were studied for their X- ray chest observations. It was found that maximum, i.e. 47 (48.5%) subjects showed no abnormality. This was followed by pleural effusion (25.8%) and consolidation (13.40%). CT head findings in the study subjects were assessed. CT was done in 15 cases out of that 9 (60%) subjects had normal CT scan results. While 2 (13.3%) subjects were found to have basal exudates, communicating hydrocephalus. ultrasound of chest and abdomen were done in 49 (50.5%) subjects. 26 (26.8%) subjects showed effusion. No abnormality was seen in 19 (19.6%) subjects while, consolidation and ascitis were seen in 2 (2.1%) and 1 (1%) case respectively. 16.49%


Introduction
Tuberculosis (TB) is an infectious bacterial disease caused by Mycobacterium tuberculosis (M. TB). Worldwide, TB is a major health problem across the world. India is one of the high TB burden countries. As per the Global TB report 2017 the estimated incidence of TB in India was approximately 28,00,000 accounting for about a quarter of the world's TB cases [1]. The actual burden of paediatric tuberculosis is not known, it is assumed that about 10% of total TB load is found in children. Globally one million cases of paediatric TB are estimated to occur every year, with more than 100,000 deaths. Childhood deaths from TB are usually caused by meningitis or disseminated disease. Mycobacterium tuberculosis remains to be one of the most significant causes of death from an infectious agent. Poor case ascertainment and limited surveillance data hamper the efforts to accurately quantify the disease burden associated with childhood TB [2].
Childhood TB is usually acquired from an infectious adult contact. High rates of transmission are sustained in TB-endemic areas due to high case density and prolonged diagnostic delay [3].As childhood TB reflects

Original Research Article
Pediatric Review: International Journal of Pediatric Research Available online at: www.medresearch.in 468|P a g e ongoing transmission, children are affected most acutely in areas where an adult TB epidemic is poorly controlled [4]. The global TB control strategy has focused predominantly on smear-positive cases and, therefore, not on childhood TB, which is usually paucibacillary and smear negative [3]. In addition, childhood TB remains neglected for various reasons, mainly the difficulty in diagnosing pulmonary TB, the lack of scientific studies on childhood TB, the largely unknown outcomes of children with TB, and the belief that childhood TB is not important for TB control [3,5].
Xpert assay is not widely recognized as a diagnostic test for TB in India specifically in context of childhood tuberculosis. Further there is paucity of data on such diagnostic sensitivity and specificity of CB-NAAT (gene-Expert) in paediatric population in Chhattisgarh state. Currently WHO approved use of CBNAAT (MTB/RIF) diagnosing pulmonary and extra-pulmonary tuberculosis to simultaneously detect M. tuberculosis and rifampicin resistance mutations in the gene in 2011.CBNAAT can provide the results within 2 hours [6]. Xpert MTB/RIF assay is an automated CBNAAT.
In paediatric population many studies have demonstrated that Xpert assay is highly sensitive and specific in diagnosing for both pulmonary and extrapulmonary TB [7,8,9,10]. Hence this study is aimed to establish the diagnostic accuracy of clinically diagnosed presumptive childhood tuberculosis of CBNAAT versus liquid culture. Sample size: Facility based time bound study so all children above mentioned study duration will included in the study. Total 97 children were enrolled for this study.

Material and Methods
Method of sample collection, procedure and transport: Under all aseptic precaution, samples Pleural fluid, cerebrospinal fluid, gastric aspirate, ascetic fluid, pus aspirate from lymph node or cold abscess) were collected as per standard protocol in sterile container and sent for CBNAAT and liquid culture analysis.
The sample was divided equally into two parts, one part to be used for the CBNAAT and second for liquid culture (MGIT).
Complete blood count, Erythrocyte sedimentation rate, PPD, AFB, Chest x-ray and Ultrasonography and CT scan investigation were done to aid diagnosis along with clinical diagnosed presumptive tuberculosis.
Inclusion criteria: All suspected case of tuberculosis cases were included in this study after screening by various clinical and conventional methods including pleural effusion, cold abscess or lymphadenopathy, ascites and sinusitis, lymph node TB, Cold Abscess, TBM and Spinal TB, Genital TB, TB Skin.
Ethical issues: approval taken from institutional ethical committee prior to study.

Data collection tools & techniques:
Data collection is divided in to three parts i.e. Interview schedule, sampling of specimen and clinical examination.
Data entry was done in Excel and analysis was done using SPSS 20.0 Wherever, possible percentage, Chisquare tests were applied.
Outcome and variables: Sensitivity, Specificity, Positive predictive value (PPV and Negative predictive value (NPV).

Original Research Article
Pediatric Review: International Journal of Pediatric Research Available online at: www.medresearch.in 469|P a g e History of tuberculosis contact & BCG immunization status in the study subjects: It was seen that out of 97 cases 68 (70.1%) cases had history of contact with tuberculosis. Immunization status in the study subjects was studied. It was found that all the subjects were immunised.
Observation of biochemical parameters: Table 1-showing mean PPD was found to be 12.65± 2.35. Mean ESR was observed to 33.82± 23.84. 29 presumptive cases had PPD more than 10mm which is suggestive of TB infection and 41 study subjects had ESR more than 25mm/hour. In subjects, minimum and maximum WBC was found to be 1100/cumm and 18000/cumm respectively with mean 9260± 5100. Mean Hb was noted to be 12.09± 2.31 gm%.  Chest X-ray, CT scan and Ultrasonography observations: The subjects were studied for their X-ray chest observations. It was found that maximum, i.e. 47 (48.5%) subjects showed no abnormality. This was followed by pleural effusion (25.8%) and consolidation (13.40%). CT head findings in the study subjects were assessed.
CT was done in 15 cases out of that 9 (60%) subjects had normal CT scan results.    (Table 4). The impact of BCG vaccination on transmission of M. tuberculosis is therefore limited [12,13]. In our study, we found that children with BCG immunization had also developed TB meningitis, which was similar to the study by Hesseling which also documented that only 86% of TB meningitis developed protection following immunization.
Out of 23 CBNNAT positive cases in our study, 18 were having active contact history. Cases of TB patients are a high-risk group for developing TB, particularly within the first year and children <5 years of age. A study by Fox GJ at el (2012 WHO consulted groupfound that Contacts of TB patients are a high-risk group for developing TB, particularly within the first year and children <5 years of age. Policy recommendations must consider evidence of the cost-effectiveness of various contact tracing strategies, and also incorporate complementary strategies to enhance case finding [14]. In our study, the history of contact with TB cases were higher in children <5years of age which was similar to other studies.
In present study Biochemical parameters PPD and ESR were observed in presumptive cases. In the study subjects, mean PPD was found to be 167. In present study various fluids were also compared with CBNAAT and Liquid culture testing. Type of fluid compared with AFB, CBNNAT and Liquid Culture shows that out of 24 CBNAAT cases 7 were sputum sample and 1 was AFB positive. Among 97 cases 25 were gastric aspirate sample out of that 4 CBNAAT and 3 Liquid cultures positive. And in same 4 gastric aspirate samples both CBNAAT and liquid culture were positive and in 1 no growth seen.
In rest of samples like Sputum, Ascitic fluid, pleural fluid and CSF, same number of CBNAAT positive and samples has seen culture growth. Diagnostic value of Gene-Xpert is significantly high as compared to Ziehl-Neelsen smear microscopy and a useful tool in early diagnosis of tuberculosis [17].
The overall sensitivity, specificity, PPV and NPV of Gene-Xpert were 86.8%, 93.1%, 78.5% and 96% respectively. Gene-Xpert has a higher sensitivity than AFB smear microscopy in respiratory samples [18]. The overall positive rate of Xpert among sputum samples was significantly higher than that of liquid culture [19].
Both the studies show similar results of increased sensitivity of CBNAAT in comparison to AFB & Liquid culture, probably due to the minimal amount of sample required for CBNAAT.
Also respiratory samples for CBNAAT are more sensitive compared to other samples.  [21].

Original Research Article
Limitation: First, one of the important strengths of the Xpert assay is its ability to detect the presence of Rifampicin resistance. The sensitivity and specificity of MTB/RIF assay to detect Rifampicin resistance in our study was not evaluated and not included in our objective as we didn't get the requisition for Rifampicin sensitivity by phenotypic method in all the positive samples. Second, as number of samples present in this study is less, further studies with a greater number of samples need to be done.

Conclusion
In this study CBNAAT shows significant association in diagnosis with other diagnostic modality i.e. liquid culture for pulmonary and extra-pulmonary TB.
Gene expert has overall sensitivity of 100 % and specificity of 98.6%. CBNAAT has highest diagnostic significance (100% sensitivity and specificity) in tubercular meningitis as compare to pulmonary tuberculosis with 100% sensitivity and 98% specificity. From present study it is concluded that in highly clinically presumptive TB cases.
What this adds to existing knowledge?
The present study will provide vital inputs on applicability of gene expert as diagnostic test used for early diagnosis of paediatric age tuberculosis and help us to allocate role of CBNAAT as a screening tool, confirmatory test or supplementary test.
The yield of CBNAAT is 23.7%. Thus, clinical evaluation cannot be excluded to treat tuberculosis patients in poor health care settings where CBNNAT and liquid culture is not available.

Author contributions
Dr. Sharja Phuljhele: Conceptualized, designed and analyzed the study.
Dr. Anil Kumar Saroj: Conducted data-collection and help in analysis and manuscript writing.
Funding: Nil, Conflict of interest: None initiated, Permission from IRB: Yes