CBNAAT: Advantage and Efficacy in Pulmonary Tuberculosis (PTB) atop on Traditional Methods for Diagnosis in a Tertiary Care Hospital in India

Background: Mycobacterium Tuberculosis (MTB) is one of the most ancient diseases of mankind. Pulmonary tuberculosis (PTB) is the most common, despite the diagnosis and treatment of TB. Many studies reported, a collaboration between PTB susceptibility. In our research study, we report meantime findings after enrolling 732 of a planned 212 participants. Study Design: A descriptive cross-sectional study. Methods: The study conducted on patients with TB in west India was conducted in the Department of Microbiology, Index Medical College; Indore Madhya Pradesh. Patients suspected of PTB were qualified for screening if their age varied from 25 to 60 years and with both gender, signs and symptoms associated with PTB such as cough for more than 2 weeks, fever, weight loss, chest pain, and abnormal chest X-ray findings in results and cartridge-based nucleic acid amplification test (CBNAAT) positive. All Patients were monitored monthly while they visited in TB and chest clinic for TB treatment. Results: A total of 937 patients were selected for the study. Out of which only 732 patients were enrolled. About 212 patents were positive for CBNAAT and 520 were found negative. The confirmed positive CBNAAT patients do not have a history of tuberculosis. In this study about 21.72% were ZN stain positive, 33.46% were culture positive and 28.96% were CBNAAT positive. Conclusion: The current scenario of traditionally AFB-negative PTB is not sensitive enough to establish the diagnosis of active tuberculosis without CBNAAT. They underdiagnose PTB and overtreat people without PTB.


INTRODUCTION
"Mycobacterium Tuberculosis (MTB) only the most prehistoric diseases of society, is one of the leading causes of mortality from a single infectious medium" [1,2]. The emergence of increasingly drug-resistant forms of tuberculosis (TB) is a considerable challenge to current and future TB prevention and care efforts. Despite recent progress in addressing the epidemic, TB persists as one of the major causes of mortality globally with an estimated 10 "MDR-TB, which results from inconsistent as well as incorrect TB treatment or direct person-to-person transmission, poses a global threat to tuberculosis control" [8]. "MDR-TB is far deadlier than drug-susceptible TB, and current treatments are expensive, time-consuming, and frequently cause severe side effects" [9]. "Patients face many financial, biological, and systemic barriers, psychosocial to treatment compliance, which constantly steer to poor results and elaboration of drug resistance" [10,11,12].
"The National Strategic Plan (2017-25) of India suggests a strong master plan with equivalent resources to rapidly diminish TB in the country by 2030. This is in pipeline with the worldwide end TB targets and defensive development goals to achieve the innovation of a TB-free India. The goal is to attain a quick diminish in the burden of TB, morbidity, and mortality while working towards the exclusion of TB in India by 2025" [13]. "Whereas WHO with its "STOP TB" strategy has given the vision to eliminate TB as a public health problem from the face of this earth by 2050" [14].
"Health-related quality of life during MDR TB treatment traditionally, the goal of tuberculosis treatment has been microbiological cure, with less emphasis on morbidity and patient-reported outcomes such as quality of life (QOL). Healthrelated quality of life is a multidimensional concept that emphasises the patient's point of view and defines health as physical, mental, and social well-being rather than simply the absence of illness" [15].
Patient quality of life and treatment retention are important factors in treatment success, and failure to follow-up represents an opportunity for intervention. Some socioeconomic factors, such as a lack of education, a low income, alcohol abuse, joblessness, and a lack of health insurance, are also linked to the failure of MDR-TB treatment [16,17].
Without proper and fact-based studies that impact the victory of MDR-TB, studies that assess the merger of extended authority have not been smoothly directed in countries with a high TB burden [18]. Hence in this research study, we sight to pinpoint the elements, particularly connected with favorable outcome treatment in high-burden MDR-TB settings in India.

MATERIALS AND METHODS
The study was conducted in the Department of Microbiology at Index Medical College Hospital and Research Centre, Indore, Madhya Pradesh (M.P.).

Sample Size
A total number of 732 samples were collected which includes sputum, bronchoalveolar lavage (BAL), and gastric aspirate. After dividing the patient base into pre-diabetic and diabetic groups, the sample size was chosen.

Study Duration
Two years (July 2019 -July 2021) including 2 years of data analysis.

Study Population
Patients visited the TB & chest clinic and were diagnosed with PTB.

Inclusion Criterion
Patients were qualified for screening if their age varied from 25 to 60 years and with both genders, signs and symptoms associated with PTB. Symptoms such as cough of more than 2 weeks, fever, weight loss, chest pain, and abnormal chest X-ray findings (patchy consolidation, poorly-defined nodules) in results and CBNAAT positive.

Exclusion Criterion
Patients were disqualified for screening if they were below 25 years and more than 60 years of age. Patients with a pre-diabetic history were also excluded from the study because their levels were not high enough to be classified as Type 2 diabetes.

Specimen Collection (PTB)
Two consecutive morning speck sputum samples will be collected from suspected PTB cases in a sterile, wide-mouthed, triple layer-leak proof plastic container according to RNTCP protocol. All patients were instructed to cough vigorously in order to produce sputum specimens that could be collected without contaminating the sample collection container. If a patient is unable to produce sputum, such as a child or the elderly, gastric aspirate and bronchoalveolar lavage fluid will be accepted for further processing.

Transport
The specimens were transported from the concerned departments to the central laboratory by maintaining a cold chain with triple-layer packaging.

I.
All specimens were processed by taking aseptic precautions and personal protective equipment (PPE) properly in the BSL-II laboratory. II.
Visually, the grade of the sputum specimen will be judged for consistency and if it carries more saliva then the specimen will be rejected and asked for a new specimen.

Smear Microscopy
All smears were prepared directly from the specimens and stained with Z-N staining. Specimens with two negative smears were documented. These patients engaged in conversation.
Research Study Performa was being filled up for those with clinicalradiological suspicions of PTB & who are willing to agree to participate in the research study. The enrolled patient's specimen was further processed. A minimum of 100 fields for acid-fast bacilli in a smear indicated the severity of PTB infection in patients. In grading, positive AFB smears were reported. as shown in

Procedure
One MCT tube pallet was cultured into Middlebrook7H9 broth. One smear will be checked by Z-N staining. The result will be recorded. Middlebrook7H9 broth supplemented with 0.8 ml OADC and PANTA will be used for liquid culture. It will be prepared as per the manufacturer's instruction Hi Media (Hi Media Pvt Ltd, Mumbai, India). 0.5 ml of the processed sample will be inoculated and tubes will be incubated at 37 +/-1°C. Readings will be taken visually twice weekly for up to 6 weeks. Positive culture with a granular appearance without significant turbidity will be noted. If growth is observed, Z-N staining will be done to confirm the presence of AFB.

CBNAAT
It is a novel rapid automated machine for the rapid diagnosis of TB. This is the cartridge-based nucleic acid amplification test (CBNAAT) that can detect TB within 2 h of the collection along with RIF's resistance directly from the pulmonary samples. Detection based on the target sequences and nucleic acid amplification by RT-PCR and reverse transcriptase. In a conical tube containing 1ml of a sample (Sputum, BAL, and gastric aspirate), 2 ml of sample reagent was added and mixed vigorously. This mixture was incubated at room temperature for 10 to 15 minutes and the treated sample was transferred into the sample cartridge chamber by using a sterile graduated or ungraduated pipette and then cartridge loaded into the GeneXpert machine. Result Interpretation is done by using GeneXpert Dx System software, which measured fluorescent signals algorithm [16].

RESULTS
A total of 937 patients were registered in the TB and chest clinic which were as have a suspicion of TB but at most 732 patients were enrolled based on age criterion and out of 732 only 212 were entitled and found verifiable positive in our research study after confirmed through CBNAAT as shown in table-2. The rest (520 patients) were found negative. In the number of 732 samples which were suspected of MTB, 212 (28.96%) samples were confirmed positive for MTB by CBNAAT (GeneXpert) comparatively with smear and culture as summarized in Table 2.
Out of 212 positive TB cases, most of the patient do not have a history of tuberculosis but positivity were high as compared to patients with a family history of TB. Suspected male patients also show a high positivity rate as compared to suspected female patients with include alcohol consumption and smoking as shown in the socio-economic demographic Table 3.
Among this, the distribution of clinical samples was (546/74.53% sputum, 140/19.18% gastric aspirate, and 46/6.27% BAL) as shown in Table  4. Clinical data at the time of demonstration of patient enrollment and radiological peculiarity found in chest X-rays of positive CBNAAT cases are outlined in Table 5.

DISCUSSION
In this research study, we have evaluated the role of GeneXpert over regular methods for MTB and Rifampicin-resistant detection in pulmonary specimens (sputum, gastric aspirate, and BAL) since PT is the foremost cause of mortality and morbidity in India. In our research study, "MTB was generally high in urban communities in comparison with the rural community, and that is similar to the study in Madurai, India in 2015 and Madhya Pradesh in 2016"; [20,21,22] [21,23]. In our research study, "73% of patients who were newly exposed to PTB in the number of all positive cases for MTB", which was compatible (71%) with other research studies shore up by Subbarao et al., in 2018 [24].

Resistant
Aside from the use of CBNAAT, an extended span of Rifampicin resistance was a clock in [25]. Some of the CBNAAT positive samples in a research study by Ikuabe et al., in 2018 [25,26] had Rifampicin resistance of 14.7%, which was nearly comparable to our research study (15.42%). However, a divergent study by Lee et al., 2013 found 5.7% resistance. Resistance to RIF in CBNAAT is thought to be a substitute indicator of MDR-TB [27,28].
Rifampicin resistance affects a specific quantity of resistance, such as mutation, co-infection with HIV, and insufficient or inappropriate anti-TB medication, as only 212 CBNAAT-positive samples were [26]. Resistance to these medications in mycobacterium strains was discovered not long after their clinical appearance. In terms of developing new chemical combinations to treat MTB, some new medications are in the works, but they are still in the preliminary clinical phase [29].

CONCLUSION
The current scenario of traditionally AFB negative PTB is insufficiently sensitive to establish the diagnosis of active tuberculosis without CBNAAT. They underdiagnose PTB and overtreat those who do not have it. PTB accounts for nearly half of all tuberculosis, and it is extremely difficult to obtain a bacteriological identification for negative TB specimens [23]. CBNAAT detects PTB with high specificity and sensitivity rather than liquid culture medium and sputum microscopy, which is why CBNAAT detects MTB quickly and correctly in less than 2 hours. Simultaneously, CBNAAT identifies RIFs for MDR-TB screening and patient prompt treatment, potentially lowering the new victim graph prevalence [30].

CONSENT
We included all the age groups and gender after taking written informed consent in our study.

SOURCE OF SUPPORT
Revised national tuberculosis control program (RNTCP), Govt. of India, State TB cells, Bhopal, Madhya Pradesh and District health society (DHS), Indore, Madhya Pradesh.

ACKNOWLEDGEMENT
Authors would like to thanks the faculties and all technical staff of the Department of Microbiology, TB and the chest Department, and RNTCP unit, Index Medical College, Indore, Madhya Pradesh and RNTCP program Bhopal, Madhya Pradesh, India for their guidance and support during this research work.