Diagnostic accuracy of BAL Gene Xpert compared to BAL AFB staining in Sputum negative Pulmonary Tuberculosis Suspects

Tuberculosis has stood the test of time over the millennia and still wreaks havoc on human life all over the world. We have studied the role of bronchoscopy in smear negative PTB suspects and have compared the results of BAL Gene Xpert with BAL AFB staining among the study population. We have also determined the microbiological pro ile in cases who are MTB negative. We retrospectively reviewed the respiratory samples (sputum and BAL) of 82 sputum negative PTB suspects from july 2018 – july 2019 for ZN stain and Gene Xpert. 25 cases were detected by Gene Xpert additionally to the 11 cases detected by BAL a b staining alone. Out of the 36 cases detected on gene Xpert 3(8%) were RIF resistant. These 5 samples which were positive on BAL AFB and negative on Gene Xpert,are suspected to be NON TB mycobacteria. Out of the remaining 41 samples, non TB culture results showed 8 Acinetobacter, 10 pseudomonas, 12 klebsiella, 2 e.coli,2 candida, 7 no growth on culture. According to our study, the bronchoscopic samples when subjected to Gene Xpert detect a higher number of cases which would go undetected otherwise compared to BAL AFB staining. Hence, bronchoscopy must be performed on such smear negative suspects andGene Xpert could replace smearmicroscopy as initial diagnostic testing. It also helps in detecting irst line drug resistance in addition.


Bronchoalveolar lavage, Gene Xpert, Tuberculosis, Microscopy
A Tuberculosis has stood the test of time over the millennia and still wreaks havoc on human life all over the world. We have studied the role of bronchoscopy in smear negative PTB suspects and have compared the results of BAL Gene Xpert with BAL AFB staining among the study population. We have also determined the microbiological pro ile in cases who are MTB negative. We retrospectively reviewed the respiratory samples (sputum and BAL) of 82 sputum negative PTB suspects from july 2018 -july 2019 for ZN stain and Gene Xpert. 25 cases were detected by Gene Xpert additionally to the 11 cases detected by BAL a b staining alone. Out of the 36 cases detected on gene Xpert 3(8%) were RIF resistant. These 5 samples which were positive on BAL AFB and negative on Gene Xpert,are suspected to be NON TB mycobacteria. Out of the remaining 41 samples, non TB culture results showed 8 Acinetobacter, 10 pseudomonas, 12 klebsiella, 2 e.coli,2 candida, 7 no growth on culture. According to our study, the bronchoscopic samples when subjected to Gene Xpert detect a higher number of cases which would go undetected otherwise compared to BAL AFB staining. Hence, bronchoscopy must be performed on such smear negative suspects and Gene Xpert could replace smear microscopy as initial diagnostic testing. It also helps in detecting irst line drug resistance in addition.

INTRODUCTION
Tuberculosis, an important preventable and treatable cause of death, is a major health issue worldwide. Detecting cases with pulmonary tuberculosis is an important component in starting early appropriate treatment and interrupting the chain of trans-mission in such cases. Although sputum smear is a low cost test implemented by RNTCP, Gene Xpert has emerged as a highly speci ic and sensitive test in detecting pulmonary tuberculosis within a matter of hours (Agrawal et al., 2016). Whereas mycobacterial culture take about 6-8 weeks time (Evans, 2011) during which valuable time is lost in initiating treatment. Bronchoscopy is one of the newer methods in obtaining samples from "sputum smear negative PTB" (Bachh et al., 2010). Reasons for sputum smear negativity include poor quality of sputum sample, de icient preparation or staining, or examination or very low bacterial load. Although persons with smear negative TB have less bacterial load their potential to transmit disease is higher in the long run as they have the propensity to go undetected for a longer duration. Conversely, correct prediction of persons who are unlikely to have TB is important as well to limit the expense and potential toxicity of empiric therapy.

1.
To study the role of bronchoscopy in smear negative PTB suspects 2. To compare the result of BAL Gene Xpert with BAL AFB staining among study population 3. To determine the microbiological pro ile in cases who are MTB negative.

MATERIALS AND METHODS
This is a Retrospective study conducted in the department of Pulmonary medicine, Saveetha medical college and hospital, Chennai between period of July 2018-july 2019. The study population included all sputum negative cases/ sputum scarce patients suspected radiologically and / or clinically to have pulmonary tuberculosis. Bronchoscopy was done and BAL was subjected to AFB staining, Gene Xpert, and Non TB culture and results were recorded. Outcome measures of the study was to compare the results of Gene Xpert over AFB staining of the Bal samples so that the suspected cases are initiated on early treatment thus reducing morbidity and mortality and hence reducing undue spread. Graph development and percentage calculation was done using spreadsheets .
Bal AFB and non TB culture was done in the department of microbiology, Saveetha medical college. Gene Xpert of the BAL samples were sent to Hitech labs poonamalee for reporting in a sterile falcon tube and transported in a cold chain box.

Inclusion Criteria
Patients with clinical suspicion of Pulmonary tuberculosis including symptoms of cough with or without expectoration for >2 weeks, weight loss, fatigue , hemoptysis and loss of appetite with/ without radiological lesions like non homogenous opacities, nodules, hilar enlargement or consolidation with air bronchogram. Subjects must have had no sputum production or two sputum AFB samples negative and ,must have been subjected to Flexible optic bronchoscopy and samples sent for AFB staining, Gene Xpert, non TB culture.

Exclusion Criteria
1. Samples for which gene Xpert was not done.
2. Patient diagnosed with other lung conditions like lung malignancies 3. Samples for which history not available.
Hence 25 cases were detected by agent Xpert in addition to the 11 cases detected by BAL a b staining alone (Figure 4). Out of the 36 cases detected on gene xpert 3(8%) were rif resistant ( Figure 5).

Figure 2: Distribution of BAL AFB Vs BAL Gene Xpert
The aim of this study is to determine the diagnostic superiority of gene xpert over traditional stain-  (Xpert, 2013). But through this study we recommend gene xpert in highly suspicious cases (either clinically or radiologically) to detect more cases.
The results of our study show that Gene Xpert has higher yield than AFB smear in sputum negative / sputum scarce pulmonary tuberculosis cases. The conventional AFB staining can miss sputum with low bacterial load as microscopy needs at least 10,000 bacilli/ml for direct microscopy to show a positive result (Mavenyengwa et al., 2017).
Gene Xpert is a simple assay which can be performed with minimal training and results can be obtained in a couple of hours (Khalil et al., 2015). Gene Xpert has helped us in identifying such cases which tend to go undetected and hence have the propensity to go untreated. Such cases can continue spreading TB in the community in the long run. By doing a FOB wash we also detect cases earlier than the time when they eventually turn to be sputum positive. These cases, before detection, have the probability to spread the disease to a few contacts.
Among the BAL samples 25 cases (31%) were detected by Gene xpert while it was missed by AFB staining. This is comparable to a study by Iyer VN et al who also had similar percentage of TB cases detected from BAL gene Xpert testing (Iyer et al., 2011).
Another important factor, RIF resistance was found among 3(8%) of the PTB positive cases. This is comparable to a study by H.Y Lee et al, where they had demonstrated Rif resistance in 2 of the 35 positive cases (Lee et al., 2013).
According to a study by Y.MOK et al in Singapore, BAL gene xpert also lessens the requirement for invasive like procedures Transbronchial biopsy to prove TB af liction. The same proved true for our study subjects who underwent bronchoscopy due to PTB suspicion (Mok et al., 2016).
Out of the remaining samples which were negative for PTB both by Gene Xpert and AFB smear, we followed up the non-Tb culture results and found Klebsiella(29%) to be the most common organism grown in culture followed by pseudomonas(24%)and Acinetobacter (20%) respectively.
We also analysed the radiological pro ile among the 36 cases detected on Gene Xpert and found that 44%had non homogenous opacity, 25 % had homogenous opacity with air bronchogram, 17% had cavitation, 8% had nodules and 6 % had hilar enlargement. In a study done by Deependra k rai et al patchy in iltrates and cavitation was observed in sputum positive compared to sputum negative cases, but in our study we could not make that comparison as we have studied only sputum negative PTB suspects (Rai et al., 2019).
Bronchoscopy contraindications are relative and if performed by an experienced bronchoscopist the risks of bleeding, injury and discomfort to patient etc can be minimized and bene its will outweigh the risks.

Limitations
Tb Culture was not performed to rule out possibility of false positive results, however Xpert / MTB/RIF have shown speci icities above 95%in previous studies (Policy statement, 2011).

CONCLUSIONS
FOB should be advised in clinically/ radiologically suspected PTB cases and the BAL should be subjected to Xpert MTB/RIF assay along with AFB staining. We have showed in our study, that Xpert MTB/RIF assay has helped us detect PTB in 31% of the suspects, which went undetected by BAL AFB staining alone. This is particularly helpful in detecting patients who could go untreated for a long period of time but meanwhile continue spreading TB in the community.