Introduction: We evaluated if ascitic fluid sample could be used for the diagnosis of tuberculosis intestinal obstruction (TBIO) by a nested polymerase chain reaction (PCR) assay and analyzed the clinical and laboratory findings in 33 patients with confirmed intestinal tuberculosis (TB) compared to 54 patients with other pathologies of intestinal obstruction. Methods: We tested ascitic fluid sample by Ziehl-Neelson staining; Löwenstein-Jensen (L-J) culture method was used for the culture and PCR was performed for targeting the IS6110 sequence. Results: We found that 87.9% (29/33) of cases with intestinal TB and 0% (0/54) with other causes who had positive results with the nested PCR assay. PCR, in principle, is a highly sensitive technique that detects DNA from a single to a few microorganisms with the overall sensitivity, specificity, and positive predictive value (PPV) of 87.87%, 100%, and 100%, respectively. Acid-fast bacillus (AFB) staining alone has positivity of only 24% and histology alone has positivity of 88%. Conclusion: PCR has a potentially important role in improving the diagnostic accuracy.
Keywords: Ascitic fluid, Mycobacterium tuberculosis (M. tuberculosis), polymerase chain reaction (PCR), tuberculosis intestinal obstruction (TBIO)
|How to cite this article:
Yadav SK, Jha NK, Sinha DK, Singla H, Kumar S, Yadav J, Ranjan R, Kumar RR, Kumar S. Evaluation of the role of ascitic fluid polymerase chain reaction targeting IS6110 of Mycobacterium tuberculosis in the diagnosis of tuberculous intestinal obstruction. Ann Trop Med Public Health 2015;8:198-201
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Yadav SK, Jha NK, Sinha DK, Singla H, Kumar S, Yadav J, Ranjan R, Kumar RR, Kumar S. Evaluation of the role of ascitic fluid polymerase chain reaction targeting IS6110 of Mycobacterium tuberculosis in the diagnosis of tuberculous intestinal obstruction. Ann Trop Med Public Health [serial online] 2015 [cited 2020 Aug 10];8:198-201. Available from: https://www.atmph.org/text.asp?2015/8/5/198/159847
In spite of the introduction of various programs for the prevention and control of tuberculosis (TB), globally it is still a leading cause of adult mortality arising from a single infectious agent and 21% of the world’s TB-infected population is in India [according to the Revised National Tuberculosis Control Programme (RNTCP) 2008 report). The global annual incidence estimate is 8.7 million, most of those affected being children (especially in endemic areas), and it leads to approximately 1.4 million deaths annually. 
In Tanzania, a report of 118 cases with intestinal obstruction found that Mycobacterium tuberculosis (M. tuberculosis) was the etiology in 22.4% of cases.  An accurate diagnosis of intestinal obstruction due to TB is crucial for surgeons since most such obstructions are likely to be treated conservatively, and a prompt effective intervention in the absence of TB may eventually yield to longer survival or better quality of living.
Several studies have demonstrated the usefulness of polymerase chain reaction (PCR) on various samples for the diagnosis of intestinal TB. , Use of PCR has considerably grown over the recent years; this technique has proven to be useful for early and accurate diagnosis of TB and for guiding preemptive therapy in patients. This method has the advantage of automatic detection of amplified products, thus avoiding potential contamination and reducing the risk of false positive results. Here, we report our results obtained during this prospective study as we analyze PCR to detect M. tuberculosis in ascetic fluid sample of patients with tuberculosis intestinal obstruction (TBIO). Additionally, we examined the clinical and laboratory findings in patients with TBIO and compared it to other etiologies.
|Materials and Methods|
Study design, patients and setting
This study was a descriptive clinical study based on prospectively collected information. The research was approved by the Research and Ethics Committee of the institution (Rajendra Institute of Medical Sciences, Ranchi, Jharkhand, India). The patients were informed about the study and written informed consent was obtained from each one of them or their closest relative. Clinical, radiological, and laboratory data were collected between July 2013 and October 2014. The study enrolled 87 patients with symptoms of intestinal obstruction with gross or minimal ascites. Ultrasonography (USG)-guided fine-needle aspiration was done in patients with minimal ascites. Diagnosis of TBIO was established after PCR, histopathological examination (HPE), and response to antitubercular chemotherapy.
Amplification of mycobacterial DNA
A pair of oligonucleotide primers targeting the insertion sequence IS6110 of 123-bp length fragment specific for M. tuberculosis complex was used in this study.
Detection of amplified products
Amplified products were resolved by agarose gel electrophoresis (2%) at 80 V for 40 min. Agarose gel was stained with ethidium bromide (0.5 μg/mL) and viewed under ultraviolet (UV)-transilluminator (VILBER -LOURMAT, France, TCP-20.M).
Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR results were evaluated against the Lφwenstein-Jensen (L J) culture method, which was the gold standard, by using bivariate two-by-two tables (binary classification method). Differences in proportions were compared by the chi-square test. The Kruskal-Wallis H test was applied to the continuous variables. We considered P < .005 as a significant value. The statistical analysis was carried out with the Statistical Package for Social Sciences version 16.0 (SPSS Inc., Chicago, Il, USA).
Total 87 patients with intestinal obstruction were enrolled. At the end of the research, 33 patients were diagnosed with TBIO based on the response to antituberculosis therapy (ATT). The remaining 54 patients had other etiologies and formed the control group.
Results of nested PCR assay for M. tuberculosis DNA on ascetic fluid samples
In 87.9% (29/33) of cases of TBIO, PCR was positive compared with no positivity (0/54) in the control group. The difference was statistically significant (Fisher’s test P– 0.00). IS6110 PCR has shown 87.87% sensitivity [range: 71.78-96.52%; confidence interval (CI): 95%] and 100% specificity (range: 93.33-100.00%; CI: 95%). Overall PPV and NPV of IS6110 PCR was observed at 100.00% (range: 87.94-100.00%; CI: 95%) and 93.10% (range: 83.26-98.05%; CI: 95%), respectively.
Comparison of PCR with other tests
In present study, acid-fast bacillus (AFB) staining of ascitic fluid had sensitivity of 24% (range: 11.12-42.26%; CI: 95%). Culture of ascitic fluid for M. tuberculosis showed sensitivity of 48.48% in our study. Out of the 18 tissue samples in the test group, caseating tubercles suggestive of TB was present in 16 and absent in two cases. Sensitivity of HPE was 88.88% (range: 65.25-98.30%; CI: 95%) and specificity was 100.00% (range: 93.33-100.00%; CI: 95%).
Clinical and laboratory findings
Sensitivity, specificity and false negative rates for AFB, Culture, PCR and HPE is shown in [Table 1]. The results of clinical and laboratory examinations performed in both groups of patients are summarized in [Table 2].
|Table 1: Sensitivity, specifity and false negative rates of various tests
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|Table 2: Value of clinical and laboratory examination in both groups
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The major challenge in the diagnosis of intestinal TB is the detection of M. tuberculosis. Conventional methods including smear and culture of ascitic fluid have poor sensitivity due to the paucibacillary load in the samples. For the smear to be AFB positive, the sample should contain at least 10,000 bacilli/mL. L-J method for culture is still considered to be the gold standard, but 10-100 viable bacilli are mandatory for culture positivity. Moreover, a longer period of time (of about 6-8 weeks) is required for positive reports. Thus, most clinical and therapeutic decisions cannot be made;  and diagnosis by the tissue sample can be established only by diagnostic laparoscopy or laparotomy. Without an affirmative answer, clinicians cannot start treatment due to the delayed diagnosis. Therefore, a high index of suspicion is necessary to make an early diagnosis, and quite often, more than one procedure is necessary for the confirmation of this diagnosis.
There is a growing number of literatures regarding the role of PCR in the diagnosis of extrapulmonary TB. But very few studies are undertaken on the role of PCR in the diagnosis and management of TBIO.
IS6110 as the target sequence for the detection of mycobacterial DNA from extrapulmonary clinical samples showed a wide variation in specificity and sensitivity. ,, Negi et al. reported 83% PCR positivity and Ogusku et al. showed 92.1% of their samples to be IS6110 positive. Amin et al. reported 38.6% positivity rate in pus, 42.1% in cerebrospinal fluid (CSF), and 46.6% in urine samples. Makeshkumar et al. showed a low number of PCR positives among the 154 body fluid samples, i.e., 27% in ascetic fluid, 22% pleural fluids, and 36% CSF samples. 
In an European study conducted by Tzoanopoulos et al.,  all the three suspected cases and a single case by Protopapas et al.  of TBIO were found positive by PCR targeting IS6110. As the sample size was very low and response to ATT was considered as the final inclusion criterion for TBIO, the sensitivity was found to be higher in these two studies that might be misleading. Uzunkoy et al.  too in their eleven case series found sensitivity of 100%. They suggest that studies having less sensitivity are due to the use of a single primer. Schwake et al. obtained a negative result in the two cases they tested. Jain et al. reported 39 PCR positive cases of tubercular ascites and 35 responded to ATT. The sensitivity, specificity, PPV, and NPV were found to be 97.1%, 88.6%, 87.2%, and 97.5%, respectively. 
In our study, PCR was positive in 29 of 33 cases (i.e., 87.9% of cases) and cases with PCR positivity having clinical features of subacute intestinal obstruction (19/29 cases) were conservatively managed. All the cases responded to ATT and are presently asymptomatic.
In our study, there were four cases who could not be detected by nested PCR (false negative results-12.12%). The reasons of lower sensitivity might be the presence of PCR inhibitors or poor extraction procedure. These four cases were operated and HPE was suggestive of M. tuberculosis; all of them responded to ATT and are asymptomatic. Of 33 cases, 12 (36.36%) with TBIO were explored. In our study, there were two tissue samples in which the histological diagnosis came out to be negative and which were preoperatively identified by PCR as positive and later responded to ATT. In the control group, all the 54 cases (100%) were explored.
In conclusion, the reports of studies that included rigorous controls for PCR specificity and our own results indicate that PCR in ascetic fluid is a useful tool in the diagnosis and management of TBIO because in most of patients with subacute obstruction, laparotomy can be avoided. This assumption is supported by the report of a comparable mortality rate in confirmed TBIO (9.0%) and nonTBIO (9.2%) cases. Based on our experience, we recommend routine use of PCR in cases with subacute obstruction and if positive, the cases should be managed conservatively. In case of PCR, negative subacute obstruction or acute obstruction intervention should be done. ATT should be started postoperatively if HPE is supportive of TB.
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Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]