Diagnosis of Extrapulmonary Tuberculosis by Smear, Culture, and PCR Using Universal Sample Processing Technology

Abstract

Definitive and rapid diagnosis of extrapulmonary tuberculosis is challenging since conventional techniques have limitations. We have developed a universal sample processing (USP) technology for detecting mycobacteria in clinical specimens. In this study, this technology was evaluated blindly on 99 extrapulmonary specimens collected from 87 patients. USP-processed specimens were submitted to smear microscopy for detection of acid-fast bacilli (AFB), culture, and two PCR tests targeting devR (Rv3133c) and IS6110 gene sequences. On the basis of clinical characteristics, histology and cytology, conventional microbiology results, and response to antitubercular therapy, 68 patients were diagnosed with tuberculosis. Although USP smear and culture were significantly superior to conventional microbiology, which was not optimized (P < 0.0001), these approaches fell short of PCR tests (P < 0.0001). The low yields by smear and culture are attributed to the paucibacillary load in the specimens. The highest sensitivity in PCR was achieved when devR and IS6110 test results were combined; the sensitivity and specificity values were 83 and 93.8%, 87.5 and 100%, and 66.7 and 75%, respectively, in pleural fluid, needle-biopsied pleural tissue, and lymph node specimens. In conclusion, the application of USP technology, together with clinicopathological characteristics, promises to improve the accuracy and confidence of extrapulmonary tuberculosis diagnosis.Significant mortality and morbidity is caused by tuberculosis in developing countries, including India (31). Tuberculous pleural effusion is the most common exudative pleural effusion prevalent in India in contrast to the west, where malignant effusions are more frequent (27). However, the disease most often remains undiagnosed and, even worse, untreated. The chief difficulty with extrapulmonary specimens is that they yield very few bacilli and consequently are associated with low sensitivity of acid-fast bacillus (AFB) smear and culture. Acid-fast staining was positive in fewer than 10% of patients in most reports, whereas pleural fluid cultures for M. tuberculosis were positive in up to 12 to 70% of cases and pleural biopsies revealed granulomas in 50 to 97% of patients with tuberculous pleural effusion (1). The role of PCR in the diagnosis of tubercular pleural effusion has been evaluated extensively as an alternative diagnostic tool and has yielded variable results, with sensitivities ranging between 42 and 100% and specificities ranging between 85 and 100% using various PCR targets such as IS6110, 65kDa, TRC4, GCRS, etc. (9,10,22,23,26,29,30). The most common form of extrapulmonary tuberculosis is tuberculous lymphadenopathy (2,17,20), and its diagnosis remains a challenge since granulomatous lymphadenopathy has an extensive differential diagnosis. Several conditions, including sarcoidosis, fungal infections, and other inflammatory conditions, can present the same cytology and/or histopathology as tuberculous lymphadenopathy. A diagnosis of tuberculosis is then confirmed by ...

A Study of Manifestations of Extrapulmonary Tuberculosis in the ENT Region

“…The age distribution shows a normal pattern with a peak of 38.4 % in the fourth decade of life. This is as per the distribution reported by Chakravarty et al [1,6]. It has been reported that usually PTB is not associated with EPTB.…”

“…This is contrary to our findings wherein majority of the EPTB cases (91.7 %) were in the ENT region. TB lymphadenitis of the cervical region is the commonest manifestation of EPTB [2,4,[6][7][8]. In the present study too cervical TB lymphadenitis accounted for 87.4 % of cases of EPTB and 95.3 % of cases of EPTB in the ENT region.…”

“…However these studies were from low disease load, developed countries. Studies done in India have reported a clear cut male preponderance [6]. The age distribution shows a normal pattern with a peak of 38.4 % in the fourth decade of life.…”

Comparison of IS6110 and ‘short fragment’ devR (Rv3133c) gene targets with phenotypic methods for diagnosis of Mycobacterium tuberculosis

“…The USP methodology followed was found to be efficient for processing all samples and was compatible with smear examination, culture and genotypic assays, as proven by similar studies. 8 In this study, the sensitivity of phenotypic methods (ZN, LJ and BACTEC 460) was 89.29%, whereas that of IS6110 was 94.64% and 'short fragment' devR (Rv3133c) was 80.36%. While genotypic assays were more sensitive compared to phenotypic methods; amongst genotypic methods, IS6110 was found to be more sensitive than 'short fragment' devR (Rv3133c) as concluded by similar studies.…”

“…56 clinical TB samples from a tertiary care apex center along with 50 control samples, excluding samples from patients already on ATT were processed by the USP methodology 8 after approval from the Hospital Ethical Committee. The sample was mixed with a solution containing 5 M guanidinium hydrochloride, 2 M TriseHCl (pH 7.5), 0.5 M EDTA (pH 8.0), 20% Sarkosyl, b-mercaptoethanol (all from SigmaeAldrich Corporation, St. Louis, Missouri 63103, USA), incubated for 10e15 min at room temperature, centrifuged, supernatant decanted, pellet resuspended in Tween 80 solution and transferred to a 1.5 ml polypropylene lead vial.…”