Diagnosis of Extra-Pulmonary Tuberculosis from Clinically Presumptive Cases by PCR

Kyi May Htwe, Aye Aye Lwin, Khine Zar Win, HlaingMya Win & Khin Saw Aye

Special Issue May 2020

Tuberculosis (TB) is one of the top ten causes of death and the leading cause from a single infectious agent (above HIV/AIDS). Millions of people continue to fall sick with TB each year. In 2017, TB caused an estimated 1.3 million deaths (range 1.2-1.4 million) among HIV-negative people and there were an additional 300,000 deaths from TB (range 266,000-335,000) among HIV-positive people. Globally, the best estimate is that 10 million people (range 9-11.1 million) developed TB disease in 2017: 5.8 million men, 3.2 million women and 1 million children. Myanmar is one of the 30 high burden countries of TB.¹ According to Global Tuberculosis Report 2015, extra-pulmonary TB (EPTB) accounts for 12.5% of all new TB cases notified in Myanmar.²Pulmonary TB is more common but EPTB remains quite severe because of unapparent and non-specific outcome. Diagnostic tests for TB disease include rapid molecular test, sputum smear microscopy and culture-based method. Rapid molecular test, the only rapid test for diagnosis of TB currently recommended by WHO, is the Xpert® MTB/RIF assay (Cepheid, USA). The major challenge in the diagnosis of EPTB is the frequently atypical clinical presentation simulating other inflammatory and neoplastic conditions, which frequently results in a delay or deprivation of treatment. 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 diagnosis. This study aimed to assess the usefulness of polymerase chain reaction (PCR) as a diagnostic tool for EPTB among clinically presumptive TB patients.

A cross-sectional descriptive study was conducted at Yangon General Hospital, Medical Unit I from 2016 to 2017. A total of 109 samples from clinically presumptive EPTB cases were tested for Mycobacterium tuberculosis (MTB) using acid-fast bacilli (AFB) smear microscopy and PCR method. MTB DNA was extracted from 47 cerebrospinal fluid samples, 32 pleural fluid samples, 29 ascitic fluid samples and 1 pericardial fluid sample using beads solution method. PCR amplification was performed to detect a 123-bp region from MTB targeting the IS6110. PCR was carried out in a 25µl reaction volume consisting of  4µl DNA, 2µl 5X reaction buffer, 0.25µl Taqpoly merase, 0.5µl dNTPs, each 0.5µl forward primer          (5'-CTGCGAGCGTAGGCGTCGGT-3') and reverse primer  (5'-CTCCTCCA GCCCCCCCT TCCC-3'), 1µl 25mM MgCl₂, 4µl Q solution, and 12.25µl RNase free water. The program was 94°C for 5 minutes denaturation  followed by 42 cycles, each consisting of 94°C for 30s, 68°C for 30s and 72°C for 30s with final extension step at 72°Cfor 10 minutes.³ The PCR products were resolved by 2% agarose gel and stained with ethidium bromide and evaluated under UV light. To avoid risks of false positive results, the PCR assays were done with strict precautions against cross contamination.

In this study, the mean (±SD) age of study population was 44.7 (±16.8) years and age ranged from 13 to 78 years. About 55% (60/109) were male gender and 45% were female. Ten percent (11/109) had history of TB contact in the family. About 16.5% (18/109) of patients had previous history of TB and among them, 55% (10/18) received anti-TB treatment. Most of the patients (70%) had low-grade fever and weight loss. All cases of EPTB were negative by AFB smear microscopy.

 MTB was detected in 3.7% (4/109) clinically presumptive EPTB cases by PCR method. All MTB PCR positive patients were male and included 3 pleural effusion cases and one alcoholic cirrhosis case. History of Koch’s lung was found in only one MTB PCR positive patient. MTB positivity was found only in pleural fluid (9.4%) and ascetic fluid (3.4%) types. In Myanmar, AFB smear microscopy and chest X-ray are used primarily for pulmonary TB because of its affordability and Xpert® MTB/RIF assay is also widely used for MTB/MDRTB. Although PCR positivity rate was low, it could detect MTB in all AFB-negative EPTB cases of the study. The result highlighted that the role of PCR in diagnosis of EPTB revealed as an alternative diagnostic tool.

MTB PCR positivity was lower than the other reports in which46.5% of smear-negative samples were positive in various samples and 12.1% of EPTB samples were positive by PCR in India.⁴ It may be probably due to type or nature of the samples like liquid/solid and volume of the samples used for PCR. In this study, all samples were most likely clear samples and less volume especially in cerebrospinal fluid sample. PCR required a minimum quantity of only 200 µl of the sample. In contrast to pulmonary specimens, the major problem of extrapulmonary specimens is insufficient volume of specimens.

PCR proved to be an effective rapid test and it was useful for the diagnosis of EPTB. The negative PCR result may be attributable to the nil or very low number of copies of MTB DNA. The possibility of variation in the DNA exaction cannot be ruled out as a reason for the false negativity. Various methods are available for DNA extraction and their sensitivities vary also. No single extraction method has been accepted as a gold standard, especially for the clinical specimens.⁵ TB bead extraction method contributes to higher sensitivity and detection rate of AFB testing.⁶Molecular techniques which are being simplified and improved continuously and rapidly appear to be the future tests of choice for most infections including TB.

As a conclusion, PCR can be useful as an alternative tool in the diagnosis of EPTB. More than one diagnostic method is necessary for the confirmation of early diagnosis and PCR results along with the clinical presentation may be adequate to initiate anti-tuberculous treatment.

Ethical consideration

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The authors declare that they have no competing interests.

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2.       Global Tuberculosis Report 2015. WHO/ HTM/ TB/2015; 22.

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