Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Mohan Z Mani

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Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



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Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
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Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2023 | Month : November | Volume : 17 | Issue : 11 | Page : DC24 - DC27 Full Version

Role of Real Time Polymerase Chain Reaction Targeting mpb64 Gene in the Diagnosis of Genitourinary Tuberculosis: A Cross-sectional Study


Published: November 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/63979.18755
Deepesh Kumar, Anita Pandey, Peetam Singh, Priyanka Chaturvedi

1. PhD Scholar, Department of Microbiology, Subharti Medical College, Meerut, Uttar Pradesh, India. 2. Professor and Head, Department of Microbiology, Subharti Medical College, Meerut, Uttar Pradesh, India. 3. Assistant Professor, Department of Microbiology, Subharti Medical College, Meerut, Uttar Pradesh, India. 4. Assistant Professor, Department of Microbiology, Subharti Medical College, Meerut, Uttar Pradesh, India.

Correspondence Address :
Dr. Anita Pandey,
Professor and Head, Department of Microbiology, Subharti Medical College, Meerut-250005, Uttar Pradesh, India.
E-mail: anipanmicro@gmail.com

Abstract

Introduction: Genitourinary Tuberculosis (GUTB) remains an important cause of infertility in India. The clinical manifestations of GUTB are non specific, resulting in delayed diagnosis and initiation of Anti Tubercular Treatment (ATT). This delay can lead to various complications such as kidney dysfunction, ureteral strictures, and a shrunken bladder.

Aim: To evaluate the diagnostic importance of Real Time Polymerase Chain Reaction (RT-PCR) targeting the mpb64 gene for the rapid and accurate diagnosis of GUTB.

Materials and Methods: A cross-sectional hospital-based observational study was conducted in the Department of Microbiology at Subharti Medical College and associated Chhatrapati Shivaji Subharti Hospital in Meerut, Western Uttar Pradesh, India. The study was conducted over a three-year period, from January 2019 to December 2021. A total of 200 genitourinary samples were collected from female patients with clinical suspicion of Tuberculosis (TB) and subjected to direct microscopy, mycobacterial culture by BacT/Alert 3D (Biomerieux, France), and RT-PCR targeting the mpb64 gene (Qiagen). Demographic details were recorded in a predesigned proforma. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) software version 26.0 (IBM Corp., Armonk, NY, USA). The p-value was calculated using the Chi-square test.

Results: RT-PCR showed a higher positivity rate of 56 (28.9%) compared to 47 (24.2%) by culture. Acid Fast Bacilli (AFB) microscopy was the least sensitive, detecting only 7 (3.6%) cases. When comparing the results of RT-PCR with conventional methods, RT-PCR had a sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) of 91.9%, 99.4%, 85.1%, and 93.8%, respectively.

Conclusion: RT-PCR targeting the mpb64 gene is a specific and effective additional test that aids in the early and accurate diagnosis of Extrapulmonary Tuberculosis (EPTB), including GUTB, compared to conventional methods. Early diagnosis facilitates timely initiation of ATT, leading to better clinical outcomes.

Keywords

Extrapulmonary tuberculosis, Molecular diagnosis of tuberculosis, Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a leading and significant cause of morbidity and mortality worldwide (1). TB accounts for approximately 90% of total cases and deaths in the developing world, with 75% of the cases occurring in the economically productive age group (2). Early diagnosis of TB is crucial for the timely initiation of ATT (3). Multidrug Resistant (MDR) or Extensively Drug Resistant (XDR) TB is a matter of concern. The diagnosis of EPTB poses a major challenge due to its pauci-bacillary nature. GUTB is a serious form of EPTB commonly observed in India, accounting for 20-73% of all EPTB cases in the general population, but it is less frequently reported in children (4). Concurrent GUTB has been observed to develop in 2-20% of patients with pulmonary TB (5). Clinical manifestations of GUTB are non specific, making diagnosis difficult and leading to delayed initiation of ATT, which can result in various genitourinary complications such as kidney dysfunction, ureteral strictures, and a shrunken bladder (6).

Although the demonstration of AFB by microscopic examination and culture on Lowenstein Jensen (LJ) medium remains the cornerstone of TB diagnosis, these conventional methods are time-consuming and have low sensitivity, especially in clinical samples with a low bacterial load (7). The availability of a quick and effective method to diagnose TB using RT-PCR has significantly reduced false positivity, as amplification and detection occur in the same reaction tube. Thus, RT-PCR can also be used to detect TB in specimens where culture yields negative results due to low bacterial load (8).

Most of the RT-PCR studies conducted on MTB diagnosis have targeted the IS6110 gene. However, authors hypothesised that targeting the mpb64 gene could also serve as an important tool in diagnosing EPTB. Since only a few studies have been conducted on the importance of RT-PCR targeting the mpb64 gene, present study further attempted to evaluate its role in the diagnosis of GUTB and contribute to the existing literature (2),(4),(8).

Considering that some strains of Mycobacterium tuberculosis circulating in the Asian and Indian population lack the insertion sequence 6110 (IS6110) (9),(10),(11), present study used RT-PCR targeting the mpb64 gene of Mycobacterium tuberculosis. Additionally, aimed to compare the diagnostic efficacy of culture and microscopy with RT-PCR. The study aimed to evaluate the role of RT-PCR targeting the mpb64 gene in the diagnosis of GUTB, while the objective was to compare culture and microscopy for diagnosing GUTB.

Material and Methods

A cross-sectional, hospital-based observational study was conducted in the Department of Microbiology at Subharti Medical College and associated Chhatrapati Shivaji Subharti Hospital in Meerut, Western Uttar Pradesh, India, over a period of three years from January 2019 to December 2021. The study included a total of 200 genitourinary samples collected from female patients suspected of having GUTB. Approval from the Institutional Ethics Committee (IEC) was obtained before the study commenced, with reference number SMC/IEC/2018/81A. Informed consent was obtained from patients after explaining the study’s purpose before collecting clinical specimens.

Inclusion criteria: The study included genitourinary samples received from female patients suspected of having GUTB.

Exclusion criteria: Genitourinary samples received from male patients were excluded from the study.

Sample size calculation: The sample size was determined using the following formula:

n=Z2 * p * (1-p)/d2

Where:
n=sample size.
Z=level of confidence (1.96 at a 5% error).
p=expected prevalence (19%).
d=precision/expected error (5%).
n=1.96 * 1.96 * 0.19 * (1-0.19)/0.05 * 0.05.

Study Procedure

The sample collection, transportation, and processing involved the following samples: Endometrial biopsy (125), endometrial curettings (30), menstrual blood (20), placenta (5), and urine (20). These samples were collected in clean sterile universal containers following aseptic precautions and transported to the testing laboratory within two hours in a cold chain (4°C).

Briefly, tissue specimens were grinded with the help of a tissue grinder in a 2 mL screw-capped tube, and silica beads were added. Decontamination of all the clinical specimens was performed using the modified Petroff’s method, which involved N-acetyl-L-cysteine and NaOH (NALC/NaOH) (12),(13),(14). Subsequently, all specimens were centrifuged in 15 mL sterile centrifuge tubes at 5,000 revolutions per minute for 20 minutes, and the resulting pellet was used for both DNA extraction and AFB smear preparation. Additionally, all decontaminated specimens underwent culture using the BacT/Alert 3D (bioMerieux, France) automated blood culture system, following the standard protocol (15).

DNAextraction: DNA was extracted using the Quick-DNA? Miniprep Kit (Catalogue Numbers: D3024, D302) and innovative Zymo-Spin? Technology, following the manufacturer’s instructions. The extracted DNA aliquot was stored at -80°C until further testing (16).

RT-PCR: The extracted DNA was then subjected to RT-PCR to detect the mpb64 gene of the Mycobacterium tuberculosis Complex (MTC). The extracted DNA was amplified using QuantiFast® SYBR® Green PCR (Qiagen) and primers (Sigma Aldrich) specific to the mpb64 gene of MTC. The amplification was performed using the 2× QuantiTect SYBR Green PCR Master Mix. The forward primer sequence was 5´-TCC GCT GCC AGT CGT CTT CC, and the reverse primer sequence was 5´-GTC CTC GCG AGT CTA GGC CA (17). The amplification process involved an initial hold at 95°C for 15 minutes, followed by cycling with denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and primer extension at 72°C for 30 seconds. This cycle was repeated for 45 rounds, and a final extension was performed at 72°C for seven minutes.

Validation and standardisation of RT-PCR: The RT-PCR validation was conducted using known positive and negative specimens, as well as specimens spiked with the American Type Culture Collection (ATCC) standard strain H37Rv. The mpb64 gene was detected in all three repeat runs and all dilutions. The analytical sensitivity of the RT-PCR was found to be as low as 10 bacteria/mL of the specimen. The cycling and melting curves of the RT-PCR are shown in (Table/Fig 1).

The diagnostic importance of RT-PCR in the diagnosis of EPTB was evaluated using appropriate statistical parameters, as mentioned in the statistical analysis, considering Mycobacterium Tuberculosis culture as the gold standard for the diagnosis of EPTB.

Statistical Analysis

The statistical analysis was performed using SPSS software version 26.0 (IBM Corp., Armonk, NY, USA), and the interpretation was based on a p-value <0.05, indicating statistical significance. The analysis aimed to calculate the sensitivity, specificity, PPV, NPV, and p-value of RT-PCR in diagnosing EPTB, considering MTB culture as the gold standard. The p-value was calculated using the Chi-square test.

Results

The overall age of the patients who participated in the study ranged from 20 to 50 years, with a mean age of 27.35±3.93 years. The majority of the patients (130 or 67.01%) were under 30 years old, indicating that genital TB is more common in relatively younger females. A total of 194 samples were included in the study, as six samples failed during DNA extraction and were excluded. Among the tested samples, 56 (28.9%) were positive by RT-PCR, compared to 47 (24.2%) by culture using BacT/Alert 3D. The lowest positivity rate was observed by microscopy, with only 7 (3.6%) positive results (Table/Fig 2).

However, 138 (71.1%) samples tested negative by RT-PCR, despite being obtained from clinically suspected cases of GUTB. The average time for detection by culture was 23.6 days. The amplification curves of positive specimens are shown in (Table/Fig 1). When comparing the results of RT-PCR with conventional methods, RT-PCR showed a sensitivity of 91.9%, specificity of 99.4%, Positive Predictive Value (PPV) of 85.1%, and Negative Predictive Value (NPV) of 93.8% (Table/Fig 3). The Receiver Operator Characteristic (ROC) curve is shown in (Table/Fig 4).

Discussion

The TB is a major public health issue, particularly in the developing world, including India, and is one of the important causes of morbidity and mortality worldwide. EPTB, though common, is often difficult to diagnose. About 5-45% of all TB cases have extrapulmonary manifestations, and out of the total EPTB cases, 30-40% involve the urogenital tract. It has been observed that about 2-20% of females can develop GUTB after a latency of 5-40 years of PTB (18). The available routine diagnostic tools do not have sufficient sensitivity to aid in the early diagnosis of EPTB. Conventional methods like AFB smear microscopy have poor sensitivity and specificity, and culture is often time-consuming, resulting in retrospective diagnosis (19).

The clinical utility of early detection of Mycobacterium tuberculosis targeting the mpb64 gene by RT-PCR, including its accuracy in diagnosing GUTB, was evaluated in the present study. Although female genital TB is a chronic disease, the symptoms are often mild. In most cases of GUTB, the fallopian tubes are affected, leading to infertility in females. Women frequently present with atypical symptoms that mimic other gynaecological conditions (20). A recent study conducted by Chaudhari KV et al., reported mpb64 RT-PCR as a highly specific test for the diagnosis of Mycobacterium tuberculosis. According to them, the sensitivity can be increased by combining IS6110 RT-PCR with mpb64 RT-PCR for the diagnosis of EPTB (21).

In the present study, RT-PCR was found to be the most sensitive method among the three used to detect MTC. The sensitivity, specificity, PPV, and NPV of RT-PCR were 91.9%, 99.4%, 85.1%, and 93.8%, respectively (Table/Fig 3). Conventional PCR can also aid in the rapid detection of MTC, but without strict quality control, it may introduce false positives and negatives (22). On the other hand, various studies have shown that RT-PCR is less prone to contamination and has reported sensitivities of 51.9%-90.3% and specificities of 81.8%-100% for diagnosing TB, depending on the target gene used [23,24]. Therefore, RT-PCR can serve as an important diagnostic test for cases of EPTB where culture and AFB microscopy yield negative results.

In the present study, two samples that were culture positive tested negative by RT-PCR. This discrepancy may be due to the misidentification of Non Tubercular Mycobacteria (NTM) as MTC, as culture and conventional biochemical identification are susceptible to subjective errors. Similar findings were reported by Raveendran R and Wattal C, where out of six samples that were microbiologically positive but PCR negative, two samples were identified as NTM upon further testing (25). However, in the present study, the authors found 11 samples that were positive by RT-PCR but negative by culture. This could be attributed to the presence of non viable mycobacteria or a low bacillary load resulting from prior treatment with ATT before sample collection.

The present study highlights that RT-PCR provides a significantly higher diagnostic yield, good sensitivity, and specificity in the diagnosis of GUTB compared to microscopy and culture. It may serve as a powerful tool for the early detection of GUTB and the timely initiation of ATT, leading to a significant reduction in morbidity, mortality, and financial burden on the community. Mycobacterial culture, despite being the gold standard method for TB diagnosis, rarely aids in the initiation of ATT due to its time-consuming nature and low sensitivity (26). Smear microscopy for AFB, although more economical and less time-consuming, suffers from low sensitivity, which is a major drawback. The present study highlights the role of RT-PCR targeting the mpb64 gene, which can contribute to the early diagnosis of EPTB, enabling prompt initiation of ATT and subsequently reducing morbidity and mortality. Further studies comparing the diagnostic utility of various target genes can aid in selecting the most effective target gene for the early and accurate diagnosis of EPTB.

Limitation(s)

The present study had a few limitations due to a lack of resources:

i) Diagnostic utility of various other target genes, such as IS6110, IS1081, hsp65, mtp40, using multiplex PCR, could not be assessed.
ii) Further testing to rule out the possibility of NTM in RT-PCR positive and culture-negative samples was not performed.
iii) This study was confined only to a limited geographical area with a relatively small sample size. Therefore, the findings cannot be generalised to the entire population.

Conclusion

RT-PCR targeting the mpb64 gene is a specific and effective additional test that helps in the early and accurate diagnosis of EPTB, including GUTB, compared to conventional methods. However, to establish its role as the sole diagnostic test for EPTB, more studies from different geographical regions with a larger sample size are needed. Furthermore, sensitivity can be improved by using multiplex PCR with genus and Mycobacterium tuberculosis-specific primers for the accurate detection of Mycobacterium spp., thereby minimising false positives and false negatives.

References

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DOI and Others

DOI: 10.7860/JCDR/2023/63979.18755

Date of Submission: Mar 09, 2023
Date of Peer Review: Apr 22, 2023
Date of Acceptance: Oct 17, 2023
Date of Publishing: Nov 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Mar 11, 2023
• Manual Googling: Oct 12, 2023
• iThenticate Software: Oct 14, 2023 (10%)

ETYMOLOGY: Author Origin

EMENDATIONS: 8

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