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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
Original article / research
Full Version
Rifampicin Resistance by Xpert MTB/RIF Assay in Pulmonary Tuberculosis- Is there a Need for Confirmation by Retesting?
Correspondence Address :
Dr. Gita Nataraj,
Department of Microbiology, 7th Floor, MSB, Seth GS Medical College and KEM Hospital, Parel, Mumbai-400012, Maharashtra, India.
E-mail: gitanataraj@gmail.com
Introduction: Xpert Mycobacterium tuberculosis/ resistance to Rifampicin (MTB/RIF) assay detects MTB complex (MTBC)and rifampicin resistance simultaneously. In high prevalence countries like India, detection of rifampicin resistance in sputum specimen of a newly diagnosed case of pulmonary TB with a low pretest probability needs to be confirmed by retesting.
Aim: To evaluate the results of retesting of rifampicin resistant specimens in newly diagnosed pulmonary TB cases.
Materials and Methods: A retrospective analysis of the data of Xpert assay was performed on specimens received in Department of Microbiology, Seth G.S. Medical College and KEM Hospital, Mumbai, Maharashtra, India. If rifampicin resistance was detected in a newly diagnosed case of Tuberculosis (TB), a second specimen was retested by Xpert assay for confirmation. Concordance of retesting was seen with results of Line Probe Assay (LPA).
Results: Total 27,429 specimens were processed by Xpert assay of which 803 specimens showed rifampicin resistance, 157 sputum specimens fulfilling criteria of Programmatic Management of Drug resistant Tuberculosis (PMDT) guidelines were retested. High, medium, low and very low bacterial load was observed in 30, 51, 34 and 42 specimens’ respectively. All specimens having high or medium bacillary load showed rifampicin resistant result on retesting. On retesting 34 sputum specimens with low bacterial load, rifampicin resistance was confirmed in 30 specimens. LPA done after growing them by liquid culture confirmed rifampicin resistance in remaining four specimens.
Conclusion: Xpert assay is recommended when the bacterial load identified by Xpert assay is very low and when there is discordance between Xpert results of rifampicin resistance and the reflex LPA testing.
Drug resistance, Molecular assay, Repeat testing
Drug resistant TB is an important challenge in the control and elimination of TB. World Health Organisation (WHO) endorsed Xpert MTB/RIF assay (Xpert assay) for the diagnosis of MTB and Rifampicin resistance (1). The geneXpert diagnostic system developed by Cepheid (Sunnyvale, CA, USA) has been in use since 2013 and has proved to be a game changer in TB diagnosis and rifampicin resistance detection especially in high burden countries. The Xpert assay uses molecular beacon technology to detect DNA sequences amplified in a heminested real-time-Polymerase Chain Reaction (PCR) assay. The test uses a cartridge-based system. All the steps required for PCR like extraction, amplification and detection of targeted sequences from the patient’s samples takes place inside the cartridge (2). The limit of detection of the assay is approximately 131 colony forming units per ml (cfu/mL) of sputum specimen (3).
Xpert MTB/RIF assay detects MTBC and rifampicin resistance simultaneously as most common mutation conferring rifampicin resistance occurs in the same 81 bp region of genome which also codes for MTBC specific Deoxyribonucleic Acid (DNA) sequence (4),(5). This assay identifies rifampicin resistance associated mutations in codon 507 to 533 of rpoB gene of Mycobacterial genome (6). It detects either absence of binding of the probe affected (negative analyte result for the affected probe) or sensing a “significant” delay in amplification of the mutated segment/s compared to wildtype segments. The delay is considered as significant if it is more than four cycles. (7). The assay also gives a semi-quantitative indication of the bacterial load in the specimen as high, medium, low and very low (2).
Rifampicin resistance is considered a surrogate marker of multidrug resistant TB (8). Timely and accurate diagnosis of rifampicin resistant TB is important as the diagnostic algorithm under PMDT guidelines depend totally on status of rifampicin resistance (9). In high prevalence countries like India, detection of rifampicin resistance in sputum specimen of a newly diagnosed case of pulmonary TB with a low pretest probability needs to be confirmed by retesting. Since June 2018, all such specimens were retested for confirmation of rifampicin resistance irrespective of their bacterial load (10). As per revised guidelines of June 2021, only specimens having low (Cycle threshold (Ct) value 22-28] and very low (Ct value >28) bacterial load needed to be retested (9). Xpert assay is an expensive test and each cartridge used for retesting creates huge burden on the National TB Elimination programme (11). Hence, this study was undertaken with the primary objective of evaluating the results of retesting of rifampicin resistant specimens in newly diagnosed pulmonary TB cases.
This retrospective analytical study was conducted in the Department of Microbiology Seth G.S. Medical College and KEM Hospital, Mumbai, Maharashtra, India. Data was collected for a period of two years, from 1st June 2018 to 31st May 2020 and was analysed in next four months (June 2020-September 2020). This study was ethically approved from the Institutional Ethical Committee (IEC) (EC/OA-174/2020) of same medical college. As this study involved analysis of results entered in laboratory register; waiver was obtained for informed consent.
Inclusion criteria: A total of 27,429 patients, either outpatient or admitted in the hospital, submitted sputum specimen to the laboratory for Xpert assay during the study period. All specimens were tested and results were entered in lab register.
Exclusion criteria: All the specimens were included in this study and there was no exclusion criterion.
Procedure: Clinically suspected cases of pulmonary tuberculosis is any person who presents with symptoms or signs suggestive of tuberculosis (12). Two sputum specimens were collected from the cases which were clinically suspected cases of pulmonary tuberculosis. Same sample ID was given to both specimens with sublabel as “A” and “B”. This assay provided the result as “MTB not detected” or “MTB detected” using three specific primers and five unique molecular probes to ensure a high degree of specificity. Positive result like “MTB Detected” is reported when minimum two probes combine and the difference between their Ct value is less than 2.0 (13). If MTB is detected, a semi-quantitative estimation of bacterial load is also provided by machine as high (Ct value <16), medium (Ct value 16-22), low (Ct value 22-28) and very low (Ct value >28) (2). Simultaneously information was obtained as “Rifampicin resistance detected’ or “Rifampicin resistance NOT detected”. If Rifampicin resistance was detected in a newly diagnosed case of TB, then second specimen was retested for confirmation as per PMDT guidelines (9). Results of testing as well as retesting were entered in the laboratory register. Results obtained by retesting were analysed and percentage of discordant results was determined. As per PMDT guidelines, additional sputum specimen was collected from the patient having discordant results and it was tested by LPA for first line anti-TB drugs (9). LPA technology involves the DNA extraction from MTB culture isolates or directly from clinical specimens. Using PCR, amplification and hybridisation was done to detect presence or absence of resistance to rifampicin in of MTBC (14). LPA was performed only for specimens showing discordance between first and repeat Xpert assay.
Statistical Analysis
Data was analysed using Microsoft excel. Total number and percentage of concordant and discordant results were calculated. Result of LPA testing was compared with result of Xpert assay.
During the study period, total 27,429 patients visited the lab and submitted same number of specimens. The age range were from 1-60 years with mean age being 39.59±10.74 years, including both male (16404) and female (11025) (Table/Fig 1). They were processed by Xpert MTB/RIF assay. Of these 27429 patients, MTB was detected in 3353 (12.22%) specimens and in this category, rifampicin resistance was detected in 803 (23.95%), but was not detected in 2454 (73.19%) and indeterminate resistance was found in 96 (2.86%) specimens. Of the 803 specimens showing rifampicin resistance results, 157 sputum specimens were from newly diagnosed patients and 646 from previously treated patients (Table/Fig 2).
All 157 newly diagnosed patients were retested. High, medium, low and very low bacterial load was observed in 30, 51, 34 and 42 specimens respectively (Table/Fig 3). All specimens having high (30, 19.11%) or medium (51, 32.48%) bacillary load showed rifampicin resistant result on retesting demonstrating 100% concordant results. However, specimens having low (34, 21.66%) or very low (42, 26.75%) bacillary load had 88.23% concordance.
On retesting 34 sputum specimens with low bacterial load by Xpert assay, rifampicin resistance was confirmed in 30 specimens. In remaining four specimens, MTB was not detected. These four specimens were tested by LPA, either directly or after obtaining mycobacterial isolate from Mycobacteria Growth Indicator Tube (MGIT) liquid culture. All four showed presence of rifampicin resistance.
Total 42 patients with rifampicin resistance and very low bacterial load were retested by Xpert assay. Rifampicin resistance was confirmed in 29 specimens by retesting. Remaining 13 patients were tested by LPA by using culture isolates obtained by growing them in MGIT 960 liquid culture system. Rifampicin resistance was confirmed in nine patients. Four patients showed absence of rifampicin resistance and responded to drug sensitive TB treatment.
In two specimens, rifampicin resistance was not detected which was confirmed by LPA. These two patients were started on treatment for rifampicin sensitive TB and they responded to it. In 11 specimens, Xpert assay could not detect MTB. LPA confirmed rifampicin resistance in nine patients, rifampicin resistance was detected in nine specimens. In two specimens, MTB was detected but rifampicin resistance was not detected.
Overall, in 140 patients with rifampicin resistance, concordant results were obtained on retesting by Xpert assay. In the remaining 17 patients, LPA confirmed rifampicin resistance in 13 patients. Four patients having very low bacterial load showed discordance between Xpert assay and LPA results (Table/Fig 4).
Of the 157 specimens retested, 17 specimens showed rifampicin resistance due to delay in hybridisation with probes while remaining 140 specimens showed rifampicin resistance due to dropout of one or more probe.
Molecular diagnosis of tuberculosis and rifampicin resistance by Xpert MTB/RIF assay is a game changer for tuberculosis control programme. The advantages of this test are that it can be performed directly on sample, give rapid results within two hours, provides information on rifampicin resistance when MTB is detected (2). Getting the results on the same day helped the clinicians to decide appropriate treatment.
Rifampicin resistance when detected in patients with a low pretest probability, a repeat testing with a fresh sample is advised. In the present study, an attempt was made to understand the necessity of repeat testing for confirmation of rifampicin resistance by comparing results of first and repeat test. Of the total 27,429 specimens tested by Xpert assay, MTB was detected in 3353 (12.22%) specimens, of which 803 (23.95%) showed presence of rifampicin resistance on first testing. As per guidelines of National TB Elimination Programme, specimens from 157 newly diagnosed TB cases were retested for confirmation of presence of rifampicin resistance (10).
Xpert assay detects rifampicin resistance by probing for point mutations in the 81 bp (27 codons) rifampicin resistance determining region of rpoB gene of MTB. It is detected by using five overlapping probes labeled as A, B, C, D and E (15),(16),(17). The Xpert assay also offers a semi-quantitative estimation of bacterial burden in form of Ct values. Ct values have been useful to predict rifampicin resistance. The difference between the first (early Ct) and last (later Ct) MTB-specific molecular beacon (delta CT Max) is the basis of rpoB mutation and rifampicin resistance detection (18),(19). In Xpert assay, to detect rifampicin resistance, delta Ct max should be >4 (17). Mutation that completely inhibits one or more probe hybridisation is defined as causing probe “dropouts” whereas rpoB mutation that permit partial probe hybridisation and produce a measurable Delta CT Max of >4 cycles is considered “delays” (20). Amount of DNA in the specimen may have some impact on detection of rifampicin resistance (21).
Xpert assay also provides information about semi-quantitative load of MTBC in the given sample as per the Ct value as high, medium, low and very low. Ct values demonstrate the number of PCR cycles that the MTB DNA goes through to reach the level of detection; higher Ct values correlate with lower bacterial loads (22). In the present study, bacterial load of rifampicin resistant specimens was found as high (30), medium (51), low (34) and very low (42) in first testing. Such variable Ct values are observed in various other studies also (23),(24). On retesting, 81 specimens having high and medium bacterial load showed 100% concordant results for MTB detection and rifampicin resistance.
On retesting of second specimen from 34 patients with low bacterial load, 30 (88.23%) specimens confirmed rifampicin resistance. In remaining four specimens, LPA testing confirmed presence of rifampicin resistance in these specimens. All of them responded to treatment and got cured. On retesting 42 specimens with very low bacterial load, rifampicin resistance was confirmed in 29 (69.04%) specimens. Of the remaining 13 patients, LPA confirmed rifampicin resistance in 11 patients. Similar finding were reported in other studies. Van Rie A et al., reported six samples as rifampicin resistant by Xpert assay. Five out of these six samples were found resistant by LPA (25). In a study by Rufai SB et al., 64.4% of rifampicin monoresistant TB cases by LPA were correctly diagnosed by the Xpert MTB/RIF assay (26).
Detection of rifampicin resistance by Xpert assay depends on two important factors such as proportion of mutants present in the sample as well as type of mutation like “dropouts” or “delays”. Some of the issues with Xpert highlighted by other studies are false rifampicin resistant results related to existence of “disputed” and silent mutations (27),(28),(29),(30),(31),(32). Blakemore R et al., evaluated the analytical performance of Xpert MTB/RIF assay in their study and tested the ability of the assay to detect the rifampicin resistant fraction of a mixed sample (MTB DNA with a wild-type rpoB sequence was mixed in various ratios with MTB DNA that contained rpoB mutations) (20). Their study showed that the proportion of mutant DNA required for the detection of rifampicin resistance was dependent on the type of mutation. Xpert assay is capable of detecting the presence of rifampicin resistance mutations down to a concentration of 40% mutant DNA (33). Chakravorty S et al., demonstrated that detection of rifampicin resistance in DNA mixtures with 10, 20, and 30% mutant DNA were indistinguishable from a sample containing 100% wild-type DNA (33). Significantly higher mutant proportions were needed (65%-100% of the total bacterial population) for a positive identification compared to the minimum 1% mutant population required for clinical resistance (20),(34). Solid media-based proportion method and even liquid media based automated testing method (MGIT 960) to a certain extent, are capable of identifying such low proportions (35).
In a country like India, cost of a test is an important factor to decide its use. Cost of a single Xpert assay cartridge is approximately Rs 1500/- and hence there are strict protocols for its use under the National TB elimination programme (10). In this study, in 140 (89.17%) specimens, same result was confirmed on retesting by Xpert assay. These patients were started on treatment for drug resistant TB and they responded well. Hence retesting of these 140 specimens did not either provide any additional information or help the clinicians to start the appropriate treatment. Instead, it delayed the report by few hours or a day and resulted in wastage of cartridges.
Limitation(s)
Phenotypic DST was not performed for confirmation of rifampicin resistance could be a limitation of the present study. Long turnaround time of phenotypic test limit its use for starting the early treatment to the patient.
Based on the results of the present study, it can be suggested that retesting by Xpert assay is recommended in situations (a) when the bacterial load identified by Xpert assay is very low and (b) when there is discordance between Xpert results of rifampicin resistance and the reflex LPA testing, a repeat Xpert assay with backup liquid culture DST should be considered.
Mumbai District Tuberculosis Control Society provided the consumables required for Xpert MTB/RIF assay under National Tuberculosis Elimination Programme.
DOI: 10.7860/JCDR/2022/55315.16413
Date of Submission: Jan 29, 2022
Date of Peer Review: Feb 18, 2022
Date of Acceptance: Apr 18, 2022
Date of Publishing: May 01, 2022
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? No
• For any images presented appropriate consent has been obtained from the subjects. NA
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