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

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Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dermatolgy,
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."



Dr Kalyani R
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.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



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 : 2024 | Month : January | Volume : 18 | Issue : 1 | Page : DC06 - DC12 Full Version

Impact of BioFire FilmArray Multiplex PCR in the Detection of Microbial Agents causing Severe Acute Respiratory Infection in the COVID-19 Era: A Cross-sectional Study from a Tertiary Care Hospital in Central India


Published: January 1, 2024 | DOI: https://doi.org/10.7860/JCDR/2024/65688.18972
Meena Mishra, Pooja Shendre, Neeta Gade, Vishal Shete, Soumyabrata Nag, SM Vibha Dutta

1. Professor and Head, Department of Microbiology, AIIMS, Nagpur, Maharashtra, India. 2. Assistant Professor, Department of Microbiology, Goverment Medical College, Nagpur, Maharashtra, India. 3. Associate Professor, Department of Microbiology, AIIMS, Nagpur, Maharashtra, India. 4. Associate Professor, Department of Microbiology, AIIMS, Nagpur, Maharashtra, India. 5. Associate Professor, Department of Microbiology, AIIMS, Nagpur, Maharashtra, India. 6. Ex-Director and CEO, Director Secretariat, AIIMS, Nagpur, Maharashtra, India.

Correspondence Address :
Dr. Meena Mishra,
Professor and Head, Department of Microbiology, AIIMS, SEZ-MIHAN, Nagpur-441108, Maharashtra, India.
E-mail: meenarishika@yahoo.co.in

Abstract

Introduction: Many viral and bacterial respiratory tract infections can present with respiratory signs and progress to complicated pneumonia. In the recent Coronavirus Disease-2019 (COVID- 19) pandemic, it is crucial to test all Severe Acute Respiratory Infection (SARI) patients for other microbial infections in addition to COVID-19, enabling timely diagnosis and treatment to reduce morbidity and mortality. The automated system, BioFire FilmArray, utilises multiplex Polymerase Chain Reaction (PCR) to rapidly detect and identify multiple respiratory pathogens, including selected Antimicrobial Resistance (AMR) genes, within an hour.

Aim: To detect bacterial and/or viral pathogens associated with hospitalised COVID-19-negative SARI patients using the BioFire FilmArray Pneumonia Panel (BFPP).

Materials and Methods: This laboratory-based cross-sectional study was conducted at All India Institute of Medical Sciences (AIIMS), Nagpur, Maharashtra, India, from June 2020 to February 2021. Respiratory samples, such as sputum, tracheal aspirate, Endotracheal (ET) secretions, and Bronchoalveolar Lavage (BAL), were collected from COVID-19-negative hospitalised SARI cases. A total of 81 patients were included in the study. The samples were tested using the BFPP (multiplex PCR) system and processed using conventional culture techniques. Patient characteristics, clinical and laboratory investigation data, and findings of respiratory viral and bacterial agents, as well as antibiotic resistance genes detected by BioFire FilmArray, were recorded using paper case reports. The data were collected and analysed using Statistical Package for Social Sciences (SPSS) software.

Results: Out of the 81 clinical samples processed, the BFPP detected 168 bacterial and 18 viral pathogens. Bacterial-viral co-detection was observed in 13 (16%) samples. Atypical bacteria were detected in 3% of cases. Among the bacterial pathogens, the AMR gene for New Delhi Metallo-beta-lactamases (NDM) was detected in 42 (25.9%) cases, followed by CTX-M beta-lactamases, VIM, and the oxacillinase group of β-lactamases.

Conclusion: The BFPP test is a valuable tool for the rapid detection of a wide range of pathogens, including associated AMR genes, with high sensitivity and specificity. This can greatly aid in treatment decisions.

Keywords

Antimicrobial drug resistance, Co-infection, Polymerase chain reaction

The SARI, which primarily affects the respiratory tract, is one of the leading causes of morbidity and mortality worldwide. The World Health Organisation (WHO) defines SARI as an acute respiratory infections with a history of fever or measured fever >38°C and cough, with onset within the last 10 days and requiring hospitalisation (1). SARI is a major cause of hospitalisation and death globally and can be caused by various viral and bacterial agents (2). In the current COVID-19 pandemic era, it is crucial to test all SARI patients for other microbial infections in addition to COVID-19. Prompt treatment with antibiotics for bacterial infections can improve outcomes, and delays in effective antimicrobial therapy are associated with increased hospital stay and treatment costs.

Conversely, in cases of acute respiratory conditions caused by viral infections, the use of antibiotics is not recommended. Unnecessary antibiotic use contributes to the rise of antibiotic-resistant infections, leading to significant morbidity and mortality (3),(4).

Rapid diagnostic testing for identifying the causative agents in SARI patients can facilitate proper management, discontinuation of unnecessary antibiotics, enhance supportive therapy, and reduce healthcare costs and hospital stay duration (5).

The aetiological diagnosis of respiratory tract infections is a challenging area in clinical microbiology due to the wide range of potential pathogens and the non sterile environment of the respiratory tract. Traditional diagnostic methods, such as Gram stain and culture, may have a low diagnostic yield (6),(7),(8).

The automated system, BioFire FilmArray, based on the principle of multiplex PCR, provides rapid detection and identification of multiple respiratory viral and bacterial pathogens, along with selected AMR genes, from respiratory samples such as sputum or BAL in individuals suspected of having Lower Respiratory Tract Infections (LRTI), within one hour (9). Considering the ongoing COVID-19 pandemic, it was important to study the aetiology of cases with similar presentations to COVID-19 but tested negative by PCR. The BFPP was an ideal platform to simultaneously detect 27 different pathogens. The BFPP can detect 18 bacteria (11 Gram-negative, 4 Gram-positive, 3 atypical), seven antibiotic resistance markers, and nine viruses known to cause pneumonia and other LRTIs within one hour. It also provides a semi-quantitative measure of the organism load present in the sample (10).

To the best of the author’s knowledge, this unique study is one of the first from Central India. Its aim was to detect bacterial and/or viral pathogens associated with hospitalised COVID-19-negative SARI patients using the BFPP. The hypothesis of the study was to evaluate the utility of BioFire in the laboratory diagnosis of COVID-19-negative SARI cases, identifying multiple pathogens using the pneumonia panel with their antimicrobial-resistant genes, including viral and atypical bacteria that are difficult to diagnose using standard tests.

Material and Methods

This study was a laboratory-based cross-sectional study conducted at AIIMS, Nagpur, tertiary care hospital in Central India. The study was conducted from June 2020 to February 2021 in the Molecular Diagnostic Laboratory of the hospital. Ethical approval for the study was obtained from the Institute Ethical Committee with letter number IEC/Pharmac/2021/238.

The study included SARI patients who were tested negative for Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) using Real-Time Polymerase Chain Reaction (RT-PCR). These patients were hospitalised in various hospitals in the Vidarbha region of Maharashtra, which consists of 11 districts: Nagpur, Amravati, Akola, Chandrapur, Buldhana, Washim, Gondia, Yavatmal, Wardha, Bhandara, and Gadchiroli. Sputum, tracheal aspirate, ET secretions, and BAL samples were collected from these COVID-19-negative SARI patients and transported to the study centre while maintaining a cold chain temperature of 2-4°C.

Inclusion criteria:

1. Patients hospitalised with symptoms of SARI in various hospitals in the Vidarbha region of Maharashtra. SARI was defined according to the WHO criteria (1) as follows:

a. Acute respiratory infections

b. Fever or measured fever >38°C

c. Cough, with onset within the last 10 days and requiring hospitalisation

Exclusion criteria:

1. Samples that tested positive for COVID-19.

2. Patients who did not fulfill the criteria for SARI definition.

During the study period, which spanned the first wave of COVID-19, a total of 81 samples were received from COVID-19-negative SARI patients who met the inclusion criteria and were included in the present study.

Methodology:

Sputum, tracheal aspirate, ET secretions, and BAL samples received from COVID-19-negative SARI cases during the study period were tested using the BFPP (multiplex PCR) system. All respiratory samples underwent gram stain and conventional culture on blood agar, MacConkey agar, and Chocolate agar as part of the Standard-Of-Care (SOC) testing. Samples were not processed if a score of 0 or 1 was detected according to the Murray Washington criteria (11) which are based on the number of squamous cells and neutrophils per low-power field on Gram’s stain of sputum samples. Detailed demographic (age, sex, address), clinical (presenting symptoms, co-morbidity, days of intubation if present), and laboratory investigation details (C-reactive protein, d-Dimer, procalcitonin, X-ray findings) were collected from all the patients.

Methodology of BFPP testing:

• Samples were tested on the BFPP according to the manufacturer’s instructions, which are as follows:
• Principle of the test:
• The BioFire FilmArray disposable pouch is a freeze-dried multiplex PCR assay that contains all the necessary reagents for sample preparation, reverse transcription PCR, PCR, and detection.
• During a test run, the system extracts and purifies all nucleic acids from the unprocessed sample. It then 7performs nested multiplex PCR in two stages. The first stage involves a single, large-volume, multiplexed reaction, while the second stage includes individual, single-plex reactions to detect the products from the first stage.
• Using endpoint melting curve data, the BioFire System software automatically analyses the results for each target on the panel. At the end of the test run, the software reports whether each pathogen is detected in the sample or not. This information is printed in an automated response at the end of the test run.
• The BFPP targets 27 pathogens, including bacteria (such as Acinetobacter calcoaceticus-baumannii complex, Enterobacter cloacae, Escherichia coli, Haemophilus influenzae, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus sp., Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes), Atypical bacteria (like Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae), and viruses {including Influenza A, Influenza B, Adenovirus, Coronavirus, Parainfluenza virus, Respiratory Syncytial Virus, Human Rhinovirus/Enterovirus, Human Metapneumovirus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV)}. It also detects seven AMR genes (ESBL-CTX-M; Carbapenemases- KPC, NDM, Oxa48-like, VIM, IMP; methicillin resistance mecA/mecC and MREJ), with an analysis time of about one hour, including sample preparation and DNA/RNA extraction.
• Each test includes two internal controls: (1) to verify nucleic acid extraction; and (2) to verify PCR performance. The run is considered valid only if both controls pass.
• Bacteria are reported semi-quantitatively at ≥107, 106, 105, and 104 copies/mL of the specimen, where 104 copies/mL is the significant cut-off limit.
• Viral and atypical bacterial agents are reported as not detected or detected.
• AMR-genes are reported as positive only in the case of simultaneous detection of a compatible pathogen, i.e., S. aureus in combination with mecA/mecC and MREJ.
• Values calculated below 104 copies/mL are reported as “not detected.”

Methodology for culture Standard of Care (SOC) test:

• All specimens are cultured conventionally using blood agar, chocolate agar, and MacConkey agar plates and are incubated at 35°C in 5% CO2. All plates are examined for growth at 18-24 hours and 48 hours of incubation (if no growth is observed at 18 to 24 hours).
• Any growth is subjected to identification by conventional methods, and antibiotic susceptibility testing is performed using the Kirby Bauer disc diffusion method on Mueller Hinton agar.

Statistical Analysis

Patient characteristics, clinical and laboratory investigation data, as well as data for all BioFire FilmArray findings of respiratory viral and bacterial agents, and antibiotic resistance genes, were collected. They were then entered into an Excel sheet and analysed using SPSS software version 28.0 to calculate percentages.

Results

A total of 81 different samples from patients with non-COVID pneumonia were tested using the BFPP. The majority of samples were sputum (67, 82.7%), followed by BAL (08, 9.8%), and ET aspirate (06, 7.5%) (Table/Fig 1).

Among the 81 patients, 38 (47%) were female and 43 (53%) were male. The mean age of the patients was 47.8 years, with the minimum and maximum ages being 14 years and 82 years, respectively. The demographic details and infection marker test results are presented in (Table/Fig 2).

Patients presented with various clinical signs and symptoms. Bilateral pneumonia was the most common clinical diagnosis in 33 (40.7%) of the patients, followed by SARI in 25 (30.8%), and other respiratory manifestations such as LRTI, Cor-pulmonale, Acute Respiratory Distress Syndrome (ARDS), etc., in 11 (13.5%) of the patients (Table/Fig 3).

Out of the 81 samples processed, 63 (78%) samples showed the presence of only bacterial pathogens, and only viruses were detected in 5 (6.2%) cases. Two or more bacterial pathogens were detected in 58 (71.6%) samples, while only a single type of bacterium was detected in 5 (6.2%) samples. Bacterial-viral co-detection was observed in 13 (16%) of the samples (Table/Fig 4).

In the 81 samples tested, the BFPP detected a total of 185 bacterial and viral pathogens. Among them, 161 (87.02%) were bacterial agents, including 6 (3%) atypical bacteria. Among all bacterial pathogens, the highest number was 39 (24.2%) Klebsiella pneumoniae, followed by 33 (20.5%) Acinetobacter calcoaceticus-baumannii complex. The distribution of pathogens isolated from non COVID SARI patients by the BFPP is shown in (Table/Fig 5).

The BFPP detects pathogens using semi-quantitative log bin values. A pathogen was marked as detected only when it was detected at ≥104 copies/mL of the sample. (Table/Fig 6) depicts the numbers of bacterial pathogens with their corresponding log bin values as detected by the BFPP.

Among the 168 bacterial pathogens detected, 167 (99.4%) were detected by BFPP, and 152 (90.4%) were detected by the SOC test. The detection rate by BFPP and SOC was 100% for Klebsiella pneumoniae group, Acinetobacter calcoaceticus-baumannii complex, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella aerogenes, and Klebsiella oxytoca. BFPP was negative in only one case of the on-panel bacterium, Enterobacter cloacae complex, which was detected by SOC but not by BFPP, and the authors could not provide justification for this discrepancy. In the case of Streptococcus pneumoniae and Serratia marcescens, 100% were detected by BFPP, but only 50% were detected by SOC tests. For the detection of Streptococcus agalactiae by the SOC test, it was considered insignificant as the growth was very scanty, but BFPP detected it with a log bin value of 104 copies/mL. Fastidious organisms like H. influenzae and atypical bacteria like Mycoplasma pneumoniae and Legionella pneumophila were detected only by BFPP, while SOC failed to detect them. This suggests that BFPP was a more sensitive method for detecting fastidious and atypical organisms that could be missed by routine conventional culture techniques. The distribution of negative and positive results for all bacteria detected by BFPP and isolated by SOC is shown in (Table/Fig 7).

A total of 162 AMR genes were detected from 161 bacterial pathogens using BFPP. Among the bacterial pathogens, the AMR gene for NDM was detected in 42 (25.9%) cases, followed by CTX-M beta-lactamases in 32 (19.8%) cases, VIM (Verona integron-encoded metallo-β-lactamase) in 29 (18%) cases, and Oxacillinase group of β-lactamases (OXA) in 27 (16.6%) cases. The detection of AMR genes was reported only if an applicable bacterium (i.e., potential carrier of AMR genes) was also detected in the sample. (Table/Fig 8) shows the distribution of AMR genes isolated from all bacterial pathogens detected by BFPP.

Discussion

The COVID-19, first recognised in Wuhan, China, in December 2019, has rapidly spread across the globe and become a public health crisis. The emergence and geographical spread of COVID-19 coincided with the influenza season in many temperate regions (12),(13). The typical clinical presentation of SARS-CoV-2 is an acute respiratory illness or viral pneumonia with rapid respiratory deterioration (14). The COVID-19 pandemic has also affected bacterial co-pathogens, which are commonly identified in respiratory tract infections and are an important cause of morbidity and mortality, necessitating timely diagnosis and antibacterial therapy (15).

Many systemic bacterial infections may present with respiratory signs or progress to complicated pneumonia. Empiric therapy for SARI often involves broad-spectrum antibiotics to cover both gram-positive and gram-negative bacteria due to the risk of infection with multidrug-resistant pathogens (16). However, the broad use of empiric antibiotic treatment for undiagnosed infections has been associated with increased antibiotic resistance globally (17). Therefore, identifying the specific pathogen(s) is crucial for prompt initiation of targeted and effective therapy.

Rapid detection of the causative agent of respiratory infections, coupled with the detection of prominent markers of antibiotic resistance, can help reduce unnecessary broad-spectrum antimicrobial treatment (18).

This study is one of the first from Central India aimed at detecting other bacterial and viral agents in COVID-19-negative SARI patients using BFPP and guiding clinicians about appropriate antimicrobial therapy during the COVID-19 pandemic. The BioFire FilmArray is a rapid method for the detection and identification of common bacterial pathogens and resistance markers in suspected SARI patients. It also identifies viral agents and atypical bacteria that are challenging to isolate using conventional culture techniques.

A total of 81 samples from SARI patients were tested in this study, of which 18 (22%) samples were positive for viral targets, including 13 (16%) specimens with bacterial-viral co-detection. Bacterial co-detection was identified in 58 (71.6%) cases. In a study by Webber DM et al., bacterial-viral co-detection was noted in 4.6% and 2.9% of sputum and BAL samples, respectively, while only virus detection was observed in 8.5% and 12.9% of samples (19). In another similar study by Yoo IY et al., co-detection of two pathogens was found in 23% of samples, three pathogens in 12% of samples, and four or more pathogens in 9% of specimens, although the authors did not provide details about the specific types of co-detection (20).

Among the total of 18 viral agents detected in the present study, 16 (89%) were Human Enterovirus/Rhinovirus, and 2 (11%) were Adenovirus. In a study by Kyriazopoulou E et al., (21), which evaluated BFPP for cases of LRTI, bacterial-viral co-infections were observed in 25.6%, higher than in this study, and bacterial co-detection was seen in 17.8%, much lower compared to this study. In another similar study, bacteria and virus co-detection were found in 14.78%, and bacterial co-detection was seen in 26.27% (22).

The most commonly detected organism in this study was Klebsiella pneumoniae, with 39 (24.2%) cases, followed by 33 (20.5%) cases of Acinetobacter calcoaceticus-baumannii complex, and 26 (16.2%) cases of Pseudomonas aeruginosa. Fastidious organisms like Haemophilus influenzae and Streptococcus pneumoniae were detected in 3.1% and 2.5% of cases, respectively. In a study by Kosai K et al., that aimed to detect pathogens of LRTI using BFPP, the most commonly detected organisms were Pseudomonas aeruginosa and Staphylococcus aureus, both with 17 (20%) cases, followed by Klebsiella pneumoniae in 10 (12%) of the detected organisms. Haemophilus influenzae 06 (7%) and Streptococcus pneumoniae 04 (4.7%) were also detected in the same study (23). These findings are almost comparable to the findings of this study.

In a study by Faron ML et al., BFPP identified 67% more total bacterial targets in BAL samples and 100% more targets in sputum samples compared to culture. Additionally, it identified multiple potential pathogens (up to 4) in 14% of BAL samples and 34% of sputum specimens, compared with 7% and 16% of routine cultures, respectively (24). Results from the FilmArray may aid in the earlier identification of respiratory pathogens and optimisation of antibiotic therapy. Respiratory bacterial pathogens impact public health by affecting healthy and immunocompromised individuals, causing postviral infections in both community and hospital settings (25).

The worldwide problem of AMR particularly hampers developing countries due to high infectious disease exposure, antibiotic overconsumption, and poor quality of antibiotics (26). Several studies have reported the emergence of Multi-Drug Resistant (MDR) bacterial pathogens from different sources, including humans, birds, and cattle, which highlights the need for routine application of antimicrobial susceptibility testing methods to screen and detect emerging MDR strains [27-30].

The present study also aimed to detect the aetiological agents and associated AMR genes (ARGs) early, so that prompt treatment could be initiated with the appropriate drug to inhibit the emergence of drug resistance. NDM production was found in 42 (25.9%) of the bacterial pathogens. Since the discovery of NDM-producing bacteria in 2008, NDM carbapenemases have been reported globally (31). NDM enzymes confer resistance to almost all β-lactam drugs (except aztreonam), including carbapenems, which are often considered the drugs of last resort for the treatment of serious infections (31). Treatment options for infections caused by NDM-producing bacteria are very limited. In a study, out of 356 clinical isolates, 160 showed carbapenem resistance, and among them, 131 displayed Metallo β-Lactamases (MBLs) production. PCR amplification confirmed 31 (23.6%) isolates carrying the blaNDM-1 gene in MBLs-producing organisms (32). This finding was comparable to the results of the present study. Apart from a significant number of isolates producing NDM, the authors in this study also found CTX-M, VIM, OXA, and IMP in 32 (19.8%), 29 (18%), 27 (16.6%), and 13 (8%) isolates, respectively. The expression of these genes limits treatment options and often leads to poor prognosis. Additionally, the sensitivity for the detection of these genes is lower than conventional methods.

Klebsiella pneumoniae Carbapenemase (KPC) has been the most frequently encountered class A carbapenemase since its first description in the eastern USA in 1996 [31,33]. KPC is often associated with hospital and nosocomial infections, resulting in high morbidity and mortality (34). In the present study, 6 (3.7%) KPC was detected out of 162 ARGs. In a study by Awoke T et al., the overall prevalence of carbapenemase production among total K. pneumoniae isolates was 28/132 (21.2%) using the modified Carbapenem Inactivation Method (mCIM) (35). Despite a high disease burden, reports from India on the prevalence of resistance mechanisms in MDR K. pneumoniae isolates are limited (36).

Among the 14 S. aureus isolates detected in this study, 13 showed the presence of mecA/mecC or I#IMREJ I?Igene. These genes encode a Penicillin-Binding Protein (PBP2a) that leads to low affinity for beta-lactam antibiotics and are carried on a chromosomally integrated mobile genetic element known as the Staphylococcal Cassette Chromosome mec (SCCmec). To distinguish between Methicillin-Resistant S. aureus (MRSA) or co-detection of Methicillin-Sensitive S. aureus (MSSA) and another Staphylococcus spp. carrying the SCCmec cassette and mecA/mecC, the BFPP contains an additional assay that detects the SCCmec Right-Extremity Junction (MREJ), which links the SCCmec cassette to the S. aureus genome and indicates MRSA. In a study by Buchan BW et al., among 18 isolates with routine susceptibility results available, the BFPP demonstrated 81.8% (9/11) sensitivity and 85.7% (6/7) specificity for the identification of MRSA (based on the detection of both mecA and mecC and the SCCMREJ) (37).

As the world has witnessed the devastating waves of COVID-19, healthcare facilities were overcrowded even in developed countries (38). The overwhelming number of cases increased the burden on frontline Healthcare Workers (HCWs) in patient-facing roles (39). Although there is no documentation stating breaches in infection control practices in such overcrowded and overburdened healthcare facilities during the COVID-19 pandemic, the combination of increased workload and reduced numbers of HCWs is likely to strain the capacity to maintain essential healthcare services (40). In such situations, the chances of nosocomial cross-contamination with MDR superbugs from one patient to another increase. In a study, it was observed that patients with Carbapenem-Resistant Enterobacteriaceae (CRE) infections were three times more likely to receive inappropriate empiric treatment than non CRE infections. In turn, inappropriate empiric treatment was associated with an adjusted increase in mortality of 12% (95% confidence interval 3% to 23%) and an excess of 5.2 days (41). To combat this, early detection of aetiological agents and associated ARGs plays an important role.

With technically low demanding preprocessing of samples and a shorter turnaround time for detection, the BFPP may provide actionable information on antimicrobial susceptibility and ARGs for key pathogens causing pneumonia.

This multiplex PCR-based test has raised the expectations of clinical microbiology laboratories and clinicians regarding the possibilities for rapid and accurate diagnosis of infective aetiology in respiratory tract infections. The upcoming years should see a continued increase in options for rapid, sensitive, and simple-to-perform molecular assays for infectious diseases.

However, despite the wide array of detection and many advantages of BFPP, there are a few limitations when using BFPP for respiratory samples:

1. The respiratory tract, being an unsterile site, has a lot of commensals, non pathogenic, or colonised flora, and sample collection is prone to oropharyngeal contamination. The challenge of interpretation from the pneumonia panel is determining if the organisms detected are clinically significant.
2. Determining whether the AMR genes that are detected are actually being expressed in the pathogen or not poses a challenge when considering treatment changes.
3. The cost of BioFire FilmArray is high compared to traditional methods and other commercially available molecular diagnostic tools.
4. Finally, the impact of new and emerging infections cannot be denied. In recent years, COVID-19 or the monkeypox virus reminded us of how quickly a new and emerging virus can appear and spread. The challenge for manufacturers is to regularly and timely accommodate potential emerging pathogens in the detection panel while maintaining cost-effectiveness.

Limitation(s)

The samples were processed only from hospitalised patients. Additionally, the sample size was low. The follow-up of the escalation or de-escalation of antibiotic therapy after the report of BFPP could not be tracked.

Conclusion

Respiratory tract infections are caused by a wide array of pathogens, including viruses, typical bacteria, and atypical bacteria. In the treatment of these infections, drug resistance among gram negative pathogens poses a risk factor for inappropriate empiric treatment, subsequently increasing the risk of mortality. The BFPP tests for a wide range of pathogens, along with their associated AMR genes, promptly and with high sensitivity and specificity. This study has an impact on the timely detection of aetiological agents and associated antimicrobial-resistant genes that cause SARI in non COVID patients through the use of BFPP.

Acknowledgement

The authors acknowledge the assistance provided by the District Collectors of Nagpur and Amravati in procuring the BioFire respiratory panel kits.

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DOI and Others

DOI: 10.7860/JCDR/2024/65688.18972

Date of Submission: Jun 01, 2023
Date of Peer Review: Jul 24, 2023
Date of Acceptance: Nov 20, 2023
Date of Publishing: Jan 01, 2024

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

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Jun 02, 2023
• Manual Googling: Aug 18, 2023
• iThenticate Software: Nov 17, 2023 (14%)

ETYMOLOGY: Author Origin

EMENDATIONS: 7

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