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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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 : 2022 | Month : March | Volume : 16 | Issue : 3 | Page : DC25 - DC30 Full Version

Evaluation of Lateral Flow Assay for the Diagnosis of Cryptococcal Meningitis and its Comparison with the Gold Standard and Other Laboratory Tests


Published: March 1, 2022 | DOI: https://doi.org/10.7860/JCDR/2022/52172.16138
Kottarathil Malavika, Thayanidhi Premamalini, Anupma Jyoti Kindo

1. PhD Scholar, Department of Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, Tamil Nadu, India. 2. Associate Professor, Department of Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, Tamil Nadu, India. 3. Professor and Head, Department of Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, Tamil Nadu, India.

Correspondence Address :
Dr. Thayanidhi Premamalini,
Associate Professor, Department of Microbiology, Sri Ramachandra Medical College
and Research Institute, SRIHER, Chennai, Tamil Nadu, India.
E-mail: drtpremamalini@gmail.com

Abstract

Introduction: Cryptococcosis is a potentially fatal fungal disease affecting both immunocompromised and immunocompetent individuals. Hence, a Point Of Care Test (POCT) is required with higher sensitivity and specificity (100%), for the rapid detection of cryptococcosis which will be life saving for the patient.

Aim: To evaluate the usefulness of Lateral Flow Assay (LFA) for rapid detection of Cryptococcal Antigen (CrAg) from Cerebrospinal Fluid (CSF) sample.

Materials and Methods: This diagnostic/pilot study was conducted in Mycology Laboratory, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India, over a period of six months (June 2019-November 2019). The CSF samples (n=37) from patients with suspected meningitis were considered for the study. Direct microscopy with gram stain, negative stain and histopathological stains was performed for all the CSF samples. Culture was done on basal medium, enriched media and special media. Cryptococcus genus specific Polymerase Chain Reaction (PCR) was also performed to confirm the isolates grown on the culture. Serological tests like Latex Agglutination Test (LAT) and LFA were also performed on all the samples. The LFA results were compared with microscopy, culture and LAT.

Results: Among the 37 suspected meningitis patients, three grew Cryptococcus in culture which was considered as the gold standard in the diagnosis of Cryptococcal Meningitis (CM). Direct microscopic techniques had 100% specificity however their sensitivity was less i.e., 67%. LAT had a sensitivity of 100% and specificity of 94%. However, LFA in present study had a very good sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) (100%).

Conclusion: The LFA for the detection of CM is considered to be an effective method when compared to the other conventional and serological methods with higher sensitivity and specificity. Hence, in present study authors evaluated the use of LFA in diagnosis of CM and found that this assay when used as a POCT, can give the results in short duration. Considering this as a pilot study, further studies including higher number of samples are essential to confirm the effectiveness of the findings.

Keywords

Cryptococcal antigen, Immunochromatography, Point of care test, Sensitivity, Specificity

Globally, the annual estimate of Cryptococcal disease-related deaths exceeds 180,000 (1). This fungal infection is preventable and treatable yet, due to the delay in diagnosis it continues to be associated with extreme morbidity and mortality (2). The infection caused by Cryptococcus spp. is collectively known as Cryptococcosis, which is a major global health problem since the advent of Human Immunodeficiency Virus (HIV) pandemic in 1980 (3). Cryptococcus spp. primarily causes pulmonary cryptococcosis as the route of entry of the propagules is by inhalation. This mainly occurs in immunocompetent individuals. In immunocompromised individuals the infection gets disseminated to the brain causing CM. The genus Cryptococcus includes three main species among which Cryptococcus neoformans (C. neoformans) is most commonly involved in causing CM (4).

The laboratory diagnosis of cryptococcosis is generally based on direct microscopic demonstration, culture, serological tests for the Cryptococcal Antigen (CrAg) detection and molecular assays. Microscopic methods are highly specific, but may have low sensitivity and require expertise for its interpretation (5). Culture though considered as the gold standard, may be time consuming. PCR offers an excellent alternative for the early diagnosis of cryptococcosis compared to conventional methods, and can detect low fungal loads (6). But PCR can be performed only in laboratories with good molecular setup and requires expertise for performance and interpretation.

Serological tests are found to have more sensitivity and specificity when compared to other tests. LAT is more sensitive but suffers from the limitation of false positivity as well as high rates of false negativity. Also, performing LAT is a tedious process, it require a rapid immunochromatographic test to detect CM with a short turnover time. LFA is instrument free, simple and a rapid diagnostic test having higher sensitivity and specificity when compared to LAT (7),(8). Thus, in this study, the aim was to evaluate the performance of LFA for diagnosing CM, by comparing the results obtained by LFA with the gold standard (culture) and other tests (microscopy and serology).

Material and Methods

This diagnostic/pilot study was conducted in the Mycology Laboratory, Sri Ramachandra Medical College and Research Institute over a period of six months (June 2019-November 2019). The study was started after obtaining clearance from Institutional Ethics Committee (IEC) (REF: CSF/19/MAY/77/156).

Inclusion criteria: The CSF samples from all patients of suspected meningitis (based on clinical presentation and biochemical analysis of CSF) which was sent to the laboratory in six months were included in the study.

Exclusion criteria: Specimens with visible lipids or any other obvious signs of contamination were excluded from the study.

Study Procedure

Sample collection and preservation of the culture: A total of 37 CSF samples from patients with suspected meningitis were considered for the study. Direct microscopy, histopathological examination, culture and serological tests were performed for all the samples. Culture positive isolates were maintained in Sabouraud Dextrose Agar (SDA) slopes and stored at room temperature.

Phenotypic characterisation: Microscopic examination of all the 37 samples was done by Gram stain, Negative stains (India Ink and Nigrosin) and Histopathological stains (Giemsa stain and Haematoxylin & Eosin). All the samples were inoculated into basal fungal culture media (SDA and Oat meal agar), special media (bird seed agar, cumin seed agar, coriander seed agar) and differential agar {Cryptococcus differential agar, Creatinine Dextrose Bromothymol blue Thymine agar (CDBT)} for culture identification. Cryptococcus spp. utilise the phenolic and polyphenolic compounds present in the special media, producing melanin which is absorbed by the yeast cell wall forming a tan to reddish-brown pigmented colonies. Differential agar helps in the identification of different species of Cryptococcus, as C. neoformans produce dry blue coloured colonies, C. gattii produces brown mucoid colonies and C. laurentii produces dry brown colonies. CDBT helps in the further identification of the serotypes of Cryptococcus spp. In CDBT agar, serotype D (Cryptococcus neoformans var. neoformans) produces orange coloured colonies, serotype A (Cryptococcus neoformans var. grubii) does not grow, whereas C. gattii produces blue green colonies.

Genotypic characterisation: The PCR was performed for the isolates which grew in culture, for the confirmation of the identification by phenotypic methods.

DNA extraction (Phenol-Chloroform method): A loopful of colony was mixed with 500 μL of lysis buffer Tris HCl EDTA Sodium chloride Sodium dodecyl sulphate (TESS buffer) in a microcentrifuge tube and kept in a water bath at 100oC for 1 minute. To this mixture 500 μL of Phenol: Chloroform (1:1) was added and vortexed. The tube was centrifuged at 10,000 rpm for 10 minutes. The aqueous layer was transferred to a new tube and equal volume of chloroform was added, vortexed and centrifuged at 10,000 rpm for 10 minutes. Equal volume of ice cold absolute Isopropyl Alcohol (IPA) was added and mixed gently and the tubes were spun at 10,000 rpm for 10 minutes. The aqueous layer was discarded and the resultant Deoxyribonucleic Acid (DNA) pellet was washed in 200 μL of 70% ethanol and centrifuged at 10,000 rpm for 10 minutes. The alcohol was discarded and the tubes were air dried. The DNA pellet was resuspended in 50 μL elution buffer/nuclease free water and stored at -20oC until use.

Cryptococcus genus specific Polymerase Chain Reaction (PCR): The PCR was performed using the genus specific primers CN4 F (5’ATC ACC CTA CCA TTC ACA CATT-3’) and CN5 R (5’GAA GGG CAT GCC AGA TGT TTG3’) along with suitable positive and negative controls. These primers flanking ITS rDNA region will generate a fragment of 136 bp.

Composition of PCR reaction mix and reaction condition: The PCR master mix was prepared containing 10 μL of PCR mix (Takara, Japan), 0.5 μL of forward (CN4) and reverse primer (CN5), 7 μL of template DNA, and the volume made upto 20 μL with sterile nuclease free water. The reaction mixtures were amplified in a thermal cycler (Veriti 96 well, Applied Biosystems, USA), with the following program: 95°C for 5 minutes, followed by 35 cycles consisting of 95°C for 30 seconds, 67°C for 30 seconds, and 72°C for 30 seconds, with a final extension period at 72°C for 7 minutes. After thermal cycling, 10 μL of the amplified product was run on a 1.5% (wt/vol) agarose gel, stained with ethidium bromide, and visualised with ultraviolet light.

Serological tests: Serological tests like LAT test and LFA were performed on all the samples.

Latex Agglutination Test (LAT): Samples were centrifuged and the supernatant was used for the latex test of C. neoformans antigen detection. The test was performed using the CALAS kit Meridian Bioscience (Lot no: 140100K308), according to the manufacturer’s instructions. Boiling the CSF specimens and then cooling them to room temperature prior to testing will limit the non specific interference. The positive control should give a positive reaction with the detection latex and a negative reaction with the control latex. LAT CSF titres of 1:4 or less are presumptive evidence of central nervous system infection by C. neoformans, still additional follow-up and culture are strongly recommended. CSF titres of 1:8 or greater (significant titre) from patients with suspected meningitis strongly suggest infection by C. neoformans.

Lateral Flow Assay (LFA): The LFA was performed using the BIOSYNEX Crypto PS (Lot no:180220 & 180221), according to the manufacturer’s instructions. It is a rapid immunochromatographic test for the semi-quantitative detection and titration of Cryptococcus spp. capsular antigens in CSF to guide the diagnosis of CM. The test does not require any pretreatment of the samples.

Interpretation: Two distinct coloured lines in the control (C) and test line (T1)-positive. Three distinct coloured lines in the control (C) and test lines (T1) and (T2)-Strong positive. A coloured line appearing in the control (C) and no lines on both the test lines-negative. The test line (T1) appears at 25 ng/mL of capsular antigen and the test line (T2) appears at 2.5 μg/mL of capsular antigen. No visible coloured line in the control line (C) but coloured lines present in both the tests (T1) and (T2) is considered invalid (9).

Statistical Analysis

The sensitivity, specificity, PPV and NPV were calculated using Statistical Package for the Social Sciences (SPSS), Version 17.0 software considering culture as gold standard.

Results

Age and gender distribution: The age distribution of individuals suspected with meningitis ranged from 0 months-85 years. The age distribution is shown in (Table/Fig 1). Among the 37 patients with suspected meningitis, 23 of them (62.16%) were male and 14 of them (37.84%) were female. Out of the three patients with proven CM, two were male and one was a female patient.

Co-morbid conditions and risk factors: Among the 37 patients with suspected meningitis diabetes mellitus (13/37), immunosuppressive therapy (6/37) and Decompensated Liver Disease (DCLD) (5/37) were the major risk factors/co-morbid conditions associated. The distribution of other risk factors/co-morbid conditions among the patients is shown in the (Table/Fig 2). Out of the three patients with proven CM one patient had DCLD and one was on immunosuppressive therapy and the third patient was with uncontrolled diabetes mellitus.

Direct Microscopy

Gram stain: Among the 37 CSF samples, only two of them showed spherical gram positive yeast cells with narrow-necked budding (Table/Fig 3).

Negative stains- India Ink and Nigrosin stain: Out of the 37 samples, only two samples showed spherical budding yeast cells with a clear halo around, which represents the presence of capsule in India Ink (Table/Fig 4)a and Nigrosin stain (Table/Fig 4)b.

Histopathological stains: Among the 37 samples processed, only two samples showed purple coloured spherical, budding yeast cells of Cryptococcus spp. in Giemsa stain (Table/Fig 5)a and a clear capsule surrounding the pale blue nucleus of Cryptococcus sp. in Haematoxylin and Eosin (H&E) stain (Table/Fig 5)b.

Phenotypic Identification

Basal media: Out of 37 samples, only three grew in basal culture media (SDA and Oat meal agar) producing creamy mucoid colonies after three days of incubation at 37oC (Table/Fig 6)a,b

Special media: Bird seed agar (BSA), Cumin agar and coriander agar: Among the 37 CSF samples, three which grew on basal media produced brown coloured colonies on BSA, Cumin agar and Coriander agar seen after 48 hrs of incubation at 37°c due to the utilisation of phenolic compounds present in the media (Table/Fig 7)a-c.

Differential Media

Cryptococcus differential agar: Out of 37 CSF samples, all the three culture positive isolates produced dry blue coloured colonies (Table/Fig 8). Hence, identified as Cryptococcus neoformans.

Creatinine Dextrose Bromothymol blue Thymine agar (CDBT): Among the 37 CSF samples, CDBT agar identified two as Cryptococcus neoformans var. neoformans (Orange colonies) (Table/Fig 9)a and one as Cryptococcus neoformans var. grubii (no growth) (Table/Fig 9)b in three culture positive isolates.

Genotypic characterisation: Genotypic characterisation was performed for the three culture positive isolates by PCR for confirmation.

Cryptococcus genus specific PCR: Cryptococcus genus specific primers resulted in specific amplification of a single DNA fragment at ~136 bp for positive control (C. neoformans ATCC 14116) and all the three culture positive isolates. Negative control (Candida albicans ATCC 90028) did not yield any amplification (Table/Fig 10).

Serological Tests

Latex Agglutination Test (LAT): Out of the 37 CSF samples, five of them showed agglutination in LAT. Among the five samples, one sample showed a titre of >1:64, two of them showed a titre of 1:16 (Significant titre: 1:8). Two samples which showed a titre of 1:8 were direct microscopy and culture negative.

Lateral Flow Assay (LFA): Out of the 37 CSF samples screened by LFA, three samples (LAT titre >1:64 and 1:16) showed strong positive result by producing three distinct coloured lines (C, T1 and T2) (Table/Fig 11)a and the rest showed a negative result (Table/Fig 11)b. All the three samples were culture positive. The LFA results were compared with conventional and other serological tests (Table/Fig 12).

Discussion

The CM is a life-threatening opportunistic infection. Management of CM still remains a challenge because of the delay in diagnosis. Starting the patient on oral fluconazole at an early stage can reduce risk of CM and its mortality (10),(11). Since for the sake of advantage, early screening and prompt initiation of pre-emptive fluconazole therapy, decreasing the time from first clinic visit to testing and afterwards treatment are crucial. Hence, in this study the performance of LFA which detects the cryptococcal capsular antigen was assessed, which can be a POCT and performed even by trained nurses in a hospital care setting.

Three patients with proven CM in present study were in the age group of 41-60 years. It has also been established in previous studies that elderly population was most commonly affected by CM (12). Ageing is a process that negatively impacts the immune system and its capability to function. Also, associated disabilities and co-morbidities are common in the elderly (13). These factors make the elderly individuals more vulnerable to infectious diseases (14). However, the characteristic presentations of infectious diseases are not always noted in elderly patients, making it difficult to make an early diagnosis leading to a delay in treatment (15). Hence, timely diagnosis and management of these age group patients acts as an important life saving measure.

Majority of the patients with suspected meningitis, and two out of the three patients with CM were men. Previous studies by Tay ST et al., and McClelland EE et al., also have reported an increased incidence of the disease among men (74/96 and 12/28 respectively) (16),(17). McClelland EE et al., in his study had also stated that, macrophages from females phagocytosed more cryptococcal cells than macrophages from males, also male macrophages had a higher fungal burden and showed increased killing by Cryptococcus spp. This may be due to an interaction of Cryptococcus with testosterone that results in increased Glucuronoxylomannan (GXM) release and Cryptococcus-mediated macrophage death (17). The above data suggested that Cryptococcus strains are adapted to different hosts and the male individuals may be more prone to cryptococcal infection.

The DCLD was identified as one of the major risk factors in one of the patients with proven CM, in present study. Decompensated liver cirrhosis was identified as an independent risk factor in previous studies for the occurrence of invasive cryptococcosis (18),(19),(20). The pathogenesis could involve multiple aspects. First, although Cryptococcus spp. typically enters the human body via inhalation through the respiratory system, people may become infected by ingesting contaminated food (19). The presence of collateral circulation in decompensated liver cirrhosis allows the ingested Cryptococcus to bypass the liver scavenger system and directly enter the circulatory system, thereby causing cryptococcaemia and being further disseminated into the central nervous system. Other reported possible risk factors include diabetes mellitus, lymphoproliferative malignancy, haematological malignancy, cancer, autoimmune diseases, and lung diseases (21). The second patient in present study with CM was on immunosuppressive therapy. Cell mediated immunity has also been shown to play a critical role in the host’s defense against C. neoformans in animal models (22),(23). The third patient with proven CM was a female patient with uncontrolled diabetes mellitus. Diabetes mellitus was present in 8.5-33% of cryptococcosis cases in reported series (24),(25). Diabetes mellitus is a relative common condition in the age group of 41-60 years and uncontrolled diabetes mellitus leads to lowering of immunity and hence predispose to infections.

The laboratory diagnosis of cryptococcosis is generally based on direct demonstration, culture, and antigen detection by LAT. In present study, direct microscopic methods like gram stain, negative stain (India ink and Nigrosin) and histopathological stains though were 100% specific, showed a low sensitivity (67%) when compared to culture, which is the gold standard test for the diagnosis of CM. This finding was similar to the finding of other studies which also stated that microscopic methods, though specific, showed a sensitivity of 50-80% (5),(26),(27). This means that there will be many false negatives and few false positives, when CM is diagnosed with this test. In the present study, the NPV for the direct microscopy was found to be 97%, which means that there is always a 3% chance of people who are tested negative to have the disease. Also, microscopy requires laboratory infrastructure, it is dependent on fungal concentration and is highly operator dependent rather than the test performance (28).

In this study, all the samples which grew on culture were positive by PCR, thereby confirming its identification. Culture is considered as the gold standard for diagnosis of CM. But still culture takes time and requires more labour and large volumes of samples. Hence, both microscopy and culture cannot be used for the early diagnosis of CM.

In present study, LAT had 100% sensitivity and 94% specificity, but a low PPV of 60%. This means that there is only a 60% probability for the patient who is tested positive to have the disease. Hence, LAT may have lot of false positive reactions. Though earlier studies also had demonstrated a good sensitivity and specificity of >99% for LAT and is less labour intensive than culture, it also requires technical expertise and laboratory infrastructure (29),(30). Culture and LAT cannot be performed in resource limited settings, thus limiting their clinical utility (31). Henceforth, a serological test which will have less false positive reactions and easy to perform without instruments would be ideal for rapid diagnosis of CM.

In the present study, LFA had higher sensitivity, specificity, PPV and NPV of 100%, when compared to the LAT. Blood, serum, or CSF can be used in LFA and is stable at room temperature, has rapid turn-around time of (<15 min), and user friendly (9). The LFA meets the World Health Organisation’s Affordable Sensitive Specific User friendly Robust and Rapid Equipment free Deliverable (ASSURED) to those who need them criteria for being affordable, sensitive, specific, rapid and robust, equipment free, user-friendly, and deliverable to end-users (32). Moreover, the present paper suggest that LFA surpass the other standard diagnostic tests that are currently been used for the detection of CrAg.

Limitation(s)

Since present study was performed with low sample size, further studies including higher number of samples need to be performed to establish the accuracy of present study findings.

Conclusion

The availability of this assay as a POCT not only in tertiary healthcare settings, but also in remote locations could have a meaningful impact on cryptococcal diagnosis, assisting in early treatment thereby reducing the morbidity and mortality rate. In view of the advantages of LFA over other tests, consideration should be given to establish the CrAg LFA as the current gold standard for the detection of cryptococcal infection.

Acknowledgement

Authors would like to express their heartfelt thanks to the Management, Sri Ramachandra Institute of Higher Education and Research (SRIHER), Chennai, Tamil Nadu, India, for providing excellent infrastructures and facilities for research.

References

1.
Lakoh S, Rickman H, Sesay M, Kenneh S, Burke R, Baldeh M, et al. Prevalence and mortality of cryptococcal disease in adults with advanced HIV in an urban tertiary hospital in Sierra Leone: A prospective study. BMC Infectious Diseases. 2020;20(1):01-07. [crossref] [PubMed]
2.
Stott KE, Loyse A, Jarvis JN, Alufandika M, Harrison TS, Mwandumba HC, et al. Cryptococcal meningoencephalitis: Time for action. Lancet Infect Dis. 2021;21(9):e259-71. [crossref]
3.
Pharkjaksu S, Kwon-Chung KJ, Bennett JE, Ngamskulrungroj P. Population diversity and virulence characteristics of Cryptococcus neoformans/C. gattii species complexes isolated during the pre-HIV-pandemic era. PLoS Neglected Tropical Diseases. 2020;14(10):e0008651. [crossref] [PubMed]
4.
Kwon-Chung KJ, Fraser JA, Doering TL, Wang ZA, Janbon G, Idnurm A, et al. Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis. Cold Spring Harbor Perspectives in Medicine. 2014;4(7):a019760. [crossref] [PubMed]
5.
Snow RM, Dismukes WE. Cryptococcal meningitis: Diagnostic value of cryptococcal antigen in cerebrospinal fluid. Archives of Internal Medicine. 1975;135(9):1155-57. [crossref]
6.
Paschoal RC, Hirata MH, Hirata RC, Melhem MD, Dias AL, Paula CR. Neurocryptococcosis: Diagnosis by PCR method. Revista do Instituto de Medicina Tropical de São Paulo. 2004;46(4):203-07. [crossref] [PubMed]
7.
Perfect JR, Bicanic T. Cryptococcosis diagnosis and treatment: What do we know now. Fungal Genetics and Biology. 2015;78:49-54. [crossref] [PubMed]
8.
Kabanda T, Siedner MJ, Klausner JD, Muzoora C, Boulware DR. Point-of-care diagnosis and prognostication of cryptococcal meningitis with the cryptococcal antigen lateral flow assay on cerebrospinal fluid. Clinical Infectious Diseases. 2014;58(1):113-16. [crossref] [PubMed]
9.
Koczula KM, Gallotta A. Lateral flow assays. Essays in Biochemistry. 2016;60(1):111-20. [crossref] [PubMed]
10.
Mfinanga S, Chanda D, Kivuyo SL, Guinness L, Bottomley C, Simms V, et al. Cryptococcal meningitis screening and community-based early adherence support in people with advanced HIV infection starting antiretroviral therapy in Tanzania and Zambia: An open-label, randomised controlled trial. The Lancet. 2015;385(9983):2173-82. [crossref]
11.
Kapoor SW, Magambo KA, Kalluvya SE, Fitzgerald DW, Peck RN, Downs JA. Six-month outcomes of HIV-infected patients given short-course fluconazole therapy for asymptomatic cryptococcal antigenemia. AIDS (London, England). 2015;29(18):2473. [crossref] [PubMed]
12.
Tsai WC, Lien CY, Lee JJ, Hsiao WC, Huang CR, Tsai NW, et al. The clinical characteristics and therapeutic outcomes of cryptococcal meningitis in elderly patients: A hospital-based study. BMC Geriatrics. 2019;19(1):01-06. [crossref] [PubMed]
13.
Gruver AL, Hudson LL, Sempowski GD. Immunosenescence of ageing. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland. 2007;211(2):144-56. [crossref] [PubMed]
14.
Lai WA, Chen SF, Tsai NW, Chang CC, Chang WN, Lu CH, et al. Clinical characteristics and prognosis of acute bacterial meningitis in elderly patients over 65: A hospital-based study. BMC Geriatrics. 2011;11(1):01-06. [crossref] [PubMed]
15.
Nau R, Djukic M, Spreer A, Ribes S, Eiffert H. Bacterial meningitis: An update of new treatment options. Expert Rev Anti Infect Ther. 2015;13(11):1401-23. [crossref] [PubMed]
16.
Tay ST, Rohani MY, Soo Hoo TS, Hamimah H. Epidemiology of cryptococcosis in Malaysia. Mycoses. 2010;53(6):509-14. [crossref] [PubMed]
17.
McClelland EE, Hobbs LM, Rivera J, Casadevall A, Potts WK, Smith JM, et al. The role of host gender in the pathogenesis of Cryptococcus neoformans infections. PloS one. 2013;8(5):e63632. [crossref] [PubMed]
18.
Singh N, Sifri CD, Silveira FP, Miller R, Gregg KS, Huprikar S, et al. Unique characteristics of cryptococcosis identified after death in patients with liver cirrhosis: Comparison with concurrent cohort diagnosed antemortem. Medical Mycology. 2017;55(3):278-84.
19.
Lin YY, Shiau S, Fang CT. Risk factors for invasive Cryptococcus neoformans diseases: A case-control study. PloS one. 2015;10(3):e0119090. [crossref] [PubMed]
20.
Cheng JH, Yip CW, Jiang YK, Zhou LH, Que CX, Luo Y, et al. Clinical predictors impacting cryptococcal dissemination and poor outcome in patients with cirrhosis. InOpen forum Infectious Diseases. 2021;8(7):ofab296. US: Oxford University Press. [crossref] [PubMed]
21.
Pfaller MA, Diekema DJ. Epidemiology of invasive mycoses in North America. Critical Reviews in Microbiology. 2010;36(1):01-53. [crossref] [PubMed]
22.
Nishimura K, Miyaji M. Histopathological studies on experimental cryptococcosis in nude mice. Mycopathologia. 1979;68(3):145-53. [crossref] [PubMed]
23.
Salkowski CA, Balish E. Role of natural killer cells in resistance to systemic cryptococcosis. Journal of Leukocyte Biology. 1991;50(2):151-59. [crossref] [PubMed]
24.
Chuang YM, Ho YC, Chang HT, Yu CJ, Yang PC, Hsueh PR. Disseminated cryptococcosis in HIV-uninfected patients. European Journal of Clinical Microbiology & Infectious Diseases. 2008;27(4):307-10. [crossref] [PubMed]
25.
Tseng HK, Liu CP, Ho MW, Lu PL, Lo HJ, Lin YH, et al. Taiwan Infectious Diseases Study Network (TIDSnet) for cryptococcosis. Microbiological, epidemiological, and clinical characteristics and outcomes of patients with cryptococcosis in Taiwan, 1997-2010. PloS one. 2013;8(4):e61921. [crossref] [PubMed]
26.
Abassi M, Boulware DR, Rhein J. Cryptococcal meningitis: Diagnosis and management update. Current Tropical Medicine Reports. 2015;2(2):90-99. [crossref] [PubMed]
27.
Binnicker MJ, Jespersen DJ, Bestrom JE, Rollins LO. Comparison of four assays for the detection of cryptococcal antigen. Clinical and Vaccine Immunology. 2012;19(12):1988-90. [crossref] [PubMed]
28.
Gal AA, Koss MN, Hawkins J, Evans S, Einstein H. The pathology of pulmonary cryptococcal infections in the acquired immunodeficiency syndrome. Archives of Pathology & Laboratory Medicine. 1986;110(6):502-07.
29.
Gitonga LK, Boru WG, Kwena A, Maritim M, Wamicwe J, Ransom J. Point of care testing evaluation of lateral flow immunoassay for diagnosis of Cryptococcus meningitis in HIV-positive patients at an urban hospital in Nairobi, Kenya, 2017. BMC Research Notes. 2019;12(1):01-06. [crossref] [PubMed]
30.
Huang HR, Fan LC, Rajbanshi B, Xu JF. Evaluation of a new cryptococcal antigen lateral flow immunoassay in serum, cerebrospinal fluid and urine for the diagnosis of cryptococcosis: A meta-analysis and systematic review. PloS one. 2015;10(5):e0127117. [crossref] [PubMed]
31.
Kozel TR, Bauman SK. CrAg lateral flow assay for cryptococcosis. Expert Opinion on Medical Diagnostics. 2012;6(3):245-51. [crossref] [PubMed]
32.
World Health Organization. Rapid advice: Diagnosis, prevention, and management of cryptococcal disease in HIV-infected adults, adolescents and children. Geneva: WHO Document Production Services; 2011:01-37.

DOI and Others

DOI: 10.7860/JCDR/2022/52172.16138

Date of Submission: Aug 31, 2021
Date of Peer Review: Nov 24, 2021
Date of Acceptance: Jan 03, 2022
Date of Publishing: Mar 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? NA
• For any images presented appropriate consent has been obtained from the subjects. NA

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