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 : 2022 | Month : March | Volume : 16 | Issue : 3 | Page : DC14 - DC19 Full Version

Diagnostic Significance of Wet Mount Microscopy- A Retrospective Observational Study


Published: March 1, 2022 | DOI: https://doi.org/10.7860/JCDR/2022/51015.16120
Abha Sharma, Poonam Sood Lomba, Bibhabati Mishra, Ashna Bhasin, Sulmaz Reshi

1. Associate Professor, Department of Microbiology, GIPMER, New Delhi, India. 2. Director Professor, Department of Microbiology, GIPMER, New Delhi, India. 3. Consultant Microbiologist, Department of Microbiology, GIPMER, New Delhi, India. 4. Senior Resident, Department of Microbiology, GIPMER, New Delhi, India. 5. Senior Resident, Department of Microbiology, GIPMER, New Delhi, India.

Correspondence Address :
Abha Sharma,
AC 61, Second Floor, Tagore Garden, New Delhi-110027, India.
E-mail: abha_sh79@rediffmail.com

Abstract

Introduction: Wet mount microscopy is a rapid and easy conventional technique that provides a quick answer when positive and it provides an approximation of the infection burden. In this era of modern medicine where almost every infection is being diagnosed, using expensive and sophisticated molecular techniques, a simple wet mount examination of a clinical sample can still be relevant in several infections and play a significant role in the early diagnosis and treatment of infectious diseases.

Aim: To focus and review the significance of wet mount examination of clinical specimens for diagnosis.

Materials and Methods: A retrospective observational study with 11 cases was conducted at a superspeciality hospital in New Delhi over a period of six months from March 2019 to August 2019. Direct microscopic demonstration of motile E. histolytica trophozoites in saline wet mount of colonic biopsy specimens was done in patients presenting with non specific gastrointestinal symptoms. Positive cases were immediately reported to the clinician for further management. In view of this study, literature search was done on PubMed and Google scholar platforms for studies on wet mount examination of clinical specimens, data was analysed and all infectious diseases were identified for which wet mount microscopy will help in decreasing the Turn around Time (TAT) for diagnosing the infection from 24 hours to 20-30 minutes in real time.

Results: Motile trophozoites of E. histolytica were seen in eight out of 11 cases studied. Amoebic serology by Enzyme-linked Immunoassay (ELISA) and histopathology for amoebic trophozoites was positive in all eight patients with 100% association between microscopic demonstrations of motile E. histolytica trophozoites in saline wet mount for amoebiasis.

Conclusion: Right test at right time for clinical management of infectious diseases requires good communication between laboratory and clinicians. Direct Wet mount microscopy of clinical samples is rapid, simple, cost-effective method to decrease TAT and guide the clinicians to rule out infections and avoid unnecessary empirical use of antimicrobial drugs.

Keywords

Clinical specimen, Diagnostic stewardship, Gastrointestinal symptoms, Infectious disease

The role of Rapid Diagnostic Tests (RDTs) and point of care tests in clinical microbiology for diagnosis of infectious diseases has been the focus of researchers in recent years and there have been significant developments also. The RDTs have the potential to affect diagnostic algorithms and therapeutic decisions (1). Many new rapid molecular diagnostic techniques for infectious diseases have been developed that provide faster results enabling rapid delivery of patient care (2). The appropriate use of laboratory tests to guide patient treatment and optimise clinical outcome is diagnostic stewardship. The ultimate goal is to limit spread of antimicrobial resistance. Diagnostic stewardship is a collaborative effort with team work between laboratory and clinician so as to order the right test at right time and result can be interpretated with less TAT in real time for early patient management (3).

However, it seems that in today’s world in order to achieve this goal the clinical laboratories are directly jumping into automation and molecular diagnostic techniques for diagnosing almost all infections. This might be the best approach in the developed countries but in developing countries with limited resources, except for viral infections, the role of old conventional techniques in diagnosis of all other infections should still have a place in clinical diagnostic microbiology. A direct simple wet mount examination of clinical specimen is facing extinction in this era of modern medicine. The aim of this study was to focus and review the significance of wet mount examination of clinical specimens in order to bring the attention of clinical microbiologists towards the role of simple, cost effective laboratory test like wet mount microscopy that can also easily guide in patient management.

Material and Methods

It was a retrospective observational study conducted at a superspeciality hospital in New Delhi over a period of six months from March 2019 to August 2019. After receiving approval from the Institutional Ethical Committee (EC/Micro/03/2/2019/10). The role of direct microscopy in the diagnosis of amoebic colitis was studied and in view of this study, literature search was done on PubMed and Google scholar platforms for studies done on wet mount examination of clinical specimens. Data was analysed and all infectious diseases were identified for which wet mount microscopy will help in decreasing the TAT for diagnosing the infection from 24 hrs to 20-30 minutes in real time.

Inclusion criteria: Referred cases presenting to the Gastroeneterology Out-patient Department of a superspecialty hospital with non specific gastrointestinal symptoms and history of bloody diarrhoea, suspected of having chronic amoebic colitis on clinical examination were included in the study. Clinical history was recorded in a proforma.

Exclusion criteria: All patients with no history of diarrhoea and no clinical history suggestive of colitis were excluded from the study.

Study Procedure

Serum sample was collected from all patients for amoebic serology by ELISA. Colonoscopic examination of all patients was perfomed for evidence of diffuse mucosal inflammation, with or without mucosal ulceration. Two colon biopsy specimens were collected during colonoscopy. One was kept in sterile container filled with formalin and sent to the pathology laboratory for histopathological examination for confirmation of invasive amebiasis in colonic tissue. The other specimen was kept in sterile container filled with normal saline and immediately transferred to the microbiology lab within 15 minutes of taking the biopsy for direct saline wet mount preparation of colonic biopsy specimen. In the microbiology laboratory, a wet mount on glass slide was prepared by taking a small drop of saline on the slide and adding a small amount of biopsy tissue from the container with the help of clean forceps. Then a cover slip was placed over the wet mount preparation on slide and the slide was examined under 400x (high power) of light microscope. Direct microscopic demonstration of motile E. histolytica trophozoites in saline wet mount of colonic biopsy specimens was done (Table/Fig 1). The positive cases were immediately reported to the clinician for further management of the cases.

Statistical Analysis

Descriptive statistics were used for analysis of the data.

Results

A total of 11 cases were presented and studied (males=7 and females=4) with mean age 52.2 years for females and 25 years for males. Motile trophozoites of E. histolytica were seen in eight cases. Amoebic serology by ELISA and histopathology for amoebic trophozoites was positive in all eight patients. There was 100% association between microscopic demonstration of motile E. histolytica trophozoites in saline wet mount of colonic biopsy and trophozoites seen in histopathologic examination of colonic biopsy and amoebic serology for serum antibodies for amoebiasis (Table/Fig 2).

Discussion

In order to prevent treatment failure and the development of antimicrobial resistance it is important that early presumptive diagnosis of infectious diseases gain attention by the researchers. For the judicious use of antimicrobials it is necessary that both over diagnosis and under diagnosis of infections be avoided. This can be achieved only by adopting appropriate diagnostic stewardship strategy for diagnosing various infections. Strategies for diagnostic stewardship are essential not only for bacterial infections but also for parasitic, viral and fungal infections. In the present study to achieve this objective real time analysis of colonic biopsy tissue was done for invasive amoebiasis by demonstrating motile E. histolytica trophozoites in saline wet mount preparation of biopsy so that presumptive diagnosis of amoebic colitis is made in real time with a TAT of 15-30 minutes. The clinician could decide whether to rule out Inflammatory Bowel Disease (IBD) and start metronidazole therapy or not immediately after receiving the microscopy report. Otherwise metronidazole would be prescribed empirically until the histopathology reports and amoebic serology reports are available TAT 24-48 hours (sometimes even more time is taken for reporting depending on workload of the laboratories).

Metronidazole is the standard empirical drug for the treatment of suspected cases of invasive amoebiasis irrespective of acute or chronic infection, whether invasive or not. Because of the indiscriminate use of metronidazole for treatment of diarrhoea, metronidazole resistance has been reported in E. histolytica from various parts of the world (4). It is essential that the specimen be transported immediately to the laboratory. Right test at right time for clinical management requires good communication between laboratory and clinicians which is definitely the need of the hour. The TAT of direct visualisation of motile trophozoites in biopsy is just 15 to 30 minutes in comparison to 12-24 hours TAT in case of histopathology of biopsy or ameobic serology also for that matter.

The wet mount preparation has the advantage of being simple, minimal infrastructure requirement, performed immediately and therefore allows immediate preventive, diagnostic, or therapeutic action where a positive result is found. Wet mount also gives a fair idea about the approximate burden of an infection. It easily helps in identifying the characteristic motility of various parasitic trophozoites and bacteria causing infections in humans. The disadvantage of wet mount microscopy is that it dries within few minutes, making it difficult to visualise or identify the live organism. For later consultation or demonstration, a fresh wet mount preparation needs to be made each time to view slides thus consuming the resources and time of the technical staff (5),(6). Various type of techniques are used for wet mount microscopy (Table/Fig 3) (7),(8). The most common wet mount technique is the saline wet mount used mainly for parasitic infections followed by Potassium Hydroxide (KOH) mount used for fungal infections. List of infections that can be easily diagnosed with simple wet mount microscopy is given in (Table/Fig 4) (9),(10),(11),(12),(13),(14),(15),(16),(17),(18),(19),(20),(21),(22),(23),(24),(25),(26),(27),(28). The TAT for these infections can be reduced to 30 minutes from 24-48 hours which is the TAT for culture and molecular methods used in their diagnosis.

In comparison to staining, culture and molecular techniques a wet mount preparation procedure is simple (Table/Fig 5) (28). A drop of saline or simple dye like India ink, methylene blue, lactophenol cotton blue or KOH solution is put on a glass slide. The sample is added and a cover slip is put and the mount is ready to be examined under the light microscope. Whereas direct smear staining methods have different multistep procedures that require technical expertise, smears need to be prepared followed by heat or chemical fixation and one or more than one dye is needed for staining. Several kinds of differential and special stains are required in staining techniques for direct examination of fixed clinical samples like Gram’s stain, Albert stain, Acid fast stain, Giemsa, fluorescent stains etc., (29). Moreover, more complex and expensive microscopes like fluorescent microscope, phase contrast microscope etc., are required to view the specially stained slides. Culture and Polymerase Chain Reaction (PCR) techniques also require a complex infrastructure, technical expertise and are time consuming and labor intensive although they have a wider scope and higher sensitivity and specificity. Similarly, for other infections also, mainly bacterial, fungal and parasitic, a wet mount microscopy will be useful in greatly decreasing the TAT for presumptive diagnosis of those infections and thus guide the empirical therapy to be given. Viral infections can also be diagnosed by direct microscopy but it requires electron microscope and complex infrastructure which is beyond the scope of this discussion.

Diagnostic Significance of Wet Mount Microscopy

Bacterial infections: Many clinicians rely on routine microscopic examination of urine without culture as indicator of probable UTI and guide their treatment based on the positive results (Table/Fig 6), (Table/Fig 7). Urine culture is the Holy Grail in diagnosis of Urinary Tract Infection (UTI) however the significance of preliminary urinalysis by wet mount microscopy cannot be neglected (30). Studies have shown that the sensitivity of urine wet mount ranges between 40-70% and specificity between 85-95% in predicting UTI (31),(32). Evaluation of suspected UTI includes history, physical examination and laboratory investigations. Urine wet mount analysis for presence of pus cells (pyuira) and bacteria (bacteruria) are important in the adequate management of UTI (31). In the absence of significant bacteriuria, presence of pyuria in a symptomatic patient (e.g., acute urethral syndrome) is an indication for treatment and hence the importance of wet mounts microscopy (32).

Vaginal fluid wet mount examination helps to assess the status of vaginal lactobacillary flora, which is an indicator of vaginal infection and pregnancy complications (33). Fatima A et al., in their study found sensitivity of 85.85%, specificity of 72.89%, positive predictive vale of 64.88% and negative predictive value of 89.55% for urine wet mount in detection of UTI (5). Leptospirosis is a wide spread zoonotic disease in the world. In India, several outbreaks have been reported especially South-India, mainly between June and October due to heavy rains and floods (34). Dark field microscopy is used to detect Leptospires in body fluids like urine. Thin, bright, actively motile spiral bacilli with characteristic rapid spinning and jerking motility are suggestive of Leptospires. The disadvantage of this technique is that it is less sensitive and specific as atleast 10 leptospires/mL must be present for one cell per field to be visible by darkfield microscopy (34).

Listeria monocytogenes is a gram-positive bacterium that is not very common but important cause of severe CNS infections. L. monocytogenes infection can lead to meningitis, supratentorial abscesses (commonly in immunocompromised individuals) and even brainstem encephalitis (rhomboencephalitis) in immunocompetent patients. Due to the high mortality and common serious sequelae in survivors, an accurate initial diagnostic approach is important for applying appropriate treatment early (35). Though early diagnosis is challenging but Listeria demonstrates “tumbling motility” in wet mounts of Cerebrospinal Fluid (CSF) and if positive can contribute towards timely patient management.

Parasitic infections: Through wet mount microscopy of centrifuged urine sediment, a wide morphological spectrum of parasites causing pyuria and haematuria may be diagnosed. The morphological awareness guides in prompt and effective management in most cases (36). Parasites commonly found in urine are Trichomonas, Schistosoma hematobium and micofilaria (9),(37). Schistosoma infection (Bilharziasis) and filarial infection is not common in India (10). Trichomonas vaginalis causing vaginitis and urethritis in females can be seen in urine and vaginal discharge or swab wet mount examination. Both spun urine (centrifuged deposits) in conjunction with vaginal fluid examination improves the detection rate of Trichomonads (11). Trichomonads also infect the male urogenital tract and are an important sexually transmitted infection. The trophozoites of T. vaginalis can be seen in urinary sediments from males as well (12). Rare reports of urinary balantidiasis have been been seen mostly in immunocompromised patients (13),(14). The trophozoites of ciliated parasite Balantidium coli can be seen in urine sediment wet mount microscopy. Even the larvae of Strongyloides stercoralis and planoconvex egg of Enterobius vermicularis have been reported in urine wet mount (36). The urine sediment wet mount examination Tetteh-Quarcoo PB et al., examined urine sample in one study microscopically for the presence of S. haematobium eggs and 18.0% urine samples were positive (15).

Parasites causing diarrhoea are present commonly in water supplies, infecting a large proportion of the human population in the developing countries (16). Stool is the most common specimen collected and examined for demonstration of parasites of the gastrointestinal tract. Usually two preparations, saline and iodine wet mount of stool is made on a single slide. The saline wet mount is made up of physiological saline and is an unstained preparation. The advantage of saline wet mount is that it demonstrates the motility of trophozoites. However, internal structures are often poorly visible in saline mount and to overcome this simple stain solutions like iodine, methylene blue, lactophenol cotton blue have been used for preparation of temporarily stained wet mounts of stool sample. Commonly iodine wet mount is prepared from stool sample however many studies have used other stains also for better results. A combination of methylene blue and glycerol in wet mount preparation of fresh stool samples was used by Khanna V et al., for the demonstration of medically important intestinal parasites (16). They found that methylene blue-glycerol combination provided an excellent contrast between the parasitic structures and the artefacts as compared to saline and iodine mount. A similar type of study was done by Parija SC and Prabhakar PK with similar results with different parasites where they evaluated the use of lactophenol cotton blue wet mount preparation as an alternative to routine saline and iodine wet mounts (17).

Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrillaris, and Sappinia sp. are pathogenic free-living amoebae. Chronic granulomatous encephalitis is caused by Acanthamoeba spp. and B. mandrillaris. Acanthamoeba spp. also skin and eye infection. Amoebic Keratitis is infection of the cornea that is associated with contact lens use or corneal trauma and can lead to loss of sight. Sappinia pedata has been identified as the cause of a nonlethal case of amoebic encephalitis (18). The direct detection of the causative agent in corneal scraping specimen is the only reliable diagnostic method for Amoebic Keratitis (19). Although culture and PCR techniques are definitely more sensitive, however, amoeba density is high in severe infection and the amoebic trophozoites can already be detected by direct microscopy of the clinical sample (19). Direct wet mount microscopy of CSF sediments after gentle centrifugation of samples is recommended to look for the trophozoites for the diagnosis of Acanthamoeba in CSF (20). Primary Amoebic Meningoencephalitis (PAM) a rare condition, caused by free-living amoeba Naegleria fowleri, is associated with fresh water exposure (swimming in ponds and lakes in the summer). The ameobae penetrate through the nasal mucosa and rapidly progressive meningoencephalitis ensues after a brief incubation period. The clinical course of N. fowleri infection is fulminant rapidly progressing to death in a median of five days. Wet mount examination of the CSF reveals motile trophozoites (21). Presumptive diagnosis of N. fowleri infection can be done via direct microscopic examination of the CSF immediately after sample collection although confirmatory diagnosis requires specialised experience, immunohistochemical staining, or Polymerase Chain Reaction (PCR) testing (22). In a study done by Capewell LG et al., PAM was diagnosed before death (or in the case of the survivors, discharge) in 27% (39 of 142) of the patients (23). In nearly all (38 of 39) cases, motile amoebas were seen in a wet mount of the CSF.

The hooklets and scolex of Echinococcus granulosus (dog tapeworm) are visible in the cystic fluid wet mount examination of aspirate from liver in case of hydatid liver disease (Table/Fig 8).

Fungal infections: Culture is the gold standard for diagnosing fungal infections but they are expensive and time consuming (requires six weeks incubation). While a 10% KOH mount of clinical samples is clinically useful for early diagnosis of common superficial fungal infections. The KOH is a cheap and readily available. It digests, and clears cellular and keratin debris but leaves fungal hyphae cell wall intact which is resistant to digestion by KOH, thus clearing the background and allows visualisation of fungal elements under a microscope. Gautam M and Bhatia S in their study showed that KOH has higher sensitivity as compared to fungal culture (70 to 100%) for cutaneous dermatophyte infections, Tinea capitis or tinea barbae, Onychomycosis, Tinea versicolor and candidiasis (6). The KOH mount examination of sputum samples help to identify Aspergillus infection by the presence of V shaped branching and septate hyphae. In case of Mucormycosis, aseptate hyphae will be seen on KOH mount of clinical sample (Table/Fig 9). Mucormycosis is a life-threatening fungal infection that occurs in immunocompromised patients. During Coronavirus Disease-2019 (COVID-19) pandemic increasing number of mucormycosis cases has been reported from India (24). Rapid diagnosis of mucormycosis is important to guide timely initiation of amphotericin B and possible surgical intervention in order to reduce morbidity. The clinical presentation, signs, and radiographic manifestations of mucormycosis are nonspecific and so direct KOH examination of clinical samples like tissue, respiratory secretions, bronchoalveolar lavage, and other body fluids is frequently done (25).

The incidence of cryptococcosis in India is on the rise thus becoming a serious threat. High morbidity and mortality are associated with cryptococcal meningitis and therefore early and timely diagnosis is essential to prevent serious complications (26). The CSF India ink examination has provides an immediate, specific diagnosis of cryptococcal meningitis within minutes of receiving the CSF sample in the laboratory (Table/Fig 10). In one study by Saha DC et al., sensitivity and specificity of India ink examination of CSF for cryptococcus was found to is 83.3% and 96.49%, respectively (27). The laboratory diagnosis of cryptococcal meningitis includes direct visualisation of cryptococci via microscopy, culture of the organism and/or the detection of cryptococcal antigens in CSF (38). Although India ink has low sensitivity and specificity yet it is still widely used for the detection of cryptococci in CSF, particularly in resource-limited countries as it is cheap, rapid and reliable (38),(39).

Limitation(s)

The limitation of this study was that only amoebic colitis cases have been studied here by direct microscopy and rest all infection list has been collected and compiled according to the literature search. Further research needs to be done by correlating the effect of reduced TAT of diagnosis by direct microscopic examination with patient outcome and management not only in amoebic colitis cases but also other infections.

Conclusion

Wet mount microscopy should not just remain a teaching tool for training microbiology residents but the significance of this simple although limited in scope diagnostic test for several infectious diseases needs to be reassessed and analysed probably in countries like India with limited resources. In resource limited settings with absence of culture and PCR facilities, a rapid, simple and cost-effective technique like microscopy of wet mount preparation of clinical specimens with a TAT of 15 to 30 minutes is the first step towards diagnostic stewardship initiative to avoid empirical use of antimicrobial agents atleast in positive or heavily infected cases where organism load is high and therefore presumptive diagnosis can easily be made on wet mount microscopy. Similar studies on larger sample size with direct impact on use of antimicrobial drugs and clinical management is the way forward.

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DOI and Others

DOI: 10.7860/JCDR/2022/51015.16120

Date of Submission: Jun 23, 2021
Date of Peer Review: Oct 20, 2021
Date of Acceptance: Feb 19, 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? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

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• iThenticate Software: Feb 03, 2022 (12%)

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