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.
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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
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,
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,
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 ones 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
(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 journalsNo manuscriptsNo 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)
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.
On April 2011

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
On Jan 2020

Important Notice

Year : 2012 | Month : May | Volume : 6 | Issue : 3 | Page : 503 - 509 Full Version

Microbiological Stool Examination: Overview

Published: May 1, 2012 | DOI:
Kotgire Santosh A.,

1. Assistant Professor Department of Microbiology, Dr. Ulhas Patil Medical College, Jalgaon, Maharashtra, India.

Correspondence Address :
Santosh Kotgire
Assistant Professor, Department of Microbiology,
Dr. Ulhas Patil Medical College, Jalgaon, Maharashtra, India.
Phone: +919922867558

Stool (faeces) is an important body substance which has to be checked for the presence of disease-causing microorganisms [1,2,3,4].
Diarrhoea: Is defined as an increase in the frequency, fluidity or the volume of the bowel movement, which is relative to the usual habit of an individual. The passage of three or more motions a day is considered as diarrhoea.
Dysentery: It is defined as the passage of blood and mucus stained stools, which is often associated with abdominal cramps and tenesmus. Gastroenteritis:
Is defined as an inflammation of the mucus membranes of the stomach and the intestines, resulting in diarrhoea which is associated with vomiting.

Examination of the stool for bacteria Bacterial agents which are responsible for diarrhoea (1)
1. Gram positive • Staphylococcus aureus • Clostridium perfringens • Clostridium difficle • Bacillus cereus 2. Gram negative • Vibrios (i) Vibrio cholera (ii) Vibrio parahemolyticus (iii) Other halophlic vibrios • Escherichia coli(ETEC, EPEC) • Salmonella (i) S .enteritidis (ii) S.typhimurium • Shigella spp • Campylobacter jejuni • Yersinia enterocolitica

Bacterial agents which are responsible for dysentery (1)
1. Shigella spp (sh. dysentriae, sh.flexneri, sh.boydii and sh.sonnei) 2. Escherichia.coli (EIEC and EHEC) 3. V.parahemolyticus 4. Campylobacter jejuni 5. Salmonella spp

Notes on pathogens 1. Staphylococcus aureus. S. aureus produces six types of enterotoxins (A to F). The type A is most often incriminated in outbreaks of staphylococcal foodReportpoisoining. Acute staphylococcal food poisoining is caused by the ingestion of preformed toxin contaminated food (often diary products). The incubation period is 2-6 hours and it is associated with an acute onset of nausea and vomiting, sometimes followed by diarrhoea [1,2].

2. Bacillus cereus It is an aerobic, spore forming, gram positive bacillus. It produces two types of toxins, one which resembles the LT of E.coli and the other which resembles staphylococcal enterotoxin. It causes two distinct clinical manifestations, one which is characterized by diarrhoea and abdominal pain (incubation period of 6-15 hours), which is similar to gastroenteritis that is caused by E.coli and the other which is characterized by nausea and vomiting, which resembles Staphylococcal food poisoning (short incubation period of 1-4 hours). Food poisoining is caused by the ingestion of preformed toxin which is usually present in fried rice or other cereals which have been cooked and stored at warm temperatures (1).

3. Clostridium perfringens This organism is widely distributed in soil and it is found in the faeces of humans and animals. Gastroenteritidis which is caused by C.perfringens is characterized by diarrhoea and abdominal cramps, following the ingestion of contaminated food (poultry products and meat). Most of cases are caused by the type A strains which produce an enterotoxin. (incubation period of 6-12 hours) [3,4].

4. Clostridium difficle It is associated with antibiotic associated diarrhoea and pesudomembrane colitis. The common antibiotics which are involved are lincomycin, clindamycin, ampicillin and the cephalosporins. There is an overgrowth of antibiotic-resistant C.difficle due to the suppression of the normal cut flora (1).

5. Vibrio cholera The classical as well as the EITor biotypes of V. cholerae cause cholera. The severe dehydration, vomiting, abdominal pain and acidosis which are associated with cholera are due to the action of the exotoxin, CT(cholera toxin), which activates cAMP, leading to the outpouring of fluid and electrolyte-diarhoea. In severe cases, the typical rice water stools (without faecal matter) are passed, necessitating urgent fluid replacement therapy to prevent collapse and death. V.Parahaemolyticus causes invasive diarrhoea and it is responsible for a majority of the food poisoning cases in many parts of the world, including Asia, Africa, Europe and America. The foods which are responsible include raw seafood (fish and shell fish). The mechanism of the diarrhoea which is produced by it is not well understood. It produces a heat labile enterotoxin like the one which E.coli produces [5,6].

6. E.coli The strains of E.coli which are recognized to cause diarrhoeal diseases include enterotoxigenic E.coli (ETEC) and enteropathogenic E.coli (EPEC). ETEC produces a heat-labile enterotoxin (LT) and a heat stable enterotoxin (ST), resulting in mild to severe diarrhoea in the developing countries. It is an important cause of travelleler’s diarrhoea. EPEC have been implicated in diarrhoea in infants, that neither produce toxins nor do they invade the gut mucosa. The exact mechanism of their action is not known. They adhere to the mucosal cells of the small intestine and multiply [5,6].

7. Shigella species S.dysenteriae, S.flexneri, S.bodyii and S.sonnei cause bacillary dysentery. The dysentery which is caused by the Shigella species is referred to as shigellosis. The Shigella spp causes at least 50% of the cases of bloody diarrhoea in young children and adults in the developing countries. S.dysenteriae type 1 is particularly virulent, causing epidemic and endemic dysentery. It is highly infectious and resistant to the commonly available antibiotics (2). 8. Salmonellae Acute gastroenteritis which is caused by the Salmonella spp is characterized by self limiting fever and diarrhoea. The incubation period is 12-36 hrs. A large majority of the outbreaks are caused by, S.typhimurium and S .enteritidis. The human infection is usually caused by the consumption of animal foods or food products. The infection leads to salmonella food poisoning and sometimes septicaemia may develop (2).

9. Campylobacter jejuni C.jejuni is the common cause of enteritis in the developing countries. It occurs in the intestinal flora of many animals, especially poultry, which probably serves as the major source of the infections in humans. The milk and waterborne outbreaks of diarrhoea have also been documented. The disease is sometimes associated with vomiting and bloody mucoid stools [1,2].

10. Yersinia enterocolitica. Y. enterocolitica has been identified as an important cause of diarrhoea. The sources of the infection are birds and animals. Foodborne outbreaks have also been reported. The predominant symptom in young children is acute watery diarrhoea. In older children and young adults, there is pain in the abdomen and fever [1,2].

Examination of the stool for parasites (7) 1. Protozoa • Entamoeba histolytica • Giardia lamblia • Intestinal Coccidian Parasites (i) Cryptosporidium parvum (ii) Cyclospora (iii) Isospora • Balantidium Coli 2. Helminthes • Nematodes: (i) Ascaris lumbricoides (ii) Trichuris trichuria (iii) Hookworm • Ancylostoma duodenale • Nectar americans

(iv) Strongyloides stercoralis • Cestodes: (i) Taenia spp • T. saginata • T.solium (ii) Hymenolepsis nana (iii) Enterobius vermicularis

Notes on pathogens 1. Entamoeba histolytica It is endemic in many parts of the tropical and sub-tropical areas. It is transmitted by the faeco-oral route. The cysts which contain four nuclei are indicative of an infective stage in humans. It produces amoebic dysentery which is characterized by large, flask shaped ulcers. It may get complicated into amoebic liver abscess and amoebic lung abscess (7).

2. Giardia lamblia The infection with Giardia lamblia may lead to explosive watery diarrhoea, foul smelling stools and steatorrhoea. In endemic areas, young children are more frequently infected than the adults, particularly those who are malnourished. It produces a severe form of infection in immuno-compromised individuals, particularly in those with AIDS (7).

3. Coccidian parasites Cryptosporidium parvum, Isospora. Belli and Cyclospora have been recognized as important agents which are responsible for the watery diarrhoea in immunocompromised patients, particularly in those with HIV-AIDS (7).

4. Balantidium coli It is an uncommon parasite which is found in humans. It commonly infects pigs and it has a world-wide distribution. It produces dysentery which is transmitted by the ingestion of infective cysts in food and water or from hands which are contaminated with pig faeces (7). 5. Ascaris lumbricoides It has a world-wide distribution and its spread occurs by the faecal pollution of the environment. A person becomes infected by ingesting its infective eggs through contaminated food or by eating with contaminated hands (7).

6. Trichuris trichiura It is common in the moist, warm climates. Its infection is spread by ingesting its infective eggs through contaminated food or by eating with contaminated fingers. In children, it can cause chronic diarrhea and intestinal ulceration with blood and mucus (7). 7. Hookworm Its infection is caused by A.duodenale and Nectar americanus, with Ancylostoma as the predominant species. The infection is spread by the faecal pollution of the soil. The infection occurs when the infective filariform larvae penetrate the skin. Hookworm resides in the intestine and sucks blood, leading to iron deficiency anaemia and chronic blood loss (7). 8. Strongyloides stercoralis It is endemic in many tropical and sub-tropical countries. The infection occurs by the penetration of skin by the infective filariform larvae. In immuno-competent hosts, it is generally asymptomaticor it produces minimal symtoms. But in immuno-compromised hosts, it produces a potentially life threatening infection called the hyperinfection syndrome (7).

9. Taenia Spp. Two species, namely T.saginata and T.solium, are responsible for the infections in humans. The infection is mainly transmitted by eating raw or insufficiently cooked beef and pork meat respectively. T.solium is not as widely distributed as T.saginata, but it can produce a serious infection called neurocysticercosis, which causes epilepsy and other central nervous disorders (7). 10. Hymenolepsis nana It is transmitted by ingesting its eggs from food or water or from hands which are contaminated with infected faeces. The eggs are infective when they are passed in the faeces. An internal auto-infection is a common problem which is caused by H.nana (7).
11. Enterobius vermicularis It has worldwide distribution, with children being more commonly infected than adults. Its transmission is by the injestion of infective eggs. The eggs are deposited on the anal skin usually during the night hours. An autoinfection is common in children because the eggs cause intense irritation and scratching in the infected anal area (7).

Examination of the stool for viruses 1. Rota virus 2. Norwalk virus 3. Adenovirus 4. Astrovirus 5. Calcivirus 6. Coronavirus

Rotavirus This virus is now considered as the most common cause of diarrhoea in infants and young children. The disease peaks at the ages of six months to two years. The mode of the infection is believed to be the faeco-oral route. The incubation period is 2-4 days. Vomiting is a predominant early symptom, which often precedes diarrhoea. The stools are watery and they are associated either mild fever or respiratory symptoms [1,4,6].

Other viruses have also been frequently incriminated in producing diarrhoea and vomiting in infants and young children [1,4,6].
Laboratory examination of faeces
Collection of the stool specimen
The faeces for the microbiological examination should be collected during the acute stage of the diarrhoeal disease [1,4,6,7,8]. • Ask the patient to pass the stool sample in a clean, dry, disinfectant free, suitable, wide–necked container or a plastic cup with a tight fitting lid. • About 20-40 grams of well-formed stool or 5-6 table spoonfuls of watery stool will suffice for a routine examination. • The ingestion of some medicines prior to the collection of the faecal sample may interfere with the detection of the micro-organisms. These include tetracyclines, sulfonamides, antiprotozoalagents, laxatives, antacids, castor oil, magnesium hydroxide, barium sulphate, bismuth kaolin compounds, hypertonic salts, etc. These should not be taken 1-2 weeks before the examination of the stool sample.

• All the specimens must be properly labelled with the patient’s name, age, sex, and the date of the sample collection. Note • Do not keep the specimen at warm temperatures. Try to keep it in cool places. • Prevent the drying of the specimen. • Prevent its contamination with urine or dirt particles. • Multiple stool examinations are required before the presence of an infection is ruled out. • The stool should not be collected from bed-pans which contain disinfectants.

Rectal swabs Only when it is not possible to obtain faeces, should a specimen be collected by using a cotton wool swab. The swab should be inserted in the rectum for about 10 seconds. Care should be taken to avoid unnecessary contamination of the specimen with bacteria from the anal skin [7,8].

The adhesive tape method This is useful for the detection of the eggs of E.vermicularis. The eggs can be collected by wrapping a strip of clear adhesive tape (e.g. cellotape, scotch tape) around the anus. After collecting the eggs, the tape should be sticked lengthways, face down on a microscope slide. Alternatively, an anal or perianal specimen can be collected by using a National Institute of Health (NIH) swab [7,8].
Transport of the specimen

• The specimen must reach the laboratory within 30 minutes of passing of the stool, since the motile organisms, for example, Vibrio and amoebic trophozoites are heat sensitive and they can die or become unrecognizable after that period. • Transport media such as the Cary-Blair medium can be used for Salmonella, Shigella and Yersinia. • When cholera is suspected, about 1 ml of specimen should be transferred into 10 ml of alkaline peptone water, which will act as an enrichment as well as transport medium. • When worms or tapeworm segments are present, these should be transferred to a container of physiological saline and sent to a laboratory for identification [1,4,6].

Macroscopic examination
Various points which have to be noted are: • Consistency: formed, unformed (soft), loose or watery. The cysts have been mostly found in the formed stools, while trophozoites have been most abundantly found in watery stools. • The presence of blood, mucus or pus. • The presence of worms, e.g. Enterobius Vermicularis, Ascaris, tapeworm segments, e.g. Taenia species. • Colour (white, yellow, brown or black). • Normal faeces appear brown and formed or semiformed. Infant faeces are yellow-green and semiformed [1,5,8,9].

Microscopic examination Methylene blue preparation Place a small fleck of the stool specimen or the rectal swab together with a small flake of mucus in a drop of 0.05% methylene blue solution on a clean glass slide and examine it for cellular exudates as follows [1,2,4]:

• clumps of pus cells of > 50 cells per high power field along with macrophages and erythrocytes are typical of shigellosis. • A smaller number of pus cells of <20 per high power field are found in salmonellosis and in infections which are caused by invasive E.coli. • Few leucocytes (< 5 cells per high power field) are present in cholera, EPEC and ETEC and viral diarrhoea.

Wet mount The simplest way of examining a bacterial suspension for motile bacteria is by doing a wet mount. Place a small drop of suspension on a slide, cover it with a coverslip and examine microscopically for motile organisms by using the 10X and the 40X objectives. Also make sure that the iris diaphragm of the condenser is sufficiently closed, to give a good contrast (2).

Hanging drop preparation Placing a drop of suspension on a cover glass and inverting this over a cavity slide or a normal slide which is supported on a ring of plasticine can also be used for observing motile organisms [1,2].

Basic fuschin smear Make a thin smear of the specimen on a slide, stain it with Basic fuschin and examine the slide by using a 100X objective. This has been shown to be a sensitive method for the presumptive diagnosis of Campylobacter spp. It appears as small, delicate, spiral curved bacteria or s-shaped forms [10,11].

Culturing the specimen MacConkey’s Agar This is useful, non-selective medium for use for general purposes and an added advantage is the formation of pink coloured colonies in case of lactose fermenters and colourless colonies in case of nonlactose fermenters. Also, it inhibits most of the gram positive organisms and the swarming growth of Proteus, which may pose a problem in mixed cultures. Salmonella, Shigella and Vibro form colourless colonies on this medium as these are nonlactose fermenters. E.coli which are lactose fermenters, form pink coloured colonies [10,11].
Xylose lysine deoxycholate (XLD) agar This selective medium has been recommended for the isolation of Salmonella and particularly Shigella from faecal samples [10,11]. Shigella forms pink-red colonies because it does not ferment xylose and lactose. Salmonella also forms pink coloured colonies with black centres because of hydrogen sulphide production.

Thiosulphate citrate bile salt sucrose (TCBS) agar
This is an excellent, selective medium for the primary isolation of V.cholerae. Prior enrichment in alkaline peptone water is recommended unless the specimen contains large number of Vibrio bacteria in the acute stage. On TCBS, Vibro produces large yellow coloured colonies because of sucrose fermentation [10,11]. Sorbitol MacConkey’s agar This MacConkey’s agar contains sorbitol instead of lactose. E.coli 0157 produces colourless colonies on this medium because it does not ferment sorbitol. Most of the other E.coli strains and other enterobacteria ferment sorbitol and produce pink colonies. So, this medium is useful for screening 0157 E.coli.

Slide agglutination test The isolation of Salmonella and Vibrio can be done by doing the slide agglutination test. A loopful of growth from the culture is emulsified in two drops of saline on a slide. One emulsion acts as a control to show that the strain is not autoagglutinable. In case of Salmonella, the ‘o’ antiserum is added to one drop of bacterial emulsion on the slide. A prompt agglutination denotes the presence of the Salmonella group [8,9].

Similarly for V.cholera, the cholera o subgroup I serum is tested. If it is found to be positive, the agglutination may be repeated by using the specific Ogawa and Inaba sera for serotyping.

Stool examination for parasites [7,12] 1. Saline wet mount: It is used to detect worms, bile stained eggs, larvae, protozoan trophozoites and cysts. In addition, it can reveal the presence of RBCs and WBCs. 2. Iodine wet mount: It is used to stain the glycogen and nuclei of the cysts. A cyst is appreciated better in an iodine preparation, but the motility of the trophozoite is inhibited in the iodine preparation.

Procedure • Place a drop of saline on the left half of the slide and one drop of iodine on the right half. • With an applicator stick, pick up a small portion of the specimen (equivalent to the size of a match head) and mix it with a saline drop. • Similarly, pick up a similar amount and mix with a drop of iodine. • Put the cover slip separately on both and examine under the microscope. • The ova, cysts, trophozoites and adult worms can be identified as per their characteristic features.

Concentration techniques [7,8,13,14] If the number of parasites in the stool specimens is low, the examination of a direct wet mount may not reveal them and hence the stool should be concentrated. Eggs, cysts and larvae can be recovered after the concentration procedure, whereas trophozoites can get destroyed during this procedure. This makes a direct wet mount examination obligatory as the initial phase of the microscopic examination.

The concentration procedures can be grouped under 2 categories: 1. Sedimentation procedures: In which the eggs and cysts settle down at the bottom. 2. Flotation procedures: In which the eggs and cysts float at the surface due to the specific gravity gradient. The basic disadvantage of the sedimentation technique is that the examination of the sediment is often difficult due to the presence of excessive faecal debris that may mask the presence of the parasites. The basic disadvantage of the flotation technique is that not all eggs and cysts float in the flotation procedures. Two commonly used concentration techniques are the formalin-ether and the saturated salt solution techniques.

The formal ether sedimentation technique Procedure [7,8,15] • Transfer half a teaspoonful of faeces into 10 ml of water in a glass container and mix it thoroughly. • Place 2 layers of gauze in a funnel and strain the contents into a 15 ml centrifuge tube. • Centrifuge for 2 minutes at about 500 g. • Discard the supernatant and resuspend the sediment in 10 ml of physiological saline. Centrifuge at 500 g and discard the supernatant. • Resuspend the sediment in 7 ml of 10% formaldehyde (1 part of 40% formalin in 3 parts of saline). • Add 3 ml of ether (or ethyl acetate). • Close the tube with a stopper and shake vigorously to mix. Remove the stopper and centrifuge at 500g for 2 minutes. • Rest the tube on a stand. Four layers now become visible; the top layer consists of ether, the second layer is a plug of debris, the third layer is a clear layer of formalin and the fourth layer is the sediment (Table/Fig 2). • Detach the plug of debris from the side of the tube with the aid of a glass rod and pour off the liquid, leaving only a small amount of formalin for the suspension of the sediment. • With a pipette, remove the sediment and mix it with a drop of iodine. Examine under the microscope.

The saturated salt flotation technique [7,8,9] • Place about one millilitre of faeces in a container which is flat bottomed and which has a diameter of less than 1½ inches and a capacity of about 15-20 ml. • Add a few drops of saturated salt solution (specific gravity 1.200) and stir it to make a fine emulsion. • Add more salt solution so that the container is nearly full, stirring the solution continuously. • Remove any coarse matter which floats up. • Place the container on a levelled surface. Do the final filling by using a dropper until a convex meniscus is formed. • A glass slide 3”x 2” is carefully laid on the top of the container so that the centre is in contact with the fluid. • This preparation is allowed to stand for 20 minutes after which the glass slide is quickly lifted, turned over smoothly to avoid the spilling of the fluid and it is examined under the microscope after putting a coverslip on it.

Staining procedures [12,13] The modified acid fast staining is a simple and effective method for the diagnosis of coccidian parasites in the stool sample by using 1% H2SO4 as a decolourizer. In unstained wet preparations, the small oocysts of C.parvum are difficult to differentiate from thoseof yeasts and other small spherical structures which are found in faeces.

The oocyst of C.parvum
Small, round to oval pink red stained bodies which measure 4-6μm.
The oocyst of Isospora belli It is oval, measuring 20-22 μm in diameter and it usually shows a granular zygote. Occasionally, the oocyst may show two sporocysts (each with four sporozoites).

Oocyst of I. belli showing two sporocysts (each with four sporozoites). B- Pink red coloured oocyst of I.belli showing a zygote Morphological features of the common parasites/eggs/ova/cysts Entamoeba histolytica trophozoite It measures 12-60 μ, it is asymmetric, it shows purposeful directional motility and it has a single spherical nucleus, a single centralkaryosome and delicate and evenly distributed chromatin. Entamoeba histolytica cyst It is spherical and it measures 10-20 μ. It is a mature cyst with four nuclei, with a compact, centrally located karyosome; the chromatin is delicate. Some cysts may have chromatoid bars. This is the infective stage of the parasite.

Giardia lamblia trophozoite It measures 9-21x5-15 μ and is pear shaped, with tapering ends. It is actively motile like a falling leaf and it has 2 centrally placed nuclei and uniform granular cytoplasm.

Giardia lamblia cyst It is oval, 8-12 μ long and 7-10 μ wide, andits nucleus has 4 karyosomes, whichtend to be eccenterically placed. There is a clear space between the cell wall and the cytoplasm. Four median bodies are present. Fertile egg of roundworms It measures 60 × 45 μ and is round or ovoid with a thick shell. It is covered by a thick albuminous coat, its inner cell is in various stages of cleavage and it is brown in colour.

Decorticated egg of roundworm The albuminous coat is lost. All other features are the same as in a fertile egg. Infertile egg of roundworm It measures 90x40 μ, it is elongated, its shell is often thin and its internal material is a mass of globules.

Hookworm egg They are oval and ellipsoid and they measure 60x40 μ. Their shells are thin walled, smooth and colourless. Their internal cleavages are well developed at the 4-8 cell stage, which pull away from the shell, leaving an empty space.

Threadworm egg (E.vermicularis) It is a planoconvex, elongate and an asymmetric egg which measures 55 x 26 μ. Its shell is thin and smooth. Fully developed larvae are seen in the eggs. Whipworm egg (Trichuris trichura) It is elongate and barrel shaped, with polar hyaline plugs. It measures 54-22 μ. Its shells is yellow to brownish and its plugs are colourless.

Tapeworm egg (Taenia spp) It is spherical, it measures 31-43μ and it has a thick shell with prominent radial striations. An embryonated oncosphere which possesses 3 pairs of hooklets within the shell is diagnostic of the genus. Species identification on the basis of morphology is not possible.


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DOI and Others

DOI: JCDR/2012/4003:2073


Date of Submission: Aug 09, 2011
Date of Peer Review: Jan 07, 2012
Date of Acceptance: Jan 18, 2012
Date of Publishing: May 01, 2012

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