Laboratory diagnosis of gastroenteritis

Preface

Gastrointestinal tract teems with a diverse flora. The heterogeneity of commensals makes the human body all the more prone to infections in diminished states of health & also poses a tough task for us to label an organism pathogenic or colonizer or commensal. The chapter here, incorporates an exhaustive list of microbes from different groups causing diarrheal diseases, which is hoped to come in handy for the diagnosis.

Commensal bacterial flora of intestines:

Lactobacilli, Streptococci, Bacteroides, Bifidobacterium, Peptostreptococcus, Clostridium, Enterobacteriacae. The bacteria are mostly anaerobes or facultative anaerobes. These commensals provide competition for nutrition & growth to pathogenic bacteria & thus, do not easily allow them to thrive.

 

Learning objectives:

1. Different types of diarrheal diseases and their etiologies.

2. How the stool sample should be transported and analyzed in laboratory?

Following are the different terminologies used to describe the diarrheal diseases.

1. Gastroenteritis: It is defined as the inflammation of  mucosa of the gastrointestinal tract (stomach - "gastro" and intestine - "entero") often leading to diarrhea, vomiting and abdominal discomfort.

2. Diarrhea: It is defined as a condition in which loose or liquid stool pass for three or more times a day. It is the most common presentation of gastroenteritis. Infective diarrhea is often considered synonymous to gastroenteritis.

3. Dysentery: It is a condition associated with frequent passage of stool containing mucus, blood or pus (exudates). It is characteristically associated with abdominal cramps, tenesmus and fever.

4. Food poisoning: It is a condition of acute gastroenteritis originating due to consumption of food containing large number of bacteria or their products like toxins; which after entry in gastrointestinal tract, without undergoing further multiplication or toxin production immediately and with very short incubation period lead to pathogenesis and clinical manifestations.

5. Colitis: Inflammatory condition of large bowel (colon) is called colitis. Many organisms may cause only colitis or enterocolitis leading to watery diarrhea or dysentery. 

Patients of infectious diarrhea have two kinds of presentations:

            1. Watery diarrhea

            2. Dysentery

 

Causative agents of watery diarrhea

A. Bacteria:

Vibrio cholerae (rice watery diarrhea)

Entero-Pathogenic Escherichia coli (paediatric diarrhea)

Entero-Toxigenic Escherichia coli (traveler's diarrhea/Montezuma’s revenge/ Delhi belly )

Campylobacter spp.

Salmonella sp. (S.typhimurium, S.enteritidis etc)

Clostridium difficile (antibiotic associated colitis)

 

B. Viruses:

Rotavirus (most common cause of paediatric diarrhea)

Norwalk virus

Calici virus

Astrovirus

Adenovirus

 

C. Parasites

Giardia intestinalis ( usually leads to fatty diarrhea )

Cryptosporidium parvum (Paediatric diarrhea or diarrhea in immuno compromised patients)

Isospora belli (diarrhea in immuno compromised patients)

Helminths: Hymenolepsis nana, Trichuris trichiura, Strongyloides stercoralis, Ascaris lumbricoides, Ancyclostoma duodenale etc.

 

D. Fungus: Candida albicans

 

 

E. Bacteria associated with food poisoning

            Bacterial food poisoning are of two types

            1. Toxic type: Bacterial toxins lead to gastroenteritis         

                        Staphylococcus aureus (Common with milk and milk products)

                        Bacillus cereus (Common with fried rice)

                        Clostridium perfringens

                        Clostridium botulinum (Common with canned food)

            2. Infective type: Direct bacteria invade mucosa and cause gastroenteritis

                        Vibrio parahaemolyticus

                        Campylobacter jejuni

                        Yersinia enterocolitica

                        Salmonella typhimurium

           

Causative agents of dysentery:

According to the aetiological agents, dysentery is divided in two types

A. Bacillary dysentery: caused by bacteria

            Shigella spp.

            Entero-Invasive Escherichia coli

            Entero-haemorrhagic Escherichia coli

B. Amoebic dysentery: Caused by Entamoeba histolytica

 

 

 

 

 

Laboratory diagnosis

A. Stool analysis:

Sample collection:

Stool is the most informative sample to evaluate aetio-pathogenesis of diarrheal diseases. In paediatric patients or critically debilitated patients with severe watery diarrhea, if it is not possible to collect sample, rectal swab may be taken. Intestinal aspiration with help of endoscope may be taken to diagnose certain parasitic infection (e.g. hook worm present in small intestine, E.histolytica present in the ulcers of large intestine).

Sample transport:

            Sample as it is should be immediately transported to laboratory.

            If there is delay in transport, according to suspected bacterial pathogens. it should be transported in following transport media,

            1. In suspected cases of Cholera:

                        Venkatraman - Ramakrishnan (VR) medium

                        Cary-Blair medium

                        Alkaline peptone water (if sample is transported within a day )

            2. In suspected cases of infection by enterobacteriacae members:

                        Glycerol buffered saline

            3. In suspected viral infections:

The sample should be refrigerated if not processed within 2 hours. Rectal swabs may be stored & transported in Modified Stuart’s medium or Viral Transport Medium.

 

Sample processing:

1. Gross examination: Direct appearance may help to evaluate infective aetiology

            - Rice watery stool is most commonly seen in Vibrio cholerae

            - Presence of gross blood or pus is indicative of dysentery

            - Foul smell suggestive of fatty diarrhea is a common presentation of giardiasis

            - Helminths like round worm, hook worm, thread worm, tape worm, whip worm may be grossly visible in the stool.

 

2. Microscopic examination:

            Stool should be examined microscopically for following findings

            1. Pus cells: Presence of pus cells is suggestive of invasive infection with marked inflammation

            2. RBCs: Presence of RBCs is suggestive of dysentery

            3. Parasites: On microscopic examination, following parasites may be seen in stool,

            Cysts or/and trophozoites of Entamoeba histolytica, Giardia intestinalis.

            Oocysts of Cryptosporidium parvum, Isospora belli.

            Ova of H. nana, hook worm, round worm, whip worm, tape worm and thread worm. Larvae of S.stercoralis.

4. Fungus: Yeast cells - Candida may be demonstrated. Its significance should be evaluated with other clinical background; generally if pseudohyphae is seen along with budding yeast cells, it is considered significant.

            5. Bacteria: A large number of bacteria are normally present in stool. It is not possible to differentiate normal bacteria from pathogenic bacteria by microscopic examination. However,             presence of Vibrio cholerae may be indicated by demonstration of its specific darting type of motility with hanging drop preparation.

            6. Other structures: i.e. Charcot-Leyden (CL) crystals are present in amoebic dysentery.

 

 

3. Culture examination:

            Stool specimen normally contains large number of commensals. These bacteria may affect selective isolation of pathogenic bacteria. Escherichia coli, a common cause of diarrhea is  also a common commensal of normal stool. So, for culture examination of stool, we need different sets of culture media containing enrichment and selective media along with simple and differential media according to suspected aetiological agents.

Culture media used:

1. Simple media: Nutrient agar

2. Differential media: Mac conkey agar

3. Enrichment media:       Selenite F broth or tetrathionate broth: For Salmonella and Shigella

                                    Alkaline peptone water: For Vibrio cholerae

4. Selective media: TCBS: For Vibrio cholerae

                                    Wilson-Blair medium: For Salmonella

                                    DCA or XLD agar: For Shigella and Salmonella

Isolated bacteria are identified and processed for antibiotic sensitivity testing. Typing of bacteria may be needed for epidemiological purpose.

 

4. Antigen Detection :

            E. coli O157:H7   -   Enzyme linked Immuno Assays (EIAs).

            Rotavirus    -    Enzyme linked Immuno Assays  & Latex Agglutination Tests (LATs).

            C. difficile  -    Toxin A and/ toxin B detection.

B. Analysis of other specimens:

            Rectal swab: In paediatric patients with severe watery diarrhea, if it is not possible to collect             stool sample, rectal swab may be taken and processed for culture examination.

            Intestinal aspiration with help of endoscope may be taken to diagnose certain parasitic infection (e.g. hook worm present in small intestine)

            Intestinal biopsy: May be taken by endoscopy which may show pathogenic organism on histopathological examination. e.g. E.histolytica present in ulcer of large intestine, H.pylori             present in gastric biopsy.

            Vomited material: Particularly useful in case of food poisoning for detection of bacterial toxins

            Food sample: Should be taken to find out agent responsible for food poisoning.

 

Exercise:

A 20 year old male patient presented with acute abdominal pain and diarrhoea. Microscopic examination of stool is suggestive of dysentry. Stool sample is required to evaluate for aetiology at the microbiology laboratory situated at distant place.

Q.1     Which culture media are required for transport of speciemen?

Q.2     For recovery of possible pathogen which culture media should be employed?

Q.3     Which are the suspected bacteria?

Q.4     Enlist the Bio-chemical test used for differential identification of enterobacteriacae member.

  & also poses a tough task for us to label an organism pathogenic or colonizer or commensal. The chapter here, incorporates an exhaustive list of microbes from different groups causing diarrheal diseases, which is hoped to come in handy for the diagnosis.

Commensal bacterial flora of intestines:

Lactobacilli, Streptococci, Bacteroides, Bifidobacterium, Peptostreptococcus, Clostridium, Enterobacteriacae. The bacteria are mostly anaerobes or facultative anaerobes. These commensals provide competition for nutrition & growth to pathogenic bacteria & thus, do not easily allow them to thrive.

 

Learning objectives:

1. Different types of diarrheal diseases and their etiologies.

2. How the stool sample should be transported and analyzed in laboratory?

Following are the different terminologies used to describe the diarrheal diseases.

1. Gastroenteritis: It is defined as the inflammation of  mucosa of the gastrointestinal tract (stomach - "gastro" and intestine - "entero") often leading to diarrhea, vomiting and abdominal discomfort.

2. Diarrhea: It is defined as a condition in which loose or liquid stool pass for three or more times a day. It is the most common presentation of gastroenteritis. Infective diarrhea is often considered synonymous to gastroenteritis.

3. Dysentery: It is a condition associated with frequent passage of stool containing mucus, blood or pus (exudates). It is characteristically associated with abdominal cramps, tenesmus and fever.

4. Food poisoning: It is a condition of acute gastroenteritis originating due to consumption of food containing large number of bacteria or their products like toxins; which after entry in gastrointestinal tract, without undergoing further multiplication or toxin production immediately and with very short incubation period lead to pathogenesis and clinical manifestations.

5. Colitis: Inflammatory condition of large bowel (colon) is called colitis. Many organisms may cause only colitis or enterocolitis leading to watery diarrhea or dysentery. 

Patients of infectious diarrhea have two kinds of presentations:

            1. Watery diarrhea

            2. Dysentery

 

Causative agents of watery diarrhea

A. Bacteria:

Vibrio cholerae (rice watery diarrhea)

Entero-Pathogenic Escherichia coli (paediatric diarrhea)

Entero-Toxigenic Escherichia coli (traveler's diarrhea/Montezuma’s revenge/ Delhi belly )

Campylobacter spp.

Salmonella sp. (S.typhimurium, S.enteritidis etc)

Clostridium difficile (antibiotic associated colitis)

 

B. Viruses:

Rotavirus (most common cause of paediatric diarrhea)

Norwalk virus

Calici virus

Astrovirus

Adenovirus

 

C. Parasites

Giardia intestinalis ( usually leads to fatty diarrhea )

Cryptosporidium parvum (Paediatric diarrhea or diarrhea in immuno compromised patients)

Isospora belli (diarrhea in immuno compromised patients)

Helminths: Hymenolepsis nana, Trichuris trichiura, Strongyloides stercoralis, Ascaris lumbricoides, Ancyclostoma duodenale etc.

 

D. Fungus: Candida albicans

 

 

E. Bacteria associated with food poisoning

            Bacterial food poisoning are of two types

            1. Toxic type: Bacterial toxins lead to gastroenteritis         

                        Staphylococcus aureus (Common with milk and milk products)

                        Bacillus cereus (Common with fried rice)

                        Clostridium perfringens

                        Clostridium botulinum (Common with canned food)

            2. Infective type: Direct bacteria invade mucosa and cause gastroenteritis

                        Vibrio parahaemolyticus

                        Campylobacter jejuni

                        Yersinia enterocolitica

                        Salmonella typhimurium

           

Causative agents of dysentery:

According to the aetiological agents, dysentery is divided in two types

A. Bacillary dysentery: caused by bacteria

            Shigella spp.

            Entero-Invasive Escherichia coli

            Entero-haemorrhagic Escherichia coli

B. Amoebic dysentery: Caused by Entamoeba histolytica

 

Laboratory diagnosis

A. Stool analysis:

Sample collection:

Stool is the most informative sample to evaluate aetio-pathogenesis of diarrheal diseases. In paediatric patients or critically debilitated patients with severe watery diarrhea, if it is not possible to collect sample, rectal swab may be taken. Intestinal aspiration with help of endoscope may be taken to diagnose certain parasitic infection (e.g. hook worm present in small intestine, E.histolytica present in the ulcers of large intestine).

Sample transport:

            Sample as it is should be immediately transported to laboratory.

            If there is delay in transport, according to suspected bacterial pathogens. it should be transported in following transport media,

            1. In suspected cases of Cholera:

                        Venkatraman - Ramakrishnan (VR) medium

                        Cary-Blair medium

                        Alkaline peptone water (if sample is transported within a day )

            2. In suspected cases of infection by enterobacteriacae members:

                        Glycerol buffered saline

            3. In suspected viral infections:

The sample should be refrigerated if not processed within 2 hours. Rectal swabs may be stored & transported in Modified Stuart’s medium or Viral Transport Medium.

 

Sample processing:

1. Gross examination: Direct appearance may help to evaluate infective aetiology

            - Rice watery stool is most commonly seen in Vibrio cholerae

            - Presence of gross blood or pus is indicative of dysentery

            - Foul smell suggestive of fatty diarrhea is a common presentation of giardiasis

            - Helminths like round worm, hook worm, thread worm, tape worm, whip worm may be grossly visible in the stool.

 

2. Microscopic examination:

            Stool should be examined microscopically for following findings

            1. Pus cells: Presence of pus cells is suggestive of invasive infection with marked inflammation

            2. RBCs: Presence of RBCs is suggestive of dysentery

            3. Parasites: On microscopic examination, following parasites may be seen in stool,

            Cysts or/and trophozoites of Entamoeba histolytica, Giardia intestinalis.

            Oocysts of Cryptosporidium parvum, Isospora belli.

            Ova of H. nana, hook worm, round worm, whip worm, tape worm and thread worm. Larvae of S.stercoralis.

4. Fungus: Yeast cells - Candida may be demonstrated. Its significance should be evaluated with other clinical background; generally if pseudohyphae is seen along with budding yeast cells, it is considered significant.

            5. Bacteria: A large number of bacteria are normally present in stool. It is not possible to differentiate normal bacteria from pathogenic bacteria by microscopic examination. However,             presence of Vibrio cholerae may be indicated by demonstration of its specific darting type of motility with hanging drop preparation.

            6. Other structures: i.e. Charcot-Leyden (CL) crystals are present in amoebic dysentery.

 

 

3. Culture examination:

            Stool specimen normally contains large number of commensals. These bacteria may affect selective isolation of pathogenic bacteria. Escherichia coli, a common cause of diarrhea is  also a common commensal of normal stool. So, for culture examination of stool, we need different sets of culture media containing enrichment and selective media along with simple and differential media according to suspected aetiological agents.

Culture media used:

1. Simple media: Nutrient agar

2. Differential media: Mac conkey agar

3. Enrichment media:       Selenite F broth or tetrathionate broth: For Salmonella and Shigella

                                    Alkaline peptone water: For Vibrio cholerae

4. Selective media: TCBS: For Vibrio cholerae

                                    Wilson-Blair medium: For Salmonella

                                    DCA or XLD agar: For Shigella and Salmonella

Isolated bacteria are identified and processed for antibiotic sensitivity testing. Typing of bacteria may be needed for epidemiological purpose.

 

4. Antigen Detection :

            E. coli O157:H7   -   Enzyme linked Immuno Assays (EIAs).

            Rotavirus    -    Enzyme linked Immuno Assays  & Latex Agglutination Tests (LATs).

            C. difficile  -    Toxin A and/ toxin B detection.

B. Analysis of other specimens:

            Rectal swab: In paediatric patients with severe watery diarrhea, if it is not possible to collect             stool sample, rectal swab may be taken and processed for culture examination.

            Intestinal aspiration with help of endoscope may be taken to diagnose certain parasitic infection (e.g. hook worm present in small intestine)

            Intestinal biopsy: May be taken by endoscopy which may show pathogenic organism on histopathological examination. e.g. E.histolytica present in ulcer of large intestine, H.pylori             present in gastric biopsy.

            Vomited material: Particularly useful in case of food poisoning for detection of bacterial toxins

            Food sample: Should be taken to find out agent responsible for food poisoning.

Laboratory diagnosis of HIV infection

 

Learning objectives:

1.    Kinetics of immune response in reference to various markers used for laboratory diagnosis

2.    Pre-requisite for HIV testing

3.    NACO (National AIDS Control Organization) strategies for HIV diagnosis in various conditions / situations

4.    Specific tests for HIV infection:

Screening tests (ERS) (antibody detection)

Rapid/ Simple test / ELISA

Supplemental tests (antibody detection)

Western blot assay

Confirmatory tests: Detection of viral RNA / proviral DNA

5.    Tests to measure prognosis / monitoring progress of HIV infection

Kinetics of immune response –:

·       Viremia- Viral entry into the body leads to a transient period of high-level viremia and p24 antigenemia.

·       Window period- 3 to 12 weeks. Virus remains in the blood but antibody does not appear in the blood.

·       Later on Humoral response is evidenced by formation of antibodies against different antigens.

 

Following care should be taken (3Cs) while performing the test for HIV.

·       Consent in written format should be taken before the test is done.

·       Confidentiality of a positive test result is must. 

·       Counselling (Pre-test and Post-test Counselling) should be provided to motivate the individual and induce behavioral changes.

 

NACO STRATEGY FOR HIV DIAGNOSIS

NACO (National AIDS Control Organization, India) has formulated a strategic plan for HIV diagnosis.

Specific tests for HIV infection:

·       Screening tests (ERS) (antibodydetection)

o   Rapid/ Simple test / ELISA

·       Supplemental tests (antibody detection)

o   Western blot assay

·       Confirmatory tests

A)  Screening tests (antibody detection tests):

·       Sample taken : blood in plain vaccute

1.    Rapid/ Simple test

Advantage:

·       Easy to perform and provide quick results (takes < 30 minutes). 

·       Do not require special equipments.

Disadvantage:

·       False positive results

·       Results of a screening test should never be used as the final .It is always subjected to confirmatory tests

          Most commonly used rapid tests in India:

a.     Dot blot assays

b.    Immunochromatography

c.     Dip stick/ comb tests

1.    ELISA

Advantage:

·       Highly sensitive and specific

·       Adaptable to large number of samples  and Cost effective.

Disadvantage:

·       Require special equipments like ELISA reader and washer

·        Require training of laboratory technician. Takes 2-4 hours.

A)  Supplemental test or confirmatory test:

1)   Western blot:

·       Principle: It detects individual antibodies in serum separately against various antigenic fragments of HIV such as, Antibody to gag gene products, pol gene productsand env gene products. The antigen antibody complexes appear as distinct bands on nitrocellulose strip.

·       Sample taken: Blood in plain vaccute

·       WHO criteria i.e. presence of at least two envelope bands (out of gp120, gp160 or gp41) with or without gag or pol bands

·       CDC criteria-presence of any two out of p24, gp 120, gp160, gp41 bands

2)   Viral RNA detection

·       The ‘gold standard’ method for confirmation of HIV diagnosis.

·       Real time RT-PCR- for estimating viral load

·       Sample taken: blood in EDTA(plasma)

·       Uses:

·       For confirmation of diagnosis of HIV/AIDS(can detects even few copies of viral RNA)

·       Diagnosis of HIV during window period (earlier than all available methods :12 days post exposure)

·       Viral load monitoring-monitoring the response to antiretroviral therapy.

Early Infant Diagnosis: Do not perform antibody estimation test . HIV DNA PCR is the test of Choice.

Sample – Blood drawn from heel and taken on filter paper and dried - DBS (dried blood spots)/plasma