Diarrhoea 2003, SGPGIMS, Lucknow Diagnostic Approach to Infective Diarrhoea in the Community and...

Diarrhoea 2003, SGPGIMS, Lucknow

Diagnostic Approach to Infective Diarrhoea in the Community and

Rational Antibiotic Therapy

Dr. A. AyyagariProf. & Head

Department of Microbiology

SGPGIMS, Lucknow (U.P.) India


Introduction

• Major public health problem in India under 5 years. 1/3 of admission and 17% of death)

• Definition “passage of loose, liquid or watery stool”.• Acute diarrhoea (GE)- sudden onset which usually last 3-

7 days, may 10-14 days. • Non inflammatory e.g. V. cholerae (no abnormal

histology)• Inflammatory- with blood and mucus e.g. Shigella


0

10

20

30

40

DiarrhoeaOthersARIVPDNeonatal

Diarrhoea 32 16

Others 34 31

ARI 13 13

VPD 15 25

Neonatal 4 13

Admission Death


Chronic Diarrhoea (>14 days)

• Inflammatory- ulcerative colitis, Crohn’s disease, radiation colitis

• Osmotic- Whipple’s disease, Celiac sprue, Pancreatic insufficiency

• Secretory- Carcinoid syndrome, ZE syndrome, VIP adenomas etc.

• Altered motility- IBS, neurologic disease, fecal impaction

• Factitious- laxative abuse


Cause of Acute diarrhoea (<14 days)

• Infectious diarrhoea• Medications• Ischemic colitis• Sup. Mesenteric arterial or venous thrombosis• Acute diverticulitis


Infectious diarrhoea- Mechanisms & causes

Mechanism Examples

Toxin Production

Preformed Toxin

Enterotoxin

Cytotoxin

B. cereus, C. perfringens, S. aureus

Aeromonas, ETEC, V. cholerae

C. difficile, E. coli O157:H7

Enteroadherence Cryptosporidium, cyclospora, EPEC, EAEC, Giardia


Infectious diarrhoea- Mechanisms & causes

Mechanism Examples

Mucosal Invasion

Minimal

Variable

Severe

Norwalk, Rota, Adeno, Calici, Corona, CMV

Aeromonas, Campylobacter, Salmonella, V. parahaemoliticus

E. histolytica, EIEC, Shigella

Systemic Infection Legionella, Listeria, Measles, Psittacosis


Diarrhoea in HIV/AIDS patient

Bacteria Virus Parasite

C. jejunii CMV Cryptosporidium

Shigella sp Enteric adeno Isospora belli

Salmonella Calici virus Cyclospora

C. difficle HIV Microsporidia

EAEC

Mycobacterium avium complex


Diagnostic approach to Infective Diarrhoea

• History

Dietary details, travel history, source of drinking water, sexual preferences

• Physical examination

BP, pulse rate, pulse volume, Abd. Exam, hepatosplenomegaly, lymphadenopathy


Lab Diagnosis- Sample collection

-collected in acute stage-before the start of the treatment-before the radiological examination-no contamination with urine water or any other infective

material -In wide mouthed leak proof screw capped container (25 ml)

with a spoon (do not soil the rim of the container)-amount 5 ml of liquid stool/pea size of formed stool-Number – max. 3 samples (2 after normal movement and 1

after cathartic)


Lab Diagnosis- Sample Transport

• Cary blair transport media (pH 8.4)- Campy., Vibrio• Buffered glycerol transport media (pH 7.0)- Shigella• V.R media (pH 8.6)- V. cholerae• Hank’s balanced salt solution- Virus• Stuart and Amies- general purpose transport media


Microscopy

Wet mount- Ova and trophozoites of parasitesWBCs indicate invasive pathogens

Phase contrast microsciopy- CampylobacterImmune electron microscopy- Viruses Staining methods -

Oocyst Acis-fast stains- Cold/hot Kinyoun modified stain, Giemsa stain, PAS stain, Direct fluorescence stain,E. histolytica- Trichrome stainMicrosporidium- ModifiedTrichromeGram stain


Lab Diagnosis- Enrichment medium

Medium Incubation timeFor Salmonella and Shigella

Gram negative broth 4-6 hr

Selenite F-broth 12 hr

Tetra thionate broth 12 hr

For V. cholerae

Alkaline peptone water 6-8 hr

Monsur’s taurocholate telllurite peptone water

6-8 hr


Formed/semiformed stool (1:10 dilution in 2-3 ml PBS or 0.1% peptone water)

Liquid stool

(Cholera suspected by characteristic Motility and immobilisation by specific sera)

Grams stain if required MacConkey Selenite F broth XLD/DCA GN broth

Typical morphology DSRA Further processing Subculture on of DCA/MAC Pure LF cononies within 6 hourAll NLF col(oxidase negative) on MacConkey, should be S/c on DCA All black centered colony on DCA

All non sorbitol fermenter colony (EHEC)


Classification of Enteropathogenic E. coli

Pathotype Clin Features Epidem. Features Virulence factors

EPEC Watery diarr., vomiting

Infants, Developing countries

Bundle forming pilus, attaching-effacing

EHEC Watery diarr., Hg. colitis

Food & water borne Shiga toxins, attaching-effacing

ETEC Watery diarr Childhood diarr., Traveler's diarr.

Pili, ST & LT entero toxins

EAEC Diarr with mucus

Childhood diarr. Pili, cytotoxins

EIEC Dysentery/ watery diarr

Food borne Cellular invasion, intra cellular motility


Antibiotic Associated Diarrhea

Most common cause of diarrhea among hospitalized patients (Range-1 in 10 to 1 in 10000)

3-5 billion annual infection annually, 3 million deaths/ year.

1.5 episodes per person/ year > 50% death: elderly Self limiting, ~ 50% within 3 days.


Antibiotics implicated in AAD

• Frequently: Ampicillin, Amoxicillin-clavulenate, 2nd & 3rd generation Cephalosporin, Clindamycin

• Uncommon: Tetracycline, sulfonamides, Quinalone, Erythromycin, Chloramphenicol, TMP,

• Antineoplastic agents > Methotrexate, Other agents (Anon, 1993)- Dexorubin, cyclophosphamide

• Tube feeding- Nosocomial CD collitis.


Lab Diagnosis of AAD

• Specimen- Stool (fresh sample), colonic biopsy• Non specific- leucocytes in stool in AAD & PMC• Colonoscopy/ sigmoidoscopy (erythema, edema,

friability, adhered yellow plaques) in PMC. Endoscopy ? Normal in mild cases

• Radiographic imaging• Surveillance of nosocomial infection-

– Swab from inanimate surroundings & hospital personnel


Stool Tests for C. difficile Infection

Test Detects Advantages Disadvantages

Cytotoxin Tox B Gold standard

sen. & specific

Tissue culture ,24-48 hr

ELISA Tox A or B Fast (2-6 hr), Easy, high specificity

Not as sensitive as cytotoxin assay

LAgg Bacterial enzyme (glutamat dehydrogenase)

Fast, Inexpensive

Easy to perform

Poor sensitivity & specificity

Culture Tox and Nontox C. difficile

Sensitive

Allows strain tytping in epidemics

Requires O2, 2-5 days

Not specific for tox producing bacteria

PCR Tox A or B gene in isolates or directly in feces

High sensitivity & specificity

Requires expertise


Viruses causing Acute Gastroenteritis

Virus

(IP)

Family EM shape Nucleic acid

Charecterization

Rota Reoviridae Wheel shaped dsRNA Gp A, B, C, multiple serotypes, classified according to outer capsid proteins (P, G)

Calici

(24-48 hr)

Caliciviridae Small round ss (+)RNA Genogroups- Norwalk like viruses and Sapporo like viruses

Astro Astroviridae Star shaped ss (+)RNA 8 serotypes

Adeno

(3-10 days)

Adenoviridae Icosahedral dsDNA 40,41,31, 42-48

Other viruses- Torovirus [ss(+)RNA], Picovirna virus [dsRNA], Enterovirus 22 [ss(+)RNA],Aichi virus [ss(+)RNA


Diagnosis of Viral diarrhoea

Non Rota- – Direct and immune Em– Antigen detection- EIA with hyper immune sera , EIA with

monoclonal antibody– Antibody detection– Culture– Hybridization probes- for adeno viruses– RT-PCR for HuCV

Rota- EIA, membrane EIA, LA, EM, culture, RT-PCR



Most cases are self limiting and subside with supportive therapyIndication of antibiotic therapy • Cholera• Febrile bloody diarrhoea• Travelers diarrhoea • extremes of age• Food handlers • Immunocompromised• Day care attendee• Residents of institutional facility• Epidemic outbreaks



Problems of empiric therapy-• Not effective in EHEC, salmonella enterocolitis• In children- most cases are viral• Emerging drug resistance• Side effects • Alteration of gut flora• Induction of disease producing phage e.g; Shigatoxin

phage induced by quinolones


Therapeutic recomendations

• Shigella- TMP-SMZ, Cipro, Norflox• Salmonella-Quinolones, Ceftrixone• V.cholerae - Doxycycline, Tetracycline, Erythromycin• E. coli-Cipro, norflox• C. difficile-Metronidazole, Vanco• Cryptosporidium- Paromomycin• Isospora- TMP-SMZ, • Cyclospora-TMP-SMZ


Control measures

WHO, UNICEF- oral rehydration therapy.

Short-term:

(a) ORT – 1978 started in 85-86 (National program), 92-93 (included in maternal and child health program)

(b) normal food intake, breast fed (c)Chemotherapy- Infective; Cholera

Toxins; Shigella, E. coli, Campylobacter

Invasive; Salmonella


Control measures

Long-term: • Nutrition• Sanitation- to stop the transmission

Oro-FaecalWater supplyFood

• Health education- environment, clean drinking water• Immunization • Fly control


Vibrio cholerae on TCBS


Salmonella on XLD


Cryptosporidium parvum in stool(Modified acid fast)


Isospora belli-Direct smear(Kinyoun stain)


Cyclospora cayetanensis-oocyst (modified acid fast)


Microsporidian spores(Modified Trichrome blue stain)


Clostridium difficile growth under UV light

Diarrhoea 2003, SGPGIMS, Lucknow Diagnostic Approach to Infective Diarrhoea in the Community and...

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