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Diagnostic Approach to Infective Diarrhoea in the Community and Rational Antibiotic Therapy Dr. A. Ayyagari Prof. & Head Department of Microbiology SGPGIMS, Lucknow (U.P.) India Diarrhoea 2003, SGPGIMS, Lucknow 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 37 days, may 10-14 days. • Non inflammatory e.g. V. cholerae (no abnormal histology) • Inflammatory- with blood and mucus e.g. Shigella Diarrhoea 2003, SGPGIMS, Lucknow 40 30 20 10 0 Admission Death Diarrhoea 32 16 Others 34 31 ARI 13 13 VPD 15 25 Neonatal 4 13 Diarrhoea Others ARI VPD Neonatal Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow Cause of Acute diarrhoea (<14 days) • • • • • Infectious diarrhoea Medications Ischemic colitis Sup. Mesenteric arterial or venous thrombosis Acute diverticulitis Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow Infectious diarrhoea- Mechanisms & causes Mechanism Examples Mucosal Invasion Minimal Variable Severe Systemic Infection Norwalk, Rota, Adeno, Calici, Corona, CMV Aeromonas, Campylobacter, Salmonella, V. parahaemoliticus E. histolytica, EIEC, Shigella Legionella, Listeria, Measles, Psittacosis Diarrhoea 2003, SGPGIMS, Lucknow Diarrhoea in HIV/AIDS patient Bacteria Virus C. jejunii CMV Shigella sp Enteric adeno Salmonella Calici virus C. difficle HIV EAEC Mycobacterium avium complex Parasite Cryptosporidium Isospora belli Cyclospora Microsporidia Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow 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) Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow Microscopy Wet mount- Ova and trophozoites of parasites WBCs indicate invasive pathogens Phase contrast microsciopy- Campylobacter Immune electron microscopy- Viruses Staining methods Oocyst Acis-fast stains- Cold/hot Kinyoun modified stain, Giemsa stain, PAS stain, Direct fluorescence stain, E. histolytica- Trichrome stain Microsporidium- ModifiedTrichrome Gram stain Diarrhoea 2003, SGPGIMS, Lucknow Lab Diagnosis- Enrichment medium Medium Incubation time For Salmonella and Shigella Gram negative broth Selenite F-broth Tetra thionate broth 4-6 hr 12 hr 12 hr For V. cholerae Alkaline peptone water Monsur’s taurocholate telllurite peptone water 6-8 hr 6-8 hr Diarrhoea 2003, SGPGIMS, Lucknow 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 hour All NLF col(oxidase negative) on MacConkey, should be S/c on DCA All black centered colony on DCA All non sorbitol fermenter colony (EHEC) Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow 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. Diarrhoea 2003, SGPGIMS, Lucknow 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. Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow 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, Not as sensitive high specificity cytotoxin assay LAgg Bacterial Culture Tox and Nontox C. Sensitive Requires O2, 2-5 days difficile Allows strain tytping in Not specific for tox epidemics producing bacteria PCR Tox A or B gene in High sensitivity isolates or directly specificity in feces enzyme Fast, Inexpensive (glutamat dehydrogenase) Easy to perform Poor sensitivity specificity as & & Requires expertise Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow 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 RotaEIA, membrane EIA, LA, EM, culture, RT-PCR Diarrhoea 2003, SGPGIMS, Lucknow Rational Antibiotic Therapy Most cases are self limiting and subside with supportive therapy Indication of antibiotic therapy • Cholera • Febrile bloody diarrhoea • Travelers diarrhoea • extremes of age • Food handlers • Immunocompromised • Day care attendee • Residents of institutional facility • Epidemic outbreaks Diarrhoea 2003, SGPGIMS, Lucknow Rational Antibiotic Therapy 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 Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow 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 Diarrhoea 2003, SGPGIMS, Lucknow Control measures Long-term: • Nutrition • Sanitation- to stop the transmission Oro-Faecal Water supply Food • Health education- environment, clean drinking water • Immunization • Fly control Diarrhoea 2003, SGPGIMS, Lucknow Vibrio cholerae on TCBS Diarrhoea 2003, SGPGIMS, Lucknow Salmonella on XLD Diarrhoea 2003, SGPGIMS, Lucknow Cryptosporidium parvum in stool(Modified acid fast) Diarrhoea 2003, SGPGIMS, Lucknow Isospora belli-Direct smear(Kinyoun stain) Diarrhoea 2003, SGPGIMS, Lucknow Cyclospora cayetanensis-oocyst (modified acid fast) Diarrhoea 2003, SGPGIMS, Lucknow Microsporidian spores(Modified Trichrome blue stain) Diarrhoea 2003, SGPGIMS, Lucknow Clostridium difficile growth under UV light Diarrhoea 2003, SGPGIMS, Lucknow