Viral gastroenteritis

Brief presentation of Viral AGENTS causing Gastroenteritis with clinical and diagnostic aspects for UG Medical and PG Students.

1. Viral Gastroenteritis Dr. Suprakash Das

2. Introduction Definition- Acute Gastroenteritis is defined as diarrheal disease of rapid onset, with or without nausea, vomiting, fever or abdominal pain. It involves increased stool frequency or altered stool consistency that is unrelated to chronic condition. History of Viral agents causing gastroenteritis- In past, the etiological agent for gastroenteritis was not diagnosed in many cases and it was suspected that non-bacterial ( i.e. viral) pathogens may be involved. The first clue of such viruses comes from studies done by Gordon, et al. in 1947 where they found bacteria free filtrate of stool from gastroenteritis cases can cause the disease. But the confirmed viral agent causing gastroenteritis was discovered in 1972 ( Kapikian , et al. ) After that many other viruses were discovered.

3. The Imortant Agents Virus Family Size Appearance on EM Nucleic Acid Detection Rotavirus Reoviridae 70 Wheel shaped dsRNA EM,EIA,ICT, RT-PCR Calcivirus Calciviridae 28-35 Small round with calices ss (+ ve ) RNA EM,EIA, RT-PCR Astrovirus Astroviridae 28-30 Star shaped ss (+ ve ) RNA DO Adenovirus Adenoviridae 70-80 Icosahedral dsDNA DO + Culture Picobirnavirus Birnaviridae 35 SRV dsRNA EM, RT-PCR Coronavirus Coronaviridae 60-200 Pleomorphic with club shaped projections ss (+ ve ) RNA EM, PCR Torovirus Coronaviridae 100-150 “Do” ss (+ ve ) RNA EM Aichi virus Picornaviridae 30 SRVs ss (+ ve ) RNA EIA, Culture

4. Astroviruses Rota virus

5. Adeno viruses

6. Epidemiology Rota viruses are the most common causative agent for gastroenteritis in < 5 years of children. Noroviruses are the most common cause of mild gastroenteritis in community effecting all age groups. Noroviruses also cause traveller’s diarrhea. In developing countries gastroenteritis accounts for >2M deaths/ year- 5 th leading cause of death in children. In developing countries most children have >3 episodes of gastroenteritis each year.

7. Pathogenesis Rotaviruses- Infects the mature enterocytes on the tips of intestinal villi . Villous epithelium atrophy + villous ischemia+ round cell infiltration of lamina propria . Compensatory repopulation of the epithelium by immature secretor cells. Secondary hyperplasia of crypts. + Stimulation of G.I nervous system. Decrease in absorption in comparison to secretory capacity. Secretory diarhhea with loss of fluid and elctrolytes in lumen. The level of brush border enzymes ( sucrase , lactase), characteristic of differentiated cells is decreased Accumulation of unmetabolized di-saccharides in gut lumen Osmotic diarrhea

8. Pathogenesis Enteric Adenoviruese Infects the mature enterocytes on the tips of intestinal villi . Villous epithelium atrophy + villous ischemia+ round cell infiltration of lamina propria . Compensatory repopulation of the epithelium by immature secretor cells. Secondary hyperplasia of crypts. Decrease in absorption in comparison to secretory capacity. Secretory diarhhea with loss of fluid and elctrolytes in lumen.

9. Pathogenesis- Noroviruses Multiple studies demonstrate that NoVs bind carbohydrates . These carbohydrates are expressed on enterocytes and secreted into the gut lumen. Furthermore, enteric bacteria can express similar carbohydrates . NoVs may bind to such carbohydrates in any of these contexts  (1) NoVs are then transcytosed across the intestinal epithelium via M cells  (2) Following transcytosis , NoVs infect dendritic cells, macrophages, and B cells  (3) Depending on the species, infection can occur in the presence or absence of carbohydrates. Free carbohydrates or bacterially expressed carbohydrates may be co- transcytosed with the virus. Immune cell infection and putative concomitant viral-bacterial antigen presentation during NoV infections could have significant consequences on the nature and magnitude of antiviral immune responses.

10. Astroviruses - Routes of Infection

11. Immunology of Viral Gastroenteritis Rotaviruses- Local intestinal immunity protects against successive infections against Rota virus. Nutralizing antibodies are directed towards VP4 & VP7 proteins. NSP4 protein induces CMI Complete protection against Rotavirus infection is not possible as the antibody response is short lived and Memory B & T cells takes time to act in re-infections but they reduce the severity. Enteric Adenoviruses- Type specific nutralizing Ab can protect against current and re-infections. Norwalk virus- Induces specific IgG , IgA & IgM serum antibody response. Increase in jejunal synthesis of IgA Some individuals have genetic predisposition to viral gastroenteritis , related to ABO, Lweis & secretor blood group phenotypes.

12. Clinical Manifestations Sudden onset Average incubation period- 24h Generally lasts for- 12-60h Nausea, vomiting (more in children) Loose watery stool without blood, mucous & WBCs (i.e. diarrhea, more in adults.) Constitutional symptoms- Headache, fever, chills & myalgia .

13. Nosocomial infections - Norovirus Transmission of NoV most often occurs through direct contact with NoV shedders, or indirectly through environmental or food contamination with human feces or vomit, especially in closed settings such as hospitals, nursing homes, cruise ships, and hotels. Transmission from chronic patients shedding NoV for a long period of time may also occur. In a healthcare facility, patients with suspected norovirus may be placed in private rooms or share rooms with other patients with the same infection. Additional prevention measures in healthcare facilities can decrease the chance of coming in contact with noroviruses : Follow hand-hygiene guidelines, and carefully washing of hands with soap and water after contact with patients with norovirus infection Use gowns and gloves when in contact with, or caring for patients who are symptomatic with norovirus Routinely clean and disinfect high touch patient surfaces and equipment with an Environmental Protection Agency-approved product with a label claim for norovirus Remove and wash contaminated clothing or linens Healthcare workers who have symptoms consistent with norovirus should be excluded from work.

14. Laboratory Diagnosis Antigen detection- Recently, a wide variety of tests for the detection of antigen in fecal specimens have been developed. These are based on- Enzyme immunoassay (EIA), Agglutination with latex particles (LA), Immunochromatography (IC) and, more recently, Chemiluminescent immunoassay (CLIA), All of which are available commercially for human calicivirus , rotavirus, adenovirus, and astrovirus . In clinical laboratory practice, rapid and reproducible antigen detection methods seem to be superior among the conventional techniques. The sensitivity is generally higher than that of conventional methods ( e.g., EM and IAHA) although lower than that of molecular methods .

15. Laboratory Diagnosis Enzyme immunoassay has been proven to be very sensitive and specific for the detection of group A and C rotaviruses in fecal specimens, especially if monoclonal antibodies are used . Immunoassay techniques are also available for the detection of astroviruses , due to the development of monoclonal antibodies against these viruses . Several EIA methods using monoclonal and polyclonal antibodies have been developed for the detection of calicivirus . In particular, EIA techniques that use monoclonal antibodies have been evaluated for their ability to detect noroviruses in stool samples.

16. Laboratory Diagnosis The LA technique is used clinically in the identification and typing of most gastrointestinal viruses. Latex particles coated with virus antibodies are agglutinated in the presence of virus antigen to produce the visible aggregates. Although the agglutination test is a more rapid method than EM or EIA, it is relatively less sensitive. IC assay is rapid, technically very simple, and showed results comparable to those achieved with EIA. Given that these tests have a high sensitivity and specificity (90%–95%), they are widely used in clinical laboratory practice. Most recently, rapid detection strip tests by IC kits have become commercially available for testing for astroviruses and noroviruses in stool specimens. The CLIA method is a diagnostic chemiluminescent immunoassay in which the virus antigen is captured by antibodies coupled with a molecule capable of emitting light during a chemical reaction. Light emission is used to measure the formation of the antigen-antibody complex. A CLIA test able to detect rotavirus in stool specimens.

17. Laboratory Diagnosis Molecular methods – Several nucleic acid amplification techniques (NAATs), particularly Polymerase chain reaction (PCR), Real-time PCR, and Multiplex PCR , are currently used in routinely in clinical laboratories. Such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. Moreover, NAATs have offered additional advantages over traditional methods by production of easily standardized protocols, thus resulting in a potential for automation with a range of options for real-time detection chemistries. The advent of fully automated systems with faster turnaround times has given clinical laboratories the tools necessary to report out accurate and sensitive results to clinicians.

18. Laboratory Diagnosis New techniques, including loop-mediated isothermal amplification (LAMP) and NASBA , are starting to be used routinely in clinical laboratories to detect gastrointestinal viruses. Recently, an astrovirus -specific NASBA assay and a RT-LAMP for the identification of norovirus was developed for rapid detection of vital RNA in large numbers of stool specimens. Conventional molecular methods (end-point PCR, nested PCR) – Currently, PCR is widely employed as a tool for the routine diagnosis of astrovirus , norovirus , sapovirus , rotavirus, and adenovirus infections. These PCR assays are highly sensitive, specific, and easy to perform. The most reliable marker for diagnosis of virus infection is the presence of viral nucleic acid in stool specimens. Therefore, the specimen of choice is stool samples from patients with diarrhea .

19. Laboratory Diagnosis The amplification of the viral genome and sequencing of the amplification products should be performed, and virus genotypes can be identified based on their sequence analysis. Therefore, PCR assays and nucleic acid sequence analysis are widely used for the detection and genotype identification of viruses causing gastroenteritis. Gradually, these techniques have replaced the traditional immunological tests and have become the gold standard for diagnosis of gastrointestinal viruses for almost two decades. Nested PCR assays were also developed to increase both sensitivity and specificity. At last, multiplex RT-PCRs have been widely described. In particular, multiplex RT-PCRs for the detection of groups A, B, and C rotaviruses and identification of G and P genotypes of group A rotaviruses have been developed.

20. Laboratory Diagnosis Real-time PCR Real-time PCR technology provides results more quickly than conventional PCR assays and shows improved sensitivity and specificity. Although reagent and instrument costs are higher for real-time PCR technology compared to conventional molecular methods, real-time PCR requires less hands-on time per specimen than traditional PCR, particularly nested PCR, which is labor intensive. Automation of the extraction process and the use of real-time PCR further reduce the hands-on time in the clinical laboratory. Moreover, real-time PCR technology offers advantages over conventional PCR by providing lower risk of false-positive results due to amplicon contamination and quantification of viral load. Real-time PCR assays that detect the most common gastrointestinal viruses in large numbers of stool specimens have been developed.

21. Main features of diagnostic approaches to viral gastroenteritis Biofire (Multiplex Real Time PCR) Biomeriux

22. Biofire (Multiplex Real Time PCR) G.I Panel- Biomeriux

23. Treatment Rotavirus gastroenteritis can lead to severe dehydration. Thus appropriate treatment should be instituted early. Standard oral rehydration therapy is successful in most children who can take oral fluids, but IV fluid replacement may be required for patients who are severely dehydrated or are unable to tolerate oral therapy because of frequent vomiting. The therapeutic role of probiotics , bismuth subsalicylate, enkephalinase inhibitors, and nitazoxanide has been evaluated in clinical studies but is not clearly defined. Antibiotics and antimotility agents should be avoided. In immunocompromised children with chronic symptomatic rotavirus disease, orally administered immunoglobulins or colostrum may resolve symptoms, but The choice of agents and their doses have not been well studied and are often empirical.

24. MILD DEHYDRATION (6% OR LESS) Mild dehydration from acute gastroenteritis can be managed at home, with oral rehydration therapy as the mainstay of treatment. No significant difference in hospitalizations or return emergency department visits between oral and intravenous rehydration and only one out of 25 children treated with an ORS will eventually require intravenous fluids. Children older than six months showed that half-strength apple juice followed by preferred fluids (regular juices, milk) reduced the need for eventual intravenous rehydration compared with a formal ORS, most likely because children were more apt to drink the preferred fluids than the ORS. After each loose stool, the World Health Organization (WHO) recommends giving children younger than two years 50 to 100 mL of fluid and Children two to 10 years of age 100 to 200 mL of fluid; Older children may have as much fluid as they want. Children may consume up to 20 mL per kg of body weight per hour.

25. MODERATE TO SEVERE DEHYDRATION (MORE THAN 6%) Treatment of moderate dehydration includes an ORS plus medication if needed to decrease vomiting and improve tolerance of the ORS. WHO now recommends its reduced osmolarity ORS, which contains 75 mEq per L of sodium and 75 mmol per L of glucose dissolved in 1 L of water. Children younger than two years should be given 1 teaspoon every one to two minutes; older children should be encouraged to take frequent sips directly from the cup. If vomiting occurs, the recommendation is to wait five to 10 minutes and then start offering the ORS again more slowly, every two to three minutes. Antiemetics - Ondansetron is commonly used when needed to prevent vomiting while drinking the ORS. \

26. MODERATE TO SEVERE DEHYDRATION (MORE THAN 6%) The typical dose of ondansetron is 2 mg for children weighing 8 to 15 kg (17 lb, 10 oz to 33 lb), 4 mg for children weighing 15 to 30 kg (33 lb to 66 lb, 2 oz), and 8 mg for children weighing more than 30 kg. The dose may be repeated if the child vomits within 15 minutes of taking the medication. Ondansetron should be avoided in patients with congenital long QT syndrome . In addition, electrolytes should always be assessed before administration because hypomagnesemia and hypokalemia increase the risk of QT prolongation.

27. If the patient wants more ORS than shown, give more. Encourage breastfeeding mothers to continue breastfeeding the child. For infants younger than six months who are not breastfed: if using the old WHO ORS solution (90 mEq per L of sodium), add an extra 100 to 200 mL of clean water; this is not necessary if using the new reduced osmolarity ORS (75 mEq per L of sodium).

28. Prevention-Rotavirus vaccines Good hygiene like handwashing and cleanliness are important, but are not enough to control the spread of the disease. Rotavirus vaccine is the best way to protect your child against rotavirus disease. Most children (about 9 out of 10) who get the vaccine will be protected from severe rotavirus disease. About 7 out of 10 children will be protected from rotavirus disease of any severity. Two rotavirus vaccines are currently licensed for infants in the United States: RotaTeq ® (RV5) is given in 3 doses at ages 2 months, 4 months, and 6 months Rotarix ® (RV1) is given in 2 doses at ages 2 months and 4 months The first dose of either vaccine should be given before a child is 15 weeks of age. Children should receive all doses of rotavirus vaccine before they turn 8 months old. Both vaccines are given by putting drops in the child’s mouth.

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