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. 1972
• Norwalk virus
• Kapikian, et al.
1973
• Rota virus
• Bishop, et al.
1975
• Astroviruses
• Mandeley, et al.

4. 1987
• Enteric Adenoviruses
• Wadell, et al.
1991
• Aichi virus
• Yamashita et al.
1993
• Toroviruses
• Koopmans, et al.

5. 1993
• Picobirnaviruses
• Grohmann, et al.
2000
• NLVs & SLVs
• Chiba, et al.
2008
• Saffold virus
• Jones, M.S et al.

6. 2009
• Salivirus
• Greninger, A.L et al.
2012
• MW polyomavirus
• Siebrasse, E.A et al.
2012
• Bufavirus
• Phan, T.G et al.

7. The Imortant Agents
Virus Family Size Appearance on
EM
Nucleic
Acid
Detection
Rotavirus Reoviridae 70 Wheel shaped dsRNA EM,EIA,IC
T, 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
Picobirnavir
us
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

8. Astroviruses
Rota virus

9. Adeno viruses

10. 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- 5th leading cause of death
in children.
In developing countries most children have >3
episodes of gastroenteritis each year.

13. 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

16. 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.

18. 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.

20. Astroviruses- Routes of Infection

21. 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.

22. 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.

26. 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.

27. 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

28. 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.

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

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

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

39. 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.

40. 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.

41. 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.

42. 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.

43. 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).

45. 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.