Noroviruses - SAVALnet PY

FAMILY CALICIVIRIDAE:
Noroviruses
Dra. Sonia Montenegro
Departamento de Pediatría
Hospital Regional GGB
5 de octubre, 2008
Temas a considerar:
Taxonomía, estructure y genoma
Enfermedad y sintomas clínicos
Diagnóstico
Transmisión
Epidemiología e Incidencia
Resistencia Ambiental
Prevención y Control (Infección Nosocomial)
Taxonomía de Norovirus
Previamente- Familia Caliciviridae
Genero:1. Norwalk-Like Viruses (NLVs)
2. Sapporo-Like Viruses (SLVs)
Recientemente- Género Noroviruses
(antes Norwalk-Like Viruses)
Morfología Viral
Sin envoltura,forma icosahedrica
27-40 nm
Estruct. única proteína capsid, 60kD
- 180 moléculas, formada de 90
dímeros
Características Especiales :
32 depresiones como conos en los ejes
del icosahedro
Estructura de Norovirus por M.E.
www.pubmed.gov PMID: 13679618
Genoma
(+) ss RNA, 7900 nt de longitud
3 Open Reading Frames:
ORF1= proteins no-estructurales
- RdRp+ helicase
ORF2= proteina de capsid estructural
ORF3= proteína peq., función desc.
ORF1
5’
RdRp
hel
ORF2
cap
ORF3
?
3’
RdRp Structure
Estructura cristal.
de www.pubmed.gov
PMID: 12706072
Estrategia de Replicacion
Replicación Típica de virus RNA positive sense ss
Wagner et al. Basic Virology.
www.netlibrary.com
Manifestaciones Enfermedad
Infecta intestino delgado y causa
gastroenteritis
Expansión de los vellosidades del ID
proximal y acortamiento microvellosidad.
- celulas epiteliales permanecen intactas
Periodo Incubación : 24-48 hours
Conocido como- “stomach flu”
Via de Infección
Wagner et al. Basic Virology.
www.netlibrary.com
Sintomatología Clínica
Diarrea No-sanguinea.
Nausea
Vómitos
Cólicos Abdominal.
Malestar
Myalgias
Dolor de cabeza
Fiebre baja
Síntomas duran 12-60 horas.
Niños tienden a sufrir de vómitos, mientras que
los adultos tienden a sufrir de diarrea.
Transmisión
Fecal-Oral
Típicamente por agua de beber contaminada
Muchas fuentes encontradas :
suministro de agua municipal de
mala calidad
pozos
lagos recreacionales
piscinas
agua de cruceros
alimento manipulado por personas
infectadas o lavada con agua
contaminada
Prevalencia de Fuentes de
Transmission
Characteristics of Noroviruses
Feature
Observation
Consequences
Low infectious dose
<102 virus particles
Droplet/person-to-person,
foodhandler spread, second. spread
Prolonged
asymptomatic
shedding
Environmental
stability
Up to 2 weeks
Increases risk of secondary spread,
problems with control of food
handlers
Survives 10 ppm
chlorine, freezing,
67°C
Hard to eliminate from
contaminated water; survives in
ice, partially cooked oysters
Great strain diversity Many genetic and
Composite diagnostics; repeated
infections with many different
antigenic types, underestimate
prevalence
antigenic types
Lack of longtime
immunity
Disease with
reinfection
Childhood exposure doesn’t
protect in adulthood; difficult to
develop vaccine with lifelong
protection
Reservoir
Humans
Only in human host— identificat.
of related animal strains
Clinical Characteristics
• Excretion of virus in stool begins a few hours
before the onset of symptoms and a maximum
of 24-72 hours after exposure
• Symptoms usually last 24-60 h; 30% of
infections may be asymptomatic
• Virus can be present in stool of infected
persons for a week or more after recovery or
even if never sick
Norovirus Susceptibility and
Resistance
Susceptibility to norovirus is related to histo-blood group
antigens (HBGAs) status:
• NV attach to carbohydrates of the histo-blood group
family on human gastroduodenal epithelial cells
(and saliva) if present
• Affinity may vary by viral genotype
• Human milk contains decoy receptors
• Oysters contain similar receptors
Factors Associated with Secondary
AGE Among Household Contacts
• Age of Contact; transmission inversely
proportional to age of contact (<2yrs 4
times more likely to transmit infection)
• Bed sharing
• Exposure to primary vomiting episode
• Member of index family
Perry et al. EID Vol. 11, No. 7, July 2005
Epidemiología e Incidencia
Norovirus hay en todo el mundo.
Carga: - aprox. 267,000,000 casos anuales
- 612,000 hospitalizaciones
- 3,000 muertes
Altamnete contagioso: menos de 100 partículas
virales pueden causar infeccion.
El 2002- un barco cricero para Alaska, brote
con 13% de los 1266 a bordo
afectados; agua de beber
contaminated
Outbreaks of Gastroenteritis Associated with
Noroviruses on Cruise Ships --- United States, 2002
During January 1--December 2, 2002, CDC's Vessel Sanitation
Program (VSP), which conducts surveillance for acute gastroenteritis
(AGE) on cruise ships with foreign itineraries sailing into U.S. ports
(1), received reports of 21 outbreaks of AGE* on 17 cruise ships.
Of the 21 outbreaks, nine were confirmed by laboratory analysis of
stool specimens from affected persons to be associated with
noroviruses, three were attributable to bacterial agents, and nine were
of unknown etiology.
Seven outbreaks were reported in 2001, and of these, four were
confirmed to be associated with norovirus (CDC, unpublished data,
2002).
This report describes five of the norovirus outbreaks that occurred
during July 1--December 2, 2002, on cruise ships.
EH Cramer, D Forney, Vessel Sanitation Program;
AL Dannenberg, MD, Div of Emergency and Environ.Health Svcs, National Center
for Environmental Health;
MA Widdowson, et al Div of Viral and Rickettsial Diseases;
E Mintz, C Stover,, Div of Bacterial Diseases, Nat Cent. of Infectious Diseas;
E Isakbaeva, et al MD, CDC.
Outbreaks of Gastroenteritis Associated with
Noroviruses on Cruise Ships --- United States, 2002
•
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•
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Cruise Ship C. On September 28, cruise ship C, owned by cruise line B, embarked
1,984 passengers and 941 crew members for a 7-day round-trip cruise from Florida
to the Caribbean.
Several passengers had AGE within 24 hours of embarkation, and by October 1, a
total of 70 (4%) passengers and two (0.2%) crew members reported illness.
On October 3, CDC investigators boarded the ship to conduct an epidemiologic and
environmental investigation.
Questionnaires completed by 1,879 (95%) passengers and 860 (91%) crew members
identified 356 (19%) passengers and 13 (1.5%) crew members who met the AGE case
definition.
The epidemiologic investigation suggested a point source of infection, followed by
cases associated with person-to-person transmission.
The investigation identified an association between illness among passengers and
lunch served at embarkation (odds ratio=2.4; 95% confidence interval=1.1--5.2; p
value=0.02).
Four of 11 stool specimens from patients were positive for norovirus by RT-PCR.
Characterization of the strain by sequence analysis of RT-PCR products matched
those from an outbreak on the same ship that occurred 3 weeks previously but was
not identical to the outbreak strain on cruise ships A and B.
CDC recommended reinforcing sanitation practices and excluding ill foodhandlers
from the work place. Cruise ship C continued service, and no new cases were
reported on subsequent cruises.
Outbreaks of Gastroenteritis Associated with
Noroviruses on Cruise Ships --- United States, 2002
Cruise Ship A. On July 18,2002 cruise ship A, owned by cruise line A, embarked
1,318 passengers and 564 crew members for a 7-day cruise from Vancouver to
Alaska.
On July 19, five passengers reported to the ship's infirmary with symptoms of
Acute Gastro Eenteritis AGE.
By July 25, a total of 167 (13%) passengers and nine (2%) crew members had
reported illness.
Among the 176 patients, the predominant symptoms were vomiting (76%) and
diarrhea (73%).
Five of 10 stool specimens from ill passengers were positive for norovirus by
reverse transcriptase polymerase chain reaction (RT-PCR).
On July 25, when passengers disembarked, the ship was disinfected in
accordance with CDC recommendations, and the same day, a new group of
passengers embarked for another 7-day cruise.
During the cruise, 189 (14%) of 1,336 passengers and 30 (5.3%) of 571 crew
members had AGE with diarrhea (91%) and vomiting (85%) .
An environmental health inspection conducted by CDC revealed no sanitary
deficiencies. Cruise line A cancelled a subsequent cruise and voluntarily took
the ship out of service for 1 week for aggressive cleaning and sanitizing. No
outbreaks were reported on subsequent cruises.
Outbreaks of Gastroenteritis Associated with
Noroviruses on Cruise Ships --- United States, 2002
Cruise Ship B. On October 1, cruise ship B, also owned by cruise line A,
embarked 1,281 passengers and 598 crew members for a 21-day
cruise from Washington to Florida.
By October 16, a total of 101 (8%) passengers and 14 (2%) crew members
reported to the infirmary with AGE symptoms.
On October 18, CDC investigators boarded the ship to conduct an
epidemiologic and environmental investigation.
Of 972 surveyed passengers, 399 (41%) met the case definition for AGE.
Investigators found no association between illness and water, specific
meals served on the ship, or with offshore excursions.
Stool specimens from 12 of 13 patients tested positive for norovirus.
Characterization of the strain by sequence analysis of RT-PCR
products matched those from cruise ship A.
Despite implementation of control measures that included disinfection of
the vessel and quarantine of ill passengers and crew members, a total
of 264 passengers and 41 crew members reported illness on three
subsequent 10-day cruises.
Cruise line A voluntarily withdrew cruise ship B from service for 10 days
for aggressive cleaning and sanitizing. No outbreaks were reported on
subsequent voyages.
Prevención y Control
Altamente estable
en el ambienteresistente a:
CONGELAMIENTO
CALENTAMIENTO A 60ºC
DESINFECCIÓN C/ CLORO
CONDICIONES DE ACIDEZ
VINAGRE
ALCOHOL
ALTA CONCENTRACIÓN DE AZÚCAR
Prevencion y Control
Infección produce IgG, IgA and IgM, pero los
anticuerpos no son protectores.
No produce inmunidad duradera ni protección a
la reinfección.
Inmunidad sólo transitoria que dura 3-4 meses.
No hay vacuna y no es probable su desarrollo.
No hay drogas antivirales,sin embargo
recuperacion completa es común.
Prevenir con lavado de manos,buena higiene,
manejo adecuado del agua y de la preparación
de alimentos.
DEFINITION
“Nosocomial infection is a localized or systemic
condition that results from adverse reaction to
the presence of an infectious agent(s) or its
toxin(s) and that was not present or incubating at
the time of admission to the hospital”
CDC definitionns of nosocomial infections. APIC Infection Control and
Applied Epidemiology: Priciples and Practice. 1996:A-1-20
Norovirus en Chile
Vidal R. Et al 2006.Novel Recombinant
Norovirus Causing Outbreaks of Gastroenteritis
in Santiago, Chile.
-The purpose of this study was to characterize
genetically the capsid of the NV strains detected
in gastroenteritis outbreaks
-Outbreaks of gastroenteritis occurring in
Santiago, between 2001 and 2003 were caused
by genetically
diverse NV strains that grouped mainly into GII.
Norovirus en Chile
Vidal, R., et al 2005 V. 2005. Caliciviruses and
foodborne gastroenteritis, Chile. Emerg. Infect.
Dis. 11:1134–37.
The purpose of this
study was to determine the
t
role of HuCVs as a cause of gastroenteritis
outbreaks from June 1, 2000, to January 30,
2003, in Santiago, Chile, by using recently
improved antigen and genome detection assays,
and to characterize genetically the circulating
strains.
PREVALENCIA Y DIVERSIDAD GENETICA DE NOROVIRUS AISLADOS
DE NIÑOS CON GASTROENTERITIS AGUDA EN CONCEPCION
PROYECTO DIUC N0 207.86207.86-0606-1
Metodología
Trujillo et al. 2006.
J Clin Microbiol
44:1405-12. Use of TaqMan real-time
reverse transcription-PCR for rapid
detection, quantification, and typing of
norovirus.
OBJETIVOS
1. Determinar prevalencia de Norovirus
por diagnsótico molecular en
deposiciones de niños con
gastroenteritis aguda.
2. Clasificación genética de aislados de
Norovirus por secuenciamiento.
3. Determinar prevalencia de Rotavirus,
Adenovirus bacterias y C jejuni spp
diagnóstico convenc.
4. Caracterización clínica y
epidemiológica de los casos por
Norovirus en niños SDA
MÉTODOS
Estudio caso-control Dic 2007 a Sept. 2008
126 niños de 0-14 años con sospecha de
SDA.
58 niños asintomáticos de 0-3 año (guardería)
Análisis de deposiciones por RT-PCR “onestep” a tiempo real, con sondas Taqman
especificas para detección y tipificación de
Norovirus Genogrupos I y II.
Presencia de Rotavirus y Adenovirus en
heces por inmunocromatografía dual kit
VIKIA™ “Rota-Adeno” de Biomerieux ,
realizado en el lab. de Microbiología del
Hospital Regional GGB
Datos del Paciente
Datos del Paciente
Ficha inicial
Datos del Paciente
Ficha inicial
Datos del Paciente
Ficha inicial
Datos del Paciente
Ficha inicial
METODOS
SECUENCIAS DE PRIMERS Y
SONDAS
Primers Norovirus Genotipo I:
COG 1R :
COG 1F :
RING 1A :
RING 1B :
Tamaño de banda: 84 bp
5' - CTT AGA CGC CAT CAT TYA - 3
5' - CGY TGG ATG CGI TTY CAT GA - 3’
5' - AGA TYG CGA TCY CCT GTC CA - 3’
5' - AGA TCG CCG TCT CCT GTC CA - 3
Primers Norovirus Genotipo II:
Tamaño de banda: 97 bp
COG 2R : 5’ - TCT ACG CCA TCT TCA TTC ACA - 3’
COG 2F :
5’ - CAR GAR BCN ATG TTY AGR TGG ATG AG - 3’
NOV RING 2 : 5’ - TGG GAG GGC GAT CGC AAT CT - 3’
METODOS
PROGRAMA TERMOCICLADOR
Norovirus RT-PCR
RT:
Denaturacion :
30 min, 50˚C
10 min, 95 ˚C
PCR :
(#45)
15 sec, 94˚C
25 sec, 56˚C
25 sec, 72˚C
RESULTADOS
Norovirus Real-time Taqman PCR
C+ 1074 GII
C+ 1074 GII
C+ KS200
C+ KS200
C+ 1293 GI
C+ 1293 GI
C+ 1057 GI
C+ 1057 GI
RESULTADOS
Amplification Plots Samples
RESULTADOS
Norovirus Real-time Taqman PCR
1. AGUA
C(T)*
2. M. 1293 G1
38
3. M. 1074 GII 30
RESULTADOS
EN 126 NIÑOS:
113 hospitalizados, 13 ambulatorios
23.8 % Norovirus: 19 % G II ; 4.8 % G I
Rotavirus 14.4 5.6% de Adenovirus.
En los niños controles se encontró 5.2%
Norovirus G1 y 1.7% Norovirus GII.
RESULTADOS
EN 90 NIÑOS DE 0-2 AÑOS
31% Norovirus: 25.6% GII y 5.6% GI.
16.85% Rotavirus y 7.86% Adenovirus
Infección Mixta en 3 niños (norovirus GI y GII)
NIÑOS HOSPITALIZADOS POR SDA 46% ( de 90)
31.25% Norovirus GII; 21.9% Rota 9.1 %Adeno.
NOSOCOMIAL? 54% HOSPITAL. OTRAS CAUSAS
26.3% Norovirus GII, 13.2% Rotavirus y 2.6% Adenovirus.
DISTRIBUCION ESTACIONAL
VERANO
70 niños
3/70 4.3%
OTOÑO
38 niños
0
INVIERNO
17niños
1/17 5.9%
Norovirus
genotipo II
6/70 8.6%
11/38
28.9%
5/17 29.4%
Rotavirus
13/70
18.6%
3/70 4.3%
2/17
11.76%
Norovirus
genotipo I
Adenovirus 3/70 4.3%
4/38 10.5% 0
CONCLUSIONES
La mayor prevalencia de Norovirus se encontró
en niños de 0-2 años con mayor frecuencia de
Norovirus genotipo II.
La prevalencia de Norovirus en niños 0-2 años
fue superior a la de Rotavirus y es posible que
un porcentaje importante sea adquirido como
infección intra hospitalaria.
Resultados
Liu Z. et al 2007 Jul; Infect &Digest Tract Dis . Clinical
manifestations of norovirus gastroenteritis in infants
and children.
RESULTS: 24.8% positive for norovirus antigen . 60.8%
October to December, seasonal preference of the virus.
91.2% positives found under 2 years of age.
Rotavirus 20.3%, co-infected by these two viruses.
Principal diferencia clinica severidad de fiebre en
Rotavirus.
18% of Noro positives admitted to hospitals with
diagnosis other than gastroenteritis symptoms of
diarrhea between 1 to 11 days after hospitalization.
Nosocomial infection (NI)
Exogenous: microorganisms originated
– Hands
– Devices
– Infusions, injections
It can be prevented by high hygiene!!!
Endogenous: microorganisms originated
– Bowel system
– Skin surface
– Mounth
– Genital system