Ignite Creation Date:
2024-05-05 @ 11:51 AM
Last Modification Date:
2024-10-26 @ 9:15 AM
Study NCT ID:
NCT00154596
Status:
UNKNOWN
Last Update Posted:
2015-03-17
First Post:
2005-09-09
Brief Title:
Etiology Study of Kawasaki Disease
Sponsor:
National Taiwan University Hospital
Organization:
National Taiwan University Hospital
Study Overview
Official Title:
Etiology Study of Kawasaki Disease--A Prospective Household and Case Control Study
Status:
UNKNOWN
Status Verified Date:
2015-03
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The purpose of this study is to investigate the infectious etiology of Kawasaki disease KD a prospective household and case control study for Kawasaki disease will be done The investigators will enroll Kawasaki disease cases who have at least five of the following manifestations
1 fever for over 5 days
2 neck lymphadenopathy
3 lip fissure andor strawberry tongue
4 skin rash
5 nonpurulent bulbar conjunctivitis
6 palmsole erythema and induration followed by desquamation or coronary artery aneurysm with less than 5 of the above manifestations atypical Kawasaki disease
The KD cases will receive virological virus isolation from the blood throat swabs and rectal swabs or stool gene chip for possible viruses from stored RNA and DNA bacterial blood throat swabs and stool bacterial culture and stored strain for further toxin or superantigen detection and serological Mycoplasma pneumoniae Chlamydiae pneumoniae ASLO HHV6 EV71 peptide library approach for auto-antibody or pathogen-related antibody stored serum for further workup workup Stored DNA from the blood will also be performed
1 fever for over 5 days
2 neck lymphadenopathy
3 lip fissure andor strawberry tongue
4 skin rash
5 nonpurulent bulbar conjunctivitis
6 palmsole erythema and induration followed by desquamation or coronary artery aneurysm with less than 5 of the above manifestations atypical Kawasaki disease
The KD cases will receive virological virus isolation from the blood throat swabs and rectal swabs or stool gene chip for possible viruses from stored RNA and DNA bacterial blood throat swabs and stool bacterial culture and stored strain for further toxin or superantigen detection and serological Mycoplasma pneumoniae Chlamydiae pneumoniae ASLO HHV6 EV71 peptide library approach for auto-antibody or pathogen-related antibody stored serum for further workup workup Stored DNA from the blood will also be performed
Detailed Description:
Patient Selection and Family Surveillance At National Taiwan University Hospital in Taiwan we will study patients who fulfill the criteria of Kawasaki disease or atypical Kawasaki disease and their household family members from 2004 to Dec 2005 Institutional Review Board approval will be obtained for this study and informed consents will be obtained from all subjects or their parents If patients at the emergent service outpatient clinic or inpatient ward had clinical syndromes suggestive of EV71 infection they and their household family members were asked to participate in the study Throat and rectal swabs for virus isolation and the first blood sample Clinical manifestations courses and outcomes were recorded If at any point the patients suspected of infection displayed clinical symptoms the other family members in the same household were asked to undergo screening by virus isolation of throat swabs and received the first blood sample Questionnaire-based interviews were used to collect information including demographic data the number of bedrooms in the household contact time pattern and presence of currentrecent signs and symptoms cough rhinorrhea sore throat rash fever abdominal pain and diarrhea and preceding contact history with extra-household people who had clinical illness Follow-up telephone interviews repeated questions about signs and symptoms at 2 4 and 8-week intervals If any household family member reported experiencing signs or symptoms suggestive of Kawasaki disease during the follow-up period the household member will receive further clinical assessment and repeated laboratory investigation for Kawasaki disease A second blood sample was obtained from the KD cases and all their household family members 4 weeks after the first blood sample was obtained Control case selection An age- and sex-matched control will be randomly selected The control will be the admitted patients in the same ward who have other diagnoses such as pneumonia UTI tonsillitis They will also receive the screen for virus and bacterial workup as the household members do
Definitions of Kawasaki disease and atypical Kawasaki disease
1 Criteria for Kawasaki disease
equal to or over five of the following fever for more than 5 days non-purulent conjunctivitis skin rash palmsole induration and erythema followed by desquamation neck lymphadenopathy strawberry tongue or lip fissurebleeding
2 Atypical Kawasaki disease less than five of the above but with coronary artery aneurysm
Laboratory Methods
1 Virus Isolation and Serotyping
Throat swabs rectal swabs or stool samples were submitted for virus isolation Samples were inoculated into human embryonic fibroblast LLC-MK2 HEp-2 and rhabdomyosarcoma RD cell cultures When cytopathic effect involved more than 50 of the cell monolayer cells were scraped and subjected to indirect fluorescent antibody staining with specific antibodies Chemicon International Inc Temecula CA or typed by specific methods according to the suspected types of viruses
2 Molecular diagnosis for viruses or other pathogens difficult to be cultivated
1 Viral RNA and RNA extraction RNA and DNA extraction will be performed by using Isolation Kit according to the manufacturers guide RNA extraction kit Qiagen 140 uL of throat swab was mixed with AVL buffer for 10 to 15 minutes at room temperature Then spin down the mixture Add 560 uL 100 alcohol into the pellet and incubated for at least 10 minutes at room temperature The mixture will be put into the spin column and centrifuged at 8000 rpm for 1 minute twice Then 500uL of AW1 buffer will be added and the mixture will be centrifuged at 80000 rpm for 1 minute and then 500uL of AW2 buffer will be added and centrifuged at 14000 rpm for 3 minutes Discard the solution and centrifuge at 14000 rpm for 2 minutes Finally 25 to 30 uL of AVE buffer will be added and centrifuged at 8000 to 10000 rpm for 1 minute
2 Reverse transcription RT will be performed with 1st strand cDNA Synthesis Kit for RT-PCR Roche Mix the previously extracted RNA with reagents including buffer MgCl2 dNTP Oligo-pdT15 primer RNase inhibitor reverse transcriptase briefly vortex incubate the reaction at 25oC for 10 minutes and then 42oC for 60 minutes Finally incubate at 99oC for 5 minutes and then cool to 4oC for 5 minutes
3 Real-time TagMan PCR for enterovirus adenovirus or other viral etiology The primers and probes for enterovirus and other virus real time PCR are in the following table Primers and probes for PanEV were selected based on highly conserved regions in the 5-untranslated region of the enterovirus genome The primers and probes for EV71 were selected based on the most diverse and specific genetic regions in the VP1 region of the enterovirus genome Other potential viruses such as adenovirus or other respiratory viruses will also be performed by PCR or RT-PCR or real-time PCR following the rules of the above procedures
4 Representational differential analysis followed by subtractive cloning
3 Bacterial cultures and toxin detection Cultures were obtained from the pharynx and rectum of patients with acute KD before the start of the immune globulin intravenous infusion and from control patients with the use of a Baxter Diagnostics Culturette system that contains a cotton swab The primary data collection sheet was completed and held at the site where specimens were obtained The plates were then examined for all รข-hemolytic group A streptococci and all S aureus that were coagulase positive by the tube test These isolates were then screened in a blinded fashion for the presence of bacterial superantigen production by immunodiffusion with antisera against known staphylococcal and streptococcal superantigens as described previously Lee PK and Schlieveret PM
4 Serological test Serological tests for antibody detection of Mycoplasma pneumoniae Chlamydiae pneumoniae ASLO will be measured by commercial kits Neutralizing antibody for enterovirus will be performed by standard protocol of the neutralization test on microtiter plates EV71 isolate TW227298 was amplified and purified as an antigen for m-capture ELISA for EV71 IgM Detection If potential pathogen is defined further serology test for the specific pathogen will be done later
5 Statistical Analysis Data were analyzed with the SAS Statistical Package Version 82 SAS Institute Cary North Carolina We used Students t test for continuous variables and c 2 test for categorical data After univariate analysis screened statistically significant variables forward stepwise multiple logistic regression analysis was performed to adjust confounders simultaneously and to calculate multivariate-adjusted odds ratios The level of model selection was set at 015 for in-and-out models P 05 was considered statistically significant
Definitions of Kawasaki disease and atypical Kawasaki disease
1 Criteria for Kawasaki disease
equal to or over five of the following fever for more than 5 days non-purulent conjunctivitis skin rash palmsole induration and erythema followed by desquamation neck lymphadenopathy strawberry tongue or lip fissurebleeding
2 Atypical Kawasaki disease less than five of the above but with coronary artery aneurysm
Laboratory Methods
1 Virus Isolation and Serotyping
Throat swabs rectal swabs or stool samples were submitted for virus isolation Samples were inoculated into human embryonic fibroblast LLC-MK2 HEp-2 and rhabdomyosarcoma RD cell cultures When cytopathic effect involved more than 50 of the cell monolayer cells were scraped and subjected to indirect fluorescent antibody staining with specific antibodies Chemicon International Inc Temecula CA or typed by specific methods according to the suspected types of viruses
2 Molecular diagnosis for viruses or other pathogens difficult to be cultivated
1 Viral RNA and RNA extraction RNA and DNA extraction will be performed by using Isolation Kit according to the manufacturers guide RNA extraction kit Qiagen 140 uL of throat swab was mixed with AVL buffer for 10 to 15 minutes at room temperature Then spin down the mixture Add 560 uL 100 alcohol into the pellet and incubated for at least 10 minutes at room temperature The mixture will be put into the spin column and centrifuged at 8000 rpm for 1 minute twice Then 500uL of AW1 buffer will be added and the mixture will be centrifuged at 80000 rpm for 1 minute and then 500uL of AW2 buffer will be added and centrifuged at 14000 rpm for 3 minutes Discard the solution and centrifuge at 14000 rpm for 2 minutes Finally 25 to 30 uL of AVE buffer will be added and centrifuged at 8000 to 10000 rpm for 1 minute
2 Reverse transcription RT will be performed with 1st strand cDNA Synthesis Kit for RT-PCR Roche Mix the previously extracted RNA with reagents including buffer MgCl2 dNTP Oligo-pdT15 primer RNase inhibitor reverse transcriptase briefly vortex incubate the reaction at 25oC for 10 minutes and then 42oC for 60 minutes Finally incubate at 99oC for 5 minutes and then cool to 4oC for 5 minutes
3 Real-time TagMan PCR for enterovirus adenovirus or other viral etiology The primers and probes for enterovirus and other virus real time PCR are in the following table Primers and probes for PanEV were selected based on highly conserved regions in the 5-untranslated region of the enterovirus genome The primers and probes for EV71 were selected based on the most diverse and specific genetic regions in the VP1 region of the enterovirus genome Other potential viruses such as adenovirus or other respiratory viruses will also be performed by PCR or RT-PCR or real-time PCR following the rules of the above procedures
4 Representational differential analysis followed by subtractive cloning
3 Bacterial cultures and toxin detection Cultures were obtained from the pharynx and rectum of patients with acute KD before the start of the immune globulin intravenous infusion and from control patients with the use of a Baxter Diagnostics Culturette system that contains a cotton swab The primary data collection sheet was completed and held at the site where specimens were obtained The plates were then examined for all รข-hemolytic group A streptococci and all S aureus that were coagulase positive by the tube test These isolates were then screened in a blinded fashion for the presence of bacterial superantigen production by immunodiffusion with antisera against known staphylococcal and streptococcal superantigens as described previously Lee PK and Schlieveret PM
4 Serological test Serological tests for antibody detection of Mycoplasma pneumoniae Chlamydiae pneumoniae ASLO will be measured by commercial kits Neutralizing antibody for enterovirus will be performed by standard protocol of the neutralization test on microtiter plates EV71 isolate TW227298 was amplified and purified as an antigen for m-capture ELISA for EV71 IgM Detection If potential pathogen is defined further serology test for the specific pathogen will be done later
5 Statistical Analysis Data were analyzed with the SAS Statistical Package Version 82 SAS Institute Cary North Carolina We used Students t test for continuous variables and c 2 test for categorical data After univariate analysis screened statistically significant variables forward stepwise multiple logistic regression analysis was performed to adjust confounders simultaneously and to calculate multivariate-adjusted odds ratios The level of model selection was set at 015 for in-and-out models P 05 was considered statistically significant
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None