Example of Bias Matched Case-Control Study Lakkana Thaikruea MD., Cert of FETP, M.S., PhD. Department of Community Medicine, Faculty of Medicine, CMU

Risk Factors for Hepatitis C Virus infection among Blood Donors in Northern Thailand Lakkana Thaikruea 1, Satawat Thongsawat 1, Niwat Maneekarn 1, David L. Thomas 2, Dale Netski 2, Kenrad E. Nelson 2, 1 Departments of Community Medicine, Medicine, and Microbiology, Chiang Mai University, Chiang Mai, Thailand 2 Departments of Epidemiology & Medicine, Johns Hopkins University, Baltimore, USA

Rationale No effective program in Thailand to prevent HCV infection Lack of information of majors route of transmission Distribution of HCV genotypes are not well understood Researchers from JHU and Chiang Mai University have conducted study about epidemiology of HCV in northern Thailand Blood donor is one of the study population besides drug users, patients, and commercial sex workers This present study focuses on blood donor

Specific aims Blood donors in northern Thailand 1. To investigate the potential risk factors for HCV infection 2. To determine HCV genotype distribution 3. To investigate relationship between HCV genotype distribution and the routes of transmission

Features of hepatitis C virus infection 15% 85% 80% 20% 75%25%

Background: Route of transmission USA: IDU 1, sex with an IDU, blood transfusion among non-IDU REDS 2 :** male, black, 30 to 49 years, < high school diploma, first /only time blood donor, blood transfusion history 1 Injection drug users ; 2 Retrovirus Epidemiology Donor Study

Specific aim 1 To investigate the potential risk factors for HCV infection among blood donors in northern Thailand

Methods: Specific aim 1 Setting Design Data collection Data analysis

Study setting Faculty of Medicine, CMU The Blood Bank - The Maharaj Nakorn Chiang Mai hospital - 1,800-bed capacity and the main referral center Blood donors - January June ≥ 18 years old at the time of donation/ recruitment - Reside in the north - Donation sites: walk-in and mobile unit Methods (cont.)

Study design: Specific aim 1 Single masked matched case- control Matched variables: age, gender, donation date, donation sites Mask: interviewers and physicians did not know HCV status of the participants Methods (cont.)

Data collection Invitation letters: 2 nd letter in 2 weeks apart Non-participants: sent their participation forms indicating their unwillingness Enrolment: explain, consent form Trained health personnel of the same gender: face-to- face interview Physical examination Counseling Laboratory tests: 30 ml blood –HCV antibodies; EIA-3, RIBA-3 –HCV RNA: RT-PCR –HCV genotypes: direct sequencing –Serum ALT Eligible Case: blood bank EIA-3 positive

Data collection (cont) PCR positive, or PCR negative with RIBA-3 positive, or PCR negative with both – positive high cut-off repeat EIA-3 (Abbot) – positive high cut-off repeat EIA-3 (Ortho) Eligible cases were defined as “confirmed cases ” If they were HCV EIA-3 positive in blood bank screening and had any of the following:

Data collection (cont.) Randomly selection: –1 – 4 eligible controls per case Matched variables: –± 15 days of case’s donation date –Age ± 5 years old –Same gender –Same donation sites Enrolment: same Control: –Negative to both screening EIA-3 and repeat EIA-3 Eligible Control: blood bank EIA-3 negative

Data collection (cont.) Spouses of cases and matched controls –Regular sexual relations with the donor for ≥ 3 months – ≥ 18 years old at the time of recruitment Enrolment: same HCV infection: –EIA-3 positive and –Either PCR or RIBA-3 positive SPOUSES

Flow of matched case-control study 102 low cut-off EIA-3 positive 28 not eligible EIA-3 positive 38,340 donors 372 EIA-3 pos Eligible cases 372 eligible cases 1234 eligible matched controls 618 non-responders 85 returned letters 903 responders 238 non-participants 67 false positive cases 12 RIBA-3 indeterminate 82 matched controls 9 cases without control

Participation Compared between participants and non- participants Cases: 254 participants (91.4%) versus 24 non-participants –No statistically significant different Controls:411 participants (65.8%) versus 214 non-participants –Participants: age in years 33.2 ( VS 31.0)* –Participants: ever donated 81.6 % (VS 61.3 %)* * P value < 0.05

Data analysis: Specific aim 1 Univariate analysis –Matched odds ratio (OR) with 95% confidence interval (95% CI) Multivariate analysis –Conditional logistic regression –Step-wise selection: alpha levels of 0.05 for entry and for remove to guide the selection of variables –Either independent variables that were likely confounders or had biological importance were forced in to the preliminary model regardless of their statistical significance Phylogenic analysis 1 –Tree was constructed from nucleic acid sequence alignments using Neighbor joining method –Sequence alignments were randomly permuted 1,000 times CONSENSE provided bootstrap values –Reference sequences: GenBank 1programs: Gofasta1.1, BioEdit 5.9, Tree View 1.6, PHYLIP 3.572c package ( SEQBOOT, DNADIST, NEIGHBOR, CONSENSE)

Results: Specific Aim 1 Demographic distribution Multivariate Spouses

Limitations: Specific aim 1 Limitations: - causal relationship; temporal ambiguity - spouses; might not represent

Strengths: Specific aim 1 - appropriate study design; sample size matched case-control confounding - minimized selection bias; asymptomatic nature lacked HCV knowledge available of HCV test only in some secondary or tertiary cares self-deferral system - minimized information bias; masked face-to-face interview physical examination - minimized misclassification of HCV status; confirmatory tests

Publications The risk factors for HCV infection among blood donors in northern Thailand: Matched case-control study. Presenter: Lakkana Thaikruea (The XV International AIDS Conference. July 10-16, Bangkok, Thailand) Risk Factors for Hepatitis C Virus Infection Among Blood Donors in Northern Thailand Lakkana Thaikruea1,6, Satawat Thongsawat3,6, Niwat Maneekarn4,6, Dale Netski 5, David L. Thomas,5 Kenrad E. Nelson2 (Transfusion, in process)

Other Related Publications Epidemiology of hepatitis C virus infection among blood donors in northern Thailand Presenter: Lakkana Thaikruea (International Centers for Tropical Disease Research Network, 12th Annal Meeting, May 13-15, 2003, NIH, Bethesda, MD, USA) Relationship between HIV and Hepatitis C Viral Genotypes and Routes of Transmission among Blood Donors in Northern Thailand. Presenter: Lakkana Thaikruea) (11th CROI retrovirus and Opportunistic Infection Conference, Feb 7-11, 2004, San Francisco, CA, USA)

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