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Journal of Clinical Microbiology, August 2000, p. 2807-2813, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Comparison and Application of a Novel Genotyping Method, Semiautomated Primer-Specific and Mispair Extension Analysis, and Four Other Genotyping Assays for Detection of Hepatitis C Virus Mixed-Genotype Infections

Yu-Wen Hu,1,* Evan Balaskas,1 Milena Furione,2 Pei-Hua Yen,1 Garry Kessler,1 Vito Scalia,1 Linda Chui,3 and Graham Sher1

Canadian Blood Services, Ottawa, Ontario, Canada K1G 4J51; Servizio Di Virologia, I.R.C.C.S. Policlinico, San Matteo, 27100 Pavia, Italy2; and Microbiology and Public Health, Edmonton, Alberta, Canada T6G 2J23

Received 18 January 2000/Returned for modification 5 April 2000/Accepted 9 May 2000

To date the true prevalence of hepatitis C virus (HCV) mixed-genotype infections has not been established mainly because currently available methods are not suitable for the detection of mixed genotypes in a viral population. A novel semiautomated genotyping method, primer-specific and mispair extension analysis (S-PSMEA), which is more reliable than other genotyping assays was developed for detection of HCV mixed-genotype infections. A genotype present at levels as low as 0.8% in a defined mix of HCV genotypes was detected, showing a 20-fold increase in sensitivity over that of direct DNA sequencing. A total of 434 HCV isolates were genotyped and analyzed for a comparative study of the accuracy between S-PSMEA and four current genotyping methods. The results showed that viruses in approximately 40% of the samples from this group determined to be infected with mixed genotypes by S-PSMEA were undetected by direct DNA sequencing due to its low sensitivity. Type-specific PCR, line probe assay, and restriction fragment length polymorphism analysis performed poorly, being able to identify only 38.5, 16.1, and 15.4% of mixed-genotype infections, respectively, that were detected by direct DNA sequencing. The prevalence of mixed-genotype infections detected by S-PSMEA was 7.9% (12 of 152 donors) among HCV-infected blood donors, 14.3% (15 of 105) among patients with chronic hepatitis C, and 17.1% (6 of 36) among thalassemia patients who had received multiple transfusions. The data lead us to conclude that HCV mixed-genotype infections are more common than previously estimated and that S-PSMEA may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections is required due to its higher level of sensitivity.

* Corresponding author. Mailing address: Canadian Blood Services, 1800 Alta Vista Dr., Ottawa, Ontario, Canada K1G 4J5. Phone: (613) 739-2439. Fax: (613) 739-2426. E-mail: yu-wen.hu{at}bloodservices.ca.

Journal of Clinical Microbiology, August 2000, p. 2807-2813, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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