Jpn. J. Infect. Dis., 53, 203-205, 2000
To see an article, click this [PDF] link.
Laboratory and Epidemiology Communications
Evaluation of ViroseqTM-HIV Version 2 for HIV Drug Resistance
Motokazu Mukaide, Wataru Sugiura1, Masakazu Matuda1, Shuuzo Usuku2, Yuuzo Noguchi2, Kazuo Suzuki3, Kaoru Kawata4, Akira Ito4, Hiroko Sagara5, Kaneo Yamada6, Makiko Kondo7 and Mitunobu Imai7*
Center for Molecular Biology and Cytogenetics, SRL Inc., Tokyo 192-8535, 1AIDS Research Center, National Institute of Infectious Diseases, Tokyo 208-0011, 2Department of Virology, Yokohama City Prefectural Public Health Laboratory, Yokohama 235-0012, 3Center for Immunology St Vincent's Hospital, Sydney, Australia, 4School of Medicine, Yokohama City University, Yokohama 236-0004, 5Yokohama City Hospital, Yokohama 240-8555, 6Laboratory of Medical Science, St. Marianna University, Kawasaki 216-8511 and 7Department of Virology, Kanagawa Prefectural Public Health Laboratory, Yokohama 241-0815
Communicated by Kunihiko Masukawa
(Accepted October 23, 2000)
We compared a new anti-HIV drug resistance detection kit, ViroseqTM-HIV version 2 (AB method, Applied Biosystems, Tokyo), with the standard method developed by the National Institute of Infectious Diseases, Japan (NIID method) for its applicability to subtype E HIV-1.
The tested materials were 24 patients' sera which were found positive for subtype E HIV-1. The subtype was determined by sequencing of C2V3 envelope region. The sera were stock frozen at -80℃, and the freeze-thaw was limited to a maximum of three times.
Detection of mutations using the AB method was performed according to the manufacturer's instructions. In short, RNA was extracted using the guanidine-thiocyanate method from 0.5 ml centrifugation-cleared sera. The 1.7 kb protease-reverse transcriptase region was reverse-transcribed, PCR-amplified in the presence of uracil DNA glycosylase and dUTP, and sequenced by using primers A, B, C, D, F, G, and H (Figure) and a Big-Dye terminator (Applied Biosystems). The nucleotide sequences were analyzed using Sequence Analysis version 3.4, and the drug resistance mutations by HIV genotyping System Software version 2.2. The NIID method has been described elsewhere (1). The differences between the two methods are summarized in Table 1.
First, the sensitivity of the two methods was compared. A patient's serum with HIV-1 titer of 2 x 106 copies/ml (measured by Amplicore HIV MONITOR version 1.5 [Roche Diagnostics, Tokyo]) was diluted serially with HIV-negative serum. The AB method detected HIV-1 genome from all 19 samples with titers higher than 103.4 copies/ml. The NIID method detected 18 of the 19 samples. Both methods detected the HIV-1 genome from 2 samples among 5 samples with titers lower than 103.4. Therefore, the sensitivities were considered comparable (Table 2).
In the AB method, as internal regions were chosen as primers for sequencing, there was a possibility of mismatch between the primer sequence and some viral template sequences. Actually, primer D was effective only in 4% of subtype E. However, the sequence of the corresponding region could be obtained by sequencing the complementary strand using a different primer. The efficiency of other primers was quite high, i.e., 100% for primer B, 95% for primers A, C, G, and H, and 91% for primer F.
The drug resistance mutation data obtained using the two methods were compared in 19 cases. The results are summarized in Table 3. As for protease inhibitor resistance, D30N, M46V, G48V, I50V, and I84V were concordant in 100% of the cases, L90M in 94.3%, and V82ATFS in 84.2%. As for nucleotide reverse transcriptase inhibitor resistance, M41L, E44D, K65R, L74V, V118I, Q151M, M184IVT, and L215FY were concordant in 100%, and T69D in 94.7%. As for non-nucleotide reverse transcriptase inhibitor resistance, K103N, V106A, V108I, V118I, Y188CLH, and G190A were all concordant.
Our data showed that the AB method and the NIID method were comparable in regard to sensitivity and the detection of drug resistance mutations. The cost for one sample is currently 8,000 yen (about US$ 80) for the NIID method but for the AB method it is 20,000 yen, more than twofold more expensive than the NIID method. The advantage of the AB method is probably its commercial availability as a kit.
We thank Dr. Koya Ariyoshi for his advice. This study was supported by the Organization of Pharmaceutical Safety and Research (OPSR) of Japan.
REFERENCE
Corresponding author: Fax: +81-45-363-1037, E-mail: imaim@d2.dion.ne.jp
Go to JJID Homepage Go to JJID 53(5)