Jpn. J. Infect. Dis., 53, 2000

Laboratory and Epidemiology Commumications

Evaluation ofViroseqTM‑HIV Version 2 for HIV Drug ResistanceMotokazu Mukaide, Watam Sugiural, Masakazu Ma山dal , Shuuzo Us血2, Ⅶuzo Noguchi2,
ヽKazuo Suzuki3, KaoruKawata4, Akira Ito4, Hiroko Sagara5, Kaneo Yamada6, Makiko Kondo7
and Mitsunobu lmai7 辛Centerfor Molecular Biology and Cytogenetics, SRL Inc. , Tob70 I92‑8535,]AIDS Resewch Center: National Institute of Infectious Diseases, Too,0 208‑0011,
2Department of ViT10logy, Yokohama CiO, Prefectural Public Health Laboraloyy, Yokohama 235‑0012,
3Centerfor Immunology St Vincent i Hospital, Sydney, Australia,
4School of Medicine, Yokohama CiOJ UniversiOJ, Yokohama 236‑0004,
5Yokohama CID, Hospital, Yokohama 240‑8555,

6Laboratopy of Medical Science, St. Marianna UniversiOr, Kawasaki 21 6‑8511 and7Department of Virology, Kanagawa Prefectural Public Health Laboratow, Yokohama 241‑0815

Communicated by Kunihiko Masukawa(Accepted October 23, 2000)

We compared a new anti‑HIV drug resistance detection

The nucleotide sequences were analyzed uslng Sequencekit, ViroseqTMIHⅣ version 2 (AB method, Applied Biosystems,

Institute of Infectious Diseases, Japan (NIID method) for its

Analysts Version 3.4, and the dmg resistance mutations byHIV genotyplng System Software version 2.2. The NIID
method has been described elsewhere (1). The differences

applicability to subtype E HIV 1.

between the two methods are summarized in Table 1.

Tbkyo), with the stan血rd method developed by the National

The tested materials were 24 patients'sera which wereFirst, the sensitivityofthe two methods was compared. Afound positive for subtype E HIV‑ I. The subtype was deter‑patient's serumwithHIV‑ 1 titer of2 X I 06 copies/ml (measuredmined by sequenclng Of C2V3 envelope reg10n. The sera werestock frozen at 180oC, and the freeze‑thaw was limited to aby Amplicore HIV MONITOR version I.5 lRoche Diagnos‑tics, Tbkyo]) was diluted serially with HIV‑negative semm.

maximum of three times.

The AB method detected HIV‑ 1 genome from all 1 9 samples

Detection of mutations uslng the AB method was performed
according to the manufacturer's instructions. In short, RNAwith titers higher than lO3･4 copies/ml. The NIID method

was extracted uslng the guanidine‑thiocyanate method fromHIV‑1 genome from 2 samples among 5 samples with titers

0.5 ml centrifugation‑cleared sera. The 1.7 kb protease‑lower than lO3･4. Therefore, the sensitivities were consideredreverse transcriptase reg10n Was reverse‑transcribed, PCR‑

comparable (Table 2).

amplified in the presence of uracil DNA glycosylase anddUTP, and sequenced by uslng PnmerS A, B, C, D, F, G, andH (Figure) and a Big‑Dye teminator (Applied Biosystems).

for sequencing, there was a possibility of mismatch between

gag

proteadetected 18 of the 19 samples. Both methods detected theInthe AB method, asintemal regions Were Chosen as prlmerS
the prlmer Sequence and some viral template sequences.

RTse(91)至; a(!T･)

AB protocol
一一:

D(4%)

トトF(91%)

一トG(95%)一｣H(95%)〜 HD protocoI

1.1Kb

Figure. Primers for sequenclng and their efrlCiency. Percentage in parenthesis indicates percent of successful sequenclng･*Co汀eSpOnding author: Fax: +81‑45‑363‑I 037, E‑mail: imaim@d2. dion.ne.jp
203Jpn. J. Infect. Dis., 53, 2000
Table I. Comparison ofAB method and NIID method

NHDmethod

ABmethodsampleplasma 50 LEI

plasma 500 LEI

methodRT+nested‑PC R

RT‑PCR

RT primer

specific

spec i fie

number of amplicon

2Iand each size

protease: 450 bp

pol: I.7 Kbp

SequenCe PnmerS

same astheinner PCR primer

specific for sequenclng¥i,000¥20,000

RT: 800 bp

estimated pricc
per sample

Table 2. Amplification of patient plasma

HIV‑1 RNA
LOG 1 0(copies/ml)

AB叩㌫rj山IJt1 T Jt T J ーTJIl▼■一TJ

E E E E E E E E 1.23T‑54.7‑8.9 1‑ .1 .1 7IT.17‑〜7.2 2 to2345678.190̲

< L 2 3 3 3 3 4 4 5 5 6 7 0 .tJ 4 7 9 0 3 4 5 6 7 0 4 6 9 .1% amplification

p nre一 + + F + + + + + + + +2 1 /24(87.5%)

22/24(9 I.7%)#only protease reg10n Was Successfully amplified.

Actually, primer D was effective only in 4% of subtype E.

Our data showed that the AB method and the NIID method

However, the sequence of the corresponding region could be

were comparable in regard to sensitivityand the detection ofobtained by sequenclng the complementary strand uslng adrug resistance mutations. TTle cost for one sample is currently

different primer. The efrlCiency of other primers Was qulte

8,000 yen (about US$ 80) for the NIID method but for thehigh, i.e., 100% forprlmer B, 95% for pnmers A, C, G, andAB method it is 20,0(氾yen, morethantwofold more expensive

H, and 91% for prlmer F.

than the NIID method. The advantage of the AB method isThe drug resistance mutation data obtained uslng the two

probably its commercial availability as a kit.

methods were compared in 19 cases. The results are summa‑

rized in Table 3.As for protease inhibitor resistance, D30N,M46V, G48V, I50V, and I84V were concordant in lOO% ofWe thank Dr. KoyaAriyoshi for his advice. This study wassupported by the Organization of Pharmaceutical Safetyand

the cases, L90M in 94.3%, and V82ATFS in 84.2%. As fわr

Research (OPSR) oH叩an.

nucleotide reverse transcriptase inhibitor resistance, M4 1 L,E44D, K65R, L74V, Vl18Ⅰ, q151M, M184IVT, andL215FY

were concordant in lO0%, and T69D in 94.7%. As fornon‑nucleotide reverse transcrlPtaSe inhibitor resistance,

K103N, V106A, VIO81, Vl18I, Y188CLH, and G190Awere
all concordant.

204

Jpn. J. Infect. Dis., 53, 2000
Table 3. Comparison of drug resistance genotyping results by AB method and NIID method
PI
SampleNo.

Method71

A
JE‑5.2resistance

RTI

77

9082

V

V

88N

L

resistance
41

M

6975

T

V

210

L

219%match

K
100.00AB

NIID

JE‑12ABNIID

JE‑16ABNIID
JE‑171 0 0 00

ABNIIDJE‑181 0 0 00

AB

NIID
JE‑1.19AB〇〇.00

NIID
JE‑5.1

1 0 0 00

ABNIIDJE‑191 0 0 00

ABNIIDJE‑2010AB0.00

NIID
JE‑2 11 0 0 00

ABNIIDllaVe age

REFERENCE

1. Sugiura, W., Matsuda, M., Abumi, H., Yamada, K., Taki,M., Ishikawa, M., Miura, T., Fukutake, K., Gouchi, K.,
Ajisawa, A., Iwamoto, A., Hanabusa, H., Mimaya, J.,
Takamatsu, J., Takata, N., Kakishita, E., Yoshioka, A.,
Kashiwagi, S., Shirahata, A.and Nagai, Y. (1999): Preva‑lence of drug resistance‑related mutations among HIV‑ls in Japan. Jpn. J. Infect. Dis., 52, 21122.

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