Comparison of the results between sequencing and PCR-SSOP-Luminex assay.

<p>Comparison of the results between sequencing and PCR-SSOP-Luminex assay.</p

Validation of PCR-SSOP-Luminex assay using plasmids as templates.

<p>(A) Agarose gel electrophoresis of amplified fragments. Lanes 1–6: Fragment a (547 bp). Lanes 7–14: Fragment c (512 bp). 1, wild type; 2, M41L-TTG; 3, M41L-CTG; 4, K65R-AGA; 5, 70R-AGA; 6, 70R-AGG; 7, wild type; 8, 103N-AAC; 9, K103N-AAT; 10, M184V-GTG; 11, T215Y-TAC; 12, T215F-TTC. (B) Median fluorescence intensities (MFIs). The plasmid in the test sample is indicated on the top of each panel. Oligoprobes used for detection are indicated at the bottom. Matched results are shown as black bars, mismatched results as white bars. Assays were performed in triplicate. The mean ± standard deviation is shown at the top of each bar.</p

Design of additional oligoprobes based on clinical samples.

<p>Design of additional oligoprobes based on clinical samples.</p

Schematic representation of PCR amplification and sequences of primers for PCR and sequencing.

<p>(A) Top: Site-directed mutagenesis using oligonucleotide is shown using K103N as an example. Desired mutations in the reverse transcriptase gene were engineered in two PCR fragments, then incorporated into a larger fragment (1050 bp, HXB2 location 2388–3425) by the second PCR, and cloned into pBluescript II SK (+) at <i>Xho</i>I-<i>BamH</i>I sites. Bottom: Negative strand cDNA was synthesized from patients’ plasma. After the first strand PCR using RT1F and RT1R as primers, Fragment a or Fragment c were amplified by nested PCR. (B) Primer sequences for PCR amplification and sequencing.</p

Sequence diversity around K65 in the 31 specimens.

a<p>R: Mixed base of A and G.</p><p>Sequence diversity around K65 in the 31 specimens.</p

Design of oligoprobes based on clade B HIV-1 sequences from Japanese surveillance database.

<p>R: Mixed base of A and G; Y: Mixed base of C and T; M: Mixed base of C and A.</p><p>Design of oligoprobes based on clade B HIV-1 sequences from Japanese surveillance database.</p

Improvement of the detection by the additional probes at K65 locus.

<p>The ordinate shows the percentage of the genotype at K65 locus determined by the PCR-SSOP-Luminex DR assay. One hundred % is the sample number (74) successfully amplified by PCR.</p

Emergence of Lamivudine-Resistant HBV during Antiretroviral Therapy Including Lamivudine for Patients Coinfected with HIV and HBV in China

<div><p>In China, HIV-1-infected patients typically receive antiretroviral therapy (ART) that includes lamivudine (3TC) as a reverse-transcriptase inhibitor (RTI) (ART-3TC). Previous studies from certain developed countries have shown that, in ART-3TC, 3TC-resistant HBV progressively emerges at an annual rate of 15–20% in patients coinfected with HIV-1 and HBV. This scenario in China warrants investigation because >10% of all HIV-infected patients in China are HBV carriers. We measured the occurrence of 3TC-resistant HBV during ART-3TC for HIV-HBV coinfection and also tested the effect of tenofovir disoproxil fumarate (TDF) used as an additional RTI (ART-3TC/TDF) in a cohort study in China. We obtained 200 plasma samples collected from 50 Chinese patients coinfected with HIV-1 and HBV (positive for hepatitis B surface antigen) and examined them for the prevalence of 3TC-resistant HBV by directly sequencing PCR products that covered the HBV reverse-transcriptase gene. We divided the patients into ART-3TC and ART-3TC/TDF groups and compared the efficacy of treatment and incidence of drug-resistance mutation between the groups. HIV RNA and HBV DNA loads drastically decreased in both ART-3TC and ART-3TC/TDF groups. In the ART-3TC group, HBV breakthrough or insufficient suppression of HBV DNA loads was observed in 20% (10/50) of the patients after 96-week treatment, and 8 of these patients harbored 3TC-resistant mutants. By contrast, neither HBV breakthrough nor treatment failure was recorded in the ART-3TC/TDF group. All of the 3TC-resistant HBV mutants emerged from the cases in which HBV DNA loads were high at baseline. Our results clearly demonstrated that ART-3TC is associated with the emergence of 3TC-resistant HBV in patients coinfected with HIV-1 and HBV and that ART-3TC/TDF reduces HBV DNA loads to an undetectable level. These findings support the use of TDF-based treatment regimens for patients coinfected with HIV-1 and HBV.</p></div

Correlation between baseline HBV DNA loads and emergence of 3TC-resistance mutations.

<p>Each dot represents the HBV DNA load of a clinical sample at baseline. The horizontal lines indicate the median values of HBV DNA loads: 2.7 × 105 IU/mL in the wild-type group, 1.1 × 108 IU/mL in the group in which 3TC-resistance mutations emerged (mutant group).</p

Flowchart depicting the selection of patients for this study.

<p>Flowchart depicting the selection of patients for this study.</p