Minority Drug-Resistant HIV-1 Variants in Treatment Naïve East-African and Caucasian Patients Detected by Allele-Specific Real-Time PCR

<div><p>Objective</p><p>To assess the presence of two major non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutations (DRMs), Y181C and K103N, in minor viral quasispecies of treatment naïve HIV-1 infected East-African and Swedish patients by allele-specific polymerase chain reaction (AS-PCR).</p><p>Methods</p><p>Treatment naïve adults (n = 191) with three epidemiological backgrounds were included: 92 Ethiopians living in Ethiopia; 55 East-Africans who had migrated to Sweden; and 44 Caucasians living in Sweden. The <i>pol</i> gene was analysed by standard population sequencing and by AS-PCR for the detection of Y181C and K103N.</p><p>Results</p><p>The Y181C was detected in the minority quasispecies of six Ethiopians (6.5%), in two Caucasians (4.5%), and in one East-African (1.8%). The K103N was detected in one East- African (1.8%), by both methods. The proportion of mutants ranged from 0.25% to 17.5%. Additional DRMs were found in all three treatment naïve patient groups by population sequencing.</p><p>Conclusions</p><p>Major NNRTI mutations can be found by AS-PCR in minor quasispecies of treatment naïve HIV-1 infected Ethiopians living in Ethiopia, in East-African and Caucasian patients living in Sweden in whom population sequencing reveal wild-type virus only. Surveys with standard sequencing are likely to underestimate transmitted drug resistance and the presence of resistant minor quasispecies in treatment naïve patients should be topic for future large scale studies.</p></div

Schematic overview of allele-specific real-time PCR primers design and setting the standard curve analysis.

<p>In presence of K103N-AAC (A1) and K103-AAT (A2) mutants, only the specific mutant amplicons will be amplified when using mutant specific forward (MSFP) and a common reverse primer (CRP), respectively. The total population of sequences in the reaction are amplified using non-specific forward primer (NSFP) and the CRP (A3). An intentional mismatch at the penultimate base (indicated with I) was introduced in the allele-specific real-time PCR primers in order to increase the specificity and minimize the risk for amplification of the wild type allele. Mutant specific (Sp) and non-specific (NSp) standard curves of K103N AAC allele (B1), K103N AAT allele (B2) and Y181C TGT allele (B3) are in parallel with each experiment. The copy number of each mutant specific and total population of sequences amplifications of clinical samples were determined using such standard curves that has been run in duplicate, parallel with each sample. The quantity of the patients' mutant specific and the total population of sequences (amplified with non-specific primer) was then determined by comparing the samples Ct value with those of the specific and non-specific standard curves derived from the standard plasmid controls using the corresponding primers. The percentage of mutant specific sequences was then determined by dividing the quantity of mutant specific sequence by the quantity of the total sequences and multiplying by 100. Positive samples were repeated at least twice. Correlation coefficients (r²) were higher than 0.99. Sp: mutant specific amplification. MSFP, mutant-specific forward primer; CRP, common reverse primer; NSFP, non-specific forward primer. NSp: non-specific amplification (amplify the total population of sequences).</p

Characteristics of treatment naïve HIV-1 infected patients included in the study.

a<p>IQR, interquartile range;</p>b<p>IVDU, intravenous drug use;</p>c<p>DRM, drug resistant mutation.</p><p>Characteristics of treatment naïve HIV-1 infected patients included in the study.</p

Detection frequency of mutant K103N and Y181C minority variants.

<p>Ninety-two treatment naive HIV patients plasma were analysed by allele-specific PCR, detecting the two major NNRTI mutations K103N and Y181C in the reverse transcriptase gene. The limit of detection for the K103 mutations (AAC and AAT) is indicated by a dashed line. The limit of detection for the Y181C mutation (TGT) is indicated by a solid line. Crosses indicate two patients who died within three months. Shaded area indicates the assay background, which was determined as a mean Ct value from eight independent runs of 100% wild type template with mutant specific primers plus three standard deviations.</p

Example of allele-specific real-time PCR amplification curves of cloned wild type and mutant TGT plasmid DNAs at different frequencies (raw data).

<p>The specificity and accuracy of the assay was determined by running allele-specific real-time PCR of mixtures of mutant and wild type DNA standards ranging from 0.01 to 100% and amplified with the mutant specific primers in the background of wild type sequence. Amplification of the total population results always in the same Ct values, regardless of the amount of mutant DNAs present in the reaction. WT, wild type.</p

Characteristics of East-African, Caucasian and Ethiopian patients with drug resistance mutations.

a<p>ND, not done.</p><p>Characteristics of East-African, Caucasian and Ethiopian patients with drug resistance mutations.</p

Demographic data of children TBE, adult severe TBE, and adult TBE cohorts.

<p>Demographic data of children TBE, adult severe TBE, and adult TBE cohorts.</p

Genotype distribution of <i>TLR3</i> rs3775291 and allele prevalence among patients with TBE, stratified by severity of disease.

<p>wt – wild type; M – Mild, Mo – Moderate, S – Severe form.</p><p>* wt/wt vs wt/mut vs mut/mut, M vs Mo vs S: p = 0.757; M vs (Mo+S): p = 0.485; wt vs mut, M vs Mo vs S: p = 0.476; M vs (Mo+S): p = 0.280.</p><p>** wt/wt vs wt/mut vs mut/mut, M vs Mo vs S: p = 0.264; M vs (Mo+S): p = 0.180; (wt/wt + wt/mut) vs mut/mut, M vs Mo vs S: p = 0.197; M vs (Mo+S): p = 0.071; wt vs mut, M vs Mo vs S: p = 0.157; M vs (Mo+S): p = 0.096.</p><p>Genotype distribution of <i>TLR3</i> rs3775291 and allele prevalence among patients with TBE, stratified by severity of disease.</p

Genotype distribution of <i>CCR5</i> and <i>Δ32</i> allele prevalence among patients with TBE, stratified by severity of disease.

<p>wt – wild type; M – Mild, Mo – Moderate, S – Severe form.</p><p><b>*</b> (wt/wt + wt/Δ32) vs Δ32/Δ32, M vs Mo vs S: p = 0.479; M vs (Mo+S): p = 0.292; wt vs Δ32, M vs Mo vs S: p = 0.571; M vs (Mo+S): p = 0.391.</p><p>** (wt/wt + wt/Δ32) vs Δ32/Δ32, M vs Mo vs S: p = 0.137; M vs (Mo+S): p = 0.202; wt vs Δ32, M vs Mo vs S: p = 0.806; M vs (Mo+S): p = 0.802.</p><p>Genotype distribution of <i>CCR5</i> and <i>Δ32</i> allele prevalence among patients with TBE, stratified by severity of disease.</p

Genotype distribution of <i>TLR3</i> rs3775291 and allele prevalence among TBE patients, Lithuanian TBEV-naive control subjects, and patients with aseptic meningoencephalitis (AME) of non-TBEV etiology.

<p>wt – wild type.</p>a<p> TBE1 vs C1+C2: p = 0.453 (Pearson's χ² test).</p>b<p> TBE2 vs C1+C2: p = 0.135 (Pearson's χ² test).</p>c<p> TBE1+TBE3 vs C1+C2: p = 0.02 (Pearson's χ² test).</p>d<p> TBE1+TBE2+TBE3 vs C1+C2: p = 0.025 (Pearson's χ² test).</p>e<p> TBE1+TBE3 vs C1+C2: OR = 1.449 (95% CI 1.085–1.936; p = 0.012).</p>f<p> TBE2 vs TBE3: p = 0.022 (Pearson's χ² test).</p>g<p> TBE2 vs TBE3: p = 0.009 (Pearson's χ² test).</p><p>Genotype distribution of <i>TLR3</i> rs3775291 and allele prevalence among TBE patients, Lithuanian TBEV-naive control subjects, and patients with aseptic meningoencephalitis (AME) of non-TBEV etiology.</p