Determination of cell expansion and surface molecule expression on anti-CD3/28 expanded CD4+ T cells.

CD4+ T cell immunotherapy has potential for treatment in HIV-infected patients. A large number of expanded CD4+ T cells and confirmation of functional-related phenotypes are required for ensuring the successful outcomes of treatment. Freshly isolated CD4+ T cells from healthy donors were activated with anti-CD3/28-coated magnetic beads at different bead-to-cell ratios and cultured in the absence and presence of IL-2 supplementation for 3 weeks. Fold expansion, cell viability, growth kinetic and lymphocyte subset identities were determined. Data demonstrated that a 1:1 bead-to-cell ratio rendered the highest expansion of 1044-fold with 88% viability and 99.5% purity followed by the 2:1 and 0.5:1 ratios. No significant difference in proliferation and phenotypes was found between non-IL-2 and IL-2 supplementation groups. Several specific surface molecule expressions of the expanded cells including chemokine receptors, adhesion molecules, co-stimulatory molecules, activation molecules, maturation markers, cytokine receptors and other molecules were altered when compared to the unexpanded cells. This optimized expansion protocol using the 1:1 bead-to-cell ratio of anti-CD3/28-coated magnetic beads and culture condition without IL-2 supplementation provided the satisfactory yield with good reproducibility. Specific surface molecule expressions of the expanded cells presented potential roles in proliferation, differentiation, homeostasis, apoptosis and organ homing.

Shift Work and Leukocyte Count Changes among Workers in Bangkok.

OBJECTIVES: Previous epidemiological evidence for the association of shift work exposure and increased leukocyte count is cross-sectional in nature, thus limiting cause-effect inference. We therefore used a longitudinal design to: (i) compare leukocyte counts at baseline between shift and day workers and (ii) examine the time trend of leukocyte counts over the follow-up period for these workers. METHODS: A retrospective cohort study was conducted among 6737 workers aged <60 years at two large organizations (a humanitarian organization and a university) in Bangkok, Thailand who had participated in at least two annual health check-ups during the period 2005-2016. Shift work exposure history was assessed by a self-administered questionnaire and categorized into day, former, and current shift workers. Data on leukocyte count were collected annually as part of worksite health screening during the observation period. Association of shift work exposure and increased leukocyte count was then examined cross-sectionally and longitudinally by using multiple linear regression and multilevel analysis of repeated measures data, respectively. In addition, trends for leukocyte count over the follow-up period and work years were examined using LOWESS smooth curves. RESULTS: Compared to day work, the current shift work was associated with increased leukocyte counts. The magnitude of percentage increase was the highest for basophil counts, followed by eosinophil and lymphocyte counts. Both cross-sectional and longitudinal evidence revealed this association, although it was less pronounced longitudinally. For total leukocyte count, the magnitude of difference was constant across the 11-year follow-up period. However, for lymphocyte and basophil counts, these discrepancies tapered over the work years until they no longer differed (for lymphocyte count) or even differed in the opposite direction (for basophil count) in later work years. CONCLUSION: This study confirmed previous cross-sectional evidence that shift work exposure-increased leukocyte counts and that this was reversible. Whether this increase in immune cell count also results in an increased immune cell activity and serves as the intermediary in the association between shift work exposure and subsequent chronic disease development needs further investigation.

Increased Risk Of Type 2 Diabetes And Abnormal FPG Due To Shift Work Differs According To Gender: A Retrospective Cohort Study Among Thai Workers In Bangkok, Thailand.

PURPOSE: The gender differential evidence of the association between shift work and type 2 diabetes risk remains scarce. This longitudinal study determines whether the association between shift-work exposure and type 2 diabetes risk and abnormal fasting plasma glucose (FPG) differs according to gender; the study aims to find the association between shift work and changes in physiological, behavioral, and psychosocial stress. PATIENTS AND METHODS: This retrospective cohort study was conducted among 5947 workers (4647 female and 1300 male) aged ≤60 years old in Bangkok, Thailand. Participants required a normal FPG level (<100 mg/dL) at baseline and at least two health check-up results from 2009 to 2016. Shift-work exposure history was assessed using a self-administered questionnaire; FPG levels were measured annually. Cox proportional hazard models were used to assess the aforementioned association. RESULTS: During the follow-up period, 1470 new abnormal FPG and 154 new type 2 diabetes cases developed. Stratified analysis of male workers' data revealed an association was significant in the unadjusted model, which tended to be stronger after adjustment for demographic data and the baseline values of anthropometric and biochemical parameters. This was the case both for type 2 diabetes [Hazard Ratio (HR) (95% Confidence Interval (CI))=2.98 (1.58-5.62)] and abnormal FPG [HR (95% CI)=1.86 (1.43-2.41)]; this association was less obvious among women. CONCLUSION: Shift work is a risk factor for type 2 diabetes and abnormal FPG; this risk is gender differential, being more pronounced in men. Preventive measures aiming at ameliorating shift work induced type 2 diabetes risk should pay more attention to men.

A closed-culture system using a GMP-grade culture bag and anti-CD3/28 coated bead stimulation for CD4+ T cell expansion from healthy and HIV-infected donors.

CD4 immunotherapy is potentially useful in immune reconstitution of CD4+ T cells for HIV-infected patients. Transfusion of anti-CD3/28 expanded CD4+ T cells is also proved to be safe and effective in both SIV-infected macaques and HIV-infected patients. However, there is no such standardized and practical protocol available for cell production in order to use in clinics. This study thus aimed to develop a closed-culture system for in vitro CD4+ T lymphocyte expansion by using a commercially available GMP-grade culture bag and anti-CD3/28 activation. Freshly isolated CD4+ T cells by immunorosette formation from healthy donors and cryopreserved CD4+ T cells from HIV-infected patients with CD4 count over 500 cells/µL were stimulated with anti-CD3/28 coated beads. The activated cells were then expanded in conventional culture flasks and GMP-grade culture bags for three weeks. Fold expansion, cell viability, growth kinetic and phenotypic characters were observed. Results revealed that purified CD4+ T cells from healthy individuals cultured in flasks showed better expansion than those cultured in bags (797-fold and 331-fold, respectively), whereas, their cell viability, growth kinetic and expanded CD4+ T cell purity were almost similar. A large-scale production was also conducted and supported consistency of cell proliferation in the closed-culture system. Frozen CD4+ T lymphocytes from the patients were able to remain their growth function and well expanded with a good yield of 415-fold, 85% viability and 96% purity of CD4+ T cells at the end of a 3-week culture in bags. This developed closed-culture system using culture bags and anti-CD3/28 coated beads, therefore, can achieve a large number of expanded CD4+ T lymphocytes with good reproducibility, suggesting a promising protocol required for adoptive immunotherapy.

Impact of Vaccination on Distribution of T Cell Subsets in Antiretroviral-Treated HIV-Infected Children.

Antiretroviral therapy (ART) is generally prescribed to patients with human immunodeficiency virus (HIV) infection with vaccination introduced to prevent disease complications. However, little is known about the influence of immunization on T cell subsets' distribution during the course of infection. This study aims to identify the impact of viral replication and immunization on naïve, effector, effector memory, and central memory T cell subpopulations in ART-treated HIV-infected children. Fifty patients were recruited and injected intramuscularly with influenza A (H1N1) 2009 vaccine on the day of enrollment (day 0) and day 28. Blood samples were collected for pre- and postvaccination on days 0 and 56 for analyzing T cell phenotypes by flow cytometry. Phenotypes of all T cell subsets remained the same after vaccination, except for a reduction in effector CD8+ T cells. Moreover, T cell subsets from patients with controllable viral load showed similar patterns to those with virological failure. Absolute CD4 count was also found to have a positive relationship with naïve CD4+ and CD8+ T cells. In conclusion, vaccination and viral replication have a little effect on the distribution of T cell subpopulations. The CD4 count can be used for prediction of naïve T cell level in HIV-infected patients responding to ART.

The Effects of Anti-CD3/CD28 Coated Beads and IL-2 on Expanded T Cell for Immunotherapy.

BACKGROUND: The activation of peripheral blood mononucleated cells (PBMCs) with anti-CD3/CD28-coated magnetic beads promotes intrinsic resistance to HIV as well as cell expansion. OBJECTIVES: The aim of this study was to define an optimal cell isolation protocol for the expansion of PBMCs using anti-CD3/CD28-coated bead stimulation, with the ultimate goal of using these cells for adoptive therapy. MATERIAL AND METHODS: PBMCs were isolated from healthy donor blood samples. To determine the effect of varying the bead-to-cell ratios on the expansion rate and phenotypic characterization of the expanded cells, one million PBMCs were stimulated by anti-CD3/CD28-coated beads at bead-to-cell ratios of 0.1 : 1, 0.5 : 1 and 1.0 : 1 in the presence of 100 U/mL exogenous IL-2; also, one million PBMCs were stimulated by anti-CD3/CD28-coated beads at a bead-to-cell ratio of 0.5 : 1 in the presence of varying concentrations of IL-2 (20, 100 and 1000 U/mL). Cell expansion was carried out for three weeks. The cell numbers, cell viability and phenotypic characterization were determined by trypan blue exclusion and flow cytometry. RESULTS: The initial experiments showed no difference in the expansion rate from cells grown with the three different bead-to-cell ratios. PBMCs can be expanded up to 1000-fold at a 0.5 : 1 bead-to-cell ratio after three weeks of cell expansion with a high viability (90%). Furthermore, in the presence of 100 U/mL IL-2, the percentages of CD3-CD16+CD56+ cells showed marked increases. CONCLUSIONS: The results demonstrate that PBMCs were stimulated with anti-CD3/CD28-coated beads. This method may provide an alternative for driving T cell expansion, which may be very useful in adoptive immunotherapy.

Production of anti-CD3/28 expanded CD4⁺ T lymphocytes from HIV-infected patients with different degrees of disease progression.

AIMS: CD4+ T lymphocytes from HIV-infected patients with different degrees of disease progression based on CD4 count were expanded in vitro using anti-CD3/28-coated beads. MATERIALS & METHODS: Purified CD4+ T lymphocytes from healthy subjects and patients were expanded for 3 weeks. Moreover, the improvement of cell expansion by IL-2 supplementation was also determined. RESULTS: Expanded CD4+ T lymphocytes from patients had lower fold expansion when compared with healthy subjects. Furthermore, patients with high CD4 counts had higher fold expansion than patients with low CD4 count, and IL-2 supplementation further increased cell expansion. CONCLUSIONS: Anti-CD3/28 activation failed to potently induce expansion of CD4+ T lymphocytes from patients. However, the cell expansion could be improved by IL-2 supplementation.

Development of a cost-effective assay for genotyping of HIV-1 non-B subtype for drug resistance.

Highly Active Antiretroviral Therapy (HAART) is the most effective way to control HIV-1 replication in infected patients. Prior to the start of therapy, genotyping of HIV-1 for mutations that confer resistance to potential drug candidates is crucial for it allows formulating an effective regimen. Ineffective drugs are excluded and potentially effective ones are included. A number of diagnostic kits are commercially available for this purpose but are tailored for HIV-1 subtype-B, a strain chiefly found in AIDS patients of Europe and America. However, AIDS patients of South-East Asia including Thailand are predominant infected with HIV-1 subtype non-B. In this study, an inexpensive assay was developed that genotypes HIV-1 non-B for drug resistance and tested it on 99 Thai AIDS patients. Results showed that 98 were infected with HIV-1 subtype non-B (or CRF01_AE) and one with subtype-B. Within the HIV-1 polymerase (pol), reverse transcriptase (RT) gene, the assay identified 18 codon mutations associated with resistance to Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs) and 17 Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). Employing a commercially available kit, parallel genotyping of patient samples confirmed results providing validation of the assay. This method approximately costs 100 US dollars compared to $300 for a commercially available test. In Thailand, the burden of cost for treating HIV-infections is high not only for the average citizen but the country's health care systems. Therefore the low cost and yet effective genotyping test for HIV-1 subtype non-B is a practical and viable solution to expensive genotyping platforms.

Immune activation and viral replication after vaccination with an influenza A H1N1 2009 vaccine in HIV-infected children receiving antiretroviral therapy.

Immunization with a pandemic influenza A H1N1 2009 was recommended for HIV-infected patients. However, there is limited information concerning the impact of immunization with this vaccine on immune activation and HIV viral replication. In this study, 45 HIV-infected children and adolescents receiving antiretroviral therapy were immunized with a 2-dose series of nonadjuvated monovalent influenza A H1N1 2009 vaccine upon enrollment and approximately 1 month later. Immunogenicity was determined by haemagglutination inhibition assay. The level of immune activation was determined by identification of CD38 and HLA-DR on CD8+ T cells. Patients were divided into 2 groups which include patients who had an undetectable HIV viral load (HIV detectable group) and patients who show virological failure (HIV nondetectable group). The results showed seroconversion rate of 55.2% in HIV nondetectable group, whereas 31.3% was found in HIV detectable group. Both groups of patients showed no major increase in immune activation after immunization. Interestingly, a decrease in the frequency of CD8+ T cells that coexpressed CD38 and HLA-DR was observed after immunization in both groups of patients. We suggested that immunization with influenza A H1N1 2009 vaccine can induce immune response to the pandemic virus without major impact on HIV viral replication and immune activation.

Trends in prevalence of HIV-1 drug resistance in Thailand 2009-2010.

BACKGROUND: Treatment failure of antiretroviral therapy in HIV-1 infection is increasing due to development of viral resistance. Trends of resistance-associated mutation lead to the ineffective treatment in HIV-infected individuals. METHODS: Extracted viral RNA from HIV-infected subjects in 2009 to 2010 was performed. The genotypic resistance testing was investigated for HIV-1 drug resistance in RT and PR genes. Frequencies of mutation were compared by a Fischer's exact test. RESULTS: Three hundred and sixty-nine samples (147 in 2009 and 222 in 2010) were genotyped. At least one mutation was found in 90.8% (335/369) in PR gene and 87.0% (321/369) in RT gene. Three sequences in PR gene, M36I, H69K, and L90M, were decreased significantly in 2010 when compared to 2009. Mutations associated with resistance to nucleoside analogue reverse transcriptase inhibitors (NRTI's) were found in 61.0% and 64.2% in nonnucleoside analogue reverse transcriptase inhibitors (NNRTI's). A total of 49.6% was found in combined NRTI and NNRTI. In 2010, M41L was increased significantly from 7.5% to 14.9%. However, there was a decrease in the frequency of the mutations at position 67, 70, and 184 between 2009 and 2010. CONCLUSIONS: In 2010, three mutations in PR gene, M36I, H69K, and L90M, were decreased significantly. However, only one mutation in RT gene, M41L was significantly increased.

Influence of cell isolation method on the optimization of CD4+ T cell expansion using anti-CD3/CD28 coated beads.

BACKGROUNDS: Activation of CD4+ T lymphocytes with anti-CD3/CD28 coated magnetic beads promotes intrinsic resistance to HIV as well as cell expansion. The propose of this study is to define the optimal cell isolation protocol for expansion of CD4+ T lymphocytes by using anti-CD3/CD28 coated bead stimulation with an ultimate goal of using these cells for adoptive immunotherapy. METHODS: CD4+ T cells were isolated from healthy donor blood samples using three different methods including immunorosette formation, negative selection and CD8 depletion using immunomagnetic beads. These cells were activated with anti-CD3/CD28 coated beads at a bead to cell ratio of 1:1 and cell expansion was carried for 3 weeks. Cell numbers, cell viability and phenotypic characterization were determined by trypan blue exclusion and flow cytometry. RESULTS: Purified CD4+ T lymphocytes which were isolated via immunorosette formation can be expanded up to 1000-fold within 3 weeks with high viability (90%and high purity of CD4+ T lymphocytes (>95%). However, cell expansion from purified CD4+ T lymphocytes which were isolated by negative selection and CD8-depletion provided approximately 300-fold expansion. CONCLUSIONS: The results demonstrate that purified CD4+ T lymphocytes from immunorosette formation provided the highest CD4+ T lymphocyte expansion when stimulated with anti-CD3/CD28 coated beads. This method can be used to obtain a large number of expanded CD4+ T cells for adoptive immunotherapy.

Impact of CCR2 and SDF1 polymorphisms on disease progression in HIV-infected subjects in Thailand.

BACKGROUND: The genotypic polymorphisms of CCR5, CCR2, and SDF1 were analyzed to determine their impact as potential confounders with regard to disease progression because of the role that host genetic factors appear to be involved in determining rates of disease progression. METHODS: Genomic DNA was extracted from Ethylenediaminetetraacetate whole blood using Qiagen DNA extraction kit. The amplification of CCR5, CCR2, and SDF1 genes was performed by PCR. RESULTS: Two hundred and twenty-one samples were genotyped for the CCR5, CCR2, and SDF1 mutation. Among these, all (100%) were identified as wild type for CCR5. All were then investigated considering the impact on CD4+ T-cell counts. Samples were divided into two groups based on the CD4+ T-cell numbers. It revealed that in the group of CD4+ T-cell counts ≥200 cells/µl, 15 were found for the homozygous for SDF1 gene (3'A/3'A) whereas one was found in the group of CD4+ T-cell counts <200 cells/µl. Homozygosity for the CCR2 polymorphisms (64I/64I) were five in the group of CD4+ T-cell counts ≥200 cells/µl and none were found in the group of CD4+ T-cell counts <200 cells/µl. These results demonstrated that there was a significant association between CD4+ T-cell numbers and CCR2 and SDF1 polymorphisms (P < 0.001). CONCLUSIONS: The mutation of CCR2 and SDF1 genes showed a significant difference in the distribution of CD4+ T-cell numbers (P < 0.001) whereas mutation of chemokine coreceptor CCR5 was not appeared to be associated with the impact of CD4+ T-cell counts.

Alteration of CD8+ T cell effector diversity during HIV-1 infection with discordant normalization in effective antiretroviral therapy.

BACKGROUND: Although the impairment of HIV-specific T lymphocytes is evident during chronic HIV-infection, it is unclear whether the increased CD8+ T cells associates with either selective or overall change of effector functional phenotype. Instead of study on HIV-specific T cells only, analyzing bulk T cell populations represent a neglected area of T cell impairment, which go far beyond HIV-specific T cells. METHODS: In this study, we determined the diversity of CD8+ T cells in term of cytolytic molecule expression (perforin, granzyme A, and granzyme B) and cytokine production ability (IFN-gamma, TNF-alpha, and IL-2) using intracellular staining and flow cytometry technique. The results were compared between healthy individuals, untreated, and antiretroviral therapy (ART) treated HIV infected patients. RESULTS: We demonstrated the presence of four different subsets of CD8+ T cells that expressed different combinations of cytolytic molecules. We also identified seven different subsets of cytokine producing cells based on different combination of IFN-gamma, TNF-alpha, and IL-2. Results showed significant alterations of these cell subsets that expressed different combination of cytolytic effector molecules or cytokines in HIV infected patients. Furthermore, cytolytic molecule expressing cell subsets are not normalized in effective ART treated patients, whereas the selective population of cytokine producing cells returned to normal value. CONCLUSIONS: The effector diversity of CD8+ T cells changed in HIV infected patients. Although effective ART altered functional diversity of these cells, long-term suppression of viral replication may be required to normalize the selected CD8+ T cell effector phenotype in HIV infected patients.

Mechanism of HIV antiretroviral drugs progress toward drug resistance.

The rapid replication rate of HIV-1 RNA and its inherent genetic variation have led to the production of many HIV-1 variants with decreased drug susceptibility. The capacity of HIV to develop drug resistance mutations is a major obstacle to long-term effective anti-HIV therapy. Incomplete suppression of viral replication with an initial drug regimen diminishes the clinical benefit to the patient and may promote the development of broader drug resistance that may cause subsequent treatment regimens to be ineffective. The increased clinical use of combination antiretroviral treatment for HIV-1 infection has led to the selection of viral strains resistant to multiple drugs, including strains resistant to all licensed nucleoside analog RT inhibitors and protease inhibitors. Therefore, it is important to understand the influence of such mutations on viral properties such as replicative fitness, fidelity, and mutation rates. Although research continues to improve our understanding of resistance, leading to refined treatment strategies and, in some cases, improved outcome, resistance to antiretroviral therapy remains a major cause of treatment failure among patients living with HIV-1.

Discordant CD38 measurement of CD8+ T lymphocytes using fluorescein conjugates in comparison with phycoerythrin conjugates.

BACKGROUND: We have previously shown that monitoring of CD38 expression can be used as a marker for antiretroviral drug efficacy in HIV infected patients. However, the detection of CD38 expression may be affected by the sensitivity of the fluorochrome conjugated reagent. OBJECTIVE: In this study, we determined the level of CD38 expression using PE and FITC conjugated anti-CD38 monoclonal antibodies in different groups of HIV infected patients. METHODS: The frequency and mean fluorescence intensity of CD38 expression using PE and FITC conjugated anti-CD38 monoclonal antibodies were detected by flow cytometry either alone or in combination with HLA-DR. A correlation between CD38 expression and CD4 count, the percentage of CD4 or viral load in antiretroviral drug naive HIV infected patients was performed. The results were compared with those for antiretroviral treated HIV infected patients who responsed to therapy and patients with virological failure. RESULTS: We found that while both reagents had the ability to detect a high frequency of CD38 expressing cells in untreated patients, only PE conjugated reagent provided correlation with markers for disease progression. More importantly, FITC conjugated reagent cannot monitor the increase in CD38 expression in patients who showed virological failure. CONCLUSIONS: The results from this study suggest that a cautious selection of fluorochrome conjugated reagents and a method for utilizing the data are extremely critical in the use of CD38 expression as a monitoring tool for ART efficacy.

HIV immune escape at an immunodominant epitope in HLA-B*27-positive individuals predicts viral load outcome.

The CTL response in HLA-B*27(+) HIV-infected individuals is characterized by an immunodominant response to a conserved epitope in gag p24 (aa 263-272, KRWIILGLNK; KK10). Mutations resulting in substitution of the arginine (R264) at position 2 of this epitope have been identified as escape mutations. Nineteen HLA-B*27(+) long-term nonprogressors were identified from an Australian cohort with an average follow-up of 16 y following infection. Viral and host genetic factors impacting on disease progression were determined at multiple time points. Twelve of 19 had wild-type sequences at codon 264 at all time points; 7 of 19 carried CTL escape variants. Median viral load and CD4(+) T cell counts were not significantly different between these groups at enrollment. Viral load, as judged by levels at their last visit (1,700 and 21,000 RNA copies/ml, respectively; p = 0.01) or by time-weighted area under the curve was higher in the escape group (p = 0.02). Escape mutants at other HLA-B*27-restricted epitopes were uncommon. Moreover, host polymorphisms, such as CCR5Δ32, CCR2-64I, and SDF1-3'A, or breadth of TCR repertoire responding to KK10 did not segregate to wild-type or escape groups. Host and viral factors were examined for a relationship to viral load. The only factor to affect viral load was the presence of the R264 escape mutations at the immunodominant epitope. CTL escape at R264 in the KK10 epitope is a major determinant of subsequent viral load in these HLA-B*27(+) individuals.

A new variant cytotoxic T lymphocyte escape mutation in HLA-B27-positive individuals infected with HIV type 1.

The immune response in HIV-infected individuals who carry HLA-B27 is characterized by an immunodominant cytotoxic T lymphocyte (CTL) response to a conserved epitope corresponding to amino acids 263-272 of HIV-1 p24 gag. The arginine at position 264 is a crucial anchor residue. Amino acid substitution at 264 from arginine (R) to glycine (G), lysine (K), or threonine (T) results in a low affinity peptide that binds to HLA-B27 inefficiently and is poorly recognized by T cells that respond to the wild-type peptide. These mutants have been characterized as CTL escape mutations. We studied the plasma virus of 20 HLA-B27 longterm nonprogressors: 14 were wild type and 6 were found to be mutant. Five of these carried known escape mutations coding for K or G at position 264. One patient demonstrated a previously undescribed R264Q mutation in 30/31 clones. This altered epitope failed to elicit an IFN-gamma response from PBMC isolated from any of four HLA-B27-positive individuals with strong responses to wild-type peptide. A peptide binding assay confirmed that the R264Q mutant peptide had 30-fold lower binding affinity to HLA-B27 compared to wild type. Therefore, the R264Q variant is a likely novel escape mutation in HLA-B27-positive individuals.

Rates of transmission of antiretroviral drug resistant strains of HIV-1.

BACKGROUND: It is clear that transmission of drug resistant HIV-1 is possible and occurs regularly. However, there is a lack of clarity concerning the true rate of this transmission in a given population, the impact of combination therapies on this rate, and the contribution of transmitted resistant virus to treatment failure either in an individual or on a population basis. OBJECTIVES: To provide a review of our current understanding of rates of transmission of drug resistant HIV-1 in various populations and to report the results of a study conducted to determine this rate in Sydney, Australia in the years 1992-2000. STUDY DESIGN: A review of the literature combined with a prospective study of antiretroviral drug resistance in 130 individuals who were diagnosed with symptomatic primary infection at St. Vincent's Hospital, Sydney, Australia between 1992 and 2000. Sequencing of reverse transcriptase (RT) and protease (PR) was performed by the TruGene HIV-1 genotyping kit (Visible Genetics Inc.). RESULTS: The results found in the Sydney population contrast with much of the literature. The prevalence of mutations that conferred primary resistance to protease inhibitors (PIs) was only 0.8% at position V82I. Secondary mutations/polymorphisms were seen in the PR at position L10I/V, K20R, M36I, L63P, A71T/V, or V77I in 60%. L63P was the most frequently found mutation (46.3%). The incidence of protease-resistant strains of HIV in primary HIV-1 infection did not change after the introduction of PIs in 1996. The distribution of the most common resistance mutations in the RT was as follows; M41L (8.5%) and T215Y (8.5%) and K70R (4.8%). The frequency of mutations associated with NRTI resistance was significantly lower in the post 1995 samples (43.9 vs. 19.1%, P < 0.05). Moreover, both M41L and K70R, but not T215Y, occurred with significantly decreased frequency in the post 1995 samples. CONCLUSIONS: In contrast to other studies we found no increase in the rate of PR resistance and a decrease in the rate of RT resistance in recently transmitted virus over the period 1992-2000. The reasons for the differences between these results and those reported from elsewhere may relate to treatment regimens used in the transmitting population and may have implications for treatment policies in this country.

No increase in protease resistance and a decrease in reverse transcriptase resistance mutations in primary HIV-1 infection: 1992-2001.

Rates of antiretroviral resistance in recently transmitted virus in Sydney, Australia fluctuated over the past decade, influenced by treatment trends. Current rates of drug resistance are not high in historical terms or compared with those reported. Rates of resistance to reverse transcriptase inhibitors peaked in the mid-1990s, fell dramatically with the introduction of combination therapy and appear to have plateaued at 10-15% over the past 3 years. Primary resistance mutations in the protease gene are still rare.