Drug resistance among newly diagnosed HIV-infected children in the era of more efficacious antiretroviral prophylaxis.

BACKGROUND: In the era of more efficacious prevention of mother-to-child transmission (PMTCT) regimens, documenting the profile of drug resistance in HIV-infected infants and young children is critical to our efforts to improve care and treatment for children. METHODS: HIV drug resistance mutations in plasma virus were ascertained using population sequencing among 230 newly diagnosed HIV-infected children under 2 years of age recruited in Johannesburg, South Africa, during 2011. By this time, more effective PMTCT regimens, including combination antiretroviral therapy for pregnant women, were being implemented. RESULTS: Two-thirds (67.4%) of HIV-infected children had been exposed to some form of maternal (89%) and/or infant (97%) PMTCT. Among PMTCT-exposed, 56.8% had nonnucleoside reverse transcriptase inhibitor (NNRTI), 14.8% nucleoside reverse transcriptase inhibitor (NRTI), and 1.3% protease inhibitor mutations. NNRTI mutations were strongly related to younger age. The remaining third (32.6%) had no reported or recorded PMTCT exposures, but resistance to NNRTI was detected in 24.0%, NRTI in 10.7%, and protease inhibitor in 1.3%. CONCLUSION: The new PMTCT strategies dramatically reduce the number of children who acquire infection, but among those who do become infected, NNRTI resistance prevalence is high. In this South African setting with high PMTCT coverage, almost a quarter of children with no reported or recorded PMTCT also have drug resistance mutations. PMTCT history is an inadequate means of ruling out pretreatment drug resistance. Our results support the use of protease inhibitor-based first-line regimens in HIV-infected infants and young children regardless of PMTCT history.

Timing of maternal HIV testing and uptake of prevention of mother-to-child transmission interventions among women and their infected infants in Johannesburg, South Africa.

BACKGROUND: By 2011, South African prevention of mother-to-child transmission (PMTCT) of HIV programs had reduced perinatal HIV transmission at 6 weeks of age to 2.7%. We investigated the profile of newly diagnosed vertically infected children and their mothers to identify shortfalls in the PMTCT program. METHODS: In this operational follow-up study, fieldworkers enrolled mothers of newly diagnosed HIV-infected children up to 2 years of age at 5 major health care facilities in Johannesburg. Structured questionnaires and clinical record reviews were conducted and analyzed to describe the population and assess factors associated with PMTCT uptake. RESULTS: Two hundred eighty-nine mother-child pairs were enrolled. Timing of maternal HIV diagnosis influenced PMTCT access and feeding choices and was associated with infants' age at HIV diagnosis (7 vs. 11 vs. 31 weeks where mothers tested before, during, or after the pregnancy, respectively; P < 0.0001). Women diagnosed before pregnancy (12%) were older (median, 31 years) than those diagnosed during the index pregnancy (53%; median, 27 years). Women diagnosed after delivery (35%) were younger (median, 25 years, P < 0.0001), of lower parity, and less likely to be South African citizens. In 81 cases (29%), late maternal diagnosis precluded any PMTCT access. Where women were diagnosed during or before pregnancy, the recommended PMTCT guidelines for mother and infant were followed in 86 (61%) pairs. CONCLUSIONS: Failure to diagnose maternal HIV infection before delivery was the main reason for missing PMTCT prophylaxis and early infant testing. Timely maternal diagnosis enables PMTCT uptake, but implementation and follow-up gaps require attention to improve infant outcomes.

Cross priming amplification: mechanism and optimization for isothermal DNA amplification.

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5' ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism - single crossing CPA - and provide details on alternative mechanisms.

Epidemiological investigation of an outbreak of pandemic influenza A (H1N1) 2009 in a boarding school: serological analysis of 1570 cases.

BACKGROUND: A large number of 2009 pandemic influenza A (H1N1) infections were localized in school populations. OBJECTIVES: To describe the epidemiology, clinical features and risk factors associated with an outbreak that occurred at a vocational boarding school in Guangzhou, P.R. China. STUDY DESIGN: Data were collected prospectively and retrospectively through the use of on-site doctors and a post-outbreak survey and blood collection. The survey was used to confirm symptoms, and to investigate a series of flu-related factors such as dormitory conditions, health habits, vaccine history and population contact history. Blood samples were taken for serological analysis. Pandemic H1N1 infection was initially confirmed by a real-time RT-PCR assay. Following the identification of the outbreak by the Guangzhou CDC on September 4, cases were diagnosed symptomatically and retrospectively by serological analysis using the hemagglutination inhibition assay and a neutralization assay. RESULTS: The infection rate was 32% (505/1570) and the attack rate was 22.2% (349/1570). The asymptomatic infection rate was 9.9% (156/1570). Sharing a classroom (OR=2.17, 95% CI: 1.62-2.91) and dormitory space (OR=2.32, 95% CI: 1.84-2.93) was associated with higher rates of infection. Opening windows for ventilation was the only control measure that significantly protected against infection. CONCLUSION: Social isolation and quarantine should be used to prevent the spread of infection. Ventilation and a control of air flow between classrooms and dorms should be implemented as possible. School closures may be effective if implemented early.

Immunogenicity associated with the routine use of an influenza A H1N1 vaccine in health care personnel in Guangzhou, China.

We present immunogenicity data on the routine vaccination of 103 health care personnel during the 2009 H1N1 national vaccination campaign. The seroprotection rate (percentage of samples with hemagglutination inhibition titers of > or =1:40) was 83.2% at 30 days postvaccination, lower than those obtained in previously published controlled trials. Low baseline antibody levels and an increase in seroprotection in a negative-control cohort suggest that the virus remains prevalent.

Application of PCR-LDR-nucleic acid detection strip in detection of YMDD mutation in hepatitis B patients treated with lamivudine.

Chronic hepatitis B virus (CHBV) infection causes cirrhosis and hepatocellular carcinoma. Lamivudine (LAM) has been successfully used to treat CHBV infections but prolonged use leads to the emergence of drug-resistant variants. This is primarily linked to a mutation in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV polymerase gene at position 204. Rapid diagnosis of drug-resistant HBV is necessary for a prompt treatment response. Common diagnostic methods such as sequencing and restriction fragment length polymorphism (RFLP) analysis lack sensitivity and require significant processing. The aim of this study was to demonstrate the usefulness of a novel diagnostic method that combines polymerase chain reaction (PCR), ligase detection reaction (LDR) and a nucleic acid detection strip (NADS) in detecting site-specific mutations related to HBV LAM resistance. We compared this method (PLNA) to direct sequencing and RFLP analysis in 50 clinical samples from HBV infected patients. There was 90% concordance between all three results. PLNA detected more samples containing mutant variants than both sequencing and RFLP analysis and was more sensitive in detecting mixed variant populations. Plasmid standards indicated that the sensitivity of PLNA is at or below 3,000 copies per ml and that it can detect a minor variant at 5% of the total viral population. This warrants its further development and suggests that the PLNA method could be a useful tool in detecting LAM resistance.

Selection of mutations to detect multidrug-resistant Mycobacterium tuberculosis strains in Shanghai, China.

Novel tools are urgently needed for the rapid, reliable detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. To develop such tools, we need information about the frequency and distribution of the mycobacterial mutations and genotypes that are associated with phenotypic drug resistance. In a population-based study, we sequenced specific genes of M. tuberculosis that were associated with resistance to rifampin and isoniazid in 242 phenotypically MDR isolates and 50 phenotypically pan-susceptible isolates from tuberculosis (TB) cases in Shanghai, China. We estimated the sensitivity and specificity of the mutations, using the results of conventional, culture-based phenotypic drug susceptibility testing as the standard. We detected mutations within the 81-bp core region of rpoB in 96.3% of phenotypically MDR isolates. Mutations in two structural genes (katG and inhA) and two regulatory regions (the promoter of mabA-inhA and the intergenic region of oxyR-ahpC) were found in 89.3% of the MDR isolates. In total, 88.0% (213/242 strains) of the phenotypic MDR strains were confirmed by mutations in the sequenced regions. Mutations in embB306 were also considered a marker for MDR and significantly increased the sensitivity of the approach. Based on our findings, an approach that prospectively screens for mutations in 11 sites of the M. tuberculosis genome (rpoB531, rpoB526, rpoB516, rpoB533, and rpoB513, katG315, inhA-15, ahpC-10, ahpC-6, and ahpC-12, and embB306) could detect 86.8% of MDR strains in Shanghai. This study lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China.