Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis.

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5'-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

Presence of HIV drug resistance in antiretroviral therapy-naive and -experienced patients from Papua New Guinea.

OBJECTIVES: The optimal benefits of antiretroviral therapy (ART) can be compromised by the emergence of HIV drug resistance (HIVDR) resulting in treatment failure. ART was introduced in Papua New Guinea (PNG) in 2004, yet biological data on HIVDR are lacking. The aim of the study was to investigate levels of HIVDR in ART-naive and -experienced patients in PNG. METHODS: We recruited, interviewed and collected blood from 108 ART-naive and 102 ART-experienced patients from two Highlands provinces of PNG. Dried blood spots were tested for HIVDR from all patients with detectable plasma viral load of ≥200 copies/mL using established in-house assays. RESULTS: The PCR amplification success was 90.6% (n = 96) and 66.7% (n = 12) using dried blood spots from ART-naive and -experienced patients, respectively. Transmitted drug resistance was detected in 2.1% (n = 2) of samples from ART-naive patients; acquired drug resistance was detected in 50% (n = 6) of samples from ART-experienced individuals. CONCLUSIONS: Our data showed that transmitted drug resistance in PNG is low and acquired drug resistance is higher with 12.7% of the ART-experienced patients failing treatment. As ART access is rapidly expanding in PNG, monitoring of drug resistance is paramount for early detection of treatment failure.

The heterosexual HIV type 1 epidemic in Papua New Guinea is dominated by subtype C.

Papua New Guinea is in the midst of a generalized HIV epidemic. As part of a larger behavioral survey aiming to further characterize the HIV epidemic occurring in PNG, samples were collected from 1175 participants from seven different provinces. Seventy-one (6%) of these samples were HIV-1 positive, and 35 (49%) successfully underwent a double nested RT-PCR that was designed to amplify the C2-V4 region of the HIV-1 envelope. Sequence analysis showed that 33 (94%) samples were subtype C and the remaining 2 (6%) were subtype B. Further phylogenetic analysis demonstrated that there was no province-specific clustering among the samples and that within the global pandemic, PNG subtype C isolates most closely resembled those from East Africa.

Factors Influencing Antiretroviral Adherence and Virological Outcomes in People Living with HIV in the Highlands of Papua New Guinea.

Adherence to antiretroviral therapy (ART) is paramount for virological suppression and positive treatment outcomes. ART has been rapidly scaled up in Papua New Guinea (PNG) in recent years, however clinical monitoring of HIV+ individuals on ART is limited. A cross-sectional study was conducted at two major sexual health clinics in high HIV prevalence provinces in the Highlands Region of PNG to assess ART adherence, factors affecting adherence and the relationship between ART adherence and virological outcomes. Ninety-five HIV+ individuals were recruited and administered a questionnaire to gather demographic and ART adherence information whilst clinical data and pill counts were extracted from patient charts and blood was collected for viral load testing. Bivariate analysis was performed to identify independent predictors of ART adherence. Fourteen percent (n = 12) of participants showed evidence of virological failure. Although the majority of participants self-reported excellent ART adherence in the last seven days (78.9%, 75/91), pill count measurements indicated only 40% (34/84) with >95% adherence in the last month. Taking other medications while on ART (p = 0.01) and taking ART for ≥1 year (p = 0.037) were positively associated with adherence by self-report and pill count, respectively. Participants who had never heard of drug resistance were more likely to show virological failure (p = 0.033). Misconception on routes of HIV transmission still persists in the studied population. These findings indicate that non-adherence to ART is high in this region of PNG and continued education and strategies to improve adherence are required to ensure the efficacy of ART and prevent HIV drug resistance.

The molecular epidemiology of HIV type 1 among Vietnamese Australian injecting drug users in Melbourne, Australia.

The proportion of human immunodeficiency virus type 1 (HIV-1) among Vietnamese injecting drug users (IDUs) in Melbourne, Australia exceeds that of the background population. To investigate the molecular epidemiology of HIV-1 among this group, the C2-V4 region of the HIV-1 envelope was directly sequenced from 11 Vietnamese Australians and 19 non-Vietnamese Australian controls. A significant difference in the distribution of the HIV-1 subtypes was demonstrated, with greater than 50% of Vietnamese Australian IDU shown to be infected with CRF01_AE-the predominant subtype in Southeast Asia, rather than subtype B, which dominates the Australian epidemic and which was found in 89.5% of the non-Vietnamese controls. The genetic diversity of the CRF01_AE epidemic in Vietnamese Australian IDUs was substantially lower that that of the background subtype B, consistent with a more recent introduction of a limited number of viral strains from Vietnam. These results support public health policy targeting Australian IDUs of Vietnamese ethnicity as a distinct vulnerable population.

Comparative performance of the Kalon and HerpeSelect enzyme-linked immunosorbant assays to determine the prevalence of herpes simplex virus type 2 in Papua New Guinea.

UNLABELLED: Background Infection with herpes simplex virus type 2 (HSV-2) is common worldwide and an important risk factor for HIV infection. Aetiological diagnosis of HSV-2 is typically determined with the use of commercially available type-specific enzyme-linked immunosorbent assays (ELISAs). This study aimed to determine the prevalence of HSV-2 among people attending sexual health clinics in the Highlands of Papua New Guinea. The study also aimed to compare the performance of two type-specific ELISA assays, the Kalon and HerpeSelect glycoprotein G2 assays, in this context. METHODS: Participants were recruited as part of a longitudinal sexual health study. Participants attended four appointments over a 12-month period and had blood taken for HSV-2 serology at each time point. Both the Kalon and HerpeSelect assays were performed as per manufacturer's instructions. RESULTS: A total of 132 participants were tested for HSV-2 using the Kalon and HerpeSelect ELISAs. HSV-2 prevalence was 52% (95% CI, 43-60) and 61% (95% CI, 52-69) with Kalon and HerpeSelect assays respectively. There was high concordance (87%, ?=0.75, P<0.001, n=115) between the two assays at the manufacturer recommended index value cut-offs. For participants with discordant results at baseline, (n=16), three sero-conversions were observed over the 12-month period when sequential sera was tested. CONCLUSIONS: A high HSV-2 prevalence was observed in this clinic-based population. Our longitudinal data indicate the higher prevalence of HSV-2 detected with the HerpeSelect ELISA was likely due to false positives rather than a higher sensitivity in the early stages of infection.

High prevalence and incidence of HIV, sexually transmissible infections and penile foreskin cutting among sexual health clinic attendees in Papua New Guinea.

UNLABELLED: Background Papua New Guinea (PNG) has one of the highest prevalences of HIV and sexually transmissible infections (STIs) in the Asia-Pacific region, and one of the highest burdens of maternal syphilis and cervical cancer globally. Despite this disease burden, only limited clinical research in sexual and reproductive health has been conducted in PNG. METHODS: A longitudinal clinical cohort study was conducted at two sexual health clinics. Participants completed a behavioural interview, clinical assessment and genital examination at baseline, and at 12, 24 and 50 weeks, including specimen collection for STI diagnostics. RESULTS: In total, 154 people attended a screening visit. Reattendance at 12, 24 and 50-weeks was 87%, 78% and 80% respectively. At baseline, HIV prevalence was 3.3%; chlamydia (Chlamydia trachomatis), 29.2%; gonorrhoea (Neisseria gonorrhoeae), 22.1%; Trichomonas vaginalis 15.6%; herpes simplex type-2 (HSV-2), 46.1%; active syphilis, 11.7%. Multiple infections were common particularly among women. The incidence of chlamydia was 27 per 100 person-years (PY); gonorrhoea, 15 out of 100 PY; T. vaginalis, 29 out of 100 PY; HSV-2, 12 out of 100 PY; syphilis, 8 out of 100 PY. No incident HIV cases were recorded. At baseline, 39% of men in Mt Hagen and 65% in Port Moresby had a penile foreskin cut, with a dorsal slit being the most common. Two men underwent penile cutting during the follow-up period. CONCLUSIONS: The prevalence and incidence of STIs, HIV and penile cutting were high among sexual health clinic attendees. High retention figures suggest that this population may be suitable for future interventions research and clinical trials.

Acquisition of HIV by African-born residents of Victoria, Australia: insights from molecular epidemiology.

African-born Australians are a recognised "priority population" in Australia's Sixth National HIV/AIDS Strategy. We compared exposure location and route for African-born people living with HIV (PLHIV) in Victoria, Australia, with HIV-1 pol subtype from drug resistance assays and geographical origin suggested by phylogenetic analysis of env gene. Twenty adult HIV positive African-born Victorian residents were recruited via treating doctors. HIV exposure details were obtained from interviews and case notes. Viral RNA was extracted from participant stored plasma or whole blood. The env V3 region was sequenced and compared to globally representative reference HIV-1 sequences in the Los Alamos National Library HIV Database. Twelve participants reported exposure via heterosexual sex and two via iatrogenic blood exposures; four were men having sex with men (MSM); two were exposed via unknown routes. Eight participants reported exposure in their countries of birth, seven in Australia, three in other countries and two in unknown locations. Genotype results (pol) were available for ten participants. HIV env amplification was successful in eighteen cases. HIV-1 subtype was identified in all participants: eight both pol and env; ten env alone and two pol alone. Twelve were subtype C, four subtype B, three subtype A and one subtype CRF02_AG. Reported exposure location was consistent with the phylogenetic clustering of env sequences. African Australians are members of multiple transnational social and sexual networks influencing their exposure to HIV. Phylogenetic analysis may complement traditional surveillance to discern patterns of HIV exposure, providing focus for HIV prevention programs in mobile populations.

First molecular epidemiology study of Mycobacterium tuberculosis in Kiribati.

Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC) and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB) cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing) was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%). Beijing strains were further differentiated in two main branches, Beijing-A (n = 8) and -B (n = 28), that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted). Many Kiribati strains (59% based on IS6110 typing of all strains) occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission), however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.

CRF01_AE dominates the HIV-1 epidemic in Indonesia.

BACKGROUND: The HIV epidemic in Indonesia remains concentrated in vulnerable populations, namely injecting drug users (IDUs), commercial sex workers (CSWs) and men who have sex with men (MSM). We aimed to determine the HIV-1 subtypes present in Indonesia and to establish the extent of the viral overlap between individuals with different risk factors. METHODS: Venous blood samples were collected from HIV-positive individuals primarily from sexually transmissible infection clinics and drug rehabilitation centres in Bali and Jakarta, and applied to filter paper. A polymerase chain reaction-based assay designed to amplify a 330-bp region of the HIV-1 envelope was used to determine HIV-1 subtype result and to perform phylogenetic analysis. RESULTS: Of the 175 individuals recruited to the study, a subtype result was obtained for 108 (62%). Four subtypes were found to exist in the population, CRF01_AE (n=96, 88.9%), B (n=10, 9.3%), C (n=1, 0.9%) and G (n=1, 0.9%). Of these 108 individuals, 65 (60%) were IDUs, and the remaining 40% were CSWs, MSM, transgender individuals, people with multiple sexual partners or those with no obvious risk factor. CRF01_AE was found to be more common among IDUs with 100% of individuals infected with this subtype. Subtype B was more common among MSM and CSWs (P=<0.001). Phylogenetic analysis revealed a lack of viral segregation between risk groups. CONCLUSIONS: In Indonesia, CRF01_AE continues to dominate the HIV epidemic, although HIV subtype B is responsible for a significant number of sexually acquired infections.

Stability of dried blood spots for HIV-1 drug resistance analysis.

The wide scale application of dried blood spots (DBS) as a collection tool for low-cost HIV drug resistance testing requires a greater understanding of the accuracy of DBS for genotype analysis and the stability of DBS under various environmental conditions. Analysis of a 50microl DBS via a single amplicon, nested PCR-based in-house assay (the Burnet genotyping assay) showed an average nucleotide concordance of 98.9% with plasma samples, although only 65% of nucleotide mixtures detected in plasma were also detected within DBS. The analysis of three DBS resulted in the detection of a greater number of nucleotide mixtures (72 and 109 mixtures detected within one and three DBS, respectively, n=10). Two DBS extraction protocols (silica particle; NucliSENS, bioMerieux and spin column extraction; High Pure, Roche) were assessed and found to be equivalent (79% and 84% recovery success respectively, n=19). FTA Elute paper (Whatman) was an inferior DBS collection medium compared to Whatman 903 paper. DBS appeared relatively tolerant to multiple freeze/thaw cycles, with 79% of DBS subjected to ten freeze/thaw cycles successfully amplified compared to 93% of DBS defrosted once (n=14). High temperature (37 degrees C) and high humidity (>90%) substantially impaired DBS recovery within two weeks of storage (38%, n=8), whilst storage at -20 degrees C or 4 degrees C adequately preserved DBS for this period (100% recovery, n=8). Therefore, whilst DBS are suitable for HIV drug resistance surveillance, the use of multiple DBS may be required to ensure accurate detection of minor HIV quasispecies and short-term storage of samples at either 4 degrees C or -20 degrees C is recommended.

Normal CD16 expression and phagocytosis of Mycobacterium avium complex by monocytes from a current cohort of HIV-1-infected patients.

Monocyte phenotype and function were measured in whole blood sampled from a current cohort of human immunodeficiency virus (HIV)-infected individuals attending a large, metropolitan, university-affiliated hospital. There was no significant difference in the prevalence of CD16+ monocytes or the capacity of monocytes to ingest heat-killed Mycobacterium avium complex between these individuals and HIV-uninfected control subjects, regardless of viral load, current CD4+ T cell count, nadir CD4+ T cell count, or time since diagnosis of HIV infection. CD16+ monocyte prevalence was, however, elevated in patients not currently receiving antiretroviral therapy. We conclude that HIV type 1 infection in the setting of highly active antiretroviral therapy is associated with normal monocyte function and phenotype.