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Objectives: To investigate the prevalence of high-risk (HR) human papillomavirus (HPV) and factors associated with HR-HPV infection among women from rural Eastern Cape, South Africa. Methods: HPV prevalence was determined by Hybrid Capture 2 assay in cervical specimens from 417 women aged >= 30 years (median 46 years) recruited from the community health clinic in the Eastern Cape. Results: HR-HPV prevalence was 28.5% (119/417), and HIV-positive women had significantly higher HR-HPV prevalence than HIV-negative women (40.6%, 63/155 vs 21.4%, 56/262, respectively; p = 0.001). HIV-positive status (odds ratio (OR) 2.52, 95% confidence interval (CI) 1.63-3.90), having >= 3 lifetime sexual partners (OR 2.12, 95% CI 1.16-3.89), having >= 1 sexual partner in the last month (OR 1.89, 95% CI 1.21-2.92), >= 4 times frequency of vaginal sex in the past 1 month (OR 2.40, 95% CI 1.32-4.35), and having a vaginal discharge currently/in the previous week (OR 2.13, 95% CI 1.18-3.85) increased the risk of HR-HPV infection. In the multivariate analysis, HIV positivity remained strongly associated with HR-HPV infection (OR 1.94, 95% CI 1.17-3.22). Conclusions: Risk factors related to sexual behaviors play a significant role in HR-HPV infection in this population. This report will inform health policymakers on HPV prevalence and contribute to discussions on the use of HPV testing as the primary cervical cancer screening test in South Africa. (C) 2020 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

The female reproductive tract (FRT) microbiome plays an important role in maintaining vaginal health. Viruses play a key role in regulating other microbial ecosystems, but little is known about how the FRT viruses (virome), particularly bacteriophages, impacts FRT health and dysbiosis. We hypothesize that bacterial vaginosis is associated with alterations in the FRT virome, and these changes correlate with bacteriome shifts. We conducted a retrospective, longitudinal analysis of vaginal swabs collected from 54 bacterial vaginosis (BV)-positive and 46 BV-negative South African women. Bacteriome analysis revealed samples clustered into five distinct bacterial community groups (CG). Bacterial alpha diversity was significantly associated with BV. Virome analysis on a subset of baseline samples showed FRT bacteriophages clustering into novel viral state types (VSTs), a viral community clustering system based on virome composition and abundance. Distinct BV bacteriophage signatures included increased alpha diversity along with Bacillus, Burkholderia and Escherichia bacteriophages. Discriminate bacteriophage-bacteria transkingdom associations were also identified between Bacillus and Burkholderia viruses and BV-associated bacteria, providing key insight for future studies elucidating transkingdom interactions driving BV-associated microbiome perturbations. In this cohort, bacteriophage-bacterial associations suggest complex interactions which may play a role in the establishment and maintenance of BV.

In this study we examined potential associations of HPV infection with the cervical microbiota. Cervical samples were collected from 87 HIV-seronegative reproductive-age Black South African women. Microbiota were characterized by Illumina sequencing of the V3-V4 hypervariable regions of the bacterial 16S rRNA gene. Thirty seven (42.5%) and 30 (34.5%) of the women had prevalent HPV and high-risk (HR)-HPV, respectively. Only 23 women (26.4%) had cervical microbiota dominated by a single Lactobacillus species (L. crispatus (2/87 (2.3%)), L. jensenii (2/87 (2.3%)), and L. iners (19/87 (21.8%)). The majority of the women (56/87 (64.4%)) had diverse cervical microbiota consisting of mainly bacterial vaginosis-associated bacteria. The remaining women (8/87 (9.2%)) had microbiota dominated by Aerococcus, Streptococcus, Chlamydia or Corynebacterium. Women with HR-HPV had significantly higher relative abundances of Aerococcaceae, Pseudomonadaceae and Bifidobacteriaceae compared to those with low-risk (LR)-HPV or no HPV-infection (LDA score >2.0, p 2.0, p 0.2). Further investigations of the bacterial taxa significantly enriched in HR-HPV-infected women are warranted., Highlights • Majority of participants (74%) had cervical microbiota not dominated by Lactobacillus. • Lactobacillus was not enriched in HPV-negative women compared to HPV-positive women. • There was no correlation between cervical microbiota diversity and HPV infection.

Background South African women of reproductive age have a high burden of sexually transmitted infections (STIs), including human papillomavirus (HPV) infection. However, there is limited information on the prevalence of sexually transmitted pathogens in women from rural Eastern Cape Province, South Africa. The study aims at determining the prevalence of sexually transmitted pathogens and co-infection with high-risk (HR) HPV among women from rural Eastern Cape Province, South Africa. Methods A total of 205 cervical specimens were collected from women aged ≥ 30 years from a rural community-based clinic. The samples were tested for a panel of pathogenic STIs [Chlamydia trachomatis (serovars A-K & L1-L3), Haemophilus ducreyi, Herpes Simplex Virus (Types 1 & 2), Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis (TV), and pathobionts [Mycoplasma genitalium (MG), Mycoplasma hominis (MH) and Ureaplasma spp. (UP)] using a multiplex PCR STD direct flow chip assay through a manual Hybrispot platform (Master Diagnostica, Granada, Spain). HR-HPV detection was performed by Hybrid Capture-2 assay. Results High-risk HPV prevalence was 32.2% (66/205) and HIV-1 prevalence was 38.5% (79/205). The overall prevalence of six pathogenic STIs was 22.9% (47/205), with TV having the highest prevalence (15.6%; 32/205). UP (70.2%, 144/205) and MH (36.6%, 75/205) were the most frequently detected pathobionts. Co-infection with ≥ 2 pathogens pathobionts was observed among 52.7% (108/205) participants. Of the six pathogenic STIs, three participants had more than one STI (1.46%) with the presence of MH and UP. HSV-2 (OR: 4.17, CI [1.184–14.690]) and HIV infection (OR: 2.11, CI [1.145–3.873]) were independent STIs associated with HR-HPV infection. Conclusions The high prevalence of pathogenic STIs underscores the need to improve syndromic management policy by implementing effective strategies of prevention, screening tests, and management. HSV-2 and HIV positive remain strongly associated with HR-HPV infection.

Background To date, the microbiota of the human penis has been studied mostly in connection with circumcision, HIV risk and female partner bacterial vaginosis (BV). These studies have shown that male circumcision reduces penile anaerobic bacteria, that greater abundance of penile anaerobic bacteria is correlated with increased cytokine levels and greater risk of HIV infection, and that the penile microbiota is an important harbour for BV-associated bacteria. While circumcision has been shown to significantly reduce the risk of acquiring human papillomavirus (HPV) infection, the relationship of the penile microbiota with HPV is still unknown. In this study, we examined the penile microbiota of HPV-infected men as well as the impact of HIV status. Results The penile skin microbiota of 238 men from Cape Town (South Africa) were profiled using Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Corynebacterium and Prevotella were found to be the most abundant genera. Six distinct community state types (CSTs) were identified. CST-1, dominated by Corynebacterium, corresponded to less infections with high-risk HPV (HR-HPV) relative to CSTs 2–6. Men in CST-5 had greater relative abundances of Prevotella, Clostridiales, and Porphyromonas and a lower relative abundance of Corynebacterium. Moreover, they were significantly more likely to have HPV or HR-HPV infections than men in CST-1. Using a machine learning approach, we identified greater relative abundances of the anaerobic BV-associated bacteria (Prevotella, Peptinophilus, and Dialister) and lower relative abundance of Corynebacterium in HR-HPV-infected men compared to HR-HPV-uninfected men. No association was observed between HIV and CST, although the penile microbiota of HIV-infected men had greater relative abundances of Staphylococcus compared to HIV-uninfected men. Conclusions We found significant differences in the penile microbiota composition of men with and without HPV and HIV infections. HIV and HR-HPV infections were strongly associated with greater relative abundances of Staphylococcus and BV-associated bacterial taxa (notably Prevotella, Peptinophilus and Dialister), respectively. It is possible that these taxa could increase susceptibility to HIV and HR-HPV acquisition, in addition to creating conditions in which infections persist. Further longitudinal studies are required to establish causal relationships and to determine the extent of the effect.

The global burden of disease caused by both human immunodeficiency virus (HIV) and human papillomavirus (HPV) is the greatest in the developing world, with the highest rates in sub-Saharan Africa. South African women not only have high rates of infection with HPV, but also have high rates of multiple concurrent infections with two or more HPV genotypes, and are among the world’s most vulnerable to developing invasive cervical cancer. HIV co-infection increases these risks. Understanding clustering patterns of concurrent HPV infections in this population has important implications for HPV screening and will help define vaccination strategies in the future as vaccines continue to be developed to target more HPV genotypes. Latent class analysis was used to identify four distinct patterns of HPV co-infection: individuals with at least one low risk HPV genotype, but no high-risk HPV (HR-HPV) infections; individuals with a disperse pattern of HR-HPV infections; individuals infected with members of the alpha-7 group, but not HPV-18; and individuals infected with HPV-16, but not HPV-18. In this analysis, although alpha-7 HPV infections were more prevalent among HIV-infected adolescents than their HIV-uninfected counterparts, overall clustering patterns were not different based on HIV status. Key words: Human papillomavirus (HPV), clustering, latent class analysis, high-risk HPV, South African adolescents.

Human papillomavirus (HPV) testing on vaginal self-collected and cervical clinician-collected specimens shows comparable performance. Self-sampling on FTA cards is suitable for women residing in rural settings or not attending regular screening and increases participation rate in the cervical cancer screening programme. We aimed to investigate and compare high-risk (HR)-HPV prevalence in clinician-collected and self-collected genital specimens as well as two different HPV tests on the clinician collected samples. A total of 737 women were recruited from two sites, a community health clinic (n = 413) and a referral clinic (n = 324) in the Eastern Cape Province. Cervical clinician-collected (FTA cards and Digene transport medium) and vaginal self-collected specimens were tested for HR-HPV using the hpVIR assay (FTA cards) and Hybrid Capture-2 (Digene transport medium). There was no significant difference in HR-HPV positivity between clinician-collected and self-collected specimens among women from the community-based clinic (26.4% vs 27.9%, p = 0.601) or the referral clinic (83.6% vs 79.9%, p = 0.222). HPV16, HPV35, and HPV33/52/58 group were the most frequently detected genotypes at both study sites. Self-sampling for HPV testing received a high positive response of acceptance (77.2% in the community-based clinic and 83.0% in referral clinic). The overall agreement between hpVIR assay and HC-2 was 87.7% (k = 0.754). The study found good agreement between clinician-collected and self-collected genital specimens. Self-collection can have a positive impact on a cervical screening program in South Africa by increasing coverage of women in rural areas, in particular those unable to visit the clinics and women attending clinics where cytology-based programs are not functioning effectively.

BackgroundLactobacillusspp. are common bacteria in the cervical and vaginal microbiota (CVM) and are thought to represent a “healthy” cervicovaginal state. Several studies have found an independent association between ethnicity/race and cervical and vaginal microbiota (CVM) composition. Women of sub-Saharan African descent appear to be significantly more likely to have non-Lactobacillus-dominated CVM compared to women of European descent. The factors contributing to these differences remain to be fully elucidated. The CVM of Black South African women and factors influencing their CVM remain understudied. In this study, we characterized the cervical microbiota of reproductive-age South African women and assessed the associations of these microbiota with participants’ metadata.MethodsThe cervical microbiota from cervical DNA of 62 reproductive-age women were profiled by Ion Torrent sequencing the V4 hypervariable region of the bacterial 16S ribosomal RNA (rRNA) gene and analyzed with the Quantitative Insights Into Microbial Ecology (QIIME), UPARSE, and metagenomeSeq tools. Associations between cervical microbiota and participants’ metadata were assessed using GraphPad Prism, R packages and an in-house script.ResultsThe cervical microbiota clustered into three distinct community state types (CSTs):Lactobacillus iners-dominated cervical microbiota (CST I (38.7%, 24/62)), unclassifiedLactobacillus-dominated cervical microbiota (CST II (4.8%, 3/62)), and diverse cervical microbiota (CST III (56.5%, 35/62)) with an array of heterogeneous bacteria, predominantly the bacterial vaginosis (BV)-associatedGardnerella,Prevotella,Sneathia, andShuttleworthia. CST III was associated with BV (p = 0.001). Women in CST I were more likely to be on hormonal contraception, especially progestin-based, compared to women in CST III (odds ratio: 5.2 (95% CI [1.6–17.2]);p = 0.005). Women on hormonal contraception had a significantly lower alpha (Shannon indices: 0.9 (0.2–1.9) versus 2.3 (0.6–2.3);p = 0.025) and beta (permutational multivariate analysis of variance (PERMANOVA) pseudo-F statistic =4.31,p = 0.019) diversity compared to non-users. There was no significant difference in the alpha (Shannon indices: 1.0 (0.3–2.2) versus 1.9 (0.3–2.2);p = 0.483) and beta (PERMANOVA pseudo-Fstatistic = 0.89,p = 0.373) diversity in women with versus without human papillomavirus infection.ConclusionsThe majority of Black women in our study had non-Lactobacillus-dominated cervical microbiota. Additional studies are needed to examine whether such microbiota represent abnormal, intermediate or variant states of health. Lastly, the association of hormonal contraception withL. inersdominance requires further in-depth research to confirm this association, determine its biological mechanism and whether it has a beneficial effect on the cervicovaginal health.

Background The indicating FTA card is a dry medium used for collection of cervical samples. HPVIR is a multiplex real-time PCR test that detects 12 high-risk human papillomavirus types (hrHPV) and provides single genotype information for HPV16, − 31, − 35, − 39, − 51, − 56, and − 59 and pooled type information for HPV18/45 and HPV33/52/58. The aim of this study was to evaluate whether a strategy with cervical samples collected on the FTA card and subsequently analysed with the HPVIR test complies with the criteria of the international guidelines for a clinically validated method for cervical screening. Methods We performed a non-inferiority test comparing the clinical sensitivity and specificity of the candidate test (FTA card and HPVIR) with a clinically validated reference test (Cobas® HPV test) based on liquid-based cytology (LBC) samples. Two clinical samples (LBC and FTA) were collected from 896 participants in population-based screening. For evaluation of the specificity we used 799 women without ≥ CIN2, and for clinical sensitivity we used 67 women with histologically confirmed ≥ CIN2. The reproducibility was studied by performing inter- and intra-laboratory tests of 558 additional clinical samples. Results The clinical sensitivity and specificity for samples collected on the FTA card and analysed using the HPVIR test were non-inferior to samples analysed with the Cobas® HPV test based on LBC samples (non-inferiority test score, p = 1.0 × 10− 2 and p = 1.89 × 10− 9, respectively). Adequate agreement of > 87% was seen in both the intra- and inter-laboratory comparisons. Conclusions Samples collected on the indicating FTA card and analysed with HPVIR test fulfil the requirements of the international guidelines and can therefore be used in primary cervical cancer screening.

Four novel human gammapapillomaviruses were characterized from penile specimens using genome amplification, cloning, and sequencing. The HPV-219 L1 gene showed 87% nucleotide identity to that of HPV-213 of species gamma-13, HPV-220 had 72% identity to L1 of HPV-212 (gamma-17), HPV-221 had 80% identity to L1 of HPV-142 (gamma-10), and HPV-222 had 73% nucleotide identity to L1 of HPV-162 (gamma-19).

Next generation sequencing technologies have enabled characterization of microbiota at an unprecedented resolution. Microbiota research methodologies are constantly improving and differing in the primer choice, sequencing technology, and bioinformatics pipeline used. These variations could impact comparability of microbiota results across studies. Here, we used 19 cervical samples to compare the performance of two metabarcoding methods that differ in the primer choice and sequencing technology used. We sequenced the V4 16S rRNA libraries with Ion Torrent PGM (IT-V4 method) and V3-V4 16S rRNA libraries with Illumina MiSeq (IM-V3/V4 method). Microbial communities were analyzed using QIIME and UPARSE. Linear correlation of the abundances of the shared genera between the IT-V4 and IM-V3/V4 datasets were computed using Pearson's correlation (r). Procrustes analysis was used to examine whether beta diversity estimations from the two methods were consistent (closeness of fit (M2) from 1000 Monte Carlo permutations 2.0). Procrustes analyses showed a statistically consistent taxonomic and functional clustering between the two methods (Count_Better = 0, M2 = 0.2–0.3, p

Summary Objectives Persistence of infection with high-risk Human papillomaviruses (HR-HPV) increases the risk of incident and progressive precancerous lesions of the cervix. Rates of HR-HPV persistence have been shown to be increased among HIV-infected adult women, however there is a paucity of literature addressing HPV persistence in the young HIV-infected population. We compared rates of HR-HPV persistence between HIV-infected and HIV-uninfected young women. Methods We obtained self-collected vaginal swabs at six-month intervals from 50 HIV-uninfected and 33 HIV-infected young women recruited through a community youth center (age 17-21 years) and compared rates of HR-HPV persistence. HR-HPV testing was conducted using the Roche's Linear Array® HPV Test. Results Eighty-three prevalent (upon baseline testing) and incident (upon subsequent testing) individual HR-HPV infections were identified among 43 members of the cohort (23 HIV-uninfected and 20 HIV-infected). At twelve months, 19% of baseline HR-HPV infections continued to be present with a statistically significant difference between HIV-uninfected and HIV-infected participants (4% versus 31%; p=0.01). Conclusions HIV-infected young women in our cohort had a seven-fold increased rate of persistence of HR-HPV overall at 12 months, indicating an increased risk for incident and progressive precancerous lesions. Identification of persistent infection with HR-HPV may complement cytological findings in determining the need for colposcopy.

Background.HIV-infected adolescents may be at higher risk for high-grade cervical lesions than HIV-uninfected adolescents. The purpose of this study was to compare the prevalence of high-risk HPV (HR-HPV) infections and Pap smear abnormalities between these two groups.Methods.In this cross-sectional study, we compared the HPV DNA and Pap smear results between 35 HIV-infected and 50 HIV-uninfected adolescents in order to determine the prevalence of HR-HPV genotypes and cervical cytological abnormalities. Comparisons were made using Pearsonχ2and independent-samplest-tests analyses, and associations between demographic and behavioral characteristics and HPV infections were examined.Results.HIV-infected participants were more likely to be infected with any HPV (88.6% versus 48.0%;P<0.001) and with at least one HR-HPV (60.0% versus 24.0%;P=0.001), and to have multiple concurrent HPV infections (68.6% versus 22.0%;P<0.001). HPV 16 and 18 were relatively underrepresented among HR-HPV infections. Abnormal Pap test results were more common among HIV-infected participants (28.8% versus 12.0%;P=0.054). A history of smoking was associated with HR-HPV infection.Conclusions.HIV-infected adolescents have an increased risk of infection with HR-HPV and of Pap test abnormalities. The majority of HR-HPV infections among our participants would not be prevented by the currently available vaccinations against HPV.

The role of African horsesickness virus (AHSV) nonstructural membrane protein NS3 in determining the effects of AHSV infection on Vero cells was examined. NS3 protein sequences are highly variable and cluster into three phylogenetic groups, alpha, beta, and gamma. Three AHSV strains, with NS3 from alpha, beta, or gamma, were shown to have quantitatively different phenotypes in Vero cells. Reassortants between these strains, in which the S10 genome segment encoding NS3 was exchanged alone or with other segments, were generated and compared to parental strains. Exchange of the NS3 gene resulted in changes in virus release, membrane permeability and total virus yield, indicating an important role for NS3 in these viral properties. Differences in the cytopathicity and the effect on cell viability between the parental strains could not be associated with NS3 alone, and it is likely that a number of viral and host factors play a role.

Infection with HIV is known to increase the risk of cervical cancer. In addition, evidence suggests that concurrent infection with multiple human papillomavirus (HPV) genotypes increases the risk of cervical dysplasia more than infection with a single HPV genotype. However, the impact of the combination of HIV coinfection and presence of multiple concurrent HPV infections on the risk of cervical dysplasia is uncertain. We compared the results of HPV testing and Pap smears between HIV-infected and HIV-uninfected young women to assess the cumulative impact of these two conditions. We found that both HIV and the presence of multiple concurrent HPV infections are associated with increased risk of associated Pap smear abnormality and that the impact of these two risk factors may be additive.

The objectives of the study were to investigate prevalence of cervical human papillomavirus (HPV) genotypes to inform HPV vaccination strategy in South Africa and to study factors associated with HPV prevalence. Sexually active, HIV-negative women, aged 16-22 years recruited from Soweto (n = 143) and Cape Town (n = 148) were tested for cervical HPV and other genital infections. Overall HPV prevalence was 66.7% (194/291) in young women. Cape Town women were more likely to have multiple HPV infections than the Soweto women (48.0%, 71/148 versus 35.0%, 50/143 respectively, p = 0.033) and probable HR-HPV types (34.5%, 51/148 versus 21.7%, 31/143 respectively, p = 0.022). The most frequently detected HPV types were HPV-16 (11.7%), HPV-58 (10.3%), HPV-51 (8.9%), HPV-66 (8.6%), HPV-18 and HPV-81 (7.6% each). HPV types targeted by the bivalent HPV vaccine (HPV-16/18) were detected in 18.6% (54/291) of women, while those in the quadrivalent vaccine (HPV-6/11/16/18) were detected in 24.7% (72/291) of women; and those in the nonavalent vaccine (HPV-6/11/16/18/31/33/45/52/58) were detected in 38.5% (112/291) of women. In a multivariable analysis, bacterial vaginosis remained significantly associated with HPV infection (OR: 4.0, 95% CI: 1.4-12.6). Women were more likely to be HPV positive if they had received treatment for STI during the past 6-months (OR: 3.4, 95% CI: 1.1-12.4) or if they had ever been pregnant (OR: 2.3, 95% CI: 1.1-5.5). Compared to women who reported only one sexual partner, those with increased number of lifetime sex partners were more likely to have HPV (4-10 partners: OR: 2.9, 95% CI: 1.1-8.0). The high prevalence of HPV types targeted by the nonavalent HPV vaccine encourages the introduction of this vaccine and catch-up HPV vaccination campaigns in South Africa. The high burden of BV and concurrent STIs also highlights the need to improve the prevention and appropriate management of sexually-acquired and other genital tract infections in South African youth.

This chapter presents a number of methods that have been successfully applied in the screening for enzymes from extremophilic microorganisms and their communities. Parameters such as temperature, pH, percentage of relative humidity, salinity, dO2, redox state, moisture content, light, local geology, weather (cloud, precipitation, wind speed), and so on, may be critical or irrelevant. Thermophilic samples, psychrophilic samples, halophilic samples, acidophilic samples, superficial sea- and freshwater samples are the issues for consideration in the sampling of various specific extreme environments. Two major extraction protocols may be used: (i) phenol extraction, where the gentle hydrophobicity of phenol makes it a good solvent for DNA extraction, and (ii) the use of commercial kits. Extremophiles are an obvious source of novel enzymes that may revolutionize the biofuels industry. Several bacterial and fungal species produce ligninases, with the enzymes produced by brown- and white-rot fungi being studied the most extensively. A report by Tuncer and his coworkers found that the ratio of carbon (C)/nitrogen (N) affected enzyme production and that a C/N ratio of 4:1 to 5.3:1 resulted in maximal enzyme production. This report also highlighted the need to grow all test strains on a range of different carbon sources, because the amount of extracellular enzymes produced varies depending on the carbon source, and it is essential to find the best medium for enzyme production. There is certainly a need for standardized methods for performing metagenomics projects, from physical-chemical description of sampling sites and sampling procedures down to the data interpretation and integration.

Infections by HIV increase the risk of acquiring secondary viral and bacterial infections and methods are needed to determine the spectrum of co-infections for proper treatment. We used rolling circle amplification (RCA) and Ion Proton sequencing to investigate the vaginal microbiome of 20 HIV positive women from South Africa. A total of 46 different human papillomavirus (HPV) types were found, many of which are not detected by existing genotyping assays. Moreover, the complete genomes of two novel HPV types were determined. Abundance of HPV infections was highly correlated with real-time PCR estimates, indicating that the RCA-Proton method can be used for quantification of individual pathogens. We also identified a large number of other viral, bacterial and parasitic co-infections and the spectrum of these co-infections varied widely between individuals. Our method provides rapid detection of a broad range of pathogens and the ability to reconstruct complete genomes of novel infectious agents.

A temperate phage, Psymv2, was isolated from an Antarctic soil bacterium, Psychrobacter sp. MV2. The morphology of Psymv2 was typical of the Siphoviridae, with an isometric head and non-contractile tail. The Psymv2 genome was found to be 35,725 bp in length, had a G + C content of 44.5 %, with 49 protein-coding genes and one tRNA gene predicted. Integration of Psymv2 occurred at an ssrA gene, with the last 27 bases of this gene directly repeated at the prophage ends. The genome was organised in a modular fashion: integration, regulation, packaging, head assembly, tail assembly, host specificity and lysis. While the genome sequence had little similarity on a nucleotide level to previously reported phage sequences, the genome architecture resembled that of Siphoviridae of low G + C Gram-positive bacteria. The closest relatives to Psymv2 were uncharacterized putative prophages within the P. arcticus 273-4 and Acinetobacter baumannii 6013113 genomes. Global alignment of the Psymv2 genome and these prophages revealed significant conservation of the structural modules despite the large spatial divergence of their hosts. A number of unique ORFs were identified in the Psymv2 genome that may contribute to phage and lysogen fitness.

Numerous gene-specific PCR methods have been developed for the cultivation-independent discovery of novel genes from complex environmental DNA samples. The recovery of full-length genes is, however, technically challenging. Here, we present an efficient and relatively simple approach that combines magnetic bead capture with subtractive hybridization for the rapid and direct recovery of full-length target ORFs. When compared with other PCR-based techniques, a higher degree of specificity is achieved through the use of larger gene fragments during hybridization followed by several high-stringency washes. Together with the recent advances in environmental nucleic acid extraction techniques, this approach should allow for the further exploration of the metagenomic sequence space.

01CD002 Studying pulmonary function in HIV positive tanzanian youth A. August; Geisel School of Medicine at Dartmouth, Hanover, NH/US Program/Project Purpose: Survival into childhood with an untreated HIV infection was once considered unusual, but has increased in recent years. This has spurred further interest in studying the health of adolescents living with HIV. There have been two major reports from Zimbabwe (Ferrand et al., 2007) and Malawi (Zverez et al., 2001) of a decrease in pulmonary function in HIV positive youth, especially if they had delayed onset of treatment. The purpose of this study is to investigate the pulmonary function of HIV positive Tanzanian children. This study will help determine the importance of monitoring lung function in HIV patients and if a pulmonary treatment regimen should be instituted. Structure/Method/Design: A cohort of 250 Tanzanian children between the ages of 8 and 18 with a diagnosis of HIV currently being treated at the DarDar Pediatric Program in Dar es Salaam, Tanzania will take part in the study. Pulmonary function (PFT) was assessed using spirometry to measure forced expiratory volume at one second (FEV1), forced vital capacity (FVC), FVC/FEV1 ratio, and forced expiratory flow 25% to 75% (FEF25-75) as well as pulse oximetry. Each participant was evaluated at rest. If recorded values for FVC and FEV 1 were > 70% of predicted, they were re-evaluated after moderate exercise and after administration of a bronchodilator. Normative data from healthy Malawian youth were used. The PFT data were analyzed in correlation with age of diagnosis and age at initiation of antiretroviral therapy. Outcomes & Evaluation: A preliminary analysis of the first 50 subjects has not shown any significant degree of pulmonary dysfunction nor any correlation with a delayed ART regimen. Of the 50 subjects, three were excluded for inconsistent PFT efforts. The remaining 47 subjects consisted of 17 females/30 males. Only 3 subjects had FEV 1 Going Forward: Ongoing challenges include obtaining consistent data from younger subjects as well as timely patient acquisition. We anticipate meeting our goal of 250 subjects by late spring 2015. Funding: This project is supported by internal funds available through the Medical Schools at Muhimbili University and Dartmouth. Funding for the author’s participation was provided by the DIHG-DICKEY International Health Fellowship and Helen’s Fund for pediatric research at the Children’s Hospital at Dartmouth.

Background Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women. Methods Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR. Results Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence’s of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6%, 12.8%, 4.6% and 8.3% respectively. Conclusions Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.