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Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

Little has been documented about partner violence among adolescent girls and young women (AGYW) who are out of school, a factor associated with HIV acquisition. To understand areas for prioritising HIV prevention intervention efforts, we explored the prevalence and correlates of partner violence among out of school AGYW in Shinyanga, Tanzania. A cross-sectional analysis of data from AGYW aged 15-23 years recruited in a cluster randomised trial conducted between October and December 2017 was used to examine correlates of partner violence. Data were collected through an Audio Computer-Assisted Self-interview. Multivariate logistic regression analysis was used to evaluate the association. 2276 (75.5%) AGYW were sexually active. Of these, 816 (35.9%) reported having experienced violence from partners in the last six months. After adjusting for other covariates, being formerly married (AOR = 1.55, 95% CI:1.02, 2.37), having children (AOR = 1.79, 95% CI:1.47, 2.16), anxiety and depression symptoms (AOR = 3.27, 95%CI: 2.15, 4.96), having engaged in sex work in the past six months (AOR = 1.92, 95% CI: 1.45, 2.53) and economic deprivation (AOR = 1.61, 95% CI: 1.34,1.92) were significantly associated with partner violence. Almost one in three sexually active AGYW had experienced partner violence in the 6 months preceding the survey. The findings underscore the need for future research to focus on understanding the reasons and dynamics underlying high level of partner violence among AGYW. Furthermore, there is a need for implementing intervention programs that aim to reduce economic deprivation among AGYWs and address social norms and structures perpetuating violence against AGYW. ClinicalTrials.gov-ID NCT03597243.

Adolescent girls and young women (AGYW) aged 15 to 24 years face disproportionately high risks of acquiring HIV and other sexually transmitted infections (STIs). A sexual health risk stratification tool can support the development and implementation of tailored HIV and STI prevention services for sub-groups of at-risk AGYW. Data were collected among sexually active AGYW aged 15 to 24 years in Tanzania between April 2015 and March 2017. Exploratory and confirmatory factor analyses were conducted to construct and assess the latent structure of a ten-item scale for rapid assessment of sexual health risks. Items with high factor loadings and minimal cross loadings were retained in the final scale. Scale performance was appraised against condomless sex (defined as unprotected vaginal or anal intercourse) reported by AGYW for construct validity. A three-factor structure of vulnerability to HIV among AGYW was supported with subscales for socioeconomic vulnerability; lack of adult support; and sexual behavioral risks. The chi-square goodness-of-fit test, root mean square error of approximation, comparative fit index, and Tucker-Lewis index indicated a strong goodness-of-fit of the three-factor scale. Cronbach alphas (0.55 for socioeconomic vulnerability, 0.55 for lack of support, and 0.48 for sexual risk) indicated sub-optimal internal consistency for all sub-scales. The factor-item and factor-factor correlations identified in these analyses were consistent with the conceptual framework of vulnerability of HIV infection in AGYW, suggesting good construct validity. The scale also demonstrated a statistically significant association with condomless sex and could be potentially used for sexual health risk stratification (OR = 1.17, 95% CI: 1.12, 1.23). The sexual health and HIV risk stratification scale demonstrated potential in identifying sexually active AGYW at high risk for HIV and other STIs. Ultimately, all AGYW in Tanzania are not at equal risk for HIV and this scale may support directing resources towards those at highest risk of HIV.

Background Newborn resuscitation is a life-saving intervention for birth asphyxia, a leading cause of neonatal mortality. Improving provider newborn resuscitation skills is critical for delivering quality care, but the retention of these skills has been a challenge. Tanzania implemented a national newborn resuscitation using the Helping Babies Breathe (HBB) training program to help address this problem. Our objective was to evaluate the effectiveness of two training approaches to newborn resuscitation skills retention implemented across 16 regions of Tanzania. Methods An initial training approach implemented included verbal instructions for participating providers to replicate the training back at their service delivery site to others who were not trained. After a noted drop in skills, the program developed structured on-the-job training guidance and included this in the training. The approaches were implemented sequentially in 8 regions each with nurses/ midwives, other clinicians and medical attendants who had not received HBB training before. Newborn resuscitation skills were assessed immediately after training and 4–6 weeks after training using a validated objective structured clinical examination, and retention, measured through degree of skills drop, was compared between the two training approaches. Results Eight thousand, three hundred and ninety-one providers were trained and assessed: 3592 underwent the initial training approach and 4799 underwent the modified approach. Immediately post-training, average skills scores were similar between initial and modified training groups: 80.5 and 81.3%, respectively ( p -value 0.07). Both groups experienced statistically significant drops in newborn resuscitation skills over time. However, the modified training approach was associated with significantly higher skills scores 4–6 weeks post training: 77.6% among the modified training approach versus 70.7% among the initial training approach ( p -value < 0.0001). Medical attendant cadre showed the greatest skills retention. Conclusions A modified training approach consisting of structured OJT, guidance and tools improved newborn resuscitation skills retention among health care providers. The study results give evidence for including on-site training as part of efforts to improve provider performance and strengthen quality of care.

Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an integration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.