Validity of dried blood spot testing for sexually transmitted and blood-borne infections: A narrative systematic review.

Testing for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) using dried blood spot (DBS) specimens has been an integral part of bio-behavioural surveillance in Canada for almost two decades, though less is known regarding the use of DBS in surveillance of other sexually transmitted and blood-borne infections (STBBI). A systematic review was conducted using a peer-reviewed search strategy to assess the current evidence regarding the validity of STBBI testing using DBS specimens. Eligibility criteria included studies reporting use of DBS specimens for STBBI testing with either commercially available or "in-house" tests in populations 15 years of age or older. Studies reporting a measure of validity such as sensitivity, specificity, positive and negative predictive values were eligible for inclusion. Quality of studies and risk of bias were assessed using the QUADAS-2 tool. A total of 7,132 records were identified. Of these, 174 met the criteria for inclusion. Among the studies that reported validity measures, a substantial proportion demonstrated high sensitivity (≥90%) in 62.5% of cases (N = 334/534 sensitivity measurements), and high specificity (≥90%) was observed in 84.9% of instances (N = 383/451 specificity measurements). However, the quality of the studies varied greatly. Our findings support the validity of the use of DBS specimens in STBBI testing where sufficient evidence was available, but validity is highly dependent on thorough method development and validation.

Differential Burden of HIV Among Adolescent Girls and Young Women by Places Associated With Sex Work: An Observational Study in Mombasa, Kenya.

BACKGROUND: The design of HIV prevention programs for adolescent girls and young women (AGYW) are informed by data on who is at highest risk and where they can be reached. Places (hotspots) associated with selling sex are an established outreach strategy for sex work (SW) programs but could be used to reach other AGYW at high risk. SETTING: This study took place in Mombasa, Kenya. METHODS: We conducted a cross-sectional, bio-behavioural survey among (N = 1193) sexually active AGYW aged 14-24 years recruited at hotspots. We compared HIV prevalence by subgroup (SW; transactional sex, TS; and non-transactional sex), stratified by hotspot type (venues and nonvenues). We examined whether associations between HIV prevalence and hotspot/subgroup remained after adjustment for individual-level risk factors, and estimated HIV prevalence ratio with and without adjustment for these individual-level factors. RESULTS: Overall HIV prevalence was 5.6%, 5.3% in venues and 7.3% in nonvenues. Overall SW HIV prevalence was 2-fold higher than among participants engaged in nontransactional sex. After adjusting for age and individual-level risk factors, HIV prevalence was 2.72 times higher among venue-based SWs (95% confidence interval: 1.56 to 4.85) and 2.11 times higher among nonvenue AGYW not engaged in SW (95% confidence interval: 0.97 to 4.30) compared with venue-based AGYW not engaged in SW. CONCLUSION: AGYW who sell sex remain at high risk of HIV across types of hotspots. The residual pattern of elevated HIV burden by AGWY subgroup and hotspot type suggests that unmeasured, network-level factors underscore differential risks. As such, hotspots constitute a "place" to reach AGYW at high risk of HIV.

A validated in-house assay for HIV drug resistance mutation surveillance from dried blood spot specimens.

Despite increasing scale-up of antiretroviral therapy (ART) coverage, challenges related to adherence and HIV drug resistance (HIVDR) remain. The high cost of HIVDR surveillance is a persistent challenge with implementation in resource-constrained settings. Dried blood spot (DBS) specimens have been demonstrated to be a feasible alternative to plasma or serum for HIVDR genotyping and are more suitable for lower resource settings. There is a need for affordable HIVDR genotyping assays which can amplify HIV-1 sequences from DBS specimens, particularly those with low viral loads, at a low cost. Here, we present an in-house assay capable of reliably amplifying HIV-1 protease and partial reverse transcriptase genes from DBS specimens, which covers the complete World Health Organization 2009 list of drug resistance mutations under surveillance. DBS specimens were prepared using whole blood spiked with HIV-1 at concentrations of 10,000, 5000, 1000, and 500 copies/mL (n=30 for each concentration). Specimens were tested in triplicate. A two-step approach was used consisting of cDNA synthesis followed by nested PCR. The limit of detection of the assay was calculated to be approximately 5000 (95% CI: 3200-10,700) copies/mL for the protease gene and 3600 (95% CI: 2200-10,000) copies/mL for reverse transcriptase. The assay was observed to be most sensitive with higher viral load specimens (97.8% [95% CI: 92.2-99.7]) for both protease and reverse transcriptase at 10,000 copies/mL with performance decreasing with the use of specimens with lower viral loads (46.7% [36.1-57.5] and 60.0% [49.1-70.2] at 500 copies/mL for protease and reverse transcriptase, respectively). Ultimately, this assay presents a promising opportunity for use in resource-constrained settings. Future work should involve validation under field conditions including sub-optimal storage conditions and preparation of DBS with fingerprick blood in order to accurately reflect real-world collection scenarios.

Temporal trends and transmission dynamics of pre-treatment HIV-1 drug resistance within and between risk groups in Kenya, 1986-2020.

BACKGROUND: Evidence on the distribution of pre-treatment HIV-1 drug resistance (HIVDR) among risk groups is limited in Africa. We assessed the prevalence, trends and transmission dynamics of pre-treatment HIVDR within and between MSM, people who inject drugs (PWID), female sex workers (FSWs), heterosexuals (HETs) and perinatally infected children in Kenya. METHODS: HIV-1 partial pol sequences from antiretroviral-naive individuals collected from multiple sources between 1986 and 2020 were used. Pre-treatment reverse transcriptase inhibitor (RTI), PI and integrase inhibitor (INSTI) mutations were assessed using the Stanford HIVDR database. Phylogenetic methods were used to determine and date transmission clusters. RESULTS: Of 3567 sequences analysed, 550 (15.4%, 95% CI: 14.2-16.6) had at least one pre-treatment HIVDR mutation, which was most prevalent amongst children (41.3%), followed by PWID (31.0%), MSM (19.9%), FSWs (15.1%) and HETs (13.9%). Overall, pre-treatment HIVDR increased consistently, from 6.9% (before 2005) to 24.2% (2016-20). Among HETs, pre-treatment HIVDR increased from 6.6% (before 2005) to 20.2% (2011-15), but dropped to 6.5% (2016-20). Additionally, 32 clusters with shared pre-treatment HIVDR mutations were identified. The majority of clusters had R0 ≥ 1.0, indicating ongoing transmissions. The largest was a K103N cluster involving 16 MSM sequences sampled between 2010 and 2017, with an estimated time to the most recent common ancestor (tMRCA) of 2005 [95% higher posterior density (HPD), 2000-08], indicating propagation over 12 years. CONCLUSIONS: Compared to HETs, children and key populations had higher levels of pre-treatment HIVDR. Introduction of INSTIs after 2017 may have abrogated the increase in pre-treatment RTI mutations, albeit in the HET population only. Taken together, our findings underscore the need for targeted efforts towards equitable access to ART for children and key populations in Kenya.

A cross-sectional survey exploring HIV and HCV prevalence among men who purchase sex in Dnipro, Ukraine.

BACKGROUND: HIV programming in Ukraine largely targets "key population" groups. Men who purchase sex are not directly reached. The aim of our study was to explore the prevalence of sexually transmitted and blood-borne infections (STBBIs) among men who purchase sex from female sex workers. METHODS: Following geographic mapping and population size estimation at each "hotspot", we conducted a cross-sectional bio-behavioural survey with men who purchase sex between September 2017 and March 2018 in Dnipro, Ukraine. Eligibility criteria included purchasing sex services at a "hotspot" and being ≥ 18 years. Participants completed a structured questionnaire, followed by HIV/HCV rapid testing and a dried blood spot (DBS) sample collection for confirmatory serology. RESULTS: The study enrolled 370 participants. The median age was 32 (interquartile range [IQR] = 27-38) and the median age of first purchase of sexual services was 22 (IQR = 19-27). Over half (56%) of participants reported ever testing for HIV; four participants (2%, N = 206) reported having tested positive for HIV, with three out of the four reporting being on ART. Forty percent of participants had ever tested for HCV, with three (2%, N = 142) having ever tested positive for HCV. In DBS testing, nine participants (2.4%) tested positive for HIV and 24 (6.5%) tested positive for ever having an HCV infection. CONCLUSION: Prevalence of HIV and HCV in this population was high. Given high rates of study enrolment and testing, efforts should be made to reach men who purchase sex with expanded STBBI programming.

Community Insights in Phylogenetic HIV Research: The CIPHR Project Protocol.

Inferring HIV transmission networks from HIV sequences is gaining popularity in the field of HIV molecular epidemiology. However, HIV sequences are often analyzed at distance from those affected by HIV epidemics, namely without the involvement of communities most affected by HIV. These remote analyses often mean that knowledge is generated in absence of lived experiences and socio-economic realities that could inform the ethical application of network-derived information in 'real world' programmes. Procedures to engage communities are noticeably absent from the HIV molecular epidemiology literature. Here we present our team's protocol for engaging community activists living in Nairobi, Kenya in a knowledge exchange process - The CIPHR Project (Community Insights in Phylogenetic HIV Research). Drawing upon a community-based participatory approach, our team will (1) explore the possibilities and limitations of HIV molecular epidemiology for key population programmes, (2) pilot a community-based HIV molecular study, and (3) co-develop policy guidelines on conducting ethically safe HIV molecular epidemiology. Critical dialogue with activist communities will offer insight into the potential uses and abuses of using such information to sharpen HIV prevention programmes. The outcome of this process holds importance to the development of policy frameworks that will guide the next generation of the global response.

Risk factors of HIV and variation in access to clean needles among people who inject drugs in Pakistan.

We identified key risk factors for HIV among people who inject drugs (PWID) in Pakistan and explored access to free clean needles. Multivariable logistic regression was used to investigate associations between HIV prevalence and demographic, behavioral, and socio-economic characteristics of PWID. Data came from the Government of Pakistan's Integrated Biological and Behavioral Surveillance (IBBS) Round 5 (2016-17; 14 cities). A secondary analysis investigated associations with reported access to clean needles. Unweighted HIV prevalence among 4,062 PWID (99% male) was 21.0%. Longer injecting duration (Odds ratio [OR] 1.06 [95% confidence interval: 1.02-1.10]; per year), higher injecting frequency (OR 1.67 [1.30-2.13]; per unit increase), and injecting heroin (OR 1.90 [1.11-3.25]) were positively associated with HIV prevalence. There was no association between using a used syringe at last injection and HIV. Having>10 years of education had lower odds of HIV than being illiterate (OR 0.58 [0.35-0.95]). Having a regular sexual partner (OR 0.74 [0.57-0.97]) or paying for sex with the opposite sex (OR = 0.62 [0.45-0.85]) had lower odds of HIV than not. Conversely, PWID paying a man/hijra for sex had higher odds of HIV (OR 1.20 [1.00-1.43]). Receipt of clean needles varied by city of residence (0-97% coverage), whilst PWID with knowledge of HIV service delivery programs had higher odds of receiving clean needles (OR 4.58 [3.50-5.99]). Injecting behaviors were associated with HIV prevalence among PWID, though risks related to paying for sex remain complicated. Geographical variation in access to clean needles suggests potential benefits of more widely spread public health services.

Key MessagesWhat is already known on this topicThe HIV epidemic in Pakistan is concentrated among key populations including people who inject drugs.What this study addsInjecting practices, sexual behaviors, and socio-economic factors are associated with HIV prevalence among people who inject drugs. Access to harm reduction services is varied in Pakistan.How this study might affect research, practice, or policyAccess to clean free needles, as well as service delivery programs, with a broad geographical reach remain important to curb the HIV epidemic among people who inject drugs in Pakistan.

HIV acquisition prior to entry into formal sex work: inference from next-generation viral sequencing.

OBJECTIVE: To infer the timing of HIV acquisition in relation to self-reported events in the sexual life course of adolescent girls and young women (AGYW) who self-identify as female sex workers (FSW) in Mombasa, Kenya. DESIGN: Next-generation viral sequencing of samples of AGYW living with HIV in the Transitions study, a cross-sectional bio-behavioural survey of AGYW aged 14-24 years in Mombasa, Kenya. METHOD: Dried blood spot specimens were collected from study participants ( n  = 37, all FSW). A portion of the HIV pol gene was sequenced using an in-house next-generation sequencing assay for HIV drug resistance mutation genotyping. Estimated time since infection (ETI) was inferred using the HIV EVO web-based tool ( https://hiv.biozentrum.unibas.ch/ETI/ ), and data on self-reported events were obtained from the survey. RESULTS: The median ETI among FSW was 3.4 (interquartile range = 1.7, 6.3) years, with a median ETI of 1.5 years prior to entry into formal sex work. We estimated that 74.1% (95% confidence interval = 53.7-88.9%) of participants living with HIV and who self-identified as FSW likely acquired HIV prior to self-identification as a sex worker. CONCLUSIONS: Findings suggest a large fraction of prevalent HIV infection among AGYW engaged in sex work stems from acquisition prior to entry into formal sex work. Current HIV prevention programs tailored for sex workers may miss key opportunities for HIV prevention as they are designed to reach women after entry into formal sex work, signaling a need for tailored programs to reach high-risk AGYW earlier on in their sexual life course.

Comparative performance data for multiplex SARS-CoV-2 serological assays from a large panel of dried blood spot specimens.

The extent of the COVID-19 pandemic will be better understood through serosurveys and SARS-CoV-2 antibody testing. Dried blood spot (DBS) samples will play a central role in large scale serosurveillance by simplifying biological specimen collection and transportation, especially in Canada. Direct comparative performance data on multiplex SARS-CoV-2 assays resulting from identical DBS samples are currently lacking. In our study, we aimed to provide performance data for the BioPlex 2200 SARS-CoV-2 IgG (Bio-Rad), V-PLEX SARS-CoV-2 Panel 2 IgG (MSD), and Elecsys Anti-SARS-CoV-2 (Roche) commercial assays, as well as for two highly scalable in-house assays (University of Ottawa and Mount Sinai Hospital protocols) to assess their suitability for DBS-based SARS-CoV-2 DBS serosurveillance. These assays were evaluated against identical panels of DBS samples collected from convalescent COVID-19 patients (n = 97) and individuals undergoing routine sexually transmitted and bloodborne infection (STBBI) testing prior to the COVID-19 pandemic (n = 90). Our findings suggest that several assays are suitable for serosurveillance (sensitivity >97% and specificity >98%). In contrast to other reports, we did not observe an improvement in performance using multiple antigen consensus-based rules to establish overall seropositivity. This may be due to our DBS panel which consisted of samples collected from convalescent COVID-19 patients with significant anti-spike, -receptor binding domain (RBD), and -nucleocapsid antibody titers. This study demonstrates that biological specimens collected as DBS coupled with one of several readily available assays are useful for large-scale COVID-19 serosurveillance.

Evaluation of the bioMérieux VIDAS HIV Duo Quick and Anti-HCV assays for dried blood spot based serosurveillance.

Serosurveillance is central to monitoring our progress towards HIV and HCV elimination targets proposed for 2030. However, serosurveillance systems are ineffective without reliable serological assays for the detection of HIV and HCV antibodies. Assays should also be compatible with dried blood spot (DBS) samples to facilitate biological sample collection. The VIDAS HIV Duo Quick and Anti-HCV assays are sold as reagents strips and processed by the automated VIDAS benchtop immunoanalyser. While both assays have shown excellent performance in serum and plasma, performance data in DBS samples is lacking. In our study, we evaluate the performance of the VIDAS HIV Duo Quick and Anti-HCV assays in DBS (n = 725) collected during a cross-sectional serosurvey (the Transitions study). The VIDAS HIV Duo quick had a sensitivity and specificity of 94.5% (95% CI 85.1%, 98.5%) and 95.7% (95% CI 93.9%, 97.0%) respectively. Likewise, the VIDAS Anti-HCV had a sensitivity and specificity of 95.6% (95% CI 91.6%, 97.8%) and 95.6% (95% CI 93.5%, 97.0%) respectively. These assays are unlikely to be helpful in low-prevalence settings due to sub-optimal performance, but their performance could likely be improved by optimizing DBS elution protocols which was, unfortunately, not possible during our study.

A scalable serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination.

Objectives: Antibody testing against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. Methods: We developed methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD) and nucleocapsid (N). We automated a surrogate neutralisation (sn)ELISA that measures inhibition of ACE2-spike or -RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. Results: Our single-point IgG-based ELISAs accurately distinguished non-infected and infected individuals. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for ≥ 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability. Conclusions: Measuring antibodies to three viral antigens and identify neutralising antibodies capable of disrupting spike-ACE2 interactions in high-throughput enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The reagents are available to enable scaling up of standardised serological assays, permitting inter-laboratory data comparison and aggregation.

Phylogeographic Assessment Reveals Geographic Sources of HIV-1 Dissemination Among Men Who Have Sex With Men in Kenya.

HIV-1 transmission dynamics involving men who have sex with men (MSM) in Africa are not well understood. We investigated the rates of HIV-1 transmission between MSM across three regions in Kenya: Coast, Nairobi, and Nyanza. We analyzed 372 HIV-1 partial pol sequences sampled during 2006-2019 from MSM in Coast (N = 178, 47.9%), Nairobi (N = 137, 36.8%), and Nyanza (N = 57, 15.3%) provinces in Kenya. Maximum-likelihood (ML) phylogenetics and Bayesian inference were used to determine HIV-1 clusters, evolutionary dynamics, and virus migration rates between geographic regions. HIV-1 sub-subtype A1 (72.0%) was most common followed by subtype D (11.0%), unique recombinant forms (8.9%), subtype C (5.9%), CRF 21A2D (0.8%), subtype G (0.8%), CRF 16A2D (0.3%), and subtype B (0.3%). Forty-six clusters (size range 2-20 sequences) were found-half (50.0%) of which had evidence of extensive HIV-1 mixing among different provinces. Data revealed an exponential increase in infections among MSM during the early-to-mid 2000s and stable or decreasing transmission dynamics in recent years (2017-2019). Phylogeographic inference showed significant (Bayes factor, BF > 3) HIV-1 dissemination from Coast to Nairobi and Nyanza provinces, and from Nairobi to Nyanza province. Strengthening HIV-1 prevention programs to MSM in geographic locations with higher HIV-1 prevalence among MSM (such as Coast and Nairobi) may reduce HIV-1 incidence among MSM in Kenya.

Quantifying rates of HIV-1 flow between risk groups and geographic locations in Kenya: A country-wide phylogenetic study.

In Kenya, HIV-1 key populations including men having sex with men (MSM), people who inject drugs (PWID) and female sex workers (FSW) are thought to significantly contribute to HIV-1 transmission in the wider, mostly heterosexual (HET) HIV-1 transmission network. However, clear data on HIV-1 transmission dynamics within and between these groups are limited. We aimed to empirically quantify rates of HIV-1 flow between key populations and the HET population, as well as between different geographic regions to determine HIV-1 'hotspots' and their contribution to HIV-1 transmission in Kenya. We used maximum-likelihood phylogenetic and Bayesian inference to analyse 4058 HIV-1 pol sequences (representing 0.3 per cent of the epidemic in Kenya) sampled 1986-2019 from individuals of different risk groups and regions in Kenya. We found 89 per cent within-risk group transmission and 11 per cent mixing between risk groups, cyclic HIV-1 exchange between adjoining geographic provinces and strong evidence of HIV-1 dissemination from (i) West-to-East (i.e. higher-to-lower HIV-1 prevalence regions), and (ii) heterosexual-to-key populations. Low HIV-1 prevalence regions and key populations are sinks rather than major sources of HIV-1 transmission in Kenya. Targeting key populations in Kenya needs to occur concurrently with strengthening interventions in the general epidemic.

Dried blood spot specimens for SARS-CoV-2 antibody testing: A multi-site, multi-assay comparison.

The true severity of infection due to COVID-19 is under-represented because it is based on only those who are tested. Although nucleic acid amplifications tests (NAAT) are the gold standard for COVID-19 diagnostic testing, serological assays provide better population-level SARS-CoV-2 prevalence estimates. Implementing large sero-surveys present several logistical challenges within Canada due its unique geography including rural and remote communities. Dried blood spot (DBS) sampling is a practical solution but comparative performance data on SARS-CoV-2 serological tests using DBS is currently lacking. Here we present test performance data from a well-characterized SARS-CoV-2 DBS panel sent to laboratories across Canada representing 10 commercial and 2 in-house developed tests for SARS-CoV-2 antibodies. Three commercial assays identified all positive and negative DBS correctly corresponding to a sensitivity, specificity, positive predictive value, and negative predictive value of 100% (95% CI = 72.2, 100). Two in-house assays also performed equally well. In contrast, several commercial assays could not achieve a sensitivity greater than 40% or a negative predictive value greater than 60%. Our findings represent the foundation for future validation studies on DBS specimens that will play a central role in strengthening Canada's public health policy in response to COVID-19.

Endocervical Regulatory T Cells Are Associated With Decreased Genital Inflammation and Lower HIV Target Cell Abundance.

Regulatory T cells (Tregs) play important roles in tissue homeostasis, but few studies have investigated tissue Tregs in the context of genital inflammation, HIV target cell density, and vaginal microbiota in humans. In women from Nairobi (n=64), the proportion of CD4+ CD25+ CD127low Tregs in the endocervix correlated with those in blood (r=0.31, p=0.01), with a higher Treg frequency observed in the endocervix (median 3.8 vs 2.0%, p<0.0001). Most Tregs expressed FOXP3 in both compartments, and CTLA-4 expression was higher on endocervical Tregs compared to blood (median 50.8 vs 6.0%, p<0.0001). More than half (34/62, 55%) of participants displayed a non-Lactobacillus dominant vaginal microbiota, which was not associated with endocervical Tregs or CD4+ T cell abundance. In a multivariable linear regression, endocervical Treg proportions were inversely associated with the number of elevated pro-inflammatory cytokines (p=0.03). Inverse Treg associations were also observed for specific cytokines including IL-1ß, G-CSF, Eotaxin, IL-1RA, IL-8, and MIP-1 ß. Higher endocervical Treg proportions were associated with lower abundance of endocervical CD4+ T cells (0.30 log10 CD4+ T cells per log10 Treg, p=0.00028), with a similar trend for Th17 cells (p=0.09). Selectively increasing endocervical Tregs may represent a pathway to reduce genital tract inflammation in women.

Elucidating Drivers for Variations in the Explosive Human Immunodeficiency Virus Epidemic Among People Who Inject Drugs in Pakistan.

BACKGROUND: Pakistan's explosive human immunodeficiency virus (HIV) epidemic among people who inject drugs (PWID) varies widely across cities. We evaluated possible drivers for these variations. METHODS: Multivariable regression analyses were undertaken using data from 5 national surveys among PWID (n = 18 467; 2005-2017) to determine risk factors associated with variations in city-level HIV prevalence. A dynamic HIV model was used to estimate the population-attributable fraction (PAF; proportion of HIV infections prevented over 10 years when that risk factor is removed) of these risk factors to HIV transmission and impact on HIV incidence of reducing their prevalence. RESULTS: Regression analyses suggested that city-level HIV prevalence is strongly associated with the prevalence of using professional injectors at last injection, heroin use in last month, and injecting ≥4 times per day. Through calibrating a model to these associations, we estimate that the 10-year PAFs of using professional injectors, heroin use, and frequent injecting are 45.3% (95% uncertainty interval [UI], 4.3%-79.7%), 45.9% (95% UI, 8.1%-78.4%), and 22.2% (95% UI, 2.0%-58.4%), respectively. Reducing to lowest city-level prevalences of using professional injectors (2.8%; median 89.9% reduction), heroin use (0.9%; median 91.2% reduction), and frequent injecting (0.1%; median 91.8% reduction) in 2020 reduces overall HIV incidence by 52.7% (95% UI, 6.1%-82.0%), 53.0% (95% UI, 11.3%-80.2%), and 28.1% (95% UI, 2.7%-66.6%), respectively, over 10 years. CONCLUSIONS: Interventions should focus on these risk factors to control Pakistan's explosive HIV epidemic among PWID, including a concomitant expansion of high-coverage needle/syringe provision, opioid substitution therapy, and antiretroviral therapy.

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific against the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS). When exposed to SARS-CoV-2 or a vaccine against SARS-CoV-2, the immune system responds by expressing antibodies at levels that can be detected and monitored to identify the fraction of the population potentially immunized against SARS-CoV-2 and support efforts to deploy a vaccine strategically. A SPR sensor coated with a peptide monolayer and functionalized with various sources of SARS-CoV-2 recombinant proteins expressed in different cell lines detected human anti-SARS-CoV-2 IgG antibodies in clinical samples. Nucleocapsid expressed in different cell lines did not significantly change the sensitivity of the assays, whereas the use of a CHO cell line to express spike ectodomain led to excellent performance. This bioassay was performed on a portable SPR instrument capable of measuring 4 biological samples within 30 minutes of sample/sensor contact and the chip could be regenerated at least 9 times. Multi-site validation was then performed with in-house and commercial ELISA, which revealed excellent cross-correlations with Pearson's coefficients exceeding 0.85 in all cases, for measurements in DBS and plasma. This strategy paves the way to point-of-care and rapid testing for antibodies in the context of viral infection and vaccine efficacy monitoring.

Findings among Indigenous participants of the Tracks survey of people who inject drugs in Canada, Phase 4, 2017-2019.

BACKGROUND: The Tracks survey of people who inject drugs (PWID) collected data in 14 sentinel sites across Canada (2017-2019). These findings describe the prevalence of human immunodeficiency virus (HIV), hepatitis C and associated risk behaviours among Indigenous participants. METHODS: Information regarding socio-demographics, social determinants of health, use of prevention services and testing, drug use, risk behaviours, and HIV and hepatitis C testing, care and treatment was collected through interviewer-administered questionnaires. Biological samples were tested for HIV, hepatitis C antibodies and hepatitis C ribonucleic acid (RNA). Descriptive statistics were calculated and reviewed by an Indigenous-led advisory group using the Two-Eyed Seeing approach. RESULTS: Of the 2,383 participants, 997 were Indigenous (82.9% First Nations, 14.9% Métis, 2.2% Inuit). Over half (54.5%) were cisgender male and the average age was 38.9 years. A large proportion (84.0%) reported their mental health as "fair to excellent". High proportions experienced stigma and discrimination (90.2%) and physical, sexual and/or emotional abuse in childhood (87.5%) or with a sexual partner (78.6%). Use of a needle/syringe distribution program (90.5%) and testing for HIV (87.9%) and hepatitis C (87.8%) were high. Prevalence of HIV was 15.4% (78.2% were aware of infection status) and 36.4% were hepatitis C RNA-positive (49.4% were aware of infection status). CONCLUSION: High rates of HIV and hepatitis C were identified. Challenges in access to and maintenance of HIV and hepatitis C care and treatment were noted. This information informs harm reduction strategies, including the need to scale-up awareness of prophylaxis in a culturally relevant manner.

Rectal microbiota diversity in Kenyan MSM is inversely associated with frequency of receptive anal sex, independent of HIV status.

OBJECTIVE: Both HIV infection and identifying as MSM have been linked to altered rectal microbiota composition, but few studies have studied sexual behavioural associations with rectal microbiota within MSM. In addition, most rectal microbiota studies in MSM have been limited geographically to Europe and North America, and replication of findings in lower and middle-income countries is lacking. DESIGN: A cross-sectional study. METHODS: We enrolled MSM from Nairobi, Kenya, and determined their HIV/sexually transmitted infection status. Rectal specimens were obtained for 16s rRNA sequencing of the rectal microbiota, and sexual behaviour was characterized using a standardized questionnaire. Microbiome differences were modelled using nonparametric statistics, Bray-Curtis ecological distance metrics and analyses of differential taxa abundance. Multivariable linear regression was used to model HIV status and recent sexual activity as predictors of alpha diversity, controlling for a range of covariates. RESULTS: Alpha diversity was consistently lower in Kenyan HIV-infected MSM (n = 80), including those on antiretroviral therapy (ART) compared with HIV-uninfected MSM. A statistical trend was observed for clustering of HIV status by Prevotella or Bacteroides dominance (P = 0.13). Several taxa were enriched in HIV-positive men, including Roseburia, Lachnospira, Streptococcus and Granulicatella. Receptive anal sex with several types of sexual partners (paying, regular, casual) was associated with lower Chao1 and Simpson diversity, independent of HIV status, while HIV infection was associated lower Chao1 (P = 0.030) but not Simpson diversity (P = 0.49). CONCLUSION: Both HIV infection and sexual behaviour were associated with rectal microflora alpha diversity, in particular richness, but not Prevotella spp. dominance, in Kenyan MSM. Associations were more robust for sexual behaviour.

HIV-1 phylodynamic analysis among people who inject drugs in Pakistan correlates with trends in illicit opioid trade.

Pakistan is considered by the World Health Organization to currently have a "concentrated" HIV-1 epidemic due to a rapid rise in infections among people who inject drugs (PWID). Prevalence among the country's nearly 105,000 PWID is estimated to be 37.8% but has been shown to be higher in several large urban centers. A lack of public health resources, the common use of professional injectors and unsafe injection practices are believed to have fueled the outbreak. Here we evaluate the molecular characteristics of HIV-1 sequences (n = 290) from PWID in several Pakistani cities to examine transmission dynamics and the association between rates of HIV-1 transmission with regards to regional trends in opioid trafficking. Tip-to-tip (patristic) distance based phylogenetic cluster inferences and BEAST2 Bayesian phylodynamic analyses of time-stamped data were performed on HIV-1 pol sequences generated from dried blood spots collected from 1,453 PWID as part of a cross-sectional survey conducted in Pakistan during 2014/2015. Overall, subtype A1 strains were dominant (75.2%) followed by CRF02_AG (14.1%), recombinants/unassigned (7.2%), CRF35_AD (2.1%), G (1.0%) and C (0.3%). Nearly three quarters of the PWID HIV-1 sequences belonged to one of five distinct phylogenetic clusters. Just below half (44.4%) of individuals in the largest cluster (n = 118) did seek help injecting from professional injectors which was previously identified as a strong correlate of HIV-1 infection. Spikes in estimated HIV-1 effective population sizes coincided with increases in opium poppy cultivation in Afghanistan, Pakistan's western neighbor. Structured coalescent analysis was undertaken in order to investigate the spatial relationship of HIV-1 transmission among the various cities under study. In general terms, our analysis placed the city of Larkana at the center of the PWID HIV-1 epidemic in Pakistan which is consistent with previous epidemiological data.