Mental health-related quality of life in mothers of children with surgically repaired congenital heart disease: a 13-year longitudinal study.

AIMS: Having a child with congenital heart disease (CHD) can affect parental health-related quality of life (HR-QoL). We investigated the long-term trajectories of mental HRQoL (m-HRQoL) in mothers of children with CHD and examined risk factors for persistent low m-HRQoL. METHODS: One hundred twenty-five mothers of children with CHD completed a standardized questionnaire on m-HRQoL (mental subscale SF-12) after the children's first open-heart surgery and subsequently when the children were 1, 4, 6, 10, and 13 years old. A z-score for m-HRQoL was calculated with national norms. Latent class growth analysis (LCGA) was used to identify subgroups of mothers with regards to their m-HRQoL trajectories over time. Regression analysis investigated predictors for chronically low m-HRQoL. RESULTS: Compared to norms, mothers of children with CHD had significantly lower m-HRQoL immediately after open-heart surgery (ß = -0.30 (CI-95: -0.44, -0.15)). Subsequently, m-HRQoL increased to a normal level (m-HRQoL compared to the norm from 1 to 13 years: ß ranges between 0.05 and 0.27). LCGA revealed two distinct groups of m-HRQoL trajectories: A group with normal m-HRQoL (75% of mothers, means z-scores range between - 0.76 and 0.62) and a group with chronically low m-HRQoL (25% of mothers, mean z-scores range between -1.32 and -0.10). Chronically, low m-HRQoL was associated with poorer social support (OR = 3.39 (CI-95: 1.40, 8.49), p = 0.008) but not with parental education, migration background, number of open-heart surgeries, diagnosis of a univentricular CHD, or low IQ. CONCLUSION: A quarter of mothers of children with CHD have chronically low m-HRQoL throughout their child's development, especially those mothers with poor social support. Further studies of family-oriented approaches are needed to identify and support these mothers and reinforce parental well-being.

Persistent Nonviral Plasmid Vector in Nasal Tissues Causes False-Positive SARS-CoV-2 Diagnostic Nucleic Acid Tests.

Biomedical personnel can become contaminated with nonhazardous reagents used in the laboratory. We describe molecular studies performed on nasal secretions collected longitudinally from asymptomatic laboratory coworkers to determine if they were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulating in the community or with SARS-CoV-2 DNA from a plasmid vector. Participants enrolled in a prospective study of incident SARS-CoV-2 infection had nasal swabs collected aseptically by study staff at enrollment, followed by weekly self-collection of anterior nasal swabs. SARS-CoV-2 diagnosis was performed by a real-time PCR test targeting the nucleocapsid gene. PCR tests targeting SARS-CoV-2 nonstructural protein 10 (nsp10), nsp14, and envelope and three regions of the plasmid vector were performed to differentiate amplification of SARS-CoV-2 RNA from the plasmid vector's DNA. Nasal swabs from four asymptomatic coworkers with positive real-time PCR results for the SARS-CoV-2 nucleocapsid targets were negative when tested for SARS-CoV-2 nsp10, nsp14, and envelope protein. However, nucleic acids extracted from these nasal swabs amplified DNA regions of the plasmid vector used by the coworkers, including the ampicillin and neomycin/kanamycin resistance genes, the promoter-nucleocapsid junction, and unique codon-optimized regions. Nasal swabs from these individuals tested positive repeatedly, including during isolation. Longitudinal detection of plasmid DNA with SARS-CoV-2 nucleocapsid in nasal swabs suggests persistence in nasal tissues or colonizing bacteria. Nonviral plasmid vectors, while regarded as safe laboratory reagents, can interfere with molecular diagnostic tests. These reagents should be handled using proper personal protective equipment to prevent contamination of samples or laboratory personnel. IMPORTANCE Asymptomatic laboratory workers who tested positive for SARS-CoV-2 for days to months were found to harbor a laboratory plasmid vector containing SARS-CoV-2 DNA, which they had worked with in the past, in their nasal secretions. While prior studies have documented contamination of research personnel with PCR amplicons, our observation is novel, as these individuals shed the laboratory plasmid over days to months, including during isolation in their homes. This suggests that the plasmid was in their nasal tissues or that bacteria containing the plasmid had colonized their noses. While plasmids are generally safe, our detection of plasmid DNA in the nasal secretions of laboratory workers for weeks after they had stopped working with the plasmid shows the potential for these reagents to interfere with clinical tests and emphasizes that occupational exposures in the preceding months should be considered when interpreting diagnostic clinical tests.

Low-frequency pre-treatment HIV drug resistance: effects on 2-year outcome of first-line efavirenz-based antiretroviral therapy.

OBJECTIVES: Assess the impact of pre-treatment high-frequency and low-frequency drug-resistant HIV variants on long-term outcomes of first-line efavirenz-based antiretroviral therapy (ART). DESIGN: Prospective observational study. METHODS: Participants' pre-treatment plasma RNA had two sections of HIV pol encoding reverse transcriptase sequenced (Illumina, MiSeq) using unique molecular identifiers to detect wild-type (pre-treatment drug-resistant variants less than 1% of viral quasispecies), low-frequency (1-9%) or high-frequency drug-resistant variants (10-100%). Associations between pre-treatment drug resistance and virologic outcomes over 24 months of efavirenz-based ART were assessed for the number and frequency of mutations by drug class and other resistance parameters. RESULTS: Virologic failure was detected in 30 of 352 (9%) and pre-treatment drug-resistant variants were detected in the viral quasispecies of 31 of 352 (9%) participants prescribed efavirenz-based ART. Survival analyses revealed statistically significant associations between pre-treatment drug resistance at low (P < 0.0001) and high (P < 0.001) frequencies, at oligonucleotide ligation assay (OLA) (P < 0.00001) and non-OLA (P < 0.01) codons, to a single-antiretroviral class (P < 0.00001), and a shorter time to virologic failure of efavirenz-based ART. Regression analyses detected independent effects across resistance categories, including both low-frequency (P < 0.01) and high-frequency (P < 0.001) drug-resistant variants. CONCLUSION: We observed that pre-treatment HIV drug resistance detected at low frequencies increased the risk of virologic failure over 24 months of efavirenz-based ART, but that most failures, regardless of drug-resistant variants' frequencies, were detected within a year of ART initiation. These observations suggest that when efavirenz-based ART is prescribed, screening for pre-treatment drug resistance by an assay capable of detecting low-frequency variants, including OLA, may guide clinicians to prescribe more effective ART.

Assessment of minority frequency pretreatment HIV drug-resistant variants in pregnant women and associations with virologic non-suppression at term.

OBJECTIVE: To assess in ART-naïve pregnant women randomized to efavirenz- versus raltegravir-based ART (IMPAACT P1081) whether pretreatment drug resistance (PDR) with minority frequency variants (<20% of individual's viral quasispecies) affects antiretroviral treatment (ART)-suppression at term. DESIGN: A case-control study design compared PDR minority variants in cases with virologic non-suppression (plasma HIV RNA >200 copies/mL) at delivery to randomly selected ART-suppressed controls. METHODS: HIV pol genotypes were derived from pretreatment plasma specimens by Illumina sequencing. Resistance mutations were assessed using the HIV Stanford Database, and the proportion of cases versus controls with PDR to their ART regimens was compared. RESULTS: PDR was observed in 7 participants (11.3%; 95% CI 4.7, 21.9) and did not differ between 21 cases and 41 controls (4.8% vs 14.6%, p = 0.4061). PDR detected only as minority variants was less common (3.2%; 95% CI 0.2, 11.7) and also did not differ between groups (0% vs. 4.9%; p = 0.5447). Cases' median plasma HIV RNA at delivery was 347c/mL, with most (n = 19/22) showing progressive diminution of viral load but not ≤200c/mL. Among cases with viral rebound (n = 3/22), none had PDR detected. Virologic non-suppression at term was associated with higher plasma HIV RNA at study entry (p<0.0001), a shorter duration of ART prior to delivery (p<0.0001), and randomization to efavirenz- (versus raltegravir-) based ART (p = 0.0085). CONCLUSIONS: We observed a moderate frequency of PDR that did not significantly contribute to virologic non-suppression at term. Rather, higher pretreatment plasma HIV RNA, randomization to efavirenz-based ART, and shorter duration of ART were associated with non-suppression. These findings support early prenatal care engagement of pregnant women and initiation of integrase inhibitor-based ART due to its association with more rapid suppression of plasma RNA levels. Furthermore, because minority variants appeared infrequent in ART-naïve pregnant women and inconsequential to ART-suppression, testing for minority variants may be unwarranted.

Low rate of SARS-CoV-2 incident infection identified by weekly screening PCR in a prospective year-long cohort study.

BACKGROUND: Asymptomatic and pre-symptomatic SARS-CoV-2 infections may contribute to ongoing community transmission, however, the benefit of routine screening of asymptomatic individuals in low-risk populations is unclear. METHODS: To identify SARS-CoV-2 infections 553 seronegative individuals were prospectively followed for 52 weeks. From 4/2020-7/2021, participants submitted weekly self-collected nasal swabs for rtPCR and completed symptom and exposure surveys. RESULTS: Incident SARS2-CoV-2 infections were identified in 9/553 (1.6%) participants. Comparisons of SARS2-CoV-2(+) to SARS2-CoV-2(-) participants revealed significantly more close contacts outside the household (median: 5 versus 3; p = 0.005). The incidence of infection was higher among unvaccinated/partially vaccinated than among fully vaccinated participants (9/7,679 versus 0/6,845 person-weeks; p = 0.004). At notification of positive test result, eight cases were symptomatic and one pre-symptomatic. CONCLUSIONS: These data suggest that weekly SARS2-CoV2 surveillance by rtPCR did not efficiently detect pre-symptomatic infections in unvaccinated participants.

Simultaneous monitoring of HIV viral load and screening of SARS-CoV-2 employing a low-cost RT-qPCR test workflow.

The COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of virologic failure and HIV-associated illness. Often this population is at high risk for exposure to SARS-CoV-2 infection, and once infected, for severe disease. Therefore, close monitoring of HIV plasma viral load (VL) and screening for SARS-CoV-2 infection are needed. We developed a non-proprietary method to isolate RNA from plasma, nasal secretions (NS), or both. The extracted RNA is then submitted to RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status (i.e., HIV virologic failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). In contrived samples, the in-house RNA extraction workflow achieved a detection limit of 200-copies per mL for HIV RNA in plasma and 100-copies per mL for SARS-CoV-2 RNA in NS. Similar detection limits were observed for HIV and SARS-CoV-2 in pooled plasma/NS contrived samples. When comparing in-house with standard extraction methods, we found high agreement (>0.91) between input and measured RNA copies for HIV LTR in contrived plasma; SARS-CoV-2 N1/N2 in contrived NS; and LTR, N1, and N2 in pooled plasma/NS samples. We further evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-positive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 combined plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house RNA extraction to those using a commercial kit (standard extraction method). The in-house extraction and standard extraction of clinical specimens were positively correlated: plasma HIV VL (R2 of 0.81) and NS SARS-CoV-2 VL (R2 of 0.95 and 0.99 for N1 and N2 genes, respectively); and pooled plasma/NS HIV VL (R2 of 0.71) and SARS-CoV-2 VL (R2 of 1 both for N1 and N2 genes). Our low-cost molecular test workflow ($1.85 per pooled sample extraction) for HIV RNA and SARS-CoV-2 RNA could serve as an alternative to current standard assays ($12 per pooled sample extraction) for laboratories in low-resource settings.

Implementation of an interactive mobile application to pilot a rapid assay to detect HIV drug resistance mutations in Kenya.

Usability is an overlooked aspect of implementing lab-based assays, particularly novel assays in low-resource-settings. Esoteric instructions can lead to irreproducible test results and patient harm. To address these issues, we developed a software application based on "Aquarium", a laboratory-operating system run on a computer tablet that provides step-by-step digital interactive instructions, protocol management, and sample tracking. Aquarium was paired with a near point-of-care HIV drug resistance test, "OLA-Simple", that detects mutations associated with virologic failure. In this observational study we evaluated the performance of Aquarium in guiding untrained users through the multi-step laboratory protocol with little supervision. To evaluate the training by Aquarium software we conducted a feasibility study in a laboratory at Coptic Hope Center in Nairobi, Kenya. Twelve volunteers who were unfamiliar with the kit performed the test on blinded samples (2 blood specimens; 5 codons/sample). Steps guided by Aquarium included: CD4+ T-Cell separation, PCR, ligation, detection, and interpretation of test results. Participants filled out a short survey regarding their demographics and experience with the software and kit. None of the laboratory technicians had prior experience performing CD4+ separation and 7/12 had no experience performing laboratory-based molecular assays. 12/12 isolated CD4+ T cells from whole blood with yields comparable to isolations performed by trained personnel. The OLA-Simple workflow was completed by all, with genotyping results interpreted correctly by unaided-eye in 108/120 (90%) and by software in 116/120 (97%) of codons analyzed. In the surveys, participants favorably assessed the use of software guidance. The Aquarium digital instructions enabled first-time users in Kenya to complete the OLA-simple kit workflow with minimal training. Aquarium could increase the accessibility of laboratory assays in low-resource-settings and potentially standardize implementation of clinical laboratory tests.

Maternal Human Immunodeficiency Virus (HIV) Drug Resistance Is Associated With Vertical Transmission and Is Prevalent in Infected Infants.

BACKGROUND: We aimed to assess if maternal human immunodeficiency virus (HIV) drug resistance is associated with an increased risk of HIV vertical transmission and to describe the dynamics of drug resistance in HIV-infected infants. METHODS: This was a case-control study of PROMISE study participants. "Cases" were mother-infant pairs with HIV vertical transmission during pregnancy or breastfeeding and "controls" were mother-infant pairs without transmission matched 1:3 by delivery date and clinical site. Genotypic HIV drug resistance analyses were performed on mothers' and their infants' plasma at or near the time of infant HIV diagnosis. Longitudinal analysis of genotypic resistance was assessed in available specimens from infants, from diagnosis and beyond, including antiretroviral therapy (ART) initiation and last study visits. RESULTS: Our analyses included 85 cases and 255 matched controls. Maternal HIV drug resistance, adjusted for plasma HIV RNA load at infant HIV diagnosis, enrollment CD4 count, and antepartum regimens, was not associated with in utero/peripartum HIV transmission. In contrast, both maternal plasma HIV RNA load and HIV drug resistance were independent risk factors associated with vertical transmission during breastfeeding. Furthermore, HIV drug resistance was selected across infected infants during infancy. CONCLUSIONS: Maternal HIV drug resistance and maternal viral load were independent risk factors for vertical transmission during breastfeeding, suggesting that nevirapine alone may be insufficient infant prophylaxis against drug-resistant variants in maternal breast milk. These findings support efforts to achieve suppression of HIV replication during pregnancy and suggest that breastfeeding infants may benefit from prophylaxis with a greater barrier to drug resistance than nevirapine alone.

Motor and visuomotor function in 10-year-old children with congenital heart disease: association with behaviour.

BACKGROUND: Children with CHD are at increased risk for neurodevelopmental impairments. There is little information on long-term motor function and its association with behaviour. AIMS: To assess motor function and behaviour in a cohort of 10-year-old children with CHD after cardiopulmonary bypass surgery. METHODS: Motor performance and movement quality were examined in 129 children with CHD using the Zurich Neuromotor Assessment providing four timed and one qualitative component, and a total timed motor score was created based on the four timed components. The Beery Test of Visual-Motor Integration and the Strengths and Difficulties Questionnaire were administered. RESULTS: All Zurich Neuromotor Assessment motor tasks were below normative values (all p ≤ 0.001), and the prevalence of poor motor performance (≤10th percentile) ranged from 22.2% to 61.3% in the different components. Visuomotor integration and motor coordination were poorer compared to norms (all p ≤ 0.001). 14% of all analysed children had motor therapy at the age of 10 years. Children with a total motor score ≤10th percentile showed more internalising (p = 0.002) and externalising (p = 0.028) behavioural problems. CONCLUSIONS: School-aged children with CHD show impairments in a variety of motor domains which are related to behavioural problems. Our findings emphasise that motor problems can persist into school-age and require detailed assessment and support.

Diagnostic Accuracy of Dried Plasma Spot Specimens for HIV-1 Viral Load Testing: A Systematic Review and Meta-analysis.

BACKGROUND: Dried plasma spot specimens may be a viable alternative to traditional liquid plasma in field settings, but the diagnostic accuracy is not well understood. METHODS: Standard databases (PubMed and Medline), conferences, and gray literature were searched until January 2019. The quality of evidence was evaluated using the Standards for Reporting Studies of Diagnostic Accuracy and Quality Assessment of Diagnostic Accuracy Studies-2 criteria. We used univariate and bivariate random effects models to determine misclassification, sensitivity, and specificity across multiple thresholds, overall and for each viral load technology, and to account for between-study variation. RESULTS: We identified 23 studies for inclusion in the systematic review that compared the diagnostic accuracy of dried plasma spots with that of plasma. Primary data from 16 of the 23 studies were shared and included in the meta-analysis, representing 18 countries, totaling 1847 paired dried plasma spot:plasma data points. The mean bias of dried plasma spot specimens compared with that of plasma was 0.28 log10 copies/mL, whereas the difference in median viral load was 2.25 log10 copies/mL. More dried plasma spot values were undetectable compared with plasma values (43.6% vs. 29.8%). Analyzing all technologies together, the sensitivity and specificity of dried plasma spot specimens were >92% across all treatment failure thresholds compared and total misclassification <5.4% across all treatment failure thresholds compared. Some technologies had lower sensitivity or specificity; however, the results were typically consistent across treatment failure thresholds. DISCUSSION: Overall, dried plasma spot specimens performed relatively well compared with plasma with sensitivity and specificity values greater than 90% and misclassification rates less than 10% across all treatment failure thresholds reviewed.

Angiotensin II receptor I auto-antibodies following SARS-CoV-2 infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with endothelial activation and coagulopathy, which may be related to pre-existing or infection-induced pro-thrombotic autoantibodies such as those targeting angiotensin II type I receptor (AT1R-Ab). METHODS: We compared prevalence and levels of AT1R-Ab in COVID-19 cases with mild or severe disease to age and sex matched negative controls utilizing multivariate logistic and quantile regression adjusted for comorbidities including hypertension, diabetes, and heart disease. RESULTS: There were trends toward increased prevalence (50% vs. 33%, p = 0.1) and level of AT1R-Ab (median 9.8 vs. 6.1 U/mL, p = 0.06) in all cases versus controls. When considered by COVID-19 disease severity, there was a trend toward increased prevalence of AT1R-Ab (55% vs. 31%, p = 0.07), as well as significantly higher AT1R-Ab levels (median 10.7 vs. 5.9 U/mL, p = 0.03) amongst individuals with mild COVID-19 versus matched controls. In contrast, the prevalence (42% vs. 37%, p = 0.9) and level (both medians 6.7 U/mL, p = 0.9) of AT1R-Ab amongst those with severe COVID-19 did not differ from matched controls. CONCLUSIONS: These findings support an association between COVID-19 and AT1R-Ab, emphasizing that vascular pathology may be present in individuals with mild COVID-19 as well as those with severe disease.

Rapid Near Point-of-Care Assay for HLA-B*57:01 Genotype Associated with Severe Hypersensitivity Reaction to Abacavir.

The nucleoside reverse transcriptase inhibitor abacavir (ABC) is used commonly to treat young children with HIV infection and is a component of the fixed-dose-combination Triumeq®. ABC can trigger a severe hypersensitivity reaction in people who are homozygous or heterozygous for HLA-B*57:01. Testing for HLA-B*57:01 before ABC initiation is standard-of-care in high-resource settings, but current tests are costly or difficult to access in resource-limited settings. To address these gaps, we developed an inexpensive simple-to-use rapid assay to detect HLA-B*57:01. We designed and optimized a multiplexed polymerase chain reaction (PCR) to amplify HLA-B*57 subtypes and the human beta-globin gene; employed probes and ligation to specifically tag the HLA-B*57:01 allele with biotin. Tagged-ligated products were detected by immunocapture in an enzyme-linked immunosorbent assay plate or lateral flow strip. Cell lines with known HLA genotypes were used to optimize the assay. The optimized assay was then compared with genotypes of clinical specimens (n = 60) determined by sequencing, with specimens enriched for individuals with HLA-B*57:01. The optimized assay utilizes 40-min 35-cycle multiplex PCR for B*57 and beta-globin; 20-min ligation reaction; and 15-min detection. Evaluation of the HLA-B*57:01 oligonucleotide ligation assay using clinical specimens had a sensitivity of 100% (n = 27/27 typed as B*57:01) and specificity of 100% (n = 33/33 typed as non-B*57:01) by visual interpretation of lateral flow strips. The cost is US$5.96/specimen. This rapid and economical assay accurately detects HLA-B*57:01 in clinical specimens. Use of this assay could expand access to HLA-B*57:01 genotyping and facilitate safe same-day initiation of ABC-based treatment.

Inexpensive workflow for simultaneous monitoring of HIV viral load and detection of SARS-CoV-2 infection.

BACKGROUND: COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of becoming virologically unsuppressed and ill. Often they are at high risk for exposure to SARS-CoV-2 infection and severe disease once infected. For this population, it is urgent to closely monitor HIV plasma viral load ( VL ) and screen for SARS-COV-2 infection. METHOD: We have developed a non-proprietary method to isolate RNA from plasma, nasal secretions ( NS ), or both. HIV, SARS-CoV-2, and human RP targets in extracted RNA are then RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status ( i . e ., HIV as VL failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). We evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-seropositive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 pooled plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house extraction to those using a commercial extraction kit. RESULTS: In-house extraction had a detection limit of 200-copies/mL for HIV and 100-copies/mL for SARS-CoV-2. In-house and commercial methods yielded positively correlated HIV VL (R 2 : 0.98 for contrived samples; 0.81 for seropositive plasma). SARS-CoV-2 detection had 100% concordant classifications in contrived samples, and in clinical NS extracted by in-house method, excluding indeterminate results, was 95% concordant (25 positives, 6 presumptive positives, and 31 negatives) to those using the commercial method. Analysis of pooled plasma/NS showed R 2 of 0.91 (contrived samples) and 0.71 (clinical specimens) for HIV VL correlations obtained by both extraction methods, while SARS-CoV-2 detection showed 100% concordance in contrived and clinical specimens. INTERPRETATION: Our low-cost workflow for molecular testing of HIV and SARS-CoV-2 could serve as an alternative to current standard assays for laboratories in low-resource settings.

Food insecurity, drug resistance and non-disclosure are associated with virologic non-suppression among HIV pregnant women on antiretroviral treatment.

We determined social and behavioral factors associated with virologic non-suppression among pregnant women receiving Option B+ antiretroviral treatment (ART). Baseline data was used from women in Mobile WAChX trial from 6 public maternal child health (MCH) clinics in Kenya. Virologic non-suppression was defined as HIV viral load (VL) ≥1000 copies/ml. Antiretroviral resistance testing was performed using oligonucleotide ligation (OLA) assay. ART adherence information, motivation and behavioral skills were assessed using Lifewindows IMB tool, depression using PHQ-9, and food insecurity with the Household Food Insecurity Access Scale. Correlates of virologic non-suppression were assessed using Poisson regression. Among 470 pregnant women on ART ≥4 months, 57 (12.1%) had virologic non-suppression, of whom 65% had HIV drug resistance mutations. In univariate analyses, risk of virologic non-suppression was associated with moderate-to-severe food insecurity (RR 1.80 [95% CI 1.06-3.05]), and varied significantly by clinic site (range 2%-22%, p <0.001). In contrast, disclosure (RR 0.36 [95% CI 0.17-0.78]) and having higher adherence skills (RR 0.70 [95% CI 0.58-0.85]) were associated with lower risk of virologic non-suppression. In multivariate analysis adjusting for clinic site, disclosure, depression symptoms, adherence behavior skills and food insecurity, disclosure and food insecurity remained associated with virologic non-suppression. Age, side-effects, social support, physical or emotional abuse, and distance were not associated with virologic non-suppression. Prevalence of virologic non-suppression among pregnant women on ART was appreciable and associated with food insecurity, disclosure and frequent drug resistance. HIV VL and resistance monitoring, and tailored counseling addressing food security and disclosure, may improve virologic suppression in pregnancy.

Association of Maternal Viral Load and CD4 Count With Perinatal HIV-1 Transmission Risk During Breastfeeding in the PROMISE Postpartum Component.

BACKGROUND: Breastfeeding mothers with HIV infection not qualifying for antiretroviral therapy (ART) based on country-specific guidelines at the time of the Promoting Maternal-Infant Survival Everywhere trial and their uninfected neonates were randomized to maternal ART (mART) or infant nevirapine prophylaxis (iNVP) postpartum. HIV transmission proportions were similar (<1%) in the 2 arms. We assessed whether maternal viral load (MVL) and CD4 cell counts were associated with breastfeeding HIV transmission. METHODS: MVL was collected at entry (7-14 days postpartum) and at weeks 6, 14, 26, and 50 postpartum. CD4 cell counts were collected at entry and weeks 14, 26, 38, and 50 postpartum. Infant HIV-1 nucleic acid test was performed at weeks 1 and 6, every 4 weeks until week 26, and then every 12 weeks. The associations of baseline and time-varying MVL and CD4 cell counts with transmission risk were assessed using time-to-event analyses by randomized treatment arm. RESULTS: Two thousand four hundred thirty-one mother-infant pairs were enrolled in the study. Baseline MVL (P = 0.11) and CD4 cell counts (P = 0.51) were not significantly associated with infant HIV-1 infection. Time-varying MVL was significantly associated with infant HIV-1 infection {hazard ratio [95% confidence interval (CI)]: 13.96 (3.12 to 62.45)} in the mART arm but not in the iNVP arm [hazard ratio (95% CI): 1.04 (0.20 to 5.39)]. Time-varying CD4 cell counts were also significantly associated with infant HIV-1 infection [hazard ratio (95% CI): 0.18 (0.03 to 0.93)] in the mART arm but not in the iNVP arm [hazard ratio (95% CI): 0.38 (0.08 to 1.77)]. CONCLUSIONS: In women receiving mART, increased MVL and decreased CD4 cell counts during breastfeeding were associated with increased risk of infant HIV-1 infection.

Immunization by exposure to live virus (SIVmne/HIV-2287) during antiretroviral drug prophylaxis may reduce risk of subsequent viral challenge.

RATIONALE/STUDY DESIGN: A major challenge in the development of HIV vaccines is finding immunogens that elicit protection against a broad range of viral strains. Immunity to a narrow range of viral strains may protect infants of HIV-infected women or partners discordant for HIV. We hypothesized that immunization to the relevant viral variants could be achieved by exposure to infectious virus during prophylaxis with antiretroviral drugs. To explore this approach in an animal model, macaques were exposed to live virus (SIVmne or HIV-2287) during prophylaxis with parenteral tenofovir and humoral and cellular immune responses were quantified. Subsequently, experimental animals were challenged with homologous virus to evaluate protection from infection, and if infection occurred, the course of disease was compared to control animals. Experimental animals uninfected with SIVmne were challenged with heterologous HIV-2287 to assess resistance to retroviral infection. METHODOLOGY/PRINCIPAL FINDINGS: Juvenile female Macaca nemestrina (N = 8) were given ten weekly intravaginal exposures with either moderately (SIVmne) or highly (HIV-2287) pathogenic virus during tenofovir prophylaxis. Tenofovir protected all 8 experimental animals from infection, while all untreated control animals became infected. Specific non-neutralizing antibodies were elicited in blood and vaginal secretions of experimental animals, but no ELISPOT responses were detected. Six weeks following the cessation of tenofovir, intravaginal challenge with homologous virus infected 2/4 (50%) of the SIVmne-immunized animals and 4/4 (100%) of the HIV-2287-immunized animals. The two SIVmne-infected and 3 (75%) HIV-2287-infected had attenuated disease, suggesting partial protection. CONCLUSIONS/SIGNIFICANCE: Repeated exposure to SIVmne or HIV-2287, during antiretroviral prophylaxis that blocked infection, induced binding antibodies in the blood and mucosa, but not neutralizing antibodies or specific cellular immune responses. Studies to determine whether antibodies are similarly induced in breastfeeding infants and sexual partners discordant for HIV infection and receiving pre-exposure antiretroviral prophylaxis are warranted, including whether these antibodies appear to confer partial or complete protection from infection.

Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays.

BACKGROUND: Detection of SARS-CoV-2 infections is important for treatment, isolation of infected and exposed individuals, and contact tracing. RT-qPCR is the "gold-standard" method to sensitively detect SARS-CoV-2 RNA, but most laboratory-developed RT-qPCR assays involve complex steps. Here, we aimed to simplify RT-qPCR assays by streamlining reaction setup, eliminating RNA extraction, and proposing reduced-cost detection workflows that avoid the need for expensive qPCR instruments. METHOD: A low-cost RT-PCR based "kit" was developed for faster turnaround than the CDC developed protocol. We demonstrated three detection workflows: two that can be deployed in laboratories conducting assays of variable complexity, and one that could be simple enough for point-of-care. Analytical sensitivity was assessed using SARS-CoV-2 RNA spiked in simulated nasal matrix. Clinical performance was evaluated using contrived human nasal matrix (n = 41) and clinical nasal specimens collected from individuals with respiratory symptoms (n = 110). FINDING: The analytical sensitivity of the lyophilised RT-PCR was 10 copies/reaction using purified SARS-CoV-2 RNA, and 20 copies/reaction when using direct lysate in simulated nasal matrix. Evaluation of assay performance on contrived human matrix showed 96.7-100% specificity and 100% sensitivity at ≥20 RNA copies. A head-to-head comparison with the standard CDC protocol on clinical specimens showed 83.8-94.6% sensitivity and 96.8-100% specificity. We found 3.6% indeterminate samples (undetected human control), lower than 8.1% with the standard protocol. INTERPRETATION: This preliminary work should support laboratories or commercial entities to develop and expand access to Covid-19 testing. Software guidance development for this assay is ongoing to enable implementation in other settings. FUND: USA NIH R01AI140845 and Seattle Children's Research Institute.

Cost-effectiveness analysis of pre-ART HIV drug resistance testing in Kenyan women.

BACKGROUND: The prevalence of pre-treatment drug resistance (PDR) to non-nucleoside reverse-transcriptase inhibitor (NNRTI) agents is increasing in sub-Saharan Africa, which may decrease the effectiveness of efavirenz-based antiretroviral therapy (ART) programs. However, due to recent safety concerns, there has been hesitancy to replace efavirenz-based ART with dolutegravir in women of reproductive potential. Our objective was to evaluate whether PDR testing for women not initiating dolutegravir-based ART would be a cost-effective strategy to address the challenges posed by PDR. METHODS: We developed an HIV drug resistance model that simulates the emergence and transmission of resistance mutations, calibrated to the Kenyan epidemic. We modeled three care strategies for PDR testing among women not initiating dolutegravir-based ART: no PDR testing, PDR testing with a low-cost point mutation assay, known as oligonucleotide ligation assay (OLA), and PDR testing with consensus sequencing. Using a health sector perspective, this model was used to evaluate the health outcomes, lifetime costs, and cost-effectiveness under each strategy over a 15-year time horizon starting in 2019. FINDINGS: OLA and CS PDR testing were projected to have incremental cost-effectiveness ratios (ICER) of $10,741/QALY gained and $134,396/QALY gained, respectively, which are not cost-effective by national income standards. Viral suppression rates among women at 12 months after ART initiation were 87·8%, 89·0%, and 89·3% with no testing, OLA testing, and CS testing, respectively. PDR testing with OLA and CS were associated with a 0.5% and 0.6% reduction in incidence rate compared to no PDR testing. Initial PDR prevalence among women was 13.1% in 2019. By 2034, this prevalence was 17·6%, 17·4%, and 17·3% with no testing, OLA testing, and CS testing, respectively. INTERPRETATION: PDR testing for women is unlikely to be cost-effective in Kenya whether one uses a low-cost assay, such as OLA, or consensus sequencing. FUNDING: National Institutes of Health, Gilead Sciences.