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.

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.

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.

Near point-of-care, point-mutation test to detect drug resistance in HIV-1: a validation study in a Mexican cohort.

OBJECTIVE: Pretreatment HIV-drug resistance (PDR, HIVDR) to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is increasing globally. NNRTIs continue to be used as first-line antiretroviral therapy (ART) in some communities due to the cost of dolutegravir-based ART or dolutegravir-associated adverse events. A simplified version of the oligonucleotide ligation assay (OLA) - 'OLA-Simple' - is a low-cost, near point-of-care assay that provides ready-to-use lyophilized reagents and reports HIVDR mutations as colored lines on lateral flow strips. Our objective was to design and validate OLA-Simple for a Mexican cohort. DESIGN: OLA-Simple probes to detect K65R, K103N/S, Y181C, M184V, and G190A were optimized for HIV Mexican sequences. Sixty clinical plasma specimens were analyzed by OLA-Simple by technicians blinded to Illumina-MiSeq sequences, and HIVDR results were compared. METHODS: Plasma RNA was tested using OLA-Simple kits. OLA-Simple lateral flow strips were read by in-house software, and were classified as mutant or wild-type at each codon. The comparison of results by OLA-Simple and Miseq was used to generate receiver-operating characteristic curves. RESULTS: OLA-Simple PCR amplified 59 of 60 specimens and successfully genotyped 287 of 295 codons, with eight of 295 (2.7%) indeterminate results. Compared to MiSeq, OLA-Simple gave five of 295 (1.7%) false-positive and four of 295 (1.4%) false-negative results. Excluding indeterminate results, OLA-Simple classified mutant with an accuracy of 97.4 and 98.8% when using thresholds at 10 and 25% mutant within an individual's HIV quasispecies, respectively. CONCLUSIONS: Compared to MiSeq, OLA-Simple detected HIVDR with high sensitivity and accuracy. OLA-Simple could expand access to affordable and rapid HIVDR testing to guide appropriate ART choices in populations using NNRTI-based ART.

Pre-treatment HIV-drug resistance associated with virologic outcome of first-line NNRTI-antiretroviral therapy: A cohort study in Kenya.

BACKGROUND: Pre-treatment HIV-drug-resistance (PDR) to WHO-recommended 1st-line non-nucleoside reverse transcriptase inhibitors (NNRTI)-based antiretroviral treatment (ART) is increasing in low-resource communities. We evaluated the risk of PDR on treatment failure if detected at single or multiple codons, at minority (2-9%) or higher (≥10%) frequencies during efavirenz- vs. nevirapine-ART. METHODS: We conducted a pooled analysis across three cohorts of Kenyans initiating 1st-line NNRTI-ART between 2006 and 2014. Mutations K103N, Y181C, G190A, M184V and K65R were detected by an oligonucleotide ligation assay (OLA) and confirmed by Sanger and next-generation sequencing (NGS). PDR was defined as detection of any mutation by OLA when confirmed by NGS. Treatment failure, defined as plasma HIV RNA ≥400 copies/mL at month-12 of ART, was compared by PDR genotypes. FINDINGS: PDR was detected in 59/1231 (4·8%) participants. Compared to wild-type genotypes, PDR in participants prescribed nevirapine-ART was associated with increased treatment failure [PDR 69·2% (27/39) vs. wild-type 10·4% (70/674); p = 0·0001], whether detected as minority [66·7% (4/6)] or higher [69·7% (23/33)] frequencies in an individual's HIV quasispecies (p = 0·002 and p < 0·0001, respectively), or mutations at single [50·0% (12/24)] or multiple [100·0% (15/15)] codons (p < 0·0001). During efavirenz-ART, PDR was also associated with increased virologic failure [PDR 25·0% (5/20) vs. wild-type 5·0% (25/498); p = 0·005], but only if detected at multiple drug-resistant codons [50·0% (3/6); p = 0·003] or high frequencies PDR [33·3% (5/15); p = 0·001]. INTERPRETATION: The risk that PDR confers for treatment failure varies by number of mutant codons and their frequency in the quasispecies, with a lower risk for efavirenz- compared to nevirapine-based regimens. PDR detection and management could extend the effective use of efavirenz-ART in low-resource settings. FUNDING: NIH, PEPFAR.

Association of Virologic Failure and Nonnucleoside Reverse Transcriptase Inhibitor Resistance Found in Antiretroviral-Naive Children Infected With Human Immunodeficiency Virus and Given Efavirenz-Based Treatment.

Among 66 antiretroviral-naive children aged <3 years with human immunodeficiency virus (HIV) or coinfected with HIV and tuberculosis and initiating efavirenz-based antiretroviral therapy (ART), non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance was detected before ART in 5 (7.6%). Virologic failure occurred in 2 of these children; they were last tested at 16 and 24 weeks of ART. Pre-ART NNRTI resistance was not associated with virologic failure.

Evaluation of the management of pretreatment HIV drug resistance by oligonucleotide ligation assay: a randomised controlled trial.

BACKGROUND: Although experts have recommended testing for pretreatment drug resistance (PDR) before antiretroviral therapy (ART) initiation, there is little evidence to support its implementation. We aimed to establish whether an inexpensive point mutation assay can improve virological suppression by identifying PDR to guide drug selection for ART in a lower-middle income country. METHODS: Investigators did an open-label, randomised controlled trial at three HIV treatment sites in Kenya: two in Nairobi and one in rural Maseno. Individuals (aged ≥2 years) were eligible to participate if they were confirmed HIV-seropositive, qualified for first-line ART, planned to reside in the area for more than 1 year, and provided informed consent. We randomly assigned participants (1:1) to either PDR testing by oligonucleotide ligation assay (OLA) to guide selection of ART or to standard of care, which did not include OLA testing. The OLA-guided therapy group had pre-ART peripheral blood mononuclear cells evaluated for drug resistance to non-nucleoside reverse transcriptase inhibitor (NNRTI) at codons Lys103Asn, Tyr181Cys, Gly190Ala, and to lamivudine at Met184Val, and when at least one drug-resistant codon was detected in a participant's pre-ART specimen, clinicians were directed to prescribe protease inhibitor-based second-line ART. Those without detected resistance and those who were randomised to standard of care received NNRTI-based first-line ART. The primary outcome was plasma HIV-1 RNA of at least 400 copies per mL at 4, 8, or 12 months after ART initiation, which defined virological failure, assessed in all participants who received treatment (data were censored for those lost-to-follow-up or who died). The study has been completed and is registered with ClinicalTrials.gov, NCT01898754. FINDINGS: We screened 1198 participants between May 28, 2013, and Nov 4, 2014, of whom 991 (83%) were enrolled (492 received OLA and 495 received standard of care; four did not begin treatment). 93 participants (prevalence 9·4%) had PDR (95% CI 7·7-11·4). 34 (8·5%) of 400 participants in the OLA group had virological failure at month 12 of ART (95% CI 6·0-11·7) compared with 39 (9·7%) of 402 (7·0-13·0) in the standard-of-care group (log-rank p=0·26). Among participants with PDR, virological failure was lower in the OLA-guided therapy group than in the standard-of-care group: five (14%) of 35 compared with 13 (50%) of 26; p=0·0020). Among those prescribed NNRTI-based ART, participants given efavirenz were less likely to have virological failure than were those receiving nevirapine (odds ratio 0·37, 95% CI 0·22-0·62; p<0·0001). The OLA-guided therapy group had 39 serious non-lethal adverse events and 34 deaths. The standard-of-care group had 34 severe adverse events and 43 deaths, differences that were not significant. Adverse events judged to potentially be due to ART were few and similar between groups, with 17 (16%) in the OLA-guided therapy group and 16 (16%) in the standard-of-care group (p=0·90). INTERPRETATION: Our finding that OLA testing for PDR reduced virological failure in only those with specific PDR mutations suggests that PDR poses less of a risk for virological failure than that predicted by past prevalence estimates, and that the value of PDR testing to reduce virological failure should be assessed for antiretroviral treatment regimens. FUNDING: US National Institutes of Health.

OLA-Simple: A software-guided HIV-1 drug resistance test for low-resource laboratories.

BACKGROUND: HIV drug resistance (HIVDR) testing can assist clinicians in selecting treatments. However, high complexity and cost of genotyping assays limit routine testing in settings where HIVDR prevalence has reached high levels. METHODS: The oligonucleotide ligation assay (OLA)-Simple kit was developed for detection of HIVDR against first-line non-nucleoside/nucleoside reverse transcriptase inhibitors and validated on 672 codons (168 specimens) from subtypes A, B, C, D, and AE. The kit uses dry reagents to facilitate assay setup, lateral flow devices for visual HIVDR detections, and in-house software with an interface for guiding users and analyzing results. FINDINGS: HIVDR analysis of specimens by OLA-Simple compared to Sanger sequencing revealed 99.6 ±â€¯0.3% specificity and 98.2 ±â€¯0.9% sensitivity, and compared to high-sensitivity assays, 99.6 ±â€¯0.6% specificity and 86.2 ±â€¯2.5% sensitivity, with 2.6 ±â€¯0.9% indeterminate results. OLA-Simple was performed more rapidly compared to Sanger sequencing (<4 h vs. 35-72 h). Forty-one untrained volunteers blindly tested two specimens each with 96.8 ±â€¯0.8% accuracy. INTERPRETATION: OLA-Simple compares favorably with HIVDR genotyping by Sanger and sensitive comparators. Instructional software enabled inexperienced, first-time users to perform the assay with high accuracy. The reduced complexity, cost, and training requirements of OLA-Simple could improve access to HIVDR testing in low-resource settings and potentially allow same-day selection of appropriate antiretroviral therapy. FUND: USA National Institutes of Health R01; the Clinical and Retrovirology Research Core and the Molecular Profiling and Computational Biology Core of the UW CFAR; Seattle Children's Research Institute; UW Holloman Innovation Challenge Award; Pilcher Faculty Fellowship.