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We present a case of a 20-year-old male who had ambiguous HIV test results after entering new provider care and whose status was later complicated by undetectable viral RNA off antiretroviral therapy (ART). Verifying HIV infection status may occasionally require sensitive DNA testing that might need to be considered in diagnostic guidelines to resolve diagnosis and ensure appropriate ART management. [ABSTRACT FROM AUTHOR]

Background The Determine™ HIV-1/2 Ag/Ab Combo (DC) rapid test can identify HIV-1 infection earlier than rapid antibody-only tests in plasma specimens. Objectives We compared the performance of DC with a laboratory-based antigen/antibody (Ag/Ab) combo assay in plasma and evaluated antigen reactivity in whole blood specimens. Study design We tested by DC 508 plasma specimens collected in a prospective study and 107 sequential plasma and simulated whole blood specimens from 20 seroconversion panels. Previous results using the ARCHITECT (ARC) Ag/Ab combo assay were compared to DC results. In seroconversion panels, the days from the first HIV1 RNA-positive test to first DC-reactive in plasma and whole blood was compared. McNemar’s and Wilcoxon signed rank tests were used for statistical analysis. Results Of 415 HIV-positive samples, ARC detected 396 (95.4%) and DC 337 (81.2%) ( p < 0.0001 ). DC was reactive in 50.0% of ARC-reactive/MS-negative, 78.6% of ARC-reactive/MS-indeterminate, and 99.6% of ARC-reactive/MS-HIV-1-positive or −undifferentiated specimens. DC antigen reactivity was higher among ARC-reactive/MS-negative than MS-indeterminate samples. In 20 HIV-1 seroconversion panels, there was a significant difference between DC reactivity in plasma (91.1%) and whole blood (56.4%) ( p < 0.0001 ). DC with whole blood showed a significant delay in reactivity compared to plasma ( p = 0.008 ). Conclusions In plasma, DC was significantly less sensitive than an instrumented laboratory-based Ag/Ab combo assay. DC in plasma was significantly more sensitive compared to whole blood in early HIV-1 infections. With the U.S. laboratory-based diagnostic algorithm, DC as the first step would likely miss a high proportion of HIV-1 infections in early stages of seroconversion. [ABSTRACT FROM AUTHOR]

Background The availability of more accurate point-of-care technology could increase the number of persons aware of their HIV status. The DPP ® HIV-1/2 assay is the first dual path platform rapid test (RT) approved in the U.S. that also received the Clinical Laboratory Improvement Amendments (CLIA) waiver for use with oral fluid and fingerstick and venous whole blood. Objective To evaluate the performance of the DPP ® HIV-1/2 assay with plasma specimens. Study design Sensitivity and specificity of the assay were calculated from 696 HIV-1 groups M (B and non-B subtypes) and O and HIV-2 (groups A and B) specimens and 505 HIV-negative specimens, respectively. Analysis of the assay performance in HIV-1 early infections was assessed by estimating the relative sensitivity of the RT before the Western blot (WB) becomes positive using a 50% cumulative frequency analysis and by comparing the reactivity with other Food and Drug Administration (FDA)-approved RTs. Results The sensitivity for established infection was 100% for HIV-1 and 100% for HIV-2. The specificity was 100%. The DPP ® HIV-1/2 assay performs similarly to most antibody-based RT approved by FDA in early HIV-1 infections. Conclusions The DPP ® technology showed no significant improvement for detecting early infections over other lateral-flow RTs used in the U.S. Without more data on the DPP ® HIV-1/2 assay, especially from whole blood and oral fluid specimens collected during the early phase of infection, its performance as point-of-care technology remains to be assessed. [ABSTRACT FROM AUTHOR]

Abstract: Background: The collection of dried blood spots (DBS) on Whatman 903 cards has facilitated for years the detection of HIV-1 in infants by DNA PCR as early as 4–6 weeks after birth in resource-limited settings (RLS), but alternate blood collection devices are proving to be necessary. Objectives: The qualitative detection of HIV-1 DNA by PCR from DBS prepared on three commercially available blood collection cards was evaluated at the Centers for Disease Control and Prevention (CDC) and in four laboratories in Africa. Study design: DBS were prepared on Ahlstrom grade 226, Munktell TFN and Whatman 903, and stored under a variety of conditions. DBS were stored at ambient temperature (RT), 37°C with high humidity, and −20°C for varying lengths of time. The presence of HIV-1 DNA was tested using Roche Amplicor HIV-1 DNA (v 1.5) weekly for 4 weeks and at weeks 8 and 12 (RT and 37°C), at weeks 4, 8, and 18 (−20°C) of storage. DBS specimens were also tested after international shipment at RT. In addition, after nearly 3 years storage at −20°C, DBS were also evaluated independently using the COBAS Ampliprep/TaqMan HIV-1 Qual and Abbott RealTime HIV-1 Qualitative tests. Results: HIV-1 DNA was detected equally well on the three blood collection cards regardless of storage conditions and PCR assay. Conclusions: Ahlstrom 226 and Munktell TFN papers were comparable to Whatman 903 for HIV-1 DNA detection and may be considered as optional blood collection devices in resource-limited countries. [Copyright &y& Elsevier]

Background: HIV antibody testing has been included in the National Health and Nutrition Examination Survey, for ages 18-49 since 1999 and for ages 18-59 years since 2009 enabling estimation of trends in HIV prevalence as part of national surveillance in the U.S. household population. Self-reported HIV testing and antiretroviral use was also included in the survey since 1999. Setting: A continuous household-based probability sample of the U.S. population. Methods: From 1999 to 2018, 29,020 participants age 18-49 years were tested for HIV antibody and 34,092 participants age 18-59 years were asked about self-report of any previous HIV testing. Results: HIV prevalence was 0.41% among those aged 18-59 in 2009-2018 with a nonsignificant trend over time among those aged 18-49 years from 1999-2002 to 2015-2018. However, significant declines in prevalence were seen among those aged 18-39 years (0.37%-0.11%), women (0.22%-0.06%) and non-Hispanic black persons (2.14%-0.80%). Participants aged 18-39 years self-reported a decline in HIV testing, whereas those aged 40-49 and 50-59 years, non-Hispanic black persons and women reported an increase in getting a HIV test. Prevalence of infection and self-reported history of HIV testing varied by demographic and risk groups. HIV testing among HIV-positive persons was 83.9%. Antiretroviral therapy among those HIV-positive was under 50%. Conclusion: Although total HIV prevalence and previous self-reported HIV testing remained stable for the last 20 years, there were significant declines in age and demographic subgroups. Prevalence for both outcomes varied by demographic and risk variables. [ABSTRACT FROM AUTHOR]

Abstract: Background: Until recently most testing algorithms in the United States (US) utilized Western blot (WB) as the supplemental test. CDC has proposed an algorithm for HIV diagnosis which includes an initial screen with a Combo Antigen/Antibody 4th generation-immunoassay (IA), followed by an HIV-1/2 discriminatory IA of initially reactive-IA specimens. Discordant results in the proposed algorithm are resolved by nucleic acid-amplification testing (NAAT). Objectives: Evaluate the results obtained with the CDC proposed laboratory-based algorithm using specimens from men who have sex with men (MSM) obtained in five metropolitan statistical areas (MSAs). Study design: Specimens from 992 MSM from five MSAs participating in the CDC's National HIV Behavioral Surveillance System in 2011 were tested at local facilities and CDC. The five MSAs utilized algorithms of various screening assays and specimen types, and WB as the supplemental test. At the CDC, serum/plasma specimens were screened with 4th generation-IA and the Multispot HIV-1/HIV-2 discriminatory assay was used as the supplemental test. NAAT was used to resolve discordant results and to further identify acute HIV infections from all screened-non-reactive missed by the proposed algorithm. Performance of the proposed algorithm was compared to site-specific WB-based algorithms. Results: The proposed algorithm detected 254 infections. The WB-based algorithms detected 19 fewer infections; 4 by oral fluid (OF) rapid testing and 15 by WB supplemental testing (12 OF and 3 blood). One acute infection was identified by NAAT from all screened-non-reactive specimens. Conclusions: The proposed algorithm identified more infections than the WB-based algorithms in a high-risk MSM population. OF testing was associated with most of the discordant results between algorithms. HIV testing with the proposed algorithm can increase diagnosis of infected individuals, including early infections. [Copyright &y& Elsevier]

Abstract: Background: Oral fluid (OF) testing is a less-invasive alternative to blood-based testing for HIV. The performance of HIV OF tests has not been extensively evaluated in serially collected paired specimens from seroconverters. Objective: To compare paired OF and plasma test performance in a cohort of HIV-1 seroconverters from Nigeria. Study design: Paired plasma and OF specimens from 14 seroconverters collected during 24 months of longitudinal follow up were included in the study. Plasma and OF were tested using Avioq HIV-1 Microelisa System, and first reactivity in plasma and OF specimens was compared. OF specimens reactive by Avioq were subsequently tested by OraSure HIV-1 Western blot. Genetic Systems HIV-1 Western blot was also performed on the corresponding plasma of the first 2 Avioq-OF positive time-points. Results: Of the 14 seroconverters, 5 (35.7%) had concordant results between plasma and OF for all time points tested, whereas 9 (64.3%) showed reactivity on plasma before OF specimens early in infection. The median delay between plasma and OF reactivity was 29 days (range: 0 day–20 months) (p <0.0039); the median overall delay for OF compared to RNA testing was 69.5 days. Delayed antibody response with OF was observed in both males and females regardless of viral load or HIV subtypes. Conclusions: Results demonstrate decreased sensitivity of OF testing compared to blood-based testing with specimens obtained early after HIV infection. Programs that utilize OF testing in populations with increased risk of incident HIV infection should understand these limitations of OF testing. [Copyright &y& Elsevier]

Abstract: Background: FDA-approved HIV Antigen/Antibody combo (4th generation) immunoassays (IAs) can identify HIV-1 infections before the Western blot (WB) becomes positive. In the US, increased detection of acute HIV infections has been facilitated by using 4th generation IAs, but there is no FDA-approved 4th generation rapid test (RT). The Alere Determine™ HIV-1/2 Ag/Ab Combo (Determine Combo) RT detects and distinguishes HIV p24 Antigen (Ag) from Antibody (Ab) to HIV-1+HIV-2 and thus has the potential to improve diagnosis of acute HIV infection. Objective: To evaluate the ability of Determine Combo RT to detect acute/early HIV-1 infections and HIV-2 antibody in well-characterized plasma specimens. Study design: In HIV-1 seroconverters from the US, Determine Combo reactivity was evaluated by performing the 50% cumulative frequency analysis and by comparing with 3rd and 4th generation IAs’ reactivity. HIV-2 plasma specimens from Ivory Coast were tested with Determine Combo. Results: The 50% cumulative frequency analysis in 17 seroconverters placed Determine Combo (Ag+/Ab−, Ag+Ab+, Ag−/Ab+) and Ab-component reactivity at 15.5 and 7 days before WB positivity, respectively. In 26 seroconverters, Determine Combo was reactive in 99.0% and 92.5% of 3rd and 4th generation IAs-reactive specimens, respectively. All HIV-2 plasma specimens were Ab-reactive/Ag-non-reactive by Determine Combo. Conclusions: Based on previous results with the same seroconversion panels, combined Ag/Ab reactivity of the Determine Combo appears between FDA-approved 4th and 3rd generation laboratory IAs. These data indicate that this RT could detect HIV-1 infection earlier than other RTs and it performs well in HIV-2 specimens. [Copyright &y& Elsevier]

Background. Understanding the period of time between an exposure resulting in infection with human immunodeficiency virus (HIV) and when a test can reliably detect the presence of that infection, that is, the test window period, may benefit testing programs and clinicians in counseling patients about when the clinician and the patient can be confident a suspected exposure did not result in HIV infection. Methods. We evaluated the intervals between reactivity of the Aptima HIV-1 RNA test (Aptima) and 20 US Food and Drug Administration-approved HIV immunoassays using 222 longitudinally collected plasma specimens from HIV-1 seroconverters from the United States. Using interval-censored survival and binomial regression approaches a multi-model framework was implemented to estimate the relative emergence of test reactivity, referred to here as an inter-test reactivity interval (ITRI).We then combined ITRI results with simulated data for the eclipse period, the time between exposure and detection of HIV virus by Aptima, to estimate the window period for each test. Results. The estimated ITRIs were shorter with each new class of HIV tests, ranging from 5.9 to 24.8 days. The 99th percentiles of the window period probability distribution ranged from 44 days for laboratory screening tests that detect both antigen and antibody to 65 days for the Western blot test. Conclusions. Our directly comparable estimates of the emergence of reactivity for 20 immunoassays are valuable to testing providers for interpreting negative HIV test results obtained shortly after exposure, and for counseling individuals on when to retest after an exposure. [ABSTRACT FROM AUTHOR]

Abstract: Background: The current algorithm for HIV diagnosis in the US involves screening with an immunoassay (IA) and supplemental testing with Western blot (WB) or immunofluorescence assay. Because of existence of more sensitive and specific FDA-approved assays that would also reduce the cost and turn-around time of testing compared to WB, several alternative algorithms have been evaluated. Recently, an alternative algorithm using a sensitive 3rd or 4th generation IA followed by an HIV-1 and HIV-2 discriminatory supplemental test on the initial IA-positive specimens was proposed. Concordant positive results indicate HIV-positive specimens and discordant results are resolved by nucleic acid amplification testing (NAAT). Objectives: To evaluate the sensitivity of assays during acute HIV infection and the performance of the current and an alternative algorithm using samples from HIV-1 seroconversion panels and persons with established HIV infections. Study design: To evaluate the algorithms in early infections, 26 HIV-1 seroconverters from the US were tested with three 3rd generation and one 4th generation IA, six rapid tests (RTs), one NAAT, and WB. Sensitivity and specificity of the algorithms were calculated by testing an additional 416 HIV-positive and 414 uninfected control samples with one 3rd generation and one 4th generation IA, four RTs, one NAAT, and WB. Results: The individual assays evaluated became positive 5 (RT) to 26 days (NAAT) before WB was positive. Among seroconverters, the alternative algorithm detected significantly more infections than the current algorithm (103–134 versus 56, p <0.0001). Furthermore, the use of a 4th generation IA instead of a 3rd generation assay as the screen resulted in significantly higher detection of acute infections (p <0.0001). In contrast, the algorithms performed equally among specimens from established HIV-1 infections. Conclusions: This study demonstrated improved sensitivity of the alternative algorithm for detecting acute HIV-1 infections, while maintaining the ability to accurately detect established HIV-1 infections. Early detection is important as individuals can be highly infectious during acute infection. In addition, the alternative algorithm should reduce turn-around time by using a RT as the supplemental test has the potential to increase the number of test results returned. [Copyright &y& Elsevier]

HIV incidence estimates are used to monitor HIV-1 infection in the United States. Use of laboratory biomarkers that distinguish recent from longstanding infection to quantify HIV incidence rely on having accurate knowledge of the average time that individuals spend in a transient state of recent infection between seroconversion and reaching a specified biomarker cutoff value. This paper describes five estimation procedures from two general statistical approaches, a survival time approach and an approach that fits binomial models of the probability of being classified as recently infected, as a function of time since seroconversion. We compare these procedures for estimating the mean duration of recent infection (MDRI) for two biomarkers used by the U.S. National HIV Surveillance System for determination of HIV incidence, the Aware BED EIA HIV-1 incidence test (BED) and the avidity-based, modified Bio-Rad HIV-1/HIV-2 plus O ELISA (BRAI) assay. Collectively, 953 specimens from 220 HIV-1 subtype B seroconverters, taken from 5 cohorts, were tested with a biomarker assay. Estimates of MDRI using the non-parametric survival approach were 198.4 days (SD 13.0) for BED and 239.6 days (SD 13.9) for BRAI using cutoff values of 0.8 normalized optical density and 30%, respectively. The probability of remaining in the recent state as a function of time since seroconversion, based upon this revised statistical approach, can be applied in the calculation of annual incidence in the United States. [ABSTRACT FROM AUTHOR]

Polymorphisms of some chemokine receptor genes and their ligands are associated with susceptibility and progression of human immunodeficiency virus infection. This study assessed whether these variants are also responsible for susceptibility to infection with human T lymphotropic virus (HTLV) type I. Frequencies of CCR5-δ32, CCR2-64I, and SDF-I-3'A genotype among 116 HTLV-I-positive and 126 HTLV-I-negative persons of African descent in Jamaica were 1.0%, 14.9 %, and 5.4%, respectively. The association of HTLV-I infection with the most common variant, CCR2-64I, was examined in 532 subjects. Thirteen (5.4%) of 241 HTLV-I-negative subjects were homozygous for CCR2-64I, versus 3 (1.0%) of 291 HTLV-I-positive subjects (P = .005). Among HTLV-I carriers, provirus load and antibody titer were not significantly different in persons with CCR2-+/64I or CCR2-+/+. These findings suggest that CCR2-641, or alleles in linkage disequilibrium with it, may affect the risk of HTLV-I infection in a recessive manner. [ABSTRACT FROM AUTHOR]

Background: In the US, one in six men who have sex with men (MSM) with HIV are unaware of their HIV infection. In certain circumstances, access to HIV testing and viral load (VL) monitoring is challenging. The objective of this study was to evaluate the feasibility of conducting laboratory-based HIV and antiretroviral (ARV) drug testing, and VL monitoring as part of two studies on self-collected dried blood spots (DBS).Methods: Participants were instructed to collect DBS by self-fingerstick in studies that enrolled MSM online. DBS from the first study (N = 1444) were tested with HIV serological assays approved by the Food and Drug Administration (FDA). A subset was further tested with laboratory-modified serological and VL assays, and ARV levels were measured by mass spectrometry. DBS from the second study (N = 74) were only tested to assess VL monitoring.Results: In the first study, the mail back rate of self-collected DBS cards was 62.9%. Ninety percent of DBS cards were received at the laboratory within 2 weeks from the day of collection, and 98% of the cards had sufficient spots for one assay. Concordance between FDA-approved and laboratory-modified protocols was high. The samples with undetectable ARV had higher VL than samples with at least one ARV drug. In the second study, 70.3% participants returned self-collected DBS cards, and all had sufficient spots for VL assay. High VL was observed in samples from participants who reported low ARV adherence.Conclusions: In these studies, MSM were able to collect and provide adequate DBS for HIV testing. The FDA-approved and laboratory-modified testing algorithms performed similarly. DBS collected at home may be feasible for HIV testing, ARV measurement, and monitoring viral suppression. [ABSTRACT FROM AUTHOR]

HIV rapid testing algorithms (RTAs) using any two orthogonal rapid tests (RTs) allow for on-site confirmation of infection. RTs vary in performance characteristics therefore the selection of RTs in an algorithm may affect identification of infection, particularly if acute. National HIV Behavioral Surveillance (NHBS) assessed RTAs among men who have sex with men recruited using anonymous venue-based sampling. Different algorithms were evaluated among participants who self-reported never having received a positive HIV test result prior to the interview. NHBS project areas performed sequential or parallel RTs using whole blood. Participants with at least one reactive RT were offered anonymous linkage to care and provided a dried blood spot (DBS) for testing at CDC. Discordant results (RT-1 reactive/RT-2 non-reactive) were tested at CDC with lab protocols modified for DBS. DBS were also tested for HIV-1 RNA (VL) and antiretroviral (ARV) drug levels. Of 6500 RTAs, 238 were RT-1 reactive; of those, 97.1% (231/238) had concordant results (RT-1/RT-2 reactive) and 2.9% (7/238) had discordant results. Five DBS associated with discordant results were available for confirmation at CDC. Four had non-reactive confirmatory test results that implied RT-1 false reactivity; one had ambiguous confirmatory test results which was non-reactive in further testing. Regardless of order and type of RT used, RTAs demonstrated high concordant results in the population surveyed. Additional laboratory testing on DBS following discordant results confirmed no infection. Implementing RTAs in the context of anonymous venue-based HIV testing could be an option when laboratory follow-up is not practicable. [ABSTRACT FROM AUTHOR]

Background: Serological assays to determine HIV incidence have contributed to estimates of HIV incidence, monitoring of HIV spread, and evaluation of prevention strategies. Two frequently used incidence assays are the Sedia HIV-1 LAg-Avidity EIA (LAg) and the Bio-Rad avidity incidence (BRAI) assays with a mean duration of recent infection (MDRI) of 130 and 240 days for subtype B infections, respectively. Little is known about how these assays perform with recombinant HIV-1 strains. We evaluated the concordance of these assays in a population infected mainly with HIV-1 CRF06_cpx. Material/Methods: Remnant serum samples (n = 288) collected from confirmed, newly-diagnosed HIV-positive persons from Estonia in 2013 were tested. Demographic and clinical data were extracted from clinical databases. LAg was performed according to the manufacturer’s protocol and BRAI testing was done using a validated protocol. Samples with LAg-pending or BRAI-invalid results were reclassified as recent if they were from persons with viral loads <1000 copies/mL or were reclassified as long-term if presenting with AIDS. Results: In total 325 new HIV infections were diagnosed in 2013 in Estonia. Of those 276 persons were tested with both LAg and BRAI. Using assay results only, the recency rate was 44% and 70% by LAg and BRAI, respectively. The majority of samples (92%) recent by LAg were recent by BRAI. Similarly, 89% of samples long-term by BRAI were long-term by LAg. After clinical information was included in the analysis, the recency rate was 44% and 62% for LAg and BRAI, respectively. The majority of samples (86%) recent by LAg were recent by BRAI and 91% of long-term infections by BRAI were long-term by LAg. Conclusions: Comparison of LAg and BRAI results in this mostly CRF06_cpx-infected population showed good concordance for incidence classification. Our finding of a higher recency rate with BRAI in this population is likely related to the longer MDRI for this assay. [ABSTRACT FROM AUTHOR]

Highlights • Geenius HIV-1/2 Supplemental assay was evaluated with HIV-1 and HIV-2 samples. • Comparison with other supplemental tests showed similar performance in HIV-1 samples. • Software v1.3 reduced the number of HIV-2 indeterminate results due to gp140. Abstract Background In the US, the HIV diagnostic algorithm for laboratory settings recommends the use of an HIV-1/HIV-2 differentiation supplemental assay after an initial reactive antigen/antibody (Ag/Ab) assay result. Since the discontinuation of the Multispot HIV-1/HIV-2 Rapid Test (MS), the Geenius HIV-1/2 Supplemental assay (Geenius) is the only FDA-approved supplemental differentiation test. Objective We compared the performance of Geenius to MS and Western Blot (WB). Study design The relative seroconversion plasma reactivity of Geenius and MS was assessed using a 50% cumulative frequency analysis from 17 HIV-1 seroconverters. In addition, previously characterized plasma specimens, 186 HIV-1 positive, 100 HIV-2 positive, and 93 Ag/Ab-positive/HIV-1 RNA-negative, were tested with Geenius v1.1 software. McNemar's test was used for paired comparison analysis. A subset of 48 specimens were retested with the upgraded Geenius v1.3 software. Results In HIV-1 seroconverters, the relative seroconversion reactivity was 2.5 and 2 days before the first positive HIV-1 WB for Geenius and MS, respectively. In HIV-1 positive samples, Geenius performed similarly to HIV-1 WB (p =0.1687) and MS (p =0.8312). In HIV-2 positive samples, Geenius underperformed compared to HIV-2 WB (p =0.0005) and MS (p =0.0012). When using the upgraded software among the HIV-1 positive and Ag/Ab-reactive/HIV-1 RNA-negative samples, gp140 reactivity decreased without affecting characterization of HIV-2 samples. Conclusions With HIV-1 samples, Geenius, WB and MS performance was similar as supplemental tests. The updated Geenius software reduced false gp140 reactivity, but had no impact on identifying true HIV-2 infections. Further evaluation will assess the impact of the Geenius software update on final diagnostic interpretations [ABSTRACT FROM AUTHOR]

Real-time PCR assays for nucleic acid testing (NAT) of hepatitis viruses A–E and for HIV-1 and HIV-2 have been developed; however, a multiplex assay that can simultaneously detect all of these agents is not yet available. Standardized TaqMan assays for detection of hepatitis viruses A–E have been described and applied to TaqMan Array Cards (TAC) which are capable of multiple pathogen detection using a single set of optimized PCR conditions. Assays for three gene regions of HIV-1 (long-terminal repeat (LTR), gag , and polymerase) and HIV-2 (overlap of LTR and gag , protease and integrase) were designed using the hepatitis assay conditions. Nucleic acid extracts of HIV-1-infected samples (44 plasma, 41 whole blood, 20 HIV-1 viral stocks) were tested on the TAC cards; 98 were reactive (92%) with 70 in multiple gene regions. Twenty-four of the 27 (89%) HIV-2 specimens (10 plasma, 1 PBMC lysate, 6 whole blood and 10 plasmids containing HIV-2 polymerase) were detected on TAC. No HIV or hepatitis virus sequences were detected in 30 HIV-negative samples (specificity 100%). Three HBV and 18 HCV co-infections were identified in the HIV-1-infected specimens. Multi-pathogen detection using TAC could provide a rapid, sensitive and more efficient method of surveying for a variety of infectious disease nucleic acids. [ABSTRACT FROM AUTHOR]

In January 2015, an outbreak of undiagnosed human immunodeficiency virus (HIV) infections among persons who inject drugs (PWID) was recognized in rural Indiana. By September 2016, 205 persons in this community of approximately 4400 had received a diagnosis of HIV infection. We report results of new approaches to analyzing epidemiologic and laboratory data to understand transmission during this outbreak. HIV genetic distances were calculated using the polymerase region. Networks were generated using data about reported high-risk contacts, viral genetic similarity, and their most parsimonious combinations. Sample collection dates and recency assay results were used to infer dates of infection. Epidemiologic and laboratory data each generated large and dense networks. Integration of these data revealed subgroups with epidemiologic and genetic commonalities, one of which appeared to contain the earliest infections. Predicted infection dates suggest that transmission began in 2011, underwent explosive growth in mid-2014, and slowed after the declaration of a public health emergency. Results from this phylodynamic analysis suggest that the majority of infections had likely already occurred when the investigation began and that early transmission may have been associated with sexual activity and injection drug use. Early and sustained efforts are needed to detect infections and prevent or interrupt rapid transmission within networks of uninfected PWID. [ABSTRACT FROM AUTHOR]

Objective FDA-approved antigen/antibody combo and HIV-1/2 differentiation supplemental tests do not have claims for dried blood spot (DBS) use. We compared two DBS-modified protocols, the Bio-Rad GS HIV Combo Ag/Ab (BRC) EIA and Geenius™ HIV-1/2 (Geenius) Supplemental Assay, to plasma protocols and evaluated them in the CDC/APHL HIV diagnostic algorithm. Methods BRC-DBS p24 analytical sensitivity was calculated from serial dilutions of p24. DBS specimens included 11 HIV-1 seroconverters, 151 HIV-1-positive individuals, including 20 on antiretroviral therapy, 31 HIV-2-positive and one HIV-1/HIV-2-positive individuals. BRC-reactive specimens were tested with Geenius using the same DBS eluate. Matched plasma specimens were tested with BRC, an IgG/IgM immunoassay and Geenius. DBS and plasma results were compared using the McNemar's test. A DBS-algorithm applied to 348 DBS from high-risk individuals who participated in surveillance was compared to HIV status based on local testing algorithms. Results BRC-DBS detects p24 at a concentration 18 times higher than in plasma. In seroconverters, BRC-DBS detected more infections than the IgG/IgM immunoassay in plasma ( p = 0 . 0133), but fewer infections than BRC-plasma ( p = 0 . 0133) . In addition, the BRC/Geenius-plasma algorithm identified more HIV-1 infections than the BRC/Geenius-DBS algorithm ( p = 0 . 0455) . The DBS protocols correctly identified HIV status for established HIV-1 infections, including those on therapy, HIV-2 infections, and surveillance specimens. Conclusions The DBS protocols exhibited promising performance and allowed rapid supplemental testing. Although the DBS algorithm missed some early infections, it showed similar results when applied to specimens from a high-risk population. Implementation of a DBS algorithm would benefit testing programs without capacity for venipuncture. [ABSTRACT FROM AUTHOR]

Background The flow-through INSTI™ HIV-1/HIV-2 Rapid Antibody (INSTI) test is a 60 s FDA-approved test for HIV-1 and HIV-2 antibody testing using whole blood and plasma. Objective We evaluated the performance of INSTI using plasma and simulated whole blood specimens. Study design INSTI’s performance in plasma specimens from commercial seroconversion panels was assessed by estimating the relative sensitivity using a 50% cumulative frequency analysis and by comparing its performance with other FDA-approved rapid tests (RTs). INSTI was further evaluated using 320 HIV-1 plasma specimens collected during a cross-sectional study and with 107 HIV-1 and 24 HIV-2 simulated whole blood specimens. Sensitivity and specificity were calculated using 615 known HIV-1 group M/O and 80 HIV-2 (Western blot (WB)-positive), and 497 HIV-negative plasma specimens, respectively. Results In HIV-1 seroconversion panels, INSTI became reactive 9 days before a positive WB. When compared to FDA-approved antibody-based lateral flow RTs, INSTI detected significantly more early infections. Among HIV-1-infected cross-sectional plasma samples, INSTI detected 23 (27%) of 85 Architect-positive/Multispot-negative or indeterminate specimens. For plasma specimens, the sensitivity was 99.84% for HIV-1 and 100% for HIV-2, and the specificity was 99.80%. Using simulated whole blood from seroconverters, INSTI performed similarly to plasma. Conclusions INSTI performed significantly better than antibody-based lateral flow RTs during early stages of seroconversion. Sensitivity and specificity were within the manufacturer’s reported ranges. Considering the observed test performance and the almost immediate results, INSTI is an accurate option to detect HIV-1/HIV-2 antibodies in point-of-care settings where lab testing is not feasible. [ABSTRACT FROM AUTHOR]

BACKGROUND: Drug resistance mutations (DRMs) can serve as distinct, nonpolymorphic markers for evaluating diversity of expressed HIV-1. We screened for DRMs during early-acute viremia and examined the diversity in reverse transcriptase (RT) relative to envelope (env) in cases of transmitted drug resistance. METHODS: We evaluated 111 longitudinal plasma samples collected every 2-7 days from 15 individuals who seroconverted for HIV-1 infection in 1994-2000. The samples were screened with sensitive polymerase chain reaction assays for the commonly transmitted M41L and K70R mutations and for K65R, which was undetected by bulk sequencing. Mutation-positive samples were further characterized by clonal sequencing of RT and env V1-V3. RESULTS: Drug resistance mutations were detected in 4 of 15 seroconverters at 5-50 days of viral nucleic acid expression; most mutations disappeared about the time of seroconversion. Clonal sequencing verified low-level K65R at frequencies of 0.4%-4.9%. In each case, K65R coexisted unlinked with variants carrying 2-5 thymidine analog mutations at frequencies of 1.6%-23.0%. In one seroconverter, variants with M184V and nonnucleoside RT inhibitor mutations were also identified at first RNA expression. Each seroconverter displayed a homogeneous V1-V3 env population. CONCLUSIONS: Reverse-transcriptase DRMs demonstrate that the breadth of variants in transmission may be greater than what is reflected in envelope sequences. [ABSTRACT FROM AUTHOR]

Background. Drug resistance mutations (DRMs) can serve as distinct, nonpolymorphic markers for evaluating diversity of expressed HIV-1. We screened for DRMs during early-acute viremia and examined the diversity in reverse transcriptase (RT) relative to envelope (env) in cases of transmitted drug resistance.Methods. We evaluated 111 longitudinal plasma samples collected every 2–7 days from 15 individuals who seroconverted for HIV-1 infection in 1994–2000. The samples were screened with sensitive polymerase chain reaction assays for the commonly transmitted M41L and K70R mutations and for K65R, which was undetected by bulk sequencing. Mutation-positive samples were further characterized by clonal sequencing of RT and env V1–V3.Results. Drug resistance mutations were detected in 4 of 15 seroconverters at 5–50 days of viral nucleic acid expression; most mutations disappeared about the time of seroconversion. Clonal sequencing verified low-level K65R at frequencies of 0.4%–4.9%. In each case, K65R coexisted unlinked with variants carrying 2–5 thymidine analog mutations at frequencies of 1.6%–23.0%. In one seroconverter, variants with M184V and nonnucleoside RT inhibitor mutations were also identified at first RNA expression. Each seroconverter displayed a homogeneous V1–V3 env population.Conclusions. Reverse-transcriptase DRMs demonstrate that the breadth of variants in transmission may be greater than what is reflected in envelope sequences. [ABSTRACT FROM AUTHOR]

Using a repeat-exposure macaque model, Walid Heneine and colleagues find that pre-exposure prophylaxis with combination antiretroviral drugs provides protection against rectal challenge with a SHIV virus. [ABSTRACT FROM AUTHOR]

• A modified Aptima HIV-1 Quant protocol can quantify RNA using 100 μL of plasma. • The standard and modified protocols had high agreement above 50 copies/mL. • Using microtainers for blood collection could expand access to treatment and care. Currently, FDA-approved HIV-1 viral load (VL) assays use venipuncture-derived plasma. The Hologic Panther system uses 0.7 mL total volume for the Aptima HIV-1 Quant Assay standard (APT-Quant-std) and dilution (APT-Quant-dil) protocols. However, smaller plasma volumes from fingerstick whole blood (FSB) collected in EDTA-microtainer tubes (MCT) could provide an easier sample collection method for HIV-1 VL testing. To evaluate the performance of the APT-Quant-std compared to the Roche CAP/CTM and Abbott m2000RT VL assays and an alternative APTQuant 1:7 dilution protocol, the latter using 100 μL of MCT-derived plasma from FSB. Linearity was determined using commercial HIV-1 RNA plasma controls. Dilutions ranging 1.56–2.95 log10 copies/mL were prepared to determine the APT-Quant-dil Limit of Quantitation (LOQ) using Probit analysis. Specificity of APT-Quant-std was calculated using 326 HIVnegative samples. To evaluate agreement, 329 plasma specimens were tested with APT-Quant-std, CAP/CTM, and m2000RT. Forty-seven matched venipuncture and MCT-derived plasma specimens were tested with APT-Quant-std and APT-Quant-dil. Among the RNA controls, specificity was 99.69 % for APT-Quant-std. The R2 values were 0.988 (APT-Quant-std/CAP/CTM), 0.980 (APT-Quant-std/ m2000RT), and 0.997 (APT-Quant-std/APT-Quant-dil). The APT-Quant-dil LOQ was estimated at 2.7 log10 copies/mL (500 copies/mL) (95 %CI 2.62–2.87). At 2.3 log10 copies/mL (200 copies/mL), the overall agreement was 91.0 % for APT-Quant-std/CAP/CTM, 85.7 % for APT-Quant-std/m2000RT, and 82.9 % for APT-Quant-std/APT-Quant-dil. Quantified APT-Quant-std results were on average 0.2 log10 copies/mL higher than CAP/CTM and m2000RT and 0.14 log10 copies/mL higher than APT-Quant-dil. APT-Quant showed similar performance compared to the CAP/CTM and m2000RT assays and remains sensitive and accurate using the dilution protocol. [ABSTRACT FROM AUTHOR]

• The MedMira G4 Reveal POC and LAB S/P assays can detect both HIV-1 and HIV-2 antibodies. • The performance of the rapid test was compared in early and established HIV infections. • The rapid test was evaluated in plasma and whole blood samples to compare performance. • Sensitive and accurate rapid tests performed in point of care settings can help increase access to HIV testing. The Reveal G4 antibody rapid test is FDA-approved for HIV-1 detection using the versions LAB S/P and POC in CLIA-moderate complexity settings with serum/plasma and whole blood, respectively. The same Reveal tests are CE-marked for HIV-1 and HIV-2 detection in laboratory and point-of-care (POC) settings. We compared the performance of G4 LAB S/P with plasma and POC with whole blood (blood) for detecting early and established HIV-1/HIV-2 infections. Matched well-characterized plasma and simulated blood were used to evaluate: sensitivity in 104 HIV-1 and 55 HIV-2 established infections, specificity in 49 HIV-negative, and reactivity in early HIV-1 infection in a performance panel (n=38) and 18 plasma panels from seroconverters (SCs, n=183). Median number of days after first RNA-positive was calculated for 13 SCs. Impact of viral suppression (VS) was evaluated in 3 SCs receiving early antiretroviral therapy (ART). Sensitivity was 100 % for HIV-1 and 98.18 % for HIV-2, while specificity was 100 %. All 38 plasma and blood become reactive by Fiebig stage V. Of 18 SCs, 10 had similar reactivity in plasma/blood, 7 showed delayed reactivity in blood, and 1 was nonreactive in plasma/blood. The median days for a G4-reactive after first RNApositive was 13 for plasma and 14 for blood. Long-term VS had no impact on G4 reactivity. Overall reactivity in early HIV-1 infections is delayed by one day in blood compared to plasma. If FDA-approved for POC settings, the G4 POC is a fast sensitive screening tool for HIV-1/HIV-2-specific IgG even during VS. [ABSTRACT FROM AUTHOR]

• Xpert HIV‐1 Viral Load is a simplified, automated, single-use quantitative assay. • Cobas and Xpert VLs were highly correlated (R2 = 0.994). • Xpert VL detected 97.9 % of established infections, and specificity was 99.80 %. • Xpert VL detected 90 % and 98.5 % of early and later acute infections, respectively. • Xpert VL performs proficiently for quantitative and qualitative uses. Cepheid's Xpert HIV‐1 Viral Load (Xpert VL), a simplified, automated, single-use quantitative assay used with the GeneXpert System, is not FDA approved. Using stored plasma, we conducted a study to assess the ability of Xpert VL to quantify viral load relative to the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 (Cobas VL) and to examine the use of the Xpert VL as a qualitative diagnostic test. Following HIV-1 viral stock dilutions, we conducted a probit analysis to identify the concentration where 95 % of specimens had quantified VLs. We also examined Xpert and Cobas log VL correlation in linearity panels; compared the proportion of 220 seroconverter specimens with virus detected using McNemar's test; and tested specimens from persons with untreated, established HIV-1 infection (n=149) and uninfected persons (n=497). Furthermore, we examined Xpert VL as a qualitative test in seroconverter specimens with early (n=20) and later (n=68) acute infections. At 1.80 log10 copies/mL, 95 % of specimens had quantifiable virus using Xpert VL. Xpert and Cobas VLs were highly correlated (R2=0.994). The proportion of seroconverter specimens with virus detected using Cobas and with Xpert VL was not statistically different (p=0.0578). Xpert VL detected 97.9 % of established infections, and specificity was 99.80 % (95 % CI 98.87%–99.99%). Xpert VL detected 90 % and 98.5 % of early and later acute infections, respectively. If approved, Xpert VL could allow U.S. laboratories that cannot bring on large, complex testing platforms to conduct HIV monitoring. An approval for diagnostic use may provide timely identification of HIV infections. [ABSTRACT FROM AUTHOR]

Prompt determination of HIV infection status is critical during follow-up visits for patients taking pre-exposure prophylaxis (PrEP) medication. Those who are uninfected can then continue safely taking PrEP, and those few who have acquired HIV infection can initiate an effective treatment regimen. However, a few recent cases have been reported of ambiguous HIV test results using common testing algorithms in PrEP patients. We review published reports of such cases and testing options that can be used to clarify true HIV status in these situations. In addition, we review the benefits and risks of 3 antiretroviral management options in these patients: (1) continue PrEP while conducting additional HIV tests, (2) initiate antiretroviral therapy for presumptive HIV infection while conducting confirmatory tests, or (3) discontinue PrEP to reassess HIV status after a brief antiretroviral-free interval. A clinical consultation resource is also provided. [ABSTRACT FROM AUTHOR]

Background: Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at -20 degrees C for prolonged periods (0.5-4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4 degrees C for 1 year.Methods: A total of 40 DBSs were prepared from residual diagnostic specimens collected from HIV subtype B-infected persons and were stored with desiccant at 4 degrees C. Total nucleic acids were extracted after 1 year using a modification of the Nuclisens assay. Resistance testing was performed using the ViroSeq HIV-1 assay and an in-house nested RT-PCR method validated for HIV-1 subtype B that amplifies a smaller (1 kb) pol fragment.Results: Using the ViroSeq assay, only 23 of the 40 (57.5%) DBS specimens were successfully genotyped; 22 of these specimens had plasma viraemia >10,000 RNA copies/mL. When the specimens were tested using the in-house assay, 38 of the 40 DBSs (95%) were successfully genotyped. Overall, resistance genotypes generated from the DBSs and plasma were highly concordant.Conclusions: We show that drug resistance genotyping from DBSs stored at 4 degrees C with desiccant is highly efficient but requires the amplification of small pol fragments and the use of an in-house nested PCR protocol with quality-controlled reagents. These findings suggest that 4 degrees C may represent a suitable temperature for long-term storage of DBSs. [ABSTRACT FROM AUTHOR]