NONO Detects the Nuclear HIV Capsid to Promote cGAS-Mediated Innate Immune Activation.

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.

Sensitivity and specificity of the new Bio-Rad HIV screening test, Access HIV combo V2.

Diagnosing of human immunodeficiency virus (HIV) types 1 and 2 requires a screening with a highly sensitive and specific enzyme immunoassay and a low detection limit for the HIV-1 p24 antigen to minimize the diagnostic window. The objective of the study was to determine the sensitivity, specificity, and p24 limit of detection of the Access HIV combo V2 assay. Retrospective part of sensitivity: 452 HIV-1 positive samples from 403 chronic (9 different HIV-1 group M subtypes, 22 different HIV-1 group M CRFs, and 3 HIV-1 group O), 49 primary HIV-1 infections, 103 HIV-2 positive samples assessed at Pitié-Salpêtrière Hospital, 600 untyped HIV-1, 10 subtype-D, and 159 untyped HIV-2 samples assessed in Bio-Rad Laboratories. Prospective part of clinical specificity: all consecutive samples in two blood donor facilities and Pitié-Salpêtrière (6,570 patients) tested with Access HIV combo V2 and respectively Prism HIV O Plus (Abbott) or Architect HIV Ag/Ab Combo (Abbott) for Ag/Ab screening, and Procleix Ultrio (Gen Probe) for HIV RNA screening. Limit of detection for p24 antigen was assessed on recombinant virus-like particles (10 HIV-1 group M subtypes/CRFs, HIV-1 group O). Sensitivity [95% confidence interval (CI)] of Access HIV combo V2 was 100% (99.63-100) for HIV-1 chronic infection, 100% (98.55-100) for HIV-2 chronic infection, and 100% (93.00-100) for HIV-1 primary infection. Specificity (95% CI) was 99.98 (99.91-100). Limit of detection for p24 antigen was around 0.43 IU/mL [interquartile range (0.38-0.56)], and consistent across the 11 analyzed subtypes/CRFs. Hence, with both high sensitivity and specificity, Access HIV combo V2 is a suitable screening assay for HIV-1/2 infection. IMPORTANCE: Bio-Rad is one of the leading human immunodeficiency virus (HIV) screening test manufacturers. This laboratory released in 2021 their new version of the Access combo HIV test. However, to date, there have been no studies regarding its performance, especially its limit of detection of the diverse p24 antigen. We present the sensitivity (chronic and primary HIV-1 infection and HIV-2 chronic infection), specificity (blood donors and hospitalized patients), and raw data for the p24/seroconversion panels the manufacturer gave to the European agencies.

HIV-2 inhibits HIV-1 gene expression via two independent mechanisms during cellular co-infection.

IMPORTANCE: Twenty-five years after the first report that HIV-2 infection can reduce HIV-1-associated pathogenesis in dual-infected patients, the mechanisms are still not well understood. We explored these mechanisms in cell culture and showed first that these viruses can co-infect individual cells. Under specific conditions, HIV-2 inhibits HIV-1 through two distinct mechanisms, a broad-spectrum interferon response and an HIV-1-specific inhibition conferred by the HIV-2 TAR. The former could play a prominent role in dually infected individuals, whereas the latter targets HIV-1 promoter activity through competition for HIV-1 Tat binding when the same target cell is dually infected. That mechanism suppresses HIV-1 transcription by stalling RNA polymerase II complexes at the promoter through a minimal inhibitory region within the HIV-2 TAR. This work delineates the sequence of appearance and the modus operandi of each mechanism.

HIV envelope tail truncation confers resistance to SERINC5 restriction.

SERINC5 is a potent lentiviral restriction factor that gets incorporated into nascent virions and inhibits viral fusion and infectivity. The envelope glycoprotein (Env) is a key determinant for SERINC restriction, but many aspects of this relationship remain incompletely understood, and the mechanism of SERINC5 restriction remains unresolved. Here, we have used mutants of HIV-1 and HIV-2 to show that truncation of the Env cytoplasmic tail (ΔCT) confers complete resistance of both viruses to SERINC5 and SERINC3 restriction. Critically, fusion of HIV-1 ΔCT virus was not inhibited by SERINC5 incorporation into virions, providing a mechanism to explain how EnvCT truncation allows escape from restriction. Neutralization and inhibitor assays showed ΔCT viruses have an altered Env conformation and fusion kinetics, suggesting that EnvCT truncation dysregulates the processivity of entry, in turn allowing Env to escape targeting by SERINC5. Furthermore, HIV-1 and HIV-2 ΔCT viruses were also resistant to IFITMs, another entry-targeting family of restriction factors. Notably, while the EnvCT is essential for Env incorporation into HIV-1 virions and spreading infection in T cells, HIV-2 does not require the EnvCT. Here, we reveal a mechanism by which human lentiviruses can evade two potent Env-targeting restriction factors but show key differences in the capacity of HIV-1 and HIV-2 to exploit this. Taken together, this study provides insights into the interplay between HIV and entry-targeting restriction factors, revealing viral plasticity toward mechanisms of escape and a key role for the long lentiviral EnvCT in regulating these processes.

Making sense of HIV innate sensing.

Innate sensing of HIV is important in host control and pathogenesis. In this issue of Immunity, Lahaye et al. (2013) demonstrate that HIV capsid-cyclophilin A interactions affect viral cDNA sensing by the DNA sensor cCAS and contribute to differential pathogenesis of HIV-1 and HIV-2.

The capsids of HIV-1 and HIV-2 determine immune detection of the viral cDNA by the innate sensor cGAS in dendritic cells.

HIV-2 is less pathogenic for humans than HIV-1 and might provide partial cross-protection from HIV-1-induced pathology. Although both viruses replicate in the T cells of infected patients, only HIV-2 replicates efficiently in dendritic cells (DCs) and activates innate immune pathways. How HIV is sensed in DC is unknown. Capsid-mutated HIV-2 revealed that sensing by the host requires viral cDNA synthesis, but not nuclear entry or genome integration. The HIV-1 capsid prevented viral cDNA sensing up to integration, allowing the virus to escape innate recognition. In contrast, DCs sensed capsid-mutated HIV-1 and enhanced stimulation of T cells in the absence of productive infection. Finally, we found that DC sensing of HIV-1 and HIV-2 required the DNA sensor cGAS. Thus, the HIV capsid is a determinant of innate sensing of the viral cDNA by cGAS in dendritic cells. This pathway might potentially be harnessed to develop effective vaccines against HIV-1.

Limited HIV-2 reservoirs in central-memory CD4 T-cells associated to CXCR6 co-receptor expression in attenuated HIV-2 infection.

The low pathogenicity and replicative potential of HIV-2 are still poorly understood. We investigated whether HIV-2 reservoirs might follow the peculiar distribution reported in models of attenuated HIV-1/SIV infections, i.e. limited infection of central-memory CD4 T lymphocytes (TCM). Antiretroviral-naive HIV-2 infected individuals from the ANRS-CO5 (12 non-progressors, 2 progressors) were prospectively included. Peripheral blood mononuclear cells (PBMCs) were sorted into monocytes and resting CD4 T-cell subsets (naive [TN], central- [TCM], transitional- [TTM] and effector-memory [TEM]). Reactivation of HIV-2 was tested in 30-day cultures of CD8-depleted PBMCs. HIV-2 DNA was quantified by real-time PCR. Cell surface markers, co-receptors and restriction factors were analyzed by flow-cytometry and multiplex transcriptomic study. HIV-2 DNA was undetectable in monocytes from all individuals and was quantifiable in TTM from 4 individuals (median: 2.25 log10 copies/106 cells [IQR: 1.99-2.94]) but in TCM from only 1 individual (1.75 log10 copies/106 cells). HIV-2 DNA levels in PBMCs (median: 1.94 log10 copies/106 PBMC [IQR = 1.53-2.13]) positively correlated with those in TTM (r = 0.66, p = 0.01) but not TCM. HIV-2 reactivation was observed in the cells from only 3 individuals. The CCR5 co-receptor was distributed similarly in cell populations from individuals and donors. TCM had a lower expression of CXCR6 transcripts (p = 0.002) than TTM confirmed by FACS analysis, and a higher expression of TRIM5 transcripts (p = 0.004). Thus the low HIV-2 reservoirs differ from HIV-1 reservoirs by the lack of monocytic infection and a limited infection of TCM associated to a lower expression of a potential alternative HIV-2 co-receptor, CXCR6 and a higher expression of a restriction factor, TRIM5. These findings shed new light on the low pathogenicity of HIV-2 infection suggesting mechanisms close to those reported in other models of attenuated HIV/SIV infection models.

Evaluation of Geenius HIV-1/2 Confirmatory Assay for the confirmatory and differential diagnosis of HIV-1/HIV-2 in Japan and reliability of the Geenius Reader in the diagnosis of HIV-2.

BACKGROUND: NEW LAV BLOT I and II (LAV I and LAV II), they were only option for human immunodeficiency virus (HIV) confirmatory test, following HIV screening test using HIV Ag/Ab combination test in Japan. We evaluated the performance of Geenius HIV-1/2 Confirmatory Assay (Geenius), both as a confirmatory test and for differentiation between HIV-1 and HIV-2, in comparison with LAV I and LAV II. METHODS: Eighty-nine HIV-1-positive plasma specimens, one anti-HIV-1 low-titer performance panel, 10 seroconversion panels, and two anti-HIV-1/2 combo performance panels were tested. The results were read with the Geenius Reader and by visual reading. RESULTS: All 89 HIV-1-positive plasma specimens were identified as HIV-1-positive using Geenius; this 100% success rate was superior to that with LAV I (95.5% using WHO criteria, 98.9% using CDC criteria). The HIV-1-positive specimens showed low cross-reactivity with HIV-2 lines in Geenius. The sensitivity of Geenius for HIV-1 detection was the same as or greater than that of LAV I, but less than that of Genscreen HIV Ag-Ab ULT, in our analysis of the commercial performance and seroconversion panels. In contrast, five of the 13 HIV-2-positive specimens that had been identified as HIV-positive untypable by visual reading because of their cross-reactivity to HIV-1 lines were successfully identified by the Geenius Reader as HIV-2-positive with cross-reactivity. CONCLUSIONS: Geenius provides strong performance for HIV confirmatory tests and HIV-1 differentiation tests. However, when visual reading is used, its performance in HIV-2 differentiation is less reliable. Because HIV-2 infection has been sporadically reported in Japan, the use of the Geenius Reader is preferable to ensure more reliable HIV-1/HIV-2 differentiation.

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 µg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 µg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 µg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

Assessing donor suitability for blood donation: Utility of Geenius HIV 1/2 confirmatory assay.

BACKGROUND: Blood donor care and blood safety require a quick and accurate decision on the presence or absence of Human Immunodeficiency Virus (HIV) infection, based on the proper selection of blood donors, serological and molecular HIV testing as well as western blot test. The aim was investigating the possibility of inclusion of Geenius HIV 1/2 Confirmatory Assay in blood donor testing algorithm in order to shorten test time and decrease the number of indeterminate results. METHODS: A total of 75 archived serum/plasma samples were tested. Their previous serological and molecular HIV results were: 3 negative samples, 7 positive samples, 65 serological indeterminate or positive but confirmatory testing and NAT negative samples. RESULTS: Geenius assay confirmed the presence of antibodies in all blood donors with HIV positive serology and Nucleic Acid Testing (NAT). HIV-1 gp160 and gp41 antibodies were detected in these donors, while p31 and p24 antibodies were not detected in two and three donors, respectively. HIV-2 antibodies gp36 and gp140 were not found. Blood donor with HIV indeterminate or positive serology but negative confirmatory testing and NAT, were negative in Geenius assay. Conclusion The results obtained are consistent with western blot results. The assay proved simple and quick to perform. Studies have confirmed the possibility of introducing Bio-Rad Geenius into a routine blood donor testing protocol.

[Experience of the National HIV-AIDS Reference Center of Turkey, in the Transition to the New HIV Diagnostic Algorithm; Comparative Analysis of Line-Immunoassay Test and Bio-Rad Geenius™ HIV-1/2 Antibody Confirmatory Assay]. / Yeni HIV Tani Algoritmasina Geçis Sürecinde Ulusal HIV-AIDS Referans Merkezi'nin Deneyimi: Line-Immunoassay Test ve Bio-Rad Geenius™ HIV-1/2 Antikor Ayirt Edici Hizli Dogrulama Testleri Karsilastirmali Analizi.

Shortly after the first detection of human immundeficiency virus (HIV) infection in USA in 1981, the number of cases have increased gradually from all around the world. Turkey's high capacity for tourism and the unique geographic location extending between Europe and Asia, provides convenience for the passage of individuals across the countries and sexually transmitted infections including HIV, as well. According to the official data of the Ministry of Health; there are 25809 HIV positive and 1958 AIDS cases as of November 30, 2020, after the epidemic started in 1985 in Turkey. Despite the decrease in the number of newly detected HIV cases as a result of serious measures taken for the transmission of infection worldwide, the increase in the number of cases still continues in our country. Shortening the reporting period and starting treatment as soon as possible in the diagnosis of infection is critical for the control of the epidemic. For this purpose, Centers for Disease Control and Prevention (CDC) published a new test algorithm in 2010, which suggested the use of the Geenius™ HIV ½ supplemental assay test instead of western blot tests, which have been used for many years to verify HIV screening test positivity. In this study, we aimed to report the experience of the National HIV-Acquiner Immundeficiency Syndrome(AIDS) and Viral Hepatitis Reference Laboratories of Turkey in the first year of transition to the new HIV algorithm and to evaluate the diagnostic performance of Geenius™ HIV ó and line immunassay (LIA) s. A total of 2090 anti-HIV positive patient sera sent to National HIV-AIDS and Viral Hepatitis Reference Laboratories of Turkey, Ankara for HIV confirmation were included in the study. All samples were retested with a fourth-generation enzyme linked immunosorbent assay (ELISA) test (VIDAS® HIV-1/2 Duo Ultra assay, BioMerieux, France) followed by the confirmatory tests; Geenius™ HIV 1/2 confirmatory assay (BioRad, Redmond, WA) and Line-immunoassay (INNO-LIA HIV ½ Score, Fujirebio, reverse transcriptase polymerase chain reaction (RT-PCR) (artus HI Virus-1 RT-PCR, Qiagen, Germany) test and in-house HIV-2 RNA and proviral DNA PCR. The sensitivity, specificity, and the agreement of the each assay were compared. Cohen's Kappa analysis was used for the evaluation of the agreement between the tests. According to the new algorithm which recommended Geenius™ test besides HIV-1 RNA test, 1707 (81.7%) HIV-1 positive samples were identified. Of these samples; 95.9% and 95.02% were identified as HIV-1 positive by GeeniusTM and INNO-LIA, respectively. However, 2.5% of the positive samples were negative with Geenius™ and 3.5% with INNO-LIA. One and a half percentage (1.5%) of these samples were detected with Geenius™ and 1.4% with INNO-LIA as indeterminant. When all the positive samples determined with ELISA were evaluated; it was detected that,1.3% were indeterminate by Geenius™ test and 2.4% by the INNO-LIA test. When the INNO-LIA test was regarded as the gold standard method; sensitivity, specificity, positive predictive and negative predictive values of the Geenius™ test were as follows; 99.7%, 96.1%, 98.9%, and 99.1%. The agreement between INNO-LIA and Geenius™ tests was found to be 98.95% (κ= 0.969; very good). When the Geenius™ and HIV-1 PCR tests were evaluated together for the confirmation; the sensitivities of Geenius™ and INNO-LIA tests were 99.8% and 98.3%, specificities were 89.8% and 85.3%, respectively. Slight positive bands were detected in the gp36 or gp140 bands, the HIV-2 specific envelope proteins, were detected in seven samples, However, the positivity disappeared after the dilution of the samples and it was accepted as false positivite reaction due to the absence of HIV-2 RNA and proviral DNA in these samples. In conclusion; we concluded that Geenius ™ and INNO-LIA tests have a perfect agreement in HIV diagnosis and due to the rapid and reliable results provided for the HIV test protocol, Geenius™ test can be used safely as an alternative to the immunoblot tests. HIV-1 RNA testing must be performed in all HIV confirmation centers in order to detect acute HIV cases in the fast and early period which are the main reason for the updates in HIV diagnosis.

eCD4-Ig Limits HIV-1 Escape More Effectively than CD4-Ig or a Broadly Neutralizing Antibody.

The engineered antibody-like entry inhibitor eCD4-Ig neutralizes every human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus isolate it has been tested against. The exceptional breadth of eCD4-Ig derives from its ability to closely and simultaneously emulate the HIV-1 receptor CD4 and coreceptors, either CCR5 or CXCR4. Here we investigated whether viral escape from eCD4-Ig is more difficult than that from CD4-Ig or the CD4-binding site antibody NIH45-46. We observed that a viral swarm selected with high concentrations of eCD4-Ig was increasingly resistant to but did not fully escape from eCD4-Ig. In contrast, viruses selected under the same conditions with CD4-Ig or NIH45-46 fully escaped from those inhibitors. eCD4-Ig-resistant viruses acquired unique changes in the V2 apex, V3, V4, and CD4-binding regions of the HIV-1 envelope glycoprotein (Env). Most of the alterations did not directly affect neutralization by eCD4-Ig or neutralizing antibodies. However, alteration of Q428 to an arginine or lysine resulted in markedly greater resistance to eCD4-Ig and CD4-Ig, with correspondingly dramatic losses in infectivity and greater sensitivity to a V3 antibody and to serum from an infected individual. Compensatory mutations in the V3 loop (N301D) and in the V2 apex (K171E) partially restored viral fitness without affecting serum or eCD4-Ig sensitivity. Collectively, these data suggest that multiple mutations will be necessary to fully escape eCD4-Ig without loss of viral fitness.IMPORTANCE HIV-1 broadly neutralizing antibodies (bNAbs) and engineered antibody-like inhibitors have been compared for their breadths, potencies, and in vivo half-lives. However, a key limitation in the use of antibodies to treat an established HIV-1 infection is the rapid emergence of fully resistant viruses. Entry inhibitors of similar breadths and potencies can differ in the ease with which viral escape variants arise. Here we show that HIV-1 escape from the potent and exceptionally broad entry inhibitor eCD4-Ig is more difficult than that from CD4-Ig or the bNAb NIH45-46. Indeed, full escape was not observed under conditions under which escape from CD4-Ig or NIH45-46 was readily detected. Moreover, viruses that were partially resistant to eCD4-Ig were markedly less infective and more sensitive to antibodies in the serum of an infected person. These data suggest that eCD4-Ig will be more difficult to escape and that even partial escape will likely extract a high fitness cost.

Routine HIV Test Results in 6 US Clinical Laboratories Using the Recommended Laboratory HIV Testing Algorithm With Geenius HIV 1/2 Supplemental Assay.

BACKGROUND: Geenius HIV 1/2 Supplemental Assay (Geenius; Bio-Rad Laboratories) is the only Food and Drug Administration-approved HIV-1/HIV-2 antibody differentiation test for the second step in the HIV laboratory testing algorithm. We characterized the occurrence of true HIV-1 and HIV-2 infections as well as false results in 6 US clinical laboratories using Geenius. METHODS: We examined routine HIV testing outcome data from the time the laboratories began using the algorithm with Geenius until September 30, 2017. We calculated the positive predictive value for Geenius HIV-1 and HIV-2 reactivity separately. RESULTS: Of 5,046,684 specimens tested, 41,791 had reactive antigen/antibody test results. Most specimens with reactive antigen/antibody results were HIV-1 antibody-positive established infections (n = 32,421), 1,865 of which also had indeterminate HIV-2 bands present. Ninety-three specimens were HIV-2 antibody positive or untypable for HIV-1/HIV-2 antibody. Acute HIV-1 infections were found in 528 specimens; 881 specimens lacked the nucleic acid test to determine the possibility of acute HIV-1 infection. False-positive antigen/antibody test results were present in 7505 specimens. Few specimens (n = 363) had false-positive antigen/antibody results with indeterminate Geenius and negative HIV-1 nucleic acid test results. The positive predictive values of Geenius reactivity were 99.4% for HIV-1 and 4.3% for HIV-2. CONCLUSIONS: Routine testing using the laboratory testing algorithm with Geenius resulted in most specimens resolving as HIV negative or HIV-1 positive. The occurrence of indeterminate HIV-2 bands with a Geenius final assay interpretation of HIV-1 positive was more common than true HIV-2 infections. Reporting indeterminate HIV-2 results in this situation may cause confusion with interpreting HIV infection status.

Performance of an Alternative Laboratory-Based HIV Diagnostic Testing Algorithm Using HIV-1 RNA Viral Load.

BACKGROUND: Since 2014, the recommended algorithm for laboratory diagnosis of HIV infection in the United States has consisted of an HIV-1/2 antigen/antibody (Ag/Ab) test followed by an HIV-1/2 antibody (Ab) differentiation test and, if necessary, a diagnostic HIV-1 nucleic acid test to resolve discordant or indeterminate results. METHODS: Using stored specimens from persons seeking HIV testing who had not received a previous diagnosis or treatment, we compared the performance of a 3-step alternative algorithm consisting of an Ag/Ab test followed by a quantitative HIV-1 RNA viral load assay and, if viral load is not detected, an Ab differentiation test, to that of the recommended algorithm. We calculated the sensitivity and specificity of 5 Ag/Ab tests and the proportion of specimens correctly classified by the alternative algorithm compared with the recommended algorithm. Results were examined separately for specimens classified as early infection, established infection, and false-reactive screening. RESULTS: Sensitivity and specificity were similar among all Ag/Ab tests. Viral load quantification correctly classified all specimens from early infection, all false-reactive screening specimens, and the majority of specimens from established infection. CONCLUSIONS: Although cost, regulatory barriers, test availability, and the ability to differentiate early from established infection must be considered, this alternative algorithm can potentially decrease the total number of tests performed and reduce turnaround time, thereby streamlining HIV diagnosis and initiation of treatment.

Rapid Testing Algorithm Performance in a Low-Prevalence Environment.

BACKGROUND: The performance of a statewide HIV rapid test algorithm (RTA) in a low-prevalence setting (0.71%) was examined for 3 years. METHODS: An initial rapid screening by HIV-1/2 Ag/Ab Combo test (RT#1) with Ab verification using a second, different rapid test (RT#2) was conducted. Clinic referral was immediate for antigen-only-positive screens. Antibody-positive screens were confirmed by RT#2. Specimens were collected following discordant RTA results (initially Ab-POS by RT#1, but negative on RT#2) and tested in accordance with the current Centers for Disease Control and Prevention/Association of Public Health Laboratories-based HIV diagnostic algorithm supplemented by a quantitative viral load whenever possible. RESULTS: Of 310,785 tests performed, 2400 preliminary positive screens were identified; 2191 (91.8%) confirmed by RT#2. Of 13 Determine Combo AG-POS results identified, only 1 confirmed positive. Of the remaining 196 discordant results, 182 (92.9%) were uninfected, including 13 with AG-POS/AB-POS results. Of 14 true positives (7.1%) identified after discordant RTA results, the average quantitative HIV-1 viral load was 277,385 copies/mL, but 5 (35.7%) of 14 had viral loads <1000 copies/mL. Among the 2191 "presumptive positive" by RTA, 3 false-positive (FP) RTAs were reported (both rapid tests having positive results, while the HIV-1/2 Ag/Ab assay and quantitative HIV-1 viral load showed negative results). CONCLUSIONS: The RTA was effective in predicting true-positive HIV test results and facilitating linkage to care. Discordant results were infrequent. Fingerstick DC Ag detection identified a single early infection. Many discordant cases that were subsequently positive were associated with viral loads <1000 copies/mL.

Comparative multi-assay evaluation of Determine™ HIV-1/2 Ag/Ab Combo rapid diagnostic tests in acute and chronic HIV infection.

In resource-limited or point-of-care settings, rapid diagnostic tests (RDTs), that aim to simultaneously detect HIV antibodies and p24 capsid (p24CA) antigen with high sensitivity, can pose important alternatives to screen for early infections. We evaluated the performance of the antibody and antigen components of the old and novel version of the Determine™ HIV-1/2 Ag/Ab Combo RDTs in parallel to quantifications in a fourth-generation antigen/antibody immunoassay (4G-EIA), p24CA antigen immunoassay (p24CA-EIA), immunoblots, and nucleic acid quantification. We included plasma samples of acute, treatment-naïve HIV-1 infections (Fiebig stages I-VI, subtypes A1, B, C, F, CRF02_AG, CRF02_AE, URF) or chronic HIV-1 and HIV-2 infections. The tests' antigen component was evaluated also for a panel of subtype B HIV-1 transmitted/founder (T/F) viruses, HIV-2 strains and HIV-2 primary isolates. Furthermore, we assessed the analytical sensitivity of the RDTs to detect p24CA using a highly purified HIV-1NL4-3 p24CA standard. We found that 77% of plasma samples from acutely infected, immunoblot-negative HIV-1 patients in Fiebig stages II-III were identified by the new RDT, while only 25% scored positive in the old RDT. Both RDTs reacted to all samples from chronically HIV-1-infected and acutely HIV-1-infected patients with positive immunoblots. All specimens from chronically infected HIV-2 patients scored positive in the new RDT. Of note, the sensitivity of the RDTs to detect recombinant p24CA from a subtype B virus ranged between 50 and 200 pg/mL, mirrored also by the detection of HIV-1 T/F viruses only at antigen concentrations tenfold higher than suggested by the manufacturer. The RTD failed to recognize any of the HIV-2 viruses tested. Our results indicate that the new version of the Determine™ HIV-1/2 Ag/Ab Combo displays an increased sensitivity to detect HIV-1 p24CA-positive, immunoblot-negative plasma samples compared to the precursor version. The sensitivity of 4G-EIA and p24CA-EIA to detect the major structural HIV antigen, and thus to diagnose acute infections prior to seroconversion, is still superior.

The use of the GeeniusTM HIV-1/2 Rapid confirmatory test for the enrolment of patients and blood donors in the WHO Universal Test and Treat Strategy in Cameroon, Africa.

BACKGROUND AND OBJECTIVE: In the WHO Universal test and treat strategy, false-positive HIV blood donors and patients may be unnecessarily put under antiretroviral treatment and false-negative subjects may be lost to follow-up. This study assessed the false positivity rate of the Cameroonian national HIV screening testing algorithm and the benefit of a confirmation test in the enrolment of patients and donors in the HIV care programme. METHODS: We included initial HIV reactive blood donors and patients in a cross-sectional study conducted in two Cameroonian hospitals. Samples were retested according to the Cameroon national algorithm for HIV diagnosis. A positive or discordant sample was retested with the Geenius Bio-Rad HIV 1&2 (Bio-Rad, Marnes-la-Coquette, France) for confirmation. The Geenius HIV-1-positive results with 'poor' profiles were retested for RNA as well as the Geenius indeterminate results. RESULTS: Of the 356 participants, 190/225 (84·4%) patients and 76/131 (58%) blood donors were declared positive with the national algorithm; 257 participants (96·6%) were confirmed HIV-1-positive. The study revealed that about 34/1000 blood donors and patients are false-positive and unnecessarily put on treatment; 89/1000 blood donors and patients declared discordant could have been included immediately in the HIV care programme if confirmatory testing was performed. The second test of the algorithm had a false-negative rate of 3%. Eleven samples (3·1%) were Geenius poor positive and NAT negative. CONCLUSION: The universal test and treat strategy may identify and refer more individuals to HIV care if a third rapid confirmatory test is performed for discordant cases.

Hepatitis A virus and hepatitis E virus prevalence relates to human immunodeficiency virus infection in Japanese male blood donors.

Hepatitis A virus (HAV) has begun to spread globally among men who have sex with men (MSM). Hepatitis E virus (HEV) also may be transmitted through sexual contact among MSM. To assess the current status of these viruses among MSM in Japan, the seroprevalence of both viruses using 503 plasma samples collected between 2009 and 2018 from human immunodeficiency virus (HIV)-positive male donors who were presumed to be mainly MSM was investigated. Our results suggested that HAV may be spreading within this population, as reported elsewhere. By contrast, the spread of HEV was confirmed only among younger HIV-positive donors.

Continuous quality monitoring in the field: an evaluation of the performance of the Fio Deki Reader™ for rapid HIV testing in South Africa.

BACKGROUND: Rapid diagnostic tests (RDTs) are a cornerstone of HIV diagnosis and rely on good quality processing and interpretation, particularly in the era of test and treat. The Deki Reader (Fio Corporation®, Toronto, Ontario, Canada) is a portable device designed specifically for analysing RDTs and was selected for evaluation in South Africa in the context of HIV RDT analysis. METHODS: This study consisted of a laboratory evaluation and two-part field evaluation of the Deki Reader v100, covering two RDT testing algorithms, and an evaluation of the continuous quality monitoring through the Fionet™ web portal. Based on user feedback from the field evaluation, the device underwent hardware and software redesign, and the Deki Reader v200 was evaluated in the laboratory. Ethics approval for this evaluation was obtained from the University of the Witwatersrand Human Research Ethics Committee: M150160. RESULTS: The intra- and inter-device laboratory precision of the Deki Reader v100 were 98.3 and 99.2% respectively, and 99.3 and 100% for the Deki Reader v200. The laboratory concordances compared to standard-of-care reporting were 99.5 and 98.0% for the two respective models, while sensitivity and specificity were 99.5 and 99.4% for the Deki Reader V100 and 100 and 93.1% for the Deki Reader V200 respectively. Screening and confirmatory concordances in the field were 99.3 and 96.5% under algorithm 1 and 99.7 and 100% under algorithm 2. Sensitivity and specificity for the field evaluation were 99.8 and 97.7%. Overall robustness of the device was acceptable and continuous quality monitoring through Fionet™ was feasible. CONCLUSIONS: The Deki Reader provides an option for improved and reliable quality assessment for rapid diagnosis of HIV using RDTs to enhance the quality of healthcare at the point-of-care. However, the introduction of new RDTs and modification of current algorithms necessitates ongoing and agile RDT reader adjustments, which will require cost modelling to ensure sustainability of devices implemented into national HIV programs.

Promoting routine syphilis screening among men who have sex with men in China: study protocol for a randomised controlled trial of syphilis self-testing and lottery incentive.

BACKGROUND: Men who have sex with men (MSM) bear a high burden of syphilis infection. Expanding syphilis testing to improve timely diagnosis and treatment is critical to improve syphilis control. However, syphilis testing rates remain low among MSM, particularly in low- and middle-income countries. We describe the protocol for a randomised controlled trial (RCT) to assess whether provision of syphilis self-testing services can increase the uptake of syphilis testing among MSM in China. METHODS: Four hundred forty-four high-risk MSM will be recruited online and randomized in a 1:1:1 ratio to (1) standard syphilis self-testing arm; (2) a self-testing arm program enhanced with crowdsourcing and a lottery-based incentive, and (3) a standard of care (control). Self-testing services include a free syphilis self-test kit through the mail at monthly intervals. Participants in the lottery incentive arm will additionally receive health promotion materials generated from an open crowdsourcing contest and be given a lottery draw with a 10% chance to win 100 RMB (approximately 15 US Dollars) upon confirmed completion of syphilis testing. Syphilis self-test kits have step-by-step instructions and an instructional video. This is a non-blinded, open-label, parallel RCT. Participants in each arm will be followed-up at three and 6 months through WeChat (a social media app like Facebook messenger). Confirmation of syphilis self-test use will be determined by requiring participants to submit a photo of the used test kit to study staff via secure data messaging. Both self-testing and facility-based testing will be ascertained by sending a secure photographic image of the completed kit through an existing digital platform. The primary outcome is the proportion of participants who tested for syphilis in the past 3 months. DISCUSSION: Findings from this study will provide much needed insight on the impact of syphilis self-testing on promoting routine syphilis screening among MSM. The findings will also contribute to our understanding of the safety, effectiveness and acceptability of syphilis self-testing. These findings will have important implications for self-testing policy, both in China and internationally. TRIAL REGISTRATION: ChiCTR1900022409 (10 April, 2019).