Fourth-generation Immunoassay Research Articles

Point-of-care nucleic acid testing - a step forward in controlling the HIV epidemic: A review.

The HIV/AIDS epidemic, with 85.6 million infections and 40.4 million AIDS-related deaths globally, remains a critical public health challenge. Current diagnostic methods, primarily fourth-generation immunoassays, have limitations due to their long window periods, and most viral load assays require centralized testing protocols that result in delays, especially in remote regions. Point-of-care (POC) nucleic acid amplification testing (NAAT) presents a transformative approach by reducing the window period for detection to one week and significantly shortening turnaround times for viral load monitoring. This review highlights the clinical utility of POC NAAT for acute HIV infection diagnosis, its role in timely combination antiretroviral therapy adjustments, and its potential to reduce the basic reproduction number (R0), a critical threshold for suppressing the epidemic. By improving early detection and facilitating faster clinical decisions, POC NAAT enhances the effectiveness of HIV prevention and treatment programmes, particularly in high-risk and remote communities, and supports the global effort to achieve the ambitious UNAIDS 95-95-95 targets.

Point-of-Care Neurofilament Light Detection for Neurological Disease Monitoring

Neurofilament light chain (NfL) is a cytoplasmic protein predominantly found in neurons. Its primary role is to provide structural support to myelinated axons during their growth and maintenance. When axons are damaged due to injury or neurodegenerative processes, NfL levels increase in various bodily fluids, including the extracellular matrix, cerebrospinal fluid (CSF), and blood. The clinical significance of NfL lies in its association with neurological conditions. High levels of NfL have been observed in several contexts: Medical Emergencies: In acute medical emergencies such as stroke and head injuries, elevated NfL levels are detected. This rapid response makes NfL a potential biomarker for assessing acute neuronal damage.Neurodegenerative Diseases: Chronic neurodegenerative conditions, including multiple sclerosis (MS) and Alzheimer's disease, are characterized by progressive neuronal loss. NfL levels are significantly elevated in these diseases, reflecting ongoing axonal damage.Monitoring Neurodegeneration: Utilizing NfL as a monitoring tool has gained attention. Its presence in easily accessible bodily fluids (such as blood) makes it valuable for creating point-of-care mechanisms. By tracking NfL levels over time, clinicians can assess disease progression, treatment response, and overall neuronal health. Current NfL monitoring techniques such as Fourth-Generation Immunoassays (SIMOA or Quanterix). These highly sensitive assays allow quantification of NfL even at low levels. They are particularly useful for measuring NfL in plasma and serum. However, they require specialized equipment and trained personnel, limiting their widespread adoption. Microfluidic Cartridge-Based Automated Immunoassay Platforms such as ELLA have also been employed. While these platforms offer automated NfL measurement more conveniently, they also require expensive equipment and proper sample transportation.In this proposal, we introduce a novel Point-of-Care (PoC) mechanism designed for the rapid detection of Neurofilament Light Chain (NfL) in blood plasma. Our approach aims to eliminate the need for sample transportation, reduce procedural delays, and improve turnaround time. We have developed an Electrochemical Impedance Spectroscopy (EIS) platform specifically tailored for quantifying NfL levels within the physiological range in plasma. By leveraging the chemical interactions between the NfL antibody and antigen, we detect changes in impedance at the electrode-electrolyte interface as a factor of the chemical interaction. To validate our platform, we conducted spike and recovery experiments. These experiments illustrate the platform's ability to detect NfL in plasma samples reliably. In summary, our proposed PoC mechanism represents a significant advancement in NfL monitoring for neurodegenerative diseases. By combining accessibility, precision, and rapidity, we aim to enhance patient care and disease management.

Internal quality control for HIV testing of blood donors - Dried tube specimen as a cost-effective alternative.

An important aspect of ensuring blood safety is the performance of mandatory serological testing for transfusion transmissible infections. The practice of internal quality control (IQC) in blood banks in India is nonuniform, especially the use of third-party materials. Cited reasons are cost, lack of access to control materials, and need for deep-freezers for storage, if prepared in-house. Validation of dried tube specimen (DTS) from HIV-positive plasma as a low-cost, stable material for use as IQC material in blood banks. Fresh-frozen plasma (FFP) prepared from four HIV-positive blood-donors were pooled. Equal numbers of seronegative FFPs were pooled. Twenty microlitre aliquots of plasma were made in micro-centrifuge tubes and air-dried overnight at room-temperature. These were stored in 2-8°C refrigerators and tested once weekly for 6 months on multiple platforms with different detection principles: Rapid tests, second-generation enzyme-linked immunosorbent assay (ELISA), fourth-generation ELISA, and fourth-generation Chemiluminescence immunoassay. The protocol was sustained over the next 6 months with decreased testing frequency to study the extended stability of DTS. A total of 139 positive-DTS and 139 negative-DTS were tested with 100% samples showing consistent results on all platforms over 1 year. There was mild deterioration in reaction strengths, which did not interfere in result interpretations. Plasma in form of DTS maintained stability when stored at 2-8°C for 1 year. This provides evidence that DTS can be a modality for the production of cost-effective, stable, in-house control material for resource-restricted countries.

Seven day pre-analytical stability of serum and plasma neurofilament light chain

Neurofilament light chain (NfL) has emerged as a biomarker of neuroaxonal damage in several neurologic conditions. With increasing availability of fourth-generation immunoassays detecting NfL in blood, aspects of pre-analytical stability of this biomarker remain unanswered. This study investigated NfL concentrations in serum and plasma samples of 32 patients with neurological diagnoses using state of the art Simoa technology. We tested the effect of delayed freezing of up to 7 days and statistically determined stability and validity of measured concentrations. We found concentrations of NfL in serum and plasma to remain stable at room temperature when processing of samples is delayed up to 7 days (serum: mean absolute difference 0.9 pg/mL, intraindividual variation 1.2%; plasma: mean absolute difference 0.5 pg/mL, intraindividual variation 1.3%). Consistency of these results was nearly perfect for serum and excellent for plasma (intraclass correlation coefficients 0.99 and 0.94, respectively). In conclusion, the soluble serum and plasma NfL concentration remains stable when unprocessed blood samples are stored up to 7 days at room temperature. This information is essential for ensuring reliable study protocols, for example, when shipment of fresh samples is needed.

HIV serology false positivity among expatriates from Africa: a screening dilemma.

HIV prevalence in Oman is low (<5 %); however, 45 % of the population are expatriates, including a portion originating from countries with high HIV prevalence (>5 %). HIV screening is performed at regional public health laboratories as part of a medical fitness programme for residency applicants. We conducted a retrospective evaluation of indeterminate serology results from 11 females of African origin, aged 21–43 years. Serology testing for HIV was conducted according to the national Oman algorithm: fourth-generation immunoassays (Bio-Rad GS HIV Combo Ag/Ab EIA, Siemens Enzygnost HIV Integral 4, Abbott ARCHITECT HIV Ag/Ab Combo, Roche Elecsys HIV Combi PT, bioMérieux VIDAS HIV DUO QUICK), confirmatory assays (Geenius HIV 1/2 Confirmatory, INNO-LIA HIV I/II Score) and PCR testing. Confirmatory testing to resolve indeterminate results was conducted with available samples for five patients using a combination of immunoassays, confirmatory assays, PCR/PERT and pro-viral DNA levels, at three external laboratories; Roche Diagnostics (Germany), Swiss National Laboratory (Switzerland) and Barts Health NHS Trust (UK). Nineteen serum, 15 plasma and two whole-blood samples were analysed. Nine of ten patients analysed on Bio-Rad and Siemens immunoassays were highly reactive; seven were highly reactive on the Abbott assay. Eight of nine patients tested with the Roche assay were negative. Three of four patients tested on the bioMérieux assay were negative. Five patients underwent confirmatory testing at external laboratories; all were negative by HIV-RNA or pro-viral DNA testing. In conclusion, HIV-RNA and pro-viral DNA testing is recommended for HIV screening of individuals from high-prevalence regions coming to low-prevalence regions.

Impact of schooling in the HIV/AIDS prevalence among Brazilian transgender women.

Objective Discrimination and bullying are common conditions among LGBT people. During schooling, these practices compromising education. The aim of this study is to evaluate educational attainment among Brazilian transgender women (TW) and how their education level affects the risk of HIV infection. Study design a cross-sectional population-based study. Subjects and methods 95 adult TW were selected. Information concerning verbal and physical aggression, school dropout, school years (SY), and educational level were assessed. HIV status was screened using a fourth-generation immunoassay followed by western blot testing. Results The mean of SY was 9.1 ± 3.8 ys. The mean age at school dropout was 16.3 ± 3.4 ys old. Verbal aggression was reported by 83%, physical by 48%, and 18% of the TW dropped out school immediately after being physically assaulted. Participants who suffered physical aggression attended school for almost 4 years less than those participants who did not suffer this abuse (OR = -3.96, p < 0.0001). A similar result was found for verbal aggression (OR = -4.35; p < 0.0001). HIV/AIDS prevalence was 18% (n = 17). The mean of SY among HIV/AIDS positive and negative individuals were 6.8 ± 43 versus 9.7 ± 3, respectively (p = 0.004). Lower education was associated with higher frequency of HIV/AIDS among TW and this relationship was sustained after adjustment for injectable drug use and sex work (OR = 0.79, p = 0.04). Conclusion Among Brazilian TW, lower education level was a risk factor associated with HIV. The reasons for low schooling among TW are multifactorial, but verbal and physical harassment strongly contribute for it.

Primary HIV Infection: Clinical Presentation, Testing, and Treatment.

The purpose of this review was to provide current data on clinical presentation, diagnosis, and treatment of primary HIV infection (PHI). In 65 to 95% of cases, PHI causes acute retroviral syndrome presenting with unspecific flu-like symptoms. Symptomatic PHI was associated with a faster clinical and immunological progression of HIV infection. Point-of-care tests remain less sensitive than fourth-generation immunoassays (IA) in PHI, especially after tenofovir-based prophylaxis use. Early antiretroviral treatment (ART) started during PHI prevents HIV transmission and decreases viral and immunological reservoir constitution. Recommended ART regimens in PHI are combinations of tenofovir and emtricitabine with either darunavir/ritonavir, or dolutegravir. Starting ART the earliest is highly recommended for clinical, virological, immunological, and public health benefits. Reducing HIV reservoir constitution in PHI may optimize potential opportunities for future functional cure.

Misdiagnosis of HIV infection during a South African community‐based survey: implications for rapid HIV testing

Introduction: We describe the overall accuracy and performance of a serial rapid HIV testing algorithm used in community-based HIV testing in the context of a population-based household survey conducted in two sub-districts of uMgungundlovu district, KwaZulu-Natal, South Africa, against reference fourth-generation HIV-1/2 antibody and p24 antigen combination immunoassays. We discuss implications of the findings on rapid HIV testing programmes. Methods: Cross-sectional design: Following enrolment into the survey, questionnaires were administered to eligible and consenting participants in order to obtain demographic and HIV-related data. Peripheral blood samples were collected for HIV-related testing. Participants were offered community-based HIV testing in the home by trained field workers using a serial algorithm with two rapid diagnostic tests (RDTs) in series. In the laboratory, reference HIV testing was conducted using two fourth-generation immunoassays with all positives in the confirmatory test considered true positives. Accuracy, sensitivity, specificity, positive predictive value, negative predictive value and false-positive and false-negative rates were determined. Results: Of 10,236 individuals enrolled in the survey, 3740 were tested in the home (median age 24 years (interquartile range 19–31 years), 42.1% males and HIV positivity on RDT algorithm 8.0%). From those tested, 3729 (99.7%) had a definitive RDT result as well as a laboratory immunoassay result. The overall accuracy of the RDT when compared to the fourth-generation immunoassays was 98.8% (95% confidence interval (CI) 98.5–99.2). The sensitivity, specificity, positive predictive value and negative predictive value were 91.1% (95% CI 87.5–93.7), 99.9% (95% CI 99.8–100), 99.3% (95% CI 97.4–99.8) and 99.1% (95% CI 98.8–99.4) respectively. The false-positive and false-negative rates were 0.06% (95% CI 0.01–0.24) and 8.9% (95% CI 6.3–12.53). Compared to true positives, false negatives were more likely to be recently infected on limited antigen avidity assay and to report antiretroviral therapy (ART) use. Conclusions: The overall accuracy of the RDT algorithm was high. However, there were few false positives, and the sensitivity was lower than expected with high false negatives, despite implementation of quality assurance measures. False negatives were associated with recent (early) infection and ART exposure. The RDT algorithm was able to correctly identify the majority of HIV infections in community-based HIV testing. Messaging on the potential for false positives and false negatives should be included in these programmes.

Relevance of cutoff on a 4th generation ELISA performance in the false positive rate during HIV diagnostic in a low HIV prevalence setting

BackgroundDespite the high specificity of fourth-generation enzyme immunoassays (4th-gen-EIA) for screening during HIV diagnosis, their positive predictive value is low in populations with low HIV prevalence. Thus, screening should be optimized to reduce false positive results. ObjectivesThe influence of sample cutoff (S/CO) values by a 4th-gen-EIA with the false positive rate during the routine HIV diagnosis in a low HIV prevalence population was evaluated. Study designA total of 30,201 sera were tested for HIV diagnosis using Abbott Architect® HIV-Ag/Ab-Combo 4th-gen-EIA at a hospital in Spain during 17 months. Architect S/CO values were recorded, comparing the HIV-1 positive results following Architect interpretation (S/CO≥1) with the final HIV-1 diagnosis by confirmatory tests (line immunoassay, LIA and/or nucleic acid test, NAT). ROC curve was also performed. ResultsAmong the 30,201 HIV performed tests, 256 (0.85%) were positive according to Architect interpretation (S/CO≥1) but only 229 (0.76%) were definitively HIV-1 positive after LIA and/or NAT. Thus, 27 (10.5%) of 256 samples with S/CO≥1 by Architect were false positive diagnose. The false positive rate decreased when the S/CO ratio increased. All 19 samples with S/CO ≤10 were false positives and all 220 with S/CO>50 true HIV-positives. The optimal S/CO cutoff value provided by ROC curves was 32.7. No false negative results were found. ConclusionsWe show that very low S/CO values during HIV-1 screening using Architect can result HIV negative after confirmation by LIA and NAT. The false positive rate is reduced when S/CO increases.

Human immunodeficiency virus: Diagnostic approach.

First descriptions of acquired immunodeficiency syndrome appeared in 1981. Four years later the causative agent was cultured which lead to development and production of tests that helped healthcare providers to identify persons living with HIV. Currently, diagnosis of HIV is performed with fourth generation immunoassays (those that detect p24 antigen together with IgM and IgG antibodies to HIV-1 and -2) which if positive need to be followed by an assay that can differentiate between HIV-1 and HIV-2 viruses. The Western blot is no longer used to confirm HIV infections per CDC guidelines. In case there is a positive fourth generation assay but negative differentiation assay, nucleic acid testing for HIV-1 should be performed. This algorithm allows for detection of acute infections. Alternatively, the World Health Organization has algorithms that use rapid testing for diagnosis of HIV infections. This review will describe the evolution of tests and diagnostic algorithms from the 1980s to the current state. Special situations regarding diagnosis will also be discussed.

Current Diagnostic Methods for HIV

Detection of HIV infection is essential for diagnosis and monitoring of the infection. There are different types of diagnostic tools available that are based on detection of HIV-specific antibodies, viral antigen or nucleic acid. Sensitivities and specificities of assays utilized for HIV detection have improved. Newer HIV testing technologies such as third-generation enzyme immunoassay which detect HIV-specific IgG and IgM antibodies, fourth-generation enzyme immunoassay which detect both anti-HIV antibodies and HIV p24 antigen and nucleic acid based tests for HIV RNA have significantly decreased the window period. This review provides an overview of current technologies for the detection and monitoring of HIV infection and recent advances in the field of HIV diagnosis.

HIV avidity index performance using a modified fourth-generation immunoassay to detect recent HIV infections.

Detecting recent HIV infections is important to evaluate incidence and monitor epidemic trends. We aimed to evaluate the diagnostic performance and accuracy of the avidity index (AI) for discriminating for recent HIV infections. We collected serum samples from HIV-1 positive individuals: A) with known date of infection (midpoint in time between last HIV-negative and first HIV-positive test); B) infected for >1 year. Samples were divided into two aliquots: one diluted with phosphate buffered saline (PBS) and the other with 1 M guanidine. Both aliquots were assayed by the Architect HIV Ag/Ab Combo 4th generation assay (Abbott). We compared AI found in recent (RI=<6 months from seroconversion) and established (EI) infections. The diagnostic accuracy was evaluated by receiver operating characteristic (ROC) curve analysis. The proportion of samples misclassified as recent (FRR) was calculated. In total, 647 samples were collected: 455 in group A (51.6% RI and 48.4% EI) and 192 in group B. Among these, sixteen samples were from elite controllers, 294 from treated patients, 328 from patients infected with non-B subtypes. Samples before antiretroviral initiation showed a mean AI significantly lower among RI compared to EI (0.66+0.28 vs. 1.00±0.12; p<0.000). The FRR was 0% using a cut-off of ≤0.70. An extremely low FRR was observed among elite controllers, samples with low VL or CD4. HIV subtype had no impact on AI misclassifications. All individuals in group A reached the AI threshold of 0.80 within 24 months after seroconversion. The AI is an accurate serological marker for discriminating recent from established HIV infections and meets WHO requirements for HIV incidence assays.

Investigation of the Pathophysiology and Diagnostic Methods Through a Case of Acute HIV Infection in which the Western Blot Method Remained Negative Over the Long Term and the 4th Generation Screening Assay Became Negative for a Certain Period of Time after Initiating Antiretroviral Therapy

A man in his early twenties previously visited a hospital because of fever in October 2012. Laboratory data revealed leukocytopenia, thrombocytopenia and increased serum ferritin, suggesting hemophagocytic syndrome (HPS). During that visit, he tested positive for a 4thGIA, but negative for HIV-1 WB and his result of HIV-1 RNA result was detected invalid because of the presence of some inhibitory material in his RNA preparation. Thereafter, he was diagnosed as having cytomegalovirus-associated HPS treatment was for which initiated. In January 2013, he developed Pneumocystis jirovecii pneumonia, and his HIV-1 RNA viral load was 7.7 × 105 copies/mL in February 2013. Acute HIV infection was suspected, because the HIV-1 WB remained negative. He was started on antiretroviral therapy in April 2013. His 4thGIA was converted to negative in May 2013 and was reconverted to positive in August 2013. HIV-1 WB, however, continued to be indeterminant until February 2014, in which it turned positive for the first time according to the CDC criteria. The genetic analyses of HIV-1 were done on the gag, env, nef and pol region of the HIV-1 gene from the patient. There was no clear element to delay antibody production on the virus side. Preserved specimens of the patient were measured with eight kinds of HIV screening assay. It was thought that the fourth generation assay was positive only by the presence of the antigen until March 2013 because the antibody had not been detected. We encountered a case of acute HIV infection in which the WB result was negative for 10 months after the first positive response of the 4thGIA. The 4thGIA is essential for the early diagnosis and early treatment of HIV infection; therefore, the 4thGIA should be strictly recommended to avoid the use of older generations of immunoassay in the diagnostic guidelines. The role of the WB test should be examined closely from various aspects for use as a confirmatory test under recent laboratory situations in which highly sensitive and specific methods, e.g. the 4th GIA, have become available. In addition, unnecessary confusion due to the diversities of antibody formation should be avoided. The antibody detection tests for HIV are still necessary and indispensable for the confirmation of the disease or the diagnosis of the acute infection stage. Therefore development of a newer antibody measuring method which could achieve an easier operation and should have a higher sensitivity and specificity for HIV confirmation is strongly expected.

Hepatitis B and C Viral Infections in Tihamet Aseer, South-Western Saudi Arabia: Are There Gender Differences?

Background:Hepatitis B virus (HBV) infection is endemic in Saudi Arabia. Many studies have shown varying results in gender differences in HBV and hepatitis C virus (HCV) infection. The objective of this study was to determine if gender differences exist in HBV and HCV infection and to elucidate any related risk factors in Tihamet Aseer, south-western Saudi Arabia.Materials and Methods:The study was a cross-sectional study of a representative sample of males and females in Tihamet Aseer, south-western Saudi Arabia. A comprehensive questionnaire was completed by all participants. Blood samples were taken and sera were tested for hepatitis B surface antigen and HCV antibodies by fourth-generation enzyme immunoassays.Results:The study included 1532 participants from the Tihamet Aseer area. An overall seroprevalence of 7.9% and 1.7% was found for HBV and HCV infections, respectively. In logistic regression analysis, no gender differences were found for HBV seroprevalence. Identified risk factors for HBV infection included a history of blood transfusion and lack of hepatitis B vaccination. On the other hand, females were more prone to become seropositive for HCV (adjusted odds ratio = 5.034, 95% confidence interval: 1.042–9.321). Other identified risk factors for HCV infection were illiteracy and a history of blood transfusion.Conclusion:The prevalence and HBV and HCV infection is high compared to the national figures. Gender differences were only observed in HCV infection. It is recommended to have an active educational and media campaign. A “catch-up” vaccination program against HBV should be introduced for adults as a strategy to achieve the herd immunity effect in the affected area.

Initiation of Antiretroviral Therapy During Acute HIV-1 Infection Leads to a High Rate of Nonreactive HIV Serology.

Third- and fourth-generation immunoassays (IAs) are widely used in the diagnosis of human immunodeficiency virus (HIV) infection. Antiretroviral therapy (ART) during acute HIV infection (AHI) may impact HIV-specific antibodies, with failure to develop antibody or seroreversion. We report on the ability of diagnostic tests to detect HIV-specific antibodies in Thai participants initiating ART during AHI. Participants with detectable plasma HIV RNA but nonreactive HIV-specific immunoglobulin G, enrolled in an AHI study, were offered immediate initiation of ART. Participants were tested at initiation and at 12 and 24 weeks following treatment using standard second-, third-, and fourth-generation IAs and Western blot (WB). Participants (N = 234) initiating ART at a median of 19 days (range, 1-62 days) from HIV exposure demonstrated different frequencies of reactivity prior to and following 24 weeks of ART depending on the IA. Third-generation IA nonreactivity prior to ART was 48%, which decreased to 4% following ART (P < .001). Fourth-generation IA nonreactivity was 18% prior to ART and 17% following ART (P = .720). Negative WB results were observed in 89% and 12% of participants prior to and following 24 weeks of ART, respectively (P < .001). Seroreversion to nonreactivity during ART was observed to at least one of the tests in 20% of participants, with fourth-generation IA demonstrating the highest frequency (11%) of seroreversion. HIV-specific antibodies may fail to develop and, when detected, may decline when ART is initiated during AHI. Although fourth-generation IA was the most sensitive at detecting AHI prior to ART, third-generation IA was the most sensitive during treatment. NCT00796146 and NCT00796263.

Acute infections, cost and time to reporting of HIV test results in three U.S. State Public Health Laboratories

In three U.S. State Public Health Laboratories (PHLs) using a fourth-generation immunoassay (IA), an HIV-1/HIV-2 differentiation antibody IA and a nucleic acid test (NAT), we characterized the yield and time to reporting of acute infections, and cost per positive specimen. Routine HIV testing data were collected from July 1, 2012-June 30, 2013 for Massachusetts and Maryland PHLs, and from November 27, 2012-June 30, 2013 for Michigan PHL. Massachusetts and Michigan used fourth-generation and differentiation IAs with NAT conducted by a referral laboratory. In Maryland, fourth-generation IA repeatedly reactive specimens were followed by a Western blot (WB), and those with negative or indeterminate results were tested with a differentiation IA and HIV-1 NAT, and if positive by NAT, confirmed by a different HIV-1 NAT. Specimens from WB-positive persons at risk for HIV-2 were tested with a differentiation IA and, if positive, with an HIV-2 WB and/or differential HIV-1/HIV-2 proviral DNA polymerase chain reaction. Among 7914 specimens from Massachusetts PHL, 6069 from Michigan PHL, and 36,266 from Maryland PHL, 0.10%, 0.02% and 0.05% acute infections were identified, respectively. Massachusetts and Maryland PHLs each had 1 HIV-2 positive specimen. The median time from specimen receipt to laboratory reporting of results for acute infections at Massachusetts, Michigan and Maryland PHLs was 8, 11, and 7 days respectively. The laboratory cost per HIV positive specimen was $336 (Massachusetts), $263 (Michigan) and $210 (Maryland). Acute and established infections were found by PHLs using fourth-generation IA in conjunction with antibody tests and NAT. Time to reporting of acute HIV test results to clients was suboptimal, and needs to be streamlined to expedite treatment and interrupt transmission.

Field Evaluation of a Dual Rapid Immunodiagnostic Test for HIV and Syphilis Infection in Peru.

Integrated prevention for HIV and syphilis is warranted because both syphilis and HIV infections have evidence-based, scalable interventions using current health care mechanisms. The advent of dual rapid point-of-care tests, single devices that can detect multiple infections using the same specimen, provides the opportunity to integrate the screening of syphilis into HIV prevention programs, potentially increasing the numbers of people tested and allowing for same-day testing and treatment. The aim of our study was to evaluate the MedMira Multiplo Rapid TP/HIV Antibody Test (MedMira Inc, Halifax, Nova Scotia, Canada), a qualitative, rapid immunoassay that detects antibodies to Treponema pallidum and HIV. The reference test for comparison to the T. pallidum component of the Multiplo TP/HIV Test was the T. pallidum particle agglutination assay. For the HIV component, the reference test included a fourth-generation enzyme immunoassay with a confirmatory Western blot test. The sensitivity and specificity for the HIV antibody component were 93.8% (95% confidence interval [CI], 69.8%-99.8%) and 100% (95% CI, 97.7%-100%), respectively. The T. pallidum component of the test had a sensitivity of 81.0% (95% CI, 68.1%-94.6%) and a specificity of 100% (95% CI, 97.6%-100%). Our study showed excellent performance of the HIV antibody component of the test and very good performance for the T. pallidum antibody component of the MedMira Multiplo Rapid TP/HIV Antibody Test, which should be considered to improve screening coverage. Use of effective dual tests will create improved access to more comprehensive care by integrating the screening of syphilis into HIV prevention programs.

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling.

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

Reactivity of routine HIV antibody tests in children who initiated antiretroviral therapy in early infancy as part of the Children with HIV Early Antiretroviral Therapy (CHER) trial: a retrospective analysis

Early antiretroviral therapy (ART) and virological suppression can affect evolving antibody responses to HIV infection. We aimed to assess frequency and predictors of seronegativity in infants starting early ART. We compared HIV antibody results between two of three treatment groups of the Children with HIV Early Antiretroviral Therapy (CHER) trial, done from July, 2005, until July, 2011, in which infants with HIV infection aged 5·7-12·0 weeks with a percentage of CD4-positive T lymphocytes of at least 25% were randomly assigned to immediate ART for 96 weeks (ART-96W) or deferred ART until clinical or immunological progression (ART-Def). We measured antibody from all available stored samples for ART-96W and ART-Def at trial week 84 using three assays: fourth-generation enzyme immunoassay HIV antigen-antibody combination, HIV-1 and HIV-2 rapid antibody test, and quantitative anti-gp120 IgG ELISA. We also assessed odds of seropositivity with respect to age of ART initiation and cumulative viral load. The CHER trial was registered with ClinicalTrials.gov, number NCT00102960. The median age of the infants from when samples were taken (184 samples from 268 infants) was 92 weeks (IQR 90·6-93·4). More specimens from the ART-96W group were seronegative than from the ART-Def group by enzyme immunoassay (ART-96W 49 [46%] of 107 vs ART-Def eight [11%] of 75; p<0·0001) and rapid antibody test (54 [53%] of 101 vs eight [11%] of 74; p<0·0001). Median anti-gp120 IgG concentration was lower in the ART-96W group (230 μg/μL [IQR 133-13 129]) than in the ART-Def group (6870 μg/μL [1706-53 645]; p<0·0001). If ART was started between 12 and 24 weeks of age, odds of seropositivity were increased 13·7 times (95% CI 3·1-60·2; p=0·001) compared with starting it between 0 and 12 weeks. All children starting ART aged older than 24 weeks were seropositive. Cumulative viral load to week 84 correlated with anti-gp120 IgG concentrations (coefficient 0·54; p<0·0001) and increased odds of seropositivity (odds ratio 1·59 [95% CI 1·1-2·3]) adjusted for ART initiation age. About half of children starting ART before 12 weeks of age were HIV seronegative by almost 2 years of age. HIV antibody tests cannot be used to reconfirm HIV diagnosis in children starting early ART. Long-term effects of seronegativity need further study. Clear guidelines are needed for retesting alongside improved diagnostic tests. Wellcome Trust, Medical Research Council, and National Institutes of Health.

Diagnosing Acute HIV Infection — Does Fourth-Generation Immunoassay Suffice?

Diagnosing acute HIV infection (AHI) is important because of the higher transmission risk during this period and the potential for early treatment to limit the HIV reservoir. Improvements in HIV testing have led to the current, fourth-generation (4thG) antigen/antibody immunoassay, which has shortened the “window period” between HIV acquisition and diagnostic-test positivity by improving the sensitivity of the antibody test and incorporating p24 antigen …