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Internet-recruited gay, bisexual, and other men who have sex with men (MSM) were offered HIV self-tests (HIVSTs) after completing baseline, 3-, 6-, and 9-month follow-up surveys. The surveys asked about the use and distribution of these HIVSTs. Among 995 who reported on their distribution of HIVSTs, 667 (67.0%) distributed HIVSTs to their social network associates (SNAs), which resulted in 34 newly identified HIV infections among 2301 SNAs (1.5%). The main reasons participants reported not distributing HIVSTs included: wanting to use the HIVSTs themselves (74.9%); thinking that their SNAs would get angry or upset if offered HIVSTs (12.5%); or not knowing that they could give the HIVSTs away (11.3%). Self-testing programs can provide multiple HIVSTs and encourage the distribution of HIVST by MSM to their SNAs to increase awareness of HIV status among persons disproportionately affected by HIV.Hombres gais, bisexuales y otros hombres que indicaron tener contacto sexual con hombres (MSM, por sus siglas en inglés) fueron reclutados por el Internet y se les ofreció autopruebas del VIH (HIVST, por sus siglas en inglés) después de completar una encuestas inicial y encuestas de seguimiento a los 3, 6 y 9 meses. Las encuestas recogieron datos sobre el uso y distribución de estas autopruebas del VIH. De los 995 MSM que indicaron distribuir las autopruebas, 667 (67.0%) distribuyeron las autopruebas a personas en sus redes sociales (SNA, por sus siglas en inglés), resultando en 34 nuevas infecciones por el VIH identificadas entre 2,301 SNA (1.5%). Las razones principales por las que algunos participantes no distribuyeron las autopruebas del VIH incluyen: el deseo de utilizar las autopruebas del VIH para sí mismos (74.9%); pensar que las SNA se enfadarían o molestarían si se les ofreciesen autopruebas del VIH (12.5%); o no saber que podían distribuir las autopruebas del VIH (11.3%). Los programas que proporcionen múltiples autopruebas del VIH podrían alentar la distribución de las autopruebas por parte de los MSM a las SNA para aumentar el conocimento sobre el estado del VIH entre personas afectadas de manera desproporcionada por el VIH.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

Background There is benefit to early HIV-1 diagnosis and treatment, but there is no Food and Drug Administration-approved quantitative assay with a diagnostic claim. We compared the performance of the Hologic Aptima HIV-1 Quant (APT-Quant) and Aptima HIV-1 Qual (APT-Qual) assays for diagnostic use and the performance of a diagnostic algorithm consisting of Bio-Rad BioPlex 2200 HIV Ag-Ab assay (BPC) followed by APT-Quant (2-test) compared with BPC followed by Geenius HIV-1/2 supplemental assay (Geenius) with reflex to APT-Qual (3-test). Methods Five hundred twenty-four plasma, which included 419 longitudinal specimens from HIV-1 seroconverters (78 were after initiating antiretroviral therapy [ART]) and 105 from ART-naive persons with established HIV-1 infections, were used to evaluate APT-Quant performance for diagnostic use. Specimens from 200 HIV-negative persons were used to measure specificity. For the algorithm comparison, BPC-reactive specimens were evaluated with the 2-test or 3-test algorithm. McNemar's test was used to compare performance. Results The APT-Quant detected more samples early in infection compared with APT-Qual. The APT-Quant specificity was 99.8%. Before ART initiation, the algorithms performed similarly among samples from different stages of infection. After ART initiation, the 3-test algorithm performed significantly better (P = 0.0233). Conclusions The APT-Quant has excellent performance for diagnostic use. The 2-test algorithm works well in ART-naive samples, but its performance decreases after the IgG response is elicited and with ART-induced suppressed viremia. Providing confirmation and viral load assay with 1 test result could be advantageous for patient care. However, additional factors and challenges associated with the implementation of this 2-test algorithm, such as cost, specimen type, and collection need further evaluation.

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.

Identifying patients at-risk for HIV infection, such as men who have sex with men (MSM), is an important step in providing HIV testing and prevention interventions. It is unknown how primary care providers (PCPs) assess MSM status and related HIV-risk factors. We analyzed data from a panel-derived web-based survey for healthcare providers conducted in 2014 to describe how PCPs in the U.S. determined their patients' MSM status. We calculated adjusted prevalence ratios (aPR) and 95% confidence intervals (CI) to describe PCP characteristics associated with systematically determining MSM status (i.e., PCP used “a patient-completed questionnaire” or “routine verbal review of sex history”). Among the 1008 PCPs, 56% determined MSM status by routine verbal review of sexual history; 41% by patient disclosure; 39% by questions driven by symptoms/history; 23% by using a patient-completed questionnaire, and 9% didn't determine MSM status. PCPs who systematically determined MSM status (n = 665; 66%) were more likely to be female (aPR = 1.16, CI = 1.06–1.26), to be affiliated with a teaching hospital (aPR = 1.15, CI = 1.06–1.25), to routinely screen all patients aged 13–64 for HIV (aPR = 1.29, CI = 1.18–1.41), and to estimate that 6% or more of their male patients are MSM (aPR = 1.14, CI = 1.01–1.30). The majority of PCPs assessed MSM status and HIV risk factors through routine verbal reviews of sexual history. Implementing a systematic approach to identify MSM status and assess risk may allow PCPs to identify more patients needing frequent HIV testing and other preventive services, while mitigating socio-cultural barriers to obtaining such information.

In the United States, an estimated 67% of new HIV diagnoses are among men who have sex with men (MSM), however 25% of HIV-positive MSM in the 2014 National HIV Behavioral Surveillance Survey were unaware of their infection. HIV self-testing (HIVST) with rapid diagnostic tests (RDTs) may facilitate access to HIV testing. We evaluated the ability of 22 MSM to conduct two HIV RDTs (OraQuick ® In-Home HIV Test and a home-use prototype of Sure Check ® HIV 1/2 Assay), interpret sample images of test results, and collect a dried blood spot (DBS) specimen. While some participants did not follow every direction, most participants were able to conduct HIVST and correctly interpret their results. Interpretation of panels of RDT images was especially difficult when the “control” line was missing, and 27% of DBS cards produced were rated as of bad quality. Modifications to the DBS instructions were necessary prior to evaluating the performance of these tests in real-world settings.

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.

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.

Since 2014, the recommended laboratory testing algorithm for diagnosing human immunodeficiency virus (HIV) infection has included a supplemental HIV-1/HIV-2 differentiation test to confirm infection type on the basis of the presence of type-specific antibodies (1). Correctly identifying HIV-1 and HIV-2 infections is vital because their epidemiology and clinical management differ. To describe the percentage of diagnoses for which an HIV-1/HIV-2 differentiation test result was reported and to categorize HIV type based on laboratory test results, 2010-2017 data from CDC's National HIV Surveillance System (NHSS) were analyzed. During 2010-2017, a substantial increase in the number of HIV-1/HIV-2 differentiation test results were reported to NHSS, consistent with implementation of the HIV laboratory-based testing algorithm recommended in 2014. However, >99.9% of all HIV infections identified in the United States were categorized as HIV-1, and the number of HIV-2 diagnoses (mono-infection or dual-infection) remained extremely low (

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 (NAT) 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 three-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 five Ag/Ab tests and the proportion of specimens correctly classified by the alternative algorithm compared to 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.

Background HIV testing guidelines provided by the Centers for Disease Control and Prevention (CDC) are continually changing to reflect advancements in new testing technology. Evaluation of existing and new point-of-care (POC) HIV tests is crucial to inform testing guidelines and provide information to clinicians and other HIV test providers. Characterizing the performance of POC HIV tests using unprocessed specimens can provide estimates for the window period of detection, or the time from HIV acquisition to test positivity, which allows clinicians and other HIV providers to select the appropriate POC HIV tests for persons who may be recently infected with HIV. Objective This paper describes the protocols and procedures used to evaluate the performance of the newest POC tests and determine their sensitivity during early HIV infection. Methods Project DETECT is a CDC-funded study that is evaluating POC HIV test performance. Part 1 is a cross-sectional, retrospective study comparing behavioral characteristics and HIV prevalence of the overall population of the Public Health–Seattle & King County (PHSKC) Sexually Transmitted Disease (STD) Clinic to Project DETECT participants enrolled in part 2. Part 2 is a cross-sectional, prospective study evaluating POC HIV tests in real time using unprocessed whole blood and oral fluid specimens. A POC nucleic acid test (NAT) was added to the panel of HIV tests in June 2018. Part 3 is a longitudinal, prospective study evaluating seroconversion sensitivity of POC HIV tests through serial follow-up testing. For comparison, HIV-1 RNA and HIV-1/HIV-2 antigen/antibody tests are also performed for participants enrolled in part 2 or 3. A behavioral survey that collects information about demographics, history of HIV testing, STD history, symptoms of acute HIV infection, substance use, sexual behaviors in the aggregate and with recent partners, and use of pre-exposure prophylaxis and antiretroviral therapy is completed at each part 2 or 3 visit. Results Between September 2015 and March 2019, there were 14,990 Project DETECT–eligible visits (part 1) to the PHSKC STD Clinic resulting in 1819 part 2 Project DETECT study visits. The longitudinal study within Project DETECT (part 3) enrolled 27 participants with discordant POC test results from their part 2 visit, and 10 (37%) were followed until they had fully seroconverted with concordant positive POC test results. Behavioral survey data and HIV test results, sensitivity, and specificity will be presented elsewhere. Conclusions Studies such as Project DETECT are critical for evaluating POC HIV test devices as well as describing characteristics of persons at risk for HIV acquisition in the United States. HIV tests in development, including POC NATs, will provide new opportunities for HIV testing programs. International Registered Report Identifier (IRRID) RR1-10.2196/16332

Background In 2016, HIV-2 nucleic acid testing (NAT) was added to a shared service program that conducts HIV-1 NAT for public health laboratories performing the recommended algorithm for diagnosing HIV. Here, we evaluate the usefulness of HIV-2 NAT in this program as compared with HIV-1 NAT. Methods Specimens eligible for HIV-1 NAT were reactive on an HIV-1/2 antibody or antigen/antibody initial test and nonreactive or indeterminate on a supplemental antibody test or were reactive for HIV-1 antigen-only on an HIV-1/2 antigen/antibody initial test. Specimens eligible for HIV-2 NAT were reactive on an initial test, HIV-2 indeterminate or HIV indeterminate on a supplemental antibody test and had no detectable HIV-1 RNA or were reactive for HIV-2 antibody on an HIV-1/2 antigen/antibody test, and this reactivity was not confirmed with a supplemental antibody assay. All specimens were tested in a reference laboratory using APTIMA HIV-1 qualitative RNA and/or a validated qualitative HIV-2 RNA real-time PCR assay. Results During 2016 to 2019, HIV-1 RNA was detected in 234 (14%) of 1731 specimens tested. HIV-2 RNA was not detected in 52 specimens tested. Median time from specimen collection to reporting of HIV-1 and HIV-2 NAT results by year ranged from 9 to 10 days and from 22 to 27 days, respectively. Two specimens with HIV-2 indeterminate results on a supplemental antibody test had detectable HIV-1 RNA. Conclusions A shared service model for HIV-1 NAT is both feasible and beneficial for public health laboratories. However, because no HIV-2 infections were detected, our data suggest that this program should reconsider the usefulness of HIV-2 NAT testing.

The performance of recently approved point-of-care (POC) HIV tests should be assessed using unprocessed specimens.To evaluate the sensitivity and specificity of four POC HIV tests using whole blood (WB) and two using oral fluid (OF) among persons recruited from health clinics in Seattle, Washington, during September 2015-September 2017.Participants were tested with the POC tests, additional plasma and serum were collected for laboratory testing, and participant- reported use of antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) was recorded. Participants testing negative on all tests could reenroll every 90 days. Specimens from persons previously diagnosed with HIV infection as well as from those who were newly diagnosed during the study were included in the sensitivity estimate. Sensitivity and specificity were calculated based on HIV status determined by laboratory testing.Of 1,256 visits, 179 were from persons with HIV infection; 120 of these were taking ART. Among 1,077 visits from participants not diagnosed with HIV, PrEP use was reported at 155 (14.4%) visits. Sensitivity was similar among POC WB tests (95.53%-97.21%; p0.05). Among participants on ART, sensitivity was lower for the same test performed on OF compared to WB (p0.003). Specificity was high for all tests (99.44%- 100.00%); we did not detect specificity differences with PrEP use.These POC tests displayed relatively high sensitivity and specificity using unprocessed specimens, suggesting their effectiveness in identifying HIV infections whenever laboratory-based testing is not feasible. Nonetheless, clients with recent risk should retest to rule out the possibility of a false-negative result.

BACKGROUND HIV testing guidelines provided by the Centers for Disease Control and Prevention (CDC) are continually changing to reflect advancements in new testing technology. Evaluation of existing and new point-of-care (POC) HIV tests is crucial to inform testing guidelines and provide information to clinicians and other HIV test providers. Characterizing the performance of POC HIV tests using unprocessed specimens can provide estimates for the window period of detection, or the time from HIV acquisition to test positivity, which allows clinicians and other HIV providers to select the appropriate POC HIV tests for persons who may be recently infected with HIV. OBJECTIVE This paper describes the protocols and procedures used to evaluate the performance of the newest POC tests and determine their sensitivity during early HIV infection. METHODS Project DETECT is a CDC-funded study that is evaluating POC HIV test performance. Part 1 is a cross-sectional, retrospective study comparing behavioral characteristics and HIV prevalence of the overall population of the Public Health–Seattle & King County (PHSKC) Sexually Transmitted Disease (STD) Clinic to Project DETECT participants enrolled in part 2. Part 2 is a cross-sectional, prospective study evaluating POC HIV tests in real time using unprocessed whole blood and oral fluid specimens. A POC nucleic acid test (NAT) was added to the panel of HIV tests in June 2018. Part 3 is a longitudinal, prospective study evaluating seroconversion sensitivity of POC HIV tests through serial follow-up testing. For comparison, HIV-1 RNA and HIV-1/HIV-2 antigen/antibody tests are also performed for participants enrolled in part 2 or 3. A behavioral survey that collects information about demographics, history of HIV testing, STD history, symptoms of acute HIV infection, substance use, sexual behaviors in the aggregate and with recent partners, and use of pre-exposure prophylaxis and antiretroviral therapy is completed at each part 2 or 3 visit. RESULTS Between September 2015 and March 2019, there were 14,990 Project DETECT–eligible visits (part 1) to the PHSKC STD Clinic resulting in 1819 part 2 Project DETECT study visits. The longitudinal study within Project DETECT (part 3) enrolled 27 participants with discordant POC test results from their part 2 visit, and 10 (37%) were followed until they had fully seroconverted with concordant positive POC test results. Behavioral survey data and HIV test results, sensitivity, and specificity will be presented elsewhere. CONCLUSIONS Studies such as Project DETECT are critical for evaluating POC HIV test devices as well as describing characteristics of persons at risk for HIV acquisition in the United States. HIV tests in development, including POC NATs, will provide new opportunities for HIV testing programs. INTERNATIONAL REGISTERED REPORT RR1-10.2196/16332

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.

Given the many options available, selecting an HIV test for a particular clinical or research setting can be daunting. Making an informed decision requires an assessment of the likelihood of acute infection in the test population and an understanding of key aspects of the tests themselves. The ability of individual tests to reliably detect HIV infection depends on the target(s) being detected, when they can be expected to be present following infection, and the concentration of stable target in test specimens – all of which are explained by the virological and serological events following infection. The purpose of this paper is to: review the timeline of HIV infection, nomenclature and characteristics of different tests; compare point-of-care and laboratory-based tests; discuss the impact of different specimens on test performance; and provide practical advice to help clinicians and researchers new to the field select a test that best suits their needs.

HIV-positive men who have sex with men (MSM) were recruited on www.Facebook.com and www.Poz.com to give HIV self-tests to their contacts. Study participants completed a baseline survey, were given two self-tests, and completed a survey 2 months later. Of 133 eligible men, 40 (30%) completed both surveys. Most participants were 30-54 years old and non-Hispanic white. Some had a detectable viral load (n = 4), had condomless anal sex with male partners of negative or unknown status (n = 17), and had met anal sex partners at gay dating websites (n = 23). Of 80 self-tests given to participants, 59 (74%) were distributed, primarily to non-Hispanic white MSM, 30-54 years old who were friends. Participants reported results from 31 distributed tests; 2 sex partners of participants had positive results. Participants indicated these two persons were unaware of their infections. Expanding recruitment websites might reach non-white MSM. Unrecognized infections were identified through online recruitment and self-test distribution via HIV-positive persons.

Cathepsin K (CatK) inhibitors exhibited chondroprotective and pain-reducing effects in animal models, however, improvements were relatively modest at dose levels achieving maximal suppression of CatK biomarkers in urine. In this report, a previously characterized CatK inhibitor (MK-1256) is utilized to explore the potential of reduced target engagement and/or suboptimal exposure (free drug) as limiting factors to the pharmacological potential of CatK inhibitors in the knee joint. Following oral administration of MK-1256 at a dose level achieving maximal inhibition of urinary biomarker (helical peptide) in dogs, full suppression of the biomarker in synovial fluid was observed. Subsequent tissue distribution studies conducted in dogs and rabbits revealed that MK-1256 levels in synovial fluid and cartilage were consistent with the free-drug hypothesis. Reasonable projection (within twofold) of drug levels in these tissues can be made based on plasma drug concentration with adjustments for binding factors. These results indicate that the previously observed efficacies in the animal models were not limited by compound distribution or target engagement in the knee tissues.

Background: Many public health laboratories adopting the U.S. HIV laboratory testing algorithm do not have a nucleic acid test (NAT), which is needed when the third- or fourth-generation HIV screening immunoassay is reactive and the antibody-based supplemental test is non-reactive or indeterminate. Objectives: Among public health laboratories utilizing public health referral laboratories for NAT conducted as part of the algorithm, we evaluated the percentage of screening immunoassays needing NAT, the number of specimens not meeting APTIMA (NAT) specifications, time to APTIMA result, the proportion of acute infections (i.e., reactive APTIMA) among total infections, and screening immunoassay specificity. Study design: From August 2012 to April 2013, 22 laboratories enrolled to receive free APTIMA (NAT) at New York or Florida public health referral laboratories. Data were analyzed for testing conducted until June 2013. Results: Submitting laboratories conducted a median of 4778 screening immunoassays; 0–1.3% (median 0.2%) needed NAT. Of 140 specimens received, 9 (6.4%) did not meet NAT specifications. The median time from specimen collection to reporting the 11 reactive NAT results was ten days, including six days from receipt in the submitting laboratory to shipment to the referral laboratory. Acute infections ranged from 0 to 12.5% (median 0%) of total infections. Third- and fourth-generation immunoassays met package insert specificity values. Conclusions: Public health referral laboratories provide a feasible option for conducting NAT. Reducing the time from specimen collection to submission of specimens for NAT is an important step toward maximizing the public health impact of identifying acute infections. Published by Elsevier B.V.

Background The capacity of HIV Antigen/Antibody (Ag/Ab) immunoassays (IA) to detect HIV-1 p24 antigen has resulted in improved detection of HIV-1 infections in comparison to Ab-only screening assays. Since its introduction in the US, studies have shown that the Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) detects HIV infection earlier than laboratory-based IgM/IgG-sensitive IAs, but its sensitivity for HIV-1 p24 Ag detection is reduced compared to laboratory-based Ag/Ab assays. However, further evaluation is needed to assess its capacity to detect acute HIV-1 infection. Objective To assess the performance of Determine Ag/Ab in serum from acute HIV-1 infections. Study design Select serum specimens that screened reactive on a laboratory-based Ag/Ab IA or IgM/IgG Ab-only IA, with a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were retrospectively tested with Determine Ag/Ab. Results were compared with those of the primary screening immunoassay to evaluate concordance within this set of algorithm-defined acute infections. Results Of 159 algorithm-defined acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance. Of 125 that were initially detected by a laboratory-based Ag/Ab IA, 81 (64.8%) were reactive by Determine Ag/Ab. A total of 34 acute specimens were initially detected by a laboratory-based IgM/IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab. Conclusions Due to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be preferred over the Determine Ag/Ab as the screening method used by laboratories conducting HIV diagnostic testing on serum and plasma specimens.

Background 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. Objectives 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. Study design 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. Results 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. Conclusions 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.

Background The use of Western blot (WB) as a supplemental test after reactive sensitive initial assays can lead to inconclusive or misclassified HIV test results, delaying diagnosis. Objective To determine the proportion of specimens reactive by immunoassay (IA) but indeterminate or negative by WB that could be resolved by alternative supplemental tests recommended under a new HIV diagnostic testing algorithm. Study design Remnant HIV diagnostic specimens that were reactive on 3rd generation HIV-1/2 IA and either negative or indeterminate by HIV-1 WB from 11 health departments were tested with the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test (Multispot) and the Gen-Probe APTIMA HIV-1 RNA Qualitative Assay (APTIMA). Results According to the new testing algorithm, 512 (89.8%) specimens were HIV-negative, 55 (9.6%) were HIV-1 positive (including 19 [3.3%] that were acute HIV-1 and 9 [1.6%] that were positive for HIV-1 by Multispot but APTIMA-negative), 2 (0.4%) were HIV-2 positive, and 1 (0.2%) was HIV-positive, type undifferentiated. 47 (21.4%) of the 220 WB-indeterminate and 8 (2.3%) of the 350 WB-negative specimens were HIV-1 positive. Conclusion Applying the new HIV diagnostic algorithm retrospectively to WB-negative and indeterminate specimens, the HIV infection status could be established for nearly all of the specimens. IA-reactive HIV-infected persons with WB-negative results had been previously misclassified as uninfected, and HIV diagnosis was delayed for those with WB-indeterminate specimens. These findings underscore the limitations of the WB to confirm HIV infection after reactive results from contemporary 3rd or 4th generation IAs that can detect HIV antibodies several weeks sooner than the WB.

An alternate HIV testing algorithm has been proposed which includes a fourth-generation immunoassay followed by an HIV-1/HIV-2 antibody differentiation supplemental test for reactive specimens and a nucleic acid test (NAT) for specimens with discordant results.To evaluate the performance of five rapid tests (Alere Clearview, Bio-Rad Multispot, OraSure OraQuick, MedMira Reveal, and Trinity Biotech Unigold) as the supplemental antibody assay in the algorithm.A total of 3273 serum and plasma specimens that were third-generation immunoassay repeatedly reactive and Western blot (WB) negative or indeterminate were tested with rapid tests and NAT. Specimens were classified by NAT: (1) HIV-1 infected (NAT-reactive; n=184, 5.6%), (2) HIV-status unknown (NAT nonreactive; n=3078, 94.2%) or by Multispot, (3) HIV-2 positive (n=5), and (4) HIV-1 and HIV-2 positive (n=6). Excluding HIV-2 positive specimens, we calculated the proportion of reactive rapid tests among specimens with reactive and nonreactive NAT.The proportion of infected specimens with reactive rapid test results and negative or indeterminate WB ranged from 30.4% (56) to 47.8% (88) depending on the rapid test. From 1% to 2% of NAT-negative specimens had reactive rapid test results.In these diagnostically challenging specimens, all rapid tests identified infections that were missed by the Western blot, but only Multispot could differentiate HIV-1 from HIV-2. Regardless of which rapid test is used as a supplemental test in the alternative algorithm, false-positive algorithm results (i.e., reactive screening and rapid test in uninfected person) may occur, which will need to be resolved during the baseline medical evaluation.

The 2016 HIV Diagnostics Conference, held in Atlanta, Georgia, was attended by public health officials, laboratorians, HIV testing program managers, surveillance coordinators and industry representatives. The conference addressed test performance data, the implementation of new testing algorithms, quality assurance, and the application of new tests in a variety of settings. With regard to the recommended Centers for Disease Control and Prevention/Association of Public Health Laboratories HIV laboratory testing algorithm, the conference featured performance data, implementation challenges such as a lack of test options for the second and third steps, as well as data needs for new tests that may be used as part of the algorithm. There are delays when nucleic acid testing is needed with the algorithm. Novel tests such as point of care nucleic acid tests are needed on the U.S. market to readily identify acute infection. Multiplex tests are being developed which allow for the simultaneous detection of multiple pathogens. CDC staff highlighted new guidance for testing in non-clinical settings. Innovative approaches to linking testing and care in some settings have led to identification of early infections, improved receipt of test results and expedited initiation of therapy. Work continues to optimize testing so that infections are accurately identified as early as possible and time to treatment is minimized to improve health outcomes and prevent transmission.

We evaluated the performance of the GS fourth-generation antigen/antibody assay and compared Centers for Disease Control and Prevention's (CDC's) proposed alternative algorithm [repeatedly reactive fourth-generation immunoassay followed by an HIV-1/HIV-2 differentiation immunoassay and, if needed, nucleic acid test (NAT)] with the current algorithm (repeatedly reactive third-generation immunoassay followed by HIV-1 western blot).A convenience sample of the following four specimen sets was acquired: 10 014 from insurance applicants, 493 known western blot-positive, 20 known western blot-indeterminate specimens, and 230 specimens from 26 HIV-1 seroconverters.Specimens were tested with the GS third-generation and fourth-generation immunoassays, the Multispot HIV-1/HIV-2 differentiation immunoassay, NAT, and western blot. We applied the two algorithms using these results.Among insurance specimens, 13 (0.13%) specimens were immunoassay repeatedly reactive: two were HIV-positive (repeatedly reactive by third-generation and fourth-generation immunoassays, and western blot and Multispot positive); two third-generation repeatedly reactive and nine fourth-generation repeatedly reactive specimens were false-positive. Third-generation and fourth-generation specificities were 99.98% [95% confidence interval (CI) 99.93-100%] and 99.91% (95% CI 99.84-99.96%), respectively.All HIV-1 western blot-positive specimens were repeatedly reactive by third-generation and fourth-generation immunoassays. By Multispot, 491 (99.6%) were HIV-1-positive and two (0.4%) were HIV-2-positive.Only eight (40%) western blot-indeterminate specimens were fourth-generation repeatedly reactive: six were Multispot and NAT-negative and two were Multispot HIV-1-positive but NAT-negative.The alternative algorithm correctly classified as positive 102 seroconverter specimens with the third-generation immunoassay and 130 with the fourth-generation immunoassay compared with 56 using the western blot with either immunoassay.The alternative testing algorithm improved early infection sensitivity and identified HIV-2 infections. Two potential false-positive algorithm results occurred with western blot-indeterminate specimens.

Summary Objective 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. Methods 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. Results 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). Conclusions 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.

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once-weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen-deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6 mg/kg, or ODN 30 mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle-treated OVX monkeys. Treatment with ODN also led to dose-dependent increases in serum 1-CTP and maintained estrogen deficiency-elevated Trap-5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L₁ to L₄) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose-dependently increased L₁ to L₄ BMD by 7% in the 6 mg/kg group (p < 0.05 versus OVX-vehicle) and 15% in the 30 mg/kg group (p < 0.05 versus OVX-vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R = 0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN-treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long-term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates.

a b s t r a c t Background: The HIV-1 Western blot (WB) and immunofluorescence assay used to confirm HIV infections are less sensitive during seroconversion than immunoassays (IAs) used for screening. An alternative diagnostic algorithm has been proposed to detect early HIV-1 infection and differentiate HIV-1 from HIV-2. Objectives: We evaluated the performance of an algorithm with a third generation IA that when reac- tive was followed by a rapid test (Multispot) that differentiates HIV-1 from HIV-2. Multispot-reactive specimens were considered HIV-infected. Multispot-negative specimens were tested with a nucleic acid amplification test (NAAT) for resolution. Study design: WB-positive specimens (serum (n = 2202), plasma (n = 1109) and peripheral blood mononu- clear cells (PBMCs) (n = 1065)) were obtained from HIV-infected persons not taking antiretrovirals. HIV-uninfected specimens (plasma (n = 1517) and PBMCs (n = 1508)) with negative IA and NAAT results were obtained from blood donors. Specimens were tested with third generation IAs (Abbott rDNA, ADVIA Centaur, GS HIV1-2 Plus O, Ortho VITROS) in singlet, Multispot, and NAAT (APTIMA (RNA) and AMPLICOR (DNA)). We calculated algorithm sensitivity and specificity and the proportion of IA-reactive specimens requiring NAAT. Results: Algorithm sensitivity was 99.95% with APTIMA and 100% with AMPLICOR. One WB-positive spec- imen reactive by all IAs and AMPLICOR was negative by Multispot and APTIMA. Algorithm specificity was 100% using APTIMA or AMPLICOR as NAAT. From 0.10% (Abbott) to 2.43% (VITROS) of IA-reactive specimens required NAAT. Conclusions: The proposed algorithm performs with high sensitivity and specificity in specimens from persons with established HIV infection and uninfected blood donors and appears to be a good alternative to the current algorithm. Published by Elsevier B.V. 1. Background

Background Worldwide, many countries test for HIV infection using combination assays that simultaneously detect p24 antigen and HIV antibodies. One such assay, the ARCHITECT® HIV Ag/Ab Combo Assay (ARCHITECT), has recently been approved by the Food and Drug Administration (FDA) for use in the United States. Objective To evaluate the performance of ARCHITECT on well-characterized specimens from four CDC-funded studies. Study design We evaluated 3386 HIV-infected, 7551 HIV-uninfected, and 58 acute HIV infection (AHI) specimens. HIV-infected specimens were repeatedly reactive by enzyme immunoassay (EIA) and Western blot (WB) or positive by nucleic acid amplification testing (NAAT). HIV-uninfected specimens were EIA- and NAAT-negative. AHI specimens were seronegative or indeterminate (using antibody-based EIAs, rapid tests or WB) and NAAT-positive. All specimens were de-identified and sent to Abbott Diagnostics for testing with ARCHITECT. ARCHITECT test results were compared to original study characterizations and were used to assess overall sensitivity and specificity and also sensitivity for AHI. ARCHITECT false-positive specimens with sufficient quantity were retested. Results Based on results from the initial ARCHITECT test, sensitivity was 99.94% (95% confidence interval [CI]: 99.79, 99.99) and specificity was 98.78% (95% CI: 98.51–99.01). Repeat testing resulted in corrected specificity of 99.50% (95% CI: 99.31, 99.64). Also, 48 AHI specimens (83%) were detected by this screening assay. Conclusion The sensitivity and specificity of the ARCHITECT combination assay are very high and most AHIs were detected by the assay. Use of Ag/Ab combination assays may improve the number of AHIs identified relative to existing FDA-approved HIV-antibody only based serologic assays, particularly in high incidence populations.

Background An immunoassay (IA) followed by Western blot (WB) or immunofluorescence assay has been the primary algorithm used to provide laboratory confirmation of the diagnosis of HIV infection in the US for more than 20 years. Recently, an alternative diagnostic algorithm was proposed to more accurately identify early HIV-1 infection and differentiate between HIV-1 and HIV-2 infection. Objectives Evaluate a sequential alternative algorithm in which reactive IAs are followed by a rapid HIV test and, if negative, a nucleic acid amplification test (NAAT). Study design Specimens from high-risk persons were tested with 4 HIV IAs, 6 rapid HIV tests and NAAT (APTIMA ® ), which are approved by the United States Food and Drug Administration. IAs were repeated in duplicate if specimen volumes were sufficient. The performance of the alternative algorithm was compared to HIV WB and NAAT. Results The original study classified 377 specimens as HIV-positive and 3070 as HIV-negative. All 4 IAs correctly identified >99.5% of HIV-positive specimens and, on initial screening, >95.8% of HIV-negative specimens. When repeated, specificity of IAs improved to >99%. Between 6.7% and 12.4% of IA-repeatedly reactive specimens required APTIMA for resolution. The alternative algorithm led to the correct classification of all IA-reactive specimens. Conclusions Regardless of screening IA and rapid test used, the alternative algorithm correctly classified the infection status of all persons with reactive screening IA results. Few specimens required NAAT for resolution, and the proportion requiring NAAT was lower when repeat IA test results were considered.

Background HIV-1 Western blot (WB) may be positive in specimens from persons with HIV-2 infection due to cross-reactive antibodies. HIV-1 and HIV-2 infections may be identified using assays designed to differentiate HIV-1 and HIV-2 antibody reactivity. Objectives To evaluate the ability of the current CDC WB criteria, alternative more stringent HIV-1 WB criteria (2 env plus one gag or pol band) and the Multispot HIV-1/HIV-2 Rapid Test to accurately differentiate HIV-1 and HIV-2 infections. Study design Two panels were used to determine the ability of each method to properly classify HIV-1 and HIV-2 infections: an HIV-2 panel (n = 114) determined to be HIV-2 antibody-positive by both Multispot and by a validated HIV-2 WB, and 2135 HIV-1/HIV-2 immunoassay repeatedly reactive (IA-RR) specimens from the New York State Department of Health Laboratory (NYS). Results By CDC WB criteria, 53 (46.5%) HIV-2 panel specimens were HIV-1 WB positive, 60 (52.6%) were indeterminate, and 1 (0.9%) was negative; the alternative WB criteria re-classified 75.5% of the positives as indeterminate. Among 2135 NYS IA-RR specimens, the alternative WB criteria increased the proportion of indeterminates by 0.8%. Only 6 (0.3%) of the NYS specimens were determined to be HIV-2 infections; all 6 were classified either as HIV-1 positive or indeterminate by both WB criteria, but were classified as HIV-2 (n = 4) or HIV-1/2 undifferentiated (n = 2) by Multispot. Conclusions The alternative WB criteria classified most of the HIV-2 specimens that were HIV-1 positive by CDC criteria as indeterminate, but also slightly increased the proportion of HIV-1 specimens classified as indeterminate. The WB indeterminate specimens would require further testing or follow-up to resolve the infection status, whereas Multispot directly distinguished HIV-1 from HIV-2.

a b s t r a c t 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 pos- itive. 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. Published by Elsevier B.V.

This study compares the metabolism of [14C]-arachidonic acid between PGE2 synthesizing (ROS 17/2.8) and nonsynthesizing (ROS 25/1) osteosarcoma cell lines. In both cell lines: (a) 90% of [14C]-arachidonic acid was taken up at 24 h. (b) More than 90% of the label was associated with phospholipids. (c) [14C]-arachidonic acid was rapidly taken up by phosphatidylcholine which reached the highest specific activity around 5 h while the labeling of other phospholipids was still increasing at 24 h. (d) Twenty-four hours after addition of [14C]-arachidonic acid only 4% of the label was associated with triacylglycerols in ROS 25/1 and 0.3% in ROS 17/2.8 cells. The calcium ionophore A23187 enhanced the release of [14C]-arachidonic acid from phospholipids in the PGE synthesizing osteoblastic cells (ROS 17/2.8 and 2/3) but had no effect in nonosteoblastic cells (ROS 24/1 and 25/1). ROS 17/2.8 and 2/3 cells converted the released arachidonic acid as well as exogeneously added arachidonic acid into PGE2. PGE2 synthesis depended on arachidonic acid concentration. Among bone resorbing agents, parathyroid hormone and 1,25(OH)2D3 had no effect on PGE synthesis, whereas thrombin and rabbit serum stimulated PGE2 production. The effect of rabbit serum was abolished by heat inactivation. The findings of this study indicate that the difference in PGE production between the osteoblastic and nonosteoblastic osteosarcoma cells are due mainly to differences in arachidonic acid conversion to PGE2.

Background. To improve clinical and public health outcomes through early human immunodeficiency virus (HIV) detection, fourth-generation antigen/antibody immunoassay (4IA) and supplemental testing results must be returned rapidly. Methods. We examined HIV testing data at Harborview Medical Center (HMC), Massachusetts General Hospital (MGH), and the Medical University of South Carolina (MUSC), which used 4IA and supplemental antibody and nucleic acid tests (NATs). At MGH and MUSC, HIV-1 Western blot (WB) and HIV-2 testing were conducted at a reference laboratory. We compared time from specimen collection to laboratory result for established (positive WB) and acute infections (reactive 4IA, negative/indeterminate WB, detectable NAT), and we calculated testing cost per positive-test result. Results. From 3731 (MUSC) to 19 774 (MGH) tests were conducted; 0.01% (MGH) to 0.05% (HMC) were acute infections. Each laboratory had reactive 4IA, WB-negative, or indeterminate specimens without NAT (ie, potential acute infections). Time to result was 1.5 (HMC) to 5.2 days (MGH) for acute and 1.0 (HMC) to 5.2 days (MGH) for established infections. Costs were $1054 (MGH) to $1521 (MUSC). Conclusions. Conducting supplemental testing in-house lowered turnaround times, which may be further reduced with rapid HIV-1/HIV-2 differentiation tests. Hospitals may benefit from quantitative NATs not requiring physician orders, so all potential acute infections receive NAT.

Rolnicy mają zapewnione określone preferencje w zakresie podatku od towarów i usług. Mogą zrezygnować ze statusu rolnika ryczałtowego i zostać czynnymi podatnikami podatku od towarów i usług. W określonych warunkach przejście na zasady ogólne w rozliczaniu VAT może się okazać korzystne dla podatnika. Celem opracowania była ocena efektywności przejścia na zasady ogólne w rozliczaniu podatku VAT przez rolników. Ocena ta opiera się na analizie wysokości zwrotu ryczałtowego dla rolnika korzystającego ze zwolnienia oraz wysokości podatku VAT rozliczanego na zasadach ogólnych. Szczególnym przedmiotem zainteresowania były efekty podwyższenia stawek podatkowych od 2011 roku. W opodatkowaniu działalności rolniczej rolnicy osiągają przewagę, dokonując sprzedaży produktów opodatkowanych stawkami niższymi niż zakupy do produkcji rolnej, co przekłada się często na ujemne saldo podatku należnego i naliczonego. Niestety, te preferencyjne warunki od roku 2011 zostały zniwelowane poprzez podwyższenie dotychczasowych stawek opodatkowania produktów rolniczych.

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.

Objective: Post-marketing surveillance was conducted to monitor the performance of the OraQuick Advance rapid HIV-1/2 antibody test (OraQuick) on whole blood and oral fluid. Design: Surveillance of routinely collected data on clients tested with OraQuick in 368 testing sites affiliated with 17 state and city health departments between 11 August 2004 and 30 June 2005. Methods: For whole blood and oral fluid, we report the median (range) health department OraQuick specificity and positive predictive value (PPV), and the number of clients with discordant results (e.g. who had a reactive rapid test not confirmed positive by Western blot or indirect immunofluorescence). At one site with lower than expected oral-fluid specificity, we evaluated whether device expiration, manufacturing lot, operator practices, or device-storage or testing-area temperatures were associated with false-positive tests. Results: During the surveillance period, 135 724 whole blood and 26 066 oral fluid rapid tests were conducted. The median health department whole blood OraQuick specificity was 99.98% (range: 99.73-100%) and PPV was 99.24% (range: 66.67-100%); the median oral fluid specificity was 99.89% (range: 99.44-100%) and PPV was 90.00% (range: 50.00-100%). A total of 124 discordant results were reported from 68 (0.05%) whole blood and 56 (0.22%) oral fluid rapid tests. The oral fluid specificity at the site with excess oral fluid false-positive tests was 98.7% (95% confidence interval: 98.18-99.11 %). The increase in false-positive tests at that site was not associated with any specific device characteristic, operator procedure or temperature condition. Conclusion: The specificity of OraQuick performed on whole blood and oral fluid during post-marketing surveillance was compatible with the manufacturer's claim within the package insert. However, one site experienced lower than expected oral fluid specificity. Sites that observe that the specificity of OraQuick is lower than the range indicated in the package insert should notify the manufacturer and evaluate quality assurance procedures.

Multinucleated bone-resorbing osteoclasts (Ocl) are cells of hematopoietic origin that play a major role in osteoporosis pathophysiology. Ocl survival and activity require M-CSF and RANK ligand (RANKL). M-CSF signals to Akt, while RANKL, like TNFalpha, activates NF-kappaB. We show here that although these are separate pathways in the Ocl, signaling of all three cytokines converges on mammalian target of rapamycin (mTOR) as part of their antiapoptotic action. Accordingly, rapamycin blocks M-CSF- and RANKL-dependent Ocl survival inducing apoptosis, and suppresses in vitro bone resorption proportional to the reduction in Ocl number. The cytokine signaling intermediates for mTOR/ribosomal protein S6 kinase (S6K) activation include phosphatidylinositol-3 kinase, Akt, Erks and geranylgeranylated proteins. Inhibitors of these intermediates suppress cytokine activation of S6K and induce Ocl apoptosis. mTOR regulates protein translation acting via S6K, 4E-BP1 and S6. We find that inhibition of translation by other mechanisms also induces Ocl apoptosis, demonstrating that Ocl survival is highly sensitive to continuous de novo protein synthesis. This study thus identifies mTOR/S6K as an essential signaling pathway engaged in the stimulation of cell survival in osteoclasts.

Cathepsin K (CatK) is essential for osteoclast-mediated bone resorption. CatK expression is also detected in breast cancer cells that metastasize to bone. Here, the CatK inhibitor L-235 dosed in prevention (10, 30, and 100 mg/kg, p.o., b.i.d.) or treatment regimen (30 mg/kg) was compared with the bisphosphonate zoledronic acid (ZOL, 7.5 μg/kg/wk, s.c.) in the intratibial injection model of MDA-MB-231 breast carcinoma in nude rats. Progression of osteolysis, skeletal tumor burden, and local metastasis was evaluated by radiography through 42 days and ex vivo μCT and histology. IHC and RT-PCR confirmed the increases in CatK protein and mRNA levels in human breast cancer primary and metastatic tumors. In the experimental model of breast cancer bone metastasis, L-235 dosed in preventive mode resulted in a dose-related reduction of osteolysis of 72%, 75%, and 87% respectively, compared with ZOL by 86% versus intact. Similarly, L-235 significantly reduced intratibial tumor volume by 29%, 40%, and 63%, respectively, compared with 56% by ZOL versus vehicle. Efficacy of L-235 and ZOL on reduction of osteolytic lesions and tumor burden was comparable in treatment versus preventive regimens. All L-235 doses inhibited cortical disruption and extraskeletal tumor growth to a level comparable with ZOL. Assessment of local metastasis demonstrated that treatment with the CatK inhibitor was more effective than ZOL in reducing breast cancer invasion. These data support the role of CatK in breast cancer skeletal growth and metastasis and CatK inhibitors may represent a novel oral therapy for treatment of metastatic breast cancer. Mol Cancer Ther; 13(12); 2898–909. ©2014 AACR.

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.To compare paired OF and plasma test performance in a cohort of HIV-1 seroconverters from Nigeria.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.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) (p0.0039); 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.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.

Objectives. The Centers for Disease Control and Prevention recommends HIV screening in U.S. health-care settings unless providers document a yield of undiagnosed HIV infections of Methods. We used a national hospital survey of HIV testing practices, linked to HIV prevalence for the county, parish, borough, or city where the hospital was located, to assess HIV screening of some or all patients by hospitals. We used multivariate logistic regression analysis to assess the association between screening practices and hospital characteristics that were significantly associated with screening in bivariate analyses. Results. Of 376 hospitals in areas of prevalence ≥0.1%, only 25 (6.6%) reported screening all patients for HIV and 131 (34.8%) reported screening some or all patients Among 638 hospitals included, screening some or all patients was significantly (pConclusion. We found that few hospitals surveyed reported screening some or all patients, and failure to screen is common across all types of hospitals in all regions of the country. Expanded reimbursement for screening may increase compliance with the recommendations.

Background Blood specimens for HIV testing are frequently collected using serum collection tubes. The Aptima HIV-1 RNA Qualitative test, originally approved for diagnostic use as a supplemental test for use with plasma, is now approved for use with serum, and may be used in new laboratory-based HIV testing algorithms which detect acute and established HIV infections. Objectives To compare the sensitivity of Aptima using serum and plasma specimens from persons with established HIV infection. Study design Parallel serum and plasma specimens were collected from 325 persons with established HIV-1 infection who had positive immunoassay (IA) and Western blot (WB) results. Samples with negative Aptima results were considered false-negative and were subjected to repeat testing. Aptima sensitivity for serum and plasma was calculated relative to IA and WB, and compared using the McNemar test. Results The sensitivity of Aptima using serum (97.23%, 95% confidence interval [CI] 94.81–98.73) was similar to that using plasma (97.54%, 95% [CI] 95.21–98.93) p =1.00. Five of ten specimens initially false-negative on either serum or plasma were reactive on repeat testing. No specimens initially classified as false-negative on both matrices were reactive on both matrices on repeat testing. Conclusions In specimens from persons with established infections, Aptima performed with similar sensitivity when used with serum or plasma. Using serum for immunoassay screening and supplemental testing may provide added convenience for laboratories.

Objective: Examine whether false-positive HIV enzyme immunoassay (EIA) test results occur more frequently among pregnant women than among women who are not pregnant and men (others). Design: To obtain a large number of pregnant women and others tested for HIV, we identified specimens tested at a national laboratory using Genetic Systems HIV-1/HIV-2 Plus O EIA from July 2007 to June 2008. Methods: Specimens with EIA repeatedly reactive and Western blot-negative or indeterminate results were considered EIA false-positive. We compared the false-positive rate among uninfected pregnant women and others, adjusting for HIV prevalence. Among all reactive EIAs, we evaluated the proportion of false-positives, positive predictive value (PPV), and Western blot bands among indeterminates, by pregnancy status. Results: HIV prevalence was 0.06% among 921,438 pregnant women and 1.34% among 1,103,961 others. The false-positive rate was lower for pregnant women than others (0.14% vs. 0.21%, odds ratio 0.65 [95% confidence interval 0.61, 0.70]). Pregnant women with reactive EIAs were more likely than others (p,0.01) to have Western blot-negative (52.9% vs. 9.8%) and indeterminate results (17.0% vs. 3.7%) and lower PPV (30% vs. 87%). The p24 band was detected more often among pregnant women (p,0.01). Conclusions: False-positive HIV EIA results were rare and occurred less frequently among pregnant women than others. Pregnant women with reactive EIAs were more likely to have negative and indeterminate Western blot results due to lower HIV prevalence and higher p24 reactivity, respectively. Indeterminate results may complicate clinical management during pregnancy. Alternative methods are needed to rule out infection in persons with reactive EIAs from low prevalence populations.

Rapid human immunodeficiency virus testing is often conducted in nonclinical settings by staff with limited training, so quality assurance (QA) monitoring is critical to ensure accuracy of test results. Rapid tests ( n = 86,749) were generally conducted according to manufacturers' instructions, but ongoing testing competency assessments and on-site QA monitoring were not uniformly conducted.

Background Reactive oral fluid and whole blood rapid HIV tests must be followed with a confirmatory test (Western blot (WB), immunofluorescent assay (IFA) or approved nucleic acid amplification test (NAAT)). When the confirmatory result is negative or indeterminate (i.e. discordant with rapid result), repeat confirmatory testing should be conducted using a follow-up specimen. Previous reports have not described whether repeat testing adequately resolves the HIV-infection status of persons with discordant results. Methodology Post-marketing surveillance was conducted in 368 testing sites affiliated with 14 state and 2 city health departments from August 11, 2004 to June 30, 2005 and one health department through December 31, 2005. For persons with discordant results, data were collected on demographics, risk behaviors, HIV test results and specimen types. Persons with repeat confirmatory results were classified as HIV-infected or uninfected. Regression models were created to assess risk factors for not having repeat testing. Principal Findings Of 167,371 rapid tests conducted, 2589 (1.6%) were reactive: of these, 2417 (93%) had positive WB/IFA, 172 (7%) had negative or indeterminate WB/IFA. Of 89/172 (52%) persons with a repeat confirmatory test: 17 (19%) were HIV-infected, including 3 with indeterminate WB and positive NAAT; 72 (81%) were uninfected, including 12 with repeat indeterminate WB. Factors associated with HIV-infection included having an initial indeterminate WB/IFA (vs. negative) (p

Osteoporosis is associated with both atherosclerosis and vascular calcification. No mechanism yet explains the parallel progression of these diseases. Here, we demonstrate that osteoclasts (OCL) depend on lipoproteins to modulate cellular cholesterol levels and that this controls OCL formation and survival. Removal of cholesterol in OCL via high-density lipoprotein or cyclodextrin treatment dose-dependently induced apoptosis, with actin disruption, nuclear condensation and caspase-3 activation. One mechanism linked to the induction of OCL apoptosis was the cell-type-specific failure to induce HMG-CoA reductase mRNA expression, suggesting an absence of feedback regulation of de novo cholesterol biosynthesis. Furthermore, cyclodextrin treatment substantially suppressed essential M-CSF and RANKL-induced survival signaling pathways via Akt, mTOR and S6K. Consistent with these findings, cholesterol delivery via low-density lipoprotein (LDL) significantly increased OCL viability. Interestingly, OCLs from the LDL receptor (LDLR)-/- mouse exhibited reduced size and lifespan in vitro. Remarkably, LDLR+/+ OCL in lipoprotein-deficient medium phenocopied LDLR-/- OCL, while fusion and spreading of LDLR-/- OCL was rescued when cholesterol was chemically delivered during differentiation. With hyperlipidemia being associated with disease of the vascular system and bone, these findings provide novel insights into the selective lipoprotein and cholesterol dependency of the bone resorbing cell. Cell Death and Differentiation (2004) 11, S108-S118. doi:10.1038/sj.cdd.4401399 Published online 12 March 2004