Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

Point-of-care HIV viral load in pregnant women without prenatal care: a cost-effectiveness analysis.

BACKGROUND: Routine cesarean delivery has been shown to decrease mother-to-child-transmission of HIV in women with high viral load greater than 1000 copies/mL; however, women presenting late in pregnancy may not have viral load results before delivery. OBJECTIVE: Our study investigated the costs and outcomes of using a point-of-care HIV RNA viral load test to guide delivery compared with routine cesarean delivery for all in the setting of unknown viral load. STUDY DESIGN: A decision-analytic model was constructed using TreeAge software to compare HIV RNA viral load testing vs routine cesarean delivery for all in a theoretical cohort of 1275 HIV-positive women without prenatal care who presented at term for delivery, the estimated population of HIV-positive women without prenatal care in the United States annually. TreeAge Pro software is used to build decision trees modeling clinical problems and perform cost-effectiveness, sensitivity, and simulation analysis to identify the optimal outcome. The average cost per test was $15.22. To examine the downstream impact of a cesarean delivery and because most childbearing women in the United States will deliver 2 children, we incorporated a second pregnancy and delivery in the model. Primary outcomes were mother-to-child transmission, delivery mode, cesarean delivery-related complications, cost, and quality-adjusted life years. Model inputs were derived from the literature and varied in sensitivity analyses. The cost-effectiveness threshold was $100,000/quality-adjusted life year. RESULTS: Measuring viral load resulted in more HIV-infected neonates than routine cesarean delivery for all due to viral exposure during more frequent vaginal births in this strategy. There were no observed maternal deaths or differences in cesarean delivery-related complications. Quantifying viral load increased cost by $3,883,371 and decreased quality-adjusted life years by 63 compared with routine cesarean delivery for all. With the threshold set at $100,000/quality-adjusted life year, the viral load test is cost-effective only when the vertical transmission rate in women with high viral load was below 0.68% (baseline: 16.8%) and when the odds ratio of vertical transmission with routine cesarean delivery for all compared with vaginal delivery was above 0.885 (baseline: 0.3). CONCLUSIONS: For HIV-infected pregnant women without prenatal care, quantifying viral load to guide mode of delivery using a point-of-care test resulted in increased costs and decreased effectiveness when compared with routine cesarean delivery for all, even after including downstream complications of cesarean delivery.

Does Economic Strengthening Improve Viral Suppression Among Adolescents Living with HIV? Results From a Cluster Randomized Trial in Uganda.

To assess the effect of a savings-led economic empowerment intervention on viral suppression among adolescents living with HIV. Using data from Suubi + Adherence, a longitudinal, cluster randomized trial in southern Uganda (2012-2017), we examine the effect of the intervention on HIV RNA viral load, dichotomized between undetectable (< 40 copies/ml) and detectable (≥ 40 copies/ml). Cluster-adjusted comparisons of means and proportions were used to descriptively analyze changes in viral load between study arms while multi-level modelling was used to estimate treatment efficacy after adjusting for fixed and random effects. At 24-months post intervention initiation, the proportion of virally suppressed participants in the intervention cohort increased tenfold (ΔT2-T0 = + 10.0, p = 0.001) relative to the control group (ΔT2-T0 = + 1.1, p = 0.733). In adjusted mixed models, simple main effects tests identified significantly lower odds of intervention adolescents having a detectable viral load at both 12- and 24-months. Interventions addressing economic insecurity have the potential to bolster health outcomes, such as HIV viral suppression, by improving ART adherence among vulnerable adolescents living in low-resource environments. Further research and policy dialogue on the intersections of financial security and HIV treatment are warranted.

Using a retention in care protocol to promote positive health and systems related outcomes.

People living with HIV can experience the full benefits of retention when they are continuously engaged in care. Continuous engagement in care promotes improved adherence to ART and positive health outcomes. An infectious disease clinic has implemented a protocol to primarily improve patient retention. The retrospective, facility-based, costing study took place in an infectious disease clinic in Washington DC. Retention was defined in two ways and over a 12-month period. Micro-costing direct measurement methods were used to collect unit costs in time series. Return on investment accounted for the cost of treatment based on CD4 strata. ROI was expressed in 2016USD. The difference in CD4 and viral load levels between the two periods of analysis were determined for active patients, infected with HIV. The year before the intervention was compared to the year of the intervention. Total treatment expenditure decreased from $2,435,653.00 to $2,283,296.23, resulting in a $152,356.77 gain from investment for the healthcare system over a 12-month investment period. The viral load suppression rate increased from 81 to 95 (p = 0.04) over the investment period. The number of patients in need of HIV related opportunistic infection prophylaxis decreased from 21 to 13 (p = 0.06). Improved immunologic, virologic and healthcare expenditure outcomes can be linked to the quality of retention practice.

Differentiated Human Immunodeficiency Virus RNA Monitoring in Resource-Limited Settings: An Economic Analysis.

BACKGROUND.: Viral load (VL) monitoring for patients receiving antiretroviral therapy (ART) is recommended worldwide. However, the costs of frequent monitoring are a barrier to implementation in resource-limited settings. The extent to which personalized monitoring frequencies may be cost-effective is unknown. METHODS.: We created a simulation model parameterized using person-level longitudinal data to assess the benefits of flexible monitoring frequencies. Our data-driven model tracked human immunodeficiency virus (HIV)-infected individuals for 10 years following ART initiation. We optimized the interval between viral load tests as a function of patients' age, gender, education, duration since ART initiation, adherence behavior, and the cost-effectiveness threshold. We compared the cost-effectiveness of the personalized monitoring strategies to fixed monitoring intervals every 1, 3, 6, 12, and 24 months. RESULTS.: Shorter fixed VL monitoring intervals yielded increasing benefits (6.034 to 6.221 discounted quality-adjusted life-years [QALYs] per patient with monitoring every 24 to 1 month over 10 years, respectively, standard error = 0.005 QALY), at increasing average costs: US$3445 (annual monitoring) to US$5393 (monthly monitoring) per patient, respectively (standard error = US$3.7). The adaptive policy optimized for low-income contexts achieved 6.142 average QALYs at a cost of US$3524, similar to the fixed 12-month policy (6.135 QALYs, US$3518). The adaptive policy optimized for middle-income resource settings yields 0.008 fewer QALYs per person, but saves US$204 compared to monitoring every 3 months. CONCLUSIONS.: The benefits from implementing adaptive vs fixed VL monitoring policies increase with the availability of resources. In low- and middle-income countries, adaptive policies achieve similar outcomes to simpler, fixed-interval policies.

A cheap and open HIV viral load technique applicable in routine analysis in a resource limited setting with a wide HIV genetic diversity.

BACKGROUND: HIV infection in Cameroon is characterized by a great viral diversity with all HIV-1 groups (M, N, O, and P) and HIV-2 in circulation. HIV group determination is very important if tailored viral load analysis and treatments are to be applied. In our laboratory, HIV viral load is carried out using two platforms; Biocentric and Abbott depending on the HIV group identified. Biocentric which quantifies HIV-1 group M is a cheap and open system useful in resource limited settings. The objective of this study was to compare the viral load analyses of serologically group-indeterminate HIV samples using the two platforms with the view of reducing cost. METHODS: Consecutive samples received between March and May 2014, and between August and September 2014 in our laboratory for HIV viral load analysis were included. All these samples were analyzed for their HIV groups using an in-house ELISA serotyping test. All HIV-1 group M samples were quantified using the Biocentric test while all other known atypical samples (HIV-1 groups N, O and P) were analyzed using the Abbott technique. HIV group-indeterminate samples (by serotyping) were quantified with both techniques. RESULTS: Among the 6355 plasma samples received, HIV-1 group M was identified in 6026 (94.82%) cases; HIV-1 group O, in 20 (0.31%); HIV-1 group M + O, in 3 (0.05%) and HIV-2, in 3 (0.05%) case. HIV-group indeterminate samples represented about 4.76% (303/6355) and only 231 of them were available for analysis by Abbott Real-Time HIV-1 and Generic HIV Viral Load techniques. Results showed that 188 (81.39%) samples had undetectable viral load in both techniques. All the detectable samples showed high viral load, with a mean of 4.5 log copies/ml (range 2.1-6.5) for Abbott Real-Time and 4.5 log copies/ml (range 2-6.4) for Generic HIV Viral Load. The mean viral load difference between the two techniques was 0.03 log10 copies/ml and a good correlation was obtained (r 2 = 0.89; P < 0.001). CONCLUSION: Our results suggest that cheaper and open techniques such as Biocentric could be useful alternatives for HIV viral load follow-up quantification in resource limited settings like Cameroon; even with its high viral diversity.

Scale-up of Routine Viral Load Testing in Resource-Poor Settings: Current and Future Implementation Challenges.

Despite immense progress in antiretroviral therapy (ART) scale-up, many people still lack access to basic standards of care, with our ability to meet the Joint United Nations Programme on HIV/AIDS 90-90-90 treatment targets for HIV/AIDS dependent on dramatic improvements in diagnostics. The World Health Organization recommends routine monitoring of ART effectiveness using viral load (VL) testing at 6 months and every 12 months, to monitor treatment adherence and minimize failure, and will publish its VL toolkit later this year. However, the cost and complexity of VL is preventing scale-up beyond developed countries and there is a lack of awareness among clinicians as to the long-term patient benefits and its role in prolonging the longevity of treatment programs. With developments in this diagnostic field rapidly evolving-including the recent improvements for accurately using dried blood spots and the imminent appearance to the market of point-of-care technologies offering decentralized diagnosis-we describe current barriers to VL testing in resource-limited settings. Effective scale-up can be achieved through health system and laboratory system strengthening and test price reductions, as well as tackling multiple programmatic and funding challenges.

Projecting the epidemiological effect, cost-effectiveness and transmission of HIV drug resistance in Vietnam associated with viral load monitoring strategies.

OBJECTIVES: The objective of this study was to investigate the potential epidemiological impact of viral load (VL) monitoring and its cost-effectiveness in Vietnam, where transmitted HIV drug resistance (TDR) prevalence has increased from <5% to 5%-15% in the past decade. METHODS: Using a population-based mathematical model driven by data from Vietnam, we simulated scenarios of various combinations of VL testing coverage, VL thresholds for second-line ART initiation and availability of HIV drug-resistance tests. We assessed the cost per disability-adjusted life year (DALY) averted for each scenario. RESULTS: Projecting expected ART scale-up levels, to approximately double the number of people on ART by 2030, will lead to an estimated 18 510 cases (95% CI: 9120-34 600 cases) of TDR and 55 180 cases (95% CI: 40 540-65 900 cases) of acquired drug resistance (ADR) in the absence of VL monitoring. This projection corresponds to a TDR prevalence of 16% (95% CI: 11%-24%) and ADR of 18% (95% CI: 15%-20%). Annual or biennial VL monitoring with 30% coverage is expected to relieve 12%-31% of TDR (2260-5860 cases), 25%-59% of ADR (9620-22 650 cases), 2%-6% of HIV-related deaths (360-880 cases) and 19 270-51 400 DALYs during 2015-30. The 30% coverage of VL monitoring is estimated to cost US$4848-5154 per DALY averted. The projected additional cost for implementing this strategy is US$105-268 million over 2015-30. CONCLUSIONS: Our study suggests that a programmatically achievable 30% coverage of VL monitoring can have considerable benefits for individuals and leads to population health benefits by reducing the overall national burden of HIV drug resistance. It is marginally cost-effective according to common willingness-to-pay thresholds.

Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection.

BACKGROUND: Nucleic acid testing (NAT) has become the standard for high sensitivity in detecting low levels of virus. However, adoption of NAT can be cost prohibitive in low-resource settings where access to extreme sensitivity could be clinically advantageous for early detection of infection. We report development and preliminary validation of a simple, low-cost, fully automated digital p24 antigen immunoassay with the sensitivity of quantitative NAT viral load (NAT-VL) methods for detection of acute HIV infection. METHODS: We developed an investigational 69-min immunoassay for p24 capsid protein for use on a novel digital analyzer on the basis of single-molecule-array technology. We evaluated the assay for sensitivity by dilution of standardized preparations of p24, cultured HIV, and preseroconversion samples. We characterized analytical performance and concordance with 2 NAT-VL methods and 2 contemporary p24 Ag/Ab combination immunoassays with dilutions of viral isolates and samples from the earliest stages of HIV infection. RESULTS: Analytical sensitivity was 0.0025 ng/L p24, equivalent to 60 HIV RNA copies/mL. The limit of quantification was 0.0076 ng/L, and imprecision across 10 runs was <10% for samples as low as 0.09 ng/L. Clinical specificity was 95.1%. Sensitivity concordance vs NAT-VL on dilutions of preseroconversion samples and Group M viral isolates was 100%. CONCLUSIONS: The digital immunoassay exhibited >4000-fold greater sensitivity than contemporary immunoassays for p24 and sensitivity equivalent to that of NAT methods for early detection of HIV. The data indicate that NAT-level sensitivity for acute HIV infection is possible with a simple, low-cost digital immunoassay.

An inexpensive, accurate, and precise wet-mount method for enumerating aquatic viruses.

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the "filter mount" method, in which samples are filtered onto costly 0.02-µm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5×10(7) viruses ml(-1). The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17×10(6) to 1.37×10(8) viruses ml(-1) when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1×10(6) viruses ml(-1)) encountered in field and laboratory samples.

Pooled nucleic acid testing to identify antiretroviral treatment failure during HIV infection in Seoul, South Korea.

BACKGROUND: There have been various efforts to identify less costly but still accurate methods for monitoring the response to HIV treatment. We evaluated a pooling method to determine if this could improve screening efficiency and reduce costs while maintaining accuracy in Seoul, South Korea. METHODS: We conducted the first prospective study of pooled nucleic acid testing (NAT) using a 5 minipool + algorithm strategy versus individual viral load testing for patients receiving antiretroviral therapy (ART) between November 2011 and August 2012 at an urban hospital in Seoul, South Korea. The viral load assay used has a lower level of detection of 20 HIV RNA copies/ml, and the cost per assay is US$ 136. The 5 minipool +algorithm strategy was applied and 43 pooled samples were evaluated. The relative efficiency and accuracy of the pooled NAT were compared with those of individual testing. RESULTS: Using the individual viral load assay, 15 of 215 (7%) plasma samples had more than 200 HIV RNA copies/ml. The pooled NAT using the 5 minipool + algorithm strategy was applied to 43 pooled samples; 111 tests were needed to test all samples when virologic failure was defined at HIV RNA ≥ 200 copies/ml. Therefore, 104 tests were saved over individual testing, with a relative efficiency of 0.48. When evaluating costs, a total of US$ 14,144 was saved for 215 individual samples during 10 months. The negative predictive value was 99.5% for all samples with HIV RNA ≥ 200 copies/ml. CONCLUSIONS: The pooled NAT with 5 minipool + algorithm strategy seems to be a very promising approach to effectively monitor patients receiving ART and to save resources.

A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus (WYMV).

BACKGROUND: Wheat yellow mosaic virus (WYMV) is an important pathogen in China and other countries. It is the member of the genus Bymovirus and transmitted primarily by Polymyxa graminis. The incidence of wheat infections in endemic areas has risen in recent years. Prompt and dependable identification of WYMV is a critical component of response to suspect cases. METHODS: In this study, a one step real-time RT-PCR, followed by standard curve analysis for the detection and identification of WYMV, was developed. Two reference genes, 18s RNA and ß-actin were selected in order to adjust the veracity of the real-time RT-PCR assay. RESULTS: We developed a one-step Taqman-based real-time quantitative RT-PCR (RT-qPCR) assay targeting the conserved region of the 879 bp long full-length WYMV coat protein gene. The accuracy of normalized data was analyzed along with appropriate internal control genes: ß-actin and 18s rRNA which were included in detecting of WYMV-infected wheat leaf tissues. The detectable end point sensitivity in RT-qPCR assay was reaching the minimum limit of the quantitative assay and the measurable copy numbers were about 30 at 106-fold dilution of total RNA. This value was close to 104-fold more sensitive than that of indirect enzyme-linked immunosorbent assay. More positive samples were detected by RT-qPCR assay than gel-based RT-PCR when detecting the suspected samples collected from 8 regions of China. Based on presented results, RT-qPCR will provide a valuable method for the quantitative detection of WYMV. CONCLUSIONS: The Taqman-based RT-qPCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of WYMV than other currently used methods.

A study of financial incentives to reduce plasma HIV RNA among patients in care.

The role of financial incentives in HIV care is not well studied. We conducted a single-site study of monetary incentives for viral load suppression, using each patient as his own control. The incentive size ($100/quarter) was designed to be cost-neutral, offsetting estimated downstream costs averted through reduced HIV transmission. Feasibility outcomes were clinic workflow, patient acceptability, and patient comprehension. Although the study was not powered for effectiveness, we also analyzed viral load suppression. Of 80 eligible patients, 77 consented, and 69 had 12 month follow-up. Feasibility outcomes showed minimal impact on patient workflow, near-unanimous patient acceptability, and satisfactory patient comprehension. Among individuals with detectable viral loads pre-intervention, the proportion of undetectable viral load tests increased from 57 to 69 % before versus after the intervention. It is feasible to use financial incentives to reward ART adherence, and to specify the incentive by requiring cost-neutrality and targeting biological outcomes.

Pooling strategies to reduce the cost of HIV-1 RNA load monitoring in a resource-limited setting.

BACKGROUND: Quantitative human immunodeficiency virus (HIV) RNA load testing surpasses CD4 cell count and clinical monitoring in detecting antiretroviral therapy (ART) failure; however, its cost can be prohibitive. Recently, the use of pooling strategies with a clinically appropriate viral load threshold was shown to be accurate and efficient for monitoring when the prevalence of virologic failure is low. METHODS: We used laboratory request form information to identify specimens with a low pretest probability of virologic failure. Patients aged ≥15 years who were receiving first-line ART had individual viral load results available were eligible. Blood plasma, dried blood spots, and dried plasma spots were evaluated. Two pooling strategies were compared: minipools of 5 samples and a 10 ×10 matrix platform (liquid plasma specimens only). A deconvolution algorithm was used to identify specimens(s) with detectable viral loads. RESULTS: The virologic failure rate in the study sample was <10%. Specimens included were liquid plasma specimens tested in minipools(n = 400), of which 300 were available for testing by matrix, and specimens tested with minipools only: dried blood spots (n = 100) and dried plasma spots (n = 185). Pooling methods resulted in 30.5%-60% fewer HIV RNA tests required to screen the study sample. For plasma pooling, the matrix strategy had the better efficiency, but minipools of 5 dried blood spots had the best efficiency overall and were accurate at a >95% negative predictive value with minimal technical requirements. CONCLUSIONS: In resource-constrained settings, a combination of preselection of patients with low pretest probability of virologic failure and pooled testing can reduce the cost of virologic monitoring without compromising accuracy.

Low-cost assays for monitoring HIV infected individuals in resource-limited settings.

Use of a combination of CD4 counts and HIV viral load testing in the management of antiretroviral therapy (ART) provides higher prognostic estimation of the risk of disease progression than does the use of either test alone. The standard methods to monitor HIV infection are flow cytometry based for CD4+ T cell count and molecular assays to quantify plasma viral load of HIV. Commercial assays have been routinely used in developed countries to monitor ART. However, these assays require expensive equipment and reagents, well trained operators, and established laboratory infrastructure. These requirements restrict their use in resource-limited settings where people are most afflicted with the HIV-1 epidemic. With the advent of low-cost and/or low-tech alternatives, the possibility of implementing CD4 count and viral load testing in the management of ART in resource-limited settings is increasing. However, an appropriate validation should have been done before putting them to use for patient testing.

Measures of site resourcing predict virologic suppression, immunologic response and HIV disease progression following highly active antiretroviral therapy (HAART) in the TREAT Asia HIV Observational Database (TAHOD).

OBJECTIVES: Surrogate markers of HIV disease progression are HIV RNA in plasma viral load (VL) and CD4 cell count (immune function). Despite improved international access to antiretrovirals, surrogate marker diagnostics are not routinely available in resource-limited settings. Therefore, the objective was to assess effects of economic and diagnostic resourcing on patient treatment outcomes. METHODS: Analyses were based on 2333 patients initiating highly active antiretroviral therapy (HAART) from 2000 onwards. Sites were categorized by World Bank country income criteria (high/low) and annual frequency of VL (> or = 3, 1-2 or <1) or CD4 (> or = 3 or <3) testing. Endpoints were time to AIDS/death and change in CD4 cell count and VL suppression (<400 HIV-1 RNA copies/mL) at 12 months. Demographics, Centers for Disease Control and Prevention (CDC) classification, baseline VL/CD4 cell counts, hepatitis B/C coinfections and HAART regimen were covariates. Time to AIDS/death was analysed by proportional hazards models. CD4 and VL endpoints were analysed using linear and logistic regression, respectively. RESULTS: Increased disease progression was associated with site-reported VL testing less than once per year [hazard ratio (HR)=1.4; P=0.032], severely symptomatic HIV infection (HR=1.4; P=0.003) and hepatitis C virus coinfection (HR=1.8; P=0.011). A total of 1120 patients (48.2%) had change in CD4 cell count data. Smaller increases were associated with older age (P<0.001) and 'Other' HIV source exposures, including injecting drug use and blood products (P=0.043). A total of 785 patients (33.7%) contributed to the VL suppression analyses. Patients from sites with VL testing less than once per year [odds ratio (OR)=0.30; P<0.001] and reporting 'Other' HIV exposures experienced reduced suppression (OR=0.28; P<0.001). CONCLUSION: Low measures of site resourcing were associated with less favourable patient outcomes, including a 35% increase in disease progression in patients from sites with VL testing less than once per year.

A new affordable flow cytometry based method to measure HIV-1 viral load.

Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub-Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV-1-5'LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)-products were captured by anti-DIG and anti-DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin-R-phycoerythrine. The primer system used recognized all HIV-1 subtypes. A coamplified internal standard warranted the functionality of the PCR and allows reproducible viral load measurements. Two drawbacks of current viral load measurements are overcome by the flow cytometry based test described hereof. First, all known worldwide relevant HIV-1 subtypes including subtypes O, N, and new CRFs are quantifiable with high sensitivity (50 to >1 x 10(6) copies per PCR). Second, the cost per test can be reduced to less than 12 US$ instead of the current 50-100 US$. Additionally, the test described in this report offers the possibility to perform complete monitoring program (CD4 T-cell count, CD4% and viral load) for the first time, with the same device for HIV-infected persons.

Food insecurity is associated with incomplete HIV RNA suppression among homeless and marginally housed HIV-infected individuals in San Francisco.

BACKGROUND AND OBJECTIVES: There is growing international concern that food insecurity may negatively impact antiretroviral (ARV) treatment outcomes, but no studies have directly evaluated the effect of food insecurity on viral load suppression and antiretroviral adherence. We hypothesized that food insecurity would be associated with poor virologic response among homeless and marginally housed HIV-positive ARV-treated patients. DESIGN: This is a cross-sectional study. PARTICIPANTS AND SETTING: Participants were ARV-treated homeless and marginally housed persons receiving adherence monitoring with unannounced pill counts in the Research on Access to Care in the Homeless (REACH) Cohort. MEASUREMENTS: Food insecurity was measured by the Household Food Insecurity Access Scale (HFIAS). The primary outcome was suppression of HIV viral RNA to <50 copies/ml. We used multivariate logistic regression to assess whether food insecurity was associated with viral suppression. RESULTS: Among 104 participants, 51% were food secure, 24% were mildly or moderately food insecure and 25% were severely food insecure. Severely food insecure participants were less likely to have adherence > or =80%. In adjusted analyses, severe food insecurity was associated with a 77% lower odds of viral suppression (95% CI = 0.06-0.82) when controlling for all covariates. In analyses stratified by adherence level, severe food insecurity was associated with an 85% lower odds of viral suppression (95% CI = 0.02-0.99) among those with < or =80% adherence and a 66% lower odds among those with >80% adherence (95% CI = 0.06-1.81). CONCLUSIONS: Food insecurity is present in half of the HIV-positive urban poor in San Francisco, one of the best resourced settings for HIV-positive individuals in the United States, and is associated with incomplete viral suppression. These findings suggest that ensuring access to food should be an integral component of public health HIV programs serving impoverished populations.

Optimizing viral load testing access for the last mile: Geospatial cost model for point of care instrument placement.

INTRODUCTION: Viral load (VL) monitoring programs have been scaled up rapidly, but are now facing the challenge of providing access to the most remote facilities (the "last mile"). For the hardest-to-reach facilities in Zambia, we compared the cost of placing point of care (POC) viral load instruments at or near facilities to the cost of an expanded sample transportation network (STN) to deliver samples to centralized laboratories. METHODS: We extended a previously described geospatial model for Zambia that first optimized a STN for centralized laboratories for 90% of estimated viral load volumes. Amongst the remaining 10% of volumes, facilities were identified as candidates for POC placement, and then instrument placement was optimized such that access and instrument utilization is maximized. We evaluated the full cost per test under three scenarios: 1) POC placement at all facilities identified for POC; 2)an optimized combination of both on-site POC placement and placement at facilities acting as POC hubs; and 3) integration into the centralized STN to allow use of centralized laboratories. RESULTS: For the hardest-to-reach facilities, optimal POC placement covered a quarter of HIV-treating facilities. Scenario 2 resulted in a cost per test of $39.58, 6% less than the cost per test of scenario 1, $41.81. This is due to increased POC instrument utilization in scenario 2 where facilities can act as POC hubs. Scenario 3 was the most costly at $53.40 per test, due to high transport costs under the centralized model ($36 per test compared to $12 per test in scenario 2). CONCLUSIONS: POC VL testing may reduce the costs of expanding access to the hardest-to-reach populations, despite the cost of equipment and low patient volumes. An optimal combination of both on-site placement and the use of POC hubs can reduce the cost per test by 6-35% by reducing transport costs and increasing instrument utilization.