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Plasma HIV viral load testing is the preferred means of monitoring antiretroviral treatment response. Dried blood spots (DBSs) hold considerable logistical advantages over EDTA samples, but they more frequently misclassify virological failure and have higher limits of detection (LoD)., Plasma HIV viral load testing is the preferred means of monitoring antiretroviral treatment response. Dried blood spots (DBSs) hold considerable logistical advantages over EDTA samples, but they more frequently misclassify virological failure and have higher limits of detection (LoD). Plasma separation cards (PSCs) may overcome these limitations. Health workers collected EDTA whole blood by venipuncture and 140 μl of finger-prick blood by capillary tube from 53 HIV-infected adults. Capillary blood was immediately transferred to PSCs. Additionally, 432 EDTA samples from HIV-infected adults were spotted onto PSCs and analyzed together with the finger-prick samples. Specificity and sensitivity of PSC with paired EDTA-PSC samples tested on a cobas 6800/8800 system with the cobas HIV-1 test (cobas HIV) was determined. LoD (3rd HIV-1 WHO International Standard) and stability at a range of temperatures and storage durations was determined using cobas HIV and cobas AmpliPrep/cobas TaqMan HIV-1 test v2.0 (CAP/CTM). Of 132 specimens with quantitative values for paired EDTA-PSC samples, the mean log10 difference between samples was 0.05 copies/ml (95% confidence interval [CI], −0.01 to 0.11). The LoD for cobas HIV was 790.2 copies/ml and for CAP/CTM was 737.9 copies/ml. At 1,000 copies/ml, PSC sensitivity was 97.0% (128/132) and specificity was 97.2% (343/353). Results correlated well with those from EDTA samples (Deming R2 = 0.90). PSC results were unaffected by temperature and storage conditions. PSC samples correlate well with plasma viral load and have adequate sensitivity and specificity. The improved performance may be as a result of a reduction in contribution from cell-associated viral nucleic acids. The card provides an alternative sample collection technology to DBSs.

BACKGROUND: A bridging study was performed to compare the safety, dose delivery, and acceptability of a prefilled plastic and user-filled paper applicator to assess whether a low-cost, user-filled, paper applicator could serve as a delivery option for tenofovir (TFV) 1% vaginal microbicide gel. METHODS: The study used a randomized crossover design with 25 healthy women randomized to begin with the prefilled or user-filled applicator. Within each study arm, participants delivered two 4.0-mL doses of TFV 1% gel vaginally for 7 days, with one dose delivered at the clinic each morning and a second dose delivered at home each evening. To assess the primary objective, applicator safety, colposcopy examinations were performed at 2 time points in each study arm. RESULTS: There were no colposcopic findings or adverse events attributable to either applicator. One case of vulvovaginal candidiasis was considered possibly related to gel use. On average, the user-filled applicator delivered 96% of the target dose, with 85% of doses falling within ± 10% of the average dose volume. Participants found both applicators comparable for ease of use, insertion, and dispensing gel, with 60% of participants preferring the user-filled applicator. CONCLUSIONS: This study suggests that both applicators are safe, and most women delivered TFV with the user-filled applicator as directed. Participants found both applicators acceptable, with a slight majority preferring the user-filled applicator. Incorporating a low-cost, user-filled, paper applicator to deliver TFV could help reduce costs and improve access to TFV 1% gel, especially in resource-limited settings heavily impacted by HIV.

Microfluidic paper-based analytical devices (μPADs) represent a promising platform technology for point-of-care diagnosis. Highly sensitive, rapid, and easy-to-perform immunoassays implemented on μPADs are desirable to fulfill the promise of the μPAD technology. This article reports the first microfluidic paper-based origami nanobiosensor (origami μPAD), which integrates zinc oxide nanowires (ZnO NWs) and electrochemical impedance spectroscopy (EIS) biosensing mechanism, for label-free, ultrasensitive immunoassays. The EIS mechanism features simple and label-free assay operations which take less than 25 min to be finished, while the ZnO NWs allow covalent bonding for immobilizing probe proteins and improve the biosensing performance with such features as high surface-area-to-volume ratios and high sensitivity to surface binding. The calibration of the device reveals an ultralow limit of detection (LOD) of 60 fg mL(-1) (>100 times lower than those of existing μPADs) for rabbit immunoglobulin G in phosphate-buffered saline. The detection of human immunodeficiency virus p24 antigen in human serum with a low LOD of 300 fg mL(-1) (>33 times lower than that of a commercial p24 antigen test kit) is also demonstrated. This novel μPAD design offers ultrahigh sensitivity, short assay time, and ease of operation, and thus possesses significant potential for low-cost, rapid molecular diagnosis of early-stage diseases.

Background: Electronic patient-reported outcomes (PRO) provide quick and usually reliable assessments of patients’ health-related quality of life (HRQL). Objective: An electronic version of the Patient-Reported Outcomes Quality of Life-human immunodeficiency virus (PROQOL-HIV) questionnaire was developed, and its face validity and reliability were assessed using standard psychometric methods. Methods: A sample of 80 French outpatients (66% male, 52/79; mean age 46.7 years, SD 10.9) were recruited. Paper-based and electronic questionnaires were completed in a randomized crossover design (2-7 day interval). Biomedical data were collected. Questionnaire version and order effects were tested on full-scale scores in a 2-way ANOVA with patients as random effects. Test-retest reliability was evaluated using Pearson and intraclass correlation coefficients (ICC, with 95% confidence interval) for each dimension. Usability testing was carried out from patients’ survey reports, specifically, general satisfaction, ease of completion, quality and clarity of user interface, and motivation to participate in follow-up PROQOL-HIV electronic assessments. Results: Questionnaire version and administration order effects (N=59 complete cases) were not significant at the 5% level, and no interaction was found between these 2 factors ( P =.94). Reliability indexes were acceptable, with Pearson correlations greater than .7 and ICCs ranging from .708 to .939; scores were not statistically different between the two versions. A total of 63 (79%) complete patients’ survey reports were available, and 55% of patients (30/55) reported being satisfied and interested in electronic assessment of their HRQL in clinical follow-up. Individual ratings of PROQOL-HIV user interface (85%-100% of positive responses) confirmed user interface clarity and usability. Conclusions: The electronic PROQOL-HIV introduces minor modifications to the original paper-based version, following International Society for Pharmacoeconomics and Outcomes Research (ISPOR) ePRO Task Force guidelines, and shows good reliability and face validity. Patients can complete the computerized PROQOL-HIV questionnaire and the scores from the paper or electronic versions share comparable accuracy and interpretation. [J Med Internet Res 2014;16(4):e115]

Dipstick and lateral-flow formats have dominated rapid diagnostics over the last three decades. These formats gained popularity in the consumer markets due to their compactness, portability and facile interpretation without external instrumentation. However, lack of quantitation in measurements has challenged the demand of existing assay formats in consumer markets. Recently, paper-based microfluidics has emerged as a multiplexable point-of-care platform which might transcend the capabilities of existing assays in resource-limited settings. However, paper-based microfluidics can enable fluid handling and quantitative analysis for potential applications in healthcare, veterinary medicine, environmental monitoring and food safety. Currently, in its early development stages, paper-based microfluidics is considered a low-cost, lightweight, and disposable technology. The aim of this review is to discuss: (1) fabrication of paper-based microfluidic devices, (2) functionalisation of microfluidic components to increase the capabilities and the performance, (3) introduction of existing detection techniques to the paper platform and (4) exploration of extracting quantitative readouts via handheld devices and camera phones. Additionally, this review includes challenges to scaling up, commercialisation and regulatory issues. The factors which limit paper-based microfluidic devices to become real world products and future directions are also identified.

Human immunodeficiency virus type 1 (HIV-1) in cultured cells, peripheral blood samples and sera were adsorbed on filter paper disks and inactivated by heat or ethanol. Two procedures, the polymerase chain reaction (PCR) and microtiter plate assay (HMPA) were used to detect the nucleic acid. The sensitivity after different heat treatments with nested PCR for HIV-1 DNA (or nested reverse transcription-PCR for HIV-1 RNA) was identical regardless of whether the samples were examined immediately or one month later. Inactivation by ethanol treatment resulted in a slight loss of sensitivity. The HMPA proved to be as reliable and specific as the conventional PCR technique. We conclude that the heat-treated filter paper disk assay is suitable for identifying HIV nucleic acid in clinical samples sent to the laboratory from a distance, e.g. in an envelope.

Blood sampling on filter paper has many advantages for the detection of perinatal human immunodeficiency virus (HIV) infection by the polymerase chain reaction (PCR). However, if the method is to be widely used, an assessment of its performance under field conditions is required. To simulate conditions in the field, 50-microliters aliquots of whole blood containing low levels of HIV proviral DNA (4 to 1,024 copies per 100,000 nucleated cells) were spotted onto filter paper; dried; and subjected to heat, humidity, and prolonged storage at room temperature. After exposure, the DNA was recovered and amplified with primers to human leukocyte antigen DQ alpha- and HIV-specific sequences. Treatment at 37 degrees C and 60% humidity for 7 days, storage for 12 weeks at 22 degrees C, and freeze-thawing twice had no adverse effect on PCR reactivity when compared with the results obtained with reference spots stored at -20 degrees C. The lower limits of HIV detection in all tests ranged from 4 to 16 HIV copies per 100,000 cells. Fixation in 70% ethanol improved the amplification of low levels of HIV DNA and reduced biohazard risks. These findings suggest that dried blood spots will provide a powerful new resource for testing for HIV by PCR, especially in remote areas where refrigeration and immediate sample processing are unavailable.

Objective: To report the nature of stroke in patients infected with human immunodeficiency virus (HIV) in a region with high HIV seroprevalence and describe HIV associated vasculopathy. Methods: Patients with first ever stroke, infected with HIV and prospectively included in the stroke register of the Groote Schuur Hospital/University of Cape Town stroke unit were identified and reviewed. Results: Between 2000 and 2006, 67 of the 1087 (6,1%) stroke patients were HIV infected. Of these, 91% (n = 61) were younger than 46 years. Cerebral infarction occurred in 96% (n = 64) of the HIV positive patients and intracerebral haemorrhage in 4% (n = 3). HIV infected young stroke patients did not demonstrate hypertension, diabetes, hyperlipidaemia or smoking as significant risk factors for ischaemic stroke. Infection as a risk factor for stroke was significantly more common in HIV positive patients (p = 0.018, OR 6.4, CI 3.1 to 13.2). In 52 (81%) patients with ischaemic stroke, an aetiology was determined. Primary aetiologies comprised infectious meningitides/vasculitides in 18 (28%) patients, coagulopathy in 12 (19%) patients and cardioembolism in nine (14%) patients. Multiple aetiologies were present in seven (11%) patients with ischaemic stroke. HIV associated vasculopathy was identified in 13 (20%) patients. The HIV associated vasculopathy manifested either extracranially (seven patients) as total or significant carotid occlusion or intracranially (six patients) as medium vessel occlusion, with or without fusiform aneurysmal dilation, stenosis and vessel calibre variation. Conclusion: Investigation of HIV infected patients presenting with stroke will determine an aetiology in the majority of patients. In our cohort, 20% of patients demonstrated evidence of an HIV associated vasculopathy.

We studied the use of dried spots of bodily fluids (plasma, whole blood, and mother's milk) on filter paper as a means of sample collection and storage for human immunodeficiency virus type 1 (HIV-1) viral load testing under stringent field conditions. Plasma placed directly in lysis buffer, which is customarily used for viral load assays, was used for comparison in all our experiments. Utilizing reconstruction experiments, we demonstrate no statistical differences between viral loads determined for plasma and mother's milk spotted on filter paper and those for the same fluids placed directly in lysis buffer. We found that the addition of whole blood directly to lysis buffer was unreliable and could not be considered a feasible option. However, viral load measurements for whole blood spotted onto filter paper correlated with plasma viral load values for both filter spots and lysis buffer (Pearson correlation coefficients, 0.7706 and 0.8155, respectively). In conclusion, dried spots of plasma, whole blood, or mother's milk provide a feasible means for the collection, storage, and shipment of samples for subsequent viral load measurement and monitoring. Virus material spotted and dried on filter paper is a good inexpensive alternative for collecting patient material to monitor the HIV-1 viral load. Measuring the HIV-1 burden from whole blood dried on filter paper provides a suitable alternative for low-technology settings with limited access to refrigeration, as can be found in sub-Saharan Africa.

Internet Web page survey responses were compared with those collected using traditional paper-and-pencil strategies to assess relative inclusion of a geographically dispersed population and comparativeness in responses. Three hundred and seven gay or bisexual men who had vacationed in a particular gay community completed Web-based and 244 paper-and-pencil questionnaires. Each questionnaire contained the same wording and question order. More Internet respondents than paper reported bisexual identity, nonmetropolitan residence, greater numbers of nonmain male partners for unprotected anal intercourse, and alcohol use than did paper-and-pencil respondents. Few other differences were identified. Assessing the reach of survey distribution can add to our knowledge base of surveys fielded using the Internet. Similarly, examining the potential biases in modes of administering convenience surveys can assist researchers to select the survey data collection method most appropriate to their research goals and design studies to assess the effect of the methods that are used.

The stability of human immunodeficiency virus type 1 (HIV-1) DNA in whole blood collected on filter paper (FTA Card) was evaluated. After >4 years of storage at room temperature in the dark our qualitative assay detected virus at a rate similar to that of our initial test (58 of 60, 97%; P = 0.16), suggesting long-term HIV-1 DNA stability.

The use of dried blood spots lends itself to widespread application in large field studies, especially in remote areas. We present experience gained during a perinatal HIV transmission study in southern Africa in which dried blood spot samples were used for polymerase chain reaction (PCR) tests. In this study, 15,810 filter paper cards with dried blood spots were collected. Infants were seen at age 6 and 12 weeks, and PCR was routinely done in duplicate on each sample. Of 186 negative controls (infants born to HIV-negative women), two (1.1%) had a single strongly reactive PCR result; the repeated duplicates were both negative. In contrast, all 24 known positive samples were strongly positive in both tests. Results were available from 1,976 duplicate tests on 1,235 infants born to HIV-infected women. Based on the PCR result on a later sample, the positive predictive value was 97.6% if both replicates were strongly positive (absorbance: 0.8 OD450 U), 100% when one of the replicates was strongly positive, and 27% when one or both replicates were weakly positive (but none strongly positive). When both replicates were negative, the negative predictive value wasor = 96.2%. Thus, when a single HIV PCR test has a strongly positive result, the infant is very likely to be infected. A positive PCR result after age 1 month was 98.9% accurate in predicting antibody positivity after 15 months. Suggestions for sample collection, storage, and PCR testing are provided.15,810 filter paper cards with dried blood spots were collected during a perinatal HIV transmission study in southern Africa to be subjected to polymerase chain reaction (PCR) testing. Infants were seen at ages 6 and 12 weeks, with PCR routinely done in duplicate on each sample. Of 186 infants born to HIV-negative mothers, two had a single strongly reactive PCR result, while the repeated duplicates were both negative. All 24 known positive samples were strongly positive in both tests. Results were available from 1976 duplicate tests on 1235 infants born to HIV-infected women. Based upon the PCR result of a later sample, the positive predictive value was 97.6% if both replicates were strongly positive, 100% when one of the replicates was strongly positive, and 27% when one or both replicates were weakly positive. When both replicates were negative, the negative predictive value was greater than or equal to 96.2%. Therefore when a single HIV PCR test has a strongly positive result, the infant is very likely infected. A positive PCR result after age 1 month was 98.9% accurate in predicting antibody positivity after 15 months.

A method for detection of proviral human immunodeficiency virus DNA in dried blood spots on filter paper by direct polymerase chain reaction (PCR) has been developed. To develop the method, a standard system was used which was prepared from cells each containing a single integrated provirus and titrated with normal donor blood. This rapid procedure provides virtually quantitative yields of nuclear DNA and exploits most of the standard methodology described for blood specimens. A nested PCR using SK38-SK39 gag as the internal primer pair was also designed; this PCR detected a single copy of provirus per filter at near theoretical frequency with SK19 probe. The utility of the procedure was demonstrated with clinical specimens. Blood spot filters from human immunodeficiency virus-infected and uninfected individuals were readily and unequivocally discriminated. The method is designed for ultimate use with large (1.5-ml) sample preparation tubes that are compatible as PCR tubes with thermal cyclers. This will permit convenient, direct single-tube PCR of dried blood specimens on filters. It should be adaptable to analysis of dried blood spots for a variety of infectious or genetic diseases.

Objectives: To determine the seroprevalence rate of toxoplasmosis, HIV, syphilis and rubella in a population of puerperal women. Methods: A prospective, cross-sectional study was performed from February 2007 to April 2008 at Hospital Geral, Universidade de Caxias do Sul in a population of 1,510 puerperal women. Women that gave birth to live born or stillborn infants were included in the study; maternal and perinatal variables were analyzed. Descriptive statistics and Pearson's chi-square with occasional Fisher's correction were used for comparisons. Alpha was set in 5%. Results: A total of 148 cases of congenital infection (9.8%) were identified: 66 cases of syphilis (4.4%), 40 cases of HIV (2.7%), 27 cases of toxoplasmosis (1.8%) and 15 cases of rubella (1.0%). In ten cases there was co-infection (four cases of HIV and syphilis, two cases of HIV and rubella, one case of HIV and toxoplasmosis, two cases of rubella and syphilis, and one case of toxoplasmosis and rubella). In a comparison between puerperal women with and without infection there was no statistical significance in relation to incidence of abortions, small for gestational age, prematurity, live births and stillbirths, and prenatal care. Need of neonatal intensive care unit (NICU), maternal schooling, maternal age higher than 35 years and drug use (alcohol, cocaine and crack) had statistical significance. Conclusion: The prevalence rate of infections was 9.8%. Need of NICU, maternal schooling lower than eight years, maternal age higher than 35 years and drug use were significantly associated with occurrence of congenital infection. Keywords: perinatal infections, congenital infections, clinical diagnosis, seroprevalence

The quantum spin properties of nitrogen-vacancy defects in diamond enable diverse applications in quantum computing and communications1. However, fluorescent nanodiamonds also have attractive properties for in vitro biosensing, including brightness2, low cost3 and selective manipulation of their emission4. Nanoparticle-based biosensors are essential for the early detection of disease, but they often lack the required sensitivity. Here we investigate fluorescent nanodiamonds as an ultrasensitive label for in vitro diagnostics, using a microwave field to modulate emission intensity5 and frequency-domain analysis6 to separate the signal from background autofluorescence7, which typically limits sensitivity. Focusing on the widely used, low-cost lateral flow format as an exemplar, we achieve a detection limit of 8.2 × 10−19 molar for a biotin–avidin model, 105 times more sensitive than that obtained using gold nanoparticles. Single-copy detection of HIV-1 RNA can be achieved with the addition of a 10-minute isothermal amplification step, and is further demonstrated using a clinical plasma sample with an extraction step. This ultrasensitive quantum diagnostics platform is applicable to numerous diagnostic test formats and diseases, and has the potential to transform early diagnosis of disease for the benefit of patients and populations. Lateral-flow in vitro diagnostic assays based on fluorescent nanodiamonds, in which microwave-based spin manipulation is used to increase sensitivity, are demonstrated using the biotin–avidin model and by the single-copy detection of HIV-1 RNA.