Biotin Interference in Point of Care HIV Immunoassay.

The use of high concentrations of biotin as a dietary supplement to improve hair, skin, and nail quality has increased in the United States over the past few years. High concentrations of biotin have been shown to interfere with some diagnostic assays that use streptavidin-biotin interactions as one of the steps in the assay. The objective of this report is to evaluate potential biotin interference on the analytical and clinical sensitivity of a point of care (POC) antigen-antibody combo HIV-1 assay. We spiked biotin at concentrations ranging from 12.5 to 400 ng/mL into serum and plasma containing HIV-1 subtype B p24 antigen derived from culture supernatant. The p24 antigen was present in the matrices at 30 pg/mL. Fifty microliters of each sample was applied to Alere Determine HIV-1/2 Ag/Ab combo assay strips in duplicate and results were read by eye after 20 to 30 min. Biotin interfered with detection of HIV-1 p24 in serum and plasma. HIV-1 p24 was not detected at 30 pg/mL p24 when biotin was present at 200 ng/mL concentration. Our study demonstrated that elevated levels of biotin in samples may interfere with POC assays. It is important to consider biotin supplements as potential sources of falsely increased or decreased test results, especially in cases wherein supplementation cannot be ruled out.

Computational design and clinical demonstration of a copper nanocluster based universal immunosensor for sensitive diagnostics.

Nanoparticle based sensors are good alternatives for non-enzymatic sensing applications due to their high stability, superior photoluminescence, biocompatibility and ease of fabrication, with the only disadvantage being the cost of the synthesis process (owing to the expensive precursors and infrastructure). For the first time, we report the design of an immunosensor employing streptavidin conjugated copper nanocluster, developed at a much lower cost compared to other nanomaterials like noble metal nanoparticles and quantum dots. Using in silico tools, we have tried to establish the dynamics of conjugation of nanocluster to the streptavidin protein, based on EDC-NHS coupling. The computational simulations have successfully explained the crucial role played by the components of the immunosensor leading to an efficient design capable of high sensitivity. In order to demonstrate the functioning of the Copper Nanocluster ImmunoSensor (CuNIS), HIV-1 p24 biomarker test was chosen as the model assay. The immunosensor was able to achieve an analytical limit of detection of 23.8 pg mL-1 for HIV-1 p24 with a linear dynamic range of 27-1000 pg mL-1. When tested with clinical plasma samples, CuNIS based p24 assay showed 100% specificity towards HIV-1 p24. With the capability of multiplexed detection and a cost of fabrication 100 times lower than that of the conventional metal nanoclusters, CuNIS has the potential to be an essential low-cost diagnostic tool in resource-limited settings.

Cross-Subtype Detection of HIV-1 Capsid p24 Antigen Using a Sensitive Europium Nanoparticle Assay.

Accurate and early detection of diverse HIV-1 subtypes using currently available p24 antigen assays have been a major challenge. We report the development of a sensitive time resolved fluorescence (TRF) europium nanoparticle immuno assay for cross subtype detection of p24 antigen using broadly cross-reactive antibodies. Several antibodies were tested for optimal reactivity with antigens of diverse HIV-1 subtypes and circulating recombinant forms. We tested HIV strains using this assay for sensitivity and quantification ability at the pico-gram per millilter level. We identified two broadly cross-reactive HIV-1 p24 antibodies C65690M and ANT-152, which detected all strains of HIV tested. These two antibodies also yielded a better signal to cutoff ratio for the same amount of antigen tested in comparison to a commercial assay. Using an appropriate combination of C65690M and ANT-152 p24 antibodies capable of detecting all HIV types and highly sensitive TRF-based europium nano particle assay platform, we developed a sensitive p24 antigen assay that can detect HIV infection of all HIV subtypes and may be useful in early detection.

Sub-picogram level sensitivity in HIV diagnostics achieved with the europium nanoparticle immunoassay through metal enhanced fluorescence.

We describe a novel application of Metal Enhanced Fluorescence (MEF) to immunoassays for boosting the signal through a single step modification of the europium nanoparticle based immunoassay with addition of gold nanoparticles. The new limit of detection was found to be 0.19 pg mL-1 which was much lower than that of the conventional assay which was around 1.80 pg mL-1, thus achieving a ten-fold increase in the limit of detection of p24, an early biomarker for HIV infections. Real world applications of the new technique were demonstrated with the commercially available Perkin Elmer Alliance kits greatly improving their sensitivity limits, thus demonstrating that the sensitivity and reproducibility of this approach are as good as those of high-end, sensitive immunoassays. The results of this study pave the way for the development of a highly sensitive screening protocol based on any fluorescent nanoparticle based immunoassay.

Streptavidin-conjugated gold nanoclusters as ultrasensitive fluorescent sensors for early diagnosis of HIV infection.

We have engineered streptavidin-labeled fluorescent gold nanoclusters to develop a gold nanocluster immunoassay (GNCIA) for the early and sensitive detection of HIV infection. We performed computational simulations on the mechanism of interaction between the nanoclusters and the streptavidin protein via in silico studies and showed that gold nanoclusters enhance the binding to the protein, by enhancing interaction between the Au atoms and the specific active site residues, compared to other metal nanoclusters. We also evaluated the role of glutathione conjugation in binding to gold nanoclusters with streptavidin. As proof of concept, GNCIA achieved a sensitivity limit of detection of HIV-1 p24 antigen in clinical specimens of 5 pg/ml, with a detection range up to1000 pg/ml in a linear dose-dependent manner. GNCIA demonstrated a threefold higher sensitivity and specificity compared to enzyme-linked immunosorbent assay for the detection of HIV p24 antigen. The specificity of the immunoassay was 100% when tested with plasma samples negative for HIV-1 p24 antigen and positive for viruses such as hepatitis B virus, hepatitis C virus, and dengue. GNCIA could be developed into a universal labeling technology using the relevant capture and detector antibodies for the specific detection of antigens of various pathogens in the future.

Femtogram Level Sensitivity achieved by Surface Engineered Silica Nanoparticles in the Early Detection of HIV Infection.

We have engineered streptavidin labelled Europium doped fluorescent silica nanoparticles which significantly increased sensitivity without compromising the specificity of the immunoassay. As a proof of concept, a time resolved fluorescence based sandwich immunoassay was developed to detect HIV-1 p24 antigen in clinical specimens. The detection range of the silica nanoparticle based immunoassay (SNIA) was found to be between 0.02 to 500 pg/mL in a linear dose dependent manner. SNIA offers 1000 fold enhancement over conventional colorimetric ELISA. Testing of plasma samples that were HIV negative showed no false positive results in the detection of HIV-1 p24 antigen. This highly sensitive p24 assay can help improve blood safety by reducing the antibody negative window period in blood donors in resource limited settings where nucleic acid testing is not practical or feasible. This technology can also be easily transferred to a lab-on-a-chip platform for use in resource limited settings and can also be easily adopted for the detection of other antigens.

Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms.

Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody.

Genotypic prediction of tropism of highly diverse HIV-1 strains from Cameroon.

BACKGROUND: The use of CCR5 antagonists involves determination of HIV-1 tropism prior to initiation of treatment. HIV-1 tropism can be assessed either by phenotypic or genotypic methods. Genotypic methods are extensively used for tropism prediction. However, their validation in predicting tropism of viral isolates belonging to group M non-B subtypes remains challenging. In Cameroon, the genetic diversity of HIV-1 strains is the broadest reported worldwide. To facilitate the integration of CCR5 antagonists into clinical practice in this region, there is a need to evaluate the performance of genotypic methods for predicting tropism of highly diverse group M HIV-1 strains. METHODS: Tropism of diverse HIV-1 strains isolated from PBMCs from Cameroon was determined using the GHOST cell assay. Prediction, based on V3 sequences from matched plasma samples, was determined using bioinformatics algorithms and rules based on position 11/25 and net charge applied independently or combined according to Delobel's and Garrido's rules. Performance of genotypic methods was evaluated by comparing prediction generated with tropism assigned by the phenotypic assay. RESULTS: Specificity for predicting R5-tropic virus was high, ranging from 83.7% to 97.7% depending on the genotypic methods used. Sensitivity for X4-tropic viruses was fairly low, ranging from 33.3% to 50%. In our study, overall, genotypic methods were less able to accurately predict X4-tropic virus belonging to subtype CRF02_AG. In addition, it was found that of the methods we used the Garrido rule has the highest sensitivity rate of over 50% with a specificity of 93%. CONCLUSION: Our study demonstrated that overall, genotypic methods were less sensitive for accurate prediction of HIV-1 tropism in settings where diverse HIV-1 strains co-circulate. Our data suggest that further optimization of genotypic methods is needed and that larger studies to determine their utility for tropism prediction of diverse HIV-1 strains may be warranted.

Development of a microchip Europium nanoparticle immunoassay for sensitive point-of-care HIV detection.

Rapid, sensitive and specific diagnostic assays play an indispensable role in determination of HIV infection stages and evaluation of efficacy of antiretroviral therapy. Recently, our laboratory developed a sensitive Europium nanoparticle-based microtiter-plate immunoassay capable of detecting target analytes at subpicogram per milliliter levels without the use of catalytic enzymes and signal amplification processes. Encouraged by its sensitivity and simplicity, we continued to miniaturize this assay to a microchip platform for the purpose of converting the benchtop assay technique to a point-of-care test. It was found that detection capability of the microchip platform could be readily improved using Europium nanoparticle probes. We were able to routinely detect 5 pg/mL (4.6 attomoles) of HIV-1 p24 antigen at a signal-to-blank ratio of 1.5, a sensitivity level reasonably close to that of microtiter-plate Europium nanoparticle assay. Meanwhile, use of the microchip platform effectively reduced sample/reagent consumption 4.5 fold and shortened total assay time 2 fold in comparison with microtiter plate assays. Complex matrix substance in plasma negatively affected the microchip assays and the effects could be minimized by diluting the samples before loading. With further improvements in sensitivity, reproducibility, usability, assay process simplification, and incorporation of portable time-resolved fluorescence reader, Europium nanoparticle immunoassay technology could be adapted to meet the challenges of point-of-care diagnosis of HIV or other health-threatening pathogens at bedside or in resource-limited settings.

Point of Care Technologies for HIV.

Effective prevention of HIV/AIDS requires early diagnosis, initiation of therapy, and regular plasma viral load monitoring of the infected individual. In addition, incidence estimation using accurate and sensitive assays is needed to facilitate HIV prevention efforts in the public health setting. Therefore, more affordable and accessible point-of-care (POC) technologies capable of providing early diagnosis, HIV viral load measurements, and CD4 counts in settings where HIV is most prevalent are needed to enable appropriate intervention strategies and ultimately stop transmission of the virus within these populations to achieve the future goal of an AIDS-free generation. This review discusses the available and emerging POC technologies for future application to these unmet public health needs.

Seroprevalence of human herpesvirus-8 in HIV-1 infected and uninfected individuals in Cameroon.

We evaluated the prevalence of HHV-8 antibodies in 516 plasma samples collected from HIV positive and negative patients from blood banks and urban areas of Cameroon. Among HIV-1 positive samples, HHV-8 seropositivity rate was 61% based on combined reactivity using both ELISA and IFA techniques. HIV negative samples showed 62% seropositivity rate for HHV-8 antibodies. Our results indicate a high HHV-8 prevalence rate in both HIV infected and uninfected individuals in Cameroon.

Sensitive detection assays for influenza RNA do not reveal viremia in US blood donors.

BACKGROUND: There have been anecdotal reports of influenza viremia since the 1960s. We present an assessment of the prevalence of seasonal and 2009 H1N1 influenza viremia (via RNA testing) in blood donor populations using multiple sensitive detection assays. METHODS: Several influenza RNA amplification assays, including transcription-mediated amplification (TMA) and 2 reverse-transcription polymerase chain reaction (RT-PCR) assays, were evaluated and used to test donor samples. Retrospective samples from 478 subjects drawn at sites with high influenza activity were tested. Prospective samples were collected from 1004 blood donors who called their donation center within 3 days of donation complaining of influenza-like illness (ILI). The plasma collected on the day of donation for these subjects was tested. RESULTS: Of the repository samples, 2 of 478 plasma samples were initially reactive but not repeat reactive by influenza TMA. Of blood donors reporting ILI symptoms postdonation, 1 of 1004 samples was TMA initially reactive but not repeat reactive; all samples were nonreactive by RT-PCR testing. CONCLUSIONS: Targeting blood donor populations most likely to have influenza infection, we failed to detect influenza RNA in 1482 donor samples, with most tested by 3 different RNA assays. Seasonal influenza does not appear to pose a significant contamination threat to the blood supply.

Anthrax edema toxin impairs clearance in mice.

The anthrax edema toxin (ET) of Bacillus anthracis is composed of the receptor-binding component protective antigen (PA) and of the adenylyl cyclase catalytic moiety, edema factor (EF). Uptake of ET into cells raises intracellular concentrations of the secondary messenger cyclic AMP, thereby impairing or activating host cell functions. We report here on a new consequence of ET action in vivo. We show that in mouse models of toxemia and infection, serum PA concentrations were significantly higher in the presence of enzymatically active EF. These higher concentrations were not caused by ET-induced inhibition of PA endocytosis; on the contrary, ET induced increased PA binding and uptake of the PA oligomer in vitro and in vivo through upregulation of the PA receptors TEM8 and CMG2 in both myeloid and nonmyeloid cells. ET effects on protein clearance from circulation appeared to be global and were not limited to PA. ET also impaired the clearance of ovalbumin, green fluorescent protein, and EF itself, as well as the small molecule biotin when these molecules were coinjected with the toxin. Effects on injected protein levels were not a result of general increase in protein concentrations due to fluid loss. Functional markers for liver and kidney were altered in response to ET. Concomitantly, ET caused phosphorylation and activation of the aquaporin-2 water channel present in the principal cells of the collecting ducts of the kidneys that are responsible for fluid homeostasis. Our data suggest that in vivo, ET alters circulatory protein and small molecule pharmacokinetics by an as-yet-undefined mechanism, thereby potentially allowing a prolonged circulation of anthrax virulence factors such as EF during infection.

Absence of detectable XMRV and other MLV-related viruses in healthy blood donors in the United States.

BACKGROUND: Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4-6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern. METHODOLOGY/PRINCIPAL FINDINGS: To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.

Susceptibility of human primary neuronal cells to xenotropic murine leukemia virus-related (XMRV) virus infection.

BACKGROUND: Xenotropic Murine Leukemia Virus-related (XMRV) virus is a recently identified mouse gammaretrovirus that has the ability to infect certain human cells. In this study, we investigated the susceptibility of primary neuronal cell types to infection with XMRV. FINDINGS: We observed that the human primary progenitors, progenitor-derived neurons, and progenitor-derived astrocytes supported XMRV multiplication. Interestingly, both progenitors and progenitor-derived neurons were more susceptible compared with progenitor-derived astrocytes. In addition, XMRV-infected Jurkat cells were able to transmit infection to neuronal cells. CONCLUSIONS: These data suggest that neuronal cells are susceptible for XMRV infection.

Failure to confirm XMRV/MLVs in the blood of patients with chronic fatigue syndrome: a multi-laboratory study.

Murine leukemia viruses (MLVs), including xenotropic-MLV-related virus (XMRV), have been controversially linked to chronic fatigue syndrome (CFS). To explore this issue in greater depth, we compiled coded replicate samples of blood from 15 subjects previously reported to be XMRV/MLV-positive (14 with CFS) and from 15 healthy donors previously determined to be negative for the viruses. These samples were distributed in a blinded fashion to nine laboratories, which performed assays designed to detect XMRV/MLV nucleic acid, virus replication, and antibody. Only two laboratories reported evidence of XMRV/MLVs; however, replicate sample results showed disagreement, and reactivity was similar among CFS subjects and negative controls. These results indicate that current assays do not reproducibly detect XMRV/MLV in blood samples and that blood donor screening is not warranted.

XMRV: usage of receptors and potential co-receptors.

BACKGROUND: XMRV is a gammaretrovirus first identified in prostate tissues of Prostate Cancer (PC) patients and later in the blood cells of patients with Chronic Fatigue Syndrome (CFS). Although XMRV is thought to use XPR1 for cell entry, it infects A549 cells that do not express XPR1, suggesting usage of other receptors or co-receptors. METHODS: To study the usage of different receptors and co- receptors that could play a role in XMRV infection of lymphoid cells and GHOST (GFP- Human osteosarcoma) cells expressing CD4 along with different chemokine receptors including CCR1, CCR2, etc., were infected with XMRV. Culture supernatants and cells were tested for XMRV replication using real time quantitative PCR. RESULTS: Infection and replication of XMRV was seen in a variety of GHOST cells, LNCaP, DU145, A549 and Caski cell lines. The levels of XMRV replication varied in different cell lines showing differential replication in different cell lines. However, replication in A549 which lacks XPR1 expression was relatively higher than DU145 but lower than, LNCaP. XMRV replication varied in GHOST cell lines expressing CD4 and each of the co- receptors CCR1-CCR8 and bob. There was significant replication of XMRV in CCR3 and Bonzo although it is much lower when compared to DU145, A549 and LNCaP. CONCLUSION: XMRV replication was observed in GHOST cells that express CD4 and each of the chemokine receptors ranging from CCR1- CCR8 and BOB suggesting that infectivity in hematopoietic cells could be mediated by use of these receptors.

Absence of detectable xenotropic murine leukemia virus-related virus in plasma or peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected blood donors or individuals in Africa.

BACKGROUND: Since the identification of xenotropic murine leukemia virus-related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV-1)-infected individuals to gain evidence of XMRV infection in this geographic region. STUDY DESIGN AND METHODS: A total of 199 plasma samples, 19 PBMNC samples, and 50 culture supernatants from PBMNCs of blood donors from Cameroon found to be infected with HIV-1 and HIV-1 patients from Uganda were screened for XMRV infection using a sensitive nested polymerase chain reaction (PCR) or reverse transcription (RT)-PCR assay. RESULTS: Using highly sensitive nested PCR or RT-PCR and real-time PCR assays capable of detecting at least 10 copies of XMRV plasmid DNA per reaction, none of the 268 samples tested were found to be XMRV DNA or RNA positive. CONCLUSIONS: Our results failed to demonstrate the presence of XMRV infection in African blood donors or individuals infected with HIV-1. More studies are needed to understand the prevalence, epidemiology, and geographic distribution of XMRV infection worldwide.

Testing strategies for detection of xenotropic murine leukemia virus-related virus infection.

Xenotropic murine leukemia virus-related virus (XMRV) is a newly identified gamma retrovirus and may be associated with prostate cancer- (PC) and chronic fatigue syndrome (CFS). Since its identification in 2006 and detection of polytropic murine lenkemia virus (MLV)-like sequences in CFS patients in 2010, several test methods including nucleic acid testing methods and serological assays have been developed for detection of XMRV and/or MLV-like sequences. However, these research assays have not yet been validated and evaluated due to the lack of well-characterized reference materials. Mouse DNA contamination should be carefully checked when testing human specimens in order to avoid false-positive detection of XMRV or MLV-like sequences.