Human herpesvirus 8 infection in HIV-1-infected and uninfected pregnant women in Ethiopia.

BACKGROUND: Human Herpes Virus (HHV-8) is related to Kaposi Saracoma, an opportunistic infection occurring with HIV infection. Little is known about the seroepidemiology of Human Herpesvirus 8 (HHV-8) infection among Ethiopian women, even though women are a major HIV risk group in Ethiopia. OBJECTIVES: This study aimed at determining the seroprevalence of HHV-8 infection in HIV-1-infected and uninfected pregnant women in five selected regions of Ethiopia. METHODS: A cross-sectional study was conducted from December 2006 to June 2007 where pregnant women were recruited after age-matching in groups. A total of 400 pregnant women were enrolled, with 200 being HIV-infected and 200 being HIV-uninfected Sera were screened for IgG lytic antibody to HHV-8 using an Indirect Fluorescence Assay (IFA) in Virology Unit of Ethiopian Health and Nutrition Research Institute (EHNR1). RESULTS: Of 400 pregnant women attending antenatal clinic (ANC) testing sites of five regions in Ethiopia, 212 (53.0%) were positive for HHV-8 IgG lytic antibody. There was a high prevalence of HHV-8 infection among HIV-1-infected pregnant women (138, 69.0%) as compared with HIV-1-uninfected pregnant women (74, 37.0%). CONCLUSION: The study shows a high prevalence of HHV-8 infection among HIV-1-infected pregnant women as compared with HIV-1-uninfected pregnant women. Therefore, creating awareness and educating women on safe sexual practice and avoiding deep kissing may be a fundamental ways to limit the roots of transmission. Moreover, initiating strong antiretroviral therapy (ART) for HIV infected women would be best treatment prior to the development of Kaposi's sarcoma (KS).

A simple and inexpensive particle agglutination test to distinguish recent from established HIV-1 infection.

OBJECTIVES: We sought to modify the Serodia HIV-1/HIV-2 particle agglutination assay (PA), a simple and cost-effective HIV assay that is used globally for the detection of HIV antibodies, as a sensitive/less sensitive test (S/LS) to identify recently infected individuals and to estimate HIV incidence. METHODS: The Serodia PA test was modified as an S/LS test (PA-LS) by using HIV antigen-coated gelatin particles at a dilution of 1:68 and a specific diluent, and calibrated using 37 HIV clade B seroconversion panels (309 samples) from Trinidad and from a commercial source that were tested at dilution intervals from 1:10 to 1:80,000. The greatest sensitivity for correctly classifying samples from recent and established infections was determined by receiver operator curve (ROC) analysis. RESULTS: At a 1:40,000 sample dilution and a days post-seroconversion cutoff of 190 days, the PA-LS test yielded a 97% sensitivity for classifying recent and established infection samples. Furthermore, at a 1:20,000 dilution, the positive predictive value for correctly identifying recently infected individuals was 99%. The PA-LS test offers a 30-44-fold cost saving over currently available S/LS tests. CONCLUSION: A modified, low cost and simple-to-perform PA test is appropriate for use in resource-limited countries, and has exhibited excellence in distinguishing recent from established HIV infection.

Assessment of Two Rapid Assays for Diagnostic Capability to Accurately Identify Infection by Treponema pallidum.

BACKGROUND: Rapid, point-of-care tests that accurately identify syphilis are gaining popularity and offer several advantages over classic tests. METHODS: The SD Bioline Syphilis 3.0 and the Chembio DPP Syphilis Screen and Confirm Assay (CB) were assessed using 1283 samples that had been characterized by reference tests. The challenge samples included 5 commercial panels (seroconversion, mixed-titer), archived samples, fresh samples, and a dilution series. Both tests detect specific anti-treponemal antibodies, and the CB additionally detects antibodies to a non-treponemal (NT) component. The evaluation was used to determine performance indices and compare with those cited by the manufacturers. RESULTS: When assessing reactivity to treponemal, the sensitivities for the 2 tests were 98.3% and 93.2%, with specificities of 100% and 99.4%, respectively. For both tests, precision, whole blood testing, and high-temperature testing produced perfect results, and there were no invalid results. Comparisons of 2 different lots of each test indicated excellent concordance (100% and 99.5%), and reproducibility was 100% and 98.0%, respectively. For the CB, the sensitivity for the NT component was between 65.3% and 80.9%, but increased to 98.5% with samples having a rapid plasma regain (RPR) titer of ≥8. The specificity for NT was found to be 100%, and the reading of results visually and when using a battery-operated reader indicated a concordance for all challenges of 95%-100%. CONCLUSIONS: Both rapid tests produced impressive results for the detection of antibodies to treponemal for all challenges and exceeded, met, or closely approached the performance characteristics as cited by the manufacturers.

Update on the laboratory diagnosis and monitoring of HIV infection.

In China, the estimated number of HIV infected cases is approaching one million. Although public education has been initiated for awareness and behavioral modification for this devastating infection, better diagnostic methods are needed to identify infected persons and manage infection. Simple and more accurate diagnostic tools have become available, particularly for early detection and to monitor treatment in those who receive anti-retroviral treatment. In this short review, we summarize some of the common and new methodologies that can be used in clinical laboratories, in the field, or in private laboratories. These range from simple antibody tests to more sophistical methods that are used to monitor disease progression and identify drug resistance. These tools can assist physicians, medical practitioners, and laboratory personnel to select suitable diagnostic tools for the diagnosis, blood screening, monitoring of disease progression, and for detection of drug resistance to anti-retroviral therapies.

A 10-minute, US Food and Drug Administration-approved HIV test.

It is now an established fact that rapid HIV tests have important applications in a number of testing situations. Currently, four rapid HIV assays have been approved by the US Food and Drug Administration, including the one discussed in this review. A rapid, lateral flow HIV assay was evaluated at the University of Maryland, USA, one of several sites involved in the clinical evaluation. Samples used in the evaluation totaled 9000 and consisted of serum, plasma and venous whole-blood sets of samples from 3000 study subjects that included population groups considered to be at high risk for HIV infection (n=1000), from HIV-positive individuals (n=1000), and from population groups considered at low risk for HIV infection (n=1000). US Food and Drug Administration-licensed screening and confirmatory assays were used as reference tests. The rapid assay exhibited a sensitivity of 100% across all three sample media and exhibited a specificity of 99.8% using whole blood and plasma and 99.7% using serum. This rapid assay is an excellent addition to the existing US Food and Drug Administration-approved rapid HIV tests, and provides versatility by allowing the testing of venipuncture and fingerstick whole-blood samples in addition to serum and plasma.

Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR.

Pathologic prion protein (PrP(Sc)), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrP(Sc). The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrP(C) was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.

HIV testing technologies after two decades of evolution.

Over the past two decades, HIV diagnostics have been essential in detecting and monitoring infection, and continue to play a major role in saving lives throughout the world. As technology evolved, screening, confirmatory, and HIV monitoring assays have been improved and offer better alternatives to address blood screening, surveillance, diagnosis, and patient management. Molecular methods are critical in detecting early infection and for managing patients on anti retroviral therapy whose viral infection may become resistant to therapy. In addition, modifications to conventional methods have introduced new assays, such as sensitive/less sensitive (detuned) assays that can estimate when someone was infected, thereby providing a useful tool for epidemiologic incidence estimates and enrollment into specific intervention programmes for recently infected persons. Many of the newly evolving technologies are essential for use in resource-limited countries because they can address cost issues, limited infrastructure, and a lack of formally trained personnel. Newer rapid HIV kits can be stored in a wide range of temperatures (2-30 degrees C) to address cold-chain issues, can use easily-collected fingerstick blood and oral fluids, and have one-step procedures that are relatively foolproof. Manual CD4 lymphocyte count assays that require only a light microscope and haemacytometer and more simple assays to estimate viral load are appropriate for developing countries where sophisticated instrumentation cannot be supported. Technologic advances with HIV diagnostics continue to address outstanding and new issues associated with diagnosis and the monitoring of infection by providing more simplified, cost-effective, and accurate testing throughout the world.

Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen.

Presently, the assay that attains maximal sensitivity and dynamic range of HIV-1 viral copy number (50 copies per milliliter) is nucleic acid amplification of HIV RNA in plasma. Enzyme-linked immunosorbent assay (ELISA) methods for quantification of HIV-1 p24 antigen have been relatively insensitive. In this report, we show data that indicate real-time immuno-polymerase chain reaction (IPCR), a combination of the ELISA and PCR techniques, is more sensitive for HIV-1 p24 antigen detection than other currently reported methods. When derived from an IPCR standard curve, a dose response was observed from patient samples with known viral loads diluted within a 3-log range (1.68-6,514 viral RNA copies per milliliter). IPCR detected 42% (22/52) of patient samples that had fewer than 50 viral RNA copies per milliliter by reverse transcriptase-PCR. IPCR shows the potential to become the most analytically sensitive test available for determination of HIV-1 viral load by the detection of HIV-1 p24 antigen.

Rapid confirmation of HIV infection.

OBJECTIVE: To investigate the utility of a prototype rapid confirmatory HIV assay which offers specific results in 5 min and has applications in a variety of important testing situations. METHODS: The performance of the rapid confirmatory assay was assessed with 849 blood samples, including serum, plasma, venipuncture whole blood, and peripheral blood collected via fingerstick. Included were over 700 HIV Western blot (WB)-confirmed antibody positive sera, and others which were classified as negative or indeterminate by WB. The analytic sensitivity of the rapid confirmatory assay was assessed using 13 HIV-1 seroconversion panels, and all results were compared to those of an FDA-licensed WB reference test. RESULTS: The rapid test exhibited 100% concordance with the reference test when testing HIV-1 WB-confirmed positive samples, and 92.3% concordance with samples having WB-inconclusive results. The sensitivity for confirming recent seroconversion was as good, or better than, the FDA-licensed HIV-1 WB in 10/13 panels. The rapid assay performed accurately with whole blood collected from fingerstick, and exhibited excellent precision and reproducibility. CONCLUSION: We conclude that this rapid HIV confirmatory assay, the first of its kind, demonstrates proof of principle for the accurate confirmation of HIV-1 infection and offers important advantages in public health and clinical testing venues.

Hepatitis C virus transfusion-transmitted infection in Brazilian cardiac surgery patients.

In the past two decades, major improvements in blood safety have been achieved, particularly for HIV and hepatitis C virus (HCV). A prospective study was carried out between 1996 and 1999 in Brazil to determine the incidence of post-transfusion infection in surgery patients caused by HCV. One hundred sixty-four patients who received a blood transfusion during cardiac surgery were followed for six months and blood samples collected before and after surgery were assessed to investigate HCV infection. Alanine aminotransferase levels were serially determined, as well as clinical data related to hepatitis. Prior to surgery, HCV infection was detected by anti-HCV ELISA III in 6 patients. Any post-surgical samples which were positive by a third generation ELISA test were confirmed by immunoblot and reverse-transcription polymerase chain reaction (RT-PCR), as were the pre-transfusion samples to exclude pre-transfusion HCV infection not detected by ELISA screening. Results indicated that one patient who was previously considered negative for HCV antibody in the pre-surgical sample was later found to be positive for HCV by RT-PCR in that sample. Seroconversion for HCV antibody after surgery was observed in two patients, one of them with clinical hepatitis; their genotypes were 1a and 1b. The overall prevalence of HCV infection was 4.26% (7/164) and the incidence rate of HCV infection after surgery was 1.27% (2/157). This study shows a high rate of HCV infection acquired post-transfusion in a cohort of surgery patients in Brazil and suggests that better screening methods such as viral RNA assessment may be effective in lowering this rate.

Risk factors for HIV, hepatitis B, and hepatitis C among persons with severe mental illness.

OBJECTIVE: Previous reports have indicated that persons with severe mental illness have an elevated risk of contracting HIV, hepatitis B, and hepatitis C compared with the general population. This study extends earlier findings by examining the factors that are most predictive of serologic status among persons with severe mental illness. METHOD: S: A total of 969 persons with severe mental illness from five sites in four states were approached to take part in an assessment involving testing for blood-borne infections and a one-time standardized interview containing questions about sociodemographic characteristics, substance use, risk behaviors for sexually transmitted diseases, history of sexually transmitted diseases, and health care. RESULTS: The greater the number of risk behaviors, the greater was the likelihood of infection, both for persons in more rural locations (New Hampshire and North Carolina), where the prevalence of infection was lower, and those in urban locations (Hartford, Connecticut; Bridgeport, Connecticut; and Baltimore, Maryland), where the prevalence was higher. Although no evidence was found that certain behaviors increase a person's risk of one blood-borne infection while other behaviors increase the risk of a different infection, it is conceivable that more powerful research designs would reveal some significant differences among the risks. CONCLUSION: S: Clinicians should be attentive to these risk factors so as to encourage appropriate testing, counseling, and treatment.

Demographic profiles of newly acquired HIV infections among adolescents and young adults in the U.S.

Understanding the demographic and risk profiles of youth with recent HIV infection offers insight for imputing the dynamics of the epidemic and targeting prevention efforts. Three hundred forty-two HIV-positive youth were tested using a Sensitive/Less Sensitive strategy; 33% were classified as recently infected; with the majority (51%) occurring within African Americans.

Immunomagnetic quantitative immuno-PCR for detection of less than one HIV-1 virion.

Methods that allow the accurate and reliable detection of ultra-low molecular levels of proteins using techniques such as quantitative immuno-PCR (qIPCR) have demonstrated numerous technical difficulties. Protein detection methods lose specificity when the protein target is immersed within a matrix of thousands of molecules having wide ranges of concentrations. In addition, sensitivities are limited because of high background signals. To validate the performance of an immunomagnetic bead qIPCR method designed to remove the 'matrix' effect for HIV-1 p24 antigen detection, regression analyses were performed using samples from patients infected with HIV-1 diluted to approximately 100-1000, 10-100, 1-10, and 0.1-1.0 HIV-1 p24 Ag molecules/reaction. The number of HIV-1 p24 Ag molecules was derived from quantified HIV-1 RNA determinations. The modified immunomagnetic qIPCR bead assay demonstrated a limit of quantification of 10-100 HIV-1 p24 molecules per reaction, with an average correlation coefficient of 0.948+/-0.028 over a 4-log dynamic range. This method detects less than one HIV-1 virion (a limit of detection unreported previously for HIV-1), and thus, has the potential to identify HIV-1 infection and monitor the dynamics of the disease course earlier than nucleic acid methods. The immunomagnetic qIPCR bead assay is a simple and inexpensive method for ultra-low protein detection of infectious agents, toxins, and cancer markers at a level unrecognized previously using any enzymatic or molecular method.

Protocol for nearly full-length sequencing of HIV-1 RNA from plasma.

Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 10(5) and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.

Calibration and validation of an oral fluid-based sensitive/less-sensitive assay to distinguish recent from established HIV-1 infections.

Sensitive/less-sensitive (S/LS) serum-based serologic methods have been developed to measure human immunodeficiency virus (HIV) incidence by distinguishing recent from established infections. Such methods require venipuncture. The goal of this study was to develop an alternative to serum-based S/LS testing using oral fluid (OF) as the testing medium. Serum/OF pairs were collected from 342 patients attending 15 Adolescent Trials Network (ATN) clinical sites. The sera were tested with the use of the dilutional Vironostika (DV; Biomerieux, Durham, NC) S/LS assay (DV(SOD=1.0)) as the reference against which an OF LS assay was calibrated using 40 of the OF pairs. Receiver operating characteristic (ROC) curve analyses pinpointed the OF LS test parameters that maximized concordance with the serum-based DV. Validation of the calibrated OF LS included testing of the remaining 302 serum/OF pairs. During calibration the maximum concordance with the DV was 95.2% and 89.5% for 21 recent and 19 established samples, respectively, at a 1:50 OF sample dilution and an optical density (OD) cutoff of 0.280. When applied to the validation sample set (N=302), the concordance was 73.6% for the recent samples and 89.6% for the established samples. The OF LS assay showed a good concordance with the serum-based reference S/LS assay. It presents an alternative to invasive specimen collection, and has the potential for increasing test compliance in young subjects. However, because of the uncertainty of the performance characteristics of the serum-based S/LS assay with which it was compared, further validation of the OF LS using seroconversion sample pairs is needed.

Prevalence of human immunodeficiency virus, hepatitis B, and hepatitis C among homeless persons with co-occurring severe mental illness and substance use disorders.

This study was undertaken to determine the prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) among homeless persons with co-occurring severe mental illness (SMI) and substance use disorders and to determine associated risk factors. As part of a longitudinal study of the effectiveness of integrated treatment for homeless persons with SMI and substance abuse or dependence, serological testing was performed to ascertain the prevalence of HIV, HBV, and HCV. At baseline, 6.2% of participants (11/172) were HIV-positive. Nearly one third of participants (37/114) had evidence of prior exposure to HBV, and 30% (34/114) were antibody positive for HCV. About 44% of participants (50/114) had a reactive test for either HBV or HCV. Having a reactive test was strongly associated with substance use, especially with a history of injection drug use. A significant threat exists to the health and well-being of homeless person with SMI due to high prevalence of blood-borne pathogens. Mental health providers need to play a proactive role in the identification of health-related needs and to assist with access to general health services for persons with SMI.

Improved classification of recent HIV-1 infection by employing a two-stage sensitive/less-sensitive test strategy.

Current serologic techniques for the classification of recent HIV-1 infection produce some misclassifications, and, together with the loss to follow-up of individuals, results in decreased enrollment of HIV-infected persons into appropriate intervention programs. We report on the development of a sensitive/less sensitive (S/LS) test strategy that includes a rapid assay to quickly identify persons most likely to have recent infection, followed by an enzyme immunoassay (EIA) with exquisite specificity. The Uni-Gold Recombigen HIV rapid assay (UG; Trinity Biotech, Dublin, Ireland) was procedurally-modified and calibrated as an LS test to differentiate recent (<133 days) from established HIV infections using 178 samples from persons with known dates of infection. This method correctly classified 83.0% of recent infections, but with a high misclassification rate of persons with established infection. By performing the rapid test followed by a modified S/LS EIA, the positive predictive value of the combined results for recent infections was increased to 100%. This two-stage testing algorithm can result in an increased efficiency for the enrollment of recent infection cases over a standard EIA S/LS method alone due to provisional enrollment during an initial testing visit, and because of an increased accuracy for identifying truly recent infections. We conclude that the rapid S/LS assay provides a tool for capturing recent infection cases quickly and is particularly valuable in resource-limited settings, and that the two-stage strategy provides a more accurate identification of persons with recent HIV infection.