Niel T. Constantine

EBV strain variation: Geographical distribution and relation to disease state

The strains of Epstein-Barr virus (EBV) were characterized in epithelial and lymphoid malignancies from geographic regions with high or low incidence. The predominant strains in nasopharyngeal carcinoma (NPC) from regions with elevated incidence were EBV type 1 in southeast Asia and Mediterranean Africa. In Alaskan Eskimos, a distinct variant of EBV type 2 was found in NPC and carcinoma of the parotid gland. This strain contained polymorphisms characteristic of the Asian EBV type 1. The strains prevalent in southeast Asia and Mediterranean Africa were also found in NPC which developed in caucasian Americans. These variants were not detected in lymphomas which developed in central Africa, Mediterranean Africa, or continental United States. These results suggest that distinct EBV strains predominate in geographic areas with elevated incidence of NPC. The detection of these distinct strains in epithelial tumors from areas of low incidence may reflect an epithelial cell tropism or pathogenicity.

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.

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.

Toxoplasma gondii Immunoglobulin G Antibodies and Nonfatal Suicidal Self-Directed Violence

OBJECTIVE The primary aim was to relate Toxoplasma gondii seropositivity and serointensity to scores on the self-rated Suicide Assessment Scale (SUAS-S). Another aim was to reevaluate the previously reported positive association between T gondii serointensity and a history of nonfatal suicidal self-directed violence. METHOD This cross-sectional, observational study compared T gondii serointensity and seropositivity in plasma from 54 adult suicide attempters (inpatients at Lund University Hospital, Lund, Sweden) and 30 adult control subjects (randomly selected from the municipal population register in Lund, Sweden) recruited between 2006 and 2010. The potential of patients and controls for self-directed violence was evaluated with the SUAS-S. Psychiatric diagnoses were made according to DSM-IV criteria. Plasma samples were tested for immunoglobulin G antibodies to T gondii, cytomegalovirus, and herpes simplex virus type 1. Data were analyzed using multivariable logistic regression to investigate the association between T gondii serointensity or seropositivity and a history of nonfatal suicidal self-directed violence; multivariable linear regression was used to explore the relationship between T gondii serointensity or seropositivity and the SUAS-S. Both regression models included sex, age, and body mass index as covariates. RESULTS Seropositivity of T gondii (adjusted odds ratio [OR] = 7.12; 95% CI, 1.66-30.6; P = .008) and serointensity of T gondii (adjusted OR = 2.01; 95% CI, 1.09-3.71; P = .03) were positively associated with a history of nonfatal suicidal self-directed violence. Seropositivity of T gondii was associated with higher SUAS-S scores, a relationship significant for the whole sample (P = .026), but not for suicide attempters only. No significant associations with other pathogens were identified. CONCLUSIONS These results are consistent with previous reports on the association between T gondii infection and nonfatal suicidal self-directed violence. Confirming these results in future large longitudinal studies and including suicide as an outcome may lead to novel individualized approaches in suicide prevention.

Detection of Kaposi's Sarcoma—Associated Herpesvirus in Oral and Genital Secretions of Zimbabwean Women

Kaposis sarcoma-associated herpesvirus (KSHV) in oral and genital secretions of women may be involved in horizontal and vertical transmission in endemic regions. Nested polymerase chain reaction assays were used to detect KSHV DNA sequences in one-third of oral, vaginal, and cervical specimens and in 42% of peripheral blood mononuclear cell (PBMC) specimens collected from 41 women infected with human immunodeficiency virus type 1 who had Kaposis sarcoma (KS). KSHV DNA was not detected in specimens from 100 women without KS, 9 of whom were seropositive for KSHV. A positive association was observed between KSHV DNA detection in oral and genital mucosa, neither of which was associated with KSHV DNA detection in PBMC. These data suggest that KSHV replicates in preferred anatomic sites at levels independent of PBMC viremia. Detection of genital-tract KSHV only among relatively immunosuppressed women may provide an explanation for infrequent perinatal transmission of KSHV.

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.

Assessment of the performance of a rapid, lateral flow assay for the detection of antibodies to HIV.

Summary: Rapid HIV assays have recently been shown to have important applications for various testing situations, including early identification of infected individuals, to allow intervention strategies in a clinically relevant time frame. A rapid, lateral flow, HIV‐1/2/O assay was evaluated using 2,000 serum or plasma samples from various risk groups and geographic locations, including HIV‐1 and HIV‐2 positive sera from five countries. Two U.S. Food and Drug Administration (FDA)‐licensed screening assays and a FDA‐licensed confirmatory assay were used as reference tests. The rapid assay exhibited a near‐perfect sensitivity (99.2%) and an excellent specificity (99.9%). Moreover, its analytical sensitivity was found to be better than most FDA‐licensed enzyme‐linked immunosorbent assays (ELISAs), detecting infection at the same time as the most sensitive ELISA in two of five seroconversion panels, and at the same time or earlier than four of five ELISAs in all five panels. We conclude that this rapid assay is a suitable test for the detection of HIV infection that could be particularly useful in developing countries where facilities may not support the use of instrumentation.

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 105 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.

Seroprevalence of human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and rapid plasma reagin in a trauma population. Discussion

We evaluated the presence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and rapid plasma reagin (RPR) among patients admitted to our trauma unit from April 15 to June 30, 1993. Of 984 patients tested, we found 255 (26%) had evidence of exposure to one or more of these agents: HIV, 4%; HBV, 20%; HCV, 14%; and RPR, 1%. Thirty-eight percent of patients had more than one positive serology, 75% of the HIV patients, 49% of the HBV patients, and 66% of the HCV patients. There was no difference between penetrating and nonpenetrating trauma with respect to any of the viruses. The risk factors for HIV-positive patients were non-White race, positive drug screen, positive alcohol screen, and city resident. Risk factors for HBV patients were non-White race, positive drug screen, and city resident. Risk factors for HBC patients were male sex, non-White race, positive alcohol screen, positive drug screen, and city resident. The risk of blood-borne infections in this group of patients is substantial.

Evaluation of two novel immunoassays designed to detect HIV antibodies in oral fluids

The testing of oral fluid samples for the detection of HIV antibodies offers several advantages over the testing of blood. Our objective was to evaluate a new generation of rapid and simple assays designed specifically to detect HIV‐1 and HIV‐2 antibodies in oral fluids (saliva). Serum and oral fluid pairs were collected from 615 high‐ and low‐risk individuals in the United States, Peru, and the Ivory Coast. Two different oral fluid collection devices and rapid assay systems included: (1) the Orapette/SalivaCard HIV‐1/HIV‐2 and (2) the Omni‐Sal/ImmunoComb II HIV‐1 and HIV‐2. The corresponding serum pairs were analyzed by conventional ELISAs, and all reactive sera were confirmed with HIV‐1 and HIV‐2 Western blots. The results indicated a 100% sensitivity for both rapid oral fluid assays, including successful detection of HIV‐2 antibodies. Specificities ranged from 99.8% to 100%. One sample produced a reactive result by the SalivaCard while being nonreactive by the other assays including the Western blots. Both assays performed excellently, indicating that antibodies to HIV can be detected reliably in oral fluids by simple and rapid assays. This combination of rapid testing technology and the use of easily collected oral fluid samples offers an efficient and accurate alternative to conventional testing and can be appropriately applied to a variety of testing situations for the laboratory diagnosis of HIV infection. J. Clin. Lab. Anal. 11:63–68.