Antibody-negative Samples Research Articles

Screening for ‘window‐period’ acute HIV infection among pregnant women in rural South Africa

The aim of this study was to evaluate the HIV-1 RNA pooled nucleic acid amplification testing (NAAT) strategy to screen pregnant women in the 'window period' of acute HIV infection (AHI) in rural South Africa. In 2007 and 2008, 750 consecutive pregnant women on their first antenatal care visit to a primary health care clinic were tested anonymously for HIV infection. HIV-1 RNA pooled NAAT was performed on HIV antibody-negative samples. All positive pools were tested individually and positive samples were classified as incident cases to calculate HIV incidence. The overall HIV prevalence was 37.3% [95% confidence interval (CI) 34.3–41.3]. Of the 467 HIV antibody-negative samples, four (0.9%) were HIV-1 RNA-positive. The mean viral load in the four samples was 386 260 HIV-1 RNA copies/mL (range 64 200–1 228130). The HIV incidence was 11.2%per year (95% CI 0.3–22.1) and all women with AHI were 21 years of age. Identifying AHI in pregnancy is important for health interventions to reduce perinatal and heterosexual transmission of HIV, and to estimate HIV incidence for epidemiological surveillance.

Validation of Rapid HIV Antibody Tests in 5 African Countries

The sensitivity and specificity of 3 rapid HIV antibody tests were assessed at 5 clinical trial sites in Africa and 1 site in the United States using a minimum of 100 HIV antibody positive samples and 100 HIV antibody negative samples at each site. The overall sensitivity and specificity for the OraSure OraQuick, Abbott Determine, and Trinity Unigold tests were 99.3%, 99.8%, and 98.5%, respectively, and 99.3%, 99.4%, and 99.5%, respectively. There were no instances at any site in which false-negative or false-positive results were obtained for the same sample on more than 1 rapid test kit. The results of this study provide assurance that for these diverse sites in Africa, the accuracy of these kits is quite good. Given the excellent accuracy, relatively fast turnaround time, and minimal infrastructure required, these rapid tests for HIV antibody provide a very attractive and accurate testing format.

Cases of HIV Type 1 Acute Infection at STI-Related Clinics in Osaka

Since 1992 we have investigated HIV antibodies in persons with a venereal disease who worked for a sex business or engaged in sexual behavior with a high risk of HIV infection at five clinics of venereology, urology, and gynecology in Osaka Prefecture. Starting at the end of 2000, we performed a nucleic acid-amplification test (NAT) on antibody-negative samples with the aim of detecting cases in the window period covering the early phase of infection. Three cases that were thought to be in this phase were found in 2006. All were confirmed to be positive in an NAT, although they were negative in initial screening for HIV performed using the IC method. Gene analysis of the env-C2V3 region of HIV-1 showed that the three samples had different origins.

Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies

There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies. To identify cell surface antigens, we developed a “conformational membrane antigen isolation and identification” strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and immune complexes are precipitated. Third, the bound surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific tyrosine receptor kinase (MuSK) and sera or plasmas from patients with a subtype of the autoimmune disease myasthenia gravis in which patients have autoantibodies against MuSK. MuSK was robustly detected as the only membrane protein in immunoprecipitates from all three patient samples tested and not from the three MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from cell extracts. The conformational membrane antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The approach would also be very suitable for the analysis of human or experimental antitumor responses.

Sexually Transmitted Infections Among Patients With Acute HIV in North Carolina

Sexually transmitted infections (STIs) have a well-established synergistic relationship with HIV infection. Co-infection with HIV and an STI can increase the probability of HIV transmission to an uninfected partner by increasing HIV concentrations in genital lesions, genital secretions, or both.[1, 2] STI infection can also increase the likelihood of HIV acquisition by interrupting mucosal barriers, increasing the access to and concentration of HIV receptor cells, and, in women, changing the vaginal microflora to favor HIV infection.[3–6] Among patients with acute HIV (AHI), the 4–6 week interval in the HIV disease course when the virus can be detected in the blood prior to seroconversion, co-infection with an STI may be common.[7] More than 70% of AHI patients had an STI co-infection in a Malawian sexually transmitted disease (STD) clinic in two separate studies.[8, 9] However, little is known about the frequency of STI and acute HIV infection co-infection outside of the STD clinic setting.

Method specific second-trimester reference intervals for thyroid-stimulating hormone and free thyroxine

Objective To determine second trimester reference intervals for TSH and FT4. Design Samples from 3102 subjects were tested for TPO and Tg antibodies. Methods Elecsys E170 reference intervals for TSH and FT4 were determined using antibody-negative samples. Results Second trimester reference intervals for TSH and FT4 were 0.18–4.07 mIU/L and 9.5–15.8 pmol/L, respectively. The Elecsys E170 TSH results were positively biased compared to ARCHITECT i2000 SR results for these same samples. Conclusions Method-specific reference intervals are required for TSH and FT4.

Incidence and Characterization of Acute HIV-1 Infection in a High-Risk Thai Population

The objective of this study was to investigate the incidence, demographics, HIV subtype, and genotypic resistance of acute HIV infections in a high-risk Thai population. Between March 2006 and September 2007, 6426 stored samples at the Thai Red Cross Anonymous Clinic were screened for acute HIV infection by 2 methods: pooled nucleic acid testing (NAT) of fourth-generation enzyme immunoassay (EIA)-negative samples (n = 5402) and subsequent first-generation EIA testing of fourth-generation EIA-positive samples (n = 1024). Eleven acute HIV-infected subjects were identified by pooled NAT (n = 7) and serial EIA (n = 4). Mean age was 28 years; 9 were male; and 60% were men who have sex with men. Median HIV RNA was 99,601 copies per milliliter (log10 viral load (VL) = 5.00). Eight samples could be genotyped: 6, CRF01_AE; 1, subtype B; and 1, CRF01_AE/B recombinant. No resistance to antiretroviral therapy was found. The HIV incidence per 100 person-years, calculated from the pooled, antibody-negative samples, was 2.7% (95% confidence interval, 2.2%-4.3%). This is the first report of antibody-negative, NAT-positive, acute HIV infection in Thailand. The majority were men who have sex with men, which reflects the current epidemic in Thailand and justifies prevention programs aimed at this group. This high-risk population may be suitable for future studies on acute HIV infection, HIV treatment, vaccine, and prevention of onward transmission strategies.

Antigen‐antibody combination assays for blood donor screening: weighing the advantages and costs

Antigen‐antibody combination assays for blood donor screening: weighing the advantages and costs

Detection of HIV antigen and cDNA among antibody-negative blood samples in Nigeria

In developing countries as many as 50% of patients for whom a transfusion is indicated are at risk of dying immediately if transfusion is withheld. It is therefore important that blood transfusion is made as safe as possible. This study was designed to assess the safety of blood transfusion in two large blood banks in Ibadan, Nigeria. Aliquots of 250 samples already screened and passed as negative for HIV-1 and -2 were collected from each of the blood banks. Samples were tested for the presence of HIV-1 antigen (ELAVIA Ag I) and the antigen-positive samples tested for the presence of specific HIV-1 antibodies by Western blot (BioRad, France). All antigen-positive samples were also subjected to PCR. HIV-1 antigen was detected in 6 (1.2%) of the 500 samples, of which 4 (0.8%) and 3 (0.6%) were Western blot-indeterminate and PCR-positive, respectively. Transfusion of HIV-contaminated blood may be contributing significantly to the spread of the virus in Nigeria. There is therefore an urgent need for an organized blood-banking system with facilities for more sensitive assays for the detection of HIV in blood to prevent transmission through transfusion.

Hepatitis C virus window‐phase infections: closing the window on hepatitis C virus

The detection of hepatitis C virus (HCV) infection is of major importance for the prevention of transfusion-transmitted hepatitis. The testing of donations by nucleic acid testing (NAT) techniques may not be feasible or economic. Combined antigen and antibody assays are now available, and the performance of two combined assays on window-phase donations is evaluated. Three panels of antibody-negative plasma samples from HCV NAT-only-positive donors were characterized for HCV status by quantitative reverse transcription-polymerase chain reaction, a commercial third-generation HCV antibody assay (Ortho), and combined antigen and antibody assays (Bio-Rad MONOLISA and Murex). All 142 plasma samples were antibody negative by Ortho third-generation HCV. A total of 112 samples (79%) were found to contain HCV RNA; 32 were detected by the Bio-Rad assay (29%), whereas 56 (50%) were detected by the Murex assay. Of 45 samples with viral loads of greater than 10(6), 32 (71%) were positive in the Bio-Rad combination assay and 44 (98%) were positive in the Murex assay. Interestingly none of the 3a genotypes were detected by the Bio-Rad MONOLISA, including eight donations that were greater than 10(6) IU per mL. Combined antigen and antibody testing provides a useful improvement on the sole reliance on antibody testing for detection of HCV infection; however, it remains less sensitive than NAT for detecting viremic donors and may be genotype susceptible.

Second-Trimester Reference Intervals for Thyroid Tests: The Role of Ethnicity

Thyroid function changes during pregnancy, complicating the diagnosis of thyroid disorders. Maternal thyroid dysfunction has been associated with a variety of adverse outcomes. We evaluated thyroid function test results by ethnicity and week of gestation during the 2nd trimester of pregnancy. We collected 3064 blood specimens in serum tubes from Asians (13%), blacks (22%), Hispanics (23%), and whites (42%). We measured thyroid-stimulating hormone (TSH), total and free thyroxine (TT4 and FT4), total and free triiodothyronine (TT3 and FT3), thyroglobulin autoantibodies (TgAb), and thyroid peroxidase autoantibodies (TPOAb) by use of an ARCHITECT i2000(SR) (Abbott Diagnostics). The TSH reference interval was calculated for samples negative for both TgAb and TPOAb and reference intervals for TT4, FT4, TT3, and FT3 in antibody-negative samples with normal TSH. Serum samples were positive for TgAb in 10.6%, 1.8%, 6.2%, 6.5%, and 5.9% of Asian, black, Hispanic, white, and combined groups, respectively. Samples were positive for TPOAb in 12.4%, 4.1%, 11.8%, 12.3%, and 10.4% of the same groups, respectively. The nonparametric reference intervals for all participants were 0.15-3.11 mIU/L (TSH), 9.3-15.2 pmol/L (0.72-1.18 ng/dL; FT4), 89.0-176.3 nmol/L (6.90-13.67 mug/dL; TT4), 3.82-5.96 pmol/L (2.48-3.87 pg/mL; FT3), and 1.82-3.68 nmol/L (118-239 ng/dL; TT3). Blacks had lower prevalences of TgAb and TPOAb positivity and of increased serum TSH. The prevalence of TgAb and TPOAb positivity was highest in Asians. Whites had the highest prevalence of increased TSH. The lower and upper reference limits of TT3 were significantly lower for Asians. Reference intervals for women in the 2nd trimester were different from those of nonpregnant individuals.

Feasibility and cost–benefit of implementing pooled screening for HCVAg in small blood bank settings

To examine the accuracy, feasibility and benefits of screening for hepatitis C virus core antigen (HCVAg) using enzyme-linked immunosorbent assay (ELISA) test in pools. Many countries cannot afford to test blood donations for hepatitis C using molecular methods. Screening individual units using the ELISA HCVAg test is an acceptable, yet still expensive, alternative, especially for small blood bank settings. This study evaluated the option of screening for HCVAg in pools. The sensitivity (Se) and specificity (Sp) of HCVAg in pools of three and six antibody-negative samples were estimated and compared with polymerase chain reaction (PCR). The feasibility and cost-benefit of the assay was assessed on 960 routine samples collected at a hospital blood bank in Gaza. Based on results for 50 PCR-positive pools and 50 and 110 PCR-negative pools of three and six, the Se of testing in pools of three and six samples is 80-82% [95% confidence interval (CI): 66.3-91.4] and Sp >or=98% (95% CI: 89.4-100.0) compared with PCR. The incidence of antigen in donors in Gaza was 0.1% (95% CI: 0-0.56). Cost analyses suggested significant benefits from implementing screening blood donations for HCVAg when the incidence rate is >4.2/10,000, leading to reduction in the expenditures needed to treat patients infected with HCV. The risk of transfusion-transmitted hepatitis C in resource-deprived developing countries can be efficiently reduced by additional screening of antibody-negative blood donations for HCVAg in pools of six.

Evaluation of procleix HIV/HCV RNA diagnostic assay

To investigate the sensitivity and specificity of Procleix HIV/HCV RNA diagnostic assay. HIV antibody positive or suspected positive plasmas of blood donors were collected from different provinces and detected with HIV antibody ELISA and HCV antibody ELISA. Samples positive for HIV by ELISA were confirmed by using HIV Blot. All the plasma samples were detected with Procleix HIV/HCV assay, HIV-1 discriminatory assay and HCV discriminatory assay, respectively. All 74 samples positive for both HIV and HCV antibody were positive and 5 samples negative for both HIV and HCV antibody were negative when detected using Procleix HIV/HCV assay; 82 of 84 supplemental HIV antibody positive samples and 6 of 12 supplemental indeterminate samples were positive for HIV RNA, and all 7 HIV antibody negative samples were negative for HIV RNA when detected by using Procleix HIV discriminatory assay. Seventy of 81 HCV antibody positive samples and 4 of 22 HCV antibody negative samples were positive for HCV RNA when detected by using Procleix HCV discriminatory assay. This reagent is more sensitive and could be used in blood screening, thereby can reduce both HIV and HCV transmission of blood in window period of HIV and HCV infection.

Antibodies to Extractable Nuclear Antigens in Antinuclear Antibody–Negative Samples

Antibodies to extractable nuclear antigens (ENAs)—SSA, SSB, U1RNP, Sm, Scl-70, and Jo-1—are clinically important in patients with systemic rheumatic diseases. Indirect immunofluorescence (IIF) with HEp-2 cells is a common initial screening test for detection of antinuclear antibodies (ANAs) and antibodies to ENAs. IIF-positive samples are further screened with more specific assays, but few studies have addressed the value of this cascade testing (1)(2). Although screening with conventional HEp-2 cells may miss some antigens, such as SSA (3) and Jo-1(4), false-negative ANA results are infrequent (1)(2). SSA-transfected cells in particular, which overexpress SSA (60 kDa), are considered highly sensitive for detection of anti-SSA antibodies (5). Some antibodies to ENAs can be missed by IIF, however (6). Hoffman et al. (6) found that of 291 ANA-negative samples, 12 were positive for antibodies to ENAs, including antibodies to SSA (Ro52 and Ro60), SSB, RNP-A, RNP-C, RNP-70, SmD, Scl-70, and Jo-1. We performed a prospective study to …

The Italian Quality Control Scheme for Crossmatching Procedures and HLA Sera Screening: The 2002 Pilot Study

The first national quality control (QC) program of histocompatibility serum testing was performed in Italy in 2002. To monitor the performance of HLA typing laboratories while meeting the accreditation requirements of the European Federation for Immunogenetics (EFI), which require HLA typing laboratories to participate in external QC of their crossmatch and antibody analyses. The Turin Transplant Immunology Service was asked to organize a QC survey of 17 HLA typing laboratories in Italy. Each laboratory received 12 serum specimens and 6 blood samples and was required to perform 36 crossmatches and 12 serum antibody specificity determinations. Data of participating centers were compared to establish whether EFI requirements were satisfied. In crossmatch analysis, the results of 32 of 36 crossmatches reached the 75% consensus target, with all the participating laboratories meeting the standards of the EFI. In antibody analysis, only 7 of 17 laboratories met the EFI standards. The first Italian QC program shows that the participating laboratories obtained consistent results in crossmatching, whereas the results were less satisfactory in the determination of serum antibody specificity, where consensus was reached only with monospecific sera and antibody-negative samples.

C-reactive protein concentration is not related to islet autoimmunity status in offspring of parents with type 1 diabetes

Autoimmunity may be associated with acute or chronic inflammation. In order to determine whether the inflammatory marker C-reactive protein (CRP) was an indicator of inflammatory events that precede, predict, or associate with islet autoimmunity or type 1 diabetes, CRP was measured in sequential antibody-negative, seroconversion, and follow-up-positive samples from 65 prospectively studied islet autoantibody-positive children. Although changes in CRP concentrations were observed in some children, overall CRP concentrations were similar in antibody-negative samples (median, 0.21 mg/L), antibody-positive samples (median, 0.26 mg/L), and samples at seroconversion (median, 0.26 mg/L). CRP concentrations at diabetes onset (median, 0.59 mg/L) were not significantly increased over antibody-negative samples ( P = 0.07). CRP concentrations did not predict diabetes development. CRP concentrations were related to age ( r = 0.26; P < 0.001) and were increased in samples obtained from October to January ( P < 0.001). These findings suggest that CRP concentrations are not a valuable marker of progression to type 1 diabetes and highlight the importance of correcting analyses for seasonal variations.

Sero-epidemiology of mumps in western Europe.

Six countries (Denmark, England and Wales, France, Germany, Italy and The Netherlands) conducted large serological surveys for mumps, in the mid-1990s, as part of the European Sero-Epidemiology Network (ESEN). The assay results were standardized and related to the schedules and coverage of the immunization programmes and the reported incidence of mumps. Low incidence of disease and few susceptibles amongst adolescents and young adults was observed in countries with high mumps vaccine coverage (e.g. The Netherlands). High disease incidence and large proportions of mumps virus antibody negative samples in adolescent and young adult age groups was noted in countries with poor vaccine coverage (e.g. Italy). The build-up of susceptibles in older children and adolescents in England and Wales, France, the former West Germany and Italy indicate the possibility of further mumps outbreaks in secondary school environments. To control mumps in western Europe, current MMR immunization programmes will need to be strengthened in a number of countries. Sero-surveillance of mumps is an important component of disease control and its usefulness will be enhanced by the development of an international mumps standard.

A Modified Ultrasensitive Assay to Detect Quantified HIV-1 RNA of Fewer Than 50 Copies per Milliliter

We compared the sensitivity and specificity of versions 1.0 and 1.5 and a modified version 1.5 of the AMPLICOR HIV-1 MONITOR ultrasensitive RNA assay (Roche, Indianapolis, IN) by using a virus stock dilution series and plasma samples from HIV-1–infected and uninfected subjects. The modified assay was linear and consistently positive down to 12 copies per milliliter vs 25 copies per milliliter for the other 2 assays. Versions 1.0, 1.5, and modified 1.5, respectively, detected 9 (23%) of 39, 11 (28%) of 40, and 43 (61%) of 71 replicates of a 4-copy-number and 11 (28%) of 40, 17 (46%) of 37, and 88 (90%) of 98 replicates of a 10-copy-number standard. Of 44 patient samples with undetectable levels using version 1.0, 32 (73%) had detectable levels on the modified assay, and 5 (25%) of 20 had detectable levels on version 1.5. None of the assays detected HIV-1 RNA in HIV-1 antibody–negative samples. The modified version 1.5 of the RNA assay is more sensitive for detecting HIV-1 RNA in significantly more patients than are versions 1.0 and 1.5.

Detection of HCV core antigen in human serum and plasma with an automated chemiluminescent immunoassay

Currently, the detection of HCV infection in blood donors relies on the ability of immunoassays to detect circulating HCV antibodies. However, a significant delay exists between the time of infection and the development of antibodies. This delay (window period) can last up to 70 days. The introduction of NAT for the detection of HCV RNA has reduced this window period dramatically. However, NAT is labor intensive, prone to contamination, and expensive as compared with standard serologic tests. An automated, microparticle-based chemiluminescent assay for the detection of HCV core antigen in human serum and plasma was developed. The specificity and sensitivity of this prototype assay were evaluated by testing a population of normal blood donors and commercially available seroconversion panels. The HCV core antigen assay exhibited a 99.9-percent specificity by detecting a single repeatably reactive sample out of 1004 normal donors tested. Assay sensitivity was determined by comparing the HCV core antigen detection rate with the antibody seroconversion profile and the rate of HCV RNA detection. Among 15 seroconversion panels examined, core antigen was detected in 69 of 70 antibody-negative and/or RNA-positive samples for a sensitivity relative to NAT of 98.6 percent. These data indicate that the automated, microparticle-based chemiluminescent HCV core antigen assay can reduce the window period for detection of potentially infected blood donors by 32.7 days, and it represents a viable alternative to HCV RNA testing.

Novel Approach To Reduce the Hepatitis C Virus (HCV) Window Period: Clinical Evaluation of a New Enzyme-Linked Immunosorbent Assay for HCV Core Antigen

The window period in hepatitis C virus (HCV) infection is still a major problem in ensuring blood safety. HCV RNA detection by nucleic acid amplification technology-based tests has contributed to reduce the infectivity of blood products, but it is expensive, time-consuming and affected by a high prevalence of false-positive results. The aim of this study was to assess the performance of a newly developed enzyme immunoassay for the detection of HCV core antigen and its suitability for use in the screening of blood units in order to identify infecting samples that do not contain specific antibodies. For evaluation of laboratory performance, different samples were selected: to evaluate specificity, we tested 2,586 sera from blood donors, 500 general population samples, and 58 "difficult sera". All samples were tested by two screening assays, and results were negative. To estimate clinical sensitivity, 103 HCV RNA-positive, anti-HCV-negative samples, 6 natural seroconversion panels, and 9 commercial seroconversion panels were tested. Intra- and interassay precision were determined on two HCV-RNA-positive, anti-HCV-negative sera. Seventeen (0.66%) blood donor samples, 2 (0.4%) general population samples, and 2 (3.44%) difficult sera were initially reactive; 3 sera were positive on repetition. These 21 samples tested by reverse transcription-PCR were negative. The clinical sensitivity calculated with seroconversion panels and seroconverted patient samples was very similar to PCR sensitivity: 95% of PCR-positive, antibody-negative samples contained detectable HCV antigen. Data on intra- and interassay precision showed dispersion indices with values of less than 10%. In conclusion, the HCV antigen assay showed high sensitivity and specificity and could become a useful means of improving the safety of blood and blood products.