Detection of recent HIV infections in African individuals infected by HIV-1 non-B subtypes using HIV antibody avidity.

BACKGROUND: To estimate HIV incidence several methods have been used to discriminate recent HIV infections from long-standing infections using a single serum sample. OBJECTIVE: To evaluate the performance of the anti-HIV avidity index (AI) for identifying recent HIV infections in individuals with a known date of seroconversion from Uganda, where the predominant HIV subtypes are A and D. STUDY DESIGN: We selected 149 repository serum samples from Ugandan HIV-positive individuals and evaluated the AI. Specimens collected < or =6 months after seroconversion were considered as recent infections, and those collected >6 months as long-standing infections. All specimens were serotyped using a V3 peptide enzyme immunoassay. RESULTS: The mean AI was 0.55+/-0.21 among the 108 patients with recent infections and 0.93+/-0.14 among the 41 samples from long-standing infections (p<0.0001). The AI test showed a sensitivity of 85.2% and a specificity of 85.4% at a cutoff of 0.80. No significant association was observed between serotype and the misclassification of samples by AI. CONCLUSIONS: The AI, which is inexpensive and easy-to-perform, can be useful in identifying recent HIV infections in countries where HIV-1 non-B subtypes are prevalent.     

Precision and accuracy of a procedure for detecting recent human immunodeficiency virus infections by calculating the antibody avidity index by an automated immunoassay-based method

We evaluated the precision and accuracy of a procedure for detecting recent human immunodeficiency virus (HIV) infections, specifically, the avidity index (AI) calculated using a method based on an automated AxSYM HIV 1/2gO assay (Abbott). To evaluate precision, we performed multiple replicates on eight HIV-positive serum samples. To evaluate the accuracy in identifying recent infections (i.e., within 6 months of seroconversion), we used 216 serum samples from 47 persons whose dates of seroconversion were known. To evaluate the sensitivity and specificity of the procedure for different AI cutoff values, we performed receiver operating characteristic (ROC) analysis. To determine the effects of antiretroviral treatment, advanced stage of the disease (i.e., low CD4-cell count), and low HIV viral load on the AI, we analyzed 15 serum samples from 15 persons whose dates of seroconversion were unknown. The precision study showed that the procedure was robust (i.e., the total variance of the AI was lower than 10%). Regarding accuracy, the mean AI was significantly lower for samples collected within 6 months of seroconversion, compared to those collected afterwards (0.68 +/- 0.16 versus 0.99 +/- 0.10; P < 0.0001), with no overlap of the 95% confidence intervals. The ROC analysis revealed that an AI lower than 0.6 had a sensitivity of 33.3% and a specificity of 98.4%, compared to 87.9 and 86.3%, respectively, for an AI lower than 0.9. Antiretroviral treatment, low CD4-cell count, and low viral load had no apparent effect on the AI. In conclusion, this procedure is reproducible and accurate in identifying recent infections; it is automated, inexpensive, and easy to perform, and it provides a quantitative result with different levels of sensitivity and specificity depending on the selected cutoff.     

Avidity Index for anti-HIV antibodies: comparison between third- and fourth-generation automated immunoassays

The development of assays for detecting recent HIV infections has become crucial for analyzing trends in infection in different populations, both for surveillance and prevention activities. The anti-HIV avidity index (AI), measured with third-generation immunoassays (which detect anti-HIV antibody), has been shown to be an accurate tool for discriminating recent HIV infections (<6 months) from established infections (≥ 6 months). We compared a third-generation immunoassay (AxSYM HIV 1/2 gO; Abbott Diagnostics) to a fourth-generation immunoassay (Architect HIV Ag/Ab Combo; Abbott Diagnostics; which detects anti-HIV antibody and p24 antigen) in terms of AI performance in distinguishing between recent and established HIV infections. A total of 142 samples from 75 HIV-infected individuals with an estimated date of seroconversion were assayed. The two assays showed the same accuracy in identifying a recent infection (91.5%), using an AI cutoff of 0.80, although Architect HIV Ag/Ab Combo was slightly more sensitive (89.4% versus 84.8%; P > 0.05) and yet less specific (93.4% versus 97.4%; P > 0.05). The correlation between assays was high (r = 0.87). When 20 specimens falling in the gray zone around the cutoff point (0.75 ≤ AI ≤ 0.84) were excluded, the accuracy of AI with Architect HIV Ag/Ab Combo was 94.7%, and the concordance between the two assays was 99.2%. The anti-HIV AI is a serological marker that accurately discriminates recent from established HIV infections. It can be successfully applied on fully automated fourth-generation HIV Ab/Ag immunoassays, which have several advantages, including increased throughput, high reproducibility, no need for specific technical skills, and easy comparability of results obtained in different settings.     

Modification of rapid human immunodeficiency virus (HIV) antibody assay protocols for detecting recent HIV seroconversion

Assay protocols of three rapid human immunodeficiency virus (HIV) assays, OraQuick-1/2, SeroStrip-1/2, and Determine-1/2, were modified to detect recent HIV seroconversion using a higher dilution of serum specimens. Optimal predilution of specimens resulted in negative test results during early periods of seroconversion (about 6 months), when antibody levels were low. A total of 269 seropositive specimens from routine HIV type 1 testing and from commercial sources (low-titer and seroconversion panels) were tested, and results were recorded as negative (score=0) or positive using intensity scores from 0.5 (weak positive) to 4 (strongly positive). The same specimens were previously tested by a less sensitive (LS) enzyme immunoassay (EIA), Abbott 3A 11-LS, and were classified as recent or long-term infections based on the standardized optical density (SOD) cutoff of 0.75. Overall concordance of >94% was observed between 3A 11-LS and modified rapid tests (RT-LSs) for detecting and distinguishing recent HIV seroconversion from long-term HIV infection (kappa statistics=0.894 to 0.901). Moreover, intensity scores on RT-LSs correlated well with median 3A 11-LS SOD values (R(2)>0.98). Our results indicate that rapid HIV tests can be modified to detect recent seroconversion with results comparable to those from less sensitive EIA.     

Improving HIV surveillance in Victoria: the role of the "detuned" enzyme immunoassay

Between 1999 and 2000, new diagnoses of HIV in Victoria (Australia) rose by 41%, from 140 to 197. In this time period, sera from new HIV diagnoses were tested using the Organon Teknika "detuned" enzyme immunoassay (EIA). We compared the results of the detuned EIA with incident infections defined by surveillance (on the basis of a previous negative or indeterminate HIV test and/or a seroconversion illness within the 12 months preceding HIV diagnosis). Of 317 specimens, 97 (31%) incident infections and 114 (36%) recent infections were detected using surveillance and detuned EIA, respectively. The detuned assay misclassified 11 cases with AIDS and 2 cases with CD4 counts < or = 200 micro3 (probable long-standing infections) as recent infections and was unable to identify 31 (32%) of 97 cases previously classified as incident cases by surveillance. The assay detected an extra 35 recent infections that were previously classified as nonincident by surveillance. By combining the detuned assay and surveillance, 132 (42%) incident infections were identified from 317 specimens, 36% more than surveillance alone. We recommend that a detuned assay or similar test become part of the routine strategy to identify incident infections in Victoria. Incident infections provide important information for targeting prevention strategies and the opportunity to interrupt ongoing viral transmission.     

Use of the serologic testing algorithm for recent HIV seroconversion (STARHS) to identify recently acquired HIV infections in men with early syphilis in Los Angeles County

BACKGROUND: Syphilis outbreaks among men who have sex with men (MSM) in the United States, many of whom are HIV infected, have prompted increased concern for HIV transmission. METHODS: To identify whether men are acquiring HIV concomitantly or within the critical period of syphilis infection, banked Treponema pallidum particle agglutination-positive serum specimens from men with early syphilis infection were screened for HIV-1 antibody. Samples that were positive for HIV antibody were then tested with a less sensitive (LS) HIV-1 antibody enzyme immunoassay (serologic testing algorithm for recent HIV seroconversion [STARHS]) to identify HIV infections that occurred on average within the previous 6 months. RESULTS: Of the 212 specimens banked from men with early syphilis, 74 (35%) were HIV-positive. Of these, 15 tested non-reactive by the LS assay. Twelve of these 15 were considered to be recent infections by the LS assay and testing history. Eleven (92%) of the recent infections were among MSM. One man had primary syphilis, 6 (50%) had secondary syphilis, and 5 (42%) had early latent syphilis. Eight men (67%) reported sex with anonymous partners, and 3 (25%) reported consistent condom use. The estimated HIV incidence was 17% per year (95% confidence interval [CI]: 12%-22%) among all men with early syphilis, and it was 26% per year (95% CI: 91%-33%) among MSM. CONCLUSIONS: Syphilis epidemics in MSM may be contributing to HIV incidence in this population. The STARHS can be applied as a surveillance tool to assess HIV incidence in various at-risk populations, but further studies are necessary for validation.     

Reactivation of Epstein-Barr virus during early infection with human immunodeficiency virus.

Reactivation of Epstein-Barr virus (EBV) in early human immunodeficiency virus (HIV) infection was investigated in 49 homosexual men who seroconverted to HIV (cases) as compared with 49 matched controls who remained seronegative to HIV during a longitudinal study. EBV infection was reactivated in cases 6 months, but not 12 months, prior to HIV seroconversion as compared with controls and remained reactivated during 18 months of follow-up after HIV seroconversion, as shown by increases in immunoglobulin (Ig) G antibody titers to EBV early antigen. Antibody titers to EBV viral capsid antigen did not differ between cases and controls prior to the time of seroconversion to HIV but were significantly increased among cases by the first seropositive study visit and remained elevated during the 18 months after HIV seroconversion. Total serum IgG levels were increased in cases at the visit of seroconversion, and during 18 months of follow-up, but did not correlate with enhanced IgG production specific for EBV antigens. Significant decreases in numbers of CD4+ cells and increases in numbers of CD8+ cells during this early phase of HIV infection were not associated with changes in patterns of EBV antibody responses. Reactivation of EBV beginning 6 months before HIV seroconversion may have implications regarding the role of this herpesvirus in the pathogenesis of HIV.     

Quantitative assessment of hepatitis B virus DNA in chronic hepatitis B: comparison of two solution hybridization assays

We compared two solution hybridization assays, the AffiProbe assay (Orion Corporation) and the Abbott HBV-DNA assay (Abbott), for quantitative measurement of hepatitis B virus (HBV) DNA in serum samples obtained from chronic hepatitis B surface antigen (HBsAg) carriers. Forty transversally collected (group 1) and 83 serially collected (group 2) serum samples from chronic hepatitis B patients were tested with both assays. The serial serum samples were obtained from 6 patients who underwent α-interferon therapy with different outcomes (nonresponse, hepatitis B e antigen [HBeAg] seroconversion, HBeAg and HBsAg seroconversion). In group 1 a good correlation (r = 0.91; P < 0.001) was found between the HBV-DNA results of the two assays. Two samples (5%) were HBV-DNA positive according to the Abbott but negative according to the AffiProbe assay; for all other samples the HBV-DNA status corresponded. In group 2 the assays gave colinear HBV-DNA results during follow-up of 5 of the 6 patients (correlation for the total group: r = 0.90; P < 0.001). Nevertheless, in both groups the AffiProbe assay yielded about 5–10 times higher HBV-DNA levels than the Abbott HBV-DNA assay (P < 0.001). These discordant results were most probably due to standardization differences of the positive control samples of the two test systems. This observation underlines the need for international standardization of HBV-DNA and uniform reference panels. © 1993 Wiley-Liss, Inc.     

Novel approach for differential diagnosis of HIV infections in the face of vaccine-generated antibodies: utility for detection of diverse HIV-1 subtypes

Because increasing numbers of HIV vaccine candidates are being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Most of the currently tested vaccines contain multiple viral components. As a result, many vaccine recipients give positive results in FDA-licensed HIV serodetection tests. We have identified conserved sequences in Env-gp41 and Gag-p6, which are recognized soon after infection but are not included in most HIV vaccine candidates. A new HIV serodetection assay, the HIV-SELECTEST, was established that distinguishes between vaccine-induced antibodies and seroconversion due to true HIV infections. It is important to make this assay globally relevant, because many clinical trials are conducted around the world where most HIV infections are due to non-B subtype HIV-1. Therefore, the current study examined the reactivity of plasma samples from >3,000 infections with diverse HIV subtypes worldwide. The HIV-SELECTEST performed at >99% specificity and sensitivity. Both recent and established infections with clades A, B, C, D, E, F, G, J, and CRFs were detected. Antibodies elicited by other vaccinations or infections endemic to the clinical trial sites did not react in this assay. Therefore, HIV-SELECTEST could be an important differential diagnostic tool for HIV vaccine trials, blood banks, and population screening worldwide.     

Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus

Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in Escherichia coli or to chemically synthesized NS1 peptides. Immune sera were obtained from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines. Seroconversion to positivity for antibodies to the NS1 protein was achieved in birds experimentally infected with multiple subtypes of influenza A virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated gradient-purified vaccines had no seroconversion to positivity for antibodies to the NS1 protein, and animals vaccinated with commercial vaccines had low, but detectable, levels of NS1 antibodies. The use of a second ELISA with diluted sera identified a diagnostic test that results in seropositivity for antibodies to the NS1 protein only in infected birds. For the field application phase of this study, serum samples were collected from vaccinated and infected poultry, diluted, and screened for anti-NS1 antibodies. Field sera from poultry that received commercial AI vaccines were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test, but they were negative by the NS1 ELISA. Conversely, diluted field sera from AI-infected poultry were positive for both AGP and NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industries.     

Spurious Hemolysis Does not Influence the Reliability of Digoxin Testing on Siemens RXL MAX and Roche Cobas e601

Little information is available on the influence of spurious hemolysis on digoxin immunoassays. Seventeen consecutive, non-hemolyzed, sodium-heparin samples were divided in three aliquots. The first was immediately centrifuged and tested for hemolysis index (HI), as well as plasma digoxin on Siemens RXL MAX using the Siemens Dimension Flex and Roche Cobas e601 by electrochemiluminescent (ECLIA) technique. The second and third aliquots were subjected to mechanical hemolysis by aspirating the blood one and two times through a thin needle. The concentration of digoxin measured on Siemens RXL MAX was significantly decreased from aliquot #A, to aliquot #B (-4%), and aliquot #C (-6%), but in none of the hemolyzed specimens the 10% bias was exceeded. No significant variation was observed by measuring plasma digoxin on Roche Cobas.     

Utility of IgM/IgG ratio and IgG avidity for distinguishing primary and secondary dengue virus infections using sera collected more than 30 days after disease onset

Dengue virus (DV) IgM/IgG ratio and IgG avidity value (AV) can reliably distinguish between primary and secondary DV infections using sera collected within 30 days of disease onset, but little is known about their efficacies using sera collected >30 days after onset. To investigate this issue, we analyzed specimens submitted to our reference laboratory for DV antibody testing. We first classified patients as having primary (n = 55) or secondary (n = 58) infections based on seroconversion patterns in a comparison of two sera collected <30 days apart. We then evaluated IgM/IgG ratios and IgG AVs of the second specimens by using receiver operating characteristic curve analysis. The IgM/IgG ratio that best discriminated primary from secondary infection was 1.32; 95% of 55 primary infections exhibited ratios of >1.32, whereas 93% of 58 secondary infections exhibited ratios of ≤1.32. The discriminatory AV was 0.39; 95% of 41 primary infections exhibited AVs of ≤0.39, whereas 95% of 38 secondary infections exhibited AVs of >0.39. We then evaluated the IgM/IgG ratios and AV for primary-infection patients whose second serum samples were collected ≥30 days after the first serum samples; only 56% of 27 sera exhibited ratios of >1.32, whereas 81% of 21 sera exhibited AVs of ≤0.39. Assuming that the first specimens were collected within a week after symptoms appeared, these findings indicate that IgG AV is superior to the IgM/IgG ratio for distinguishing primary from secondary DV infections when using samples collected more than 5 weeks after disease onset.     

Performances of fourth generation HIV antigen/antibody assays on filter paper for detection of early HIV infections

BackgroundPoint-of-care testing and diagnosis of HIV acute infections play important roles in preventing transmission, but HIV rapid diagnosis tests have poor capacity to detect early infections. Filter paper can be used for capillary blood collection and HIV testing using 4th generation immunoassays.ObjectivesAntigen/antibody combined immunoassays were evaluated for their capacity to identify early HIV infections using filter paper in comparison with rapid test.Study designThirty nine serum samples collected from HIV seroconverters were spotted onto filter paper and tested by the Roche Elecsys^® HIV Combi PT test and the DiaSorin Liaison XL Murex HIV Ab/Ag assay.ResultsFourth generation immunoassays identified 34 out of 39 HIV early infections using dried serum spot, whereas the Determine™ HIV-1/2 rapid test detected 24 out of 39 HIV positive serum (87.2% vs 61.5% respectively, p = 0.009). p24 antigen was detected by the Liaison XL in 19 dried serum samples (48.7%). In the group characterized by a negative western blot, 7 out of 8 (87.5%) and 6 out of 8 (75.0%) samples were found positive for HIV using the Elecsys and the Liaison XL, respectively. None of these eight samples classified in this group of early acute infections were found positive by the rapid test.ConclusionFourth generation Ag/Ab immunoassays performed on dried serum spot had good performance for HIV testing during the early phases of HIV infection. This method may be useful to detect HIV early infections in hard-to-reach populations and individuals living in remote areas before rapid tests become positive.     

Performance of a Cytomegalovirus IgG Enzyme Immunoassay Kit Modified To Measure Avidity

The measurement of cytomegalovirus (CMV) IgG avidity accurately discriminates recent and past CMV infections. We sought to determine if the Wampole Laboratories CMV IgG enzyme immunoassay (EIA) could be modified to measure avidity. The evaluation panel consisted of 156 serum samples we used in 2002 to validate a laboratory-developed EIA, in which 78 serum samples exhibited low avidity, 7 exhibited intermediate avidity, and 71 exhibited high avidity. The qualitative agreement between the two avidity assays was 94% (147/156); all 9 sera with discordant results exhibited intermediate avidity in one of the assays. The avidity index (AI) values in the two assays showed excellent correlation (r = 0.96, P < 0.0001). The definition of high avidity was verified for the Wampole assay by demonstrating high avidity in 91/93 (98%) recently collected CMV IgG-positive/IgM-negative serum samples. The performance of the Wampole avidity assay in a reference laboratory setting was assessed using 470 consecutive serum samples submitted for CMV IgG avidity testing. Surprisingly, 101 serum samples were negative when screened for CMV IgG using the Wampole kit per the package insert; 98 of these 101 serum samples were tested using a CMV IgG chemiluminescent immunoassay, and only 5 were positive. Of the 369 CMV IgG-positive samples, 6% exhibited low IgG avidity, 6% exhibited intermediate avidity, and 88% exhibited high avidity; CMV IgM detection rates were inversely related to AI levels. These findings show that (i) the Wampole CMV IgG EIA can be modified to measure CMV IgG avidity, (ii) many samples are apparently submitted for avidity testing without knowledge of their CMV IgG status, and (iii) most CMV IgG-positive sera submitted for avidity testing exhibit high avidity.     

Substances interfering with direct detection of Mycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serum albumin.

Interfering substances have been reported to inhibit PCR assays for the direct detection of Mycobacterium tuberculosis in clinical specimens. Using an internal control, we determined that 52% of respiratory specimens interfered with our PCR assay. On the basis of these findings, we tried to circumvent the problem by simply diluting prepared sediments. With sediment from a routinely processed sputum known to be inhibitory to PCR, one aliquot was prepared in a routine manner for PCR. Remaining sediment was diluted in phosphate-buffered saline, Middlebrook 7H10 broth, or BACTEC 12B broth; an internal control was added to all reaction mixtures and controls. Internal control was detected only in the sample diluted with BACTEC 12B medium. Components of the BACTEC 12B medium including PANTA reagent (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin), reconstituting fluid, 0.2% glycerol, 0.05% Tween 80, and 0.05% bovine serum albumin (BSA) were tested in a similar manner. Only 0.05% BSA resulted in amplification of the internal control DNA. Varying concentrations of BSA were added to 11 aliquots of a respiratory sediment known to be inhibitory to the PCR. Internal control was detected in all reaction mixtures containing 0.00038 to 0.1% BSA. To determine the ability of BSA to override inhibition, respiratory specimens were run in triplicate: undiluted, diluted 1:2 with BACTEC 12B medium, or diluted with 0.026% BSA. For 21 of 22 inhibitory specimens, BSA was able to override the presence of interfering substances. These data suggest that the presence of BSA in a PCR assay is critical for the direct detection of M. tuberculosis in respiratory specimens.     

Subclass distribution of rubella virus-specific immunoglobulin G

An enzyme-linked immunosorbent assay was used to study the subclass distribution of rubella virus-specific immunoglobulin G (IgG) in 97 serum samples from healthy donors and from patients with recent or remote rubella infections. Plastic beads coated with rubella antigen were incubated with test serum and then with monoclonal antibodies to the four human subclass of IgG. Rubella virus-specific IgG1 was present in all serum samples containing rubella virus-specific IgG antibodies. Rubella virus-specific IgG2 was present in 1 of 35 samples from healthy donors that also contained specific IgG1. Rubella virus-specific IgG3 was found in serum samples from patients with recent rubella infections but had disappeared by 6 months after the onset of symptoms. Rubella virus-specific IgG4 was found in low amounts in 7 of 35 samples from healthy immune donors. Of 20 serum samples that were negative by other serological techniques, 8 gave absorbances above cutoff levels in the assays for rubella virus-specific total IgG and IgG1. In 1 of 20 serum samples, the assays for total IgG and IgG2 were positive. High absorbance in the assay for rubella virus-specific IgG4 was found in one serum. This serum was negative in all other assays for rubella virus-specific antibodies.     

Use of seroconversion panels to estimate delay in detection of anti-human immunodeficiency virus antibodies by enzyme-linked immunosorbent assay of pooled compared to singleton serum samples

The transfusion of unsafe blood worldwide accounts for 5 to 15% of new human immunodeficiency virus (HIV) infections, most of which occur in sub-Saharan Africa. While developed countries now apply PCR testing of pooled samples, some developing countries still do not have universal screening policies. More efficient low-cost procedures for the screening of pooled samples have the potential to encourage mass screening efforts in resource-poor settings. The aim of this study was to estimate the delay in the detection of HIV antibodies in pooled serum samples compared to that in singleton serum samples by enzyme-linked immunosorbent assay (ELISA) and to evaluate the risk of transfusion-transmitted HIV infection during the window period. Serial blood samples obtained from five HIV seroconversion panels were mixed with HIV-seronegative blood samples to create pools of 6, 12, 16, 24, 32, and 48 samples. The delay in detection of the first anti-HIV antibody-positive sample in tests with pooled samples was calculated for each pool size and compared to that obtained by testing of singleton samples and statistically evaluated by a robust log-linear regression analysis. The risk of a false-negative (FN) result caused by dilution was estimated by use of the incidence risk/window period model. The additional risk of transmission related to ELISA screening of pooled samples for HIV did not exceed 9% of the current risk of an FN result (estimated to be 1/1,067,000). The countries with virus prevalence rates in donors of less than 15% are expected to save up to 30% in the number of tests. ELISA screening of pooled samples could be considered in settings where the testing of blood supplies for HIV is not routinely done.     

Multicenter evaluation of a rapid and convenient method for determination of cytomegalovirus immunoglobulin G avidity

An easy, rapid, and reproducible test to distinguish residual cytomegalovirus (CMV) immunoglobulin M (IgM) antibodies from antibodies produced in primary infection could be useful, especially for pregnant women. The CMV avidity of IgG antibodies with the VIDAS automated enzyme-linked fluorescent assay and 6 M urea was evaluated in a multicenter study to differentiate between primary CMV infections and past infections or reactivations. A total of 416 serum specimens were tested: 159 specimens were from follow-up of primary infections, and 257 were from past infections. All of the specimens from primary infections collected within 4 months (17 weeks) after the onset of the infection had an avidity index lower than 0.8. An avidity index higher than 0.8 excludes a recent primary infection of less than 4 months. However, an avidity index higher than 0.8 cannot confirm all past infections, since 48 specimens (18%) from past infections had an avidity index lower than 0.8 (between 0.5 and 0.8). The exclusion capacity could be improved (96.9%) by using a cutoff of 0.7, but this index would decrease the specificity of the technique, since the avidity index was found to be between 0.7 and 0.8 in two patients with recent primary infection. All specimens from primary infections obtained more than 4 months after the onset of infection had an avidity index more than 0.2. In this study, an avidity index less than 0.2 confirms the presence of a recent primary infection of less than 4 months. The VIDAS CMV IgG avidity test is a rapid, reproducible test with very good performance.     

An immunoblot assay for detection of immunoglobulin M antibody to human herpesvirus 6

We identified the human herpesvirus 6 (HHV-6)-dominant immunoglobulin M (IgM)-reactive virion protein as being the same 101-kDa protein (101K) previously identified as the major IgG immunoreactive protein and a specific serologic marker of HHV-6 infection. An immunoblot assay (IB) to detect HHV-6-specific IgM antibodies against the 101K protein in human serum samples was developed. The assay was validated by using acute- and convalescent-phase serum collected from children under 2 years of age in which we previously detected IgG seroconversion to the HHV-6 101K protein. Of 32 serum pairs which previously demonstrated IgG seroconversion to the 101K protein, 29 had IgM reactivity to the same protein in the acute-phase sample and the remaining 3 had reactivity in the convalescent-phase sample. We also detected HHV-6 IgM activity in sera collected from individuals > or =4 years of age who were also IgM seropositive to measles or rubella. Results of cross-adsorption studies using measles virus-, rubella virus-, and HHV-6-infected cells as the adsorbing antigen indicated no cross-reactivity between measles or rubella IgM and HHV-6 IgM in human serum samples. The IgM IB detected HHV-6-specific IgM antibody to the 101K protein in 78% (63 of 81) of tested acute-phase serum collected from young children with an undifferentiated rash illness by using a single serum dilution.