Evaluation of Modified Two-Tiered Testing Algorithms for Lyme Disease Laboratory Diagnosis Using Well-Characterized Serum Samples.

Standard two-tiered testing (STTT) is the recommended algorithm for laboratory diagnosis of Lyme disease (LD). Several limitations are associated with STTT that include low sensitivity in the early stages of disease, as well as technical complexity and subjectivity associated with second-tier immunoblotting; therefore, modified two-tiered testing (MTTT) algorithms that utilize two sequential first-tier tests and eliminate immunoblotting have been evaluated. Recently, a novel MTTT that uses a VlsE chemiluminescence immunoassay followed by a C6 enzyme immunoassay has been proposed. The purpose of this study was to evaluate the performance of the VlsE/C6 MTTT using well-characterized serum samples. Serum samples from the CDC Lyme Serum Repository were tested using three MTTTs, VlsE/C6, whole-cell sonicate (WCS)/C6, and WCS/VlsE, and three STTTs (immunoblotting preceded by three different first-tier assays: VlsE, C6, and WCS). Significant differences were not observed between the results of the MTTTs assessed; however, the VlsE/C6 MTTT resulted in the highest specificity (100%) when other diseases were tested and the lowest sensitivity (75%) for LD samples. Significant differences were present between the results for various MTTTs and STTTs evaluated. Specifically, all MTTTs resulted in higher sensitivities than the STTTs for all LD groups combined and were significantly more accurate (i.e., higher proportion of correct classifications) for this group, with the exception of the WCS/ViraStripe STTT. Additionally, when other diseases were tested, only the results of the VlsE/C6 MTTT differed significantly from those of the WCS/ViraStripe STTT, with the VlsE/C6 MTTT resulting in a 6.2% higher accuracy. Overall, the VlsE/C6 MTTT offers an additional laboratory testing algorithm for LD with equivalent or enhanced performance compared to that of the other MTTTs and STTTs evaluated in this study.

Comparison of a commercial dengue IgM capture ELISA with dengue antigen focus reduction microneutralization test and the Centers for Disease Control dengue IgM capture-ELISA.

Dengue (DENV) infection is caused by an arbovirus that is a member of the family Flaviviridae, genus Flavivirus. The diagnosis of acute dengue infection using clinical signs and symptoms can be difficult since the manifestations cannot be readily differentiated from other infections. Therefore the diagnosis of acute dengue infection depends upon laboratory assays. Dengue virus ELISAs have been designed for the detection of IgM and IgG antibodies in addition to nonstructural 1 (NS1) antigens. The InBios IgM Dengue ELISA was compared to the Antigen Focus Reduction Microneutralization Test (FRµNT90) and Centers for Disease Control Dengue IgM Capture-ELISA (CDC MAC-ELISA). The calculated sensitivity, specificity and agreement of the InBios ELISA compared to FRµNT90 and CDC MAC-ELISA was 88.7% (C.I. 81.4-93.7), 93.1% (C.I. 89.1-95.8) and 91.5% (C.I. 86.3-95.0), respectively. In summary the InBios IgM Dengue ELISA sensitivity and specificity is comparable to other commercially available IgM Capture-ELISAs.

Surveillance of human papilloma virus using reference laboratory data for the purpose of evaluating vaccine impact.

Nationwide positivity rates of high-risk human papillomavirus for the United States before and since the introduction of a Human Papillomavirus (HPV) vaccine in 2006 would provide insight into the population impact of HPV vaccination. Data for high-risk HPV testing results from January 1, 2004 to June 1, 2013 at a national reference laboratory were retrospectively analyzed to produce 757,761 patient records of women between the ages of 14 and 59. Generalized linear models and finite mixture models were utilized to eliminate sources of bias and establish a population undergoing standard gynecological screening. Unadjusted positivity rates for high-risk HPV were 27.2% for all age groups combined. Highest rates occurred in women aged 14 to 19. While the positivity rates decreased for all age groups from 2004 to 2013, the higher age categories showed less downward trend following vaccine introduction, and the two age categories 20 to 24 and 25 to 29 showed a significantly different downward trend between pre- and post-vaccine time periods (-0.1% per year to -1.5% per year, and 0.4% per year to -1.5% per year, respectively). All other age groups had rates of change that became less negative, indicating a slower rate of decline.

Seropositivity rates for measles, mumps, and rubella IgG and costs associated with testing and revaccination.

Retrospective analysis of IgG test results and patterns for measles, mumps, and rubella revealed generally high seropositivity rates, with that of mumps being the lowest. A simplified cost analysis shows that when there is a suspicion of nonimmunity, serological testing may be cheaper than vaccination.

A spectrophotometric method to precisely determine endpoint titers in complement fixation assays.

Since the early 20th century, complement fixation (CF) testing has been used to quantify the humoral response to various pathogens. The qualification of a positive result is based on a subjective determination of 30% lysis of sheep red blood cells, which can lead to variability in the analysis. A spectrophotometric reading of a standard with a known 30% lysis was used to standardize the currently used CF method and tested with controls and patient sera for various fungal assays. By utilizing this method a precise and non-subjective determination of endpoint titers was achieved.

Antituberculosis IgG antibodies as a marker of active Mycobacterium tuberculosis disease.

Anti-Mycobacterium tuberculosis IgG antibodies may aid in the diagnosis of active M. tuberculosis disease. We studied whether anti-M. tuberculosis IgG antibodies are elevated in active M. tuberculosis disease and assessed factors contributing to false-positive and -negative results. A retrospective study of 2,150 individuals tested by the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay was conducted at the University of Utah, ARUP Laboratories, November 2008 to December 2010. All samples were tested with the InBios Active TbDetect antituberculosis (anti-TB) IgG antibody assay. Of 1,044 patients with a positive QFT-GIT, 59 (5.7%) were positive for M. tuberculosis antibodies. Fourteen of 1,106 (1.3%) with a negative or indeterminate QFT-GIT were positive for M. tuberculosis antibodies. M. tuberculosis antibody tests were positive in 61.5% with confirmed active M. tuberculosis disease and other mycobacterial infections. Over half of the false-negative M. tuberculosis antibody tests occurred in patients ≥ 90 years of age. False positives were seen in 12.9% of autoimmune patients. The odds ratio of being positive by the QFT-GIT and the InBios TB IgG assay increased with confirmed M. tuberculosis disease or highly suspected M. tuberculosis disease and was 86.7 (95% confidence interval [CI], 34.4 to 218.5) in these two groups compared to patients negative by both tests. Although anti-M. tuberculosis antibodies can be detected in patients with active M. tuberculosis disease, caution should be used with patients where immunoglobulin levels may be decreased or patients with autoantibodies.

Comparison of Western immunobloting to an enzyme-linked immunosorbent assay for the determination of anti-Bordetella pertussis antibodies.

During Bordetella pertussis infection, it has been established that an increase of anti-pertussis toxin (PT) and anti-filamentous hemagglutinin (FHA) antibodies occurs. Immunoblots from two manufacturers using FHA and PT antigens were compared with an enzyme-linked immunosorbent assay (ELISA) that used both FHA and PT. One manufacturer used two concentrations of PT bands for the IgG immunoblot, calibrated to the World Health Organization standard for PT in international units (IU/ml), 100 IU/ml (PT-100) and 8 IU/ml (PT). The second immunoblot kit measured antibodies to a single calibrated PT band. Both kits measured IgA antibodies, and one additionally measured IgM antibodies. Two of 41 (5%) ELISA IgM positives were confirmed positive by IgM immunoblotting, suggesting poor specificity of the IgM ELISA. The agreements of the IgG and IgA immunoblots with the ELISA ranged from 72.5% to 85.3%, with only 38 to 51% of IgA positives confirmed by immunoblotting and only 61 to 68% of IgG positives confirmed by immunoblotting. The two immunoblots correlated well with each other, with 91.7% and 94.3% agreement for IgG and IgA, respectively. When the FHA band was used with the PT band as the criterion for positivity, significant differences existed in specificity compared to the ELISA (IgG, 84.1% versus 33.3%; IgA, 82.4% versus 71.0%). When the positive IgA immunoblots (evidence of natural recent infection) were compared to the positive PT-100 IgG immunoblots (evidence of recent infection or vaccination), the PT-100 blot showed a 71% sensitivity in detecting natural recent infection. B. pertussis immunoblots, alone or in combination with ELISAs, can aid in the diagnosis of B. pertussis infection.

A comparison of Brucella IgG and IgM ELISA assays with agglutination methodology.

Despite brucellosis having a low incidence rate in developed nations, it still remains the leading zoonotic disease in the world. Culturing of Brucella spp. provides good specificity but in cases where the fever is intermittent, sensitivity is problematic. This has led to the development of serological methods of detection. Brucella agglutination methods have been considered the serological gold-standard since their inception, although commercial Brucella IgG and IgM enzyme-linked immunosorbent assays are available to potentially aid in the diagnosis of the disease. In our study, anti-Brucella IgG and IgM assays were compared with agglutination. Individually the IgG assay tested had an accuracy of 56% and the IgM assay had an accuracy of 77%. These poor accuracies reinforce Centers for Disease Control's conclusion that nonagglutination tests should not be used to confirm brucellosis.

Evaluation of two immunoblot assays and a Western blot assay for the detection of antisyphilis immunoglobulin g antibodies.

In the present study, two immunoglobulin G (IgG) immunoblot assays and one IgG Western blot assay were compared to the rapid plasma reagin test (RPR), the fluorescent treponemal antibody absorption test (FTA-ABS), and the Treponema pallidum particle agglutination assay (TP-PA). The agreement levels of the Viramed, Virotech, and MarDx assays were 97.0%, 96.4%, and 99.4%, and the agreements of samples inconclusive by FTA-ABS and resolved by TP-PA were 91.7%, 83.3%, and 69.4%, respectively.

An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies.

The envelope glycoprotein G of rabies virus in vaccines induces the production of neutralizing antibodies important in the protection against the disease. The measurement of anti-envelope glycoprotein antibodies is a good predictor of the degree of humoral immunity in people during anti-rabies treatment or after vaccination. Several assays exist for the serological determination of antibody protection against rabies virus infection. Antibody neutralization by the rapid fluorescent focus inhibition test (RFFIT) or the fluorescent antibody virus neutralization (FAVN) test is currently the gold standard. Performance of the highly complex RFFIT and FAVN tests, however, requires specialized reference laboratories with expertise with this assay. Although not widely used, ELISA test kits are available and may be an additional option for testing that is more accessible. The aim of the present study was to evaluate available ELISA assays for the determination of anti-rabies antibodies. We compared the Bio-Rad Platelia Rabies II ELISA, DRG Rabies Virus IgG Ab ELISA and Focus Diagnostics Rabies Antibody Detection by ELISA to RFFIT. Bland-Altman plots comparing the Bio-Rad Platelia assay and the Focus Diagnostics assay to RFFIT showed a low degree of variability between the ELISA assays and RFFIT results except in samples with high RFFIT values. The agreement, sensitivity and specificity of Bio-Rad Platelia Rabies II ELISA when compared to RFFIT were 95.1 %, 94.1 % and 95.8 %, respectively. The DRG Rabies assay compared to RFFIT had an agreement of 77.7 %, a sensitivity of 86.7 % and a specificity of 69.4 %. The agreement, sensitivity and specificity of Focus Diagnostics Rabies Detection by ELISA when compared to RFFIT were 82.2 %, 91.7 % and 73.0 %, respectively. Overall, the Bio-Rad Platelia assay showed higher accuracy and specificity than either the DRG or Focus assays. All of these ELISAs, however, measure all antibody types and do not discriminate the neutralizing antibodies as measured by functional assays (RFFIT and FAVN) and cannot be relied upon to predict the neutralizing activity of the sera. The results of this study offer insight into the availability of alternative, less-complex methods to monitor rabies antibody titres in at-risk individuals following vaccination.

Comparison of a multiplexed herpes simplex virus type-specific immunoglobulin G serology assay to immunoblot, Western blot, and enzyme-linked immunosorbent assays.

The human herpes simplex virus (HSV) is highly pathogenic, with infections caused by two distinct antigenic types, HSV-1 and HSV-2. Differentiation of antibodies to these specific antigens can provide useful information for the diagnosis of subclinical or undiagnosed HSV-2 infections, as well as for reducing the risk of maternal transfer of HSV to the neonate. In this study, a multiplex assay capable of concurrent detection of HSV-1 and -2 immunoglobulin G (IgG) antibodies was compared to immunoblot, Western blot, and enzyme-linked immunosorbent assays. Agreement of the multiplex assay was 95% or greater (n = 332) for both HSV-1 and -2 compared to the three assays. Sensitivities for HSV-1 ranged from 94.9 to 97.9%, with specificities of 93 to 97%. For HSV-2, the sensitivity and specificity ranges were 92.6 to 98.9% and 98.3 to 98.7%, respectively. Our studies show that the multiplexed microsphere-based assay offers a sensitive and specific alternative method for the detection HSV-1 and -2 type-specific antibodies. Advantages of the multiplex assay include multiple results per assay, the inclusion of internal controls for each specimen, and higher throughput of results.

Assessment of three commercially available serologic assays for detection of antibodies to Mycobacterium tuberculosis and identification of active tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major world disease, with approximately 9 million new cases each year. Identification and treatment of active disease are essential for TB control. Serology may offer increased detection of active disease in patients with a positive tuberculin skin test (TST) or QuantiFERON-TB (QFT-G). The InBios Active TbDetect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), IBL M. tuberculosis IgG ELISA, and Anda Biologics TB ELISAs were evaluated for the ability to detect M. tuberculosis antibodies in patients with active disease. Agreement, sensitivity, and specificity for each ELISA were determined and compared to those for culture or amplified direct detection and M. tuberculosis low-risk control patients. The InBios Active TbDetect ELISA had an agreement of 96.2%, a sensitivity of 83.3%, and a specificity of 98.9%. The IBL M. tuberculosis ELISA had an agreement of 84.0%, a sensitivity of 5.6%, and a specificity of 100.0%. The agreement, sensitivity, and specificity of the Anda Biologics TB ELISA were 74.2%, 83.3%, and 72.0%, respectively. The sensitivity for detecting M. tuberculosis antibodies in human immunodeficiency virus-associated TB was 50% for both the InBios Active TbDetect ELISA and the Anda Biologics TB ELISA and 0% for the IBL M. tuberculosis ELISA. The positivity rates for InBios Active TbDetect ELISA, IBL M. tuberculosis ELISA, and Anda Biologics TB ELISA in latently infected individuals positive by TST and/or QFT-G were 5.1%, 0.0%, and 30.8%, respectively. It can be concluded that the InBios Active TbDetect IgG ELISA is superior to the other ELISAs in accurately detecting active TB.

Evaluation of a new commercial enzyme immunoassay for the detection of IgM antibodies to West Nile virus using a ratio method to eliminate nonspecific reactivity.

As West Nile virus (WNV) has become endemic in the United States, following the first reported cases in New York during the summer of 1999, the demand for specific serology has increased. Several IgM capture ELISA assays for the detection of WNV-specific IgM have been approved by the Food and Drug Administration for in vitro diagnostic testing, including kits from Focus Diagnostics and InBios International, Inc. The Focus Diagnostics IgM capture ELISA has a background subtraction protocol and the InBios IgM capture ELISA implements a ratio method to detect nonspecific reactivity due to rheumatoid factor, heterophile antibodies, and other interfering substances. We compared the InBios IgM capture ELISA with the Focus Diagnostics capture ELISA. Agreement, sensitivity, and specificity of the InBios IgM capture ELISA were 99, 98, and 100%, respectively. Samples that originally tested positive on the Focus Diagnostics IgM capture ELISA without the subtraction protocol and were then determined negative following the subtraction protocol agreed 100% with the InBios IgM capture ELISA. We conclude that a method to eliminate background reactivity is a necessary portion of any anti-WNV IgM assay in order to eliminate false-positive results.

Rapid immunochromatographic strip test for detection of anti-K39 immunoglobulin G antibodies for diagnosis of visceral leishmaniasis.

InBios International has developed an immunochromatographic rapid strip for the detection of visceral leishmaniasis that requires minimal equipment and only a small amount of blood to run a test. We compared the InBios rapid strip test with the CDC immunofluorescent antibody assay, and the agreement, sensitivity, and specificity were 98%, 90%, and 100%, respectively.