Heather Jost

2009 Pandemic Influenza A (H1N1): Pathology and Pathogenesis of 100 Fatal Cases in the United States

In the spring of 2009, a novel influenza A (H1N1) virus emerged in North America and spread worldwide to cause the first influenza pandemic since 1968. During the first 4 months, over 500 deaths in the United States had been associated with confirmed 2009 pandemic influenza A (H1N1) [2009 H1N1] virus infection. Pathological evaluation of respiratory specimens from initial influenza-associated deaths suggested marked differences in viral tropism and tissue damage compared with seasonal influenza and prompted further investigation. Available autopsy tissue samples were obtained from 100 US deaths with laboratory-confirmed 2009 H1N1 virus infection. Demographic and clinical data of these case-patients were collected, and the tissues were evaluated by multiple laboratory methods, including histopathological evaluation, special stains, molecular and immunohistochemical assays, viral culture, and electron microscopy. The most prominent histopathological feature observed was diffuse alveolar damage in the lung in all case-patients examined. Alveolar lining cells, including type I and type II pneumocytes, were the primary infected cells. Bacterial co-infections were identified in >25% of the case-patients. Viral pneumonia and immunolocalization of viral antigen in association with diffuse alveolar damage are prominent features of infection with 2009 pandemic influenza A (H1N1) virus. Underlying medical conditions and bacterial co-infections contributed to the fatal outcome of this infection. More studies are needed to understand the multifactorial pathogenesis of this infection.

A pilot study of host genetic variants associated with influenza-associated deaths among children and young adults.

Low-producing MBL2 genotypes may have increased risk for MRSA co-infection.

A comparison of the analytical level of agreement of nine treponemal assays for syphilis and possible implications for screening algorithms

Objective The serological diagnosis of syphilis requires the detection of two distinct antibodies, the non-treponemal and trepomenal. Center for Disease Control and Prevention (CDC) recommends screening first with a non-treponemal test such as (Rapid Plasma Reagin/Venereal Disease Research Laboratory), and then confirming those results with one of the several treponemal tests (Fluorescent Treponemal Antibody-Absorption (FTA-ABS), Enzyme Immunoassay, chemiluminescence, treponema pallidum particle agglutination (TP-PA) or Point of Care). Owing to the high volume of samples processed by some laboratories using automated systems, the screening with treponemal assays and confirming with non-treponemal tests is becoming the established norm. The purpose of this study was to evaluate eight treponemal assays using TP-PA as the predicate assay. Methods 290 stored serum samples were tested qualitatively according to the manufacturers directions. Results Concordance with specimens tested as reactive or non-reactive using TP-PA was: FTA-ABS 94.5–100%, Trep-Sure 100–98.9%, BioELISA 100–98.9%, INNO-LIA 99.1–99.4%, BIOLINE 100–98.9%, CAPTIA IgG 100–97.2%, Trep-ID 100–100% and LIAISON 100–99.4%. In order to properly evaluate the performance of these assays, the analytical sensitivity was determined by endpoint titration of serial dilutions of the reactive serum samples in normal sera. The median endpoint titre varied from 1:4 for FTA-ABS to 1:512 for Trep-Sure. Conclusions The performance of the treponemal serological assays was comparable while using medium and high-titre sera. However, the varying performance on specimen dilutions suggests that there may be differences in sensitivity with low-titre sera that are more prevalent in primary and late syphilis cases.

Diagnosis of Influenza from Respiratory Autopsy Tissues Detection of Virus by Real-Time Reverse Transcription-PCR in 222 Cases

The recent influenza pandemic, caused by a novel H1N1 influenza A virus, as well as the seasonal influenza outbreaks caused by varieties of influenza A and B viruses, are responsible for hundreds of thousands of deaths worldwide. Few studies have evaluated the utility of real-time reverse transcription-PCR to detect influenza virus RNA from formalin-fixed, paraffin-embedded tissues obtained at autopsy. In this work, respiratory autopsy tissues from 442 suspect influenza cases were tested by real-time reverse transcription-PCR for seasonal influenza A and B and 2009 pandemic influenza A (H1N1) viruses and the results were compared to those obtained by immunohistochemistry. In total, 222 cases were positive by real-time reverse transcription-PCR, and of 218 real-time, reverse transcription-PCR-positive cases also tested by immunohistochemistry, only 107 were positive. Although formalin-fixed, paraffin-embedded tissues can be used for diagnosis, frozen tissues offer the best chance to make a postmortem diagnosis of influenza because these tissues possess nucleic acids that are less degraded and, as a consequence, provide longer sequence information than that obtained from fixed tissues. We also determined that testing of all available respiratory tissues is critical for optimal detection of influenza virus in postmortem tissues.

Evaluation of treponemal serum tests performed on cerebrospinal fluid for diagnosis of neurosyphilis.

OBJECTIVES We evaluated the use of treponemal serum tests in cerebrospinal fluid (CSF) to diagnose neurosyphilis since CSF-Venereal Disease Research Laboratory (VDRL) is specific but lacks sensitivity. METHODS We tested CSF specimens using the following treponemal serum tests: INNO-LIA, Treponema pallidum particle agglutination (TP-PA), Trep-Sure, and Maxi-Syph. The reference standard to calculate sensitivity and specificity was having two or more reactive/positive tests on CSF. RESULTS The reference standard group included 11 cases that fulfilled the definition of neurosyphilis (reactive CSF-VDRL plus symptoms) and three cases that did not fulfill the definition: two cases had neurologic symptoms but a nonreactive CSF-VDRL, and one had several positive CSF syphilis tests (reactive VDRL and positive treponemal and syphilis polymerase chain reaction) but no history (referred sample). Controls included 18 patients in whom a CSF-VDRL was performed the same week as patients in the reference group. The sensitivity was 85.7% (12/14) for CSF-VDRL, 92.9% (13/14) for Trep-Sure, 100% (10/10) for Maxi-Syph, 92.3% (12/13) for INNO-LIA, and 83.3% (10/12) for TP-PA. Specificity was 100% for all tests. CONCLUSIONS Treponemal serum tests performed on CSF were useful in identifying two patients with nonreactive CSF-VDRL.

Effect of multiple freeze and thaw cycles on the sensitivity of IgG and IgM immunoglobulins in the sera of patients with syphilis.

We describe the effects of multiple freeze and thaw cycles on the sensitivity of the immunoglobulins IgG and IgM measured by enzyme-linked immunoassays in the sera of patients with syphilis. Stored frozen sera can withstand repeated freezing and thawing cycles with a minimal detrimental effect on the sensitivity of the sera.

Correlation of Treponemal Immunoassay Signal Strength Values With Reactivity of Confirmatory Treponemal Testing

ABSTRACT Automated treponemal immunoassays are used for syphilis screening with the reverse-sequence algorithm; discordant results (e.g., enzyme immunoassay [EIA] reactive and reactive plasma reagin [RPR] nonreactive) are resolved with a second treponemal test. We conducted a study to determine automated immunoassay signal strength values consistently correlating with reactive confirmatory treponemal testing. We conducted a cross-sectional analysis of four automated immunoassays (BioPlex 2200 microbead immunoassay [MBIA], Liaison chemiluminescence immunoassay [CIA], Advia-Centaur CIA, and Trep-Sure EIA) and three manual assays (Treponema pallidum particle agglutination [TP-PA], fluorescent treponemal antibody absorption [FTA-ABS] test, and Inno-LIA line immunoassay). We compared signal strength values of automated immunoassays and positive and negative agreement. Among 1,995 specimens, 908 (45.5%) were true positives (≥4/7 tests reactive) and 1,087 (54.5%) were true negatives (≥4/7 tests nonreactive). Positive agreement ranged from 86.1% (83.7 to 88.2%) for FTA-ABS to 99.7% (99.0 to 99.9%) for Advia-Centaur CIA; negative agreement ranged from 86.3% (84.1 to 88.2%) for Trep-Sure EIA to 100% for TP-PA (99.6 to 100%). Increasing signal strength values correlated with increasing reactivity of confirmatory testing (P < 0.0001 for all automated immunoassays by Cochran-Armitage test for trend). All automated immunoassays had signal strength cutoffs corresponding to ≥4/7 reactive treponemal tests. BioPlex MBIA and Liaison CIA had signal strength cutoffs correlating with ≥99% and 100% TP-PA reactivity, respectively. The Advia-Centaur CIA and Trep-Sure EIA had signal strength cutoffs correlating with at least 95% TP-PA reactivity. All automated immunoassays had signal strength cutoffs correlating with at least 95% FTA-ABS reactivity. Assuming that a 95% level of confirmation is adequate, these signal strength values can be used in lieu of confirmatory testing with TP-PA and FTA-ABS.

Laboratory evaluation of the Chembio Dual Path Platform HIV-Syphilis Assay

Background Use of rapid diagnostic tests for HIV and syphilis has increased remarkably in the last decade. As new rapid diagnostic tests become available, there is a continuous need to assess their performance and operational characteristics prior to use in clinical settings. Objectives In this study, we evaluated the performance of the Chembio Dual Path Platform (DPP®) HIV–Syphilis Assay to accurately diagnose HIV, syphilis, and HIV/syphilis co-infection. Method In 2013, 990 serum samples from the Georgia Public Health Laboratory in Atlanta, Georgia, United States were characterised for HIV and syphilis and used to evaluate the platform. HIV reference testing combined third-generation Enzyme Immunoassay and Western Blot, whereas reference testing for syphilis was conducted by the Treponema pallidum passive particle agglutination method and the TrepSure assay. We assessed the sensitivity and specificity of the DPP assay on this panel by comparing results with the HIV and syphilis reference testing algorithms. Results For HIV, sensitivity was 99.8% and specificity was 98.4%; for syphilis, sensitivity was 98.8% and specificity was 99.4%. Of the 348 co-infected sera, 344 (98.9%) were detected accurately by the DPP assay, but 11 specimens had false-positive results (9 HIV and 2 syphilis) due to weak reactivity. Conclusion In this evaluation, the Chembio DPP HIV–Syphilis Assay had high sensitivity and specificity for detecting both HIV and treponemal antibodies. Our results indicate that this assay could have a significant impact on the simultaneous screening of HIV and syphilis using a single test device for high-risk populations or pregnant women needing timely care and treatment.

P5.091 Head-Head Comparison of Reactivity and Signal Strength Value For Reactivity Among Seven Treponemal Assays: A Preliminary Report

Background Automated immunoassays (AI) for detection of T. pallidum antibodies are increasingly used for syphilis screening in the United States. These assays demonstrate fast performance, reduced labour requirements, and high throughput with walk-away capability. Limited data are available about the relative seroreactivity among commercial treponemal assays, especially in low risk populations. Additionally, it is unknown to what extent the AI signal strength values, used to assess reactivity, are associated with non-AI treponemal reactivity. We compared concordance of seroreactivity among 7 treponemal tests and assessed AI signal strength values associated with reactivity. Methods Previously identified reactive and nonreactive sera (n = 566) were obtained from Kaiser Northern and Southern California regional laboratories. All sera were tested with AIs: BioPlex 2200 Syphilis IgM/IgG (BioRad), treponemal LIAISON (DiaSorin), Advia-Centaur syphilis (Siemens), and non-AIs (INNOLIA syphilis score (INNOGENETICS), TrepSure (Phoenix Biotech), Treponemal Pallidum Particle Agglutination (TP-PA) (Fujirebio), and Fluorescent Treponemal Antibody-Absorption (FTA-ABS) (Zeus Scientific) tests. Reactivity was interpreted according to manufacturers’ instructions. Results Seroreactivity ranged from 40.5 – 43.9% for AIs, and 33.0–42.2% for non-AIs. In all 7 tests, 30% (167/566) were reactive, and positive agreement among assays was 82.3%. The overall seroreactivity among AIs was 38.9% (220/566) and positive agreement was 92.6%. Minimum signal strength values of 11.72 (Centaur, range: 1.1–45), 4.4 (BioPlex, range: 1.1–8) and 9.4 (Liaison, range: 1.1–70) correlated 100% with TPPA reactivity. The proportion of AI-seroreactive specimens that were also TP-PA reactive were: 86.5% (198/229) for BioPlex, 85.2% (202/237) for ADVIA-Centaur, and 81.6% (200/245) for LIAISON. Conclusion Although there is some variation in seroreactivity among the 7 tests, there is good correlation. A large proportion of AI tests with a minimal signal-to-cutoff ratio were associated with a positive TP-PA, suggesting that a second treponemal test may not be necessary to confirm AI-reactive, RPR-nonreactive sera.

Evaluation of a Digital Flocculation Reader for the Rapid Plasma Reagin Test for the Serological Diagnosis of Syphilis

We described the ASiManager-AT digital flocculation reader to demonstrate concordance between visual and digital readings of the rapid plasma reagin test for detection of antibodies in the serum of patients with syphilis. A qualitative and quantitative rapid plasma reagin was performed on each serum samples giving a concordance of 98.6% and 99.7%, respectively, for reactives and 100% for nonreactives.