Syphilis Assay Research Articles

Current trends in donor testing to detect syphilis infection.

Potential organ and tissue donors are tested to detect infection with T. pallidum, the etiologic agent of syphilis. Important considerations for testing potential donors include available specimen type and volume, turnaround time, and ability to distinguish between past and current infection. Aspects of syphilis infection that inform organ and tissue donor assay selection and interpretation and the principles underlying available assays are described. Serologic assays for syphilis are the methods most commonly used in donor testing. The two categories of serologic assays, treponemal and nontreponemal, have advantages and limitations for testing potential donors. Knowledge of the common syphilis-testing algorithms used in clinical diagnostic testing is useful for assay selection in the organ and tissue donor setting.

Plasmonic ELISA for the ultrasensitive detection of Treponema pallidum

In this report, we have developed a plasmonic ELISA strategy for the detection of syphilis. Plasmonic ELISA is an enzyme-linked immunoassay combined with enzyme-mediated surface plasmon resonance (SPR) of gold nanoparticles (AuNPs). Immune response of the Treponema pallidum (T. pallidum) antibodies triggers the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine to produce abundant thiocholine. The positive charged thiol, in turn, alters the surface charge distribution the AuNPs and leads to the agglomeration of the AuNPs. The induced strong localized SPR effect of the agglomerate AuNPs can, thus, allow the quantitative assay of T. pallidum antibodies due to the remarkable color and absorption spectral response changes of the reaction system. The plasmonic ELISA exhibited a quasilinear response to the logarithmic T. pallidum antibody concentrations in the range of 1pg/mL–10ng/mL with a detection limit of 0.98pg/mL. Such a low detection limit was 1000-fold improvements in sensitivity over a conventional ELISA. The results of plasmonic ELISA in syphilis assays of serum specimens from 60 patients agreed with those obtained using a conventional ELISA method. The plasmonic ELISA has characteristics (analyte specific, cost-effective, ease of automatic, low limit of detection) that provide potential for diagnosis and therapeutic monitoring of syphilis.

Syphilis detection using the Siemens ADVIA Centaur Syphilis treponemal assay

BackgroundTreponemal tests for detecting syphilis should be sufficiently sensitive and specific, especially when used as the first-line method in reverse-algorithm testing. We compared the Siemens ADVIA Centaur® Syphilis assay to 2 other commercial assays in use by the Star-MDC laboratory to evaluate its performance and usability. MethodsAgreement between the Siemens ADVIA Centaur Syphilis assay, Siemens IMMULITE® 2000 Syphilis Screen, and Biokit bioelisa Syphilis 3.0 assay was evaluated using 1251 patient samples (50 from known positives, 701 from patients referred for syphilis testing, and 500 from pregnant women). Reactive samples (i.e., reactive according to at least two of the three treponemal methods) were further evaluated using Western blot IgG and IgM, and Venereal Disease Research Laboratory (VDRL) testing. ResultsOverall, positive and negative agreement was 100% between the Centaur and IMMULITE assays. In this study, overall agreement was 99.92% between either of the Siemens assays and the Biokit assay; positive agreement was 99%, and negative agreement was 100%. Overall, 0.88% (11/1251) of the samples were interpreted as positive/reactive based on the combined positive results by the ADVIA Centaur, IMMULITE 2000, and bioelisa assays; a positive Euroline anti-Treponema pallidum IgM blot; and a VDRL result of ≥1:8. In this study, no false-reactive samples were identified using this method. ConclusionThe Centaur Syphilis assay performance is comparable to the other 2 commercial assays.

Clinical Prediction and Diagnosis of Neurosyphilis in HIV-Infected Patients with Early Syphilis

The diagnosis of neurosyphilis (NS) is a challenge, especially in HIV-infected patients, and the criteria for deciding when to perform a lumbar puncture (LP) in HIV-infected patients with syphilis are controversial. We retrospectively reviewed demographic, clinical, and laboratory data from 122 cases of HIV-infected patients with documented early syphilis who underwent an LP to rule out NS, and we evaluated 3 laboratory-developed validated real-time PCR assays, the Treponema pallidum particle agglutination (TPPA) assay, the fluorescent treponemal antibody absorption (FTA-ABS) assay, and the line immunoassay INNO-LIA Syphilis, for the diagnosis of NS from cerebrospinal fluid (CSF) samples of these patients. NS was defined by a reactive CSF-VDRL test result and/or a CSF white blood cell (WBC) count of >20 cells/μl. Thirty of the 122 patients (24.6%) had early NS. Headache, visual symptoms, a CD4 cell count of <500 cells/μl, and viremia, as defined by an HIV-1 RNA count of ≥50 copies/ml, were associated with NS in multivariate analysis (P = <0.001 for each factor). Blood serum rapid plasma reagin (RPR) titers were not associated with early NS (P = 0.575). For the diagnosis of NS, the PCR, FTA-ABS, TPPA, and INNO-LIA assays had sensitivities of 58%, 100%, 68%, and 100%, specificities of 67%, 12%, 49%, and 13%, and negative predictive values of 85%, 100%, 84%, and 100%, respectively. Visual disturbances, headache, uncontrolled HIV-1 viremia, and a CD4 cell count of <500 cells/μl were predictors of NS in HIV-infected patients with early syphilis, while blood serum RPR titers were not; therefore, RPR titers should not be used as the sole criterion for deciding whether to perform an LP in early syphilis. When applied to CSF samples, the INNO-LIA Syphilis assay easily helped rule out NS.

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

ObjectiveThe 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...

Rapid Multiplexed Immunoassay for Simultaneous Serodiagnosis of HIV-1 and Coinfections

Diagnosis of opportunistic infections in HIV-infected individuals remains a major public health challenge, particularly in resource-limited settings. Here, we describe a rapid diagnostic system that delivers a panel of serologic immunoassay results using a single drop of blood, serum, or plasma. The system consists of disposable cartridges and a simple reader instrument, based on an innovative implementation of planar waveguide imaging technology. The cartridge incorporates a microarray of recombinant antigens and antibody controls in a fluidic channel, providing multiple parallel fluorescence immunoassay results for a single sample. This study demonstrates system performance by delivering antibody (Ab) reactivity results simultaneously for multiple antigens of HIV-1, Treponema pallidum (syphilis), and hepatitis C virus (HCV) in a collection of clinical serum, plasma, and whole-blood samples. By plotting antibody reactivity (fluorescence intensity) for known positive and negative samples, empirical reactivity cutoff values were defined. The HIV-1 assay shows 100% agreement with known seroreactivity for a collection of 82 HIV Ab-positive and 142 HIV Ab-negative samples, including multiple samples with HCV and syphilis coinfection. The treponema-specific syphilis assay correctly identifies 67 of 68 T. pallidum Ab-positive and 100 of 102 T. pallidum Ab-negative samples, and the HCV assay correctly identifies 59 of 60 HCV Ab-positive and 120 of 121 HCV Ab-negative samples. Multiplexed assay performance for whole-blood samples is also demonstrated. The ability to diagnose HIV and opportunistic infections simultaneously at the point of care should lead to more effective therapy decisions and improved linkage to care.

Evaluation of a new Syphilis assay on Vitros® 5600 Integrated System

Introduction. A new homogeneous immunoassay for detection of primary infection of Treponema Pallidum (TP) on Vitros® 5600 Integrated System was evaluated.The scope of the study was to verify analytical performances and diagnostic accuracy in comparison to commercial methods (Immunoblotting test, ELISA test, Immunoturbidimetric test). Methods. The new Syphilis assay from SENTINEL CH. SpA, is an immunoturbidimetric assay, using microparticles coated with TP fixed on the surface of polystyrene latex particles which agglutinate by an antigen-antibody reaction when anti-TP antigen is present in the specimen. The assay was implemented on Vitros® 5600 Integrated System. Modified CLSI protocols were adopted. Acceptance criteria for total imprecision were 5% for negative samples (or SD 0.5 U/mL) and 4% for positive samples. In comparison to commercial methods, sensitivity must be 99.5% and specificity 99.5%. Results. Total imprecision (22 days) gave SD at 6 U/mL lower than 0.5 U/mL, and CV% at 10 U/mL and 45 U/mL lower than 4%. Low quantitation limit is 5 U/mL. No prozone up to 13000 U/mL was found. In the on-board calibration stability study no drift was found up to 4 weeks. 153 samples were tested vs immunoblotting method and specificity was 100%, sensitivity was 100%. 495 samples were tested vs ELISA method and test specificity and sensitivity were 99.6% and 100% respectively. 521 samples were tested vs immunoturbidimetric method and specificity was 99.8%, sensitivity was 100%. Interference from Bilirubin (20 mg/dL), Hemoglobin (500 mg/dL) and Triglycerides (1000 mg/dL) was not detected.All the sample collection tubes tested (K2EDTA, SST, LH PST II, LH, NH) did not interfere with the assay. Conclusion. Performances of the new SENTINEL Syphilis assay on Vitros® 5600 Integrated System meet the requirements for its use as screening tool in blood bank, thus allowing consolidation with general chemistry on a single high volume chemistry analyzer, which is highly valuable for optimizing workflow and efficiency in today’s laboratories.

A 44‐year‐old woman with cutaneous bullae and extensive skin necrosis

Five weeks before presentation, the patient had experi-enced a cough and mild dyspnea. She was diagnosed withpneumonia, completed a 7-day course of ciprofloxacin anddoxycycline, and her respiratory symptoms resolved.Three weeks before presentation, the patient had reportedfevers, chills, diaphoresis, arthralgias, and myalgias, anddeveloped a small erythematous lesion on her left breast.The lesion subsequently enlarged and became hemor-rhagic. She simultaneously developed additional lesionson her chest, abdomen, arms, and back. The largest ofthese, located on her left arm, was 15 cm in diameter. Allof the lesions were exquisitely tender. She was hospital-ized at an outside hospital for 5 days and given a diagnosisof cocaine-induced vasculitis. Upon discharge, she wasinstructed to abstain from cocaine and was prescribedtrimethoprim/sulfamethoxazole for a possible cellulitis in-volving the skin lesions.Ten days after discharge from the outside hospital, thepatient presented to our institution for pain managementand further evaluation. The rash had progressed since herearlier discharge. She had continued to use cocaine, her-oin, and prescription opioids. She had last smoked co-caine 3 days before presentation and last used intranasalheroin 1 week before admission. She reported acquiringcocaine from a new supplier 2 weeks before her rash hadbegun.

Levamisole Adulterated In Cocaine Causing Agranulocytosis, Vasculopathy and Acquired Protein S Deficiency

AbstractAbstract 1421More than 2 million Americans use cocaine each month. Levamisole is a veterinary antihelminthic agent that has been used as a cutting agent in cocaine and adds bulk to powdered cocaine. It has been used for the treatment of colon cancer, rheumatoid arthritis and nephrotic syndrome. According to the Drug Enforcement Administration (DEA) as of July 2009, 69% of seized cocaine lots coming into the United States contained levamisole as an added agent. Levamisole has recently been reported to cause neutropenia in cocaine abusers. We report a case of a severe cutaneous necrotizing vasculopathy, agranulocytosis with acquired protein S deficiency caused by tainted cocaine in an active abuser.A 44-year-old active cocaine abusing woman presented with a day of acute worsening cutaneous lesions, subjective fever and arthralgias. She last inhaled three bags of cocaine the day prior to admission. On examination, she had multiple tender palpable purpuric plaques with central necrosis on her thighs, lower extremities, chest, abdomen, arms, and bilateral ear helices. Urine toxicology screen was positive for cocaine. Laboratory data showed neutropenia of 1.4 × 103/uL with mild anemia (haemoglobin 12g/dl) and normal platelet count. She had anemia of chronic disease with negative Coombs test. She also has normal kidney and liver functions. Vasculitides and rheumatological workup showed positive antineutrophil cytoplasmic antibodies (ANCAs) directed against proteinase-3 (PR-3) and myeloperoxidase (MPO) but negative for ANA or rheumatoid factor and normal complement levels. Assays for syphilis, hepatitis B and C, and the human immunodeficiency virus were negative. A skin biopsy showed small vessel thrombo-vasculopathy with organizing thrombus and mixed cell infiltrate. Subsequent workup for hypercoagulable states revealed a positive lupus anticoagulant and a low protein S activity. The patient was treated with steroids. Her skin lesions and neutropenia improved gradually in two weeks. [Display omitted] [Display omitted] [Display omitted] The acute onset of necrotic skin lesions following cocaine use, ANCA positivity, histopathology and neutropenia strongly suggest levamisole-induced vasculopathy. Although levamisole levels were not obtained, exposure from cocaine contamination is very likely. Levamisole is known to have immunostimulating effects with the production of autoantibodies like ANCA. Life-threatening agranulocytosis and a positive lupus anticoagulant have been reported in a few cases of prolonged levamisole use. The pathogenesis of agranulocytosis is hypothesized to either be a direct toxin effect or immune-mediated. Lupus anticoagulant has also been reported to cause a similar pattern of cutaneous necrosis. Clinicians should be aware of this kind of clinical presentation in active cocaine abusers and consider it as a significant emerging public health problem. Disclosures:No relevant conflicts of interest to declare.

Balancing agendas in donor testing

Balancing agendas in donor testing

Serology of Treponema pallidum infection: methodological considerations and seroprevalence in selected Italian populations

An increase of syphilis incidence in recent years has been reported throughout Europe and also in Italy. The advent of automated serological assays for the detection of anti-Treopnema pallidum antibodies increases the sensitivity and the reproducibility of antibody detection: one of such methods (the chemiluminescent Architect Syphilis TP assay) has been evaluated in a multisite study, that involved 22 Italian laboratories, on 62,036 serum specimens obtained from blood donors (n= 44,884), from hospital in- or outpatients at low (n= 16,482) or high (n= 284) risk for treponemal infection and from 386 patients already diagnosed with syphilis at different stages. The Architect Syphilis assay showed a good reproducibility both on controls (718 replicates, CV of 14.4% on negative and of 12.7% on positive control) and on clinical specimens (93 samples, CV of 3.61%). The sensitivity on syphilis cases was 99.73% compared to 99.43% by other EIAs, and the specificity on 44,898 blood donors was 99.67%.The positive predictive value was related to the signal of the Architect assay (4.3% for sample to cutoff (S/CO) values 3). The positivity rates for anti-Treponema pallidum antibodies were 0.15% in blood donors, 3.46% among low risk subjects and 16.90% among patients at high risk for syphilis infection. The high prevalence in low risk groups enhances the need for a surveillance of syphilis, that appears to increase in the general population, with sensitive first-level assays. An appropriate algorithm for confirmation of low positive results by Western blot testing should be envisioned.

The Architect Syphilis assay for antibodies to Treponema pallidum: an automated screening assay with high sensitivity in primary syphilis

Objectives:To determine the sensitivity and specificity of the Architect Syphilis Chemiluminescence Assay (CLIA): a new highly automated screening test for syphilis.Methods:To establish the sensitivity of the Architect Syphilis assay we...

The sensitivity of syphilis assays in detecting different stages of early syphilis

Our aim was to determine the sensitivity of the Murex ICE enzyme immunoassay (EIA) as a screening test for early syphilis and to determine how many additional cases of infection were detected by performing additional tests when requested on clinical grounds. This was an observational study on consecutive patients diagnosed with syphilis in the Department of Genitourinary Medicine, Edinburgh between January 1st 2004 and April 1st 2005. Additional tests were performed on sera that gave a positive or equivocal EIA on screening as well as by clinical request on sera from contacts of syphilis, and those with clinical signs of syphilis. Additional tests included a Venereal Diseases Research Laboratory (VDRL) carbon antigen test, a Treponema pallidum particle agglutination (TPPA) test, INNO-LIA line immunoblot assay, and an EIA specific for anti-treponemal IgM.A total of 105 patients were diagnosed with syphilis: primary (50), secondary (26), early latent (8), and of unknown duration (21). The TPPA was the most sensitive test in primary syphilis and had a sensitivity of 96% (48/50), which was significantly higher (P <0.05) than the sensitivity of 84% (42/50) for the screening EIA: seven of the EIA negatives were detected by EIA-IgM, six by TPPA, five by immunoblot, and two by VDRL. EIA-IgM was negative in six primaries; all were positive by TPPA and immunoblot. We conclude that, in order to maximize the serological detection of primary syphilis a specific EIA-IgM test and a TPPA test should be performed whenever there is a clinical suspicion of primary infection. This is particularly important when an EIA such as Murex ICE is used as a single screening test as it is less sensitive than the TPPA in primary infection.

Managing Syphilis in the HIV-infected Patient.

Syphilis has re-emerged in the United States and elsewhere, and clinicians caring for HIV-infected patients are challenged with syphilis diagnosis and management decisions. HIV alters the natural history of syphilis to an extent that is poorly understood, and initial presentation may be more varied in coinfected patients. Although commonly available diagnostic assays for syphilis should be interpreted as usual, such tests rely on antibody measurement and may be an imperfect indicator of active infection. Assessment of all available clinical and risk behavior data remains critically important in the diagnosis of syphilis in coinfected patients. Treatment of syphilis in such patients requires stage-appropriate therapy, with careful serologic monitoring to assess response. Clinicians must have heightened appreciation of the role of frequent risk assessment, serologic screening, symptom recognition, and follow-up of treated patients, as well as an understanding of public health functions such as sex partner treatment and communicable disease reporting.

Evaluation of INNO-LIA syphilis assay as a confirmatory test for syphilis.

We evaluated the sensitivity and specificity of a new confirmatory test for treponemal antibodies, INNO-LIA Syphilis (Innogenetics NV, Ghent, Belgium), on a large number of sera from a clinical laboratory. This multiparameter line immunoassay (LIA) uses recombinant and synthetic polypeptide antigens derived from Treponema pallidum proteins. In a single-blinded cross-sectional retrospective study, 289 seronegative sera, 219 seropositive sera, and 23 sera with an indeterminate serological status for syphilis were analyzed. All sera were tested by the T. pallidum hemagglutination assay (TPHA), the immunoglobulin (IgG)-fluorescent T. pallidum absorption assay (IgG-FTA-ABS), and the Venereal Disease Research Laboratory (VDRL) test. In addition, some seropositive samples were analyzed by the 19S-IgM-FTA-ABS test, an enzyme immunoassay (IgM-EIA), and the MarDx immunoblotting assay. Based on a consensus diagnosis derived from conventional serology, all of the sera were classified as positive, negative, or indeterminate, and the results were compared with the findings of the INNO-LIA Syphilis assay. The sensitivity and specificity of the LIA were 100% (219 of 219) and 99.3% (286 of 288), respectively. Compared to TPHA and IgG-FTA-ABS, the new test gave a significantly higher number (P = 0.021 and P < 0.0001, respectively) of correct results than either of the other two tests. The multiparameter INNO-LIA Syphilis assay is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques.

Enzyme-linked immunospot assay for the diagnosis of active Treponema pallidum infection during the various stages of syphilis.

Specific serologic assays for syphilis cannot differentiate current infections from past infections and are inefficient to monitor efficacy of antibiotic therapy. To develop a new immunologic assay for the identification of active Treponema pallidum infection during the various stages of syphilis. Peripheral blood mononuclear cells obtained from patients with syphilis in an STD clinic were tested for T. pallidum-specific circulating antibody-secreting cells (ASC) by an enzyme-linked immunospot assay (ELISPOT). Specific ASC were demonstrated in all six patients with primary syphilis and in 14 of 16 patients diagnosed with secondary syphilis. ASCs were undetectable in five patients 8 to 16 days after appropriate therapy, but persisted in one case that was considered treatment failure. Among the 13 patients diagnosed with latent syphilis, six (46%) demonstrated ASC, reflecting antigenic stimulation. The ELISPOT assay is effective for the diagnosis of primary and secondary syphilis. The presence of circulating ASC suggests persistent active infection in some patients during the latent disease stage.

Novel recombinant-antigen enzyme immunoassay for serological diagnosis of syphilis.

Enzyme immunoassay (EIA) is an ideal method for screening large numbers of patients for syphilis. We evaluated a novel immune-capture EIA (ICE Syphilis; Murex Diagnostics) that uses three recombinant Treponema pallidum antigens (TpN15, TpN17, and TpN47) and compared the results with those obtained by the native T. pallidum antigen EIA (Captia SelectSyph-G; Centocor) that we currently use for the serodiagnosis of syphilis. Specificity was evaluated by screening 1,184 unselected serum specimens in parallel by the ICE Syphilis and SelectSyph-G assays, while sensitivity was tested with a panel of 101 serum specimens containing antitreponemal antibodies (treated and untreated) from patients with various stages of infection. The specificity of the ICE Syphilis EIA (99.8%) on screening was significantly higher (P < 0.02) than that of the SelectSyph-G EIA (99.2%). The sensitivity of the ICE Syphilis EIA was significantly higher (P < 0.01) than that of the SelectSyph-G EIA on both initial (99 versus 91.4%) and repeat (100 versus 92.4%) testing. The ICE Syphilis EIA was also significantly more sensitive (P < 0.01) than the fluorescent treponemal antibody-abs (92.4%) but not the T. pallidum hemagglutination assay (97.1%). Sera containing antitreponemal antibodies gave a much higher antibody index (absorbance of test serum/kit cutoff) by the ICE Syphilis EIA than by the SelectSyph-G EIA. This combined with the overall high sensitivity makes the ICE Syphilis EIA an ideal test for excluding or detecting treponemal infection in human immunodeficiency virus (HIV)-infected patients. The ICE Syphilis EIA was positive with sera from all 15 HIV-infected patients in the study, whereas sera from 3 HIV-infected patients were negative by the SelectSyph-G EIA. We conclude that the high sensitivity and specificity of the ICE Syphilis EIA and its suitability for automation make it an ideal screening test.

Recurrent disseminated intravascular coagulation and fulminant intra hepatic thrombosis in a patient with the anti‐phospholipid syndrome

We describe a patient with the lupus anti-coagulant who had recurrent episodes, over a 2 year period, of a severe and disseminated intravascular coagulopathy. This patient also had positive serological assays for syphilis and anti-cardiolipin antibodies. Associated with the coagulopathy were co-expressed episodes of liver disease, ultimately terminating in fulminant liver failure. At autopsy the features were characteristic of the Budd-Chiari syndrome. This is the first report to document how consumptive coagulopathy may present as a dominant feature of the anti-phospholipid syndrome. It also clearly describes an immune mediated thrombotic mechanism as a cause of hepatic veno-occlusive disorders. Furthermore, this case highlights the varied clinical spectrum of the anti-phospholipid syndrome and suggests that a high index of suspicion is required to ensure its diagnosis.

A lack of justification for routine screening assays for syphilis in general hospital psychiatric patients

Of 136,035 Yale-New Haven Hospital patients who had screening tests for syphilis, 1.2% were VDRL (Venereal Disease Research Laboratory) positive, and 0.6% were FTA-Abs (Fluorescent Treponemal Antibody Absorption) positive. Of this total sample, 2037 were psychiatric inpatients, 15 of whom were VDRL positive (0.7%), and only 4 (0.2%) were FTA-Abs positive. The authors discuss the merits of routine screening based on this and other surveys. The results of screening the psychiatric subsample were of particular interest since these patients might be at risk for misdiagnosis and brain dysfunction. Of this psychiatric inpatient cohort: (1) the number of syphilitics identified was 3.8-fold lower than that in the total sample, (2) strong consideration could be given to screening only those DSM-III ( Diagnostic and Statistical Manual of Mental Disorders [ed 3]) diagnostic subgroups at risk, and (3) these were major affective disorders or delusional dementia. Further studies of other samples, psychiatric and nonpsychiatric, to define subgroups at risk are suggested.