Urinary Antigen Detection Assay Research Articles

Incidence, aetiology and serotype coverage for pneumococcal vaccines of community-acquired pneumonia in adults: a population-based prospective active surveillance study in Brazil

ObjectivesTo determine the incidence, aetiology and pneumococcal serotype distribution of community-acquired pneumonia (CAP) in Brazilian adults during a 2-year period.DesignProspective population-based surveillance study.SettingPatients from two emergency hospitals in Brazil were...

23-Valent Pneumococcal Polysaccharide Vaccination Does Not Prevent Community-Acquired Pneumonia Hospitalizations Due to Vaccine-Type Streptococcus pneumoniae.

Controversy exists regarding the clinical effectiveness of the 23-valent pneumococcal polysaccharide vaccine (PPSV23) for the prevention of serotype-specific community-acquired pneumonia (CAP). The objective of this study was to define the effectiveness of PPSV23 for the prevention of CAP hospitalizations due to vaccine-contained serotypes. This secondary analysis was a nested case–control, test-negative study design of adult patients hospitalized for CAP between 1 June 2014 and 31 March 2017. Cases included patients with CAP due to a S. pneumoniae serotype contained in the PPSV23. Urinary antigen detection of the 23 serotypes was performed. In the study, PPSV23 vaccination alone and no other pneumococcal vaccination was the primary exposure of interest. Vaccine effectiveness was calculated as (1-OR) × 100. Adjusted estimates were obtained from a logistic regression model that controlled for confounding variables. A total of 3686 patients were included in the analysis. The PPSV23 vaccination was documented in 608 (16%) patients, and the PPSV23-serotype CAP was detected in 48 (8%) PPSV23-vaccinated patients and in 288 (9%) non-vaccinated patients. Unadjusted vaccine effectiveness for preventing PPSV23-serotype CAP was 17% (95% CI: −13% to 40%). Adjusted estimates for preventing PPSV23-serotype CAP was 14% (95% CI: −17% to 38%). In this study, PPSV23 vaccination offered no protection against PPSV23-serotype CAP hospitalization in adults. This is the first PPSV23 vaccine effectiveness study from United States that utilized a urinary antigen detection assay as the main method for S. pneumoniae serotyping. This study highlights the need for more effective vaccines in the prevention of hospitalization due to S. pneumoniae CAP.

Epidemiology of community-acquired pneumonia in the era of extended serotype-covering multivalent pneumococcal conjugate vaccines

BackgroundSouth Korea has been providing 10-valent pneumococcal conjugate vaccine/(PCV10)/13-valent pneumococcal conjugate vaccine (PCV13) to children and 23-valent pneumococcal polysaccharide vaccine (PPSV23) to older adults as part of a national immunization program. MethodsFrom September 2015 to August 2017, a prospective cohort study was conducted for adults aged ≥19 years with community-acquired pneumonia (CAP) at four university hospitals. All-cause and pneumococcal CAP incidence and mortality rates were evaluated on the basis of hospital catchment population. Serotype distribution of pneumococcal CAP was also evaluated. ResultsAmong 2669 patients with CAP, 252 cases (9.4%) were pneumococcal CAP cases. The annual incidences of all-cause and pneumococcal CAP were 194.3 cases and 18.3 cases respectively, per 100,000 persons. Serotyped Streptococcus pneumoniae was identified in 107 cases (42.5%) through culture or a serotype-specific urinary antigen detection assay. Pneumococcal CAP caused by the PCV13 and PPSV23 serotypes were 50 cases (46.7% of serotyped pneumococcal CAP and 19.8% of pneumococcal CAP), and 83 cases (77.6% of serotyped pneumococcal CAP and 32.9% of pneumococcal CAP), respectively. The most prevalent serotype was 3 (n = 21, 19.6% of serotyped pneumococcal CAP), followed by 19A (n = 10, 9.3% of serotyped pneumococcal CAP) and 11A (n = 10, 9.3% of serotyped pneumococcal CAP). Compared with non-pneumococcal CAP patients, pneumococcal CAP patients were more likely to have a higher CURB-65 scores (P = 0.002). The overall 30-day mortality rate of pneumococcal CAP was higher than that of non-pneumococcal CAP (6.3% versus 5.6%; odds ratio [OR], 1.15; 95% confidence interval [CI], 0.67–1.96), but this trend was reversed in patients aged 65–74 years (4.2% versus 8.6%; OR, 0.47; 95% CI, 0.14–1.54). ConclusionsThe disease burden of PCV13-serotype pneumococcal CAP remains significantly high in Korean adults, particularly among elderly people, even after a high uptake of pediatric PCVs.

Qualification and Clinical Validation of an Immunodiagnostic Assay for Detecting 11 Additional Streptococcus pneumoniae Serotype-specific Polysaccharides in Human Urine.

BackgroundIdentifying Streptococcus pneumoniae serotypes by urinary antigen detection (UAD) assay is the most sensitive way to evaluate the epidemiology of nonbacteremic community-acquired pneumonia (CAP). We first described a UAD assay to detect the S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, covered by the licensed 13-valent S. pneumoniae conjugate vaccine. To assess the substantial remaining pneumococcal disease burden after introduction of several pneumococcal vaccines, a UAD-2 assay was developed to detect 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F) in individuals with radiographically confirmed CAP.MethodsThe specificity of the UAD-2 assay was achieved by capturing pneumococcal polysaccharides with serotype-specific monoclonal antibodies, using Luminex technology. Assay qualification was used to assess accuracy, precision, and sample linearity. Serotype positivity was based on cutoffs determined by nonparametric statistical evaluation of urine samples from individuals without pneumococcal disease. The sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation, using urine samples obtained from a large study that measured the proportion of radiographically confirmed CAP caused by S. pneumoniae serotypes in hospitalized US adults.ResultsThe UAD-2 assay was shown to be specific and reproducible. Clinical validation demonstrated assay sensitivity and specificity of 92.2% and 95.9% against a reference standard of bacteremic pneumonia. In addition, the UAD-2 assay identified a S. pneumoniae serotype in 3.72% of nonbacteremic CAP cases obtained from hospitalized US adults. When combined with bacteremic CAP cases, the proportion of pneumonias with a UAD-2 serotype was 4.33%.ConclusionsThe qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of nonbacteremic S. pneumoniae CAP and for assessing the efficacy of future pneumococcal conjugate vaccines that are under development.

Pneumococcal conjugate serotype distribution and predominating role of serotype 3 in German adults with community-acquired pneumonia

IntroductionImplementation of the 7-valent pneumococcal conjugate vaccine (PCV7) in infant vaccination programs has substantially reduced the burden of PCV7 serotypes also in adult community-acquired pneumonia (CAP). Currently, it is unclear, if this extensive herd protection effect can be extrapolated to the additional 6 serotypes included in the 13-valent pneumococcal conjugate vaccine (PCV13), which replaced PCV7 in Germany in 2010. ObjectivesWe investigated changing trends for PCV13 serotypes in adult CAP patients between three to seven years after implementation of PCV13 infant immunization in Germany. MethodsBetween December 2012 and January 2017, urine samples from German adult patients with radiologically confirmed CAP were prospectively collected by the multi-center cohort study CAPNETZ and analyzed by the serotype-specific multiplex urinary antigen detection assay (SSUAD) allowing for the detection of PCV13 serotypes. ResultsPCV13 serotypes were found in 59 of 796 (7.4%) patients with all-cause CAP, most prevalent was serotype 3 (30 of 59 patients, 50.8%). All patients with serotype 3-CAP were admitted to hospital and the majority required oxygen at admission (83.3% of patients with serotype 3-CAP versus 50.9% of patients with pneumococcal CAP by other serotypes, p = 0.005). Compared to SSUAD testing, conventional microbiological workup missed 27 of 30 (90.0%) serotype 3-CAP cases. We could not observe a time trend in the proportions of PCV13 serotypes and serotype 3 in all-cause CAP between 2013 and 2016 (OR trend per year 0.84, 95% CI 0.64–1.11 for PCV13 serotypes and OR trend per year 0.95, 95% CI 0.70–1.28 for serotype 3). Conclusions: Conventional methods underestimate serotype 3-CAP that can cause severe disease. Changes in overall PCV13 coverage were not detected during the years 2013 to 2016, mostly driven by a high proportion of serotype 3.

244. Development, Qualification, and Clinical Validation of an Immunodiagnostic assay for the Detection of 11 Additional S. pneumoniae Serotype-Specific Polysaccharides in Human Urine

BackgroundIdentifying Streptococcus pneumoniae (Sp) serotypes by urinary antigen detection assay (UAD) is the most sensitive and specific way to evaluate the changing epidemiology of non-bacteremic community-acquired pneumonia (CAP) and efficacy of pneumococcal vaccines. We first described an UAD to detect the Sp serotypes 1,-3,-4,-5,-6A,-6B,-7F,-9V,-14,-18C,-19A,-19F,-23F covered by the 13-valent Sp conjugate vaccine PCV13. To assess the pneumococcal disease burden of additional serotypes, a UAD-2 assay was developed to diagnose 11 additional Sp serotypes (-2,-8,-9N,-10A,-11A,-12F,-15B,-17F,-20,-22F,-33F).MethodsUAD-2 specificity was achieved by capturing highly purified pneumococcal polysaccharides with serotype-specific monoclonal antibodies using Luminex technology. Assay qualification assessed accuracy, precision, and sample linearity. Serotype positivity was based on cutoffs determined by non-parametric statistical evaluation of urine samples from individuals without pneumococcal disease. Clinical sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation.ResultsThe UAD-2 was shown to be specific and reproducible. Clinical validation using urine samples from invasive disease patients demonstrated assay sensitivity and specificity of 92.2% and 95.9%, respectively compared with a gold standard of isolating and typing (by Quellung) Sp bacteria from patient samples. Analysis of 11,087 CAP patients showed a UAD-2 and UAD-1 serotype prevalence of 4.33% and 4.60%, respectively (bacteremic and non-bacteremic CAP combined).ConclusionThe qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of nonbacteremic Sp CAP as well as assessing vaccine efficacy of future pneumococcal conjugate vaccines.Disclosures All authors: No reported disclosures.

Impact of pneumococcal vaccination in children on serotype distribution in adult community-acquired pneumonia using the serotype-specific multiplex urinary antigen detection assay

The aim of the study was to compare the distribution of the vaccine-serotypes covered by pneumococcal conjugate vaccines (PCV7 and PCV13) in adult patients with pneumococcal community-acquired pneumonia in Germany between the periods 2002–2006 and 2007–2011 using a novel serotype-specific multiplex urinary antigen detection assay (SSUA). Vaccination of children started with PCV7 in 2007, which was replaced by PCV13 in 2010. Following confirmation of the accuracy of SSUA in long-term stored urine samples from 112 patients with confirmed pneumonia and known pneumococcal serotype, urine samples of 391 CAPNETZ patients with documented pneumococcal pneumonia (i.e. positive BinaxNOW® Streptococcus pneumoniae urine antigen test) but unknown serotype were tested for the 13 vaccine-serotypes using SSUA. The proportion of PCV7-serotypes significantly decreased in adult patients with pneumonia from 30.6% (2002–6) to 13.3% (2007–11, p<0.001); in bacteremic pneumonia, PCV7-serotypes completely disappeared (3/14 versus 0/19, p=0.058). Conversely, pneumococcal serotypes included by PCV13 remained stable during study period with a coverage of 61.5% (2002–06) and 59.7% (2007–11) in non-bacteremic pneumonia and 79% (for both periods) in bacteremic pneumonia, mainly due to an increase in pneumococcal serotypes 1, 3 and 7F during the second period. Thus, implementation of PCV7 in children in Germany in 2007 was associated with a significant decrease in vaccine-serotypes covered by PCV7 in adult patients with non-bacteremic pneumococcal pneumonia and with an elimination of PCV7 vaccine-serotypes in bacteremic pneumococcal pneumonia. PCV13 coverage remained high up to 2011, mainly due to an increase in serotypes 1, 3 and 7F.German Clinical Trials Register: DRKS00005274.

New Pneumococcal Diagnostics---further ahead or more confused?

Invited Lecture AbstractsISPPD-0205 New Pneumococcal Diagnostics---further ahead or more confused?: USEFULNESS OF SEROTYPE SPECIFIC URINARY ANTIGEN DETECTION (UAD) ASSAY TO DETECT PNEUMOCOCCAL PNEUMONIA IN ADULTSW. Albrich1, M.W. Pride2, S.A. Madhi3, J. Callahan4, P.V. Adrian3, R. French5, N. van Niekerk6, S. Sebastian2, V. Souza2, K.U. Jansen2, K.P. Klugman31Division of Infectious Diseases and Hospital Epidemiology, Cantonal Hospital St. Gallen, St. Gallen, Switzerland; 2Pfizer Vaccine Research and Early Development, Pfizer Inc., Pearl River, USA; 3Respiratory and Meningeal Pathogens Research Unit/Medical Research Council, National Institute for Communicable Diseases, Johannesburg, South Africa; 4Callahan Associates Inc, Callahan Associates Inc, La Jolla, USA; 5Biotechnology Clinical Development Statistics, Pfizer Inc., Pearl River, USA; 6Respiratory and Meningeal Pathogens Research Unit/Medical Research Council, Respiratory and Meningeal Pathogens Research Unit/Medical Research Council, Johannesburg, South AfricaBackground and Aims: A urinary antigen detection assay (UAD) for 13 pneumococcal serotypes was recently reported as a useful diagnostic tool for pneumococcal pneumonia. We tested this assay in a well-defined cohort of HIV-infected South African adults with X-ray confirmed pneumonia.Methods: A Luminex xMAP bead-based multiplex UAD detecting the PCV13 serotypes was tested retrospectively in HIV-infected South African adults with pneumonia. UAD positivity cut-offs were established in HIV-infected South Africans without respiratory disease. Pneumonia was considered pneumococcal if a composite diagnostic including any of sputum Gram stain, sputum culture, blood culture or urinary Binax was positive.Results: The UAD identified 6/7 (85.7%) patients with bacteremia due to a PCV13 serotype and did not misclassify any of 3 patients with bacteremia due to a non-PCV13 serotype. Even though the UAD detects only 13 serotypes, it detected more cases of presumed pneumococcal pneumonia among X — ray confirmed cases than the serotype-independent urine Binax (106/235 (45.1%) vs. 52/235 (22.1%); p < 0.001). The UAD was positive in 52/71 (73.2%) patients with a positive composite diagnostic suggesting that 73% of disease was due to PCV13 vaccine types and overall detected significantly more cases than the composite (106/235 (45.1%) vs 71/235 (30.2%); p < 0.001). No correlation was detected between nasopharyngeal colonization density measured by lytA rtPCR and quantitative UAD values.Conclusions: The UAD compared more favourably with other diagnostic tests for pneumococcal pneumonia in adults and may become a new standard for detection of pneumococcal pneumonia in adults.Conflict of interestISPPD-0569 New Pneumococcal Diagnostics---further ahead or more confused?: DISSECTING THE COMPLEXITY OF CARRIAGE: MOLECULAR MINING BY MICROARRAYJ. Hinds11Bacterial Microarray Group, St George’s University of London, London, United KingdomNasopharyngeal carriage represents the natural host reservoir from which Streptococcus pneumoniae may achieve transmission to a new host or cause invasive disease. The biology of the pneumococcus has evolved for successful colonization of this complex niche, including strategies to avoid host defences and co-exist with other microbes. The dynamic microbial community of the nasopharynx is in constant flux and presents opportunities for genetic exchange, both impacting on the effectiveness of pneumococcal vaccines and antimicrobial resistance. Diagnostic methods have revealed key insights into carriage of co-colonizing species and pneumococcal serotypes. As nasopharyngeal carriage is a precursor to invasive pneumococcal disease and has a role in herd immunity, monitoring the effect of vaccines on carriage is especially important. In the era of polysaccharide conjugate vaccines, serotyping has been a key technique to study the impact upon circulating vaccine and non-vaccine serotypes. The microarray-based molecular serotyping method has shown high specificity and sensitivity for detection of multiple pneumococcal serotypes in carriage samples. Enhanced detection of additional serotypes at low abundance may affect metrics used in assessing vaccine efficacy, such as acquisition and duration of carriage, and also case:carrier ratios. Furthermore, the ability to detect serotype variants and non-typeables along with closely related species, antimicrobial resistance determinants and pneumococcal genotype provide valuable supplementary information. This combines to help dissect the complex nasopharyngeal niche and also contextualise an individual sample within other local or global studies.Conflict of interest

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Validation of an immunodiagnostic assay for detection of 13 Streptococcus pneumoniae serotype-specific polysaccharides in human urine.

To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres. Positivity for each serotype was based on positivity cutoff values calculated from a standard curve run on each assay plate together with positive- and negative-control urine samples. The assay is highly specific, since significant signals are detected only when each PnPS was paired with its homologous MAb-coated microspheres. Validation experiments demonstrated excellent accuracy and precision. The UAD assay and corresponding positivity cutoff values were clinically validated by assessing 776 urine specimens obtained from patients with X-ray-confirmed CAP. The UAD assay demonstrated 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. Importantly, the UAD assay identified Streptococcus pneumoniae (13 serotypes) in a proportion of individuals with nonbacteremic CAP, a patient population for which the pneumococcal etiology of CAP was previously difficult to assess. Therefore, the UAD assay provides a specific, noninvasive, sensitive, and reproducible tool to support vaccine efficacy as well as epidemiological evaluation of pneumococcal disease, including CAP, in adults.

Identification and Diagnostic Utility of Leishmania infantum Proteins Found in Urine Samples from Patients with Visceral Leishmaniasis

Despite the clear need to control visceral leishmaniasis (VL), the existing diagnostic tests have serious shortcomings. Here, we introduce an innovative approach to directly identify Leishmania infantum antigens produced in vivo in humans with VL. We combined reverse-phase high-performance liquid chromatography (RP-HPLC) with mass spectrometry and categorized three distinct L. infantum proteins presumably produced in bone marrow/spleen/liver and excreted in the urine of patients with VL. The genes coding for these proteins (L. infantum iron superoxide dismutase, NCBI accession number XP_001467866.1; L. infantum tryparedoxin, NCBI accession number XP_001466642.1; and L. infantum nuclear transport factor 2, NCBI accession number XP_001463738.1) were cloned, and the recombinant molecules were produced in Escherichia coli. Antibodies to these proteins were produced in rabbits and chickens and were used to develop a capture enzyme-linked immunosorbent assay (ELISA) designed to detect these L. infantum antigens in the urine of VL patients. Specificity of the antibodies was confirmed by a Western blot analysis using both recombinant proteins and whole parasite extract. Importantly, a urinary antigen detection assay assembled with pairs of antibodies specific for each of these antigens identified 17 of 19 patients with VL. These results indicate that an improved antigen detection assay based on L. infantum proteins present in the urine of patients with VL may represent an important new strategy for the development of a specific and accurate diagnostic test that has the potential to both distinguish active VL from asymptomatic infection and serve as an important tool to monitor therapy efficacy.

Evaluation of Streptococcus pneumoniae urinary antigen test in healthy children with nasopharyngeal pneumococcal carriage

We evaluated the influence of colonization with pneumococci on the results of Streptococcus pneumoniae urinary antigen detection assay by testing 23 healthy children aged 1-3 years in one nursery. Nasopharyngeal swab specimens for culture and urine samples for antigen detection test were obtained. 7 of 12 children who were nasopharyngeal carriers of pneumococci had a positive result of the urine antigen test. 3 of 11 children without pneumococci in the nasopharynx also had a positive result of the urine antigen test, who were diagnosed as having acute pneumonia within one month before this study. Thus we found that 58.3% of the children with pneumococcal carriage and 27.3% of non-carriers had false-positive test results. This test is not likely to be useful for diagnosing the etiology of childhood acute pneumococcal pneumonia.

Characterisation of Legionella pneumophila antigen in urine of guinea pigs and humans with Legionnaires' disease

To study and develop the urinary antigen detection assay, urine was obtained from aerosol infected guinea pigs. The appearance of Legionella pneumophila antigen in guinea pig urine was then compared with that detected in urine from human Legionnaires' Disease (LD) cases. This study demonstrated that the antigen expressed in the experimental model of LD was of identical molecular weight, reacted almost identically in an ELISA based assay and in Western blots with the antigen found in human urine. The guinea pig urine can therefore be used as a reliable, consistent and controllable source of antigen for use in the urinary antigen assay.