Preliminary Evaluation of an lytA PCR Assay for Detection of Streptococcus pneumoniae in Urine Specimens from Hospitalized Patients with Community-Acquired Pneumonia

Streptococcus pneumoniae is an encapsulated, Gram-positive coccoid shaped, facultative anaerobic bacterium. In addition, S. pneumoniae is alpha-hemolytic under aerobic conditions and beta-hemolytic under anaerobic conditions. It can be distinguished from other Streptococcus species using optochin and bile susceptibility tests, although there are exceptions. The organism is somewhat fastidious, and thus only 20-30% of pneumococcal pneumonia cases are blood culture positive [1, 2]. Due to its fastidious nature and other factors, such as autolysin production in the stationary growth phase [3, 4], culture remains difficult. There are 90 serotypes of S. pneumoniae, but a much smaller subset of these cause significant human disease, making vaccine development a successful endeavor in reducing disease in at-risk populations [5, 6].

Reportable Diseases and Conditions list, and tracking drug resistance patterns remains important. As of 2013, the ABCs report indicated that among 2898 isolates of S. pneumoniae, 2.2% were resistant to penicillin, 28.2% were resistant to erythromycin and 10.3% were resistant to tetracycline [8].
Laboratory diagnosis remains challenging for S. pneumoniae, however. Available diagnostic testing methods fall into three major categories at this time, including microscopy with microbiological culture, urine antigen detection assays and molecular testing such as PCR on cerebrospinal fluid (CSF), sputum or whole blood; however, no gold standard has been agreed upon. Molecular diagnostic testing is attractive, due to increased speed and sensitivity. One promising PCR target is the lytA gene of S. pneumoniae because the lytA gene is specific to S. pneumoniae [1]. This gene encodes a well-documented virulence factor and has little genetic variation within the species [10].
While lytA has been published as a promising PCR target for whole blood, sputum, serum samples, middle ear fluid and CSF specimen types [1,2], it has not been tested extensively on urine samples. Our aim was to assess the feasibility of fully validating a rapid and sensitive PCR assay on the Luminex ARIES ® platform using a non-invasive specimen type in order to detect S. pneumoniae from patients with a clinical diagnosis of pneumonia. The design of this study was to use archived urine samples from hospitalized patients with Community-acquired pneumonia (CAP) [11] for PCR analysis using the lytA gene target with Multicode ® primers. Because previous laboratory results were available for these cases, the selected urine samples were either from patients with S. pneumoniae blood culture positive results or from patients, who by laboratory analysis, were negative for S. pneumoniae. In addition, urine samples from healthy volunteers were used as negative controls for this study. Preliminary results are promising for this assay and warrant further research and development.

Methods and Materials
Archived Urine Specimens: Sixty archived urine specimens were obtained from the University of Louisville Respiratory Biorepository in the Division of Infectious Diseases. Urine specimens selected for this study were collected between 2014-2016 [11]. Briefly, the original urine samples from patients hospitalized with Community-acquired pneumonia (CAP) were held at 4°C, then processed within 28 hours of collection by adding 0.5 mL of 0.5M PIPES buffer (VWR, Radnor, PA, Part Number BB-121-250 mL) to 9.5 mL of urine. After mixing well, aliquots were frozen at -80°C in the Biorepository until thawed once for lytA PCR and BinaxNOW ® S. pneumoniae antigen card testing. Aliquots of 200 μL of archived urine samples were used from 30 pneumococcus negative patients (having no laboratory evidence of Streptococcus pneumoniae infection) and 30 patients having Streptococcus pneumoniae blood culture positive results.
Fresh Urine Specimens (Negative Controls): An additional 30 fresh urine samples were collected from healthy volunteers. These urine samples were collected and stored at 4°C less than 28 hours prior to processing by adding 0.5 mL of 0.5M PIPES buffer to 9.5 mL of urine. They were tested first after refrigerating, and again after freezing once at -20°C for 1-4 weeks, with no change in lytA PCR or BinaxNOW ® test results. ARIES ® Instrument, ReadyMix ® , Cassettes and PCR Analysis: The universal thermal profile settings on the ARIES ® instrument were used for PCR. Cassettes (Part Number 50-10026) and ReadyMix ® (Part Number 3697) were purchased from Luminex (Austin, TX). The ARIES ® software automatically generated the amplification and melt curves and assigned the corresponding cycle threshold (Ct) and melting temperature (Tm) values. Specimens were considered positive only if lytA and the specimen processing control Mouse Hepatitis Virus-2 (MHV2) were detected in the Ct and Tm ranges established for this assay (see Table 1). A sample was called "Invalid" if the MHV2 amplification failed.
Reagents: MultiCode ® primer pairs for the Streptococcus pneumoniae lytA gene were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). The MultiCode ® primer sequence for lytA forward primer is /56-FAM//iMe-isodC/A CGC AAT CTA GCA GAT GAA GCA and the reverse primer is CTC CCT GTA TCA AGC GTT TTC GGC, based on a previous publication [1]. The MHV2 primer set (proprietary primer sequences, Luminex, Part Number 3803) was used as the sample processing control (SPC) in order to confirm that PCR inhibitors were not present. Furthermore, melt curve (Tm) data allows for calibration of detected melting temperatures for both lytA and MHV2 PCR amplifications. The lytA primer set was assigned to the FAM channel and MHV2 primer set was assigned to the AP525 channel.
Primer Pool Preparation: 50 μL of 5 µM lytA forward primer, 50 μL of 5 µM lytA reverse primer and 100 μL of MHV2 primer set were mixed together. Frozen aliquots of the primer mix were stored at -20°C. From these aliquots, 4 μL of the primer pool (giving a final concentration of 200 nM) were used per ReadyMix ® vial.
Control Material: Titered Streptococcus pneumoniae (ZeptoMetrix, Buffalo, NY, Part Number 0801439) was purchased and used for accuracy and analytical sensitivity studies by diluting it in negative urine specimens at various concentrations. Urine samples were screened by the BinaxNOW ® S. pneumoniae antigen card and lytA PCR assay prior to use to ensure specimens were negative by both methods.
Accuracy: For the accuracy study, 11 known negative urine samples and 16 known positive samples, made up of various concentrations of Streptococcus pneumoniae that were spiked into negative urine (screened by BinaxNOW ® S. pneumoniae antigen card and lytA PCR assay). Samples were blinded and ULJRI Vol 1, (4) 2017

Clinical Criteria
Invasive Pneumococcal (Streptococcus pneumoniae) Disease, or IPD, causes many clinical syndromes, depending on the site of infection (e.g., bacteremia, meningitis.)

Laboratory Criteria for Diagnosis
Supportive: Identification of S. pneumoniae from a normally sterile body site by a culture independent diagnostic test [CIDT] without isolation of the bacteria. Confirmatory: Isolation of S. pneumoniae from a normally sterile body site.

Comments
The use of CIDTs as stand-alone tests for the direct detection of S. pneumoniae from clinical specimens is increasing. Data regarding their performance indicate variability in the sensitivity, specificity, and positive predictive value of these assays depending on the manufacturer and validation methods used. It is therefore useful to collect information on the laboratory conducting the testing, and the type and manufacturer of the CIDT used to diagnose each IPD case. Culture confirmation of CIDTpositive specimens is still the ideal method of confirming a case of IPD.
tested by staff members that did not prepare the accuracy panel.
Box 1 Council of State and Territorial Epidemiologists (CSTE) Case Definition [12] Data Analysis: Mean and standard deviation were calculated using Microsoft Excel. The MEDCALC Diagnostic Test Evaluation tool was used to determine analytic sensitivity and specificity [13]. Luminex SYNCT ® software was used to analyze the PCR data. The highest Ct value for an lytA true positive was 40.4, so the lytA target was set at 40.5 ( Table 1). The mean of the Tm for an lytA true positive was 79.21°C, and was used as the melt curve window ± 2, and 20% of deepest Tm Δ RFU (change in relative fluorescence units) was set as Tm peak threshold cutoff ( Table 1). The SPC MHV2 cutoff was set the same way as the S. pneumoniae lytA. After completing PCR assay data analysis, cut off values were fixed on the SYNCT ® software ( Table 1) assay file and locked on the ARIES ® instrument for subsequent specimen testing. Alere BinaxNOW ® S. pneumoniae Antigen Card: For this study, 90 urine specimens were tested by BinaxNOW ® , 30 from each group described in "Specimens" section. Urine samples were screened following the manufacturer's instructions [14]. Briefly, the swab provided with the Alere kit was dipped into the urine specimen to be tested, the swab was placed in the BinaxNOW ® device, and three drops of reagent were added to swab. Result was read at 15 minutes. Per the package insert (14), the limit of detection if 10 5 cells/mL or 1:250 dilution of a known positive patient urine.

Results
The analytical sensitivity for Streptococcus pneumoniae in urine with this PCR assay was 10 CFU/mL. Results are represented in Table 2 as mean ± standard deviation, calculated from three independent experiments. No mean and standard deviation were calculated for the 1 CFU/mL concentration, which was only positive in one out of three runs (33%). Inter-assay precision, as illustrated by the small standard deviations for Ct values and Tm values ( Table 2), was excellent. There was little variation among three independent runs at the 1000 CFU/mL, 100 CFU/mL and 10 CFU/mL dilutions.  The accuracy of the assay was tested using a blinded panel of negative urine samples spiked with known quantities of S. pneumoniae. There were no false negative results, but one apparent "false" positive result. This was from a sample spiked with 1 CFU/mL, which is below the established LOD, but S. pneumoniae was present in sample. We found that this level could be detected 33% of the time ( Table 2). Based on results shown in   Specificity studies were not done in this preliminary study due to lytA PCR specificity data being available from three previously published studies also using the lytA target used in this report. Carvalho et al. [1] used an extensive strain collection from the CDC Streptococcus Laboratory to assess specificity of the lytA primer set. Carvalho demonstrated that using 5 ng/µL DNA extracted from 67 strains, representing 44 different serotypes of S. pneumoniae isolates, and 104 non pneumococcal isolates, there was 100% specificity for S. pneumoniae. In another study demonstrating specificity, Sheppard et al. [2] used a panel of 59 isolates including 18 species of streptococci and 21 other closely related species or common pathogens and found no crossreactivity with an lytA primer set. Finally, McAvin et al. used 44 organisms from diverse genera and 26 negative control organisms, including 7 Streptococcus species, and also observed 100% specificity with no cross-reactivity with an lytA PCR assay [10].

Discussion
The goal of this preliminary study was to assess the feasibility of fully validating the performance of an lytA PCR assay in urine specimens from patients diagnosed with CAP [11] for use in a future, prospective study. The lytA target was chosen due to several publications indicating high specificity of the autolysin gene for S. pneumoniae [1]. Using the Luminex ARIES ® platform that automates DNA extraction, PCR amplification and real-time detection, we found that the MultiCode ® lytA assay could reliably detect 10 CFU/mL in urine. Accuracy studies using negative urine samples spiked with known quantities of S. pneumoniae demonstrated 16/16 (100%) lytA positive for known positive samples and 10/11 (90.9%) lytA negative for known negative samples. With archived clinical samples, we observed 43.3% lytA positive urine samples from S. pneumoniae blood culture positive CAP patients and 10% lytA positive urine samples from CAP patients with no laboratory evidence of S. pneumoniae infection. None of the urine samples from 30 healthy volunteers were positive for lytA. All 90 urine specimens were tested by the BinaxNOW ® S. pneumoniae antigen card test as well as by PCR after one freeze-thaw cycle. The only group with any positive BinaxNOW ® S. pneumoniae antigen results was the blood culture positive patients. The BinaxNOW ® S. pneumoniae antigen card did not perform as well as stated in the package insert [14] in our study, however. The urine antigen detection assay BinaxNOW ® for S. pneumoniae has a stated sensitivity of 86% and specificity of 94% [14] in adults, based on a retrospective study of urine specimens from blood culture positive patients. The package insert does not specify either the length of time or freezer temperature storage conditions that ensure the test results are valid for urine, thus it is possible that long-term storage at -80°C affected assay performance. As noted in our results, there were five discrepant results out of 30 CAP patients with S. pneumoniae positive blood cultures, but overall the two assays detected similar numbers of positive results in this group ( Table 4). Three of these were lytA positive with the lytA PCR assay, but BinaxNOW ® negative, while two patients were lytA negative but BinaxNOW ® positive. Another explanation for the discrepant results (besides long-term storage at -80°C) is that the targets of each assay are different (DNA versus antigens). Further study of these patient specimens would be required to truly understand these interesting differences, as well as completing a prospective study with similar groups using fresh urine specimens rather than long-term frozen urine specimens.
To place these results in context of other standard laboratory diagnostic tests, only 15-30% of patients with CAP due to S. pneumoniae have positive blood cultures [1,2]. While numerous lytA PCR assays have been reported for use with multiple specimen types, urine has not been evaluated extensively. Whole blood was evaluated in one study for patients with either pneumococcal septicemia or with pneumococcal pneumonia with positive blood cultures [2]. For the septicemic patient group, 51.7% of the whole blood specimens were lytA positive, while for the pneumonia patient group, 39.5% were positive [2]. Additional testing by this group found that for 105 blood culture positive samples, 42.9% were positive for lytA [2]. This is in contrast to 100% of CSF specimens, sputum specimens and lung biopsy tissue giving positive results with the lytA PCR assay [2]. In a study by Murdoch et al. [15], using the pseudolysin (ply) gene instead of lytA, throat swabs and sputum specimens demonstrated 55% and 81% PCR positivity, respectively. Results for plasma, buffy coat and urine were 2% using the same ply PCR assay [13].
Despite the findings of these previous studies, urine remains an attractive specimen type due to its non-invasive collection method for patients with CAP. Urine antigen assays are widely used because they are rapid and perform well on adults [2] despite the highly variable carriage rate in healthy adults [5]. The BinaxNOW ® S. pneumoniae urine antigen assay is not recommended pediatric patients, however, due to welldocumented issues with S. pneumoniae nasopharyngeal carriage in children [16]. The potential benefit of PCR assays is the ability to quantify the amount of DNA present and to set thresholds for carriage versus infection rates. Future studies could address the utility of a quantitative PCR assay with urine from children suspected of having streptococcal infections.
This preliminary study has some limitations. The first is that only the lytA autolysin gene target was used for PCR analysis in urine. Future studies may involve S. pneumoniae psaA MultiCode primer sets, or other published gene targets specific to S. pneumoniae, to serve as a confirmatory test after screening positive with the lytA PCR assay. A second limitation is that the urine samples were archived from patients hospitalized with CAP, stored at -80°C since January 2014. Repeated freeze thawing of certain sample types is known to reduce sensitivity of PCR assays and the effect of long term storage at -80°C on the BinaxNOW ® antigen test remains unclear. Specimen stability experiments will need to be performed in a prospective study to address these variables. Finally, the urine sample sizes are small due to the availability of archived urine samples that met the criteria established for this study (i.e. urine had to be from patients with CAP having a positive blood culture versus those patients having all negative laboratory results for S. pneumoniae). If the preliminary data hold true when testing additional urine from patients with CAP, then future studies could be performed to validate other MultiCode ® primers on the ARIES ® platform. Upon completion of the full validation study, a cost comparison of the lytA PCR assay and the urinary antigen test would be useful information. The potential to develop MultiCode ® primer sets to detect key antibiotic resistance markers in addition to identifying Streptococcus pneumoniae would be an advance in laboratory diagnostics for patients with CAP.