Decline in Decreased Cephalosporin Susceptibility and Increase in Azithromycin Resistance in Neisseria gonorrhoeae, Canada.

Antimicrobial resistance profiles were determined for Neisseria gonorrhoeae strains isolated in Canada during 2010-2014. The proportion of isolates with decreased susceptibility to cephalosporins declined significantly between 2011 and 2014, whereas azithromycin resistance increased significantly during that period. Continued surveillance of antimicrobial drug susceptibilities is imperative to inform treatment guidelines.

Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae.

The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

Molecular Assay for Detection of Ciprofloxacin Resistance in Neisseria gonorrhoeae Isolates from Cultures and Clinical Nucleic Acid Amplification Test Specimens.

We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism.

Unique combined penA/mtrR/porB mutations and NG-MAST strain types associated with ceftriaxone and cefixime MIC increases in a 'susceptible' Neisseria gonorrhoeae population.

OBJECTIVES: To determine which mutations in penA, mtrR and porB are implicated in increasing minimum MICs of ceftriaxone and cefixime in a susceptible gonococcal population and to ascertain associations with gonococcal strain types (STs). METHODS: One hundred and forty-six Neisseria gonorrhoeae isolates formed two extended-spectrum cephalosporin susceptibility groups: group 1 isolates with cefixime and ceftriaxone MICs of 0.0005-0.016 mg/L; and group 2 isolates with cefixime MICs of 0.03-0.125 mg/L (n = 24) and ceftriaxone MICs of 0.03-0.06 mg/L (n = 23). Mutation patterns in penicillin-binding protein 2 (PBP2; penA), multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB) were ascertained by DNA sequence and bioinformatic analysis. STs were determined using N. gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS: Most isolates carried PBP2 mutation pattern IX (D345a, F504L, A510V, A516G and P551L; 50/146, 34.2%), a G45D substitution in MtrR (37.7%) and a wild-type (WT) sequence for PorB (43.2%). Group 2 gonococcal isolates were significantly associated with: penA pattern IX; dual mutations in the promoter (A-) and DNA dimerization domain (H105Y) of MtrR; and G120K;A121D substitutions in PorB. There were 50 combined penA/mtrR/porB mutation patterns, with corresponding patterns I/WT/WT and IX/G45D/G120K;A121D predominating. Gonococci susceptible to ceftriaxone and cefixime were significantly associated with NG-MAST ST 25 (33/36; 92%) and the combined penA/mtrR/porB mutation pattern I/WT/WT. No combined mutation pattern or specific ST was associated with elevated ceftriaxone MICs. NG-MAST ST 3654 was significantly associated with the pattern IX/G45D/G120K;A121D and cefixime group 2 isolates. CONCLUSIONS: Specific single or combined mutation patterns in penA, mtrR and porB and specific STs were associated with differences in susceptibility to ceftriaxone and cefixime.

Erratum to "What is open-access publishing and what it means for the forensic enterprise" [Forensic Sci. Int.: Synergy (2019) 290-293].

[This corrects the article DOI: 10.1016/j.fsisyn.2019.06.045.].

Evaluation of three automated nucleic acid amplification systems for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in first-void urine specimens.

A total of 500 first-void urine specimens were tested for the presence of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids using ProbeTec ET reagents on a Viper platform (BD Diagnostics, Mississauga, Ontario, Canada), Aptima Combo 2 reagents on a Tigris platform (Gen-Probe, Inc., San Diego, CA), and Abbott RealTime CT/NG reagents on an m2000 platform (Abbott Molecular Diagnostics, Des Plaines, IL). The performance of the three assays for detection of N. gonorrhoeae was comparable, but detection of C. trachomatis by the three assays showed more variation. All three platforms were suitable for the detection of C. trachomatis and N. gonorrhoeae, but additional factors, such as maximum daily specimen throughput, are important in evaluating automated systems for C. trachomatis and N. gonorrhoeae detection in high-volume laboratories.

The use of a confirmatory assay to increase the sensitivity and specificity of the Chlamydiazyme test.

The blocking assay was used to reexamine 15,662 patient specimens (2,565 male specimens, 13,097 female specimens) that were submitted for Chlamydia detection using the Chlamydiazyme EIA assay (Abbott Laboratories, North Chicago, IL). Specimens that gave optical density (OD) readings between 1.99 to cutoff and between cutoff to 3 times the negative control in the Chlamydiazyme EIA assay were analyzed further by the blocking assay during the phase 1 study. In the phase 2 study, another 1,120 specimens (473 male specimens and 647 female specimens) that had the above mentioned OD range in the Chlamydiazyme assay were tested with the blocking assay and the direct fluorescent antibody test using the cytospin method. Significant finding from phase 1 study demonstrated that 42.3% of the male specimens with optical density between cutoff to three times the negative control can be blocked by blocking assay (confirmed positive), whereas only 50% of the female specimens with OD range between cutoff to .5 were blocked by the blocking assay. In the phase 2 study, similar results were obtained with the blocking assay. The direct fluorescent antibody test showed excellent correlation with the blocking assay. These data showed that both blocking or direct fluorescent antibody tests can be used for confirmation purposes to increase the sensitivity and specificity of the assay. However, for specimens with OD values below the cutoff to 3 times the negative control, it was necessary to reassess the specimens by either of the methods. This was true especially with the male specimens.

A comparison of the Genie and western blot assays in confirmatory testing for HIV-1 antibody.

The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic-peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie-negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio-immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies.

Comparison of the Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay with the original standard assay and cell culture.

The new Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay was evaluated by comparison with the original standard assay and cell culture. A total of 853 paired male and female genital tract specimens was tested with both Sanofi Chlamydia Microplate EIA shortened and standard assays and the results were compared with those of cell culture. For confirmation, a blocking assay run in the shortened format was used. Discrepancies between the three methods were resolved by a direct fluorescent antibody (DFA) test on the EIA samples or the culture retentate, or both. After resolution of discrepant results, the standard assay had a sensitivity, specificity, positive predictive value and negative predictive value of 98.5%, 100%, 100% and 99.9%, respectively. The shortened assay results were 100%, 100%, 100% and 100%, respectively. The shortened assay takes approximately 1.5 h less time than the standard assay and this study demonstrated that they have equivalent sensitivity and specificity. The improvement in turnaround time enables results to be reported on the same day.

Minimal inhibitory effect of male urine on detection of Chlamydia trachomatis by Roche Amplicor PCR.

A total of 1120 specimens of fresh urine from male patients was tested for Chlamydia trachomatis with the Roche Amplicor PCR Kit and an in-house PCR assay. The in-house PCR had an internal control to monitor inhibitory effects of clinical specimens on the PCR assay. All urine samples were processed within 24-48 h of collection and DNA was extracted on the same day that the assays were performed. Specimens that gave discrepant PCR results were tested by a reference laboratory with both the Roche Amplicor kit and their in-house PCR assay. Of the 1120 samples, 174 gave positive results in both assays and 942 gave negative results in both assays. Only one specimen showed an inhibitory effect on the in-house PCR assays, as indicated by failure to produce the internal control PCR product. This specimen gave negative results by both assays. There were four discrepant results in the two PCR assays. One was a false negative result obtained with the Roche Amplicor kit and the remaining three discrepant results could not be resolved because there was an insufficient quantity of specimen. This study demonstrated that the Roche Amplicor kit could effectively detect C. trachomatis in urine specimens from this population of male patients with negligible inhibition of PCR.

Hypophosphataemia in general practice patients.

We compared plasma phosphate concentrations in general practice patients and hospital inpatients and outpatients over an 8-month period. The distribution of results in all three groups was similar and 12-16% of results were at or below 0.8 mmol/L. In general practice patients, 8.3% of results from males and 12.1% from females were below the lower limit of their respective reference ranges. Eighteen of these patients (0.2% of results) had plasma phosphate concentrations < or = 0.4 mmol/L. On follow-up, only two of these patients had any attributable cause for their severe hypophosphataemia; in the remainder, it was unexpected and unexplained. Hypophosphataemia in outpatients and general practice patients is more common than has previously been appreciated. We present a strategy for further investigation of these patients.

Performance characteristics of the Becton Dickinson ProbeTec System for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in male and female urine specimens in comparison with the Roche Cobas System.

OBJECTIVE: The Becton Dickinson BDProbeTec ET System is a new semiautomated system using strand displacement amplification technology that simultaneously amplifies and detects Chlamydia trachomatis and Neisseria gonorrhoeae DNA. The strand displacement amplification products are hybridized with a fluorescent detector probe and are captured by a chemiluminescent assay in a microwell format. An amplification control is also included to monitor assay inhibition. This study evaluated the performance of the BDProbTec ET system in detecting C trachomatis and N gonorrhoeae in male and female urine specimens, calculated its ability to process large volumes of specimens, and determined the inhibition rate. MATERIALS AND METHODS: Eight hundred twenty-five male and 399 female urine specimens were tested for both C trachomatis and N gonorrhoeae with the BDProbeTec ET system, and results were compared with those of the Roche Amplicor Cobas system. All urine specimens were processed on both assays on the same day they were received, according to the manufacturers' instructions. Discrepant results were resolved by in-house polymerase chain reaction assays. Internal or amplification controls were also used in each specimen assay to monitor inhibition. The throughput of the BDProbTec ET system was further tested with 150 urine specimens on an 8-hour shift for 2 days. RESULTS: The overall sensitivity, specificity, positive predicative value, and negative predicative value for for detection of chlamydia were 95.3%, 99.3%, 95.9%, and 99.2% for strand displacement amplification and 95.9%, 98.3%, 90.6%, and 99.3% for the Roche Amplicor system. For detection of gonorrhea, these values were 100%, 99.7%, 88.2%, and 100% and 96.7%, 98.9%, 69%, and 99.9%, respectively. The overall inhibition rates for both strand displacement amplification and Roche Amplicor were less than 3.5%. The BDProbTec ET system was able to produce 150 results each for chlamydia and gonorrhea and the internal control within the 8-hour shift. CONCLUSIONS: The performance characteristics of the BDProbeTec ET assay are similar to those of the Roche Amplicor polymerase chain reaction for detection of chlamydia and gonorrhea in male and female urine specimens. The system was able to produce 300 results in an 8-hour shift.

Comparison of the effectiveness of polymerase chain reaction and enzyme immunoassay in detecting Chlamydia trachomatis in different female genitourinary specimens.

BACKGROUND: In high-volume laboratories, enzyme immunoassay (EIA) is the most commonly used method of detecting Chlamydia trachomatis. The optimal specimen for detecting C trachomatis is a combined urethral and cervical swab. OBJECTIVE: To compare EIA with the combined urethral and cervical swab with polymerase chain reaction (PCR) on urine alone and urine mixed with cervical cells. PATIENTS AND METHODS: Phase 1 of the study included 752 sets of specimens used for comparison. In phase 2, another 212 samples of urine and urine plus cervical cells were added to the study for comparison of the 2 specimen types using PCR. RESULTS: In phase 1, 648 samples were negative and 76 were positive by all 3 methods and specimen combinations. Enzyme immunoassay was able to detect 81 positive samples (10.8%), whereas PCR on urine alone detected 97 positive samples (12.9%) and PCR on urine plus cervical cells detected 102 positive samples (13.6%), giving a sensitivity of 75%, 93.3%, and 98. 1% respectively. In phase 2, PCR on urine alone detected 119 positive samples (12.3%) and PCR on urine plus cervical cells detected 127 positive samples (13.1%), with a sensitivity of 92.2% and 98.5%, respectively. CONCLUSION: Polymerase chain reaction on urine alone or urine plus cervical cells is superior to EIA on combined cervical and urethral swabs. There is a slight advantage of adding cervical cells to the urine compared with the urine specimen alone when PCR is used as the assay for detection. The total inhibition rate in our female population is only 3.1% when PCR is used.

Comparison of four latex kits for detection of E. coli O157.

OBJECTIVE: To compare four Escherichia coli O157 test kits for detection of E. coli O157:H7 isolated from clinical specimens. DESIGN: One hundred two Escherichia coli O157:H7 isolates obtained from stored specimens and 99 non-sorbitol fermenting enterobacteriaceae isolates from current clinical specimens were tested against four latex kits: Wellcolex, RIM, Prolex, and Oxoid. Each isolate was tested against all four kits on the same day. SETTING: Provincial Laboratory of Saskatchewan, Canada. PATIENTS: Patients from Saskatchewan with diarrhea submitted stool specimens through their family physicians to the Provincial Laboratory for detection of enteric pathogens including E. coli O157:H7. RESULTS: The sensitivity and specificity of each test kit were: Wellcolex 100%, 99%; RIM 100%, 99%; Prolex 99%, 100%; Oxoid 100%, 100%. The Prolex kit failed to detect one E. coli O157:H7 isolate. CONCLUSION: All kits tested were able to identify E. coli O157 isolated from stool specimens. Further study with Prolex is needed to assess the significance of the one missed E. coli O157 isolate.

Lessons to be learned: a case study approach: severe hyponatraemia induced by primary hypothyroidism and associated with possible increased hepatic sensitivity to thyroxine replacement.

The case is presented of a 74 year-old woman who was admitted with severe hypo-osmolar hyponatraemia associated with inappropriately raised urinary osmolality, and who was subsequently discovered to have primary hypothyroidism. A normal serum sodium concentration was restored by means of judicious fluid restriction and thyroid hormone replacement. Low dose thyroxine therapy led to rapid but modest increases in the serum activities of alanine aminotransferase (ALT) and alkaline phosphatase (ALP); both returned to normal over a period of three weeks. These sub-clinical enzyme changes may indicate tissue 'hyperthyroidism'; and in this case, the fact that they occurred acutely at only low doses of thyroxine possibly suggests an increased hepatic sensitivity to the hormone.

Use of immunoglobulin G avidity assays for differentiation of primary from previous infections with West Nile virus.

Since its introduction in 1999, West Nile virus (WNV) infections have spread rapidly across the North American continent. Diagnosis of acute WNV infection by detection of WNV-specific immunoglobulin M (IgM) is complicated by the persistence of detectable IgM for more than 1 year in some patients. IgG antibody avidity testing was assessed as a supplemental assay in the diagnosis of current infections. Three groups of serum samples were assayed in parallel by two different IgG avidity test systems (indirect immunofluorescence test [IIFT] and prototype enzyme-linked immunosorbent assay [ELISA]; EUROIMMUN, Luebeck, Germany). Group I (40 sera taken between 2 and 9 days after the onset of influenza-like symptoms) and group II (40 sera taken between 10 and 43 days after onset) were acute and convalescent specimens from patients with a positive anti-WNV IgM test (ELISA; Focus Diagnostics, Cypress, CA). Group III consisted of 43 patient sera collected between 6 and 12 months after infection. IgG antibodies specific for WNV were detected in 38% (ELISA) and 50% (IIFT) of group I sera, in 90% (ELISA and IIFT) of group II sera, and in 100% (ELISA and IIFT) of group III sera. Low-avidity IgG antibodies were demonstrated in 86% (ELISA) and 95% (IIFT) of IgG-positive patient samples taken between 2 and 43 days after the onset of symptoms (groups I and II). High-avidity IgG antibodies were detected in 100% of group III sera obtained 6 months or more after the onset of symptoms (ELISA and IIFT). IgG avidity tests for WNV infections are rapid and simple to perform. The determination of IgG avidity provides additional diagnostic certainty in differentiating between recently acquired and previous infections with WNV.

Evaluation of Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay and comparison with cell culture and Syva Chlamydia MicroTrak II EIA in high- and low-risk populations.

Seven hundred thirty-two female urogenital samples were collected for Chlamydia trachomatis testing by both the Sanofi Diagnostics Pasteur (Chaska, Minn.) Chlamydia Microplate EIA by the shortened protocol and the Syva (San Jose, Calif.) MicroTrak II EIA, and the results were compared with those obtained by cell culture. For the analysis of samples from female patients, the patients were divided into high- and low-risk categories. An additional 121 male urethral samples were collected and tested by the Sanofi Microplate EIA and cell culture; for the analysis of samples from male patients, the patients were divided into asymptomatic and symptomatic categories. All specimens positive by enzyme immunoassay (EIA) were confirmed by a blocking assay following the respective manufacturer's instructions. Specimens negative by EIA that fell within a gray zone 30% below the cutoff and negative cultures with one or more corresponding positive EIA results were tested further by cytocentrifugation and direct immunofluorescent assay. The overall sensitivity, specificity, positive predictive value, and negative predictive value for Syva versus culture were 94, 98.8, 85.5 and 99.6%, respectively. After resolution, the results were 94.5, 99.6, 94.5, and 99.6%, respectively. The parallel results for the Sanofi Microplate EIA versus culture were 94.0, 98.7, and 83.9, and 99.6%, respectively, and after being resolved, the results were 94.9, 100, 100, and 99.6%, respectively. In the small male population tested, the resolved results of the Sanofi Microplate EIA versus culture demonstrated sensitivity, specificity, positive predictive value, and negative predictive value of 100, 100, 100, and 100%, respectively. The present study demonstrated that the Sanofi Microplate EIA shortened protocol is highly sensitive and specific in comparison with cell culture and the Syva MicroTrak II EIA.