Evaluation of the Hologic Aptima Combo 2 Assay for Detection of Neisseria gonorrhoeae from Joint Fluid Specimens.

Gonorrhea is a sexually transmitted bacterial infection caused by Neisseria gonorrhoeae. Nucleic acid amplification testing is the preferred method for routine diagnosis of gonorrhea from urogenital specimens, but culture is commonly used for diagnosis of disseminated infections, including gonococcal arthritis. The Hologic Aptima Combo 2 (AC2), a transcription-mediated amplification assay, is FDA and Health Canada licensed for detection of N. gonorrhoeae and Chlamydia trachomatis from urogenital, rectal, and pharyngeal specimens, but not joint fluid. In the current study, we compared the performance of microscopy, culture, and the AC2 for detection of N. gonorrhoeae from 170 joint fluid specimens. A total of five specimens were culture-positive, whereas 14 were AC2-positive. Gram-negative diplococci, characteristic of Neisseria, were observed in only two joint fluid specimens. Complementary testing confirmed the presence of N. gonorrhoeae in seven discordant (i.e., culture-negative/AC2-positive) specimens. These results indicate that the AC2 is more sensitive than culture for the diagnosis of gonococcal arthritis.

Evaluation of three MALDI-TOF mass spectrometry libraries for the identification of filamentous fungi in three clinical microbiology laboratories in Manitoba, Canada.

Matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) is commonly used by clinical microbiology laboratories to identify bacterial pathogens and yeasts, but not for the identification of moulds. Recent progress in extraction protocols and the composition of comparative libraries support potential application of MALDI-TOF MS for mould identification in clinical microbiology laboratories. We evaluated the performance of the Bruker Microflex™ MALDI-TOF MS instrument (Billerica, MA, USA) to identify clinical isolates and reference strains of moulds using 3 libraries, the Bruker mould library, the National Institutes of Health (NIH) library and the Mass Spectrometry Identification (MSI) online library, and compared those results to conventional (morphological) and molecular (18S/ITS; gold standard) identification methods. All 3 libraries demonstrated greater accuracy in genus identification (≥94.9%) than conventional methods (86.4%). MALDI-TOF MS identified 73.3% of isolates to species level compared to only 31.7% by conventional methods. The MSI library demonstrated the highest rate of species-level identification (72.0%) compared to NIH (19.5%) and Bruker (13.6%) libraries. Greater than 20% of moulds remained unidentified to species level by all 3 MALDI-TOF MS libraries primarily because of library limitations or imperfect spectra. The overall identification rate of each MALDI-TOF MS library depended on the number of species and the number of spectra representing each species in the library.

Bacterial presence on flexible endoscopes vs time since disinfection.

AIM: To correlate the length of endoscope hang time and number of bacteria cultured prior to use. METHODS: Prospectively, we cultured specimens from 19 gastroscopes, 24 colonoscopes and 5 side viewing duodenoscopes during the period of 2011 to 2015. A total of 164 results had complete data denoting date of cleansing, number of days stored and culture results. All scopes underwent initial cleaning in the endoscopy suite utilizing tap water, and then manually cleaned and flushed. High level disinfection was achieved with a Medivator© DSD (Medivator Inc., United States) automated endoscope reprocessor following manufacturer instructions, with Glutacide® (Pharmax Limited, Canada), a 2% glutaraldehyde solution. After disinfection, all scopes were stored in dust free, unfiltered commercial cabinets for up to 7 d. Prior to use, all scopes were sampled and plated on sheep blood agar for 48 h; the colony count was obtained from each plate. The length of endoscope hang time and bacterial load was analyzed utilizing unpaired t-tests. The overall percentage of positive and negative cultures for each type of endoscope was also calculated. RESULTS: All culture results were within the acceptable range (less than 200 cfu/mL). One colonoscope cultured 80 cfu/mL after hanging for 1 d, which was the highest count. ERCP scopes cultured at most 10 cfu, this occurred after 2 and 7 d, and gastroscopes cultured 50 cfu/mL at most, at 1 d. Most cultures were negative for growth, irrespective of the length of hang time. Furthermore, all scopes, with the exception of one colonoscope which had two positive cultures (each of 10 cfu/mL), had at most one positive culture. There was no significant difference in the number of bacteria cultured after 1 d compared to 7 d when all scopes were combined (day 2: P = 0.515; day 3: P = identical; day 4: P = 0.071; day 5: P = 0.470; day 6: P = 0.584; day 7: P = 0.575). There was also no significant difference in the number of bacteria cultured after 1 day compared to 7 d for gastroscopes (day 2: P = 0.895; day 3: P = identical; day 4: P = identical; day 5: P = 0.893; day 6: P = identical; day 7: P = 0.756), colonoscopes (day 2: P = 0.489; day 4: P = 0.493; day 5: P = 0.324; day 6: P = 0.526; day 7: P = identical), or ERCP scopes (day 2: P = identical; day 7: P = 0.685). CONCLUSION: There is no correlation between hang time and bacterial load. Endoscopes do not need to be reprocessed if reused within a period of 7 d.

Evaluation of MRSASelect (™) chromogenic medium for the early detection of methicillin-resistant Staphylococcus aureus from blood cultures.

INTRODUCTION: Staphylococcus aureus bacteremia is associated with considerable morbidity and mortality. In theory, reducing the turnaround time in reporting of methicillin-resistant S aureus (MRSA) among patients with bactermia could assist with the rapid optimization of antimicrobial therapy. OBJECTIVE: To evaluate the sensitivity and specificity of MRSASelect (Bio-Rad Laboratories, USA), a chromogenic medium, in the early detection of MRSA from blood cultures growing Gram-positive cocci in clusters, and to confirm that routine use of this medium would, in fact, reduce turnaround time for MRSA identification. METHODS: The present study was conducted at three microbiology laboratories in Manitoba. Between April 2010 and May 2011, positive blood cultures with Gram-positive cocci in clusters visualized on Gram stain were subcultured to both MRSASelect and routine media. MRSA isolates were identified using conventional microbiological methods from routine media and using growth with the typical colony morphology (pink colony) on MRSASelect medium. RESULTS: A total of 490 blood cultures demonstrating Gram-positive cocci in clusters on Gram stain were evaluated. S aureus was recovered from 274 blood cultures, with 51 S aureus isolates (51 of 274 [18.6%]) identified as MRSA. MRSASelect medium had a sensitivity of 98%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 99.8% for the recovery and identification of MRSA directly from positive blood culture bottles. In addition, use of MRSASelect medium was found to improve turnaround time in the detection of MRSA by almost 24 h relative to conventional methods. DISCUSSION: These data support the utility of MRSASelect medium for the rapid identification of MRSA from positive blood cultures. Further clinical studies are warranted to determine whether the improvement in turnaround time will result in a measurable reduction in suboptimal antimicrobial therapy and/or improvement in patient outcome.

HISTORIQUE: La bactériémie à Staphylococcus aureus s'associe à une morbidité et une mortalité considérables. En théorie, la réduction du délai à confirmer un S aureus résistant à la méthicilline (SARM) chez les patients ayant une bactériémie pourrait contribuer à l'optimisation rapide de la thérapie antimicrobienne. OBJECTIF: Évaluer la sensibilité et la spécificité du MRSASelect (Bio-Rad Laboratories, États-Unis), un milieu de culture chromogène, pour déceler rapidement le SARM dans des cultures sanguines contenant des coccobacilles à Gram positif en grappes et confirmer que l'utilisation systématique de ce milieu de culture réduit véritablement le délai de dépistage du SARM. MÉTHODOLOGIE: Les chercheurs ont mené la présente étude dans trois laboratoires de microbiologie du Manitoba. Entre avril 2010 et mai 2011, les cultures sanguines positives aux coccobacilles à Gram positif en grappes visualisées sur une coloration de Gram ont été repiquées à la fois dans un milieu de culture MRSASelect et dans un milieu de culture habituel. Les chercheurs ont repéré des isolats de SARM au moyen des méthodes microbiologiques classiques dans les milieux de culture habituels et des milieux de culture ayant la morphologie classique des colonies (colonie rose) sur les milieux de culture MRSASelect. RÉSULTATS: Au total, les chercheurs ont évalué 490 cultures sanguines démontrant des cocccobacilles à Gram positif en grappes sur une coloration de Gram. Ils ont prélevé le S aureus sur 274 analyses sanguines et déterminé que 51 isolats de S aureus (51 sur 274 [18,6 %]) étaient un SARM. Le milieu de culture MRSASelect avait une sensibilité de 98 %, une spécificité de 100 %, une valeur prédictive positive de 100 % et une valeur négative prédictive de 99,8 % à l'égard de la récupération et du dépistage du SARM directement dans les fioles de culture sanguine. De plus, l'utilisation du milieu de culture MRSASelect raccourcissait de près de 24 heures le délai de dépistage du SARM par rapport aux méthodes classiques. EXPOSÉ: Ces données appuient l'utilité du milieu de culture MRSASelect pour dépister rapidement le SARM dans les cultures sanguines positives. D'autres études cliniques s'imposent pour déterminer si l'obtention plus rapide des résultats s'associera à une réduction mesurable de la thérapie antimicrobienne sous-optimale ou à une amélioration de l'issue des patients.

The use of TaqMan PCR assay for detection of Bordetella pertussis infection from clinical specimens.

OBJECTIVE: The routine clinical laboratory detection of Bordetella pertussis is through culture, which can require 5 to 7 days for the bacteria to grow. Using a polymerase chain reaction (PCR) assay can shorten this detection time while increasing the sensitivity of detection with similar specificity. This study compared culture with TaqMan PCR for detection of B pertussis in clinical specimens and the turnaround time for each assay during the pertussis season. MATERIALS AND METHODS: Nasopharyngeal swabs in Regan-Lowe transport media were collected from 1556 persons who had symptoms of whooping cough or who had had contact with infected persons; the swabs were submitted for B pertussis detection during the pertussis season. A single nasopharyngeal swab from each patient was submitted for both culture and TaqMan PCR detection. Upon receipt of the specimens, the swabs were inoculated onto Regan-Lowe agar for culture and incubated for up to 7 days. The same swab was processed for PCR detection using TaqMan PCR assay. A second nested PCR was used on positive specimens for resolution purposes. The TaqMan PCR assay was performed 3 to 5 days a week, whereas the culture was performed 6 days a week. All specimens were processed on the same day or earliest possible working day for TaqMan or culture, and specimens queued for resolution by nested PCR were batched. RESULTS: There were a total of 275 PCR positives and 28 culture positives. After resolution with the second nested PCR, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 97.4%, 87.6%, and 100% for TaqMan PCR and 11.6%, 100%, 100%, and 85.7% for culture. The average turnaround time for positive culture was 5.1 days, and the average turnaround time for PCR was 2.3 days. CONCLUSION: The TaqMan PCR assay has superior sensitivity and shorter turnaround time over culture because it can be finished within one working day. Furthermore, the same swab can be used for culture of the bacteria for antibiotic susceptibility testing. The early detection of pertussis using TaqMan PCR assay allows early intervention on the spread of the disease and the ability to culture the bacteria from the same swab, thereby eliminating the need for a second swab and allowing for detection of antibiotic resistance.