Marion J. Tuohy

Multicenter Evaluation of the BDProbeTec ET System for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine Specimens, Female Endocervical Swabs, and Male Urethral Swabs

ABSTRACT The performance of the Becton Dickinson BDProbe Tec ET SystemChlamydia trachomatis and Neisseria gonorrhoeaeAmplified DNA Assays (BD Biosciences, Sparks, Md.) was evaluated in a multicenter study. Specimens were collected from 2,109 men and women, with or without symptoms, attending sexually transmitted disease, family planning, and obstetrics and gynecology clinics. Both swab and urine samples were collected, and the results obtained from 4,131 specimens were compared to those from culture and the LCx nucleic acid amplification test (Abbott Industries, Abbott Park, Ill.). PCR and cytospin of the culture transport medium with chlamydia direct fluorescent antibody staining were used to adjudicate chlamydia culture-negative results. Sensitivity and specificity were calculated both with and without use of the amplification control (AC), with little apparent difference in the results. Without the AC result, sensitivity for C. trachomatis and N. gonorrhoeae were 92.8 and 96.6%, respectively, for cervical swabs and 80.5 and 84.9% for urine from women. C. trachomatis and N. gonorrhoeae sensitivities were 92.5 and 98.5%, respectively, for male urethral swabs and 93.1 and 97.9% for urine from men. This amplified DNA system for simultaneous detection of chlamydial and gonococcal infections demonstrated superior sensitivity compared to chlamydia culture and has performance characteristics comparable to those of other commercially available nucleic acid-based assays for these organisms.

Rapid Identification of Staphylococcus aureus and the mecA Gene from BacT/ALERT Blood Culture Bottles by Using the LightCycler System

ABSTRACT One hundred BacT/ALERT blood culture bottles growing gram-positive cocci in clusters were cultured and studied by LightCycler PCR for the sa442 and mecA genes. PCR was 100% sensitive and specific for detecting Staphylococcus aureus and methicillin resistance in S. aureus but was less accurate for methicillin resistance in coagulase-negative staphylococci.

Detection and Differentiation of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Isolates by Real-Time PCR

ABSTRACT Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35°C (63.27 to 65.42°C); M. kansasii, 59.20°C (58.07 to 60.33°C); M. avium, 57.82°C (57.05 to 58.60°C); M. intracellulare, 54.46°C (53.69 to 55.23°C); M. marinum, 58.91°C (58.28 to 59.55°C); rapidly growing mycobacteria, 53.09°C (50.97 to 55.20°C) or 43.19°C (42.19 to 44.49°C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.

Evaluation of the Verigene Gram-positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants.

ABSTRACT Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.

Improved Detection of Biofilm-formative Bacteria by Vortexing and Sonication : A Pilot Study

Bacteria such as staphylococci commonly encountered in orthopaedic infections form biofilms and adhere to bone implants and cements. Various methods to disrupt the biofilm and enhance bacterial detection have been reported. We will describe the effectiveness of vortexing and sonication to improve the detection of biofilm-formative bacteria from polymethylmethacrylate by conventional quantitative bacterial culture and real-time quantitative PCR. We used a single biofilm-formative Staphylococcus aureus strain and 20 polymethylmethacrylate coupons as an in vitro biofilm model; four coupons were used for each of two control groups or three experimental sonication times (1, 5, and 30 minutes). Vortexing the cement without sonication increased the yield of adherent bacteria to a considerable extent. The combination of vortexing and sonication further enhanced the yield regardless of the duration of sonication. Quantitative conventional cultures correlated with quantitative PCR assay. The combination of vortexing and sonication to disrupt the bacterial biofilm followed by quantitative PCR and/or culture seems to be a sensitive method for detecting bacteria adherent to bone cement.

Improving clinical significance of PCR: Use of propidium monoazide to distinguish viable from dead staphylococcus aureus and staphylococcus epidermidis

Molecular techniques, such as the polymerase chain reaction (PCR) have high sensitivity when used to diagnose infection, but may detect DNA, RNA, and proteins from dead, as well as viable, bacteria. Propidium monoazide (PMA) is a DNA binding agent, that has the ability to penetrate only dead cells with compromised membranes and has been used in conjunction with real‐time PCR to distinguish intact from dead bacterial cells. In this study, intact, heat‐inactivated (dead), and intact/dead admixed Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) were treated with PMA or left untreated before DNA extraction. We quantified levels of 16S rDNA and tuf gene by real‐time quantitative PCR (qPCR), to test the ability of PMA to distinguish intact from dead bacteria. Our results indicated that PMA inhibited detection of dead bacteria, and the qPCR results reflected the number of intact bacteria without being impacted by the presence of the dead bacteria. This approach of combining qPCR with and without PMA treatment has promise to limit false‐positive PCR results when used to diagnose infections, but needs to be further validated in clinical samples.

Antimicrobial susceptibility of Abiotrophia adiacens and Abiotrophia defectiva

The susceptibilities of 27 Abiotrophia adiacens (proposed reclassification Granulicatella adiacens comb.nov., Collins & Lawson, 2000) and 12 Abiotrophia defectiva isolates were tested by microdilution in pyridoxal hydrochloride and lysed horse blood supplemented Mueller-Hinton broth. According to NCCLS interpretative criteria for Streptococcus spp. not Streptococcus pneumoniae, the susceptibilities of A. adiacens and A. defectiva were, respectively: penicillin, 55% and 8%; amoxicillin, 81% and 92%; ceftriaxone, 63% and 83%; meropenem, 96% and 100%; and 100% for both species with clindamycin, rifampin, levofloxacin, ofloxacin, quinupristin/dalfopristin, and vancomycin.

Brief ultrasonication improves detection of biofilm-formative bacteria around a metal implant.

Biofilms are complex microenvironments produced by microorganisms on surfaces. Ultrasonication disrupts biofilms and may make the microorganism or its DNA available for detection. We determined whether ultrasonication could affect our ability to detect bacteria adherent to a metal substrate. A biofilm-formative Staphylococcus aureus strain was used for an in vitro implant infection model (biofilm-formative condition). We used quantitative culture and real time-polymerase chain reaction to determine the influence of different durations of ultrasound on bacterial adherence and viability. Sonication for 1 minute increased the yield of bacteria. Sonication longer than 5 minutes led to fewer bacterial colonies by conventional culture but not by polymerase chain reaction. This suggests short periods of sonication help release bacteria from the metal substrate by disrupting the biofilm, but longer periods of sonication lyse bacteria prohibiting their detection in microbiologic cultures. A relatively short duration of sonication may be desirable for maximizing detection of biofilm-formative bacteria around implants by culture or polymerase chain reaction.

Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide

Aims:  The DNA‐intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells by selectively penetrating through the damaged membrane of dead cells and blocking the DNA amplification during the polymerase chain reaction (PCR). We optimized and tested the assay in vitro using Staphylococcus aureus and Staphylococcus epidermidis cultures to distinguish viable from dead bacteria, with the goal of reducing false positive PCR results.

Unexpected Challenges in Treating Multidrug-Resistant Gram-Negative Bacteria: Resistance to Ceftazidime-Avibactam in Archived Isolates of Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of β-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination. The isolates were systematically analyzed through a variety of genetic, biochemical, genomic, and microbiological methods to understand how resistance manifests to a unique drug combination that is not yet clinically released. We discovered that avibactam was able to inactivate different AmpC β-lactamase enzymes and that blaPDC regulatory elements and penicillin-binding protein differences did not contribute in a major way to resistance. By using carefully selected combinations of antimicrobial agents, we deduced that the greatest barrier to ceftazidime-avibactam is membrane permeability and drug efflux. To overcome the constellation of resistance determinants, we show that a combination of antimicrobial agents (ceftazidime/avibactam/fosfomycin) targeting multiple cell wall synthetic pathways can restore susceptibility. In P. aeruginosa, efflux, as a general mechanism of resistance, may pose the greatest challenge to future antibiotic development. Our unexpected findings create concern that even the development of antimicrobial agents targeted for the treatment of multidrug-resistant bacteria may encounter clinically important resistance. Antibiotic therapy in the future must consider these factors.