Multicenter Clinical Evaluation of Vitek 2 Meropenem-Vaborbactam for Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

The carbapenem/beta-lactamase inhibitor meropenem-vaborbactam (MEV) used to treat complicated urinary tract infections and pyelonephritis in adults was approved in 2017 by the U.S. Food and Drug Administration (FDA). Here, we evaluated Vitek 2 MEV (bioMérieux, Durham, NC) compared to the reference broth microdilution (BMD) method. Of 449 Enterobacterales isolates analyzed per FDA/CLSI breakpoints, the overall performance was 98.2% essential agreement (EA), 98.7% category agreement (CA), and 0% very major errors (VME) or major errors (ME). For 438 FDA intended-for-use Enterobacterales isolates, performance was 98.2% EA, 98.6% CA, and 0% VME or ME. Evaluable EA was 81.0%, but with only 42 on-scale evaluable results. Individual species demonstrated EA and CA rates of ≥90% without any VME or ME. When evaluated using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, overall Vitek 2 MEV performance for Enterobacterales and Pseudomonas aeruginosa demonstrated 97.3% EA, 99.2% CA, 2.3% VME, and 0.6% ME (after error resolution: 97.3% EA, 99.4% CA, 2.2% VME, and 0.4% ME) compared to the reference BMD method. Performance for P. aeruginosa included 92.2% EA, 97.4% CA, 0% VME, and 3.0% ME (after error resolution: 92.2% EA, 98.7% CA, 0% VME, and 1.5% ME). Performance for Enterobacterales included 98.2% EA, 99.6% CA, 3.0% VME, and 0.2% ME. Evaluable EA was 80.6% but was based on only 67 evaluable results. These findings support Vitek 2 MEV as an accurate automated system for MEV susceptibility testing of Enterobacterales and P. aeruginosa and could be an alternate solution to the manual-labor-intensive reference BMD method.

Multicenter evaluation of the VITEK MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry system for identification of bacteria, including Brucella, and yeasts.

The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has proven to be rapid and accurate for the majority of clinical isolates. Some gaps remain concerning rare, emerging, or highly pathogenic species, showing the need to continuously expand the databases. In this multicenter study, we evaluated the accuracy of the VITEK MS v3.2 database in identifying 1172 unique isolates compared to identification by DNA sequence analysis. A total of 93.6% of the isolates were identified to species or group/complex level. A remaining 5.2% of the isolates were identified to the genus level. Forty tests gave a result of no identification (0.9%) and 12 tests (0.3%) gave a discordant identification compared to the reference identification. VITEK MS is also the first MALDI-TOF MS system that is able to delineate the four members of the Acinetobacter baumannii complex at species level without any specific protocol or special analysis method. These findings demonstrate that the VITEK MS v3.2 database is highly accurate for the identification of bacteria and fungi encountered in the clinical laboratory as well as emerging species like Candida auris and the highly pathogenic Brucella species.

Asymptomatic Patient Testing After 10:1 Pooling Using the Xpert Xpress SARS-CoV-2 Assay.

OBJECTIVES: Pool testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) preserves testing resources at the risk of missing specimens through specimen dilution. METHODS: To determine whether SARS-CoV-2 specimens would be missed after 10:1 pooling, we identified 10 specimens with midrange (ie, 25-34 cycles) and 10 with late (ie, >34-45 cycles) crossing threshold (Ct) values and tested these both neat and after 10:1 pooling. Final test results and Ct changes were compared. RESULTS: Overall, 17 of 20 specimens that contained SARS-CoV-2 were detected after 10:1 pooling with the Xpert Xpress SARS-CoV-2 Assay (Cepheid), rendering an 85% positive percentage of agreement. All 10 of 10 specimens with an undiluted Ct in the mid-Ct range were detected after 10:1 pooling, in contrast to 7 of 10 with an undiluted Ct in the late-Ct range. The overall Ct difference between the neat testing and the 10:1 pool was 2.9 cycles for the N2 gene target and 3 cycles for the E gene target. The N2 gene reaction was more sensitive than the E gene reaction, detecting 16 of 20 positive specimens after 10:1 pooling compared with 9 of 20 specimens. CONCLUSIONS: An 85% positive percentage of agreement was achieved, with only specimens with low viral loads being missed following 10:1 pooling. The average impact on both reverse transcription polymerase chain reactions within this assay was about 3 cycles.

Cerebral Paragonimiasis Presenting with Sudden Death.

A 58-year-old Korean-born woman with a history of seizures and psychiatric issues was found dead at home. Autopsy was notable for large, calcified nodules that had nearly replaced her right temporal lobe. Histologic examination revealed the presence of Paragonimus eggs. This case demonstrates a rare manifestation of an aberrantly migrated lung fluke that resulted in epilepsy and sudden death years after the initial infection.

Cystoisospora belli Infection of the Gallbladder in Immunocompetent Patients: A Clinicopathologic Review of 18 Cases.

Cystoisospora belli, previously known as Isospora belli, is an obligate intracellular coccidian parasite that is most often associated with gastrointestinal disease in immunocompromised patients. In this study, we detail the clinicopathologic features of 18 cases of Cystoisospora infection affecting the gallbladder in immunocompetent individuals and compare them with a control group. Each case was reviewed for cholecystitis (none, acute, chronic), epithelial disarray, presence of intraepithelial lymphocytes (none, rare [≤5 per 20 epithelial cells], present [>5 per 20 epithelial cells]), architectural distortion, intramucosal eosinophilia, and mural thickening/serositis. The mean age of patients with Cystoisospora infection was 33 years and the male to female ratio 1:4.3. Cholecystectomy was performed for biliary dyskinesia (n=7), abdominal pain (n=7), suspected cholelithiasis (n=5), and cholecystitis (n=3). In 2 cases, Cystoisospora was found in donor gallbladders resected at the time of liver transplantation. Each case was characterized by eosinophilic, oval or banana-shaped intraepithelial parasites within perinuclear parasitophorous vacuoles. Most cases showed epithelial disarray and minimal intraepithelial lymphocytosis. Of the 11 cases with an average follow-up of 15 months, none had evidence of disease related to Cystoisospora infection within the biliary tract or elsewhere in the gastrointestinal tract. We present the largest series of gallbladder cystoisosporiasis in immunocompetent patients to date. Cystoisospora infection is underrecognized in the gallbladders of immunocompetent patients, in part due to the subtle findings in routine cholecystectomy specimens. On the basis of the clinical follow-up, gallbladder cystoisosporiasis in immunocompetent individuals appears to be a self-limited infection.

Skin Sterility After Application of a Vapocoolant Spray Part 2.

INTRODUCTION: Refrigerant sprays have been used for pain relief at the time of minor office procedures. However, their sterility remains in question. This study investigates the microbiologic effect of this vapocoolant when sprayed after 70 % isopropyl alcohol skin preparation. MATERIALS AND METHODS: In 50 healthy volunteers, three skin culture samples were collected: Group 1 prior to alcohol application; Group 2 after preparation with alcohol, and Group 3 after preparation with alcohol followed with vapocoolant spray. Samples were cultured in a blinded fashion and analyzed after 5 days of incubation. Gram staining was performed when cultures were positive. RESULTS: Bacterial growth was found in 98 % of samples prior to any skin preparation. This was reduced to 54 % after alcohol use (Group 2). Spraying with the skin refrigerant further reduced bacterial growth to 46 % (Group 3). The results showed a significant reduction in the number of positive bacterial cultures following skin preparation with alcohol and when alcohol prep was followed by vapocoolant spray (p < 0.001) compared to initial cultures. No statistical difference was observed between Groups 2 and 3 (p = 0.74). CONCLUSIONS: The use of the vapocoolant spray does not compromise the sterility of the skin following alcohol prep. Both 70 % isopropyl alcohol antiseptic preparation and skin preparation followed by vapocoolant spray significantly reduce skin colonization when compared to unprepared skin (p < 0.001).

Unexpected challenges in treating multidrug-resistant Gram-negative bacteria: resistance to ceftazidime-avibactam in archived isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of ß-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel ß-lactam/ß-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 ß-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.

Interobserver variability and feasibility of polymerase chain reaction-based assay in distinguishing ischemic colitis from Clostridium difficile colitis in endoscopic mucosal biopsies.

Polymerase chain reaction (PCR)-based assays using stool samples are currently the most effective method of detecting Clostridium difficile. This study examines the feasibility of this assay using mucosal biopsy samples and evaluates the interobserver reproducibility in diagnosing and distinguishing ischemic colitis from C difficile colitis. Thirty-eight biopsy specimens were reviewed and classified by 3 observers into C difficile and ischemic colitis. The findings were correlated with clinical data. PCR was performed on 34 cases using BD GeneOhm C difficile assay. The histologic interobserver agreement was excellent (κ= 0.86) and the agreement between histologic and clinical diagnosis was good (κ = 0.84). All 19 ischemic colitis cases tested negative (100% specificity) and 3 of 15 cases of C difficile colitis tested positive (20% sensitivity). C difficile colitis can be reliably distinguished from ischemic colitis using histologic criteria. The C difficile PCR test on endoscopic biopsy specimens has excellent specificity but limited sensitivity.

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

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.

Two cases of Cokeromyces recurvatus in liquid-based Papanicolaou tests and a review of the literature.

We present 2 cases of Cokeromyces recurvatus in routine, liquid-based Papanicolaou tests (ThinPrep). Patient 1 is a healthy, asymptomatic, 26-year-old woman with no pertinent past medical history. Patient 2 is a healthy, asymptomatic, 47-year-old woman with no pertinent past medical history. The Papanicolaou tests from both patients showed many fungal-like elements as globose, yeastlike forms measuring 10 to 30 µm in diameter with multiple, narrowly attached apparent "daughter" buds. This morphology was consistent with Paracoccidioides brasiliensis. However, broad-range fungal polymerase chain reaction and deoxyribonucleic acid sequence analysis performed with GenBank Basic Local Alignment Search Tool showed an exact match for C recurvatus. Our cases highlight the importance of molecular techniques to prevent misdiagnosis of C recurvatus as P brasiliensis, based on morphology alone. There have been 8 previously published cases of C recurvatus infection in humans, 3 of which were reported in the female genital tract.

Clinical utility of DNA amplification and sequencing to identify a strain of Mycobacterium avium in paraffin-embedded, formalin-fixed biopsies from an immunosuppressed child.

Nontuberculous mycobacterial (NTM) infections are serious, though rare, in patients with severe combined immunodeficiency who have received bone marrow transplants. A 5-year-old female patient underwent stem cell/bone marrow transplant with disseminated NTM. Real-time polymerase chain reaction (PCR) using a fluorescence resonance energy transfer (FRET) probe for detection and identification of NTM was performed. The FRET-based real-time PCR assay amplified mycobacterial DNA, and the postamplification melt curve analysis classified the organism as a NTM. The pyrosequence of the hypervariable region A definitively identified the infecting organism as Mycobacterium avium. Real-time PCR along with melt curve analysis and pyrosequencing provides faster, definitive identification of mycobacteria, as compared to bacterial culture. In this case report, we emphasize the importance of utilizing molecular means for fast and accurate diagnosis.

Performance of nucleic acid amplification following extraction of 5 milliliters of whole blood for diagnosis of Mycobacterium tuberculosis bacteremia.

To investigate the performance of a nucleic acid amplification test (NAAT) for the diagnosis of Mycobacterium tuberculosis bacteremia, 5-ml aliquots of blood were inoculated into bioMérieux mycobacterial (MB) bottles and incubated, and 5-ml aliquots of blood were extracted and tested by real-time PCR. Of 25 samples from patients with M. tuberculosis bacteremia, 9 (36.0%) were positive and 1 (1.5%) of 66 control samples was positive by NAAT. The NAAT shows promise, but modifications should focus on improving sensitivity.

Progression to bacteremia in critical care patients colonized with methicillin-resistant Staphylococcus aureus expressing Panton-Valentine leukocidin.

The role of Panton-Valentine leukocidin (PVL) in methicillin-resistant Staphylococcus aureus (MRSA) infections is unclear. PVL has been long associated with soft tissue infections and necrotizing pneumonia, but inconsistently with other site infections or mortality. The retrospective cohort study explores the association between PVL and bacteremia in colonized medical intensive care unit (ICU) patients with surveillance isolates and blood cultures. A total of 840 patients were screened by nasal swab, with 266 patients found to be colonized and 46 with bacteremia. Colonization by PVL(+) MRSA increased the odds of bacteremia (odds ratio, 2.40; confidence interval, 1.23-4.57), and invasive infection developed earlier in these patients (relative risk, 0.44; confidence interval 0.25-0.85) compared to those colonized with PVL(0) MRSA. PVL was not associated with infections at other sites, length of ICU stay, or mortality. PVL decreases the time to bacteremia in colonized patients but does not otherwise contribute to disease course or clinical outcome.

Nontuberculous mycobacterial blood stream and cardiac infections in patients without HIV infection.

Nontuberculous mycobacteria rarely cause bacteremia in HIV-negative patients. We describe 16 cases, including the first Mycobacterium neoaurum endocarditis. Nine cases were line related. Most patients were immunocompromised secondary to hematologic malignancy or other comorbid conditions. Amikacin had the most reliable in vitro activity. Combination therapy was frequently used. Mortality was 25%.

Successful pyrosequencing of GC-rich DNA sequences by partial substitution of deoxyguanosine with deoxyinosine.

We experienced significant problems while developing a PyrosequencingTM (Biotage, Uppsala, Sweden) assay to characterize the rifampin resistance-determining region of Mycobacteriumtuberculosis, a target with high guanosine-cytosine content. This paper describes the successful use of a modified pyrosequencing protocol through partial substitution of deoxyguanosine triphosphate with deoxyinosine triphosphate in the polymerase chain reaction.

Distinction between intact and antibiotic-inactivated bacteria by real-time PCR after treatment with propidium monoazide.

One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent false-positive results, possibly due to the detection of dead bacteria. We recently showed that the use of DNA-binding agent propidium monoazide (PMA) could distinguish viable from heat-inactivated bacteria, and, in this study, we investigated whether the same technique can be applied to bacteria killed by two antibiotics with distinctly different mechanisms of action, a test of greater clinical relevance than thermal inactivation. Staphylococcus aureus and S. epidermidis were inactivated by vancomycin and gentamicin and treated with PMA or left untreated before DNA extraction. The threshold cycle difference of antibiotic-treated bacteria with and without PMA pretreatment was investigated with PCR primers for the 16S rDNA and tuf genes. Our results indicated that PMA effectively inhibited detection by PCR of bacteria, which had been inactivated by either vancomycin or gentamicin. The effect was statistically significant at 24 h after treatment (C(t) difference consistently >3; p < 0.05) and after 10 days of treatment (C(t) difference >4; p < 0.01), when compared to viable cells (C(t) difference 1-2). Vancomycin had a stronger effect on the C(t) value than gentamicin, reflecting the different mechanism of action of each antibiotic. Techniques of this type may help reduce clinically false-positive PCR results caused by the detection of dead bacteria, and may be especially useful in patients who have received antibiotics, such as patients undergoing the second stage of a two-stage revision for infected arthroplasty.

Pyrosequencing for rapid detection of Mycobacterium tuberculosis resistance to rifampin, isoniazid, and fluoroquinolones.

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.

Limiting false-positive polymerase chain reaction results: detection of DNA and mRNA to differentiate viable from dead bacteria.

We compared 2 methods for determining Escherichia coli viability in vitro. A 16S rDNA polymerase chain reaction (PCR) assay detected bacteria irrespective of viability. A groEL mRNA reverse transcriptase PCR was positive for 72 h but later became negative. Detecting mRNA holds promise but is tedious, and groEL may not be the best target.

Bilateral periprosthetic joint infection caused by Salmonella enterica serotype Enteritidis, and identification of Salmonella sp using molecular techniques.

Salmonella septic arthritis is rare. Our objective was to identify bacterial species from joint fluid using a broad-range real-time PCR and pyrosequencing technique. We describe a case of bilateral Salmonella enterica serotype Enteritidis infection of right and left total knee arthroplasties. DNA was extracted from the joint fluid of the left knee, amplified by PCR, and the amplicons were evaluated by pyrosequencing. The patient was treated with ciprofloxacin, and the polyethylene liners were replaced in both knees. The results of pyrosequencing detected a Salmonella species. To the best of our knowledge, this is the first report describing the detection of Salmonella in joint fluid by universal PCR followed by pyrosequencing.

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.