Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays.

In December 2019, a novel coronavirus (SARS-CoV-2) was first isolated from Wuhan city, China and within three months, the global community was challenged with a devastating pandemic. The rapid spread of the virus challenged diagnostic laboratories to rapidly develop molecular diagnostic methods. As SARS CoV-2 assays became available for testing on existing molecular platforms, laboratories devoted unprecedented energy and resources into evaluating the analytical performance of the new tests and in some cases developed their own diagnostic assays under FDA-EUA guidance. This study compares the validation of three different molecular assays at the Johns Hopkins Molecular Virology laboratory: the RealStar® SARS-CoV-2 RT-PCR, ePlex® SARS-CoV-2, and the CDC COVID-19 RT-PCR tests. Overall, our studies indicate a comparable analytical performance of the three assays for the detection of SARS-CoV-2.

Differentiating Streptococcus pseudoporcinus from GBS: could this have implications in pregnancy?

BACKGROUND: Streptococcus agalactiae (GBS) is a common pathogen known to cause neonatal and maternal infectious morbidity. Streptococcus pseudoporcinus (S pseudoporcinus) is a separate, recently identified ß-hemolytic gram-positive coccus that can cause false-positive results on standard GBS agglutination testing assays. OBJECTIVE: To determine the prevalence and clinical implications of Streptococcus pseudoporcinus colonization in pregnancy. MATERIALS AND METHODS: This is a 2-year retrospective cohort study comparing pregnant women colonized with GBS to those colonized with S. pseudoporcinus. A proteomics method of identification, namely, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, was used to distinguish between S. pseudoporcinus and GBS colonization. Antibiotic susceptibility testing was carried out on all specimens. Maternal and neonatal chart reviews were conducted to identify predictors of S. pseudoporcinus colonization and to compare maternal and neonatal outcomes. RESULTS: S. pseudoporcinus colonization occurred in 1.6% of all pregnancies. A total of 2.5% of all GBS-positive results by agglutination assay were false positive, instead reflecting S. pseudoporcinus colonization. Clindamycin resistance among S. pseudoporcinus isolates is uncommon. S. pseudoporcinus colonization in pregnancy is independently associated with African American race, tobacco use, and body mass index ≥35. Preterm premature rupture of membranes or spontaneous preterm birth was more common in patients colonized with S. pseudoporcinus. CONCLUSION: Although the prevalence of S. pseudoporcinus colonization is low, it primarily occurs in African American women and is associated with preterm premature rupture of membranes or spontaneous preterm birth when compared to individuals colonized with GBS.

Validation of Autoclave Protocols for Successful Decontamination of Category A Medical Waste Generated from Care of Patients with Serious Communicable Diseases.

In response to the Ebola outbreak in 2014, many hospitals designated specific areas to care for patients with Ebola and other highly infectious diseases. The safe handling of category A infectious substances is a unique challenge in this environment. One solution is on-site waste treatment with a steam sterilizer or autoclave. The Johns Hopkins Hospital (JHH) installed two pass-through autoclaves in its biocontainment unit (BCU). The JHH BCU and The Johns Hopkins biosafety level 3 (BSL-3) clinical microbiology laboratory designed and validated waste-handling protocols with simulated patient trash to ensure adequate sterilization. The results of the validation process revealed that autoclave factory default settings are potentially ineffective for certain types of medical waste and highlighted the critical role of waste packaging in successful sterilization. The lessons learned from the JHH validation process can inform the design of waste management protocols to ensure effective treatment of highly infectious medical waste.

Characteristics and Treatment Outcomes of Propionibacterium acnes Prosthetic Shoulder Infections in Adults.

Background. Prosthetic joint infections (PJIs) significantly complicate joint arthroplasties. Propionibacterium acnes is an increasingly recognized PJI pathogen, yet limited clinical and therapeutic data exist. We sought to examine characteristics of P. acnes shoulder PJIs and compare surgical and nonsurgical management outcomes. Methods. A retrospective analysis of P. acnes shoulder PJIs was conducted at an academic center in Baltimore, Maryland from 2000 to 2013. Results. Of 24 cases of P. acnes shoulder PJIs, 92% were diagnosed after extended culture implementation; 42% in the delayed and 46% in the late postsurgical period. Joint pain and diminished function were the predominant presenting clinical signs. Erythrocyte sedimentation rate and C-reactive protein elevations occurred in 47% and 44%, respectively. All tested isolates were susceptible to ß-lactams, moxifloxacin, vancomycin, and rifampin. Clindamycin resistance was identified in 6%. Of the antibiotic-only treated cases, 67% had a favorable clinical outcome compared with 71% (P = 1.0) of cases with a combined antibiotic-surgical approach. Favorable outcome with and without rifampin therapy was 73% and 60% (P = .61), respectively. Conclusions. Propionibacterium acnes PJI diagnoses increased with extended culture. Inflammatory markers were elevated in a minority of cases. Isolates maintained broad antimicrobial susceptibility. Compared to combined antibiotic-surgical approaches, antibiotic-only approaches were similarly successful in selected cases.

Practical Utility and Accuracy of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Corynebacterium Species and Other Medically Relevant Coryneform-Like Bacteria.

BACKGROUND: Corynebacterium species and gram-positive coryneform-like bacteria (coryneforms) are increasingly reported causes of opportunistic infections in immunocompromised patients. Biochemical identification methods for these bacteria are often inaccurate. We evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for routine Corynebacterium and coryneform identification. METHODS: A total of 286 Corynebacterium species and coryneforms recovered from patients were identified by MALDI-TOF MS analysis using the Bruker Microflex instrument, Biotyper software version 3.0, and database version 3.1.66 (Bruker Daltonics, Billerica, MA) following formic acid-based, direct on-plate extraction. The spectral cutoff scores used for interpretation were 2.0 or more for species-level identification and 1.7 or more for genus level. Scores lower than 1.7 were considered as no reliable identification. The results were compared with API Coryne (bioMérieux, Durham, NC) and sequencing of 16S ribosomal RNA genes. RESULTS: Of the 231 Corynebacterium (19 species), 99.6% were correctly identified to the genus level and 88.7% to the species level. Of the 55 coryneforms (14 genera), 90.9% were correctly identified to the genus level and 67.3% to the species level. API Coryne was able to identify 89.2% of Corynebacterium species (species level) and 63.6% of coryneforms (genus level). CONCLUSIONS: Rapid on-plate testing yielded identification of more Corynebacterium species and related bacteria than biochemical methods.

Recognition of Streptococcus pseudoporcinus Colonization in Women as a Consequence of Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Group B Streptococcus Identification.

During a 14-month period of using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for group B streptococcus (GBS) identification, we recovered 32 (1%) Streptococcus pseudoporcinus isolates from 3,276 GBS screening cultures from female genital sources (25 isolates from pregnant women and 7 from nonpregnant women). An additional two S. pseudoporcinus isolates were identified from a urine culture and a posthysterectomy wound culture. These isolates were found to cross-react with three different GBS antigen agglutination kits, PathoDx (Remel) (93%), Prolex (Pro-Lab Diagnostics) (38%), and Streptex (Remel) (53%). New approaches to bacterial identification in routine clinical microbiology laboratories may affect the prevalence of S. pseudoporcinus.

Impact of Toxigenic Clostridium difficile Colonization on the Risk of Subsequent C. difficile Infection in Intensive Care Unit Patients.

BACKGROUND: Clostridium difficile infection (CDI) in hospitalized patients is generally attributed to the current stay, but recent studies reveal high C. difficile colonization rates on admission. OBJECTIVE: To determine the rate of colonization with toxigenic C. difficile among intensive care unit patients upon admission as well as acquired during hospitalization, and the risk of subsequent CDI. METHODS: Prospective cohort study from April 15 through July 8, 2013. Adults admitted to an intensive care unit within 48 hours of admission to the Johns Hopkins Hospital, Baltimore, Maryland, were screened for colonization with toxigenic C. difficile. The primary outcome was risk of developing CDI. RESULTS: Among 542 patients, 17 (3.1%) were colonized with toxigenic C. difficile on admission and an additional 3 patients were found to be colonized during hospitalization. Both colonization with toxigenic C. difficile on admission and colonization during hospitalization were associated with an increased risk for development of CDI (relative risk, 10.29 [95% CI, 2.24-47.40], P=.003; and 15.66 [4.01-61.08], P<.001, respectively). Using multivariable analysis, colonization on admission and colonization during hospitalization were independent predictors of CDI (relative risk, 8.62 [95% CI, 1.48-50.25], P=.017; and 10.93 [1.49-80.20], P=.019, respectively), while adjusting for potential confounders. CONCLUSIONS: In intensive care unit patients, colonization with toxigenic C. difficile is an independent risk factor for development of subsequent CDI. Further studies are needed to identify populations with higher toxigenic C. difficile colonization rates possibly benefiting from screening or avoidance of agents known to promote CDI.

The Bacteroides fragilis toxin gene is prevalent in the colon mucosa of colorectal cancer patients.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) produces the Bacteroides fragilis toxin, which has been associated with acute diarrheal disease, inflammatory bowel disease, and colorectal cancer (CRC). ETBF induces colon carcinogenesis in experimental models. Previous human studies have demonstrated frequent asymptomatic fecal colonization with ETBF, but no study has investigated mucosal colonization that is expected to impact colon carcinogenesis. METHODS: We compared the presence of the bft gene in mucosal samples from colorectal neoplasia patients (cases, n = 49) to a control group undergoing outpatient colonoscopy for CRC screening or diagnostic workup (controls, n = 49). Single bacterial colonies isolated anaerobically from mucosal colon tissue were tested for the bft gene with touch-down polymerase chain reaction. RESULTS: The mucosa of cases was significantly more often bft-positive on left (85.7%) and right (91.7%) tumor and/or paired normal tissues compared with left and right control biopsies (53.1%; P = .033 and 55.5%; P = .04, respectively). Detection of bft was concordant in most paired mucosal samples from individual cases or controls (75% cases; 67% controls). There was a trend toward increased bft positivity in mucosa from late- vs early-stage CRC patients (100% vs 72.7%, respectively; P = .093). In contrast to ETBF diarrheal disease where bft-1 detection dominates, bft-2 was the most frequent toxin isotype identified in both cases and controls, whereas multiple bft isotypes were detected more frequently in cases (P ≤ .02). CONCLUSIONS: The bft gene is associated with colorectal neoplasia, especially in late-stage CRC. Our results suggest that mucosal bft exposure is common and may be a risk factor for developing CRC.

Use of the "RAM" susceptibility testing method for rapid detection of clarithromycin resistance in the Mycobacterium avium complex.

Standard susceptibility testing of the Mycobacterium avium complex (MAC) can require 7 to 14 days from initial isolation. We evaluated a high-performance liquid chromatography-based susceptibility test for rapid determination of clarithromycin (CLR) resistance in MAC. This method can be completed in 72 h of incubation. A total of 110 MAC strains were tested using the following concentrations of CLR: 4, 16, and 64 microg/mL, for a total of 330 tests. Microbroth dilution was used as the reference method. Rapid analysis of mycolic acid ("RAM") concordance with the reference method for CLR susceptibility was 98% (254/258) and 100% for CLR resistance (72/72). The 4 discordant results occurred with 2 strains, which demonstrated intermediate resistance with an MIC of 16 microg/mL. This study demonstrates that "RAM"-based susceptibility testing for determination of CLR resistance in MAC is both rapid and accurate, providing a significant reduction in turn-around-time from 7 to 14 days to 72 h of incubation.