Sanchita Das

Modified Carbapenem Inactivation Method for Phenotypic Detection of Carbapenemase Production among Enterobacteriaceae

ABSTRACT The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. All existing methods have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cutoff that best discriminated between CP-CRE and members of the family Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole-genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [95% CI], 93% to 100%), and the specificity was 100% (95% CI, 82% to 100%). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 h and excellent reproducibility across laboratories.

Impact of Detection, Education, Research and Decolonization without Isolation in Long-term care (DERAIL) on methicillin-resistant Staphylococcus aureus colonization and transmission at 3 long-term care facilities

We tested infection prevention strategies to limit exposure of long-term care facility residents to drug-resistant pathogens in a prospective, cluster randomized 2-year trial involving 3 long-term care facilities (LTCFs) using methicillin-resistant Staphylococcus aureus (MRSA) as a model. We hypothesized that nasal MRSA surveillance using rapid quantitative polymerase chain reaction and decolonization of carriers would successfully lower overall MRSA colonization. In year 1, randomly assigned intervention units received decolonization with nasal mupirocin and chlorhexidine bathing and enhanced environmental cleaning with bleach every 4 months. Newly admitted MRSA nares-positive residents were decolonized on admission. Control units were screened but not decolonized. All units received periodic bleach environmental cleaning and instruction on hand hygiene. In year 2, all units followed intervention protocol caused by failure of the cluster randomized approach to sufficiently segregate patients. MRSA colonization was monitored using point prevalence testing every 4-6 months. Colonization status at admission and discharge was performed 1 quarter per year to determine acquisition. Fisher exact test was used for statistical analysis. Baseline MRSA colonization rate was 16.64%. In year 1, the colonization rate of intervention units was 11.61% (P = .028) and 17.85% in control units (P = .613) compared with baseline. Intervention unit rate difference compared with the controls was significant (P = .001). In year 2, the colonization rate was 10.55% (P < .001) compared with baseline. The transmission rates were 1.66% and 3.52% in years 1 and 2, respectively (P = .034). The planned interventions of screening and decolonization were successful at lowering MRSA colonization.

Total Laboratory Automation and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Improve Turnaround Times in the Clinical Microbiology Laboratory: a Retrospective Analysis

ABSTRACT Technological advances have changed the practice of clinical microbiology. We implemented Bruker matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and BD Kiestra total laboratory automation (TLA) 4 and 3 years ago, respectively. To assess the impact of these new technologies, we compared turnaround times (TATs) for positive and negative urine cultures before and after implementation. In comparison I, TATs for 61,157 urine cultures were extracted for two periods corresponding to pre-TLA and post-TLA, both using MALDI-TOF MS for organism identification. In comparison II, time to organism identification (ID) and antimicrobial susceptibility (AST) reports were calculated for 5,402 positive culture reports representing four different periods: (i) manual plating and conventional biochemical identification (CONV), (ii) manual plating and MALDI-TOF MS identification (MALDI), (iii) MALDI-TOF MS identification and early phase implementation of TLA (TLA1), and (iv) MALDI-TOF MS identification and late phase implementation of TLA (TLA2). By the comparison I results, median pre- and post-TLA TATs to organism IDs (18.5 to 16.9 h), AST results (41.8 to 40.8 h), and preliminary results for negative cultures (17.7 to 13.6 h), including interquartile ranges for all comparisons, were significantly decreased post-TLA (P < 0.001). By the comparison II results, MALDI significantly improved TAT to organism ID compared to CONV (21.3 to 18 h). TLA further improved overall TAT to ID (18 to 16.5 h) and AST (42.3 to 40.7 h) results compared to MALDI (P < 0.001). In summary, TLA significantly improved TAT to organism ID, AST report, and preliminary negative results. MALDI-TOF MS significantly improved TAT for organism ID. Use of MALDI-TOF MS and TLA individually and together results in significant decreases in microbiology report TATs.

Nasal Carriage of Epidemic Methicillin-Resistant Staphylococcus aureus 15 (EMRSA-15) Clone Observed in Three Chicago-Area Long-Term Care Facilities

ABSTRACT The spread of pandemic methicillin-resistant Staphylococcus aureus (MRSA) clones such as USA300 and EMRSA-15 is a global health concern. As a part of a surveillance study of three long-term care facilities in the Greater Chicago area, phenotypic and molecular characterization of nasal MRSA isolates was performed. We report a cluster of pandemic EMRSA-15, an MRSA clone rarely reported from the United States, detected during this study.

The new frontier of diagnostics: Molecular assays and their role in infection prevention and control

&NA; Recent advances in technology over the last decade have propelled the microbiology laboratory into a pivotal role in infection prevention and control. The rapid adaptation of molecular technologies to the field of clinical microbiology now greatly influences infectious disease management and significantly impacts infection control practices. This review discusses recent developments in molecular techniques in the diagnosis of infectious diseases. It describes the basic concepts of molecular assays, discusses their advantages and limitations, and characterizes currently available commercial assays with respect to cost, interpretive requirements, and clinical utility.

Active Surveillance and Decolonization Without Isolation Is Effective in Preventing Methicillin-Resistant Staphylococcus aureus Transmission in the Psychiatry Units

Background  Control of methicillin-resistant Staphylococcus aureus (MRSA) is difficult in select populations. We used molecular typing to study the effect of universal surveillance and decolonization of carriers, without isolation, on MRSA transmission in a specialized unit. Methods  Patients admitted to the unit were screened for nasal MRSA at admission and discharge. Those who acquired MRSA during their stay were identified and linked to carriers with shared time in unit. Molecular typing of isolates was performed to identify transmission. Results  Of 3285 admissions, 82% were tested for MRSA nasal carriage; the discharge screening compliance was 64.7%. Admission prevalence was 2.3% among patients screened, and 7 (0.42%) acquired nasal MRSA during their stay. All patients who acquired MRSA shared time in the unit with a colonized patient. There were 3.9 MRSA acquisitions per 1000 at-risk days. Isolates from 5 patients that acquired MRSA during their stay as well as their potential donors (11 donor: recipient patient pairs) were available for typing. Pulsed-field gel electrophoresis matched 1 acquisition isolate to a colonized patient isolate. There were no MRSA infections during the study period. Conclusions  Despite less than perfect nasal screening compliance and exemption from traditional isolation precautions, acquisition of MRSA was 0.42% in this patient population over a course of 4.75 years, including a single case of acquisition, genetically similar to a known potential donor source. Screening for MRSA colonization and decolonizing of carriers was sufficient in reducing transmission in this vulnerable population.

Utilization and Performance of a Laboratory Developed Nucleic Acid Amplification Test for the Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in a Low Prevalence Area: A 14 Year Study.

Abstract Background Tuberculosis (TB) is a significant global health problem. Nucleic acid amplification tests (NAATs) are valuable in reducing delays to initiation of therapy and infection control protocols. A retrospective study was performed to assess the utilization and performance of a laboratory developed Mycobacterium tuberculosis complex (MTBC) PCR assay (TBPCR) for diagnosis of pulmonary (PTB) and extrapulmonary (EPTB) tuberculosis. Methods Study site was a 4 hospital system in suburban Chicago. All culture confirmed TB specimens with complete laboratory data from January 2002 to December 2016 were included. Patient records were accessed using an electronic data warehouse, following approval from Institutional Review Board. Standard microbiology procedures were followed for smear and culture of MTBC. A lab-developed real time PCR targeting a 123 bp region of the IS6110 insertion sequence of MTBC was performed on smear positive specimens or if ordered by physician. Clinical and laboratory data was compared with TBPCR results for all culture confirmed cases. Results There were 151 culture positive patients and 2186 TBPCR performed. Median age of patients at diagnosis was 49 years (IQR 33–66), 74 (49%) were female and 14 were on immunosupressive therapy. The mean number of samples tested per patient was 2. Of culture positive specimens, 59% were from a respiratory source and 3 were MDR; ordering of TBPCR was higher in specimens from PTB source (58.4%) as compared with EPTB source (37%). Combined sensitivity of the TBPCR on all specimen types was 86.6% (95% CI 76.3–93.1); 90.3% for PTB specimens alone (95% CI 78.2–96.4). Specificity was 100% (95% CI 99.5–100), PPV 100% (95% CI 90.5–100%) and NPV 99.5% (95% CI 98.8–99.8%), and were similar for all specimen types. Sensitivity of TBPCR was 97% in smear positive and 79% in smear negative PTB specimens. The median time to culture positivity was 7 days longer in specimens that were TBPCR negative compared with those that were positive (P = 0.14, NS), however, TBPCR shortened time to diagnosis by 13 days. Conclusion We found TBPCR to be underutilized in both PTB and EPTB although it was found to be a rapid and reliable method for early diagnosis. Education regarding utility of NAATs could be useful in low burden areas where paucibacillary disease is more common, especially in EPTB. Disclosures All authors: No reported disclosures.

Multicenter Evaluation of the Modified Carbapenem Inactivation Method and the Carba NP for Detection of Carbapenemase-Producing Pseudomonas aeruginosa and Acinetobacter baumannii

ABSTRACT The purpose of this study was to develop the modified carbapenem inactivation method (mCIM) for the detection of carbapenemase-producing Pseudomonas aeruginosa (CP-PA) and carbapenemase-producing Acinetobacter baumannii (CP-AB) and perform a multicenter evaluation of the mCIM and Carba NP tests for these nonfermenters. Thirty P. aeruginosa and 30 A. baumannii isolates previously characterized by whole-genome sequencing from the CDC-FDA Antibiotic Resistance Isolate Bank were evaluated, including CP isolates (Ambler class A, B, and D), non-carbapenemase-producing (non-CP) carbapenem-resistant isolates, and carbapenem-susceptible isolates. Initial comparison of a 1-μl versus 10-μl loop inoculum for the mCIM was performed by two testing sites and showed that 10 μl was required for reliable detection of carbapenemase production among P. aeruginosa and A. baumannii. Ten testing sites then evaluated the mCIM using a 10-μl loop inoculum. Overall, the mean sensitivity and specificity of the mCIM for detection of CP-PA across all 10 sites were 98.0% (95% confidence interval [CI], 94.3 to 99.6; range, 86.7 to 100) and 95% (95% CI, 89.8 to 97.7; range, 93.3 to 100), whereas the mean sensitivity and specificity among CP-AB were 79.8% (95% CI, 74.0 to 84.9; range, 36.3 to 95.7) and 52.9% (95% CI, 40.6 to 64.9; range, 28.6 to 100), respectively. At three sites that evaluated the performance of the Carba NP test using the same set of isolates, the mean sensitivity and specificity of the Carba NP test were 97.8% (95% CI, 88.2 to 99.9; range, 93.3 to 100) and 97.8% (95% CI, 88.2 to 99.9; range, 93.3 to 100) for P. aeruginosa and 18.8% (95% CI, 10.4 to 30.1; range, 8.7 to 26.1) and 100% (95% CI, 83.9 to 100; range, 100) for A. baumannii. Overall, we found both the mCIM and the Carba NP test to be accurate for detection of carbapenemase production among P. aeruginosa isolates and less reliable for use with A. baumannii isolates.

Leishmania donovani Tryparedoxin Peroxidases: functional involvement during infection and drug treatment

ABSTRACT The parasite Leishmania donovani causes visceral leishmaniasis, a potentially fatal disease. The parasites survive within mammalian macrophages and express a unique set of enzymes, the tryparedoxin peroxidases, for their defense against oxidative stress generated by the host. In this study, we demonstrate different roles of two distinct enzymes, the mitochondrial tryparedoxin peroxidase (mTXNPx) and the cytosolic tryparedoxin peroxidase (cTXNPx), in defending the parasites against mitochondrial and exogenous oxidative stress during infection and drug treatment. Our findings indicate a greater increase in cTXNPx expression in response to exogenous oxidative stress and a higher elevation of mTXNPx expression in response to mitochondrial or endogenous stress created by respiratory chain complex inhibitors. Overexpression of cTXNPx in Leishmania showed improved protection against exogenous stress and enhanced protection against mitochondrial stress in parasites overexpressing mTXNPx. Further, parasites overexpressing cTXNPx infected host cells with increased efficiency at early times of infection compared to control parasites or parasites overexpressing mTXNPx. The mTXNPx-overexpressing parasites maintained higher infection at later times. Higher mTXNPx expression occurred in wild-type parasites on exposure to miltefosine, while treatment with antimony elevated cTXNPx expression. Parasites resistant to miltefosine or antimony demonstrated increased expression of mTXNPx, as well as cTXNPx. In summary, this study provides evidence of distinct roles of the two enzymes defined by virtue of their localization during infection and drug treatment.