Rapid Detection of Carbapenemase-Producing Multidrug-Resistant (MDR) Pathogens by Modified Carba NP Test in Ventilator-Associated Pneumonia (VAP) in Elderly Patients.

Background Ventilator-associated pneumonia (VAP) is defined as pneumonia that develops 48 hours or more after endotracheal intubation or tracheostomy and is brought on by infectious organisms that are not present or incubating during mechanical ventilation. Multidrug-resistant organisms originate primarily from the hospital environment and significantly contribute to ventilator-associated pneumonia. These organisms pose a severe threat, leading to a higher mortality rate due to their resistance to more potent antibiotics. Methods The study aims to assess the efficacy of the modified Carba NP test in detecting carbapenemase-producing bacteria in geriatric VAP patients. Results Forty (38 gram-negative and 2 gram-positive) pathogens were isolated from VAP patients. The isolates were identified using standard laboratory protocol; Acinetobacter spp. (n=16; 40% ), followed by Klebsiella pneumoniae (n=13; 32.5%), is the most common organism isolated. Seventeen (44.73%) were multi-drug resistant gram-negative bacteria. The carbapenemase producers were detected by the Kirby-Bauer disc diffusion method and compared with the modified Carba NP test with a turnaround time of 12-18 hrs in comparison to the disk diffusion test which requires additional 12hrs. Carbapenemase production was seen in 12 (70.59%) MDR isolates (7-Acinetobacter spp, 3-Klebsiella pneumonia, 1-Escherichia coli, and 1-Pseudomonas aeruginosa).  Conclusion Modified Carba NP can be used as a rapid test to detect carbapenemase production, and it can replace the traditional disk diffusion method of detecting carbapenemase production. This test plays a crucial role in the management of critical patients by saving 12-18 hours to determine the most appropriate and effective antibiotic treatment. This timely decision is essential in preventing sepsis caused by localized infections.

Evaluation of phenotypic detection of carbapenemase-producing Pseudomonas spp. from clinical isolates.

Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. Although the main mechanism of carbapenem-resistance in Pseudomonas aeruginosa is the loss of OprD porin, carbapenemases continue to be a problem worldwide. The aim of this study was to evaluate the performance of phenotypic tests (Carba NP, Blue Carba, and mCIM/eCIM) for detection of carbapenemase-producing Pseudomonas spp. in Brazil. One hundred twenty-seven Pseudomonas spp. clinical isolates from different Brazilian states were submitted to phenotypic and molecular carbapenemase detection. A total of 90 carbapenemase-producing P. aeruginosa and 5 Pseudomonas putida (35, blaVIM-2; 17, blaSPM-1; 2, blaIMP-10; 1, blaVIM-24; 1, blaNDM-1; 39, blaKPC-2). The phenotypic Carba NP, Blue Carba, and mCIM/eCIM showed sensitivity of 94.7%, 93.6%, and 93.6%, and specificity of 90.6%, 100%, and 96.8%, respectively. However, only the Carba NP presented the highest sensitivity and showed the ability in differentiating the carbapenemases between class A and class B using EDTA. Blue Carba failed to detect most of the class B carbapenemases, having the worst performance using EDTA. Our results show changes in the epidemiology of the spread of carbapenemases and the importance of their detection by phenotypic and genotypic tests. Such, it is essential to use analytical tools that faithfully detect bacterial resistance in vitro in a simple, sensitive, rapid, and cost-effective way. Much effort must be done to improve the current tests and for the development of new ones.

Intestinal Colonization Due to Carbapenem-Resistant Enterobacteriaceae Among Hematological Malignancy Patients in India: Prevalence and Molecular Charecterisation.

Faecal carriage of Carbapenem-resistant Enterobacteriaceae (CRE) is being observed as an important risk factor for bacteremia among patients with hematological malignancies. A prospective surveillance study was conducted among these patients to determine the gut colonization of CRE. Rectal/perianal swabs were collected to isolate CRE. Carbapenem resistance was detected by disk diffusion, modified-Hodge, Carba-NP test, and PCR for bla NDM-1, bla KPC, bla OXA-48, bla VIM, bla IMP genes. A total of 209 CRE isolates were identified from 151 patients. E. coli was the most common (83.2%) CRE identified, followed by Klebsiella spp. (9.6%). The majority of CRE were observed resistant to ertapenem (86%). bla NDM-1 was the most common gene (57.3%), followed by bla OXA-48 (37.8%). 26.8% isolates found to carry both bla NDM-1 and bla OXA-48 genes. CRE is increasingly observed to cause bacteremia among hematological malignancy patients due to increased colonization. Screening for gut CRE colonization is necessary to guide empirical therapy and apply infection control measures among these patients.

Performance Evaluation of Diagnostic Assays for Detection and Classification of Carbapenemase-Producing Organisms.

Rapid and accurate detection can help optimize patient treatment and improve infection control against nosocomial carbapenemase-producing organisms (CPO). In this study, a total of 217 routine clinical isolates (Enterobacterales and A. baumannii), including 178 CPOs and 39 non-CPOs, were tested to evaluate the performance of six phenotypic carbapenemase detection and classification assays, i.e., BD Phoenix CPO detect panel, Rapidec Carba-NP, O.K.N detection kit, and three carbapenem inactivation methods (CIMs; mCIM, eCIM, sCIM). The overall detection sensitivity and specificity were 98.78% (95.21-99.79%) and 79.49% (63.06-90.13%), respectively, for the BD phoenix CPO P/N test; 91.93% (86.30-95.45%) and 100% (88.83-100%), respectively, for the Rapidec Carba-NP; 98.06% (94.00-99.50%) and 97.44% (84.92-99.87%), respectively, for mCIM; and 96.89% (92.52-98.85%) and 94.87% (81.37-99.11%), respectively, for sCIM. The classification sensitivity and specificity for the BD phoenix CPO Ambler test, the O.K.N detection kit, and the mCIM and eCIM were 56.71% (48.75-64.34%) and 94.87% (81.37-99.11%), 99.28% (95.43-99.96%) and 100% (88.83-100%), and 92.90% (87.35-96.23%) and 97.44% (84.92-99.87%), respectively. All detection assays were reliable in detecting carbapenemase. However, the Rapidec Carba-NP and mCIM were insufficient in detecting OXA-48-like enzymes. The BD phoenix CPO detect panel had a strong ability to detect carbapenemase but failed to classify 48/59 (81.36%) KPC, 8/52 (15.38%) NDM, 8/22 (36.36%) OXA-23-like, and 6/11 (54.55%) dual enzymes. The O.K.N detection kit accurately detected and differentiated KPC, NDM, and OXA-48-like enzymes existing alone or in combination. The results of this study will support reliable laboratory work tools and promote therapeutic and infection control decisions.

Performance of distinct phenotypic methods for carbapenemase detection: The influence of culture media.

We evaluated the performance of five phenotypic tests [Modified Hodge Test (MHT); combined-disk test (CDT) using phenylboronic acid, EDTA, and cloxacillin; CarbaNP and CarbAcinetoNP; Blue-Carba, Carbapenembac™ and Carbapenembac Metallo™] for carbapenemase detection in Gram-negative bacilli (GNB). A total of 73 carbapenemase producers and 27 non-carbapenemase producers were tested. All GNB were subcultured onto Müeller-Hinton agar (MHA), MacConkey agar (MAC), and sheep blood agar (SBA). High sensitivity (100%) and specificity (100%) was observed for MHA using CarbaNP, Blue-Carba, and Carbapenembac™. The sensitivity and specificity of CarbaNP (98.6%/100%), Blue-Carba (97.1%/91.0%), and Carbapenembac™ (100%/96.5%) were slightly lower for SBA. In contrast, unacceptable sensitivity rates of CarbaNP (71.1%) and Blue-Carba (66.6%), but not Carbapenembac™ (97.3%), were observed for MAC. The colorimetric methods showed high sensitivity and specificity to detect carbapenemase production from isolates grown on MHA or SBA. However, colonies obtained from MAC must not be tested for carbapenemase detection by colorimetric methods.

Improved blood culture workflow for faster identification of KPC-producing Enterobacterales.

Carba-NP original report for blood cultures described the need of subculture and mechanical lysis before testing, reaching the turnaround time of approximately 4 hours for sample preparation. We tested 100 consecutive blood cultures positive for Gram-negative bacilli on the Gram stain from a large clinical laboratory. Bacterial pellets were prepared by centrifugation and submitted to Carba-NP and Blue-Carba tests and used further to prepare smears for Vitek MS. Results obtained with colonies grown on sheep blood agar using the same methodologies were used as the gold standard. Carbapenemase genes were confirmed by PCR and DNA sequencing. Vitek MS identified correctly 86% of the samples. Of note, 7% of the samples were incorrectly reported by the instrument as containing a single isolate. KPC-2 was the predominant carbapenemase detected. There was 100% concordance for both negative and positive results for Carba-NP. In contrast, for Blue-Carba the concordance for positive results was 92.8%, and 41% of strains negative for carbapenemases presented a yellowish color on control well turning the test non-interpretable. The turnaround time for sample preparation for preparing the pellet was 13 min, and no subculture or mechanical lysis is needed when detecting KPC production in Enterobacterales.

Rectal Carriage of Carbapenem-Resistant Enterobacteriaceae: A Menace to Highly Vulnerable Patients.

BACKGROUND: Bloodstream infection (BSI) due to carbapenem-resistant enterobacteriaceae (CRE) is the leading cause of morbidity and mortality in patients with hematological malignancy. These patients receive chemotherapy during treatment, which lead to severe mucositis of gastrointestinal tract and myelosuppression. It was hypothesized that the gut colonizer translocate into the blood circulation causing BSI. Colonization rate with CRE among these patients in India is unknown. AIM: This study aims to determine the carriage rate of CRE in cancer patients. SETTING AND DESIGN: A prospective study was conducted in a tertiary care hospital of India. MATERIALS AND METHODS: Rectal swab of 93 patients were collected and processed as per the Center for Disease Control and Prevention protocol for detection of CRE. The isolate CREs were identified by standard phenotypic tests and confirmed for carbapenem resistance by disk diffusion test using carbapenem disk (imipenem, meropenem, doripenem, and ertapenem), Carba-NP test and modified Hodge test. Resistant to any of the carbapenem disc is considered as CRE. RESULTS: A total of 86 isolates were detected from 93 patients. Seventy-six isolates were identified as CRE, and 10 isolates were Gram-positive cocci and other Gram-negative bacilli. Acute myeloid leukemia was the most common clinical presentation followed by acute lymphoid leukemia. Thirty-nine out of 93 patients were on chemotherapy. Sixty-seven out of 76 isolates of CRE were observed positive for carbapenemase production by Carba-NP test. CONCLUSION: This study highlights very high rate of CRE carriage among the hematological malignancy patients; who are highly vulnerable to infection. This confirms the need of infection control prevention activities among the hematological malignancy patients.

Carba NP as a simpler, rapid, cost-effective, and a more sensitive alternative to other phenotypic tests for detection of carbapenem resistance in routine diagnostic laboratories.

PURPOSE: Resistance to carbapenems due to carbapenemases has been increasingly noticed in Enterobacteriaceae. Clinical and Laboratory Standards Institute (CLSI) has recommended the latest Carba NP (CNP) test as a confirmatory test for carbapenemase production in Enterobacteriaceae. Low sensitivity of disk diffusion (DD) and modified Hodge test (MHT) may result in missing out of resistant strains which can adversely affect clinical management. The present study compares three phenotypic tests - CNP test, DD, and MHT for detection of carbapenemase production. MATERIALS AND METHODS: Four hundred consecutive, nonduplicate Enterobacteriaceae isolates were tested for carbapenem resistance using ertapenem disc (10 µg) by Kirby-Bauer DD method, MHT, and CNP. These tests were performed and interpreted as per the CLSI standards. CNP was considered to be the reference test for comparison. Sensitivity, specificity, and accuracy rates for ertapenem DD and MHT were calculated. RESULTS: One hundred and six out of 400 strains were positive by CNP test. Of the 294 CNP-negative strains, 28 were resistant by DD and 18 were resistant by MHT. Of the 106 CNP-positive strains, 82 were resistant and 16 were intermediate by DD while 76 were positive by MHT ertapenem DD had a sensitivity and specificity of 66.04% and 90.48%, respectively. Sensitivity and specificity of MHT were 54.72% and 93.88%, respectively. There was considerable discordance between all the three tests. CONCLUSION: As a rapid, simple, and cost-effective test with a greater capability greater to detect carbapenemase producers, CNP can be implemented in routine diagnostic laboratories, thereby benefiting patient care and antimicrobial stewardship.

Performance of CarbaNP and CIM tests in OXA-48 carbapenemase-producing Enterobacteriaceae.

This study applied two phenotypic tests, namely "Carbapenemase Nordmann-Poirel" (CarbaNP) test and "Carbapenem Inactivation Method" (CIM), against the isolates carrying the carbapenem resistance genes. The study included 83 carbapenem-resistant Enterobacteriaceae isolates producing oxacillinase-48 (OXA-48) and 30 carbapenem-sensitive Enterobacteriaceae isolates. Out of the total isolates studied, 77 isolates (92.77%) were identified as Klebsiella pneumoniae and six isolates (7.23%) were identified as Escherichia coli by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. Polymerase chain reaction (PCR) method used to detect resistance genes found that 74 isolates (89.16%) produced OXA-48 carbapenemase, whereas nine isolates (10.84%) produced both OXA-48 and New Delhi metallo-beta-lactamase-1 (NDM-1). The isolates producing both OXA-48 and NDM-1 were found to be positive by both phenotypic tests. Among isolates carrying only blaOXA-48 gene alone, nine isolates (13.04%) for CarbaNP test and two isolates for CIM test (2.90%) displayed false negative results, respectively. The sensitivity of CarbaNP and CIM tests was found to be 89.16% and 97.59%, respectively, whereas the specificity was determined to be 100% for both tests. These findings suggest that CarbaNP and CIM tests are useful tools to identify the carbapenemase producers. Molecular methods like PCR are recommended to verify false negative tests predicted to have OXA-48 activity.

A Pilot Study on Carbapenemase Detection: Do We See the Same Level of Agreement as with the CLSI Observations.

INTRODUCTION: Rapid identification of carbapenemase producing organisms is of great importance for timely detection, treatment and implementation of control measures to prevent the spread. The Modified Hodge Test (MHT) and Carba NP test is recommended by CLSI for the detection of carbapenemases in Enterobacteriaceae. However, MHT may give false positive results or fail to detect metallo ß-lactamases (MBLs). In the US, MHT is the most widely used test for detection of carbapenemases and has been found to have a sensitivity and specificity of >90% for bla KPC producers. However, in India, the prevalence of bla NDM is higher than bla KPC producers. AIM: To evaluate the usefulness of CarbaNP in an Indian setting. MATERIALS AND METHODS: A total of 260 isolates of carbapenem resistant E.coli (n=57), Klebsiella spp. (n=85), Pseudomonas aeruginosa (n=60), and Acinetobacter baumannii (58) isolated from clinical specimens between 2012-2014 at the Christian Medical College, Vellore were included in the study. All the carbapenem resistant isolates were subjected to CarbaNP, MHT and multiplex PCR for detection of carbapenemase genes. RESULTS: CarbaNP was found to be positive in 88% (n=50/57), 81% (n=69/51), 38% (n=23/60) and 81% (n=47/58) for E.coli, Klebsiella spp., P. aeruginosa, and A. baumannii respectively. While in MHT it showed, 89% (n=51/57) and 81 % (n=69/85) for E.coli and Klebsiella spp. respectively. In P.aeruginosa, synergy testing of imipenem plus cloxacillin showed that, 65% of CarbaNP negatives were ampC producers. Overall, the sensitivity and specificity of CarbaNP was found to be 94% and 100 for bla NDM; 77% and 100 % for bla OXA-48 like producers and 81% and 100% for CarbAcinetoNP respectively. CONCLUSION: This observation was more than what was reported in CLSI guidelines. Therefore, it is advisable to evaluate an assay for better laboratory diagnosis at respective regions.

The implications of endemic IMP-4 carbapenemase for clinical laboratory susceptibility testing.

A local predominance of carbapenemase producing Enterobacteriaceae with low minimum inhibitory concentrations (MIC) to meropenem prompted a review of methods available for carbapenemase detection. We report on results using two selective media, temocillin discs, CarbaNP test, GeneXpert Carba-R assay and an in-house PCR assay.

First report of OXA-48-producing Klebsiella pneumoniae strains in Iran.

Carbapenem-resistant Enterobacteriaceae are increasingly reported worldwide and cause therapeutic problem in health care facilities. In this study 28 imipenem-resistant K. pneumoniae were examined for expression of carbapenemases by phenotypic and genotypic methods. Modified Hodge Test (MHT), CarbaNP test were used for phenotypic detection, and PCR using specific primers for the detection of bla OXA-48 -, bla KPC -, bla NDM - and bla VIM -type carbapenemases with specific primers were performed. MHT and CarbaNP tests were positive for all of imipenem-resistant K. pneumoniae. The bla OXA-48 gene was detected in 27/28 isolates. One isolate was positive for the presence of the bla VIM-4 gene. According to our results NP test and MHT have high sensitivity and specificity for detection of those carbapenemases. This study reports the first cases of OXA-48-producing K. pneumoniae in Iran.