Rapid and economical detection of eight carbapenem-resistance genes in Enterobacteriaceae, Pseudomonas spp, and Acinetobacter spp directly from positive blood cultures using an internally controlled multiplex-PCR assay

To the Editor—The World Health Organization (WHO) has recognized carbapenem-resistant Enterobacteriaceae (CRE), Pseudomonas aeruginosa (CRPsA), and Acinetobacter baumannii (CRAB) as critical pathogens that cause significant morbidity and mortality in patients with bloodstream infections (BSIs), especially in healthcare settings. ^{1 , 2} Carbapenem resistance challenges empirical and targeted antibiotic treatment involving β-lactam antibiotics, including carbapenems, and it results in expensive and complicated alternative anti-infective strategies with polymyxin-B, colistin, tigecycline, and fosfomycin. ³ Despite their high cost (∼US$62 per test), commercially available PCR-based tests have been used to screen clinically important carbapenemases, known as the “Big 5” genes: metallo-β-lactamases (MBLs) (ie, NDM, IMP, and VIM), KPC, and the OXA-48 family. These tests use rectal swab samples with short turnaround times (TATs), which contribute significantly to infection control efforts. ^{2 , 4 , 5} Few validated tests are available to meet the acute need in BSI cases. ^{2 , 4} A rapid, inexpensive, and accessible testing tool for carbapenemase producing-carbapenem resistant organisms (CP-CRO) as pathogens of BSI is of paramount importance for early institution of effective antimicrobial therapy and infection prevention and its control. In the present study, a predesigned multiplex PCR assay was validated that targets the OXA carbapenemases (OXA-23, OXA-24/-143, and the OXA-58 family) commonly harbored by Acinetobacter spp, as well as with the “Big 5” genes, in DNA extracts of gram-negative bacilli positive blood-culture (BC) broths.

In this prospective study, consecutive BC broth samples (n = 415) were collected from patients (n = 278) with laboratory-confirmed BSIs at Tata Medical Center, Kolkata, India, from July 2017 to September 2018. Blood culturing was performed using the BacT/Alert 3D system (bioMérieux, Marcy-l’Étoile, France), and identification and susceptibility testing of BC isolates were performed using the Vitek2 system (bioMérieux). Carbapenem susceptibility results were interpreted following Clinical Laboratory Standard Institute (CLSI) guidelines. ⁶

DNA was extracted from positive BC broths using the alkali wash and heat lysis method. ⁷ Additionally, 20 µL McFarland 3 turbidity standard of A. baumannii ATCC19606 was added to 500 µL BC broth before extraction as an internal extraction control (IEC). Two multiplex PCRs were then performed to detect 8 carbapenemase genes (as described earlier) with a modification: CARBA (KPC, 353 base pairs [bp]; NDM, 603 bp; VIM, 437 bp; IMP, 387 bp; OXA-48 family, 265 bp) and OXA (OXA-23 family, 330 bp; OXA-24/143 family, 271 bp; OXA-58 family, 688 bp; with Acinetobacter-derived AmpC [ADC] as the IEC and PCR control, 1,059 bp). ⁸ The PCR amplicons were visualized on 3% prestained agarose gels. Bidirectional sequencing was performed using a 3500 DNA analyzer (Applied Biosystems, Foster City, CA) for confirmation. Analytical specificities of multiplex PCRs were validated using bacterial and fungal reference strains and clinical isolates. For assay standardization, cultured reference strains were suspended in normal saline to a density of 0.5 McFarland turbidity standard (∼1.5 × 10⁸ CFU/mL) using a Densimat (bioMerieux) followed by preparation of 10-fold serial dilutions ranging from 10⁷to 10² CFU/mL. Known negative BC samples (450 µL) incubated for 5 days were spiked with bacterial dilutions (50 µL), and DNA extraction followed by PCR was preformed to check analytical sensitivity.

Statistical performance indicators of the multiplex PCRs were determined using MedCalc (MedCalc Software, Mariakerke, Belgium) using carbapenem susceptibilities of Enterobacteriaceae, Pseudomonas spp, and Acinetobacter spp (EPA) as references. Non-EPA, intrinsically CRO, and uncultivable organisms were considered negative controls. Interrater agreement using Cohen’s κ was used to measure the concordance between culture and multiplex PCRs.

Of 153 CARBA-OXA multiplex PCR–positive samples, 122 had a single target (NDM, 48; OXA-48 family, 66; KPC, 2; VIM, 2; and OXA-23 family, 4) and 31 had double targets (NDM/OXA-48 family, 20; NDM/OXA-58 family, 5; KPC/VIM, 2; NDM/OXA-23 family, 2; NDM/VIM, 1; and OXA-48/OXA-58 family, 1) (Table 1). The analytical specificities of the PCRs were 100% for detecting the 8 carbapenemases; the sensitivities ranged between 500 and 5000 CFU/mL for different carbapenemases using spiked BC samples. Among 415 BC samples, accuracy of the assay was marginally improved (95.9% vs 96.9%) using both the “Big 5” CARBA and extended OXA panels (Supplementary Table 1 online). Discrepant results between the culture method and multiplex-PCRs were observed in 13 samples. The TAT for the detection of carbapenemases was <4 hours using BC positive broth, including DNA extraction (∼50 minutes), PCR amplification (∼120 minutes), and agarose-gel electrophoresis and documentation (∼30 minutes). The cost of consumables and reagents for running the assays was only ∼US$10 per sample.

Table 1. Distribution of Different Carbapenemases in Enterobacteriaceae, Pseudomonas spp, and Acinetobacter spp Isolated From Blood Samples

Note: ND, not detected by polymerase chain reaction assay.

Early and rapid detection of carbapenemases in common gram-negative bacilli is of paramount importance for optimizing antimicrobial therapy, as a tool for antimicrobial stewardship programs, for hospital infection control, and for improving patient outcomes with regard to morbidity, mortality, reducing length of hospitalization, and minimizing the cost of health care. Compared with commercial assays (eg, Carba R GeneXpert from Cepheid, Sunnyvale, CA), the cost of the current assay was significantly less with a convenient TAT (same-day result) using this noncommercial DNA extraction method and end-point multiplex PCR format. ^{4 , 5 , 9} In this study, we validated the diagnostic performance of internally controlled multiplex-PCRs targeting 8 carbapenemases in BC-positive samples. In the wake of increased carbapenem-based antimicrobial therapies, multiplex PCR may prove to be a useful tool for detecting carbapenem resistance and thus facilitating early infection control actions in clinical settings. The strengths of the current study include (1) demonstration of the molecular epidemiology of carbapenem resistance genes among a group of oncology patients in eastern India, (2) development of a rapid and cost effect test suitable for implementation in resource constrained settings; (3) application of molecular tests for gram negative bacteremia to optimize antibiotic therapy; (4) identification of potential targets for developing new drugs to treat NDM and OXA positive GNB infections, and (5) use of OXA-23/-24/-58 PCRs, which has been rarely investigated for diagnostic purposes apart from its importance as a marker in CRAB associated with outbreaks (OXA-23). ⁵ The implementation of this new, low-cost tool in resource-limited settings may enable better management of gram-negative sepsis. ¹⁰