Evaluation of the NG-Test CARBA 5 Lateral Flow Assay with an IMP-27-Producing Morganella morganii and Other Morganellaceae

Abstract

An isolate of Morganella morganii (MMOR1) that tested susceptible to 3^rd/4^th-generation cephalosporins and intermediate to meropenem was characterized as positive for NDM and IMP carbapenemases by NG-Test CARBA 5. Our objective was to further investigate this result, given the inconsistent susceptibility profile and unusual epidemiological profile for our region. The MMOR1 isolate was retested for antimicrobial susceptibilities and characterized for carbapenemase production. MMOR1 tested susceptible to ceftazidime, ceftriaxone, cefepime, aztreonam, and ertapenem, and intermediate to meropenem and imipenem. The isolate tested positive by carbapenem inactivation method (CIM) and CIM+EDTA (eCIM) testing, indicating metallo-β-lactamase production. The isolate tested negative for all carbapenemase genes on Xpert Carba-R, but positive for IMP on repeat testing of NG-Test CARBA 5. Whole-genome sequencing revealed MMOR1 contained bla_IMP-27, but no other carbapenemase genes. Additional testing with NG-Test CARBA 5 revealed a false-positive NDM band when the assay was overloaded with test inoculum. Supplementary isolates were tested with an overloaded inoculum (n = 6 M. morganii; n = 1 P. mirabilis; n = 1 IMP-27-producing P. rettgeri; n = 1 IMP-1-producing E. coli; n = 1 K. pneumoniae), and two non-carbapenemase-producing carbapenem non-susceptible M. morganii also generated a false-positive NDM band; though, this was not universal among this species. A dual IMP+/NDM+ M. morganii is an unusual result that should prompt additional investigation, especially in nonendemic regions and when the susceptibility profile is incompatible. IMP-27 is not detected by Xpert Carba-R but is variably detected by NG-Test CARBA 5. The microorganism inoculum used for NG-Test CARBA 5 must be carefully controlled for accurate results. IMPORTANCE The detection of carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) is an important function of the clinical microbiology laboratory, where positive identifications have immediate implications for infection control and surveillance strategies in the inpatient setting and can inform appropriate selection of therapy among the various novel anti-CP-CRE agents. NG-Test CARBA 5 is a relatively new lateral flow assay used for detection of carbapenemases in CP-CRE. Here, we describe the characterization of a Morganella morganii isolate that generated a false-positive NDM carbapenemase detection by this assay, and perform bacterial test inoculum experiments with additional isolates to further investigate a cause of false-positive results using the NG-Test CARBA 5. While a lateral flow assay like the NG-Test CARBA 5 is a very desirable test format for clinical laboratories, there are pitfalls to avoid when performing this test and interpreting results, including recognizing an overloaded test assay, which could lead to false-positive results.

Keywords: Enterobacterales; IMP-27; Morganella morganii; NG-Test CARBA 5; carbapenemase; lateral flow assay; metallo-β-lactamase.

FIG 1

Workflow for M. morganii MMOR1 isolate evaluation and NG-Test CARBA 5 evaluation. The submitting clinical laboratory initially characterized the MMOR1 isolate by AST and the NG-Test CARBA 5, where it originally tested as IMP+/NDM+ by NG-Test CARBA 5. The isolate was referred to our facility, where follow-up testing included phenotypic methods (AST; CIM and eCIM; and repeat NG-Test CARBA 5) and genotypic methods (Xpert Carba-R and WGS). Additional testing on NG-Test CARBA 5 was performed using varying starting inoculum sizes, including an “extra heavy” inoculum of MMOR1 and other isolates sharing features in common with MMOR1. This extra heavy inoculum led to a faint false-positive NDM band (indicated in red) in MMOR1 and two of 10 additional isolates that were tested. AST, antimicrobial susceptibility testing; CRO, ceftriaxone; CAZ, ceftazidime; FEP, cefepime; ETP, ertapenem; IPM, imipenem; MEM, meropenem; CIM, carbapenem inactivation method; eCIM, CIM with EDTA; WGS, whole-genome sequencing. False results are indicated in red. Bacteria, petri dish, PCR, and DNA icons were obtained from The Noun Project (https://thenounproject.com).

FIG 2

NG-Test CARBA 5 results for the MMOR1 isolate tested with different inoculum sizes. The NG-Test CARBA 5 lateral flow assay was performed according to the package insert, or the test inoculum was varied; representative images of inoculum and corresponding results are shown. (A and B) light inoculum (touch 1 area of growth with a 10-μL loop), (C and D) standard inoculum (touch 3 areas of growth with a 10-μL loop), (E and F) heavy inoculum (touch 6 areas of growth with a 10-μL loop), (G and H) extra heavy inoculum (1/2 of a 10-μL loop). (I) extra heavy inoculum tested on a separate day with a different lot number of NG-Test CARBA 5 (biological replicate). (J) same test cartridge in panel (I) after several minutes extra incubation (i.e., read beyond the standard 15 min incubation time, to demonstrate how the cartridge background becomes more “typical” appearing if the test is read after the correct incubation window). The red asterisk highlights the false-positive NDM band that appeared when an extra heavy inoculum was tested.

FIG 3

Representative NG-Test CARBA 5 results for other isolates tested with an “extra heavy” inoculum. Additional isolates were tested with an extra heavy inoculum (1/2 of 10-μL loop) on NG-Test CARBA 5, and the following representative examples are shown: (A) NDM-positive K. pneumoniae ATCC BAA-2146 (standard inoculum testing of a true-positive NDM isolate is shown for visual comparison to other LFAs), (B) IMP-positive E. coli NCTC 13476, (C) IMP-27-positive PR1, (D) KPC-positive MMOR6, (E) non-carbapenemase-producing MMOR5, and (F) technical replicate of MMOR5, (G) non-carbapenemase-producing MMOR3, and (H) technical replicate of MMOR3. The red asterisk highlights the false-positive NDM band that appeared in two of 10 other isolates tested with an extra heavy inoculum.