The emergence and prevalence of carbapenem-resistant gram-negative bacteria (CR-GNB) are gradually becoming a threat to public health, with carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Acinetobacter baumannii being classified as the highest-priority (i.e., critical) pathogens by the World Health Organization (WHO) (1). Infections caused by CRE usually result in high mortality and poor prognosis due to the ability of carbapenemases to hydrolyze all β-lactam antibiotics resulting in few antibiotics retaining activity against CRE (2). Therefore, without timely early treatment and infection control, patients will have an increased risk of developing mortality. And for specific high-volume areas, it will only increase the difficulty of treatment, prolong hospitalization, and aggravate the pressure of hospital infection prevention and control, which will continue to deteriorate week after week. Thus, it is clinically critical to diagnose carbapenemase infections as soon as possible. So, we need simple, rapid, effective and inexpensive techniques to screen infected patients for the presence of these pathogens.

Researchers systematically categorized the carbapenemases into four groups: A, B, C, and D. Group A included most of the KPC strains; group B included many VIM, IMP, and NDM strains with carbapenemase activity detected from gram-negative bacteria; group C included AmpC β-lactamase, but it can play a role in the hydrolysis of carbapenemase only under special circumstances (osmotic abnormality); and group D included the OXA-48 type of Enterobacteriaceae bacteria (1). In summary, under normal conditions, classes A, B, and D are able to exert carbapenemase activity, thereby achieving resistance.

The modified Carbapenem Inactivation Method combined with the EDTA Carbapenem Inactivation Method (mCIM/eCIM) recommended by the Clinical and Laboratory Standardization Institute (CLSI) guidelines has excellent precision in carbapenemase assays but at the expense of a lengthy turnaround time (TAT) (3). Currently, most of the tests for carbapenemases in clinics and laboratories are biochemical and molecular, including phenotypic analysis of carbapenemase activity, polymerase chain reaction (PCR) assays of carbapenemase genes, commercial microarray assays (1), whole-genome sequencing (WGS), and assay techniques such as Xpert Carba-R and NG-Test Carba 5. Compared with NG-Test Carba 5, conventional assay technologies still have significant drawbacks.

In addition, the NG-Test Carba 5 is a novel immunochromatographic assay evaluated for use in many studies (4–7) that qualitatively detects carbapenemases such as KPC, NDM, IMP, VIM, OXA-48, etc., through the particular binding of the antigen to monoclonal antibodies; detects carbapenemase typing, which requires only 20 min; and is characterized by ease of operation, quick detection speed, and other qualities. According to a study by Han et al. (2), the NG-Test Carba 5 performed well overall, with a sensitivity and specificity ranging from 95.3% to 100% and 97.3% to 100%, respectively, on colonies of bacteria and positive cultures of blood. The NG-Test Carba 5 was shown to be more efficient in terms of turnaround time, initial and ongoing expenses, and sensitivity in a study by Kanahashi et al. (4). The Xpert Carba-R assay was not as effective as the NG-Test Carba 5. For certain strains that test negative, there is a chance that the Carba 5 NG-Test will go undetected.

To conduct evidence-based research and provide a thorough assessment of the detection ability of carbapenemase-producing gram-negative bacteria (CP-GNB) by the NG-Test Carba 5, a meta-analysis was performed in this study. The research group searched PubMed for meta-analysis papers related to this topic, but there were no such papers. This is the first study to apply evidence-based medical evidence, such as the sensitivity and specificity of the NG-Test Carba 5 for the clinical detection of CP-GNB, and we hope that this study offers fresh perspectives on the rapid clinical detection of GNB resistance by the NG-Test Carba 5 and contributes to the development of the field.

It is worth mentioning that the novelty of this study compared with the existing researches is that the researchers designed three subgroups, namely, detection outcome (Group A), bacterial species (Group B) and bacterial isolation methods (Group C). For the first time, we provided a statistical reference for the bacterial isolation methods of NG-Test Carba 5 in clinical application, as well as a comprehensive analysis of the accuracy and differences between different subgroups, so that NG-Test Carba 5 can further improve its own detection performance to provide a certain evidence-based basis.

Currently, because there are few effective antibiotics available and because of the significant rates of morbidity and mortality, infections caused by CR-GNB are becoming more severe and are posing a serious threat to human health. According to one study (39), Asia and Africa are the two regions in the globe where carbapenem-resistant Enterobacteriaceae are most prevalent. Early diagnosis and intervention of CR-GNB can significantly reduce mortality as well as economic burden. As of this study, there is no meta-analysis evaluating evidence-based medical evidence, such as the sensitivity and specificity of the NG-Test Carba 5 for the clinical detection of CR-GNB. Therefore, this meta-analysis focused on the accuracy of the NG-Test Carba 5 for the rapid clinical identification of CR-GNB, aiming to provide strong evidence for early clinical diagnosis, infection control and mortality reduction.

In this study, researchers obtained data from 9,153 samples from 37 articles. The diagnosis of carbapenemase-type KPC, NDM, VIM, IMP, and OXA-48-like strains was performed by the immunochromatographic method NG-Test Carba 5 using the gold standard for PCR. Researchers found that the pooled sensitivity, specificity, PLR, NLR, and DOR for the NG-Test Carba 5 method were 0.97 [95% CI (0.97, 0.98)], 0.99 [95% CI (0.99, 1.00)], 65.38 [95% CI (36.73, 116.39)], 0.03 [95% CI (0.02, 0.05)], and 2734.42 [95% CI (1,464.05, 5,107.12], respectively. This indicates that the NG-Test Carba 5 has a high degree of sensitivity and specificity. Based on this gold standard, the area under the SROC curve, i.e., the AUC, was 0.9976, which is close to 1, and the Q-index was 0.9824 (SE = 0.0029), suggesting that the NG-Test Carba 5 has high accuracy in diagnosing carbapenemase typing. This is consistent with the findings of Saito et al. (5), Mendez-Sotelo et al. (8), Khoo et al. (6), Josa et al. (7), Hopkins et al. (40). Based on the analysis of the above data, it can be concluded that the NG-Test Carba 5 may be a reliable method for detecting carbapenemases regardless of enzyme typing or bacterial genus.

Our additional analysis of the publication bias of the included papers revealed no publication bias or strong symmetry in the Deeks funnel plot. In addition, the SROC curve was not characterized by a “shoulder-arm” distribution, demonstrating the absence of a threshold effect in the included publications. Researchers then performed meta-regression analyses for investigator, country, nucleic acid extraction technique, and assay using Meta-DiSk 1.4 software, which showed all P > 0.05, suggesting that these factors were unable to account for the heterogeneity across the included studies. Additional unidentified factors contributed to the heterogeneity among studies.

The highlight of the present study, compared to recent publications, is that a multigroup subgroup analysis was performed. Group A was categorized into five main groups according to the assay endpoints. From the study data, it was observed that the detection rate of NG-Test Carba 5 for the five carbapenemases was generally high (0.90–1.00), which is in line with previous reports (10–12). Nevertheless, when the detection endpoint was IMP, the sensitivity was significantly lower than that of the other detection endpoints (e.g., KPC, NDM, and OXA-48) for reasons that are not known to us but may be related to several factors. First, the design of the NG-Test Carba 5 assay kit manufacturer did not target a certain IMP phenotype, which led to a lower detection rate (14). Second, the possible presence of cross-reactive proteins in Acinetobacter baumannii (6) as well as the loss of CMY-2 AmpC β-lactamase and the loss of pore proteins (OmpF/OmpC) in Enterobacteriaceae spp. (12) may cause false-negative or false-positive results. Third, the diversity of gene sequences within the IMP family, mutability (40), and low expression (9, 11) may be the cause of assay failure. In the aforementioned studies, the NG-Test Carba 5 yielded failed test outcomes for some IMP types, such as IMP-8, IMP-13, IMP-14, and so on. This finding is the same as the statistical results we obtained for evidence-based medicine.

Notably, the results of the NG-Test Carba 5 assay for IMP enzymes revealed many inconsistencies. Some of the IMPs detected in the studies of Potron et al. (27) and Hopkins et al. (40) were false-negative, whereas Khoo described multiple false-positive assays for IMP in Acinetobacter baumannii in his study (6). Since it was not possible to determine the root cause of this result, more isolates from different regions are needed to validation. For false-positive results, Tarlton et al. (30) found that overloading of the LFA resulted in false positives and that instructions should be followed, and suggested that the developers of the NG-Test Carba 5 could have added inoculum schematics to circumvent the occurrence of false-positive results. Interestingly, in terms of false-negative results, one study (19) revealed the relationship between the bacterial concentration in blood cultures and assay sensitivity. For the NG-Test Carba 5 to recognize carbapenemase, there is a certain minimum concentration limit, below which the bacterial concentration may lead to negative test results with reduced sensitivity. This factor may have led to a certain amount of heterogeneity, but few articles have verified this idea.

In group B, this study focused on the differences in sensitivity and specificity between different bacterial species. The results showed that the detection outcomes for E. coli, Klebsiella pneumoniae and Enterobacter cloacae did not differ significantly, with NG-Test Carba 5 showing high sensitivity for Enterobacter cloacae despite the low number of previous studies on this bacterium alone. In contrast, the poor identification efficiency of NG-Test Carba 5 against carbapenemase-producing Pseudomonas aeruginosa is consistent with what was described by Mendez-Sotelo et al. (8), and the prevalent environment in which the bacteria are found may contribute to this result. However, Potron et al. (27) recommended the use of an upcoming version of the NG-Test Carba 5, which will be launched in 2019 in countries where carbapenemase-positive Pseudomonas aeruginosa strains are prevalent, which would help to improve the degree of accuracy of IMP detection. As mentioned in the study, the carbapenemase types of P. aeruginosa are predominantly IMP and VIM, and their resistance is associated with defects in their pore proteins (OprD), which are non-transferable mechanisms located on the plasmid.

In addition, Potron et al. (41) reported that Pseudomonas aeruginosa also produces SPM and GIM types of β-lactamases, but these rare enzymes have not been tested by the NG-Test Carba 5. Therefore, as described in the IMP detection results above, Pseudomonas aeruginosa with a predominance of the IMP type can cause poor detection outcomes of the NG-Test Carba 5, and some of the specific types of enzymes are not detected, leading to false-negative results. However, it is worth noting that in the research by Volland et al. (42), it was mentioned that NG-Test Carba 5v2 is an important advancement over the original version because it can detect all different kinds of IMP without affecting its identification of the other four carbapenemases and is particularly suitable for areas where carbapenemase-producing Pseudomonas aeruginosa is prevalent, a conclusion that is consistent with the findings of this conclusion and is consistent with the forthcoming version of NG-Test Carba 5 mentioned by Potron et al. (27).

New versions of the NG-Test Carba 5 continue to be introduced in several types of studies to provide new solutions. Tarlton et al. (30) argue that it is necessary to compare the results of carbapenemase-specific assays with the phenotypic AST profiles of the isolates and the local epidemiology, and that if these are inconsistent, additional testing is required. This also leads to the potential impact of geographic variation in CR-GNB prevalence on instrument performance. When researchers are familiar with the local phenotyping of carbapenemase resistance assays, and when the NG-Test Carba 5 gives results that are unprecedented in local epidemiologic phenotypes, we should be more sensitive to the limitations of the assay and ask questions, which could point to certain directions in future instrumentation research. This points to a certain direction for future instrumental research.

Group C of this study, which is rarely synthesized and analyzed in current publications, was targeted at bacterial isolation methods. From the isolation culture methods included in the literature, researchers categorized five types: blood culture, blood agar culture, direct clinical blood test, MacConkey agar culture and Mueller-Hinton agar culture. The results showed that the sensitivities were all in the range of 0.95–1.00, and the specificities were all in the range of 0.99–1.00, which suggested that the NG-Test Carba 5 performed better in terms of detection across all isolation techniques. In our comparison, we found that the sensitivity of 0.95 for the direct detection of clinical blood was lower than that of other bacterial isolation methods, possibly because the NG-Test Carba 5 was not originally designed for the direct detection of clinical samples (23). Nevertheless, Nishida et al. (10) reported that direct testing of clinical samples can be used as a point-of-care test (POCT) for the rapid detection of CR-GNB. Moreover, a study (15) has shown that direct testing of clinical samples, such as rectal swabs or even fecal samples, can yield more rapid and effective results for the rapid detection of gastrointestinal colonization by the majority of gram-negative bacteria that produce carbapenemase. It is significant to note that the statistics on the Mueller-Hinton agar culture method used in this study are limited and insufficient to calculate its SROC value, and more data are needed to validate this method.

In today’s prevalent assays, Xpert Carba-R is an impressive player. However, Tarlton et al. (30) found that IMP-27 is a known test limitation of Xpert Carba-R and can be detected by NG-Test Carba 5. Moreover, Kanahashi et al. (4) found that the sensitivity of the assay for Xpert Carba-R under the same metrics was 53.8%, and the sensitivity of NG-Test- Xpert Carba-R is based on an automated real-time PCR platform, which is relatively complex and requires specialized personnel to operate, but NG-Test Carba 5 is easy to operate, without complex equipment, suitable for rapid screening, which takes less time and is suitable for initial diagnosis. Based on the potentially better performance and more compatible with the screening requirements, NG-Test Carba 5 shows a more fascinating charm.

There are several limitations to this study. First, the assay endpoints in this study did not cover subgroup analyses of complex carbapenemases or the OXA-48 family, which could result in altered performance of the NG-Test Carba 5 assay. Second, the subgroup analysis included only two data sets from the Group C Mueller-Hinton agar culture method. Therefore, we are unable to determine with certainty how sensitive the NG-Test Carba 5 is to this assay, and more studies applying this method are needed to provide reliable data to validate the present conclusions. Finally, the limitations of the NG-Test Carba 5 assay equipment led to misinterpretation of some specific carbapenemases, such as GES and IMI, which may have caused heterogeneity in the included literature.

In summary, the findings of the above meta-analysis showed that the NG-Test Carba 5 can quickly, easily, and accurately detect carbapenemase, and its high sensitivity and specificity provide novel approaches for clinical diagnosis and infection control, especially in severe environments where carbapenem-resistant bacterial infections are becoming increasingly serious in China and effective antibiotic types are scarce. However, a larger sample size and additional field methods are still needed to confirm the above conclusions.