Nationwide epidemiologic and genetic surveillance of hepatitis E in Japan, 2014-2021.

Hepatitis E virus (HEV) is an emerging causative agent of acute hepatitis. To clarify the epidemiology of HEV and characterize the genetic diversity of the virus in Japan, nationwide enhanced surveillance and molecular characterization studies of HEV in Japan were undertaken from 2014 to 2021. In total, 2770 hepatitis E cases were reported, of which 88% were domestic cases, while only 4.1% represented cases following infection abroad. In addition, 57% of domestic infections occurred in males aged in their 40s-70s. For domestic cases, infection via pork meat consumption continued to be the most reported route. Analysis of the 324 sequences detected between 2016 and 2021 showed that the majority of domestic HEV strains belong to Genotype 3a (G3a) and G3b. In contrast, six of eight cases of G1 HEV reflected infection abroad. Our results suggest that HEV is circulating widely in Japan, with genotypes G3a and G3b being most prevalent. Continued surveillance is necessary to monitor future trends and changes in the epidemiology of HEV in Japan.

Isolation of OXA-48-like carbapenemase-producing Escherichia coli susceptible to piperacillin/tazobactam in a Japanese patient without a history of travel abroad.

Oxacillinase (OXA)-48-like ß-lactamases are the most common carbapenemases in Enterobacterales in certain regions of the world and are being introduced on a regular basis into regions of non-endemicity. Japan has been characterized by low rates of carbapenemase-producing Enterobacterales, and among them, OXA-48-like carbapenemase-producing isolates are extremely rare. Here we describe a Japanese medical worker, without a history of travel abroad, who was diagnosed as having a community-acquired urinary tract infection, and whose urine sample was found to be positive for OXA-48-like carbapenemase-producing Escherichia coli. None of her close contacts had a history of foreign travel, and the same drug-resistant organism was not observed in other patients who had been hospitalized and undergone environmental culture tests in the same medical institution. This isolate was resistant to penicillins, narrow-spectrum cephalosporins, fluoroquinolones, and cefmetazole, but was susceptible to broad-spectrum cephalosporins, piperacillin/tazobactam, and meropenem and displayed reduced susceptibility to imipenem. The modified carbapenem inactivation test supported carbapenemase production, but inhibitor-based synergistic tests yielded negative results of carbapenemase production. Multiplex polymerase chain reaction revealed the presence of the carbapenemase gene (blaOXA-48) blaTEM and AmpC ß-lactamase gene (blaDHA). Singleplex polymerase chain reaction targeting the blaOXA-48 region amplified a product sequencing to nearly the full length (722 bp) and matching 100% with OXA-48. The present case highlights a new concern regarding OXA-48-like carbapenemase-producing Enterobacterales, which remain challenging to detect for clinical laboratories in regions of non-endemicity, and may already be latent in Japan.

Phylogeny and Immunoreactivity of Norovirus GII.P16-GII.2, Japan, 2016-17.

During the 2016-17 winter season in Japan, human norovirus GII.P16-GII.2 strains (2016 strains) caused large outbreaks of acute gastroenteritis. Phylogenetic analyses suggested that the 2016 strains derived from the GII.2 strains detected during 2010-12. Immunochromatography between 2016 strains and the pre-2016 GII.2 strains showed similar reactivity.

A patient with severe fever with thrombocytopenia syndrome and hemophagocytic lymphohistiocytosis-associated involvement of the central nervous system.

Severe fever with thrombocytopenia syndrome (SFTS), a severe infectious disease caused by novel bunyavirus, SFTS virus (SFTSV), is endemic to China, Korea, and Japan. Most SFTS patients show abnormalities in consciousness. Pathological findings in the central nervous system (CNS) of SFTS patients are not reported. A 53-year-old Japanese man was admitted to Uwajima City Hospital with an 8-day history of fever and diarrhea. Laboratory tests revealed leukopenia, thrombocytopenia, and liver enzyme elevation. He was diagnosed as having severe fever with thrombocytopenia syndrome (SFTS) following detection of the SFTSV genome in his blood. Bone marrow aspiration revealed hemophagocytic lymphohistiocytosis. He suffered progressive CNS disturbance and died on day 13 from onset of first symptoms. The SFTSV genome load in blood and levels of certain cytokines increased over the disease course. Necrotizing lymphadenitis with systemic lymphoid tissues positive for nucleocapsid protein (NP) of SFTSV was revealed by immunohistochemical (IHC) analysis. SFTSV-NP-positive immunoblasts were detected in all organs examined, including the CNS, and in the vascular lumina of each organ. Parenchymal cells of all organs examined were negative for SFTSV-NP on IHC analysis. Microscopic examination of the pons showed focal neuronal cell degeneration with hemosiderin-laden macrophages around extended microvessels with perivascular inflammatory cell infiltration and intravascular fibrin deposition. Autopsy confirmed this patient with SFTS was positive for systemic hemophagocytic lymphohistiocytosis including in the CNS. This patient's neurological abnormalities may have been caused by both functional and organic abnormalities. These novel findings provide important insights into the pathophysiology of SFTS.

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 106 and 17.6 × 106 CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed.

Seroprevalence of severe fever with thrombocytopenia syndrome (SFTS) virus antibodies in humans and animals in Ehime prefecture, Japan, an endemic region of SFTS.

Severe fever with thrombocytopenia syndrome (SFTS) was first identified as an emerging tick-borne infectious disease caused by the SFTS virus (SFTSV) in China and has also been found to be endemic to Japan and South Korea, indicating that SFTS is of great concern in East Asia. The aim of the present study was to determine the seroprevalence of SFTSV antibodies in humans and animals in SFTS-endemic regions of Japan. One of 694 (0.14%) healthy persons over 50 years of age and 20 of 107 (18.7%) wild and domestic animals in Ehime prefecture of western Japan were determined to be seropositive for SFTSV antibodies by virus neutralization test and ELISA, respectively. The seropositive person, a healthy 74-year-old woman, was a resident of the southwest part of Ehime prefecture engaged in citriculture and field work. This woman's sample exhibited neutralizing activity against SFTSV although she had neither a clear experience with tick bites nor SFTS-like clinical illness. These findings indicate that most people living in the endemic regions are not infected with SFTSV and suggest that most of the SFTS patients reported so far do not reflect the tip of an iceberg of people infected with SFTSV, but at the same time, that SFTSV infection does not always induce severe SFTS-associated symptoms. These findings also suggested that SFTSV has been maintained in nature within animal species and ticks.

Predicting genotype compositions in norovirus seasons in Japan.

Noroviruses cause acute gastroenteritis. Since multiple genotypes of norovirus co-circulate in humans, changing the genotype composition and eluding host immunity, development of a polyvalent vaccine against norovirus in which the genotypes of vaccine strains match the major strains in circulation in the target season is desirable. However, this would require prediction of changes in the genotype composition of circulating strains. A fitness model that predicts the proportion of a strain in the next season from that in the current season has been developed for influenza A virus. Here, such a fitness model that takes into account the fitness effect of herd immunity was used to predict genotype compositions in norovirus seasons in Japan. In the current study, a model that assumes a decline in the magnitude of cross immunity between norovirus strains according to an increase in the divergence of the major antigenic protein VP1 was found to be appropriate for predicting genotype composition. Although it is difficult to predict the proportions of genotypes accurately, the model is effective in predicting the direction of change in the proportions of genotypes. The model predicted that GII.3 and GII.4 may contract, whereas GII.17 may expand and predominate in the 2015-2016 season. The procedure of predicting genotype compositions in norovirus seasons described in the present study has been implemented in the norovirus forecasting system (NOROCAST).

[Genetic Investigation of bla(CTX-M)-typing in Extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae from Clinical Specimens and Commercially Available Chicken Liver].

A genetic investigation consisting of the bla(CTX-M) typing was performed using 40 extended spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates from chicken liver and 43 ESBL-producing E. coli and 42 ESBL-producing Klebsiella pneumoniae isolates from patients. The types were determined using a sequence analysis. In 31 isolates in the bla(CTX-M-1) group, there were 13 with the bla(CTX-M-1) and all were from chicken liver. Nine E. coli isolates from chicken liver and one E. coli isolate from patients were found to be bla(CTX-M-55). In the bla(CTX-M-15), there were 6 E. coli isolates and one K. pneumoniae isolate from patients. All 39 isolates in the bla(CTX-M-2) group had the blac(CTX-M-2). Fifty-five isolates were found in the bla(CTX-M-9) group, the highest detection frequency, with 36 possessing bla(CTX-M-14) : 20 E. coli and 13 K. pneumoniae from patients and 3 E. coli from chicken liver. There were 17 bla(CTX-M-27) isolates, including 10 E. coli and 7 K. pneumoniae from patients. One bla(CTX-M-90) K. pneumoniae isolate and one bla(CTX-M-9) E. coli isolate were also obtained from patients. These results indicate that the E. coli isolates from chicken liver consist of bla(CTX-M-1), bla(CTX-M-2) and bla(CTX-M-55) ; the E. coli isolates from patients consist of bla(CTX-M-14) and bla(CTX-M-27) ; and the K. pneumoniae isolates from patients consist of bla(CTX-M-2), bla(CTX-M-14) and bla(CTX-M-27). Therefore, the bla(CTX-M) type differs in isolates from chicken liver and those from humans. These results suggest that it is unlikely that ESBL-producing E. coli from chicken liver are firmly established in the human intestinal tract.

Listeria arpJ gene modifies T helper type 2 subset differentiation.

BACKGROUND: Although the T-cell subset differentiation pathway has been characterized extensively from the view of host gene regulation, the effects of genes of the pathogen on T-cell subset differentiation during infection have yet to be elucidated. Especially, the bacterial genes that are responsible for this shift have not yet been determined. METHODS: Utilizing a single-gene-mutation Listeria panel, we investigated genes involved in the host-pathogen interaction that are required for the initiation of T-cell subset differentiation in the early phase of pathogen infection. RESULTS: We demonstrate that the induction of T helper types 1 and 2 (Th1 and Th2) subsets are separate phenomena and are mediated by distinct Listeria genes. We identified several candidate Listeria genes that appear to be involved in the host-Listeria interaction. Among them, arpJ is the strongest candidate gene for inhibiting Th2 subset induction. Furthermore, the analysis utilizing arpJ-deficient Listeria monocytogenes (Lm) revealed that the tumor necrosis factor (TNF) superfamily (Tnfsf) 9-TNF receptor superfamily (Tnfrsf) 9 interaction inhibits the Th2 response during Lm infection. CONCLUSIONS: arpJ is the candidate gene for inhibiting Th2 T-cell subset induction. The arpJ gene product influences the expression of Tnfsf/Tnfrsf on antigen-presenting cells and inhibits the Th2 T-cell subset differentiation during Listeria infection.

[A Fatal Case of Severe Fever with Thrombocytopenia Syndrome Complicated by Hemophagocytic Lymphohistiocytosis].

Severe fever with thrombocytopenia syndrome (SFTS) is a recently identified emerging viral infectious disease in China that is caused by a novel phlebovirus in the family Bunyaviridae, SFTS virus, with an average case fatality rate of 12-30%. A cytokine storm with abnormally expressed cytokine profiles is associated with the disease severity. Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and lifethreatening syndrome associated with excessive immune activation. We report herein on a fatal case of SFTS complicated by HLH. Consecutive plasma exchange and immunomodulatory therapy was ineffective in our case. The pathognomonic histological feature was necrotizing lymphadenitis with massive hemophagocytosis of systemic lymphoid tissues with SFTS viruses and SFTS-RNA copies. No specific treatment of SFTS is available, and an effective treatment strategy for patients with rapidly progressing SFTS has not been established. Appropriate immunomodulatory therapy is necessary for SFTS patients complicated by HLH.

Genotypic analysis of Pseudomonas aeruginosa isolated from ocular infection.

Pseudomonas aeruginosa is the causative pathogen of keratitis, conjunctivitis, and dacryocystitis. However little is known about their clinical epidemiology in Japan. In this study we investigated the genotypic characterization and serotype of P. aeruginosa isolates from ocular infections. Thirty-four clinical P. aeruginosa isolates were characterized according to infection type, the type III secretion system (TTSS), serotype, and multilocus sequence typing (MLST). We divided the isolates into four clinical infection types as follows: Contact lens (CL)-related keratitis (CL-keratitis; 15 isolates), non CL-related keratitis (non CL-keratitis; 8 isolates), conjunctivitis (7 isolates), and dacryocystitis (4 isolates). Regarding the TTSS classification and serotyping classification, no significant differences were found among the infection types. Two clusters (I, II) and three subclusters (A, B, C) were classified according to MLST. CL-keratitis isolates with exoU positivity were clustered in II-B, and conjunctivitis was clustered in cluster I. Some linkage was found between the genetic background and CL-keratitis or conjunctivitis.

Identification and characterization of D-hydroxyproline dehydrogenase and Delta1-pyrroline-4-hydroxy-2-carboxylate deaminase involved in novel L-hydroxyproline metabolism of bacteria: metabolic convergent evolution.

L-hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, D-hydroxyproline dehydrogenase and Δ(1)-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. D-hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (D-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α(4)ß(4)γ(4)), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αßγ of the heterotrimeric unit. These results suggested that the L-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on L-hydroxyproline (as well as D-hydroxyproline) but not L- and D-proline, indicating that this pathway is related only to L-hydroxyproline degradation, which is not linked to proline metabolism.

Distribution of Human Sapovirus Strain Genotypes over the Last Four Decades in Japan: a Global Perspective.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

Different Ca²âº-sensitivities between the EF-hands of T- and L-plastins.

Plastins are Ca(2+)-regulated actin-bundling proteins, and essential for developing and stabilizing actin cytoskeletons. T-plastin is expressed in epithelial and mesenchymal cells of solid tissues, whereas L-plastin is expressed in mobile cells such as hemopoietic cell lineages and cancer cells. Using various spectroscopic methods, gel-filtration chromatography, and isothermal titration calorimetry, we here demonstrate that the EF-hand motifs of both T- and L-plastin change their structures in response to Ca(2+), but the sensitivity to Ca(2+) is lower in T-plastin than in L-plastin. These results suggest that T-plastin is suitable for maintaining static cytoskeletons, whereas L-plastin is suitable for dynamic rearrangement of cytoskeletons.

Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells.

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.

Increased Transmissibility of the SARS-CoV-2 Alpha Variant in a Japanese Population.

To assess the relative transmissibility of the SARS-CoV-2 Alpha variant compared to the pre-existing SARS-CoV-2 in Japan, we performed a cross-sectional study to determine the secondary attack rate of COVID-19 in household contacts before and after the Alpha variant became dominant in Osaka. We accessed 290 household contacts whose index cases were diagnosed between 1 and 20 December 2020 (the third epidemic group), at a time when Osaka was free of the Alpha variant. We also accessed 398 household contacts whose index cases were diagnosed between 20 April and 3 May 2021 (the fourth epidemic group), by which time the Alpha variant had become dominant. We identified 124 household contacts whose index case was determined positive for the Alpha variant (Alpha group) in this fourth group. The secondary attack rates in the fourth group (34.7%) and the Alpha group (38.7%) were significantly higher than that in the third group (19.3%, p < 0.001). Multivariable Poisson regression analysis with a robust error variance showed a significant excess risk in the fourth group (1.90, 95% CI = 1.47-2.48) and the Alpha group (2.34, 95% CI = 1.71-3.21). This finding indicates that the SARS-CoV-2 Alpha variant has an approximately 1.9-2.3-fold higher transmissibility than the pre-existing virus in the Japanese population.

Subtype Screening of blaIMP Genes Using Bipartite Primers for DNA Sequencing.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

Detection of Jingmenviruses in Japan with Evidence of Vertical Transmission in Ticks.

Jingmen tick virus (JMTV) and the related jingmenvirus-termed Alongshan virus are recognized as globally emerging human pathogenic tick-borne viruses. These viruses have been detected in various mammals and invertebrates, although their natural transmission cycles remain unknown. JMTV and a novel jingmenvirus, tentatively named Takachi virus (TAKV), have now been identified during a surveillance of tick-borne viruses in Japan. JMTV was shown to be distributed across extensive areas of Japan and has been detected repeatedly at the same collection sites over several years, suggesting viral circulation in natural transmission cycles in these areas. Interestingly, these jingmenviruses may exist in a host tick species-specific manner. Vertical transmission of the virus in host ticks in nature was also indicated by the presence of JMTV in unfed host-questing Amblyomma testudinarium larvae. Further epidemiological surveillance and etiological studies are necessary to assess the status and risk of jingmenvirus infection in Japan.

Isolation and plasmid characterisation of Salmonella enterica serovar Albany harbouring mcr-5 from retail chicken meat in Japan.

The emergence of plasmid-mediated colistin resistance genes (mcr), which is occurring in numerous countries, is a worldwide concern, primarily because colistin is a last-resort antibiotic. Compared to E. coli, prevalence of mcr genes in Salmonella is unclear in Japan. Here we screened for mcr-1-5 genes in our collection of Salmonella strains isolated from retail meat products collected in Japan from 2012 through 2016. We found that Salmonella Albany strain 27A-368 encodes mcr-5 and that mcr genes were undetectable among the remaining 202 isolates. The resistance plasmid p27A-368 was transferred by conjugation to S. Infantis and was stably retained as a transconjugant. Whole-genome sequencing revealed that mcr-5 resided on a 115 kb plasmid (p27A-368). The plasmid backbone of p27A-368 is more similar to that of pCOV27, an ESBL-encoding plasmid recovered from avian pathogenic E. coli, rather than pSE13-SA01718 of S. Paratyphi B that encodes mcr-5. Further, mcr-5 is located on a transposon, and its sequence is similar to that of pSE13-SA01718. A phylogenetic tree based on single nucleotide variants implies a relationship between 27A-368 and S. Albany isolated in Southeast Asian countries.

Single-Tube Multiplex Polymerase Chain Reaction for the Detection of Genes Encoding Enterobacteriaceae Carbapenemase.

A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: blaKPC, blaIMP, blaNDM, blaVIM, blaOXA-48-like, and blaGES. Of 70 blaIMP variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.