Genetic and serological characterization of capsular antigen untypeable Vibrio parahaemolyticus strains reveal novel K serotypes and epidemiological characteristics in Shandong, China.

Vibrio parahaemolyticus, which is commonly found in marine and estuarine environments worldwide and isolated from aquatic products, is one of the most important food-borne pathogens. Among the various typing methods, serotyping is widely accepted and utilized by infectious disease specialists and infection control agencies for the detection and epidemiological investigation of this pathogen. Thus far, 13 O serotypes and 71 K serotypes have been defined; however, untypeable strains are frequently isolated during routine detection, and some new O and/or K antigens have been identified and characterized. During a serotyping survey in Shandong province, China from 2016 to 2018, we collected 411 clinical V. parahaemolyticus strains and found that nine of them are untypeable K antigen strains. In this study, we identified three K serotypes of V. parahaemolyticus through in-depth genetic analysis of the K antigen gene cluster, serological tests, and the production of antisera. Among the nine strains, seven possess K untypeable 2 (KUT2) antigens, which have been reported recently by another group. However, two new O and K combinations (O3:KUT2 and O11:KUT2) were first characterized by us, with the remaining two each representing a novel K serotype. Moreover, through comparative genomic analysis, we showed that the Shandong KUT2 strains exhibit different virulence profiles compared to their identical K serotype partners from Zhejiang province, another Chinese coastal province; however, strains from these two regions are clustered into the same linage and may have evolved from a recent common ancestor. Additionally, one isolate, SD2016062, was phylogenetically similar to the strains associated with several local gastroenteritis outbreaks, with similar toxin patterns, suggesting its potential to cause sporadic occurrences of disease or even local pandemics. Finally, we developed a sero-specific PCR assay targeting the three novel K serotypes, which can monitor the V. parahaemolyticus spectrum for clinical and epidemiological purposes. Thus, we identified and characterized novel strains of V. parahaemolyticus and proposed a new technique for tracking the diversity of strains, which can help manage this food-borne pathogen.

A new emerging serotype of Vibrio parahaemolyticus in China is rapidly becoming the main epidemic strain.

OBJECTIVES: During surveillance, we found a new type of Vibrio parahaemolyticus named 'O4:KUT-recAin' and studied the phenotypic, pathogenic and epidemiological characteristics of O4:KUT-recAin. METHODS: V. parahaemolyticus were isolated from acute diarrhoeal patients in coastal hospitals of China. Serum agglutination test, specific PCR assay, growth curves under different conditions and rabbit diarrhoeal models were using to characterize O4:KUT-recAin. RESULTS: The O4:KUT-recAin strain has a new type of K antigen and a 25 043-bp-large fragment encoding 20 proteins inserted in the housekeeping gene recA. Retrospective analysis found that only one O4:KUT-recAin strain was detected in 563 V. parahaemolyticus strains in 2014; then the proportion increased rapidly and reached 17.8% (105/590) in 2016 and 31.1% (224/721) in 2017, making O4:KUT-recAin the second dominant serotype following O3:K6. O4:KUT-recAin strains (100%, 14/14) exhibited increased acid resistance and could reproduce in medium at pH 4.9, while 92.9% (13/14) of the O3:K6 strains could not grow at this pH value. O4:KUT-recAin could cause diarrhoea and small intestinal tissue lesions in infant rabbits, but its diarrhoeal (93.1%, 27/29) and mortality (78.6%, 22/28) rates were slightly lower than those of O3:K6 (100% 16/16, 100% 16/16). Based on diarrhoea patients, there were no significant differences in the two groups for most clinical symptoms and laboratory results, except media age, haemoglobin and the number of red blood cells in stool samples. CONCLUSIONS: O4:KUT-recAin had enhanced acid resistance, was capable of causing infectious diarrhoea in both rabbits and humans, and has become widespread during a short period of time in China.

Further Spread of a blaKPC-Harboring Untypeable Plasmid in Enterobacteriaceae in China.

The wide spread of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae is great threat to public health in China. Plasmids are among the major factors mediating blaKPC gene dissemination. A total of 156 carbapenem-resistant Enterobacteriaceae (CRE) isolates were identified in a tertiary hospital in China. Six KPC-producing isolates, namely, E. coli (n = 2), E. asburiae (n = 1), C. freundii (n = 1), C. portucalensis (n = 1), and C. koseri (n = 1), tested positive for the pCKPC18-1-like untypeable plasmid, which was described recently in C. freundii. All 6 plasmids could be easily transferred into E. coli by chemical transformation or conjugation and were confirmed by sequencing to harbor blaKPC-2. Multilocus PCRs and EcoRI-RFLP revealed that the 6 untypeable plasmids belonged to 2 isoforms. High-throughput sequencing of representative plasmids (pCP40 and pEC86) led to the identification of 2 plasmids that shared the common backbone genes repA, DnaJ, StpA, and yafB, which were characteristic of the untypeable plasmid, and had similar blaKPC-2 genetic contexts of the Tn3-Tn4401 chimera. Nucleotide comparison revealed high sequence identity of the 2 plasmids with previously reported blaKPC-2-carrying untypeable plasmids. In particular, the pCP40 plasmid from C. portucalensis and the pHS062105-3 plasmid from K. pneumoniae differed by only 20 single-nucleotide polymorphisms (SNPs). To the best of our knowledge, this is the first report of a blaKPC-harboring untypeable plasmid spread into E. coli, E. asburiae, and C. koseri strains in China.

Untypeable hepatitis C virus subtypes in Pakistan: A neglected section.

Diagnostically untypeable subtypes contribute a considerable percent of hepatitis C virus (HCV) subtypes in Pakistan. In the present study, chronically infected HCV patients with known viremia were subjected to HCV genotyping. Among the total retrieved samples, 92.7% (64/69) were found typeable while 7.24% (5/69) were diagnostically untypeable. In conclusion, the presence of large number of untypeable HCV subtypes emphasizes the need of an updated type-specific genotyping assay and consideration of primers for proportionally rare subtypes to minimize the number of untypeable HCV subtypes.

Comparative evaluation of the Bio-Rad Geenius HIV-1/2 Confirmatory Assay and the Bio-Rad Multispot HIV-1/2 Rapid Test as an alternative differentiation assay for CLSI M53 algorithm-I.

INTRODUCTION: The CLSI-M53-A, Criteria for Laboratory Testing and Diagnosis of Human Immunodeficiency Virus (HIV) Infection; Approved Guideline includes an algorithm in which samples that are reactive on a 4th generation EIA screen proceed to a supplemental assay that is able to confirm and differentiate between antibodies to HIV-1 and HIV-2. The recently CE-marked Bio-Rad Geenius HIV-1/2 Confirmatory Assay was evaluated as an alternative to the FDA-approved Bio-Rad Multispot HIV-1/HIV-2 Rapid Test which has been previously validated for use in this new algorithm. METHODS: This study used reference samples submitted to the Canadian - NLHRS and samples from commercial sources. Data was tabulated in 2×2 tables for statistical analysis; sensitivity, specificity, predictive values, kappa and likelihood ratios. RESULTS: The overall performance of the Geenius and Multispot was very high; sensitivity (100%, 100%), specificity (96.3%, 99.1%), positive (45.3, 181) and negative (0, 0) likelihood ratios respectively, high kappa (0.96) and low bias index (0.0068). The ability to differentiate HIV-1 (99.2%, 100%) and HIV-2 (98.1%, 98.1%) Ab was also very high. CONCLUSION: The Bio-Rad Geenius HIV-1/2 Confirmatory Assay is a suitable alternative to the validated Multispot for use in the second stage of CLSI M53 algorithm-I. The Geenius has additional features including traceability and sample and cassette barcoding that improve the quality management/assurance of HIV testing. It is anticipated that the CLSI M53 guideline and assays such as the Geenius will reduce the number of indeterminate test results previously associated with the HIV-1 WB and improve the ability to differentiate HIV-2 infections.