Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), a type of Klebsiella pneumoniae (KP) that exhibits hypervirulence and carbapenem resistance phenotypes, can cause severe infections, both hospital- and community-acquired infections. CR-hvKP has brought great challenges to global public health and is associated with significant morbidity and mortality. There are many mechanisms responsible for the evolution of the hypervirulence and carbapenem resistance phenotypes, such as the horizontal transfer of the plasmid carrying the carbapenem resistance gene to hypervirulent Klebsiella pneumoniae (hvKP) or carbapenemase-producing Klebsiella pneumoniae (CRKP) acquiring a hypervirulence plasmid carrying a virulence-encoding gene. Notably, KP can evolve into CR-hvKP by acquiring a hybrid plasmid carrying both the carbapenem resistance and hypervirulence genes. In this review, we summarize the evolutionary mechanisms of resistance and plasmid-borne virulence as well as the prevalence of CR-hvKP.

Plasmodium ookinete surface proteins as post-fertilization target antigens are potential malaria transmission-blocking vaccine (TBV) candidates. Putative secreted ookinete protein 25 (PSOP25) is a highly conserved ookinete surface protein, and has been shown to be a promising novel TBV target. Here, we further investigated the TBV activities of the full-length recombinant PSOP25 (rPSOP25) protein in Plasmodium berghei, and characterized the potential functions of PSOP25 during the P. berghei life-cycle. We expressed the full-length P. berghei PSOP25 protein in a prokaryotic expression system, and developed polyclonal mouse antisera and a monoclonal antibody (mAb) against the recombinant protein. Indirect immunofluorescence assay (IFA) and Western blot were used to test the specificity of antibodies. The transmission-blocking (TB) activities of antibodies were evaluated by the in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). Finally, the function of PSOP25 during Plasmodium development was studied by deleting the psop25 gene. Both polyclonal mouse antisera and anti-rPSOP25 mAb recognized the PSOP25 proteins in the parasites, and IFA showed the preferential expression of PSOP25 on the surface of zygotes, retorts and mature ookinetes. In vitro, these antibodies significantly inhibited ookinetes formation in an antibody concentration-dependent manner. In DFA, mice immunized with the rPSOP25 and those receiving passive transfer of the anti-rPSOP25 mAb reduced the prevalence of mosquito infection by 31.2 and 26.1%, and oocyst density by 66.3 and 63.3%, respectively. Genetic knockout of the psop25 gene did not have a detectable impact on the asexual growth of P. berghei, but significantly affected the maturation of ookinetes and the formation of midgut oocysts. The full-length rPSOP25 could elicit strong antibody response in mice. Polyclonal and monoclonal antibodies against PSOP25 could effectively block the formation of ookinetes in vitro and transmission of the parasites to mosquitoes. Genetic manipulation study indicated that PSOP25 is required for ookinete maturation in P. berghei. These results support further testing of the PSOP25 orthologs in human malaria parasites as promising TBV candidates.

Background: This study aimed to evaluate the diagnostic accuracy of pleural fluid (PF) lactate dehydrogenase (LDH) to adenosine deaminase (ADA) (LDH/ADA) ratio for tuberculous pleural effusion (TPE). Especially to explore whether the LDH/ADA ratio provides added diagnostic value to ADA. Methods: The diagnostic accuracy of PF LDH/ADA ratio and ADA for TPE was evaluated in two cohorts, named the BUFF (Biomarkers for patients with Undiagnosed pleural eFFusion) cohort (62 with TPE and 194 with non-TPE) and the SIMPLE (a Study Investigating Markers in PLeural Effusion) cohort (33 with TPE and 177 with non-TPE). Receiver operating characteristic (ROC) curve and decision curve were used to measure the diagnostic accuracy of the PF LDH/ADA ratio. The added diagnostic value of the LDH/ADA ratio to ADA was evaluated with net reclassification improvement (NRI) and integrated discrimination improvement (IDI). Results: The area under the ROC curves (AUCs) of PF ADA and LDH/ADA ratio in the BUFF cohort were 0.76 and 0.74, respectively. In the SIMPLE cohort, the AUCs of PF ADA and LDH/ADA ratio were 0.80 and 0.85, respectively. The decision curves of PF LDH/ADA and ADA were close in both the BUFF and SIMPLE cohorts. The NRI and IDI analyses did not reveal any added diagnostic value of LDH/ADA to ADA. Conclusions: PF LDH/ADA ratio has moderate diagnostic accuracy for TPE. It does not provide added diagnostic value beyond ADA. The current evidence does not support LDH/ADA ratio for diagnosing TPE.

The screening performance of urine flow cytometry parameters (e.g., white blood cell and bacteria) for urinary tract infection (UTI) has been widely recognized. The majority of previous studies, however, investigated the screening performance of Sysmex UF-1000i urine flow cytometer. This study aimed to investigate the screening performance of Sysmex UF-5000 analyzer, a third-generation urinary flow cytometer, for UTI and its novel parameter named Gram flag for discriminating gram-positive and negative pathogens. Urine specimens sent to the clinical microbiology laboratory of our hospital for bacterial culture between September 13, 2021, and November 15, 2021, were prospectively and consecutively collected. The Sysmex UF-5000 analyzer was used to determine urine white blood cell (WBC) and bacteria simultaneously. A chemical strip was used to assess urine nitrate. UTI was defined as positive urine bacterial culture > 10⁴ CFU /ml. The receiver operating characteristics (ROC) curve, nomogram, decision tree, and decision curve were used to determine the screening performance of urine WBC, nitrate, and bacterial. A total of 246 UTIs and 425 non-UTIs were enrolled. The areas under the ROC curve (AUCs) for WBC and bacterial were 0.74 and 0.86, respectively. The decision curve showed that urine bacteria had a higher benefit than WBC. The nomogram indicated that urine bacterial had the largest effect on the probability of UTI. The sensitivity and specificity of the decision tree were 0.69 and 0.95, respectively. The flag of Gram-negative had a positive predictive value (PPV) of 0.93 in patients with urine bacteria > 1367 /μl. Therefore, we conclude that urine bacteria determined by the Sysmex UF-5000 had higher screening performance and greater benefit than WBC. The decision tree can be used to improve the screening performance of routine urinary parameters. The flag of Gram-negative is a reliable indicator to confirm gram-negative bacteria infection in UTI patients.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains present a significant global public health threat due to their high mortality rates. This study investigated the genomic characteristics of seven ST11-K1 CR-hvKP isolates harboring highly homologous KPC-2-encoding multidrug-resistance plasmids. The strains were isolated from a Chinese tertiary hospital between 2017 and 2020. Whole-genome sequencing and bioinformatic analysis revealed various antibiotic resistance genes (ARGs) and virulence determinants. The bla _KPC-2 -bearing plasmids that contain multiple antibiotic-resistance genes were also identified in these strains. ISfinder and Orifinder were applied to identify insertion sequences (IS) and conjugation-related factors among these bla _KPC-2 -bearing plasmids. The bla _KPC-2 was highly consistent in seven bla _KPC-2 -bearing plasmids (IS Kpn6-bla _KPC-2 -IS Kpn27 -IS Yps3 -IS 26 ). In addition, we found a region composed of IS IR , Tn 5393 , and IS 26 . It was located upstream of the bla _CTX-M-15 gene and presented in six bla _KPC-2 -bearing plasmids, with pCR-hvKP221-KPC-P3 as an exception. Conjugation experiments demonstrated the horizontal transfer of resistance plasmids pCR-hvKP128-KPC-P1 and pCR-hvKP132-KPC-P1 across species. Notably, pLVPK-like virulence plasmids carrying virulence gene clusters pCR-hvKP173-Vir-P1, and pCR-hvKP221-Vir-P1 were also detected. A fusional plasmid pCR-hvKP221-Vir-P2, which carries virulence gene clusters and ARGs, was also identified. Five CR-hvKP strains displayed enhanced biofilm formation and high virulence in vivo infection models. Phylogenetic and single nucleotide polymorphism (SNP) analyses indicated a close genetic relationship among the isolates, suggesting a subclade. These findings highlight the complex genetic profiles and potential transmission mechanisms of CR-hvKP strains. IMPORTANCE: We reported seven CR-hvKP strains all carried a highly homologous bla _KPC-2 integrated IncFⅡ-resistant plasmid, and two strains harbored virulence plasmids. Conjugation experiments confirmed the transferability of these plasmids, indicating a potential for resistance spread. Phylogenetic analysis clarified the relationship among the CR-hvKP isolates. This study provides insights into the phenotypic and genomic characteristics of seven ST11-K1 CR-hvKP strains. The high prevalence and potential for local outbreaks emphasize the need for effective control measures.

Transmission-blocking vaccines (TBVs) are a promising strategy for malaria control and elimination. However, candidate TBV antigens are currently limited, highlighting the urgency of identifying new antigens for TBV development. Using a combination of bioinformatic analysis and functional studies in the rodent malaria model Plasmodium berghei, we identified a conserved Plasmodium protein PbPH (PBANKA_041720) containing a pleckstrin homology (PH) domain. The expression of PbPH was detected by Western blot and indirect immunofluorescence assay (IFA). The function of PbPH was tested by genetic knockout. The TB activity was confirmed by in vitro ookinete conversion assay and mosquito feeding. PbPH was detected in Western blot as highly expressed in sexual stages (gametocytes and ookinetes). IFA revealed localizations of PbPH on the surface of gametes, zygotes, and ookinetes. Deletion of the pbph gene did not affect asexual growth, but significantly reduced the formation of gametocytes, ookinetes, and oocysts, indicating that PbPH protein is required for parasite sexual development. Recombinant PbPH expressed and purified from bacteria elicited strong antibody responses in mice and the antibodies significantly inhibited exflagellation of male gametocytes and formation of ookinetes in a concentration-dependent manner. Mosquito feeding experiments confirmed that mosquitoes fed on mice immunized with PbPH had 13 % reduction in the prevalence of infection and almost 48 % reduction in oocyst density. Pbph is a highly conserved Plasmodium gene and is required for parasite sexual development. PbPH protein is expressed on the surface of gametes and ookinetes. Immunization of mice against the recombinant PbPH protein induced strong antibody responses that effectively reduced the formation of male gametes and ookinetes in vitro and blocked transmission of the parasites to mosquitoes. These results highlight PbPH as a potential TBV candidate that is worth future investigations in human malaria parasites.