BACKGROUND: Early and appropriate antibiotic treatment improves the clinical outcome of patients with sepsis. There is an urgent need for rapid identification (ID) and antimicrobial susceptibility testing (AST) of bacteria that cause bloodstream infection (BSI). Rapid ID and AST can be achieved by short-term incubation on solid medium of positive blood cultures using MALDI-TOF mass spectrometry (MS) and the BD M50 system. The purpose of this study is to evaluate the performance of rapid method compared to traditional method.
METHODS: A total of 124 mono-microbial samples were collected. Positive blood culture samples were short-term incubated on blood agar plates and chocolate agar plates for 5 ∼ 7 h, and the rapid ID and AST were achieved through Zybio EXS2000 MS and BD M50 System, respectively.
RESULTS: Compared with the traditional 24 h culture for ID, this rapid method can shorten the cultivation time to 5 ∼ 7 h. Accurate organism ID was achieved in 90.6% of Gram-positive bacteria (GP), 98.5% of Gram-negative bacteria (GN), and 100% of fungi. The AST resulted in the 98.5% essential agreement (EA) and 97.1% category agreements (CA) in NMIC-413, 99.4% EA and 98.9% CA in PMIC-92, 100% both EA and CA in SMIC-2. Besides, this method can be used for 67.2% (264/393) of culture bottles during routine work. The mean turn-around time (TAT) for obtaining final results by conventional method is approximately 72.6 ± 10.5 h, which is nearly 24 h longer than the rapid method.
CONCLUSIONS: The newly described method is expected to provide faster and reliable ID and AST results, making it an important tool for rapid management of blood cultures (BCs). In addition, this rapid method can be used to process most positive blood cultures, enabling patients to receive rapid and effective treatment.

BACKGROUND: Plasmodium vivax is the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuous in vitro culture system, highlighting the need to establish a biobank of clinical isolates with multiple freezes per sample for use in functional assays. Different methods for cryopreserving parasite isolates were compared and subsequently the most promising one was validated. Enrichment of early- and late-stage parasites and parasite maturation were quantified to facilitate assay planning.
METHODS: In order to compare cryopreservation protocols, nine clinical P. vivax isolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-term in vitro culture was measured via slide microscopy. Enrichment of late-stage parasites by magnetic activated cell sorting (MACS) was measured. Short and long-term storage of parasites at either - 80 °C or liquid nitrogen were also compared.
RESULTS: Of the four cryopreservation mixtures, one mixture (glycerolyte:serum:RBC at a 2.5:1.5:1 ratio) resulted in improved parasite recovery and statistically significant (P < 0.05) enhancement in parasite survival in short-term in vitro culture. A parasite biobank was subsequently generated using this protocol resulting in a collection of 106 clinical isolates, each with 8 vials. The quality of the biobank was validated by measuring several factors from 47 thaws: the average reduction in parasitaemia post-thaw (25.3%); the average fold enrichment post KCl-Percoll (6.65-fold); and the average percent recovery of parasites (22.0%, measured from 30 isolates). During short-term in vitro culture, robust maturation of ring stage parasites to later stages (> 20% trophozoites, schizonts and gametocytes) was observed in 60.0% of isolates by 48 h. Enrichment of mature parasite stages via MACS showed good reproducibility, with an average of 30.0% post-MACS parasitaemia and an average of 5.30 × 105 parasites/vial. Finally, the effect of storage temperature was tested, and no large impacts from short-term (7 days) or long-term (7-10 years) storage at - 80 °C on parasite recovery, enrichment or viability was observed.
CONCLUSIONS: Here, an optimized freezing method for P. vivax clinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays.

Carbapenemase resistance in Enterobacterales is a global public health problem and rapid and effective methods for detecting these resistance mechanisms are needed urgently. Our aim was to evaluate the performance of a MALDI-TOF MS-based \"Klebsiella pneumoniae carbapenemase\" (KPC) detection protocol from patients\' positive blood cultures, short-term cultures, and colonies in healthcare settings. Bacterial identification and KPC detection were achieved after protein extraction with organic solvents and target spot loading with suitable organic matrices. The confirmation of KPC production was performed using susceptibility tests and blaKPC amplification using PCR and sequencing. The KPC direct detection (KPC peak at approximately 28.681 Da) from patients\' positive blood cultures, short-term cultures, and colonies, once bacterial identification was achieved, showed an overall sensibility and specificity of 100% (CI95: [95%, 100%] and CI95: [99%, 100%], respectively). The concordance between hospital routine bacterial identification protocol and identification using this new methodology from the same extract used for KPC detection was ≥92%. This study represents the pioneering effort to directly detect KPC using MALDI-TOF MS technology, conducted on patient-derived samples obtained from hospitals for validation purposes, in a multi-resistance global context that requires concrete actions to preserve the available therapeutic options and reduce the spread of antibiotic resistance markers.

The importance of circulating tumor cells (CTC) is well recognized. However, the biological characteristics of CTC in the bloodstream have not yet been examined in detail, due to the limited number of CTC cell lines currently available. Thirty-nine CTC cell lines were reported by 2021. For successful cell culturing, these CTC cell lines were reviewed. Previous studies on short-term cultures of CTC also analyzed approaches for establishing the long-term culture of CTC. Negative selection, hypoxic conditions, three-dimensional conditions, and careful management are preferable for the long-term culture of CTC. However, the establishment of CTC cell lines is dependent on the specific characteristics of each cell type. Therefore, a method to establish CTC cell lines has not yet been developed. Further efforts are needed to resolve this issue.

Short-term culture followed by MALDI-TOF MS is one of the most widely used methods for fast identification of microorganisms from blood cultures. The method identifies the vast majority of bloodstream infection pathogens in 2-6 h after positive blood culture. Transport time of blood culture bottles to laboratories is a major problem affecting total turnaround time. Therefore, many central laboratories establish satellite blood culture systems in other clinics and hospitals to allow blood culture bottles to be incubated immediately after sampling. However, positive blood culture bottles still need to be transported to the clinical microbiology laboratory for analysis. The aim of this study was to investigate how delayed analysis of positive blood culture bottles would affect the short-term culture followed by MALDI-TOF MS method.
To simulate the effect of transportation and delayed analysis of blood culture bottles, 51 simulated blood culture bottles were incubated for 0, 2, 4 and 24 h at room temperature. After each time interval, a 2 to 4 h short-term culture followed by MALDI-TOF MS was performed. In addition, 257 prospective clinical positive blood culture bottles were analysed with the same method after a 24 h incubation at room temperature.
In simulated samples, all (120/120) Gram-negative bacteria and 77/84 (91.6%) Gram-positive bacteria were accurately identified at species-level after a 2 h short-term culture, regardless of the duration of simulated transport time. In the clinical samples, 100/116 (86.2%) Gram-negative, and 44/141 (31.2%) Gram-positive bacteria were accurately identified at species-level after a 2 h short-term culture. After contaminants were excluded, 39/71 (54.9%) Gram-positive bacteria could be identified after 2 h. After a 4 h short-term culture, 112/116 (96.6%) Gram-negative, and 108/141 (76.6%) Gram-positive bacteria were accurately identified at species-level. Of the clinically relevant Gram-positive bacteria, 68/71 (95.8%) were identified at species-level after 4 h.
Short-term culture followed by MALDI-TOF MS can provide fast and accurate results for identification of clinically relevant bacteria, despite long transportation times from satellite laboratories. The present data shows that the method can be used for identification of microorganisms from positive blood cultures transported from satellite blood culture systems.

Parasitic nematodes of humans, animals and plants have a major, adverse impact on global health and agricultural production worldwide. To cope with their surrounding environment in and the immune attack from the host, excretory-secretory (ES) proteins are released by nematodes to orchestrate or regulate parasite-host interactions. In the present study, we characterised the ES products from short-term (12 h) in vitro culture of different developmental stages/sexes of Haemonchus contortus (one of the most important parasitic nematodes of livestock animals worldwide) using a high throughput tandem mass-spectrometry, underpinned by the most recent genomic dataset. In total, 878 unique proteins from key developmental stages/sexes (third-stage and fourth-stage larvae, and female and male adults) were identified and quantified with high confidence. Bioinformatic analyses showed noteworthy ES protein alterations during the transition from the free-living to the parasitic phase, especially for proteins which are likely involved in nutrient digestion and acquisition as well as parasite-host interactions, such as proteolytic cascade-related peptidases, glycoside hydrolases, C-type lectins and sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7 (= SCP/TAPS) proteins. Our findings provide an avenue to better explore interactive processes between the host and this highly significant parasitic nematode, to underpin the search for novel drug and vaccine targets. SIGNIFICANCE: The present study represents a comprehensive proteomic analysis of the secretome of key developmental stages/sexes of H. contortus maintained in short-term in vitro culture. High throughput LC-MS/MS analysis of ES products allowed the identification of a large repertoire of proteins (secretome) and the establishment of a new proteomic database for H. contortus. The secretome of H. contortus undergoes substantial changes during the nematode\'s transition from free-living to parasitic stages, suggesting a constant adaptation to different environments outside of and within the host animal. Understanding the host-parasite relationship at the molecular level could assist significantly in the development of intervention strategies (i.e. novel drugs and vaccines) against H. contortus and related nematodes.

OBJECTIVE: The recent identification of acid sensing ion channels (ASICs) in vascular beds suggests their possible involvement in modulating vasomotor tone. Therefore, we investigated the gene expression profiles of ASIC subtypes in the middle cerebral artery (MCA) of Wistar rats and the functional implication of ASICs in acidosis-induced relaxation as well as maintenance of resting tension.
METHODS: Real time PCR was employed to study the pattern of ASIC mRNA expression in the MCA wall in comparison with (i) matching brain tissue samples and (ii) arteries cultured for 24 h and 48 h. The functional implication regarding vasomotor response to acidosis and maintenance of resting tension was assessed using in vitro myography.
RESULTS: A robust mRNA expression of ASIC-1, -2 and -4 was found in brain tissue samples and to a lower extent in freshly isolated MCA. In the MCA wall, short term culture induced a down-regulation of ASIC-1 and -2 expression without any remarkable change in ASIC-4 expression. Acidosis induced a pH-related relaxation of freshly isolated MCA ring segments, being more pronounced after short term culture. Incubation with the ASIC blocker amiloride moderately enhanced acidosis-induced relaxation, in cultured MCAs somewhat stronger than in freshly isolated vessels. In addition, amiloride resulted in a decrease of resting tension, albeit only in freshly isolated MCA.
CONCLUSIONS: Our results comprehensively describe ASIC subtype composition in the rat MCA in physiological and pathological conditions and strongly suggest the involvement of ASICs in the modulation of vasomotor responses under conditions of normal or decreased pH values.

Our objective was to evaluate the apoptosis incidence in immature mouse testicular tissue after two different protocols of vitrification and short-term culture. Testes of 7-day-old Naval Medical Research Institute mice were isolated and distributed into control and vitrification groups. In vitrification 1 group, testes were vitrified using a combination of ethylene glycol and DMSO in three steps, and in vitrification 2 group, testes were vitrified using a combination of ethylene glycol and sucrose in five steps. Then, fresh and vitrified-warmed testis fragments were cultured for 20 hours. Morphology, cell viability, apoptosis incidence, and apoptosis gene expression (BAX, BCL2, Caspase 3, Fas, Fas ligand, p53) were evaluated at 0, 3, and 20 hours of culture by light microscopy, flow cytometry, and real-time polymerase chain reaction, respectively. Significant decrease of early apoptosis (annexin V+/PI- cells in vitrification 1 and 2 groups at 0 hours of culture, 37.34 ± 0.91 and 30.72 ± 2.2, and at 20 hours of culture, 1.46 ± 0.28 and 0.76 ± 0.11, respectively), increase of late apoptosis (annexin V+/PI+ cells in vitrification 1 group at 0 hours of culture, 14.46 ± 0.86, and at 20 hours of culture, 37.18 ± 2.34), and BAX/BCL-2 ratio (in vitrification 1 and 2 groups at 0 hours of culture, 7.31 ± 0.31 and 6.83 ± 1.38, and at 20 hours of culture, 24.08 ± 4.32 and 9.35 ± 1.91, respectively) were observed in vitrification groups during culture period. Caspase 3 expression was significantly decreased in all groups after 3 hours of culture (in control, vitrification 1, and vitrification 2 groups at 0 hours of culture, 1.00 ± 0.0, 1.56 ± 0.09, and 0.79 ± 0.06, and at 20 hours of culture, 0.37 ± 0.0, 0.96 ± 0.10, and 0.12 ± 0.03, respectively). Expression of p53 was significantly lower in vitrification 1 (0.32 ± 0.02) and control (0.50 ± 0.03) groups in 20 hours of culture as compared with vitrification 2 (0.88 ± 0.14) group. Fas (in vitrification 1 and 2 groups at 0 hours of culture, 2.29 ± 0.23 and 1.14 ± 0.15, and at 20 hours of culture, 12.43 ± 0.46 and 6.7 ± 0.48, respectively) and Fas Ligand (in vitrification 1 and 2 groups at 0 hours of culture, 1.2 ± 0.28 and 5.24 ± 0.32, and at 20 hours of culture, 21.75 ± 2.00 and 25.82 ± 2.15, respectively) expressions significantly increased in vitrification groups after 20 hours of culture. Although both vitrification protocols cause cell death via apoptotic and necrotic pathway, it seems that vitrification 1 protocol induces cell death more via apoptotic pathway than via necrosis. The apoptosis incidence after vitrification may have occurred independent of p53.

Because dendritic cells (DCs) play a critical role in the regulation of adaptive immune responses, they have been ideal candidates for cell-based immunotherapy of cancers and infections in humans. Generally, monocyte-derived DCs (MDDCs) were generated from purified monocytes by multiple steps of time-consuming physical manipulations for an extended period cultivation. In this study, we developed a novel, simple and rapid method for the generation of type-1 helper T cell (Th1)-stimulating human DCs directly from bulk peripheral blood mononuclear cells (PBMCs). PBMCs were cultivated in the presence of 20 ng/ml of granulocyte-macrophage colony-stimulating factor, 20 ng/ml of interleukin-4 (IL-4) and 1,000 U/ml of interferon-β for 24 h followed by 24 h maturation with a cytokine cocktail containing 10 ng/ml of tumor necrosis factor-α (TNF-α), 10 ng/ml of IL-1β and 1 μg/ml of prostaglandin E2. The phenotype and biological activity of these new DCs for induction of allogeneic T cell proliferation and cytokine production were comparable to those of the MDDCs. Importantly, these new DCs pulsed with inactivated HIV-1 could generated HIV-1-reactive CD4(+) T cell responses in humanized mice reconstituted with autologous PBMCs from HIV-1-negative donors. This simple and quick method for generation of functional DCs will be useful for future studies on DC-mediated immunotherapies.