Retrospective analysis of molecular characteristics, risk factors, and outcomes in carbapenem-resistant Klebsiella pneumoniae bloodstream infections.

BACKGROUND: Klebsiella pneumoniae (KP) is the second most prevalent Gram-negative bacterium causing bloodstream infections (BSIs). In recent years, the management of BSIs caused by KP has become increasingly complex due to the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP). Although numerous studies have explored the risk factors for the development of CRKP-BSIs, the mortality of patients with KP-BSIs, and the molecular epidemiological characteristics of CRKP, the variability in data across different populations, countries, and hospitals has led to inconsistent conclusions. In this single-center retrospective observational study, we utilized logistic regression analyses to identify independent risk factors for CRKP-BSIs and factors associated with mortality in KP-BSI patients. Furthermore, a risk factor-based prediction model was developed. CRKP isolates underwent whole-genome sequencing (WGS), followed by an evaluation of microbiological characteristics, including antimicrobial resistance and virulence genes, as well as epidemiological characteristics and phylogenetic analysis. RESULTS: Our study included a total of 134 patients with KP-BSIs, comprising 50 individuals infected with CRKP and 84 with carbapenem-susceptible Klebsiella pneumoniae (CSKP). The independent risk factors for CRKP-BSIs were identified as gastric catheterization (OR = 9.143; CI = 1.357-61.618; P = 0.023), prior ICU hospitalization (OR = 4.642; CI = 1.312-16.422; P = 0.017), and detection of CRKP in non-blood sites (OR = 8.112; CI = 2.130-30.894; P = 0.002). Multivariate analysis revealed that microbiologic eradication after 6 days (OR = 3.569; CI = 1.119-11.387; P = 0.032), high Pitt bacteremia score (OR = 1.609; CI = 1.226-2.111; P = 0.001), and inappropriate empirical treatment after BSIs (OR = 6.756; CI = 1.922-23.753; P = 0.003) were independent risk factors for the 28-day mortality in KP-BSIs. The prediction model confirmed that microbiologic eradication after 6.5 days and a Pitt bacteremia score of 4.5 or higher were significant predictors of the 28-day mortality. Bioinformatics analysis identified ST11 as the predominant CRKP sequence type, with blaKPC-2 as the most prevalent gene variant. CRKP stains carried multiple plasmid-mediated resistance genes along with some virulence genes. Phylogenetic analysis indicated the presence of nosocomial transmission of ST11 CRKP within the ICU. CONCLUSIONS: The analysis of risk factors for developing CRKP-BSIs and the association between KP-BSIs and 28-day mortality, along with the development of a risk factor-based prediction model and the characterization of CRKP strains, enhances clinicians' understanding of the pathogens responsible for BSIs. This understanding may help in the timely administration of antibiotic therapy for patients with suspected KP-BSIs, potentially improving outcomes.

A novel quantitative double antigen sandwich ELISA for detecting total antibodies against Candida albicans enolase 1.

PURPOSE: This study aimed to develop a double antigen sandwich ELISA (DAgS-ELISA) method for more efficient, accurate, and quantitative detection of total antibodies against Candida albicans enolase1 (CaEno1) for diagnosing invasive candidiasis (IC). METHODS: DAgS-ELISA was developed using recombinant CaEno1 and a monoclonal antibody as the standard. Performance evaluation included limit of detection, accuracy, and repeatability. Dynamic changes in antibody levels against CaEno1 in serum from systemic candidiasis mice were analyzed using DAgS-ELISA. Patient serum samples from IC, Candida colonization, bacterial infections, and healthy controls were analyzed with DAgS-ELISA and indirect ELISA. RESULTS: DAgS-ELISA outperformed indirect ELISA in terms of linear range and test background. In systemic candidiasis mice, a distinctive 'double-peak' pattern in dynamic antibody levels was observed. Additionally, there was a high level of consistency in the positive rates of CaEno1 antibodies detected by both DAgS-ELISA and indirect ELISA. While the positivity rates differed among patient groups, no significant variations in antibody levels were detected among the various positive patient groups. CONCLUSIONS: DAgS-ELISA offers a reliable novel approach for IC diagnosis, enabling rapid, accurate, and quantitative detection of CaEno1 antibodies. Further validation and optimization are needed for its clinical application and effectiveness.

Quantitatively detecting Candida albicans enolase1 with a one-step double monoclonal antibody sandwich ELISA assay.

Invasive candidiasis (IC) is often a cause of severe concern for the hospitalized patients, particularly those who are critically sick. However management of this disease is challenging due to a lack of effective laboratory diagnostic techniques. Hence, we have developed a one-step double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using a pair of specific monoclonal antibodies (mAbs) for the quantitative detection of Candida albicans enolase1 (CaEno1), which is considered as an important diagnostic biomarker for IC. The diagnostic efficiency of the DAS-ELISA was evaluated by using a rabbit model of systemic candidiasis and compared with other assays. The method validation results demonstrated that the developed method was sensitive, reliable, and feasible. The findings of the rabbit model plasma analysis indicated that the diagnostic efficiency of the CaEno1 detection assay was better in comparison to the (1,3)-ß-D-glucan detection and blood culture. CaEno1 is present in the blood of infected rabbits for a brief period and at relatively low levels and thus the combination of CaEno1 antigen and IgG antibodies detection could aid to increase diagnostic efficiency. However, to improve the clinical application of CaEno1 detection in the future, efforts should be made to increase the detection limit of the test by promoting technical developments and by optimizing the protocol for the clinical serial determinations.

Genotype-specific reference interval of haptoglobin tests in a Chinese population on the BN II System.

The distribution of Haptoglobin (HP) subtypes differs according to race and geography. It was also confirmed that the serum HP concentration was substantially affected by the HP subtypes. This study aimed to investigate the HP subtypes in northern Chinese and to establish reference intervals for the major HP subtypes using the BN II system. 1195 individuals were included in the study, grouped by haptoglobin subtype, and tested for concentrations by BN II System. Analysis of reference range was performed according to the EP28-A3c guideline. The need to establish reference ranges for subtype, gender, and age groupings was confirmed by the Z-test. The 2.5th and 97.5th percentiles were used as the upper and lower limits of the reference interval, respectively. In the population we investigated, the HP2-2 subtype had the highest proportion, accounting for 49.3%, followed by HP2-1 (38.0%), HP1-1 (7.2%). In addition, about 5.5% of individuals had HPdel-related subtypes. The concentrations of the major subtypes (HP1-1, HP2-1, HP2-2) were significantly different, and it was necessary to establish reference ranges by grouping according to the results of the Z-test. The reference intervals were as follows: HP1-1, 0.37-2.19 g/L; HP2-1, 0.38-2.12 g/L; HP2-2, 0.12-1.51 g/L. Significant differences in HP concentrations between genders and ages were found, however, it was not necessary to establish separate reference interval since the results of the Z-test was negative. We have established reference ranges of serum haptoglobin concentrations based on subtypes, which are necessary for the clinical application of haptoglobin.

Rapid preparation of Candida genomic DNA: combined use of enzymatic digestion and thermal disruption.

Nucleic acid based molecular technologies are the most promising tools for the early diagnosis of Candida infection. A simple and effective DNA preparation method is of critical for standardizing and applying molecular diagnostics in clinic laboratories. The goal of this study was to develop a Candida DNA preparation method that was quick to do, easy to perform, and bio-safe. Snailase and lyticase were screened and combined in this work to enhance the lysis of Candida cells. The lysis solution composition and metal bath were optimized to boost amplification efficiency and biosafety. A duplex real-time PCR was established to evaluate the sensitivity and specificity of the preparation method. Using the supernatant from the rapid preparation method as templates, the duplex PCR sensitivities for five common Candida species were determined to be as low as 100 CFUs. When compared to conventional preparation methods, the samples prepared by our method showed higher PCR detection sensitivity. PCR identification and ITS sequencing were 100% consistent, which was better than biochemical identification. This study demonstrates a rapid method for Candida DNA preparation that has the potential to be used in clinical laboratories. Meanwhile, the practical application of the method for clinical samples needs to be proven in future investigations.

[Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity].

OBJECTIVE: To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. METHODS: Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. RESULTS: Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). CONCLUSION: DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

Evaluation of a Novel Laboratory Candiduria Screening Protocol in the Intensive Care Unit.

BACKGROUND: Since urine cultures are only guaranteed for patients with obvious urinary symptoms in most cases, most of candiduria episodes are ignored in clinic. OBJECTIVE: This study aimed to design a screening protocol to improve diagnostic efficiency of candiduria, and provide information of Candida species and drug susceptibility. METHODS: All patients, who were admitted to the intensive care unit (ICU) of our hospital during December 1, 2018 and October 1, 2019, were enrolled in this study. Urinalysis was performed every three days for each subject from the first day of ICU admission. Urine specimens were sampled for fungal culture with either condition: (1) yeast-like cell counting (YLCC) ≥200; (2) positive YLCCs were observed in two consecutive tests, and at least one YLCC ≥100. RESULTS: The screening protocol dramatically improved the candiduria diagnostic rate of ICU patients from 2.28% to 17.27%. However, compared to the historical control, the screening protocol has no time-saving advantage in candiduria diagnosing. Higher percentage of C. albicans in screening protocol-identified candiduria patients was observed, although there was no statistical difference. Our results indicated that female gender, pneumonia, diabetes and infarction/hemorrhage patients were more prone to develop candiduria. Non-candiduria patients showed a better tendency for survival and shorter ICU stay length. Multisite colonization was common in the surveyed candiduria patients, who were up to 70.83% showed Candida positive cultures in sputum. CONCLUSION: The screening protocol established in the study was a convenient and practical tool for early warning and feasible management of candiduria and IC.

Progress in the Study of Epidemiologic Characteristics and Influencing Factors of Asymptomatic Malaria Infection in Africa.

Malaria is caused by protozoan parasitic Plasmodium infections. Plasmodium falciparum is common in Africa; P ovale, P malaria and P vivax infections are less prevalent and globally confined, contributing to major causes of global mortality and morbidity, particularly in children in sub-Saharan African countries. In 2018, the total incidence of malaria increased from 221 million to 229 million, with an estimated 503 000 deaths reported. Sub-Saharan Africa has the highest number of cases of malaria and highest mortality rate compared with other countries, like southeastern Asia, east Pacific, western, and America with an estimated 213 million cases. In addition, continuous exposure to Plasmodium parasites results in the production of partial immunity to guard against more problems, resulting in asymptomatic carriers. The diagnosis of asymptomatic malaria is not simple because of the apparent absence of clinical factors and sometimes low levels of parasites. The most basic concept appears to be parasitemia and a lack of malaria signs, primarily fever (axillary temperature <37.5° C). Thus, a better awareness of asymptomatic malaria epidemiology in affected countries will help improve strategies to reduce the local burden of malaria and its health consequences. Therefore, the objective of this study was to determine the magnitude of asymptomatic malaria pathology and related risk factors with epidemiologic characteristics in individuals on the African continent.

Nocardia Infection in Nephrotic Syndrome Patients: Three Case Studies and A Systematic Literature Review.

OBJECTIVE: The multicenter literature review and case studies of 3 patients were undertaken to provide an updated understanding of nocardiosis, an opportunistic bacterial infection affecting immunosuppressed nephrotic syndrome (NS) patients receiving long-term glucocorticoid and immunosuppressant treatment. The results provided clinical and microbiological data to assist physicians in managing nocardiosis patients. METHODS: Three cases between 2017 and 2018 from a single center were reported. Additionally, a systematic review of multicenter cases described in the NCBI PubMed, Web of Science, and Embase in English between January 1, 2001 and May 10, 2021 was conducted. RESULTS: This study described three cases of Nocardia infection in NS patients. The systematic literature review identified 24 cases with sufficient individual patient data. A total of 27 cases extracted from the literature review showed that most patients were > 50 years of age and 70.4% were male. Furthermore, the glucocorticoid or corticosteroid mean dose was 30.9 ± 13.7 mg per day. The average time between hormone therapy and Nocardia infection was 8.5 ± 9.7 months. Pulmonary (85.2%) and skin (44.4%) infections were the most common manifestations in NS patients, with disseminated infections in 77.8% of patients. Nodule/masses and consolidations were the major radiological manifestations. Most patients showed elevated inflammatory biomarkers levels, including white blood cell counts, neutrophils percentage, and C-reactive protein. Twenty-five patients received trimethoprim-sulfamethoxazole monotherapy (18.5%) or trimethoprim-sulfamethoxazole-based multidrug therapy (74.1%), and the remaining two patients (7.4%) received biapenem monotherapy. All patients, except the two who were lost to follow-up, survived without relapse after antibiotic therapy. CONCLUSIONS: Nephrotic syndrome patients are at high risk of Nocardia infection even if receiving low-dose glucocorticoid during the maintenance therapy. The most common manifestations of nocardiosis in NS patients include abnormal lungs revealing nodules and consolidations, skin and subcutaneous abscesses. The NS patients have a high rate of disseminated and cutaneous infections but a low mortality rate. Accurate and prompt microbiological diagnosis is critical for early treatment, besides the combination of appropriate antibiotic therapy and surgical drainage when needed for an improved prognosis.

Management of candiduria in hospitalized patients: a single-center study on the implementation of IDSA guidelines and factors affecting clinical decisions.

Candiduria are common findings in clinic especially in hospitalized patients, while its significance remains undetermined. Since there are few criteria to follow, physicians tended to make decisions by personal experience in many cases in clinical practice. The present study was designed to unveil the present situation of candiduria management in hospitalized patients in clinical practice. A total of 251 hospitalized candiduria patients were retrospectively enrolled in the study. Clinical data on patient demographics, basic conditions, catheter using, urinary symptoms, laboratory data, and antifungal therapies were obtained from electronic medical records. The high rate of the candiduria cases were managed inappropriately after the introduction of the Infectious Diseases Association of America (IDSA) evidence-based recommendations, both in the management of urinary catheter and antifungal agents. Overtreatment was common in asymptomatic candiduria patients. For symptomatic patients, improper drug selections were not rare. In addition, a part of candiduria patients did not receive antifungal therapies although the IDSA recommends. A statistically significant difference was only found in hospital charges of symptomatic candiduria patients managed following IDSA or not. The recurrence rate, mortality, and hospital stay length were similar in candiduria patients regardless of the clinical management. Physicians tend to start empiric antifungal therapy for candiduria patients with pneumonia, multisite of Candida colonization, higher urine Candida CFUs, and long hospital stay. Candiduria has not received special attention today, and empirical antifungal treatment is common. IDSA guidelines are important to standardize the management of candiduria in clinic; however, the significance of the guidelines needs to be further clarified in future multicenter investigations.

Dihydroartemisinin Prevents Progression and Metastasis of Head and Neck Squamous Cell Carcinoma by Inhibiting Polarization of Macrophages in Tumor Microenvironment.

BACKGROUND: Polarized M2 macrophages are an important type of tumor-associated macrophage (TAM), with roles in the growth, invasion, and migration of cancer cells in the tumor microenvironment. Dihydroartemisinin (DHA), a traditional Chinese medicine extract, has been shown to inhibit the progression and metastasis of head and neck squamous cell carcinoma (HNSCC); however, the effect of DHA on cancer prevention, and the associated mechanism, has not been investigated in the tumor microenvironment. MATERIALS AND METHODS: First, human Thp-1 monocytes were induced and differentiated into M2 macrophages using phorbol 12-myristate 13-acetate (PMA), interleukin-6 (IL-6), and interleukin-4 (IL-4). Induction success was confirmed by cell morphology evaluation, flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR). Then, DHA was applied to interfere with M2 macrophage polarization, and conditioned medium (CM), including conditioned medium from M2 macrophages (M2-CM) and conditioned medium from M2 macrophages with DHA (M2-DHA-CM), was obtained. CM was applied to Fadu or Cal-27 cells, and its effects on cancer invasion, migration, and angiogenesis were evaluated using transwell, wound-healing, and tube formation assays, respectively. Finally, Western blotting was used to evaluate the relationship between signal transducer and activator of transcription 3 (STAT3) signaling pathway activation and M2 macrophage polarization. RESULTS: Human Thp-1 monocytes were successfully polarized into M2-like TAMs using PMA, IL-6, and IL-4. We found that M2-like TAMs promoted the invasion, migration, and angiogenesis of HNSCC cells; however, DHA significantly inhibited IL-4/IL-6-induced M2 macrophage polarization. Additionally, as DHA induced a decrease in the number of M2-like TAMs, M2-DHA-CM inhibited the induction of invasion, migration, and angiogenesis of Fadu and Cal-27 cells. Finally, DHA inhibited M2 macrophage polarization by blocking STAT3 pathway activation in macrophages. CONCLUSION: DHA inhibits the invasion, migration, and angiogenesis of HNSCC by preventing M2 macrophage polarization via blocking STAT3 phosphorylation.

PCR-detectable Candida DNA exists a short period in the blood of systemic candidiasis murine model.

Invasive candidiasis is a major challenge to clinical medicine today. However, traditional fungal diagnostic techniques and empirical treatments have shown great limitations. Although efforts are necessarily needed in methodology standardization and multicenter validation, polymerase chain reaction (PCR) is a very promising assay in detecting fungal pathogens. Using a "heat-shock" DNA preparation method, a rapid and simple PCR protocol for quantification of the Candida albicans (C. albicans) ribosomal DNA was established. The PCR assay could detect Candida DNA as low as 10 CFU/mL in samples prepared by the heat-shock protocol, without any cross-reaction with DNA prepared from other Candida spp. and bacterial pathogens. For simulated blood samples, the PCR test sensitivity of whole blood samples was better than that of plasma and blood cells. In the systemic candidiasis murine model, detectable DNA was only observed within 24 h after C. albicans SC5314 injection, which is much shorter than that observed in the kidney.

Candiduria in hospitalized patients: an investigation with the Sysmex UF-1000i urine analyzer.

BACKGROUND: Candiduria is common in hospitalized patients. Its management is limited because of inadequate understanding. Previous epidemiological studies based on culture assay have been limited to small study populations. Therefore, data collected by automated systems from a large target population are necessary for more comprehensive understanding of candiduria in hospitalized patients. METHODS: To determine the performance of the Sysmex UF-1000i in detecting candiduria, a cross-sectional study was designed and conducted. A total of 203 yeast-like cell (YLC)-positive and 127 negative samples were randomly chosen and subjected to microbiologic analysis. The receiver operating characteristic curve (ROC) was used to evaluate the ability of YLC counts as measured by the Sysmex UF1000i to predict candiduria. Urinalysis data from 31,648 hospitalized patients were retrospectively investigated, and statistical analysis was applied to the data collected. RESULTS: Using a cutoff value of 84.6 YLCs/µL, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the yeast like cell (YLC) counts to predict candiduria were 61.7%, 84.1%, 88.6% and 66.3%, respectively. C. glabrata (33.6%) and C. tropicalis (31.4%) were more prevalent than C. albicans (24.3%) in the present study. Of the investigated hospitalized patients, 509 (1.61%) were considered candiduria-positive. Age, gender and basic condition were associated with candiduria in hospitalized patients. In the ICU setting, urinary catheterization appeared to be the only independent risk factor contributing to candiduria according to our investigation. Although antibiotic therapy has been reported to be a very important risk factor, we could not confirm its significance in ICU candiduria patients because of excessive antibiotic usage in our hospital. CONCLUSIONS: The YLC measured by Sysmex UF-1000i is a practical and convenient tool for clinical candiduria screening prior to microbiologic culture. Candiduria is common in hospitalized patients, and its incidence varies according to age, gender and the wards where it is isolated. Candiduria had no direct connection with mortality but might be considered a marker of seriously ill patients who need particular attention in the clinic.

[Detection and analysis of serum level of anti-Candida enolase IgG antibody in patients with autoimmune diseases].

Objective To detect IgG antibody against Candida enolase in the sera of patients with autoimmune diseases. Methods Using purified recombinant Candida enolase as the coating antigen, an ELISA was established for enolase IgG antibody detection and the reactive conditions were optimized. The enolase IgG antibody in the sera from patients with autoimmune diseases and healthy controls were detected by ELISA. The specificity of the positive sera was confirmed by Western blotting. Results The study collected 70 serum samples from the patients with autoimmune diseases and 44 from the healthy individuals. ELISA showed anti-Candida enolase IgG antibody in 19 cases of the autoimmune disease group and and 3 cases of the healthy control group, the positive rates of which were 27.14% (19/70) and 6.82% (3/44), respectively. In the autoimmune disease group, the positive rate of anti-Candida enolase IgG antibody in the systemic lupus erythematosus patients was 45.8% (11/24), significant higher than that in the rheumatoid arthritis patients (11.8%, 2/17). Western blotting validated the specificity of the positive sera. Conclusion The positive rate of anti-Candida enolase IgG antibody in patients with autoimmune disease is high, which would be an interference factor in the application of IgG antibody detection for the diagnosis of invasive candidiasis.

Development of a Lateral Flow Immunoassay for the Rapid Diagnosis of Invasive Candidiasis.

Early and accurate diagnosis of invasive candidiasis (IC) is very important. In this study, a lateral flow immunoassay (LFIA) was developed to detect antibody against Candida albicans enolase (Eno). Colloidal gold particle labeled mouse anti human IgG (1.0 mg/L) was used as the detector reagent. Recombinant enolase (rEno, 1.0 mg/L) and goat anti IgG (1.0 mg/L) were immobilized in test and control lines, respectively, of a nitrocellulose membrane, acting as the capture reagents. The LFIA was used to detect anti Eno in 38 sera from clinically proven IC patients, as well as in 50 healthy control subjects. Compared with an indirect ELISA designed as a reference test, the specificity and sensitivity of the LFIA were 98.2 and 84.8%, respectively. Excellent agreement between the results obtained by ELISA and the LFIA (κ = 0.851) was observed in this study. In addition, the agreement between the blood culture results and LFIA test is strong (κ = 0.658). The data presented in the study indicate that the LFIA test is a suitable tool for the serological surveillance of IC in the field or in poorly equipped laboratories.

Serological response and diagnostic value of recombinant candida cell wall protein enolase, phosphoglycerate kinase, and ß-glucosidase.

There are no specific signs and symtoms for invasive candidiasis (IC), which makes its diagnosis a challenge. Efforts have been made for decades to establish serological assays for rapid diagnosis of IC, but none of them have found widespread clinical use. Using a systemic candiasis murine model, serological response to recombinant proteins of enolase (rEno1), phosphoglycerate kinase (rPgk1), and ß-glucosidase (rBgl2) were evaluated and rEno1 was found to possess the strongest immunoreactivity, followed by rPgk1 and rBgl2. Likewise, IgG antibody titers to rEno1, rPgk1, and rBgl2 in the positive sera of proven IC patients were determined by ELISA. Results show anti-rEno1 antibody possesses the highest titer, followed by rPgk1 and rBgl2. Antibodies against rEno1, rPgk1, and rBgl2 were detected by ELISA tests in a group of 52 proven IC patients or 50 healthy subjects, The sensitivity, specificity, positive and negative predictive values were 88.5, 90.0, 90.2, and 88.2% for anti-rEno1 detection, 86.5, 92.0, 91.8, and 86.8% for anti-rPgk1 detection, and 80.8, 90.0, 89.4, and 81.8% for anti-rBgl2 detection, respectively. The data clearly demonstrate that the recombinant proteins of Eno1, Pgk1, and Bgl2 are promising candidates for IC serodiagnosis. There's great possibility that the recombinant Eno1 will be more applicable in serodiagnosis and vaccine research on account of its strong serological response.

[Whole blood leukocyte phagocytosis assay for Candida albicans based on flow cytometry].

OBJECTIVE: To establish a whole blood leukocyte phagocytosis assay for Candida albicans (C.albicans) based on flow cytometry (FCM). METHODS: C.albicans of mid-logarithmic growth phase was labeled by fluorescence probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), and then added into CD45-PC5 pre-stained human whole blood cells at a 10:1 multiplicity of infection (MOI) in 37DegreesCelsius. The cells were incubated for 10, 30 and 60 minutes. Phagocytosis rate of C.albicans by the CD45 positive cells in the blood was determined by FCM. RESULTS: In yeast extract peptone dextrose medium (YPD) and under the conditions of 37DegreesCelsius and 50 mL/L CO2, the logarithmic growth phase of C.albicans SC5314 was from the 5th to 11th hour. C.albicans were well stained by 10 mmol/L CFDA-SE after 30-minute incubation. After 10-, 30- and 60-minute incubation with SC5314 C.albicans with CD45⁺ cells, the phagocytosis rates measured by FCM were (80.1 ± 6.1)%, (83.8 ± 7.7)% and (92.3 ± 11.2)% for the neutrophils, (11.2 ± 3.6)%, (15.8 ± 4.4)% and (27.7 ± 6.8)% for the monocytes and (0.9 ± 0.3)%, (0.8 ± 0.4)% and (5.2 ± 1.6)% for the lymphocytes. CONCLUSION: The method for measuring whole blood leukocyte phagocytosis of C.albicans based on FCM is successfully established, and 30 minutes are the proper incubation time for the phagocytosis assay.

Increased production of suilysin contributes to invasive infection of the Streptococcus suis strain 05ZYH33.

Streptococcus suis serotype 2 (SS2) is widely recognized in the veterinary world as the cause of rapidly progressive and fatal sepsis in infant pigs, manifested with meningitis, polyarthritis and pneumonia. It has evolved into a highly infectious strain, and caused two large-scale outbreaks of human epidemic in China, characterized bytypical toxic-shock syndrome and invasive infection. However, the molecular basis of virulence of this emerging zoonotic pathogen is still largely unknown. The present study shows that the sequence type (ST)7 epidemic strain S. suis 05ZYH33 causes higher mortality, higher necrosis of polymorphonuclear neutrophils and a significantly higher damage to human umbilical vein endothelial cells compared to the non-epidemic strain S. suis 1940. These differences appear to associate with the enhanced secretion of suilysin (sly) by S. suis 05ZYH33 compared to the non-epidemic strain 1940. Inclusion of additional strains confirmed that the epidemic ST7 strains produce more sly protein (mean, 1.49 g/ml; range, 0.76­1.91 g/ml) than non­epidemic strains (mean, 0.33 g/ml; range, 0.07-0.94 g/ml), and this difference is significant (P<0.001). The nonpolar mutant strain S. suis Δsly, constructed from the epidemic ST7 strain S. suis 05ZYH33 confirmed the role of sly on the enhanced virulence of S. suis ST7 strains. These findings suggest that increased sly production in S. suis 05ZYH33 facilitates penetration to the epithelium and its survival in the bloodstream, thereby contributing to the invasive infection.

[Prokaryotic expression and immunogenicity analysis of phosphoglycerate kinase 1 of Candida albicans].

OBJECTIVE: To construct prokaryotic expression plasmids of Candida albicans gene phosphoglycerate kinase 1 (pgk-1) and examine the immunogenicity of the recombinant protein. METHODS: The full-length coding sequence of pgk-1 was amplified by PCR and cloned into the prokaryotic expression vector pET30a. The 6×His-tagged protein was induced by IPTG in E.coli BL-21(DE3) and the recombinant protein was identified by SDS-PAGE and Western blotting. The BALB/c mice were immunized with the purified recombinant protein to evaluate the antigenicity of the recombinant protein by ELISA. RESULTS: The full-length pgk-1 gene was cloned from SC5314 genome and pET-30a-pgk-1 was successfully constructed. The recombinant protein PGK-1 was highly expressed in E.coli with a relative molecule mass of 54 810. ELISA indicated that the titer of the antibody was about 1:1024. CONCLUSION: PGK-1 was successfully expressed by prokaryotic expression system and the recombinant protein showed favorable immunogenicity in mice.