Novel automated antifungal susceptibility testing system for yeasts based on dual-detection algorithm of turbidimetry and colorimetry.

Introduction. The increasing prevalence and growing resistance of fungi present a significant peril to public health. There are only four classes of antifungal medicines available today, and few candidates are in clinical trials.Hypothesis/Gap Statement. Rapid and sensitive diagnostic techniques are lacking for most fungal pathogens, and those that do exist are expensive or hard to obtain.Aim. This study aimed to evaluate the feasibility of a novel automated antifungal susceptibility testing system, Fungus AST, in comparison to the broth microdilution method (BMD) recommended by the Clinical and Laboratory Standards Institute (CLSI).Methodology. A total of 101 clinical Candida spp. isolates were collected from the Zengcheng Branch of Nanfang Hospital and subjected to antifungal susceptibility testing. Antifungal susceptibility was assessed using the Fungus AST method and the BMD.Results. In this study, we introduce a novel automated antifungal susceptibility testing system, Fungus AST, which detects the turbidity and/or colour intensity of microdilution wells using a four-wavelength detection technology in real time and is designed to match the growth characteristics of strains over time. Based on our analysis, all reportable ranges of Fungus AST were suitable for clinical fungal isolates in PR China. Within ±twofold dilutions, reproducibility was 100 %. Considering the BMD as a referenced method, ten antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole, voriconazole, posaconazole, itraconazole, amphotericin B, 5-flucytosine and nystatin) showed an essential agreement of >95 %. The category agreement of five antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole and voriconazole) was excellent at >90 %. One Candida albicans isolate and voriconazole showed a major error (ME) (1.7 %), and no other ME or very ME agents were found.Conclusion. Given the above, it can be argued that the utilization of Fungus AST is a discretionary automated approach. More improvements are needed in Fungus AST compared to the BMD system for a wider range of clinical isolates, including different types of fungi.

Clinical role of M. pneumoniae typing antibody detected by chemiluminescent immunoassay in the diagnosis of Mycoplasma pneumoniae pneumonia in children.

OBJECTIVE: The levels of serum M. pneumoniae typing antibodies in children with community-acquired pneumonia (CAP) were detected by chemiluminescent immunoassay (CLIA) to explore the clinical role of M. pneumoniae typing antibody (MP-IgM, MP-IgG) in M. pneumoniae pneumonia. METHODS: A total of 387 Child patients with CAP diagnosed at the Pediatric outpatient department of Zengcheng Branch, Nanfang Hospital, Southern Medical University, were enrolled between January 2020 to December 2021 and divided into M. pneumoniae pneumonia (MPP) group (n = 210) and non-M. pneumoniae pneumonia (NMPP) group (n = 177). Firstly, Clinical data, full blood count (WBC, NEU%, LYM%, MONO%, EOS%, BASO%, RBC, HGB, PLT) and biochemical tests (AST, LDH, ɑ-HBDH, CK, CKMB, CRP, PCT, IL-6) as well as laboratory diagnostic tests (MP-IgM, MP-IgG) were compared between the two groups. Secondly, we assessed the correlation between the average level of M. pneumoniae typing antibody detected by CLIA and the titer of anti-M. pneumoniae antibody (MP-Ab) tested by passive agglutination (PA) method. Thirdly, receiver operating characteristic (ROC) curve for the MP-IgM and MP-IgG was examined to evaluate the value of diagnosing M. pneumoniae pneumonia. Finally, we follow-up 120 cases of MPP group and analysis medication results. RESULTS: (1) Mean age, runny nose, expectoration, LYM%, NEU%, HGB, AST, MP-IgM and MP-IgG were statistically significant in the MPP group and NMPP group (all P < 0.05). (2) Correlation analysis showed that MP-IgM average level was linearly associated with MP-Ab titer (R2 = 0.84) and MP-IgG average level was exponentially correlated with MP-Ab titer under 1:640 (R2 = 0.96). (3) The ROC curve of MP-IgM and MP-IgG were significantly different (both P < 0.001). A serum MP-IgM level above 1 S/CO and MP-IgG level above 14.15 AU/mL were significant predictors for M. pneumoniae pneumonia: area under the curve (AUC) of 0.810, 0.815; standard error (SE) of 0.021, 0.022; 95 % confidence interval (CI) of 0.768-0.852, 0.773-0.858; the diagnostic sensitivity of 74.3 %, 62.1 %; and specificity of 72.9 %, 87.0 %; respectively. (4) Of the 120 children with M. pneumoniae pneumonia followed up, 79 (65.8 %) cases took azithromycin and 68 (86.1 %) cases were recovered. CONCLUSIONS: A series of our studies shown that, CLIA, speedy and automated clinical examination method, has higher specificity and sensitivity for the quantitative detection of MP-IgM and MP-IgG, playing an important role of early diagnosis as well as prompt intervention to reduces macrolide-resistant strains and sequelae of children with M. pneumoniae pneumonia.

Preparation and application of yellow fever virus NS1 protein-specific monoclonal antibodies.

Yellow fever is an acute infectious disease that is common in Africa and South America and causes thousands of deaths annually. However, there are very few studies on yellow fever virus (YFV) antigen detection kits. As a detection target, the nonstructural protein 1 (NS1) has been successfully used in the early diagnosis of dengue virus (a member of the Flaviviridae family) infection. In this study, we used monoclonal antibody technology to prepare anti-YFV NS1 monoclonal antibodies (MAbs) and identified their immunological properties. Next, we used two mouse MAbs that can recognize different epitopes of YFV NS1 as capture and detection antibodies to establish a YFV NS1 antigen-capture enzyme-linked immunosorbent assay (ELISA). The antigen-capture ELISA displayed exclusive specificity to YFV without cross-reaction with other related members of the flavivirus family, including the dengue virus, West Nile virus, Japanese encephalitis virus. Additionally, the detection sensitivity towards the YFV culture supernatant was 103 TCID50/mL and the detection positivity rate was 95% compared with reverse transcription-polymerase chain reaction. In conclusion, this newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of YFV infection in animals or humans.

A survey of clinical and laboratory characteristics of dengue fever epidemic from 2014 to 2018 in Guangzhou, China.

BACKGROUND: In 2014, a serious dengue outbreak occurred in Guangzhou, South China. In this study, the clinical and laboratory characteristics of dengue fever (DF) group and other febrile illnesses (OFI) group in Guangzhou were described. METHODS: Clinical and laboratory data collected by studying 1,792 patients from Nanfang Hospital, Southern Medical University during 2014 and 2018. Laboratory data was analyzed by SPSS 22.0 statistical software including Full blood counts (SYSMEX XE-5000), Laboratory Biochemical tests (Roche Cobas 8000), Dengue virus RNA (RT-PCR-Fluorescence Probing) and Dengue IgG/IgM Antibody (Colloidal Gold), Dengue Virus NS1 (ELISA). RESULTS: In the DF group and OFI group, gender ratios were 1.08:1 (male/female, P>0.05) and 1.45:1 (male/female, P<0.05). Adults aged 25-34 years old (29.4%) with the main peak appeared in the DF group, and the same main peak appeared in the OFI group: 25-34 years old (25.13%). Patients were from Medical emergency (41.2% DF group, 29.4% OFI group). The distribution of fever days before treatment was mainly focused within 5 days, with a main peak in the 2 fever days before treatment (24.6%) in the DF group and the main peak in 1 fever day before treatment (46.9%) in OFI group. The major symptoms of the DF group were presented with were fever (100%), myalgia (34.77%), pharyngeal hyperemia (31.33%), headache (25.65%), adenoids (19.62%), and rash (13.25%). In the OFI group, Pharyngeal hyperemia was the most common clinical symptom, accounting for 27.24%, and the next symptom was adenoids (21.26%). The sensitivity and specificity of DV RNA were 61.54%, 100%, respectively, compared to the DF Nonstructural protein 1 (NS1). Dengue virus (DENV) Immunoglobulin M (IgM) IgM in both groups was statistically significant, with DENV-IgM in the DF group were stronger (Z=-7.863, P<0.001), and DENV immunoglobulin G (IgG) were no statistically significant (Z=-1.212, P=0.226). In DF group, 37.14% of serum samples had elevated Alanine transaminase (ALT) levels, 76.85% of them had elevated aspartate aminotransferase (AST) levels, 32.08% of them had elevated creatine kinase (CK) levels, and 2.67% of them had elevated C-reaction protein (CRP) levels, compared with 13.51% of them had elevated ALT levels, 30.65% of them had elevated AST levels, 6.06% of them had elevated CK levels and 69.35% of them had elevated CRP levels of the OFI patients. The prominent manifestations were thrombocytopenia (occurring in 28.07% of the DF group, compared to 5.18% of OFI group) and leucopenia (occurring in 43.27% of DF group and 3.63% of OFI group). The DF incidence of all fever cases was 49.0% within three months in 2014, compared with 1.4% in 2015, 0% in 2016, 0.9% in 2017 and 6.4% in 2018 (P<0.001). DF and OFI can occur in any age and sex. DF occurred in the young and the old, OFI occurred in children and youth. The clinical symptoms of myalgia, headache, rash, weak, Chills, follicular hyperplasia in both groups were statistically significant (P<0.001). CONCLUSIONS: IgM can be easily recognized for early diagnoses, DENV-RNA had lower sensitivity and higher specificity, and DF NS1 enzyme-linked immunosorbent assay (ELISA) has a higher sensitive and specificity. DF is a serious public health problem and an emerging continuous threat in Guangzhou. In high-prevalence areas, effective epidemic monitoring and prevention measures need to be undertaken. After the unprecedented outbreak in 2014, on account of the government and citizen paying more attention to the DF epidemic, the cases of DF were decreased significantly from 2015 to 2018.

Comparison of chemiluminescence immunoassay, enzyme-linked immunosorbent assay and passive agglutination for diagnosis of Mycoplasma pneumoniae infection.

OBJECTIVE: This study aimed to compare the performance of chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), and passive agglutination (PA) method in detecting Mycoplasma pneumoniae (MP) infection. METHODS: This study enrolled a total of 280 patients who were consecutively seen at the Nanfang Hospital of the Southern Medical University in Guangdong Province, China, between August and December 2016. Serum was collected and examined by CLIA, ELISA, and PA, respectively. RESULTS: There were 180 positive (64.3%) and 100 negative cases (35.7%) by PA, 184 positive (65.7%) and 96 negative cases (34.3%) by CLIA MP-immunoglobulin (Ig) M, 89 positive (31.8%) and 191 negative cases (68.2%) by CLIA MP-IgG, 196 positive (70%) and 84 negative cases (30%) by ELISA MP-IgM, and 114 positive (40.7%) and 166 negative cases (59.3%) by ELISA MP-IgG. Patients were allocated to two groups based on PA results. In PA-negative group (≤1:40), the positive rates of MP-IgM by CLIA were 22.8% and 51.2% and by ELISA were 33.3% and 53.5%, respectively. In the PA-positive group (1:80 to ≥1:1,280), MP-IgM negative cases showed a decreasing trend: 40%, 18%, 14.3%, 10%, and 6.7% (CLIA), and 43.3%, 8%, 14.3%, 5%, and 6.7% (ELISA). The consistency between CLIA/ELISA MP-IgM, -IgG, and -IgG+MP-IgM was >92% for negative cases and >75% for positive cases, resulting in an overall consistency rate >88%. The kappa coefficients were 0.804, 0.763, and 0.806, respectively. CONCLUSION: CLIA and ELISA have a higher sensitivity compared with PA. CLIA has a high concordance with ELISA. Moreover, CLIA has a higher specificity and sensitivity for the detection of IgM and IgG and should be used for the clinical diagnosis of MP infection.

[Modified Bethesda, modified Nijmegen and blank assays for coagulation factor VIII inhibitor detection and factors affecting the results].

OBJECTIVE: To evaluate the clinical value of modified Bethesda assay, modified Nijmegen assay and blank assay for detection of coagulation factor VIII (FVIII) inhibitors in patients with hemophilia A and analyze the factors that affect FVIII inhibitor detection. METHODS: The levels of FVIII inhibitors in 257 patients with hemophilia A were detected using modified Bethesda assay, modified Nijmegen assay and blank assay (in which the buffer or FVIII-deficient plasma in the control mixture was replaced by deionized water). The 3 methods were compared for positivity rates and FVIII inhibitor titers based on the positive cut-off value of FVIII inhibitors ≥0.60 BU/mL. RESULTS: The positive rates of modified Bethesda assay, modified Nijmegen assay and blank assay were 79.38%, 85.21% and 72.37%, respectively. A strong correlation was found between the results by modified Bethesda assay and modified Nijmegen assay (r=0.996, P<0.001), and FVIII inhibitor titers (P<0.001) but not the positive rates (P=0.105) detected by the two methods differed significantly. The correlation coefficients between modified Nijmegen assay and blank assay was 0.994 (P<0.001), and a significant difference was found in FVIII inhibitor titers (P<0.001) but not the positivity rates (P=0.079) detected by the two methods. The correlation coefficient between modified Nijmegen assay and blank assay was 0.994 (r=0.994), and the two methods yielded significantly different FVIII inhibitor titers and positivity rates (P=0.001). CONCLUSION: The modified Bethesda assay has a lower sensitivity than modified Nijmegen assay but has a higher sensitivity than blank assay. The consistency level of coagulation factors in the reaction system and stable buffer system are important factors that affect FVIII-inhibitor detection.