Analysis of factors with low positive predictive value in the diagnosis of urinary tract infection by flow cytometry.

PURPOSE: Culture-negative urine specimens can be rapidly screened by urine flow cytometry (UFC), while low positive predictive value (PPV) limits the clinical application. We explored the factors associated with a low PPV. METHODS: A total of 5095 urine specimens were analyzed with UFC and culture. Diagnostic performance of leukocytes, bacteria, and BACT-info flags was evaluated by sensitivity, specificity, PPV, and negative predictive value (NPV). The association of contaminated culture and squamous epithelial cell count and BACT-info flag was performed by logistic regression analysis. RESULTS: The NPVs of parallel combination of bacteria and leucocytes were 98.9% in males and 97.9% in females, and PPVs of serial combination were 86.6% and 77.8% in men and women, respectively. The PPV of Gram-negative flag was higher than that of Gram-positive flag. The proportions of contamination in the urine culture results of false positive specimens were 86.9% in males and 98.5% in females at the cutoff points of the serial combination, and these parameters were 53.2% in males and 85.6% in females for the Gram-positive flag. There was a statistically significant association between contaminated cultures and squamous epithelial cells count in females, but not in males. Associations between contaminated cultures and Gram-positive flags or Gram-pos/-neg flags were statistically significant, but there was no association between contaminated cultures and Gram-negative flags. CONCLUSIONS: A serial combination of leukocytes and bacteria may maximize PPV in the diagnosis of bacterial urinary tract infection by urine flow cytometry, and contamination is the main reason for a low PPV.

Immune Checkpoint Blockade in Breast Cancer Therapy.

Cancer immunotherapy is emerging as the most promising novel strategy for cancer treatment. Cancer immunotherapy is broadly categorized into three forms: immune checkpoint modulation, adoptive cell transfer, and cancer vaccine. Immune checkpoint blockade is demonstrated as the most clinically effective treatment with low immune-related adverse events (irAE). Blockade of PD-1/PD-L1 and CTLA-4 has achieved remarkable success in treating various types of tumors, which sparks great interests in this therapeutic strategy and expands the role of immune checkpoint blockade in treating tumors including breast cancer. Based on the notable results obtained from clinical trials, the United States' Food and Drug Administration (FDA) has approved multiple CTLA-4 monoclonal antibodies as well as the PD-1/PD-L1 monoclonal antibodies for treatment of different types of tumors. The theories of immunoediting, T-cell exhaustions, and co-stimulatory/co-inhibitory pathways are immunological foundations for immune checkpoint blockade therapy. Breast cancers such as triple negative breast cancer and HER-2 negative breast cancer respond to immune checkpoint blockade therapy due to their high immunogenicity. PD-1/PD-L1 blockade has just received FDA approval as a standard cancer therapy for solid tumors such as breast cancer. Development of immune checkpoint blockade focuses on two directions: one is to identify proper biomarkers of immune checkpoint blockade in breast cancer, and the other is to combine therapies with PD-1/PD-L1 blockade antibodies to achieve optimal clinical outcomes.

A survey of five broad-host-range plasmids in gram-negative bacilli isolated from patients.

OBJECTIVES: To learn the prevalence of the primary classical broad-host-range (BHR) IncA/C, IncN, IncP, IncQ, and IncW plasmids in dominant gram-negative bacilli from inpatients in a teaching hospital in southern China. METHODS: A multiplex polymerase chain reaction based on the replicons of BHR IncA/C, IncN, IncP, IncQ, and IncW plasmids was developed and used to determine these BHR plasmids. The difference in prevalence rates among the different species from three specimens was evaluated by a binary logistic regression model and the differences between multidrug-resistant organisms (MDRO) and non-MDRO were assessed using a chi-square test. RESULTS: The average positive detection percentages of the replicons were 4.3%, 3.7%, 3.0%, 2.6%, and 1.9%, respectively, for IncN, IncP, IncQ, IncW, and IncA/C in descending order. The distribution of all five BHR plasmids did not differ significantly between specimens collected from wounds and urine, although both were significantly higher than those of sputum. The prevalence rates of all five BHR plasmids in MDROs were significantly higher than those in non-MDRO for Enterobacteriaceae; however, no significant difference was seen in non-fermenting gram-negative bacilli (NFGNB). CONCLUSIONS: BHR IncA/C, IncN, IncP, IncQ, and IncW plasmids, which occur more often in bacilli from wound and urine specimens than those of sputum, are widespread in Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Pseudomonas aeruginosa, and Acinetobacter baumannii strains isolated from inpatients. The prevalence rates in MDRO are higher than those in non-MDRO for Enterobacteriaceae but not significantly different for NFGNB.

Quantitative Value of Bacteria Associated with Leukocytes in Differential Diagnosis of Lower Respiratory Tract Infection in Children, in Comparison to Sputum Culture and Procalcitonin.

BACKGROUND: The mortality and morbidity rates in children with lower respiratory tract infection (LRTI) remain high. OBJECTIVE: To describe the number of bacteria that is associated with leukocytes in differential diagnosis of bacterial, mycoplasma, and viral LRTI in children. METHODS: Sputum smears were Gram stained for counting single-morphology bacteria associated with leukocytes. The differential diagnostic values of bacterial number were assessed in children with LRTI. RESULTS: The area under the receiver operating characteristic (ROC) curve was 0.95 for bacterial number in the differential diagnosis of bacterial infection from mycoplasma and viral infections. The area under the ROC curve was 0.62 for procalcitonin and 0.94 for bacterial number in the differential diagnosis of bacterial infection from mycoplasma infection. CONCLUSION: The number of bacteria associated with leukocytes in sputum was valuable and rapid in differential diagnosis of bacterial infection in children with suspected bacterial, mycoplasma, and viral LRTI.

Arterial instillation of rapamycin in treatment of rabbit hepatic xenograft tumors and its effects on VEGF, iNOS, HIF-1α, Bcl-2, Bax expression and microvessel density.

Hepatocellular carcinoma is one of the leading causes of malignant tumor related death word wide with poor prognosis. Chemotherapy and TACE are main treatment methods for advanced stage cases. Rapamycin, a macrolide compound that initially used to coat coronary stents, can inhibit the growth of a variety of cancer cells especially hepatocellular carcinoma. Twenty-four healthy adult New Zealand white rabbits underwent CT-guided puncture to prepare a model of VX2 liver xenograft tumor. The rabbits were randomly divided into four groups with six in each group and received the following treatments: APR-TACE1: arterial perfusion of high-dose rapamycin combined with TACE; APR-TACE2: arterial perfusion of low-dose rapamycin combined with TACE; TACE: TACE alone; and IVR-TACE: intravenous injection of rapamycin combined with TACE. Two weeks after TACE treatment, the rabbits received CT scan and DSA angiography examination, and then killed by air embolism. The non-necrotic region and surrounding tissues were obtained from the peripheral tumor for iNOS, HIF-1α, VEGF, Bcl-2, and Bax protein expression analysis. Protein expression of iNOS, HIF-1α, VEGF, and Bcl-2 in APR-TACE1 were significantly lower than those in groups APR-TACE2, TACE, and IVR-TACE (p < 0.05). iNOS, HIF-1α, and VEGF in APR-TACE2 were lower than those in TACE (p < 0.05). iNOS and VEGF in APR-TACE2 were significantly lower than those in IVR-TACE (p < 0.05). iNOS in IVR-TACE was significantly lower than that in TACE (p < 0.05). The expression levels of Bcl-2 and Bax were statistically significant between APR-TACE2 and TACE (p < 0.05). The MVD of the tumor tissue in APR-TACE1 was lower than that of groups APR-TACE2, TACE, IVR-TACE with statistical difference (p < 0.05). However, MVD of APR-TACE2 was lower than that of groups TACE, IVR-TACE with significant statistical difference (p < 0.05). Arterial instillation of rapamycin+TACE in treatment of rabbit hepatic xenograft tumors can reduce tumor neovascularization and inhibit iNOS, HIF-1α, VEGF, Bcl-2, and Bax protein expression.

Comparing the diagnostic value of bronchoalveolar lavage fluid galactomannan, serum galactomannanan, and serum 1,3-ß-d-glucan in non-neutropenic respiratory disease patients with invasive pulmonary aspergillosis.

ABSTRACT: The incidence of invasive pulmonary aspergillosis (IPA) is increasing higher in non-neutropenic patients. This study aimed to assess the diagnostic performance of bronchoalveolar lavage fluid (BALF). Galactomannan (GM), serum GM, and 1,3-ß-d-glucan (BDG) in non-neutropenic respiratory disease patients with IPA.A total of 333 non-neutropenic patients suspected IPA were recruited from Xiamen University Zhong Shan hospital between January 2016 and February 2019. One, 33, and 92 cases were diagnosed with proven, and possible IPA.BALF and serum GM were both elevated in the possible IPA group and the probable/proven IPA group (p < 0.001). BALF and serum GM showed a fair correlation in the possible IPA group (r = 0.286, p = 0.008), and moderate correlation in the probable/proven IPA group (r = 0.466, p = 0.005). When the cutoff value was 0.5, the sensitivity and negative likelihood ratio of BALF GM were superior to serum GM (78.3% vs 47.8%, 96.7% vs 91.6%). The specificity and positive likelihood ratio of BALF GM were slightly weaker than serum GM (91.8% vs 95.4%, 56.7% vs 85.0%). When the cutoff value was 1.0, the sensitivity and negative predictive value of BALF GM were better than serum GM (73.9% vs 26.1%, 94.5% vs 88.8%), and the specificity of were equivalent (99.2%). The optimal cutoff value of BALF GM was 0.6, wherein the sensitivity reached 78.3% and the specificity reached 95.4%. Given the extremely low sensitivity of serum BDG at different cutoff values (≥10 µg/mL = 5.3%, ≥20 µg/mL = 2.1%), it cannot be used as a preferred biomarker.The diagnostic performance of BALF GM was superior to other biomarkers and the optimal cutoff value was 0.6.

Integrated histological and molecular analyses of rebiopsy samples at osimertinib progression improve post-progression survivals: A single-center retrospective study.

BACKGROUND: This single-center retrospective cohort study sought to investigate the impact of rebiopsy analysis after osimertinib progression in improving the survival outcomes. METHODS: Eighty-nine patients with EGFR T790M-positive advanced NSCLC who received second- or further-line osimertinib between January 2017 and July 2019 were included in this study. The co-primary study endpoints were post-progression progression-free survival (pPFS), defined as the time from osimertinib progression until progression from further-line treatment, and post-progression overall survival (pOS), defined as the time from osimertinib progression until death or the last follow-up date. RESULTS: Pairwise analysis revealed that receiving targeted therapy as further-line treatment after osimertinib progression did not statistically improve the pPFS (P = 0.285) or the pOS (P = 0.903) compared to chemotherapy. However, patients who submitted rebiopsy samples at osimertinib progression for histological and molecular analyses, particularly those who had actionable markers and received highly matched therapy, had significantly longer pPFS and pOS as compared to those who received low-level matched therapy (pPFS = 10.0 m vs. 4.1 m, P = 0.005; pOS = 19.4 m vs. 10.0 m, P = 0.023), unmatched therapy (pPFS = 10.0 m vs. 4.7 m, P = 0.009; pOS = 19.4 m vs. 7.0 m, P = 0.001), and those without rebiopsy data (Rebiopsy vs Non-rebiopsy; pPFS = 6.1 m vs. 3.3 m, P = 0.014; pOS = 11.7 m vs. 6.8 m, P = 0.011). CONCLUSION: Our real-world cohort study demonstrates that integrated histological and molecular analyses of rebiopsy specimens after osimertinib progression could provide more opportunities for individualized treatments to improve the post-progression survival of patients with advanced NSCLC. Our findings provide clinical evidence that supports the inclusion of NGS-based analysis of rebiopsy specimens as standard-of-care after osimertinib progression and warrants further prospective evaluation.

Evaluation of Real-Time PCR Coupled With Multiplex Probe Melting Curve Analysis for Pathogen Detection in Patients With Suspected Bloodstream Infections.

Background: This study aimed to evaluate real-time polymerase chain reaction coupled with multiplex probe melting curve analysis (PCR-MCA) for pathogen detection in patients with suspected bloodstream infections (BSIs). Methods: A PCR-MCA assay was developed for simultaneous identification of 28 kinds of the most common pathogens and two resistance genes within a few hours. The diagnostic performance of the PCR-MCA assay was determined and compared to the results of blood culture. Results: A total of 2,844 consecutive new episodes of suspected BSIs in 2,763 patients were included in this study. There were 269 episodes of pathogens identified by blood culture. For all the pathogens tested, the PCR-MCA assay exhibited a sensitivity of 88.8% (239/269), specificity of 100% (2,575/2,575), and agreement of 98.9% (2,814/2,844). For the pathogens on the PCR-MCA list, the PCR-MCA results had a sensitivity of 99.2% (239/241), specificity of 100% (2,575/2,575), and agreement of 99.9% (2,814/2,816) compared with the results of blood culture. For seven samples with multiple pathogens identified simultaneously during one blood culture investigation, the PCR-MCA assay verified the results of the blood culture, with an agreement rate of 100% for each. Conclusion: The PCR-MCA assay could discover 88.8% of the pathogens in clinical practice, showing excellent diagnostic performance vs. that of blood culture for pathogen detection in patients with suspected BSIs, and would contribute to rapid diagnosis and correct antibiotic administration.

Arterial Infusion of Rapamycin in the Treatment of Rabbit Hepatocellular Carcinoma to Improve the Effect of TACE.

BACKGROUND: Hepatocellular carcinoma is one of the leading causes of cancer-related death. Hepatic transcatheter arterial chemo-embolization (TACE) is commonly used clinically for advanced hepatocellular carcinoma treatment. AIM: The aim of this study was to evaluate whether arterial infusion of rapamycin can improve the effect of TACE in treatment of rabbit hepatocellular carcinoma. MATERIAL AND METHODS: Eighteen healthy New Zealand white rabbits weighing 2.6 ± 0.3 kg were used in a standardised hepatocellular carcinoma model and randomly divided into three groups of 6 rabbits. Group A: the rabbits were treated with rapamycin and TACE by administering arterial perfusion of 2 mg/kg rapamycin + 1 mg/kg epirubicin, 0.2 mg/kg mitomycin, and lipiodol emulsion embolization. Group B: rapamycin was reduced to 1 mg/kg. And for Group C, the rabbits received only TACE treatment. 14 days post operation, CT scan and digital subtraction angiography (DSA) was performed to examine TACE efficacy. The rabbits were killed by air embolism and the expression of HIF-1a, VEGF, iNOS, and CD34 were measured in an immunohistochemical assay of thetumor tissue. RESULTS: HIF-1a, VEGF and iNOS protein expression in Group A was significantly lower than that of Group B and Group C (P<0.05). The tumor MVD in group C was significantly higher than that of group A and group B (P<0.05); and the tumor MVD of group B was significantly higher than group A (P<0.05). CONCLUSION: Arterial infusion of rapamycin combined with TACE can improve treatment efficacy by decreasing HIF-1a, VEGF, iNOS and CD34 expression.

Adiponectin is valuable in the diagnosis of acute heart failure with renal insufficiency.

Acute heart failure (AHF) is a major public health issue due to its high incidence and poor prognosis; thus, efficient and timely diagnosis is critical for improving the prognosis and lowering the mortality rate. Amino-terminal pro-brain natriuretic peptide (NT-proBNP) is widely used in the diagnosis of AHF; however, its efficacy is controversial in diagnosing AHF with renal insufficiency. There were numerous studies reporting the association of adiponectin (ADPN) and heart diseases. Therefore, the present study aimed to investigate whether ADPN is helpful in identifying AHF with renal insufficiency. A total of 407 participants (218 AHF patients and 189 controls) were enrolled into the current study. The plasma levels of ADPN and NT-proBNP were measured using a sandwich enzyme-linked immunosorbent assay and an electrochemiluminescence immunoassay, respectively. In addition, these levels were compared among the various New York Health Association classes, as well as the ischemic and non-ischemic AHF cases. The correlation between the two biomarkers and the renal function was analyzed by Spearman's correlation, while the diagnostic efficiency of ADPN and NT-proBNP was evaluated in AHF patients with and without renal insufficiency. The results revealed that NT-proBNP exhibited a higher diagnostic efficiency as compared with ADPN in patients without renal insufficiency [area under the receiver operating characteristic curve (AUC), 0.905 vs. 0.775]. By contrast, the ADPN presented a better diagnostic value in comparison with NT-proBNP in AHF with renal insufficiency (AUC, 0.872 vs. 0.828). Therefore, a combination of these two biomarkers may provide an excellent efficacy in the diagnosis of AHF with renal insufficiency (AUC, 0.904; sensitivity, 71.2%; specificity, 98.3%). In conclusion, ADPN is a valuable biomarker for diagnosing AHF, particularly in patients with impaired renal function.

Dissemination and Molecular Characterization of Staphylococcus aureus at a Tertiary Referral Hospital in Xiamen City, China.

Staphylococcus aureus is a global epidemic pathogen that causes heavy disease burden. The aim of this study was to determine which globally known S. aureus lineages are currently present in a hospital of Xiamen. Therefore, the 426 S. aureus strains were detected by Melting Curve Analysis (MCA) and genotyped by Pulsed Field Gel Electrophoresis (PFGE) as well as Multicolor Melting Curve Analysis-Based Multilocus Melt Typing (MLMT). In addition, Multilocus Sequence Typing (MLST) was used to identify 108 representative strains. In light of eighteen antibiotics except for Vancomycin (by Broth Dilution Method), we used the Kirby-Bauer disc diffusion method to assess antibiotic susceptibility of 426 S. aureus strains. Finally, PFGE analysis revealed 14 different patterns with three major patterns (C10, C8, and C11) that accounted for 69.42% of all S. aureus strains, and MT-1~MT-5 occupied most part of the strains by MLMT. MLST revealed 25 different STs with the predominant types being ST239, ST59, and ST188. There have been 8 antibiotics that showed more than 50% resistance of all S. aureus strains. In summary, we found several of the lineages are predominant in our hospital. And antibiotic resistance is still a severe problem that needs to be controlled in clinic.

Probing minority population of antibiotic-resistant bacteria.

The evolution and spread of antibiotic-resistant pathogens has become a major threat to public health. Advanced tools are urgently needed to quickly diagnose antibiotic-resistant infections to initiate appropriate treatment. Here we report the development of a highly sensitive flow cytometric method to probe minority population of antibiotic-resistant bacteria via single cell detection. Monoclonal antibody against TEM-1 ß-lactamase and Alexa Fluor 488-conjugated secondary antibody were used to selectively label resistant bacteria green, and nucleic acid dye SYTO 62 was used to stain all the bacteria red. A laboratory-built high sensitivity flow cytometer (HSFCM) was applied to simultaneously detect the side scatter and dual-color fluorescence signals of single bacteria. By using E. coli JM109/pUC19 and E. coli JM109 as the model systems for antibiotic-resistant and antibiotic-susceptible bacteria, respectively, as low as 0.1% of antibiotic-resistant bacteria were accurately quantified. By monitoring the dynamic population change of a bacterial culture with the administration of antibiotics, we confirmed that under the antimicrobial pressure, the original low population of antibiotic-resistant bacteria outcompeted susceptible strains and became the dominant population after 5hours of growth. Detection of antibiotic-resistant infection in clinical urine samples was achieved without cultivation, and the bacterial load of susceptible and resistant strains can be faithfully quantified. Overall, the HSFCM-based quantitative method provides a powerful tool for the fundamental studies of antibiotic resistance and holds the potential to provide rapid and precise guidance in clinical therapies.