Laboratory testing for common pathogens of respiratory tract infection in pediatrics / 中华检验医学杂志

Currently, the respiratory tract infection (RTI) is still one of the most common disease that seriously threatens children′s life and health. Timely detection of pathogens in clinical specimens of children with RTI is helpful to accurate diagnosis and reduce the irrational usage of antibiotics. It is also an important strategy to achieve the best clinical management of RTI in children. In recent years, in addition to the traditional staining and microscopy, culture and antigen detection, the polymerase chain reaction, syndromic approach testing and metagenomic next-generation sequencing have also been used for the diagnosis of pathogens in children with RTI, showing a good application prospect. This review aims to systematically summarize the classification of clinical specimens and the detection methods of common pathogens in the diagnosis process of childhood respiratory infections. This not only expands the understanding of pediatric medicine, but also provides more enlightenment for related research work.

Optimization of Microbiological Laboratory Detection Strategy for Patients in A Designated Hospital Treating Novel Coronavirus Pneumonia in Anhui Province

Novel coronavirus pneumonia (NCP) is an emerging, highly contagious community acquired pneumonia (CAP) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Highly efficient and accurate microbiological laboratory assay is essential to confirm the SARS-CoV-2 infection, rule out other pathogens that can cause CAP, and monitor secondary infections. Here, we enrolled and provide microbiological analysis for 129 suspected and 52 transferred confirmed NCP patients hospitalized in the First Affiliated Hospital of University of Science and Technology of China (USTC) from Jan 21 to Feb 29, 2020. By analyzing the dual swab samples (sputum and pharyngeal) from 129 suspected patients with realtime RT-PCR, we confirmed 33 SARS-CoV-2 infections, with two co-infection cases with adenovirus or rhinovirus. We also used multiplex PCR to detect 13 common respiratory tract pathogens in 96 non-NCP patients, and found that 30 patients (31.25%) were infected with at least one respiratory tract pathogen that may cause CAP. Further, we performed bacterial and fungal cultures as well as fungal serologic tests and found that there is no secondary bacterial/fungal infections in confirmed NCP patients. Our studies suggest that, during the epidemic of NCP in Anhui province, there was a certain proportion of infection and co-infection of other common pathogens of CAP, and the secondary bacterial and fungal infection is not detectable in NCP patients. In comparison with SARS-CoV-2 detection alone, this optimized strategy combining multiple pathogen detection for identification of NCP and other CAP patients as well as cultures and serologic tests for confirmed patients increases the diagnosis efficiency and facilitates the personalized medication.

The role of polymorphonuclear leukocyte in Staphylococcus aureus Panton-Valentine leukocidin-induced acute lung inflammation and injury / 中华传染病杂志

ObjectiveTo explore the role of polymorpbonuclear leukocyte (PMN) in PantonValentine leucocidin (PVL)-induccd acute lung inflammation and injury. Methods Fifteen New Zealand rabbits were divided into 3 groups with five rabbits in each group.The controls were treated with pbosphate buffer solution (PBS),the rabbits with normal granulocyte in rPVL group were treated with endotracheal instillation of rPVL,the granulocytopenia rabbits in vincristine (VCR) +rPVL group were firstly treated with VCR,thcn with endotracheal instillation of rPVL.Nine hours after injection,the peripheral blood and bronchoalveolar lavage fluid (BALF) were collected for counting PMN.The lactate dehydrogenase (LDH) activity in BALF,lung permeability index (LPI),PMN apoptosis and necrosis and the release of reactive oxygen species (ROS)in BALF were measured.After the rabbits sacrificed,the lung tissue samples were collcctcd for dctcrmining wet/dry (W/D) ratio and histopathological examination.The comparison among groups was done by t test.ResultsThe PMN count in the peripheral blood was (2.69=0.34) × 10 mL in rPVL group,which was significantly lower than control group [(3.63 ± 0.38) × 105/mL] (t =4.12,P＜0.05).The PMN counts in BALF in control group,rPVL group and VCR+rPVL group were (0.57±0.01 ) ×106/mL,(3.01±0.02) × 106/mL and (0.10±0.02) × 106/mL,respectively; that in rPVL group was significantly higher than those in control group (t=254.39,P＜0.05).The LDH activity,LPI and W/D ratio in rPVL group were all significantly higher than control group,while those in VCR+rPVL group were not significantly different from control group.The PMN apoptosis rate and necrosis rate in VCR+rPVL groupwere (1.17±0.24)％ and (1.13±0.17)％,respectively.The releases of ROS (meanfluorescence intensity) in rPVL group,control group and VCR+rPVL group were 1.56±0.39,0.41±0.03 and 0.39±0.02,respectively,and that in rPVL group was significantly higher (t=6.58,P＜0.05).Histopathological examination of the lung showed the diffuse infiltration of inflammatory cells,hemorrhage and edema in rPVL group,wbile there was only thimbleful infiltration of inflammatory cells observed in surrounding bronchia and alveolar septun in VCR-rPVL group.ConclusionsrPVL can induce lung inflammation and injury in rabbits with normal granulocyte,but not in neutropenic rabbirs.Lung inflammation and injury may be the result of recruitment,aggregation and subsequent lysis and/or activation of PMN,which can damage the lung by releasing the contents of cytotoxic granules and/or reactive oxygen metabolites.

Effect of PDTC on the NF-κB activation and the expression of inflammatory cytokines in THP-1 macrophages induced by rPVL / 中华微生物学和免疫学杂志

Objective To investigate effect of PDTC on the NF-κB activation and the expression of inflammatory cytokines in THP-1 macrophages induced by rPVL.Methods The study was divided into three groups:PBS-treated control group,rPVL-treated group and PDTC group which was given 100 μmol/L PDTC at 60 min before rPVL exposure.Immunohistochemistry method was used to test the translocation of NF-κB protein; the expression of NF-κB and IκB protein was analyzed by Western blot; RT-PCR and ELISA was performed to test expression of IL-8 and L-6 in THP-1 macrophages.Results Compared with rPVL-treated group,the activation of NF-κB and the expression of IL-8 and L-6 in PDTC group was significantly decreased.The protein secretions of IL-8 and IL-6 were reduced to 6.78 ng/ml,3.88 ng/ml,receptively(P ＜0.05).Conclusion The inhibitor of NF-κB,PDTC,could significantly decrease the secretion of pro-inflammatory in THP-1 macrophages by rPVL,and it suggested that PDTC played an important role in protecting tissues from damage induced by rPVL.

The role of NF-κB signaling pathway protein and cytokines in Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury / 中华微生物学和免疫学杂志

ObjectiveTo explore the role of NF-κB signaling pathway protein and cytokines in Panton-Valentine leukocidin (PVL)-induced acute lung inflammation and injury.MethodsThirty rabbits were distributed randomly into two groups,each group had fifteen rabbits.Group rPVL were directly treated with endotracheal instillation of rPVL,normal control were treated with PBS.Then five rabbits chosen at random from each group were killed at 3,6,or 9 h postinfection.The lung was removed from the rabbits to determine histopathology studies.ELISA was performed to evaluate levels of IL-6,IL-8,IL-10 and TNF-α.NF-κB p65 protein of the lung tissue was assessed by immunohistochemistry method.ResultsIn group rPVL histopathology study showed symptoms of severe illness:diffuse infiltration of inflammatory cells,hemorrhage,edema and other manifestations of lung injury.Levels of IL-6,IL-8 and TNF-α were increased gradually,and the level of IL-10 was increased at 9 h postinfection.The expression of NF-κB p65 protein was increased gradually with the infection time.ConclusionNF-κB activation and cytokines release play an important role in PVL-related lung injury.It may be an important path to down regulate the counts of NF-κB activation.

Panton-valentine leukocidin induce THP-1 macrophages IL-8 and IL-6 response through nuclear TLR4/NF-κB pathway / 中华微生物学和免疫学杂志

Objective To investigate the influence of panton-valentine leucocidin (PVL) on expression of Toll like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signals and IL-8,IL-6 in THP-1 macrophages,and to study the mechanism of PVL-related lung tissue damage.Methods THP-1 cells were cultured in the presence of 100 nmol/L phorbol-12-myristate 3-acetate (PMA) for 48 h to induce monocytemacrophage differentiation.rPVL-F and rPVL-S were induced and expressed from the recombinant plasmid,respectively purified with chromatographic column. After that,THP-1 macrophages were incubated with rPVL,and then ELISA was performed to test expression of IL-8 and L-6 in supernatants fluid; RT-PCR was performed to detect expression of IL-8,L-6 and TLR4 ; NF-κB was analyzed by Western blot and immunohistochemistry method.Results PVL was able to induce expression of IL-8 and IL-6 in THP-1 macrophages in time-and concentration-dependent manners.PVL could also significantly promote the activation of TLR4/NF-κB signals.Conclusion PVL can activate the expression of TLR4/NF-κB signals,and increased the high expression of inflammatory cytokines.Maybe it's the mechanism of action of PVL exerts the function of lung tissue damage.