Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

BACKGROUND: Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples. METHODS: Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system. RESULTS: The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanning from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits. CONCLUSION: The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

The Zuo Jin Wan Formula increases chemosensitivity of human primary gastric cancer cells by AKT mediated mitochondrial translocation of cofilin-1.

Resistance to cisplatin (DDP)-based chemotherapy is a major cause of treatment failure in human gastric cancer (GC). It is necessary to identify the drugs to re-sensitize GC cells to DDP. In our previous research, Zuo Jin Wan Formula (ZJW) has been proved could increase the mitochondrial apoptosis via cofilin-1 in a immortalized cell line, SGC-7901/DDP. Due to the immortalized cells may still difficult highly recapitulate the important molecular events in vivo, primary GC cells model derived from clinical patient was constructed in the present study to further evaluate the effect of ZJW and the underlying molecular mechanism. Immunofluorescent staining was used to indentify primary cultured human GC cells. Western blotting was carried out to detect the protein expression. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation. Flow cytometry analysis was performed to assess cell apoptosis. ZJW inhibited proliferation and induced apoptosis in primary DDP-resistant GC cells. Notably, the apoptosis in GC cells was mediated by inducing cofilin-1 mitochondrial translocation, down-regulating Bcl-2 and up-regulating Bax expression. Surprisingly, the level of p-AKT protein was higher in DDP-resistant GC cells than that of the DDP-sensitive GC cells, and the activation of AKT could attenuate ZJW-induced sensitivity to DDP. These data revealed that ZJW can increase the chemosensitivity in DDP-resistant primary GC cells by inducing mitochondrial apoptosis and AKT inactivation. The combining chemotherapy with ZJW may be an effective therapeutic strategy for GC chemoresistance patients.

[Prospective cohort study on the relationship between pathogenic bacteria in the nasal middle meatus and acute bacterial respiratory infection in children].

OBJECTIVE: To study the relationship between pathogenic bacteria in the nasal middle meatus and acute bacterial respiratory infection in children. METHODS: Three hundred and twenty eight children with respiratory infection (mean age 8 years) were included into the prospective cohort study. The mucosal fluid specimens from the nasal middle meatus were collected under an endoscope for bacterial culture. The patients with bacterial culture positive were defined as the Exposed group and those with bacterial culture negative as the Non-exposed group. The grouping of the patients was blinded to the patients, patients' parents and physicians. Both groups received anti-virus and symptomatic treatments, without antibiotic administration. Five days later, the patients were evaluated as to whether they had bacterial infection based on the leucocyte count and CRP results. RESULTS: Of the 328 patients, 168 had a positive nasal bacterial culture. The incidence of bacterial respiratory infection in the Exposed group [51.2% (86/168)] was significantly higher than in the Non-exposed group [13.1% (21/160)] (P < 0.01). The relative risk of bacterial respiratory infection occurrence in patients with nasal bacterial culture positive was 3.9002. CONCLUSIONS: The children with respiratory infection who had potential pathogenic bacteria in the nasal middle meatus were more prone to develop bacterial respiratory infection.