Quantitative detection of the KRAS gene mutation in circulating tumor DNA for patients with colorectal cancer / 基础医学与临床

Objective To establish a liquid biopsy technique of KRAS gene G12D mutation and to assess its diagnostic value. Methods KRAS G12D mutation was analyzed by ddPCR in plasma DNA from 52 colorectal cancer patients and compared that of to 80 healthy subjects. KRAS gene sequencing in cancerous tissue of colorectal cancer patient being set as a golden standard, we evaluated the accuracy of ddPCR and analyzed the correlation between G12D mutation rate, plasma concentration;and their clinical manifestations in CRC. Results ddPCR indicated that KRAS G12D mutation rate and concentration(26.92％, 81.5 copies/mL) in the plasma samples of colorectal cancer patients were significantly higher than that of healthy subjects (8.75％, 16 copies/mL). Colorectal cancer patients with highly differentiated adenocarcinoma showed a significantly higher number of mutant copies than medium and low differentiated adenocarcinoma(P＜0.05);M2 patients had a significantly higher number of mutant copies than N1 and NO patients (P＜0.05);The concordance rate of KRAS gene mutation between cancerous tissue and plasma ctDNA was 87.50％ in CRC.Conclusions ddPCR is a fast, noninvasive and accurate method for plasma testing of ctDNA, and the test results could be used to monitor the course of the disease and as clinical guidelines.

Association of HLA-G 14bp gene polymorphisms and plasma sHLA-G level with susceptibility to EV71 infection in children / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Explore the relationship between the HLA-G 14bp insertion/deletion polymorphism and the infection of Enterovirus 71 (EV71) for children.</p><p><b>METHODS</b>We genotyped HLA-G 14bp insertion/deletion polymorphism of 125 severe HFMD children infected with EV71 and 133 normal controls by PCR-PAGE;detected the plasma sHLA-G level of 66 heavy type and 15 critical type and 133 normal controls by ELISA.</p><p><b>RESULTS</b>Frequencies of the genotype 14 bp - / - ,14 bp + / - and 14 bp + / + were 49.6% , 42.4% and 8.0% for the severe HFMD children infected with EV71, and 34.6%, 48.9% and 16.5% for the normal controls, respectively. A significant difference was observed for the frequencies of the HLA-G 14bp genotype between the two groups(chi2 = 7.850, P = 0.020). And for the allele frequencies. The plasma sHLA-G levels in heavy type were dramatically higher than that in normal controls (Z = -9.692, P = 0.000). The plasma sHLA-G levels in children with critical HFMD were dramatically higher than that with heavy type (Z = -2.420, P = 0.016).</p><p><b>CONCLUSION</b>There was a relationship between the HLA-G 14 bp insertion/deletion polymorphism and the susceptibility to the severe HFMD children infected with EV71 and the plasma sHLA-G might be considered as a index for auxiliary diagnosis the severe HFMD infected with EV71.</p>

Detection of adenoviruses in feces of infants with diarrhea by Real-time PCR / 中华流行病学杂志

Objective A Real-time PCR method was established to study the infection of adenovirus (Ad) in infants with sporadic diarrhea in Wenzhou. Methods According to hexon gene of adenovirus, one prime pair was designed as universal primes and applied to detect adenovirus DNA by Real-time PCR. It was also compared with immunochromatographic assay. 157 fecal specimens from diarrhea infants were tested while positive specimens were sequenced and identified by isolate culture and restriction endonucleases. Results A rapid and specific Real-time PCR assay for detection adenovirns was set up. The positive rates of adenovirns in fecal specimens by immunochromatographic assay and Real-time PCR were 1.91% (3/157) and 3.18% (5/157), respectively. Out of the 154 specimens with negative result from immunochromatographic assay, 2 showed positive by Real-time PCR. 5 positive specimens, identified by Real-time PCR, were sequenced as Ad3 (3/157, 1.91% ) and Ad7 (2/157, 1.27% ). 2 of the 5 positive specimens were proved to be Ad3 by cell culture and restriction endonucleases.Conclusion Real-time PCR combined with sequence analysis seemed more sensitive and specific so could be used for identifying types of adenovirus in clinical specimens. Ad3 and Ad7 were important pathogens which caused infant sporadic diarrhea in Wenzhou during February and April in 2008.

Three cases of enterovirus 71 infection with pulmonary edema or pulmonary hemorrhage as the early clinical manifestation / 中华儿科杂志

<p><b>OBJECTIVE</b>To recognize the clinical features of the enterovirus 71 (EV71) infection with pulmonary edema or pulmonary hemorrhage as a fulminant and often fatal illness.</p><p><b>METHODS</b>We retrospectively reviewed the medical records of the three cases with EV71 infection for clinical manifestation, laboratory data, medications, outcome etc.</p><p><b>RESULTS</b>All the cases were infants and they all died. These infants had no skin or mucosal lesions, however, they had sudden onset of cyanosis and tachypnea 1 to 2 days after the onset of the febrile disease with vomiting. All these 3 cases were misdiagnosed and were treated for shock on admission. Pulmonary hemorrhage was not considered in any of the cases on admission. All the cases received tracheal intubation when foamy secretions were discharged from mouth and nose of the patients and notable cyanosis was noted. After intubation, all had pink foamy fluid flew out from the endotracheal tube. The patients had hyperglycemia and limb weakness, two had tachycardia, and hypertension was found in one case. Chest X-ray showed bilateral or unilateral widespread air space opacity, but the cardiac size and shape were normal. All the patients had leucocytosis. EV71 infection was confirmed by detection of specific sequences of the virus in throat swab and tracheal secretions samples and in one case in cerebrospinal fluid sample.</p><p><b>CONCLUSION</b>Pulmonary edema or pulmonary hemorrhage occurred in the 3 cases with EV71-infected infants. The initial presentation was often nonspecific with fever and vomiting, and sudden appearances of cyanosis, tachypnea, tachycardia, hypertension or hypotension, limb weakness may suggest pulmonary edema or hemorrhage. Excessive fluid resuscitation may deteriorate the illness, on the contrary, fluid restriction and inotropic agents, and early intubation with positive pressure mechanical ventilation may be the proper treatment.</p>