Clinical characteristics and drug sensitivity in children with invasive pneumococcal disease: a multicenter study / 中国当代儿科杂志

OBJECTIVE@#To study the clinical characteristics, drug sensitivity of isolated strains, and risk factors of drug resistance in children with invasive pneumococcal disease (IPD).@*METHODS@#The clinical characteristics and drug sensitivity of the isolated strains of 246 hospitalized children with IPD in nine grade A tertiary children's hospitals from January 2016 to June 2018 were analyzed.@*RESULTS@#Of the 246 children with IPD, there were 122 males and 124 females. Their ages ranged from 1 day to 14 years, and among them, 68 (27.6%) patients were less than 1 year old, 54 (22.0%) patients were 1 to 2 years old, 97 (39.4%) patients were 2 to 5 years old, and 27 (11.0%) patients were 5 to 14 years old. Pneumonia with sepsis was the most common infection type (58.5%, 144/246), followed by bloodstream infection without focus (19.9%, 49/246) and meningitis (15.0%, 37/246). Forty-nine (19.9%) patients had underlying diseases, and 160 (65.0%) had various risk factors for drug resistance. The isolated Streptococcus pneumoniae strains were 100% sensitive to vancomycin, linezolid, moxifloxacin, and levofloxacin, 90% sensitive to ertapenem, ofloxacin, and ceftriaxone, but had a low sensitivity to erythromycin (4.2%), clindamycin (7.9%), and tetracycline (6.3%).@*CONCLUSIONS@#IPD is more common in children under 5 years old, especially in those under 2 years old. Some children with IPD have underlying diseases, and most of the patients have various risk factors for drug resistance. Pneumonia with sepsis is the most common infection type. The isolated Streptococcus pneumoniae strains are highly sensitive to vancomycin, linezolid, moxifloxacin, levofloxacin, ertapenem, and ceftriaxone in children with IPD.

Investigation of etiology of massive infection with porcine pseudorabies virus in Henan and neighboring Provinces / 病毒学报

In early 2011, the serious outbreak of porcine pseudorabies virus (PRV) infection suddenly recurred in Henan and neighboring Provinces. To investigate the etiology of massive infection with PRV, 16 800 serum samples, 905 porcine epidemic diarrhea virus (PEDV) back-feeding tissues, and 56 PR gene deleted live vaccines were colleted from January 2011 to May 2013 to detect PRV field infection using a PRV gE antibody test kit. The gE and TK genes of 11 new epidemic PRV strains were sequenced by PCR, and their molecular characteristics were analyzed. Moreover, virus titer determination, protective test against PRV, and vaccine potency testing were performed. The results showed that the detection rate of PRV field infection-positive pig farms was 68.06%, and the overall positive rate of PRV field infection in serum was 38.47%; the positive rates in breeding sows, breeding boars, reserve pigs, and commercial pigs were 40.12%, 30.88%, 54.67%, and 26.52%, respectively. The new epidemic strains were in the same evolutionary branch and belonged to the virulent strain group. Compared with the classical PRV strain, the virulence of new epidemic strains changed a little. The length of gE gene was 1 787 bp, and the length of TK gene was 963 bp. The nucleotide homologies of gE and TK genes to Chinese reference strains were 98.2%-99.8% and 98.90%-99.6%, respectively, and the amino acid homologies were 97.1%-99.8% and 97.5%-99.4%, respectively. Commercial vaccine had a 100% protective effect against the new epidemic strains. The positive rate of PRV field infection was 0% in vaccine and 40.44% in back-feeding tissues. The results confirmed that PRV field infection rates were rising sharply among pigs in Henan and neighboring Provinces after 2011. The main virulence genes of new epidemic PRV strains did not change significantly over the years. PR gene deleted live vaccines had no PRV field infection and could completely resist the attack of new strains. The virus carriage of breeding boars and reserve pigs and the serious PRV field infection in PEDV back-feeding tissues were the main causative factors for massive infection with PRV and epidemic outbreak in Henan and neighboring Provinces from 2011 to 2013.

Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks / 病毒学报

Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark.

Dynamic distribution of the avian infectious bronchitis virus isolate strain Jin-13 in SPF chickens / 病毒学报

This study aimed to understand the dynamic distribution of infectious bronchitis virus (IBV) Jin-13 strain in SPF chickens. Ninety-day-old SPF chickens were inoculated with Jin-13, a virulent strain, and dissected at day 1, 4, 7, 10, 14, 21, 28 or 35 post-inoculation (dpi). Samples of heart, liver, spleen, lung, trachea, kidney and duodenum were collected and the N gene was detected by Sybr Green I real-time quantitative RT-PCR assays. The established method had a good linear correlation from 7.77 x 10(8) to 10(0) copies/microL. SPF chickens developed typical clinical signs of IBV at the 4th dpi, and the IBV viral concentration of tissues and organs gradually increased with a peak of up to 7.13 x 10(4) copies/microL. The viral concentration of most organs decreased by the 10th dpi, but those of the kidney, trachea and lung remained positive for IBV at 28 dpi and the heart was still positive for IBV at > 35 dpi. The results of this study, showed that the Jin-13 strain can cause prolonged virus excertion in chickens with severe renal damage.

Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4+ T cells and IFN-gamma-producing CD8+ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3+, CD3+CD4+CD8-, and CD3+CD4-CD8+ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

Clinical features and molecular characteristics of methicillin-resistant Staphylococcus aureus in children / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the clinical and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) infection in children.</p><p><b>METHOD</b>A total of 37 MRSA strains were isolated from hospitalized patients in Children's Hospital of Fudan University from March 2009 to November 2011. The clinical characteristics were investigated by a cohort study. Furthermore, the mecA, Panton-Valentine leucocidin (PVL) genes were detected by polymerase chain reaction (PCR), and the genotypes of SCCmec were determined by multiplex PCR.</p><p><b>RESULT</b>(1) Among the 37 MRSA isolates, infections with 21 were acquired from hospital (HA-MRSA), and 16 isolates were acquired from community (CA-MRSA). (2) In the study, MRSA frequently caused respiratory tract infection, and most of the strains were isolated from intensive care unit (ICU). (3) CA-MRSA was most frequently associated with skin and soft tissue infections (SSTI), suppurative tonsillitis, even pneumonia and septicemia. HA-MRSA infection was more aggressive, most frequently associated with pneumonia, septicemia, and central nervous system (CNS) infections, such as meningitis. In children with fever caused by HA-MRSA or CA-MRSA infection, HA-MRSA showed a longer duration of fever, for 10.5 days. C-reactive protein (CRP) level caused by HA-MRSA (63.00 mg/L) was higher than CA-MRSA (9.50 mg/L) , and there were statistically significant differences between the groups (t = 2.5670, P < 0.05). However, there were no statistically significant differences between the groups in white blood cell count (WBC) or procalcitonin (PCT) level. (4) Among 37 MRSA isolates, the whole isolates were mecA gene positive (100%). SCCmec genotyping results showed that the most frequent SCCmec types were type III, 17 isolates, the others including type IV 8 isolates, type II1 isolates, nontypable 11 isolates, type I and type V were not found in this group. Therein, among 21 HA-MRSA isolates, SCCmec III was the most common, 15 isolates, type IV 1 isolates, nontypable 5 isolates; among 16 CA-MRSA isolates, SCCmec type IV was the most common, 7 isolates, type III 2 isolates, type II 1 isolate, nontypable 6 isolates. (5) Among the 37 MRSA isolates, 28 were PVL gene positive; and among 21 HA-MRSA isolates, 17 were PVL gene positive; Among 16 CA-MRSA isolates, 11 were PVL gene positive; There were no statistically significant differences between the groups (χ(2) = 0.735, P > 0.05) .</p><p><b>CONCLUSION</b>Compared with CA-MRSA, HA-MRSA infection was more aggressive, and induced higher C reactive protein; the dominant epidemic strains of CA-MRSA was SCCmec type IV, and HA-MRSA was SCCmec type III; the positive rate of PVL gene was high.</p>

Assessment and application of a molecular diagnostic method on the detection of four types of diarrheagenic Escherichia coli / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish and evaluate a molecular diagnostic method for routine monitoring of four types of diarrheagenic Escherichia (E.) coli (DEC)and to study the distribution of four types of DEC isolated from diarrheal patients in Shanghai.</p><p><b>METHODS</b>DEC-PCR standard operation procedure(SOP)had been developed for DEC detection and isolation, using the Statens Serum Institute (SSI) DEC PCR kits with multiplex PCR technique after verification tests on reference strains. Diarrhea specimens from 3 clinical hospitals in Shanghai were tested from June to September, 2012.</p><p><b>RESULTS</b>Specificity of the PCR kit was 100% by verification on the 26 DEC reference strains. A total number of 218 DEC isolates, including 181 fermented lactose and 37 unfermented lactose were identified from the 1887 stool specimens of diarrhea patients, with positive rate as 11.6%. The most common pathogen(54.1%, 118/218)was enteropathogenic E. coli(EPEC), followed by enterotoxigenic E. coli(ETEC, 41.3%, 90/218), enteroinvasive E. coli (EIEC, 4.1%, 9/ 218) and Shigatoxin-producing E. coli(STEC, 0.5%, 1/218)in addition to 18 Shigella isolates. ETEC dominated in diarrhea patients with foreign residency, as well as 1/3 were perinatal stage of neonatal ETEC of all diarrhea cases under the age of 5, while EPEC dominated in the Chinese diarrhea patients especially among young kids under the age of 2.</p><p><b>CONCLUSION</b>Data was reliable after assessment on this molecular diagnostics and seperation procedures used for the routine monitoring on four types of DEC, while the diagnosis and reference ability of DEC regarding the laboratories net-working on food-borne pathogens need to be built up and improved.</p>

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus field strains in central China during 2010-2012 outbreaks / 病毒学报

Since late 2010, porcine epidemic diarrhea virus (PEDV) has been re-emerging in central China. To explore the possible reason of the PEDV outbreaks, twelve PEDV field strains were isolated from different swine breeding farms in central China during 2010-2012, and molecular diversity, phylogenetic relationships of these strains with other PEDV reference strains were investigated. Sequence analysis of S, M and ORE3 genes revealed that the central China PEDV isolates had several specific nucleotides and amino acids which were different from PEDV reference strains. In addition, the entire S genes of eleven central China PEDV isolates were found to be nine nucleotides longer in length than CV777 and large number of amino acid variations was accumulated in the N-terminal region of S gene. Phylogenetic analysis showed that the central China PEDV isolates had close relationship with Korea strains (2007-2009), Thailand strains (2007-2008), Vietnam strains (2009-2010), Japan strains (2010), and other prevailing strains from other parts of China (2010-2012). However, they differed genetically from European strains (CV777, Brl/87), China strains (2003-2007) and the vaccine strains (CV777) used in China. These results imply that a rapid variation and evolution of central China PEDV strains has occurred in recent years, and a more efficient vaccine strain should be selected to prevent and control outbreaks of PEDV in China.

Characterization of erythromycin-resistant Streptococcus pneumoniae isolates causing invasive diseases in Chinese children / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Erythromycin-resistant Streptococcus pneumoniae isolates that causing invasive pneumococcal diseases (IPD) in Chinese children remain uncharacterized. This study aims to identify the resistance genes associated with erythromycin resistance and to determine the genetic relationships of IPD isolates in Chinese children.</p><p><b>METHODS</b>A total of 171 S. pneumoniae strains were isolated from 11 medical centers in China from 2006 to 2008. All the isolates were characterized via serotyping and antibiotic susceptibility determination. The erythromycin-resistant isolates were further characterized via ermB and mefA gene detection, multi-locus sequence typing analysis, and pulsed-field gel electrophoresis.</p><p><b>RESULTS</b>A total of 164 (95.9%) isolates showed resistance to erythromycin, of which 162 strains with high high-level resistance (MIC ≥ 256 µg/ml). A total of 104 (63.4%) isolates carry the ermB gene alone, whereas 59 (36.0%) harbor both ermB and mefA genes. Of the 59 strains, 54 were of serotypes 19A and 19F and were identified as highly clonal and related to the Taiwan(19F)-14 clone.</p><p><b>CONCLUSIONS</b>The erythromycin resistance rate in IPD isolates is significantly high and is predominantly mediated by the ermB gene. Isolates that carry both ermB and mefA genes are predominantly of serotypes 19A and 19F.</p>

The development and application of a SYBR Green I real-time PCR assay for detection of infectious bursal disease virus / 病毒学报

To meet the needs of detection of infectious bursal disease virus (IBDV) under high efficient culture, a SYBR Green I real-time RT-PCR (qRT-PCR) was developed using a pair of primers specific to the conserved region of VP4 gene of IBDV and compared with TCID50 method by monitoring the proliferation dynamics of IBDV in DF-1 cell line adherent to micro carrier in tubular reactor. The results showed that the RT-PCRassay was linear in the range of 4. 03 X 10(1)-10(9) copies/microL. The IBDV RNA detection limit was 40 copies/microL, which was 1 000 times more sensitive than conventional PCR. No cross-reactions with other viruses was observed. The intra-assay coefficient of variation was less than 0.05%. There was a parallel correlation of IBDV proliferation dynamics in DF-1 cell under Micro carrier suspension and static adherent culture by the qRT-PCR assay and TCID50 method. The detection results of the IBDV samples from tubular and flask culture showed the differences of the micro carrier and adherent culture by both methods. In conclusion, the qRT-PCR assay is more rapid and sensitive than the TCID50 method, which is more appropriate for the real time detection of IBDV.

Molecular characteristics of methicillin resistant Staphylococcus aureus isolated from Chinese children / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from Chinese children in seven cities.</p><p><b>METHOD</b>A total of 134 MRSA isolates were collected from nine hospitals. Multilocus sequence typing and spa typing were analyzed by polymerase chain reaction (PCR), and staphylococcal chromosomal cassette mec (SCCmec) type was analyzed by multiplex PCR. The Panton-Valentine leukocidin (pvl) gene was also detected.</p><p><b>RESULT</b>Most MRSA strains were isolated from pneumonia and skin and soft tissue infection (SSTIs) patients, accounting for 82.1%. Overall, 16 sequence types (STs) were obtained, and CC59 (51.7%) was found to be the most prevalent, which included ST 59 and ST 338, followed by ST239 (16.4%). SCCmec types II, III, IV, and V were also identified in the current study. SCCmec type IV was the most predominant type at 50.0%, followed by SCCmec type V at 23.9% and III at 23.9%. SCCmec subtypes IVa, IVc, and IVg were found among SCCmec type IV strains, whereas IVa was the main subtype at 77.6%. Twenty-six spa types were also identified, among which the predominant type was t437 (47.8%). The prevalence of pvl genes and the SCCmec type of strain was relevant, and the pvl gene positive rate was higher in SCCmec type IV and V-type strains than in SCCmec type II and III strains (58.6% vs. 14.3%, P < 0.05); there was a significant difference between them. In the strains isolated from pneumonia and SSTIs, ST59-MRSA-IVa(t437) was the predominant clone. There were five clones detected from the strains isolated from septicemia, with ST59-MRSA-IVa(t437) and ST59-MRSA-V(t437) as the main clones (57.1%). Various predominant clones existed in different regions. ST59-MRSA-IVa(t437) was the prevalent clone in the Guangzhou, Beijing, Chongqing, and Shenzhen areas, whereas ST239-MRSA-III(t037) was the prevalent clone in the Shanghai area. Fifty percent of the isolates from the Wenzhou area belonged to ST910-MRSA-V(t318), whereas three clinical strains isolated from the Shenyang region belonged to three different types.</p><p><b>CONCLUSION</b>The results indicate that MRSA isolates from Chinese children are largely associated with the ST59-MRSA-IV(t437) and ST239-MRSA-III(t037) clones. These two may belong to community-acquired MRSA and hospital-acquired ones, respectively. Different prevalent clones were detected in different diseases and different regions. Therefore, there is a need to conduct further research on clinical isolates, which can guide the choice of antibiotic treatment and the examination of MRSA prevalence.</p>

Investigation of nosocomial infection in the neonatal intensive care unit / 中国当代儿科杂志

<p><b>OBJECTIVE</b>A perspective study was conducted to describe the epidemiologic profile of nosocomial infection in the neonatal intensive care unit (NICU).</p><p><b>METHODS</b>The newborn infants who were admitted in the NICU for more than 48 hrs were enrolled from February 2006 to January 2007. The clinical data were collected. The rate of nosocomial infection was calculated according to the CDC surveillance system. The risk factors of nosocomial infection were investigated by multivariate regression analysis.</p><p><b>RESULTS</b>A total of 1 159 neonates were recruited. A total of 169 nosocomial infections occurred, with a cumulative rate for nosocomial infection of 14.58%. The incidence of nosocomial infection was 19.52 per 1 000 patient-days. Ninety-two cases of pneumonia, including 38 cases of ventilator-associated pneumonia (VAP), were reported, with a nosocomial infection rate of 7.94%, which was the most common nosocomial infection in the NICU. Among these infants the rate of VAP was 48.8 per 1 000 ventilator days. The major microorganisms isolated from the infected patients were Acinetobacter baumannii, Klebsiella pneumoniae, Coagulase negative staphylococcus, and aeruginosus Bacillus. Birth weight (OR 2.130, 95%CI 1.466-3.094), mechanical ventilation (OR 7.038, 95%CI 3.901-12.698), chest tube drainage (OR 7.004, 95%CI 1.841-26.653) and ibuprofen therapy (OR 2.907, 95% CI 1.303-6.487) were the risk factors for the development of nosocomial infection.</p><p><b>CONCLUSIONS</b>Pulmonary infection is the most common nosocomial infection in the NICU, and the Gram-negative bacillus is the main pathogen. Low birth weight, mechanical ventilation, chest tube drainage and ibuprofen therapy are independent risk factors for nosocomial infection in the NICU.</p>

Bifidobacterium DNA upregulates Th1 type response of umbilical cord blood mononuclear cell / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction.</p><p><b>METHODS</b>Bifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 humidified incubator. These cells were divided into four groups, control group: without any stimulant; bDNA group: stimulated with 25 microg/ml bDNA; d-bDNA group: stimulated with 25 microg/ml d-bDNA; hDNA group: stimulated with 25 microg/ml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay (ELISA); cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot (ELISPOT) assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Six hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation (P < 0.05). No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-gamma secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells (T-bet) mRNA expression was conspicuously enhanced as compared to the other three groups (P < 0.05). GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation.</p><p><b>CONCLUSION</b>bDNA could promote secretion of Th1 type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supernatant was decreased. bDNA could stimulate secretion of IFN-gamma by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation. bDNA could upregulate Th1 type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.</p>