Penetration of erythromycin through Staphylococcus epidermidis biofilm.

BACKGROUND: The catheter related infection caused by Staphylococcus epidermidis biofilm is increasing and difficult to treat by antimicrobial chemotherapy. The properties of biofilms that give rise to antibiotic resistance are only partially understood. This study aimed to elucidate the penetration of erythromycin through Staphylococcus epidermidis biofilm. METHODS: The penetration ratio of erythromycin through Staphylococcus epidermidis biofilms of 1457, 1457-msrA, and wild isolate S68 was detected by biofilm penetration model at different time points according to the standard regression curve. The RNA/DNA ratio and the cell density within the biofilms were observed by confocal laser microscope and transmission electromicroscope, respectively. RESULTS: The penetration ratios of erythromycin through the biofilms of 1457, 1457-msrA, and S68 after cultivation for 36 hours were 0.93, 0.55 and 0.4, respectively. The erythromycin penetration ratio through 1457 biofilm (0.58 after 8 hours) was higher than that through the other two (0.499 and 0.31 after 24 hours). Lower growth rate of the cells in biofilm was shown, with reduction of RNA/DNA proportion observed by confocal laser microscope through acridine orange stain. Compared with the control group observed by transmission electrmicroscope, the cell density of biofilm air face was lower than that of agar face, with more cell debris. CONCLUSIONS: Erythromycin could penetrate to the Staphylococcus epidermidis biofilm, but could not kill the cells thoroughly. The lower growth rate of the cells within biofilm could help decreasing the erythromycin susceptibility.

Clinical application of RT-nested PCR integrated with RFLP in Hantavirus detection and genotyping: a prospective study in Shandong Province, PR China.

The aim of the present study was to evaluate the clinical usefulness of applying RT-nested PCR along with RFLP as a method for diagnosis and genotypic differentiation of Hantavirus in the acute-stage sera of HFRS patients as compared to the ELISA technique. A prospective study of patients with suspected HFRS patients was carried out. Sera were collected for serological evaluation by ELISA and RT-nested PCR testing. Primers were selected from the published sequence of the S segment of HTNV strain 76-118 and SEOV strain SR-11, which made it possible to obtain an amplicon of 403 bp by RT-nested PCR. The genotypic differentiations of the RT-nested PCR amplicons were carried out by RFLP. Sequence analyses of the amplicons were used to confirm the accuracy of the results obtained by RFLP. Of the 48 acute-stage sera from suspected HFRS patients, 35 were ELISA-positive while 41 were positive by RT-nested PCR. With Hind III and Hinf I, RFLP profiles of the RT-nested PCR amplicons of the 41 positive sera exhibited two patterns. 33 had RFLP profiles similar to the reference strain R22, and thus belonged to the SEOV type. The other 8 samples which were collected during October-December had RFLP profiles similar to the reference strain 76-118, and thus belonged to the HTNV type. Sequence phylogenetic analysis of RT-nested PCR amplicons revealed sdp1, sdp2 YXL-2008, and sdp3 as close relatives of HTNV strain 76-118, while sdp22 and sdp37 as close relatives of SEOV strain Z37 and strain R22 located in two separate clusters in the phylogenetic tree. These results were identical to those acquired by RFLP. RT-nested PCR integrated with RFLP was a rapid, simple, accurate method for detecting and differentiating the genotypes of Hantavirus in the acute-stage sera of suspected HFRS patients. In Shandong province, the main genotypes of Hantavirus belonged to the SEOV types, while the HTNV types were observed during the autumn-winter season.

[Study on the molecular epidemiological characteristics of norovirus in acute gastroenteritis of Beijing].

OBJECTIVE: To study the prevalence, genotypes and molecular characteristics of norovirus (NoV) in acute gastroenteritis. METHODS: RT-PCR was used to determine the molecular epidemiology of NoV. RESULTS: Out of 685 samples, 66 positive specimens were identified and the prevalence was 9.6% (66/685), 9.9% in males and 9.4% in females, respectively, with no significant difference. The prevalence rates showed no differences between age groups or between inpatients and outpatients. NoV gastroenteritis did not present any seasonal distribution. 43 out of the 66 specimens were classified, with 10 (22.7%) belonged to GI including 2 GI.3, 1 GI.4, 4 GI.5 and 3 GI.7. Other 33 (77.3%) belonged to GII genogroup, including GII.4 accounted for 60.6% (20/33) and followed by 7 GII.12, 2 GII.6, 1 GII.2, 1 GII.3, 1 GII.5. Six specimens mixed with GI and GII and 3 specimens were classified as GI.3/GII.7, GI.5/GII.5 and GI.4/GII.4. CONCLUSION: The main symptoms of acute gastroenteritis were abdominal pain, nausea, vomit and fever. There were many genotypes identified in our study and the main genotypes were GII.4/2006a and 2006b. GI and GII could be coinfected with each other.

Consistency of the key genotypes of Orientia tsutsugamushi in scrub typhus patients, rodents, and chiggers from a new endemic focus of northern China.

Scrub typhus is one of the most common infectious diseases of rural south and southeastern Asia and the western Pacific. It emerged in Shandong Province in northern China from autumn to winter of 1986. Since then, the "autumn-winter type scrub typhus" has been found in many areas of northern China. However, the principle genotypes of Orientia tsutsugamushi still remain unknown. This study was undertaken to identify the genotypes of O. tsutsugamushi obtained from scrub typhus patients, chigger mites and rodents from the focal point of the problem in Shandong Province. Forty-four isolates from patients, rodents, and chiggers, 47 blood clots from patients during the acute phase, 10 eschars from patients during the convalescence phase and 16 pools of larval chiggers were tested for the scrub typhus antigen 56-kD protein (Sta56) gene by nested PCR methodology and additional sequence analysis including DNA sequence alignment and phylogenetic analysis. Based on nested PCR, ninety-five initial PCR-positive samples produced amplicons using Kawasaki strain-specific primers, while the other two (the FXS4 and LHGM2 strains) produced amplicons using Karp strain-specific primers. The partial Sta56 gene sequence analysis indicated that the sequence homologies of 3 selected isolates (the B16, FXS2, and XDM2 strains) and 7 eschars out of the 95 samples, which were nested PCR-positive using Kawasaki strain-specific primers, were 94-98% to that of Kawasaki strain. The sequence homology of the FXS4 and LHGM2 strains to that of the Karp strain was respectively 83 and 96%. These findings implied that the key genotypes of O. tsutsugamushi in patients, rodents, and chiggers in Shandong Province were identical and similar to Kawasaki strains.

Perinatal outcomes and congenital abnormalities in the newborns of women affected by the 2009 pandemic influenza A (H1N1) in Beijing, China.

OBJECTIVE: To investigate the pregnancy complications, perinatal outcomes, and congenital abnormalities (CAs) that occurred in Beijing, China, when pregnant women became infected with the 2009 pandemic influenza A (H1N1) (H1N1 pdm). METHODS: Pregnancy complications, perinatal outcomes, and CAs were compared among 3 groups of pregnant women. The 23 women in group 1 were confirmed to harbor viral RNA; the 23 in group 2 had serum levels of virus-specific antibodies against H1N1 pdm, meaning that they were suspected of being infected with the virus; and the 93 in group 3 had no detectable virus-specific antibodies. RESULTS: Perinatal outcomes and pregnancy complications were not significantly different in groups 1 and 3. Higher percentages of stillbirths (12.0%) and placental disorders (13.0%) were observed in group 2 than in group 3. Many women in group 2 (62.5%) experienced symptoms of having a cold during pregnancy and most took no medication. Two cases of CA occurred in group 1, in the offspring of women infected in the second trimester. CONCLUSION: When left untreated, infection with the 2009 H1N1 pdm virus during pregnancy appears to have increased fetal mortality and morbidity. Because CAs are traumatic for all concerned, their possible association with the virus should be further evaluated.

Viral shedding in Chinese young adults with mild 2009 H1N1 influenza.

BACKGROUND: The duration of viral shedding and the transmission of 2009 H1N1 influenza among individuals, especially among the younger population with mild illness, are not well understood now. The aim of this study was to determine the viral shedding of the young adult patients with mild 2009 H1N1 influenza in China. METHODS: From September 2009 to January 2010, the clinical data and serial nasopharyngeal swabs of 67 patients with 2009 H1N1 influenza and 37 patients with seasonal influenza aged from 18 years to 35 years were collected. The nasopharyngeal swab samples were detected by real time RT-PCR to determine the viral shedding. All the patients did not receive the antiviral therapy but Chinese medicine for detoxicating. RESULTS: Among the patients with H1N1 virus infection, 82.1% (55/67) patients presented with fever symptom, while more patients with high fever (≥ 39°C) were found in seasonal influenza patients (P < 0.05). For the H1N1 patients, the median interval between the symptom onset and the undetectable RNA was six days (4 - 10 days). But viral shedding was still found in 31.3% patients after 7 days following illness onset. The median interval between disappearance of fever and an undetectable viral RNA level was three days (2 - 8 days), and 17.9% patients were found to be viral shedding 6 days later after normalization of body temperature. For the seasonal influenza patients, 94.6% patients were detected out viral RNA within 7 days. The median interval of seasonal influenza between the symptom onset and the undetectable RNA was four days (3 - 8 days). The median interval between disappearance of fever and an undetectable viral RNA level was three days (2 - 6 days). CONCLUSION: It suggests that 7 days isolation period from the illness onset or 24 hours after the resolution of fever and respiratory symptoms are not long enough to cut off the transmission among Chinese young adults with mild illness.

[Colloidal gold and dot-ELISA rapid tests for screening influenza A virus].

OBJECTIVE: To evaluate the diagnostic efficiency of colloidal gold and dot ELISA rapid tests in clinical screening of influenza A virus. METHODS: The pharyngeal swabs were collected from 297 outpatients suspected of influenza between June and October, 2009 for detection with colloid gold and dot ELISA rapid test, with real-time PCR as the golden methods. The discrepant results of colloid gold and dot ELISA methods were confirmed by sequencing, and the diagnostic efficiency of the two methods was evaluated. RESULTS: Among the 166 samples with influenza A virus infection as confirmed by real-time PCR and sequencing, the diagnostic sensitivity of dot ELISA and colloid gold methods was 54.82% (91/166) and 4.22% (7/166), respectively. The total concordance rate with PCR was 66.67% (Kappa value of 0.35). Among the 133 samples negative for influenza A virus, the specificity of dot ELISA and colloid gold methods was 81.68% (107/131) and 98.47% (129/131), respectively, with a total concordance rate with PCR of 45.79% (Kappa value 0.02). Of the 99 H1N1 influenza samples confirmed by real-time PCR, the detection rate of dot ELISA was 67.3%, whereas that of colloid gold was 5.1%. Out of the 107 dot ELISA-positive but colloid gold-negative samples, 84 were confirmed to be influenza A virus-positive by real-time PCR and sequencing. One sample negative for dot ELISA but positive for colloid gold test was confirmed to be influenza A virus-negative. The detection rate and diagnostic concordance rate for influenza A virus by dot ELISA were significantly higher than those of colloid gold (P<0.05). CONCLUSION: Dot ELISA is better than colloid gold in influenza A virus detection and shows great prospect in clinical screening.

Characteristics of pediatric scrub typhus in a new endemic region of northern China.

BACKGROUND: Scrub typhus emerged in northern China in 1986. Our objective was to document epidemiologic, clinical and laboratory features of pediatric scrub typhus in this new endemic area. METHODS: The pediatric patients diagnosed with scrub typhus during the 12-year period from 1995 through 2006 in Feixian County, Shandong province were enrolled in the study. The cases were diagnosed based on either specific antibody detection using the indirect immunofluorescent assay or detection of partial Orientia tsutsugamushi gene by polymerase chain reaction. RESULTS: Seventy pediatric scrub typhus cases were included in the study. The cases occurred from September through November. The common clinical manifestations included headache (100%), skin rash (91%), eschar (84%), lymphadenopathy (61%), and gastrointestinal signs (56%). None of the patients had neurologic involvement or thrombocytopenia. All children responded well to treatment with chloramphenicol. The patients who presented with abnormal chest radiography took significantly longer time to defervescence (Z[r] = 2.528, P = 0.011). Three strains of O. tsutsugamushi were isolated and all were identified as Kawasaki type. CONCLUSIONS: The manifestations of pediatric scrub typhus cases in the novel endemic region of northern China may be less severe than in other regions. Careful examination of skin eschars is helpful for the clinical diagnosis.

Clinical characteristics of the autumn-winter type scrub typhus cases in south of Shandong province, northern China.

BACKGROUND: Before 1986, scrub typhus was only found endemic in southern China. Because human infections typically occur in the summer, it is called "summer type". During the autumn-winter period of 1986, a new type of scrub typhus was identified in Shandong and northern Jiangsu province of northern China. This newly recognized scrub typhus was subsequently reported in many areas of northern China and was then called "autumn-winter type". However, clinical characteristics of associated cases have not been reported. METHODS: From 1995 to 2006, all suspected scrub typhus cases in five township hospitals of Feixian county, Shandong province were enrolled. Indirect immunofluorescent assay (IFA) was used as confirmatory serodiagnosis test. Polymerase chain reaction (PCR) connected with restriction fragment length polymorphism (RFLP) and sequence analyses were used for genotyping of O. tsutsugamushi DNAs. Clinical symptoms and demography of confirmed cases were analyzed. RESULTS: A total of 480 scrub typhus cases were confirmed. The cases occurred every year exclusively between September and December with a peak occurrence in October. The case numbers were relatively higher in 1995, 1996, 1997, and 2000 than in other years. 57.9% of cases were in the group aged 21-50. More cases occurred in male (56%) than in female (44%). The predominant occupational group of the cases was farmers (85.0%). Farm work was reported the primary exposure to infection in 67.7% of cases. Fever, rash, and eschar were observed in 100.0%, 90.4%, and 88.5% of cases, respectively. Eschars formed frequently on or around umbilicus, abdomen areas, and front and back of waist (34.1%) in both genders. Normal results were observed in 88.7% (WBC counts), 84.5% (PLT counts), and 89.7% (RBC counts) of cases, respectively. Observations from the five hospitals were compared and no significant differences were found. CONCLUSION: The autumn-winter type scrub typhus in northern China occurred exclusively from September to December with a peak occurrence in October, which was different from the summer type in southern China. In comparison with the summer type, complications associated with autumn-winter type scrub typhus were less severe, and abnormalities of routine hematological parameters were less obvious.

[Clinical manifestations and epidemic factors of autumn-winter type scrub typhus in children from northern new endemic area].

OBJECTIVE: Scrub typhus is an infectious disease due to Orientia tsutsugamushi transmitted by infected chigger mites. Scrub typhus has long been recognized to occur in southern areas of China, but has recently been increasingly often reported from the north since the first case was reported in Mengyin County, Shandong Province in 1986. The key objectives of the present study were to investigate the clinical manifestations and epidemic factors of scrub typhus in children from the northern new natural foci. METHODS: The case records of 56 children with scrub typhus who were admitted to the 5 hospitals of Fei County from September 1993 to January 2004 were reviewed. Orientia tsutsugamushi (Ot) was isolated from the cases. Based on ecological observations on the composition, seasonal fluctuation of animal hosts and chigger mites, Ot was isolated from rodents and chiggers. IgG antibodies to Ot was detected by IFA. Genotypes of the Ot isolates were also identified by nested PCR. RESULTS: Among 56 children scrub typhus cases, 46 were male, 10 were female; 96% exhibited typical eschars or ulcers, 100% cases had high fever, skin rashes were observed in 55 cases (98%), and regional lymphadenopathy occurred in 48 cases (86%). All cases came from countryside, and all had histories of exposure to the crop field. fifty-one serum samples of suspected patients with scrub typhus were collected, 48 were positive for antibodies to Ot. The serotypes were Gilliam types. The cases only appeared in September to December with the peak at mid and late October. Leptotrombidium (L.) scutellare was the most important vector causing scrub typhus in the foci. Apodemus (A.) agrarius was the main host animals of Ot in the crop field. Totally 26 strains were isolated from patients, rodents, and chigger mites. The serotypes of 24 out of the 26 isolates were Gilliam types, while the genotypes of these isolates were Kawasaki types. The serotypes of the other 2 isolates were identical and both were Karp types. CONCLUSION: Children scrub typhus patients were frequently seen in the new natural foci of Shandong province. Exposure history, typical eschars or ulcers, and presence of IgG antibody were the important indexes to diagnose the disease.

[Diagnostic and prognostic value of procalcitonin and common inflammatory markers combining SOFA score in patients with sepsis in early stage].

OBJECTIVE: To study the diagnostic and prognostic value of procalcitonin (PCT), common inflammatory markers combining with scores for estimating organ failure of infection related organs (SOFA) in patients with sepsis in early stage. METHODS: Patients were observed continuously in a perspective study with diagnostic tests. According to the definition of ACCP/SCCM Consensus Conference, patients were classified into 5 groups, including non-systemic inflammatory response syndrome (SIRS) (control) group, SIRS group, sepsis group, severe sepsis group and septic shock group. Indexes of inflammation, SOFA and concentration of PCT were determined at 24 hours, and their correlation was analyzed. RESULTS: Two hundred and eight patients were enrolled, including 59 in non-SIRS group, 57 in SIRS group, 52 in sepsis group, 28 in severe sepsis group and 12 in septic shock group. PCT concentrations were positively correlated with the severity of sepsis. Spearman's correlation coefficient was 0.909 (P=0.000). According to the receiver operating characteristic curves (ROC-curves) analysis principle, ROC curves were drawn and areas under these curves (AUC) was calculated. In the diagnosis of sepsis, AUC values were 0.936+/-0.020 for PCT, 0.973+/-0.011 for SOFA (both P=0.000). The best cutoff values in the diagnosis of sepsis were 0.375 microg/L for PCT, and 3. 5 for SOFA score. The Youden index of PCT and SOFA scores was 0.808 and 0.801, respectively. Binary Logistic regression analysis confirmed that PCT and SOFA score were highly correlated with sepsis (OR=84.794,10.761, respectively, both P=0.000) after eliminating confusion factors including age and C-reactive protein (CRP) etc.. PCT and SOFA score could be used to predict the incidence of sepsis. SOFA score was the best prognostic indicator of sepsis (OR=2.084, P=0.0002). CONCLUSION: The traditional inflammatory markers and CRP are useful parameters to differentiate SIRS from non-SIRS, but are not reliable indicators for the early diagnosis in patients with sepsis. PCT is more specific indicator in early diagnosis of sepsis to differentiate from SIRS. PCT combining with SOFA score can be used to predict the incidence of sepsis. SOFA score can be used to define objectively the severity of sepsis according to PCT level and is helpful for estimation of prognosis in patients with sepsis.

[Amplification and restriction fragment length polymorphism analysis on the complete sequence of Sta56 gene of Orientia tsutsugamushi isolated from Shandong area].

OBJECTIVE: To analyze the genetic differences of Orientia tsutsugamushi (Ot) Sta56 gene between Shandong isolates and other strains deposited in GenBank. METHODS: PCR and restriction fragment length polymorphism (RFLP) were used to amplify the complete sequence of Ot-Sta56 gene. RFLP profiles of Ot were predicted by a computer program according to their complete sequences of Ot-Sta56 gene. PCR amplicon from XDM2 strain was sequenced and analyzed by Clustal X (1.8) and PHYLIP software. RESULTS: The complete sequences (about 1.6 kbp) of Ot-Sta56 gene were amplified from B16 strain (isolated from patients), FXS2 strain (isolated from A. agrarius) and XDM2 strain. Four species of restriction endonucleases (Hha I, Hinf I, Hae III, Pst I) were used to digest the PCR amplicons from the 3 isolates. When comparing with the RFLP profiles of prototype Ot, the RFLP profiles of PCR amplicons from the 3 isolates were similar to those of Japan Kawasaki strain, but were quite different from the international reference strains Gilliam, Karp, Kato. Results from DNA sequence analysis showed that the complete sequence of Ot-Sta56 gene homology to Japan Kawasaki strain of XDM2 strain was 97%, and deduced amino acid sequence was 92%. CONCLUSION: Data from the complete sequence of Sta56 gene indicated that the genotypes of Ot isolates in Shandong province were similar, but with distinction from the Kawasaki strain.

[Sterilizing effect of atmospheric pressure plasma jet on microbes].

To study the sterilizing effect and mechanism of APPJ on microbes preliminarily, three representative bacteria, Staphylococcus aureus, Escherichia coli, Bacillus subtilis var. niger were treated by two kinds of plasma, DBD (dielectric barrier discharge) and APPJ. The survival curves of different microbes were compared and analyzed; Furthermore, the morphological change to cell walls and cell membranes were studied by scanning electric microscope. These results demonstrated that in the beginning phase the sterilizing effect of two kinds of plasmas on three microorganisms was stronger than in the later phase, and APPJ was more effective than DBD (D value of DBD on Bacillus subtilis var. niger was 70 s, whereas APPJ's was 4 s, much more efficient). Meanwhile, the gross morphological damage of E. coli cells under SEM after APPJ treatment was observed. These gave the obvious evidence that APPJ can destroy the microbes very efficiently, and more likely through the damage of cell walls and membranes of microbes treated by APPJ.

[Hypothesis on generating and tracer gas study regarding transmission of severe acute respiratory syndrome through ventilation system in a general hospital].

OBJECTIVE: By measuring airflow and ventilation distribution of ward building, to explore and verify the hypothesis of airborne transmission and risk factor of severe acute respiratory syndrome (SARS) nosocomial infection. METHODS: Tracer gas (perfume of plant oil) was emitted to the bathroom of wards when SARS index patient lived. Six different experimental situations were designed to control the status of exhaust fan in bathrooms, exhaust fan in the top of building and fresh air exchange system. The concentration of perfume was separately measured by 4 groups of lab workers and recorded blindly by the scores of "tenth degree". RESULTS: Tracer gas was detected from the wards of 8th to 13th floor. CONCLUSION: Architecture and ventilation system of the inpatient building in the hospital contributed to the aerodynamic condition of SARS nosocomial infection through airborne transmission. The distribution of tracer gas in the wards was associated with SARS patients in this building. It was possible that SARS could have been transmitted to for distance by aerosol or other carriers.

[Preliminary result on the nosocomial infection of severe acute respiratory syndrome in one hospital of Beijing].

OBJECTIVE: To study the transmission route of severe acute respiratory syndrome (SARS) nosocomial infection. METHODS: Ten identified SARS patients were selected from a general hospital in March. Survey was carried out through a standardized questionnaire provided by Chinese Center for Disease Control and Prevention. Contents of the questionnaire would include: history of contact with SARS patient, route of infection, methods used for protection and so on. RESULTS: (1) Distribution os SARS patients were confined to 3 wards: 4, 5, and 6 on the 7, 8, 12, 13 and 14 floors in the west unit of the inpatient building. Most of the inpatients were elderly and having severe original diseases. (2) Index patients were the first generation source of transmission and they infected inpatients and medical staff, making them the second generation. People with latent infection who had close contact with SARS patients might also serve as the possible source of transmission. (3) The major transmission routes were: near distant droplet infection and close contact infection. There was also a clue to the probability of aerosol or droplet nuclei infection through air-conditioning and ventilation system. CONCLUSION: Nosocomial infection appeared to be the main characteristic of the SARS epidemic in the early stage of this hospital. Other than close contact and near space airborne transmission of SARS virus, the possibility of long-distance aerosol transmission called for further epidemiological and experimental studies in the future.