Identification of Group A Streptococci by Direct Fluorometry

A simple direct fluorometric method for rapid identification of group A streptococci is described. The method permits the detection of the organism in mixed cultures without the aid of a microscope and is amenable to automated processing of specimens. Experience with the indirect fluorometric method revealed that nontrypsinized cells from a 10-fold dilution of overnight broth cultures could be stained with uniform brilliance with fluorescent antibody (1:15 dilution) and that fluorescent antibody dissociated from such cells at 55 C for 20 min gave serologically specific fluorometric values. With this information, it was possible to develop a simpler fluorometric test which gave results comparable to those obtained by conventional cultural-precipitin grouping techniques. In the direct test described, cultures from throat swabs were incubated overnight, and cells from a 10-fold dilution were stained with specific fluorescent antibody (1:50 dilution) and then rinsed. The stained specimens were transferred to a continuous-filter paper strip (Whatman 3 MM) and read serially in a Turner 110 fluorometer with Corning 5840 and Wratten 2A filters in place. The reagents used required careful standardization and testing to assure that fluorometric readings above a specified value would be indicative of the presence of group A streptococci.     

Antibiotic sensitivity of beta-hemolytic streptococci isolated from throat swabs and purulent material

The aim of this study was to evaluate the prevalence and susceptibility of beta-hemolytic streptococci isolated from throat swabs (142--29.9%) and purulent material (333--70.1%) taken from patients treated at University Hospital dr. A. Jurasz in Bydgoszcz Collegium Medicum. L. Rydygier in Bydgoszcz, Nicolaus Copernicus University in Torun in 2005-2009. Of the 475 tested strains, 156 (32.8%) were identified as S. pyogenes. This species accounted for 38.8% of strains isolated from purulent material and 19.0% of swabs from the throat. Among the strains isolated from throat swabs of 62 (43.7%) were identified as Streptococcus group C. Only 5.1% strains were identified as Streptococcus group F. All strains of beta-hemolytic streptococci were susceptible to ampicillin or penicillin, fluoroquinolones, vancomycin and linezolid. Erythromycin-susceptible strains was 83.8%, and 89.1% for clindamycin. A total of 51.3% of erythromycin resistance strains had the cMLS(B) phenotype (63.3% for strains from throat swabs and 46.3% of the purulent materials). Sensitivity to tetracycline was characterized by 51.2% of strains of beta-hemolytic streptococci. The percentage of strains susceptible to this antibiotic among isolates from throat swabs was 63.1%, and purulent material--48.0%. The lowest percentage of strains susceptible to tetracycline (14.1%) were found among S. agalactiae and Streptococcus group G (33.6%) strains. During the study time, saw an increase in the percentage of strains susceptible to tetracycline and erythromycin.     

Evaluation of a kit for rapid detection of group A streptococci in a pediatric emergency department.

We evaluated a kit for the rapid detection of group A streptococci from throat swabs (Culturette Brand 10-Minute Group A Strep ID, Marion Scientific, Division of Marion Laboratories, Inc., Kansas City, Missouri) in the laboratory and in a busy pediatric emergency department. The sensitivity of the kit in the laboratory was 80% for all specimens and 94% for specimens with more than 10 colony-forming units of group A streptococci; the specificity was 99%. After initial training, emergency department pediatricians and nurses achieved sensitivities of 72% and 69% respectively. The specificity achieved by the pediatricians was 76% initially but 96% after further training. Untrained residents achieved a sensitivity of 58%. We conclude that this kit is potentially useful in the hands of adequately trained personnel, but without training the accuracy of the results is unacceptable. We recommend that the kit be used by designated staff trained and monitored by laboratory personnel.     

Treatment of streptococcal pharyngitis with amoxycillin once a day.

OBJECTIVE--To evaluate treatment of group A beta haemolytic streptococcal pharyngitis with amoxycillin once daily compared with phenoxymethylpenicillin three or four times a day. DESIGN--Randomised controlled study of consecutive patients presenting with symptoms suggestive of group A beta haemolytic streptococcal pharyngitis in whom culture of a throat swab yielded positive results. SETTING--Five family medicine practices. SUBJECTS--157 patients aged over 3 years who required treatment with antibiotics. MAIN OUTCOME MEASURES--Clinical response, bacteriological response, days at work and school lost, and compliance. RESULTS--During the period of the study 393 patients presented with symptoms suggesting streptococcal pharyngitis; 157 of them had throat swabs that yielded positive results on culture. Eighty two were treated with phenoxymethylpenicillin and 75 with amoxycillin. No difference was observed in the clinical response, days at work and school lost (139 days for 64 patients taking phenoxymethylpenicillin v 100 days for 57 patients taking amoxycillin; p > 0.2), or residual positive cultures after two days (6 (7.3%) v 3 (4%); p > 0.5). A significant difference in the bacteriological response was found after 14 days (5 (6.1%) v 0; p < 0.04) with no positive cultures observed in the amoxycillin group. CONCLUSIONS--These findings support the hypothesis that amoxycillin once daily is as effective as phenoxymethylpenicillin in the treatment of group A beta haemolytic streptococcal pharyngitis.     

Magnitude of colonization and sepsis by group B streptococci in newborn infants

Infants admitted to the Neonatal Intensive Care Unit at Tampa General Hospital, Tampa, Florida, were cultured for group B streptococci (GBS). Culture swabs were quantified for GBS to determine the magnitude of colonization in infected infants. Thirty-seven (17%) of the 217 infants cultured were positive for GBS. Six of these colonized infants developed sepsis, with blood cultures positive for GBS. Septic infants generally were colonized by large numbers of GBS (10⁵ bacteria/culture swab) at two or more external skin sites, in comparison to aseptic infants, who were lightly colonized with GBS. The data suggest a possible correlation between magnitude of colonization by GBS at external skin sites and development of GBS sepsis in newborn infants.     

Periurethral aerobic microflora of pregnant and non-pregnant women.

Seventy-two pregnant and 88 non-pregnant women were examined to see whether the periurethral region had been colonised with group B streptococci (Streptococcus agalactiae), enterococci, and Gram-negative rods belonging to the Enterobacteriaeceae. A semi-quantitative method was used for periurethral sampling, and paired urethral swabs were also collected to compare the isolation rates of group B streptococci from the two sites and with the two sampling methods. A higher isolation rate was found with periurethral sampling. Most specimens showed no or scanty growth of Gram-negative rods. Pregnancy was often associated with heavy growth of enterococci. Sampling performed during menstruation and while oral contraceptives were being used produced high isolation rates of group B streptococci. These results seem to suggest that the periurethral area might protect against genital colonisation with group B streptococci as it does against urinary tract infection and that hormonal factors influence the carriage of these organisms.     

Rapid detection of Haemophilus influenzae by hel gene polymerase chain reaction

AIMS: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. METHODS AND RESULTS: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65.9%) of 91 samples in contrast to 62 (68.12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. CONCLUSIONS: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. SIGNIFICANCE AND IMPACT OF THE STUDY: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods.     

咽拭子快速培养在肺炎支原体感染中的临床应用价值

目的:探讨咽拭子快速培养在肺炎支原体感染中的临床应用价值。方法:收集2014年2月~2016年2月期间我院收治的呼吸道感染患儿220例,用肺炎支原体专用液体培养基进行肺炎支原体快速培养,用胶体金法检测肺炎支原体MP-Ig M。比较两种方法的阳性率。结果:咽拭子培养快速培养阳性率与血清MP-Ig M检测阳性率比较,差异无统计学意义(P0.05)。MP-Ig检测显示,≤1岁阳性率最低,其阳性率随年龄增加不断增高(P0.05)。肺炎支原体咽拭子培养显示,≤1岁阳性率最高,2~8岁最低(P0.05)。病程≤7 d患者肺炎支原体咽拭子培养阳性率(34.21%)显著高于肺炎支原体MP-Ig检测阳性率(14.04%)(P0.05)。病程7 d患者肺炎支原体咽拭子培养阳性率(11.32%)显著低于肺炎支原体MP-Ig检测阳性率(52.83%)(P0.05)。肺炎支原体咽拭子培养的灵敏度性以及特异性显著高于肺炎支原体MP-Ig检测,差异具有统计学意义(P0.05)。结论:咽拭子快速培养对肺炎支原体感染的早期诊断有一定临床应用价值,方法简单,无创伤,值得临床进一步研究和应用。     

Cytological diagnosis of endometritis in the mare: investigations of sampling techniques and relation to bacteriological results

Aim of this study was to compare uterine smears made using the Knudsen catheter, the cytology brush and a uterine culture swab with regard to diagnostic usefulness and the occurrence of neutrophils. Additionally correlation between culture results and the occurrence of neutrophils in uterine smears was investigated. Samples were collected from 340 mares, 81.5% of which were in estrus. Smears made using the cytology brush yielded more endometrial cells per high-power field than those made using the other two instruments (p<0.0001), and a larger proportion had PMNs compared with smears made using the uterine swab (p<0.0001). For smears made with the cytology brush, cultures of β-hemolytic streptococci were more often (p=0.002) accompanied by PMNs than cultures of bacteria other than β-hemolytic streptococci, and there was a positive correlation (r(s)=0.2 p=0.01) between the number of PMNs in smears and the number of colonies of β-hemolytic streptococci. The cytology brush was superior to the other methods because it generated a larger proportion of diagnostic useful smears and the occurrence of PMNs in smears was significantly correlated with the occurrence of cultures of β-hemolytic streptococci.     

荧光定量RT-PCR检测甲型H1N1病毒核酸的临床意义

目的观察荧光定量RT-PCR技术在检测甲型H1N1病毒核酸的临床意义。方法对135例经确诊为甲型H1N1的感染患者的咽式子采用荧光定量PCR技术检测H1N1病毒核酸,同时对40例健康体检者的咽式子做为对照组一同进行甲型H1N1病毒核酸检测。结果 135例确诊为甲型H1N1的患者经荧光定量PCR检测阳性129例,符合率为96.99%。40例健康体检者结果全阴性。结论荧光定量PCR检测甲型H1N1病毒核酸具有快速、特异性高等特点,在采用此方法诊断甲型H1N1时,阳性即可确诊,阴性者要结合临床。     

Identification of beta-hemolytic streptococci isolated from hospitalized children in Baghdad.

Two hundred and twenty four hospitalized children in Baghdad aged between 1 month and 10 years were examined for Streptococcal infections. Thirty-four percent of the throat and saliva specimens were positive for beta-hemolytic streptococci. Males were more susceptible to infection with group A streptococci than females. Streptococcus of group A was isolated from 39.5% of the positive cases while group G was 47.4%. The etiological significance of the latter group in tonsillitis and otitis media is to be further investigated. Ninety six percent of the isolated streptococci were T typable and 13.3% of the strains were M typable. A high frequency of type T-11 was found in streptococcal infections. T type 3875 was found to be a new provisional type. All isolates were M untypable, and antiopacity factor negative except for two isolates of T type 4 which were positive in both typings.     

The appropriateness of swab cultures for the release of human allograft tissue

Surgeries utilizing human allograft tissues have increased dramatically in recent years. With this increase has come a greater reliance on the use of swab culturing to assess allograft tissues for microbial contamination prior to distribution. In contrast to the typical industrial microbiological uses for swabs, the tissue banking industry has relied on swab cultures as a sterility release method for allograft tissues. It has been reported in the literature that swabs have limitations, both in sensitivity and reproducibility, so their suitability as a final sterility release method was evaluated in this study. Two different swab-culturing systems were evaluated (COPAN, EZ Culturette) using human allograft tissues spiked with low levels of multiple bacterial and fungal microorganisms. The average microbial recoveries for all challenge microorganisms for each tissue type and each swab system were calculated. Percent recoveries for each challenge microorganism were also calculated and reported. The results indicated that both swab systems exhibited low and highly variable recoveries from the seeded allograft tissues. Further analysis indicated there was no statistical difference (∝=0.05) between the two swab systems. It is the recommendation of the authors that swab culturing not be used to assess relatively low levels of microbial contamination on allografts. Instead, alternative validated microbial detection methods with improved sensitivity and reproducibility should be employed and validated for this critical task.     

Rapid Diagnosis of Diarrhea Caused by Shigella sonnei Using Dipsticks; Comparison of Rectal Swabs,Direct Stool and Stool Culture

Background

We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs.

Methodology/Principal Findings

The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10⁶ CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%–98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%–88.6%) and 100 %, respectively.

Conclusion

This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.     

Confirmatory identification of group D streptococci by slide co-agglutination

Group D streptococci were identified by slide co-agglutination and the results obtained were compared with conventional methods for the presumptive (bile esculin medium, salt tolerance) and confirmatory (Lancefield precipitin test, latex agglutination) identification of these bacteria. Of 137 clinical specimens examined, the co-agglutination procedure showed a 100% specificity and a 96.5–100% sensitivity (depending on the method of testing) in the identification of group D streptococci. The slide co-agglutination test was easy to perform and interpret and offers a valuable alternative to other less rapid techniques for the confirmatory identification of group D streptococci.     

肺炎支原体液体培养法检测的实验评价

目的比较液体培养法和固体培养法平行检测肺炎支原体结果的一致性;评价液体培养法检测肺炎支原体的可靠性。方法采用液体培养基和固体琼脂培养基平行检测1 648份临床标本的肺炎支原体,比较同一份标本在2种培养基上的检测结果。结果液体培养法阳性296例,阳性率为18%;固体培养法阳性244例,阳性率为14.8%;液体培养法阳性而固体培养法阴性57例;固体培养阳性而液体培养法为阴性5例。2种方法的阳性检出率比较差异有统计学意义(P<0.05)。结论肺炎支原体快速液体培养法与固体培养有较好的一致性,具有方便、简单、准确且可以用于早期检测等优点,适合临床大批量标本筛查。需结合患者临床症状等排除真菌和耐药菌造成的假阳性。     

Evaluation of the usefulness of selected commercial systems for identification of Streptococcus species

The usefulness was assessed of three commercially available systems for rapid identification of streptococcal strains. The studied material comprised 68 strains of streptococci and enterococci (including 24 standard strains) belonging to serological groups: A (14 strains), B (10), C (11), D (10), F (3) and G (10), as well as 10 S. pneumoniae strains. The strains were isolated from throat, nasal, wound swabs, blood, pus of inpatients and throat and nasal swabs of outpatients. For the identification of streptococci 3 commercially available systems were used: API 20 STREP (bioMerieux, France), rapid ID 32 Strep (bioMerieux, France), Streptoplast PPL 18 (HTL, Poland). The determinations were done according to producer's instructions. The highest percent of correctly identified strains was obtained with the rapid ID 32 Strep--80.9%, with the API 20 STREP--76.4% strains were identified correctly and with the PPL 18--61.8%. The study showed that the API 20 STREP and rapid ID 32 STREP are suitable for the identification of streptococcal strains from groups: A, B, C, D, F and enterococci--group D. The proportions of correctly identified strains from these groups with the Streptoplast PPL 18 were lower than those determined with the bioMerieux systems. Using of three identification systems streptococci from group G and S. pneumoniae strains cannot be identified.     

Evaluation of an improved rapid coagglutination method for the serological grouping of beta-hemolytic streptococci.

Grouping of beta-hemolytic streptococci was performed with the Phadebact Streptococcus Test, a coagglutination method, and the results compared with serological grouping by the standard Lancefield precipitin method. Of 171 clinical specimens examined, 169 (98.8%) were grouped correctly by the Phadebact Test after 24 h of continuous growth in Todd-Hewitt broth. In a parallel study, 96.9% of specimens that grew after only 4 h of incubation in broth were grouped correctly by the coagglutination method. In both studies, the accuracy of the coagglutination test was increased significantly by elimination of multiple-agglutination reactions through centrifugation of cultures and utilization of the supernatant fluid in the Phadebact Test.     

Protective immunity evoked by locally administered group A streptococcal vaccines in mice

The present studies were undertaken to determine the pathogenicity of group A streptococci introduced intranasally (i.n.) into mice in an attempt to mimic mucosal infections in humans and to determine the efficacy of streptococcal vaccines administered via the mucosal route. The LD50 of type 24 streptococci (M24 strep) administered i.n. was 3 x 10(4) CFU. Throat cultures were performed in M24 strep-inoculated mice. Of 11 mice that died, 9 had positive throat cultures 3 or 4 days after i.n. challenge, and of 9 mice that survived, only 1 had a positive throat culture, indicating an association between mucosal infection and death. Postmortem examination performed on 35 mice that died after i.n. challenge showed that all had evidence of disseminated infections, and group A streptococci were recovered from the cervical lymph nodes, blood, spleen, liver, and brain. To determine vaccine efficacy, heat-killed M24 strep or pep M24 were administered i.n. to groups of mice. Whole, heat-killed streptococci and pep M24 administered locally protected mice against death from i.n. challenge infections with homologous M24 strep. The whole cell vaccine also protected against i.n. challenge infections with heterologous type 6 streptococci. Our data suggest that streptococcal vaccines administered locally evoke protective immunity against streptococcal infections.     

2007年北京地区儿童手足口病病原的初步筛查

2007年4～6月儿童手足口病流行期间,对北京地区51例皮损症状典型、伴/不伴发热、无重症合并症的手足口病患儿采样,建立RT-PCR方法,以5'非编码区(5'UTR)肠道病毒通用引物、CA16和EV71 VP1区特异性引物直接对82份临床标本进行了初步筛查,肠道病毒阳性率达70.6%。检测病例中CA16阳性25例(25/51)、EV71阳性4例(4/51)、非CA16和EV71的肠道病毒阳性病例7例(7/51),三者比例约为6:1:2。2007年北京地区儿童轻症手足口病主要病原包括CA16和EV71,同时还存在一定比例其它肠道病毒。部分EV71毒株经测序验证及系统进化分析显示为C4基因亚型。