Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples

Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colony-forming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3% (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis.     

应用16S rRNA宽范围聚合酶链反应快速检测临床无菌体液感染

目的应用16S rRNA宽范围聚合酶链反应（PCR）快速检测临床无菌体液感染,并与传统培养方法进行比较分析。方法收集94份临床无菌体液（血液、脑脊液、胸腹水、关节液）标本,提取细菌基因组DNA,应用16S rRNA宽范围PCR扩增,并将PCR阳性产物测序,然后将测序结果在美国国家生物技术信息中心（NCBI）上进行BLAST比对,从而确定菌种。同时,应用常规培养方法检测94份无菌体液标本,并对2种方法的结果进行比较。结果 16S rRNA宽范围PCR敏感性高,最低扩增浓度为103 CFU/mL,且该技术特异性高,其阳性扩增产物经测序比对后结果和培养结果完全吻合。另外,94例临床标本中应用培养方法培养出阳性例数为9例,阳性率为9.6%,而应用宽范围PCR,除培养阳性的9例均为阳性外,另外扩增出6例阳性标本,阳性率为15.9%,而此6例经测序证实分别为4株铜绿假单胞菌、1株黏质沙雷菌、1株肺炎链球菌。结论 16S rRNA宽范围PCR与培养技术比较起来,敏感性高,特异性强,有望成为临床无菌体液病原体感染快速筛查的方法。     

Detection ofUreaplasma urealyticum in urethral swab samples from asymptomatic men and men with urethritis by a polymerase chain reaction-based assay

We compared a polymerase chain reaction (PCR)-based assay with primers specific for the 16S rRNA gene ofUreaplasma urealyticum with culture techniques for detectingU. urealyticum from urethral swab samples obtained from 256 asymptomatic men. Of 24 samples positive forU. urealyticum by culture, 23 samples were positive by the PCR-based assay, whereas 2 of 232 samples with a negative culture were positive by the PCR-based assay. The sensitivity and specificity for the PCR-based assay compared to culture were 95.8% and 99.1%, respectively. Our results confirmed the validity of the PCR-based assay for identifying this pathogen from urethral swab samples. In this study, we also examined urethral swab samples obtained from 195 men with urethritis for the detection ofU. urealyticum by this assay.U. urealyticum was detected in 1 (3.4%) of 29 men with gonococcal urethritis and 23 (13.9%) of 166 men with nongonococcal urethritis. This assay may be a relevant tool to diagnose urethralUreaplasma infections.     

Real-time broad-range PCR versus blood culture. A prospective pilot study in pediatric cancer patients with fever and neutropenia

Materials and methods In a pilot study, results of real-time broad-range (16S rRNA) polymerase chain reaction (PCR) performed on 45 blood samples of pediatric cancer patients with fever and neutropenia were compared with blood culture results. Results The PCR assay used, having proven a high sensitivity in artificially spiked blood samples, was positive in only three of ten blood culture-positive samples, and it was positive in 10 of 35 (29%) culture-negative samples. Conclusion This broad-range PCR assay, which may identify not-grown bacteria potentially contributing to fever, needs improvement in sensitivity, and different reasons for positive PCR in negative blood culture samples need to be assessed before clinical application. Provisional results of this study were presented in part at the 36th conference of the International Society of Pediatric Oncology (SIOP) in Oslo, September 2004 (Abstract PD.017). Submission as Short Communication to Supportive Care in Cancer     

弥散黏附性大肠埃希菌多重PCR检测方法的建立及其在感染性腹泻患者中的流行情况

目的 建立一种快速检测弥散黏附性大肠埃希菌（DAEC）的普通多重PCR方法,了解DAEC在腹泻患者中的流行情况。方法 根据DAEC黏附基因afaB、afaC、afaD、daaE及16S rRNA基因rrs,建立4重PCR反应体系,对PCR引物、反应体系和反应条件进行优化,评价敏感性和特异性,并应用于389份腹泻患者粪便标本的筛查。结果 4重PCR反应可以特异性扩增DAEC菌株afaB/C、afaD、daaE基因片段,除2株肠集聚性大肠埃希菌（EAEC）外,检测引物对其他致泻性大肠埃希菌及常见肠道病原菌均无特异性扩增,此多重PCR方法的检测下限可达3.20 101 CFU/反应（8.01 103 CFU/ml）。利用本研究建立的多重PCR方法对腹泻患者粪便标本进行检测,发现DAEC菌株分离率为6.2%（24/389）。结论 本研究建立的多重PCR方法可用于DAEC菌株的快速鉴别,也可用于人粪便标本DAEC的初步筛查。     

Rapid diagnosis of acute bacterial meningitis: role of a broad range 16S rRNA polymerase chain reaction

Background: Acute bacterial meningitis is a significant cause of morbidity and mortality throughout the world. It can be difficult to diagnose, as the symptoms and signs are often non-specific. Study Objective: To evaluate the performance of an in-house semi-nested polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Eubacteria for the rapid diagnosis of acute bacterial meningitis using cerebrospinal fluid (CSF) specimens. Methods: A total of 112 CSF samples from 112 patients were used in the study. Among these, 32 samples were obtained from confirmed cases of Streptococcus pneumoniae, six samples were obtained from confirmed cases of Haemophilus influenzae, one sample from a confirmed case of Neisseria meningitidis, and 10 cases of clinically suspected acute bacterial meningitis. The remaining 63 CSF samples were obtained from patients with non-infectious illnesses (n = 47) of the central nervous system (CNS) and autopsy-confirmed tuberculous meningitis (n = 16). Results: The assay had an overall sensitivity of 93% (95% confidence interval [CI] 0.81–0.98, negative predictive value = 95%) and a specificity of 98% (95% CI 0.92–1.0, positive predictive value = 98%). Conclusion: These preliminary findings suggest that the semi-nested PCR assay targeting the 16S rRNA gene may be used as a rapid test for the diagnosis of acute bacterial meningitis.     

Use of quantitative 16S rRNA PCR to determine bacterial load does not augment conventional cerebrospinal fluid (CSF) cultures among children undergoing treatment for CSF shunt infection

The aim of this study was to develop a quantitative 16S rRNA assay for determination of bacterial nucleic acid load in cerebrospinal fluid (CSF) shunt infection and to compare quantitative 16S rRNA polymerase chain reaction (PCR) findings to those of conventional bacterial culture in patients treated for CSF shunt infection. We developed a quantitative 16S rRNA PCR assay that detected bacterial load across a range of 2.5 × 10⁹ down to 2.5 × 10⁴ 16S copies/mL CSF under experimental conditions for numerous Gram-positive and Gram-negative organisms. However, when applied to archived CSF samples from 25 shunt infection episodes, correlations between positive bacterial culture and 16S rRNA levels were seen in only half of infections, and 16S rRNA levels dropped precipitously after an initial peak on the first day of sample collection. Bacterial load measured using 16S rRNA PCR does not provide sufficient information beyond bacterial culture to inform CSF shunt infection treatment.     

重症急性胰腺炎合并细菌感染的快速诊断——附17例检测分析

目的:探讨以16S rRNA基因为基础的细菌PCR法诊断重症急性胰腺炎早期合并细菌感染的价值.方法:17例疑有细菌感染的重症急性胰腺炎病人,术前B超引导下行胰周渗液细针穿刺检查,分别行以16S rRNA基因为基础的细菌PCR法检测和细菌培养.比较细菌PCR法检测结果和细菌培养结果.结果:17例中,PCR检测9例阳性,细菌培养10例阳性,该10例病人行手术治疗,术中所取胰周渗液行细菌培养证实为阳性.PCR检测重症急性胰腺炎合并细菌感染的敏感度为90%,特异度为100%.PCR法和细菌培养法分别需时5 h和3 d.结论:以16S rRNA基因为基础的细菌PCR法能快速、准确地诊断重症急性胰腺炎早期合并细菌感染,为手术治疗提供可靠依据.     

Usefulness of a direct 16S rRNA gene PCR assay of percutaneous biopsies or aspirates for etiological diagnosis of vertebral osteomyelitis

We performed a prospective study to evaluate the clinical usefulness of a direct 16S rRNA gene (16S rDNA) PCR assay of percutaneous biopsies or aspirates for the etiological diagnosis of vertebral osteomyelitis. During May 2009 to December 2010 and November 2011 to August 2012, consecutive patients with suspected vertebral osteomyelitis who underwent a percutaneous biopsy or aspiration were enrolled. Of 45 patients with vertebral osteomyelitis, 16S rDNA PCR was positive in 24 (53.3%), whereas culture was positive in 13 (28.9%) (P = 0.027). Three of PCR-positive cases (12.5%, 3/24) and 1 of culture-positive case (7.7%, 1/13) were considered to be false-positives. Of 16 patients without prior antimicrobial exposure, 75% of cases (12/16) were positive by either culture (7/16, 43.8%) or PCR (9/16, 56.3%). A 16S rDNA PCR assay with sequencing was more sensitive than routine culture for the etiological diagnosis of vertebral osteomyelitis.     

Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection

Introduction

Analysis of joint aspirate is the standard preoperative investigation for diagnosis of periprosthetic joint infection (PJI). We compared the diagnostic performance of culture and multiplex polymerase chain reaction (PCR) of synovial fluid for diagnosis of PJI.

Evaluation of two detection methods of microorganisms in platelet concentrates

Background: The performance of a bacterial 16S ribosomal DNA real‐time polymerase chain reaction (PCR) assay was evaluated and validated with an automated culture system to determine its use for screening of platelet concentrates (PCs). Study Design and Methods: PCs were spiked with suspensions of Escherichia coli, Serratia marcescens, Staphylococcus epidermidis and St. aureus at 1, 10, and 100 colony‐forming units (CFUs) mL and stored for 5 days. DNA amplification was performed using real‐time PCR. The BacT/ALERT was used as a reference method and samples were inoculated into an aerobic culture bottle; for the PCR assay, aliquots were drawn from all (spiked) PCs on days 0 to 5 of storage. Results: Real‐time PCR detected only the gram‐positive bacteria in PCs spiked with low bacterial titres (1 CFU mL) after 48 h; however, it was able to detect all positive samples in PCs spiked with 10 CFU mL of either gram‐positive or gram‐negative bacteria after 48 h. In addition, real‐time PCR detected all positive samples in PCs spiked with high gram‐positive bacterial titres (100 CFU mL) after 24 h. On the other hand, the BacT/ALERT system showed positive results in all samples within 24 h. Conclusion: The BacT/ALERT method is more sensitive and should continue to be the gold standard for identifying bacterial contaminations in blood samples. The real‐time PCR approach can be used for the screening of PCs for microbial detection before they are released from blood centres or shortly before they are used in blood transfusion, and thus allow an extended shelf life of the platelets.     

Identification of oral bacteria by 16S rRNA gene analysis in elderly persons requiring nursing care

After incubation of saliva from 58 semi-bedridden elderly persons, the cultures were identified based on the 16S rRNA gene base sequence to compare the identification by the conventional culture method. As a result, the 16S rRNA gene base sequence of 198 strains identified by the culture method showed 98.5% or more homology in some of the Human Oral Microbiome database, and the identification of bacterial species and genus was possible. When an organism identified by the 16S rRNA gene sequencing method was compared with that by the culture method, the concordance rates were 54.5% at the genus level and 35.9% at the species level. Streptococcus mitis strains most frequently isolated from saliva that were identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method (32/35), and all the 11 Streptococcus salivarius strains identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method. All the strains identified as Streptococcus anginosus group by the culture method and 8 of the 9 strains identified as Prevotella species by the culture method were identified as the same group and genus by the 16S rRNA gene sequencing method. When an oral microbial flora test with saliva samples from elderly persons is performed, the 16S rRNA gene sequence identification enables us to identify major indigenous bacteria and pathogenic bacteria and is considered useful as a means of supplementing the conventional culture method.     

Detection of Mycobacterium avium complex DNA directly in clinical respiratory specimens: opportunities for improved turn-around time and cost savings

We developed, evaluated, and implemented a Taqman multiplex real-time polymerase chain reaction (PCR) assay for the detection of Mycobacterium avium complex (MAC), targeting the 16S-23S rRNA internal transcribed spacer, which we have combined with an existing Mycobacterium tuberculosis complex assay for use directly in clinical respiratory specimens. Evaluation of the performance of this assay for MAC detection included 464 clinical respiratory specimens tested prospectively. This real-time PCR assay was found overall to have a sensitivity of 71.1%, a specificity of 99.5%, a positive predictive value of 98.0%, and a negative predictive value of 90.2% for MAC. The assay provides results prior to the availability of cultured material and identification, most within 24 h of specimen receipt, and may reduce the need to culture MAC-PCR–positive specimens when susceptibility testing is not requested. Additionally, we have found significant cost savings of approximately $21.00 per specimen and staff time reductions of 3.75 h per specimen with implementation of this assay.     

检测气单胞菌属的SYBR Green荧光PCR方法的建立和评价

目的建立一种检测气单胞菌的SYBR Green荧光PCR方法。方法使用文献报道的气单胞菌属16S rRNA基因扩增引物,建立SYBR Green荧光PCR检测方法,评价其特异性、灵敏度和实际应用价值。结果SYBR Green荧光PCR方法的检测下限为10–7 ng/ml,检测的21株气单胞菌均为阳性,检测的弧菌、大肠埃希菌、沙门菌、小肠结肠炎耶尔森菌、志贺菌均为阴性。 针对104份健康人群粪便样本,8.65%的样本分离到气单胞菌,28.85%的样本SYBR Green荧光PCR方法检测阳性。 其中8份标本采用2种方法检测均为阳性。 样本核酸检测阳性后再进行病原菌的分离,会漏检1份样本（11.11%）,但可以减少71.15%（74/104）的分离培养及鉴定工作。结论SYBR Green荧光PCR方法具有很好的特异性和敏感度,用于大样本的早期筛查可以减少70%左右的工作量,适合应用于气单胞菌感染和携带的监测。     

Epidemiology and diagnosis of Lyme borreliosis

The multisystem disease Lyme borreliosis is the most frequent tick‐transmitted disease in the northern hemisphere. In Europe Lyme borreliosis is most frequent in Central Europe and Scandinavia (up to 155 cases per 100,000 individuals) and is caused by the species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. The recently detected genospecies A14S may also play a role in skin manifestations. Microbiological diagnosis in European patients must consider the heterogeneity of borreliae for development of diagnostic tools. According to guidelines of the USA and Germany, serological diagnosis should follow the principle of a two‐step procedure (enzyme‐linked immunosorbent assay (ELISA) as first step, if reactive; followed by immunoblot). The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens (p100, p58, p41i, VlsE, OspC, DbpA) including those expressed primarily in vivo (VlsE and DbpA) instead of whole cell lysates. VlsE is the most sensitive antigen for IgG antibody detection, OspC for IgM antibody detection. At present, detection rates for serum antibodies are 20%–50% in stage I, 70%–90% in stage II, and nearly 100% in stage III Lyme disease. Detection of the etiological agent by culture or polymerase chain reaction (PCR) should be confined to specific indications and specialized laboratories. Recommended specimens are skin biopsy specimens, cerebrospinal fluid (CSF) and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50%–70%) and synovial tissue or fluid (50%–70% with PCR). CSF yields positive results in only 10%–30% of patients except when the duration of symptoms is shorter than 2 weeks (50% sensitivity). Methods which are not recommended or adequately documented for diagnosis are antigen tests on body fluids, PCR of urine, and lymphocyte transformation tests.     

Rapid diagnosis of septic arthritis using 16S rDNA PCR: a comparison of 3 methods

Few studies address the utility of molecular techniques for diagnosis of infection in synovial fluid (SF). We evaluated 3 different methods using 16S rDNA polymerase chain reaction (PCR) on 63 specimens for the diagnosis of joint infection. SF samples were classified as normal, inflammatory, or septic based on the patient's clinical and laboratory results. Samples were analyzed by conventional PCR using primers for the bacterial 16S rDNA gene and by real-time PCR utilizing 2 different sets of primers for the target gene 16S rDNA. PCR results were compared to culture results. All inflammatory and normal SF samples were culture negative. There was concordance with 10 of the 16 septic samples by 2 of the PCR methods. When comparing 3 methods for rapid detection of septic arthritis, real-time PCR using SYBR-Green I and conventional PCR demonstrated favorable test characteristics, but need further study.     

Antibody levels correlate with detection of Trypanosoma cruzi DNA by sensitive polymerase chain reaction assays in seropositive blood donors and possible resolution of infection over time

BACKGROUND: The clinical significance of anti‐Trypanosoma cruzi low‐level reactive samples is incompletely understood. Polymerase chain reaction (PCR)‐positive rates and antibody levels among seropositive blood donors in three countries are described. STUDY DESIGN AND METHODS: Follow‐up samples were collected from T. cruzi–seropositive donors from 2008 through 2010 in the United States (n = 195) and Honduras (n = 58). Also 143 samples from Brazil in 1996 to 2002, originally positive by three serologic assays, were available and paired with contemporary follow‐up samples from these donors. All samples were retested with Ortho enzyme‐linked immunosorbent assay (ELISA). PCR assays were performed on coded sample panels by two laboratories (Blood Systems Research Institute [BSRI] and American Red Cross Holland Laboratory [ARC]) that amplified kinetoplast minicircle DNA sequences of T. cruzi. RESULTS: PCR testing at BSRI yielded slightly higher overall sensitivity and specificity (33 and 98%) compared with those at the ARC (28 and 94%). Among seropositive donors, PCR‐positive rates varied by country (p < 0.0001) for the BSRI laboratory: Brazil (57%), Honduras (32%), and the United States (14%). ELISA signal‐to‐cutoff ratios (S/CO) were significantly higher for PCR‐positive compared to PCR‐negative donors (p < 0.05 for all comparisons). Additionally, PCR‐negative Brazilian donors exhibited greater frequencies of antibody decline over time versus PCR‐positive donors (p = 0.003). CONCLUSION: For all three countries, persistent DNA positivity correlated with higher ELISA S/CO values, suggesting that high‐level seroreactivity reflects chronic parasitemia. Significant S/CO declines in 10% of the PCR‐negative Brazilian donors may indicate seroreversion after parasite clearance in the absence of treatment.     

Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR,and the VITEK2 system for identification of Acinetobacter clinical isolates

Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and bla_OXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter.