Direct immunofluorescent detection of Legionella pneumophila in respiratory specimens.

Respiratory secretions from patients with clinically suspected Legionnaires pneumonia were examined by direc immunofluorescent tests at the Medical Center Hospital of Vermont and at the Center for Disease Control. No fluorescent bacteria were found by either laboratory in eight specimens from eight patients who were seronegative. Twenty specimens were obtained from seven patients who had serologically confirmed Legionnaires disease. Four of the seven cases were identified at the Medical Center Hospital of Vermont, and six of the seven were identified at the Center for Disease Control. Of 20 specimens, 8 were positive at the Center for Disease Control (six or more bacilli per slide), and 7 specimens were suspicious (one to five bacilli per slide); at the Medical Center Hospital of Vermont, 4 of 20 specimens were positive, and 2 were suspicious. The inclusion of a rhodamine-conjugated counterstain at the Center for Disease Control facilitated the examination and may have improved the sensitivity. Smears from transtracheal aspirates, bronchoscopic aspirates, transcutaneous lung aspirates, pleural fluids, and tracheal aspirate-expectorated sputum produced positive results. Several specimens contained fluorescing bacilli when stained for serogroup 2 as well as serogroup 1, perhaps reflecting the presence of cross-reacting antigens in vivo.     

实时荧光PCR快速检测嗜肺军团菌的研究

目的 建立TaqMan-MGB探针实时荧光PCR快速检测嗜肺军团菌技术,为临床和环境样品检测嗜肺军团菌提供可实用工具.方法 在对嗜肺军团菌mip序列进行分析、比较基础上,设计一对特异性引物和TaqMan-MGB探针,通过实时荧光PCR反应条件和反应体系的优化,实现对嗜肺军团菌的快速检测;用克隆到pMD-19T载体上的嗜肺军团菌mip基因阳参片段和不同菌株验证方法的敏感性和特异性.结果 当用热裂解法提取DNA,25μl的反应体系中包括上、下游引物(20μmol/L)各0.6μl,探针(20μmol/L)0.4μl,模板DNA 6.0μl,反应条件为预变95℃20 S,变性95℃10 s,退火50℃ 40 s,40个循环时,TaqMan-MGB探针实时荧光PCR技术对嗜肺军团菌mip基因阳参片段最低检测浓度为0.71拷贝/μl,其循环阈值(Ct值)与模板浓度具有极好的对应关系(r=0.999);1株嗜肺军团菌标准株、12株嗜肺军团菌分离株的Ct值在13.23～16.04之间,而包括金黄葡萄球菌、鼠伤寒沙门菌、副溶血性弧菌、大肠埃希菌、铜绿假单胞菌、痢疾志贺菌共计76株其他菌PCR Ct值均大于30;整个检测过程仅需1.5 h.结论 TaqMan-MGB探针的嗜肺军团菌实时荧光PCR检测方法具有特异性和敏感性、易操作、结果准确可靠等优点,可用于嗜肺军团菌检测.     

Real-time PCR assay targets the 23S-5S spacer for direct detection and differentiation of Legionella spp. and Legionella pneumophila

A real-time PCR for the ABI Prism 7000 system targeting the 23S-5S spacer of Legionella spp. was developed. Simultaneous detection and differentiation of Legionella spp. and Legionella pneumophila within 90 min and without post-PCR melting-curve analysis was achieved using two TaqMan probes. In sputum samples from 23 controls and 17 patients with legionellosis, defined by positive culture, urinary antigen testing, or seroconversion, 94% sensitivity and 100% specificity were observed.     

Evaluation of commercial amplification kit for detection of Legionella pneumophila in clinical specimens.

A commercial kit (EnviroAmp) designed to detect the DNA of Legionella species in environmental water samples using PCR and reverse dot hybridization was applied to clinical specimens. Results correlated well with culture for bronchoalveolar lavages. In addition, this test was easy to perform and showed good sensitivity.     

Detection of Legionella pneumophila by real-time PCR for the mip gene

A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L. pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for L. pneumophila.     

Detection and differentiation of Bordetella spp. by real-time PCR

Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/mul, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/mul, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.     

Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR

We describe rapid PCR-biprobe identification of Campylobacter spp. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture.     

A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens

Objective: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made.
Method: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested.
Results: The PCR was specific for L. pneumophila and no non- Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3–7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure.
Conclusions: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.     

Application of real-time PCR and melting curve analysis in rapid detection of Ael and Bel blood types

The ABO blood group is the most important blood group system in transfusion medicine. In addition to the normal levels of ABO antigen expression, Ael and Bel represent the two major blood types that have a weak expression of the A or B antigens on red blood cells. Due to the fact that typing of Ael and Bel by conventional serological methods is time consuming and sometimes gives false-positive and false-negative results, it is warranted to develop an additional technique for the identification of Ael and Bel individuals. Through genetic analysis we have previously identified Ael as possessing an A allele with IVS6+5G-->A mutation (Transfusion 2003;43:1138-1144) and Bel as possessing a B gene with 502C-->T mutation in Taiwan (Vox Sanguinis 2003;85:216-220). Hence, real-time PCR-based genotyping methods were developed in this study to facilitate the detection of Ael and Bel. For genotyping of Ael and Bel, the region of mutations was PCR amplified and subjected to the LightCycler (LC) real-time PCR assay using LC Red640-labeled hybridization probe. Melting curve analysis was performed to determine the melting temperature Tm that was used for genotype detection of Ael and Bel blood types. For Ael genotyping, the melting curve of the normal control appears as one peak at 59.19+/-0.07 degrees C (mean+/-SE) and that of Ael appears as 2 peaks at 59.21+/-0.07 degrees C and 64.39+/-0.07 degrees C, corresponding to the O and Ael alleles, respectively. For Bel genotyping, the melting curve of the normal control appears as one peak at 67.99+/-0.11 degrees C and that of Bel appears as 2 peaks at 59.99+/-0.12 degrees C and 68.1+/-0.13 degrees C, corresponding to the Bel and O alleles, respectively. This genotyping method was shown to be accurate, based on automated sequencing of the PCR-amplified products. It takes only 90 min to perform this genotyping test. Detecting the Ael and Bel blood types by combined LC-PCR and melting-curve analysis is a rapid, reliable, and easy method.     

Development and clinical evaluation of an internally controlled,single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species

A multiplex real-time PCR assay for detection of Legionella pneumophila and Legionella spp. and including an internal control was designed. Legionella species, L. pneumophila, and the internal control were detected simultaneously by probes labeled with 6-carboxy-fluorescein, hexachlorofluorescein, and indodicarbocyanine, respectively. Therefore, no postamplification analysis was required in order to distinguish the targets. The sensitivity of both assays was 2.5 CFU/ml, and from analysis of 10 culture-positive and 74 culture-negative samples from patients investigated for legionellosis, 100% agreement was observed by both assays in comparison to culture. Four additional positives were found by the multiplex real-time PCR assay in the Legionella culture-negative samples.     

Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region

Although the majority of cases of Legionnaires’ disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.     

Specific and sensitive detection of Neisseria gonorrhoeae in clinical specimens by real-time PCR

Early diagnosis of Neisseria gonorrhoeae infections is important with regard to patients' health and infectivity. We report the development of a specific and sensitive TaqMan assay for the detection of N. gonorrhoeae in clinical samples. The target sequence is a 76-bp fragment of the 5' untranslated region of the opa genes that encode opacity proteins. A panel of 448 well-defined N. gonorrhoeae isolates was used to evaluate and optimize the assay. The method employs two minor-groove binding probes, one of them recognizing a newly identified sequence in the opa genes. Testing a large panel of related and unrelated microorganisms revealed that other Neisseria strains and other microorganisms tested negative in the opa test. With a lower detection limit of one genome per reaction, the opa test appeared more sensitive than both the COBAS AMPLICOR (Roche Diagnostics Nederland BV, Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of a panel of 122 COBAS AMPLICOR-positive samples revealed that 68% were negative in both the 16S rRNA test and the opa assay (confirming that the COBAS AMPLICOR test produces false positives), while 30% were positive in both assays. Three samples were opa positive and 16S rRNA negative, which may be due to the higher sensitivity of the opa assay. We conclude that the opa gene-based real-time amplification assay offers a sensitive, specific, semiquantitative, and reliable assay suitable for the detection of N. gonorrhoeae in clinical specimens and/or for confirmation of less specific tests.     

Detection and differentiation of Cryptosporidium spp. in human clinical samples by use of real-time PCR

Real-time PCR has the potential to streamline detection and identification of Cryptosporidium spp. in human clinical samples. In the present article, we report the first such assay to allow not only detection and differentiation of the most common human pathogens, Cryptosporidium hominis and Cryptosporidium parvum, but also simultaneous amplification of a region of the small subunit (SSU) rRNA gene, permitting direct sequence analysis to identify any Cryptosporidium species. An internal control is incorporated to identify the presence of PCR inhibitors. Analytical sensitivity was determined to be as low as 200 oocysts per gram of feces processed, equivalent to 2 oocysts per PCR. The C. hominis and C. parvum PCRs specifically detected only species/genotypes in their respective target clades. Diagnostic sensitivity and specificity, evaluated against a widely used conventional nested SSU rRNA gene PCR as a nominated gold standard using a panel of 258 (151 positive and 107 negative) samples, were 100% and 99.1%, respectively. The assay agreed with PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene for 134 of 136 (98.5%) samples tested prospectively and typed two additional isolates. The real-time PCR assay was sensitive, specific, and reproducible and significantly improved laboratory work flow and turnaround times.     

Enhanced primary isolation of Legionella pneumophila from clinical specimens by low-pH treatment.

During the 12-month study period, 919 clinical specimens were submitted to the laboratory for culture of Legionella pneumophila. All specimens were plated onto nonselective and semiselective media both directly and after treatment with a KCl-HCl solution, pH 2.2. Of the 51 specimens (5.5%) positive for L. pneumophila and Legionella-like organisms, 20 were recovered by acid pretreatment only, 5 were recovered by direct plating only, and 26 were recovered by both acid pretreatment and direct inoculation. These data demonstrated that acid pretreatment substantially improved the recovery of L. pneumophila and Legionella-like organisms from the clinical specimens examined.     

Detection of Legionella pneumophila using a real-time PCR hybridization assay

A real-time PCR hybridization assay for Legionella pneumophila is described; the assay uses LightCycler (Idaho Technology) methodology to specifically detect 2.5 CFU/reaction, equivalent to 1,000 CFU/liter of starting water sample. The assay, including DNA extraction and confirmation of product identity, is completed within 90 min of receipt of a sample.     

Development of conventional and real-time PCR assays for detection of Legionella DNA in respiratory specimens

The development and validation of a PCR assay based on the use of new 16S ribosomal DNA (rDNA)-targeted primers to detect Legionella DNA in respiratory specimens are described. The assay was originally developed as conventional PCR followed by electrophoretic detection and was then adapted to Lightcycler format with SYBR Green I detection and melting curve analysis. The 73 Legionella pneumophila strains tested were amplified with both applications. In addition, 21 and 23 out of 27 other Legionella strains were found positive by conventional and real-time PCR assays, respectively, including the clinically important species L. micdadei, L. bozemaniae, and L. dumoffii. Two DNA purification methods were compared using artificially seeded clinical specimens: a standard organic extraction method and a commercial kit based on adsorption of DNA to silica particles. The detection limit of the assay varied from 2 CFU to >200,000 CFU per ml of clinical specimen, depending on the background sample (i.e., pooled sputa or BAL fluids) and the DNA purification method, the silica method achieving lower detection limits. Analysis of 77 clinical samples (66 bronchoalveolar lavage fluid and 11 sputum samples) by conventional PCR yielded results that were consistent with Legionella culture results. The melting curve analysis in the Lightcycler system readily detected the specific amplification products. However, run-to-run variations in the measured melting temperatures required normalization against the standard sample in each run. The results obtained with the clinical specimens were similar to those obtained with conventional PCR, but more samples are required to determine whether the system can be applied to routine screening of samples for the presence of Legionella DNA.     

Low genomic diversity of Legionella pneumophila within clinical specimens

ObjectivesLegionella pneumophila is the leading cause of Legionnaires' disease, a severe form of pneumonia acquired from environmental sources. Investigations of both sporadic cases and outbreaks rely mostly on analysis of a single to a few colony pick(s) isolated from each patient. However, because of the lack of data describing diversity within single patients, the optimal number of picks is unknown. Here, we investigated diversity within individual patients using sequence-based typing (SBT) and whole-genome sequencing (WGS).MethodsTen isolates of L. pneumophila were obtained from each of ten epidemiologically unrelated patients. SBT and WGS were undertaken, and single-nucleotide polymorphisms (SNPs) were identified between isolates from the same patient.ResultsThe same sequence type (ST) was obtained for each set of ten isolates. Using genomic analysis, zero SNPs were identified between isolates from seven patients, a maximum of one SNP was found between isolates from two patients, and a maximum of two SNPs was found amongst isolates from one patient. Assuming that the full within-host diversity has been captured with ten isolates, statistical analyses showed that, on average, analysis of one isolate would yield a 70% chance of capturing all observed genotypes, and seven isolates would yield a 90% chance.ConclusionsSBT and WGS analyses of multiple colony picks obtained from ten patients showed no, or very low, within-host genomic diversity in L. pneumophila, suggesting that analysis of one colony pick per patient will often be sufficient to obtain reliable typing data to aid investigation of cases of Legionnaires' disease.     

Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR

Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (> or = 0.1 microg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens.     

A real-time PCR for specific detection of the Legionella pneumophila serogroup 1 ST1 complex

ObjectiveLegionella pneumophila serogroup 1 (Lp1) sequence type (ST) 1 is globally widespread in the environment and accounts for a significant proportion of Legionella infections, including nosocomial Legionnaires' disease (LD). This study aimed to design a sensitive and specific detection method for Lp ST1 that will underpin epidemiological investigations and risk assessment.MethodsA total of 628 Lp genomes (126 ST1s) were analyzed by comparative genomics. Interrogation of more than 900 accessory genes revealed seven candidate targets for specific ST1 detection and specific primers and hydrolysis probes were designed and evaluated. The analytical sensitivity and specificity of the seven primer and probe sets were evaluated on serially diluted DNA extracted from the reference strain CIP107629 and via qPCR applied on 200 characterized isolates. The diagnostic performance of the assay was evaluated on 142 culture-proven clinical samples from LD cases and a real-life investigation of a case cluster.ResultsOf seven qPCR assays that underwent analytical validation, one PCR target (lpp1868) showed higher sensitivity and specificity for ST1 and ST1-like strains. The diagnostic performance of the assay using respiratory samples corresponded to a sensitivity of 95% (19/20) (95% CI (75.1–99.9)) and specificity of 100% (122/122) (95% CI (97–100)). The ST1 PCR assay could link two out of three culture-negative hospitalized LD cases to ST1 during a real-time investigation.ConclusionUsing whole genome sequencing (WGS) data, we developed and validated a sensitive and specific qPCR assay for the detection of Lp1 belonging to the ST1 clonal complex by amplification of the lpp1868 gene. The ST1 qPCR is expected to deliver an added value for Lp control and prevention, in conjunction with other recently developed molecular assays.     

Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.