Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.     

Direct detection of Streptococcus pneumoniae in positive blood cultures by real-time polymerase chain reaction

We developed a real-time polymerase chain reaction specific for Streptococcus pneumoniae to be applied directly from blood culture bottles without previous DNA extraction step. For the 128 blood culture bottles tested, the assay had 94% and 98.4% sensitivity and specificity, respectively. This assay provides rapid and accurate identification of this pathogen.     

Detection and identification of Candida spp. in human serum by LightCycler real-time polymerase chain reaction

The aim of this work was to develop LightCycler real-time polymerase chain reaction method to allow rapid detection and identification of Candida spp. in human serum with panfungal primers (internal transcribed spacer [ITS] and L18). Melting-curve analysis of the ITS sequences showed that each amplicon presented a specific melting point and enabled identification of 5 Candida spp. After parameters optimization, 58 sera were preliminary analyzed from 23 patients. For L18 primers, the LightCycler system enabled detection of DNA in 92% of patients with positive blood culture. These primers were not able to differentiate between species of Candida. By using ITS primers, the LightCycler system enabled detection of DNA in sera from 76.9% of patients with positive blood culture. With ITS primers, the species responsible for the infection was identified for 11 patients. These data revealed the LightCycler as a potential tool for early detection and identification of Candida.     

Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood

Universal 16S rRNA gene polymerase chain reaction (PCR) is a promising means of detecting bacteremia. Among other factors, the PCR reagents play a prominent role for obtaining a high sensitivity of detection. The reagents are ideally optimized with respect to the amplifying activity and absence of contaminating DNA. In this study, it was shown in a universal 16S rDNA real-time PCR assay that commercial PCR reagents can vary greatly among each other in these characters. Only 1 of the 5 reagents tested met the criteria of sensitive detection of pathogen DNA with a minimum of false-positive results. The reagent was validated by the detection of pathogens at low titers using bacterial DNA extracted from blood that was spiked with various Gram-positive and Gram-negative bacteria.     

Use of real-time polymerase chain reaction for identification of methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles

The utility of real-time polymerase chain reaction (RT-PCR) testing for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from positive blood culture bottles was evaluated. One hundred forty-two blood cultures showing Gram-positive cocci in clusters were detected. Each blood culture sample was tested for the presence of MRSA by PCR analysis (SmartCycler) via detection of the mecA and orfX genes. In parallel, they were plated on standard media for identification and characterization. PCR analysis directly from the blood culture bottle required a total time of 120 min (45 min for preparation and 75 min for the reaction). By comparison, conventional laboratory procedures required between 48 and 72 h. The overall test accuracy was 97% with a high positive likelihood ratio and a low negative likelihood ratio.     

细菌DNA的聚合酶链反应扩增及反相杂交初步分型

目的探讨聚合酶链反应（ＰＣＲ）加反相杂交技术在细菌ＤＮＡ检测中的应用。方法以１６ＳｒＲＮＡ基因为靶序列，设计引物及寡核苷酸探针，采用ＰＣＲ法加反相杂交检测标准菌株及临床标本的细菌ＤＮＡ。结果对２４株不同标准菌株进行ＰＣＲ扩增，均出现３７１ｂｐ长度的ＤＮＡ片段，敏感性试验可检测出１０－１２ｇ的细菌ＤＮＡ，与人类基因组ＤＮＡ、真菌及病毒无交叉反应；２２例血培养阳性标本及４例脑脊液培养阳性标本均扩增出３７１ｂｐ长度ＤＮＡ条带，反相杂交法区分革兰阳性／阴性细菌与培养结果相符。结论１６ＳｒＲＮＡ基因ＰＣＲ加反相杂交技术检测细菌ＤＮＡ，具有特异、敏感、快速、准确的特点，为细菌感染的临床诊断提供了科学的依据     

Cost-efficient sequence-specific priming–polymerase chain reaction screening for blood donors with rare phenotypes

BACKGROUND: Transfusion support for patients with irregular antibodies to red blood cell (RBC) antigens of high frequency may be hampered by lack of appropriate antigen-negative RBC units. Often, this perceived lack is due to the low number of typed donors. We developed a simple multiplex polymerase chain reaction (PCR) method to screen for donors with rare blood group phenotypes.
STUDY DESIGN AND METHODS: A multiplex PCR with sequence-specific priming predicting Yt^a, Co^a, Lu^b, and Kp^b antigens was developed based on a commercially available system (Extract-N-Amp, Sigma-Aldrich) that obviates the DNA purification step. PCR amplicons were analyzed by size fractionation in a 2 percent agarose gel. Samples representing rare phenotypes were identified by the lack of one of the four visible bands. Donors of blood phenotype O D– ccddee were screened.
RESULTS: Excluding the preparation of the reaction mixture and the gel, the whole procedure consisted of five pipetting steps. Hands-on time was 102 minutes for 91 donors. After optimization, interpretable results were obtained in 85 percent of samples without repetition. Among 3422 donors tested, 1 Kp(b–), 6 Co(a–), 10 Yt(a–), and 5 Lu(b–) donors were detected.
CONCLUSIONS: Multiplex PCR is a simple, versatile, and cost-efficient method for the screening for donors with rare phenotypes who may be identified by their genotype. If such donor screening is introduced on a broad basis, transfusion support for patients with anti-Co^a, anti-Yt^a, or anti-Lu^b will be considerably improved.     

Automated and simultaneous identification of microsatellite instability by fluorescence-based polymerase chain reaction (PCR) in four loci

Genomic instability is sometimes due to impairment of DNA repair systems, which results in a change in the number of microsatellite repeats in tumor cells, produced by slippage during DNA replication. Such abnormal repeats are manifested as microsatellite instability (MSI). We have devised a simple assay using four-color fluorescence for the detection of MSI by an automatic sequencer. Using this method, MSI and loss of heterozygosity (LOH) at four microsatellite loci can be identified simultaneously. We have also developed an algorithm and software for automated analysis of MSI and LOH with this method. Using our method for the detection of MSI in four microsatellite loci and the algorithm and software that we developed, 18 (94.7%) of 19 patients with hereditary nonpolyposis colorectal cancer (HNPCC), meeting the Amsterdam Minimum Criteria, were found to exhibit MSI.     

Direct identification of Yersinia enterocolitica in blood by polymerase chain reaction amplification

Primers based on the nucleotide sequence of the virF gene in the pYV plasmid and the chromosomal ail gene were used in polymerase chain reaction (PCR) amplifications to directly identify Yersinia enterocolitica in blood. Approximately 500 bacteria seeded into 100 microL of blood can be extracted and amplified by PCR to yield positive results. PCR analyses of seven Y. enterocolitica isolates previously implicated in blood contaminations showed that only one isolate harbored the plasmid-borne virF gene; however, all seven isolates were identified effectively by the PCR product amplified from the chromosomal gene. The PCR assay has the potential for use in the identification of Y. enterocolitica contamination in stored units of blood or in the rapid diagnosis of transfusion-related bacteremia caused by Y.     

应用PCR—SSP技术对粒细胞抗原NA进行基因分型

根据ＮＡ基因序列,合成ＮＡ特异性引物,通过ＰＣＲ－ＳＳＰ技术,特异性地扩增ＮＡ１和ＮＡ２,建立ＮＡ基因分型方法,同时对１２０例浙江汉族人群进行ＮＡ基因型的检测。结果表明ＰＣＲ－ＳＳＰ技术对ＮＡ的基因分型方法简单、翰林折汉族人群的ＮＡ基因型ＮＡ１为０．５２５,ＮＡ２为０．４７５。     

A novel real-time polymerase chain reaction for identification of Salmonella enterica subspecies enterica

Salmonella enterica subspecies enterica (subspecies I) causes the majority of infections in humans and homeothermic animals. We present a real-time polymerase chain reaction assay targeting the hilA gene that demonstrates 97.9% specificity and 99.9% sensitivity for rapid and reliable identification of subspecies I, offering savings in time and labor over traditional methods.     

Rapid identification of Bartonella henselae by real-time polymerase chain reaction in a patient with cat scratch disease

We report a localized submandibular lymph node infection in a patient with cat scratch disease. Directly performing real-time polymerase chain reaction assay on the biopsy sample, Bartonella henselae DNA was simultaneously detected and identified.     

Multicolor real-time polymerase chain reaction genotyping of six human platelet antigens using displacing probes

BACKGROUND: Several genotyping methods for six clinically relevant human platelet antigens (HPAs) have been reported. A four-color real-time polymerase chain reaction (PCR) method using displacing probes for genotyping of the six HPAs is described. STUDY DESIGN AND METHODS: Primers and four differently fluorophor-labeled displacing probes were designed and synthesized to detect single-nucleotide polymorphisms responsible for each of the HPA-1, -2, -3, -4, -5, and -15 genotypes. Two HPA systems were analyzed in a single PCR procedure. After validation with samples of known genotypes, a total of 150 blood samples from healthy donors were genotyped. The results were compared with PCR with sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), and/or direct DNA sequencing. The frequencies of each HPA allele were calculated. RESULTS: Unequivocal real-time PCR genotyping results were obtained with minimal manual manipulation and carryover contamination. All 150 blood samples were correctly genotyped as confirmed by PCR-SSP, PCR-RFLP, and/or direct DNA sequencing. The allelic frequencies of HPA-1 through -5 and -15 among the Chinese population in Xiamen were comparable with those previously reported with Chinese living in other territories. For each specimen, genotyping of all six HPA biallelic systems was achieved in three tubes of PCR within 90 minutes and with material cost of no more than $1. CONCLUSION: Genotyping of HPA with real-time PCR using displacing probes is more rapid and reliable compared with PCR-SSP and PCR-RFLP methods and is more affordable than existing real-time PCR-based HPA genotyping assays. Thus, our approach is more suitable for routine HPA analysis and ideal for both urgent clinical testing and high-throughput screening.     

NA gene frequencies in the German population, determined by polymerase chain reaction with sequence-specific primers

BACKGROUND: The granulocyte antigens NA1 and NA2 are often targets of granulocyte antibodies causing immune neutropenia. Currently, NA typing relies on the properties of the typing sera or antibodies and the techniques used. Therefore, the technique of polymerase chain reaction with sequence-specific primers (PCR-SSP) was adapted for DNA-based NA typing and was used for determining the NA gene frequencies in the German population. STUDY DESIGN AND METHODS: The genomic DNA of 160 unrelated healthy individuals was typed for NA1 and NA2 by PCR-SSP. In 60 granulocyte samples, the NA phenotype was additionally determined by the antigen capture assay and the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results. RESULTS: Results of the antigen capture assay and PCR-SSP correlated precisely, whereas nine individuals were typed heterozygous only by GIFT. The gene frequencies were 0.35 for NA1 and 0.65 for NA2. CONCLUSION: The NA2 gene is more frequent in the German population than the NA1 gene, as determined by genotyping using PCR-SSP. In contrast to GIFT, which showed an error rate for NA typing of 15 percent, PCR-SSP and the antigen-capture assay are more reliable methods of NA typing of granulocytes.     

A polymerase chain reaction (PCR) protocol for the specific detection of Chlamydia spp

The polymerase chain reaction is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. This technique was used to detect and differentiate Chlamydia trachomatis and Chlamydia psittaci in laboratory samples of infected McCoy cells. The polymerase chain reaction was shown to be both sensitive, detecting in the order of one chlamydial DNA molecule in 10(5) cells, and specific. No cross reaction (amplified product) was detected when a variety of mammalian cell and bacterial DNAs were used as template with the Chlamydia-specific oligonucleotide primers.     

A novel semiquantitative fluorescence-based multiplex polymerase chain reaction assay for rapid simultaneous detection of bacterial and parasitic pathogens from blood

A multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (T(m)). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals.     

聚合酶链反应法测定幽门螺杆菌的vacA基因型

以幽门螺杆菌ＤＮＡ为模板,采用聚合酶链反应设计了５个特异性寡核苷酸引物的一个共引物,分析检测了６２株幽门螺杆菌空泡形成细胞毒素基因Ｓ１ａ、Ｓ１ｂ、Ｍ１型和Ｍ２型,检测结果均为Ｓ１ａ／Ｍ２实验显示胃十二指肠疾病与空泡形成细胞毒素基因无相关关系。     

聚合酶链反应法测定幽门螺杆菌的vacA基因型

以幽门螺杆菌DNA为模板,采用聚合酶链反应设计了5个特异性寡核苷酸引物和一个公共引物,分析检测了62株幽门螺杆菌空泡形成细胞毒素基因S1a、S1b、M1型和M2型,检测结果均为S1a/M2型.实验显示胃十二指肠疾病与空泡形成细胞毒素基因无相关关系.     

A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood

Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples.