Last data update: Apr 29, 2024. (Total: 46658 publications since 2009)
Records 1-22 (of 22 Records) |
Query Trace: Wolff BJ[original query] |
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Genomic analysis of Chlamydia psittaci from a multistate zoonotic outbreak in two chicken processing plants
Wolff BJ , Waller JL , Benitez AJ , Gaines A , Conley AB , Rishishwar L , Chande AT , Morrison SS , Jordan IK , Diaz MH , Winchell JM . J Genomics 2023 11 40-44 Four Chlamydia psittaci isolates were recovered from clinical specimens from ill workers during a multistate outbreak at two chicken processing plants. Whole genome sequencing analyses revealed high similarity to C. psittaci genotype D. The isolates differed from each other by only two single nucleotide polymorphisms, indicating a common source. |
Multiplex Real-time PCR Assay for the Detection of all Chlamydia Species and Simultaneous Differentiation of C. psittaci and C. pneumoniae in Human Clinical Specimens.
Wolff BJ , Gaines A , Conley AB , Norris E , Rishishwar L , Chande AT , Yang E , Diaz MH , Winchell JM . Ann Lab Med 2023 43 (4) 375-380 We developed and assessed the performance of a new multiplex real-time PCR assay for the detection of all Chlamydia species and simultaneous differentiation of Chlamydia psittaci and Chlamydia pneumoniae-two important human respiratory pathogens-in human clinical specimens. Next-generation sequencing was used to identify unique targets to design real-time PCR assays targeting all Chlamydia species, C. psittaci, and C. pneumoniae. To validate the assay, we used a panel of 49 culture isolates comprising seven C. psittaci genotypes, eight C. pneumoniae isolates, seven other Chlamydia species, and 22 near-neighbor bacterial and viral isolates, along with 22 specimens from external quality assessment (EQA) panels and 34 nasopharyngeal and oropharyngeal swabs and cerebrospinal fluid, stool, and sputum specimens previously identified as positive or negative for C. psittaci or C. pneumoniae. The assays were 100% specific, with limits of detection of 7.64- 9.02 fg/μL. The assay results matched with historical assay results for all specimens, except for one owing to the increased sensitivity of the new C. psittaci assay; the results of the EQA specimens were 100% accurate. This assay may improve the timely and accurate clinical diagnosis of Chlamydia infections and provide a greater understanding of the burden of disease caused by these agents. |
Use of Real-Time PCR for Chlamydia psittaci Detection in Human Specimens During an Outbreak of Psittacosis - Georgia and Virginia, 2018.
McGovern OL , Kobayashi M , Shaw KA , Szablewski C , Gabel J , Holsinger C , Drenzek C , Brennan S , Milucky J , Farrar JL , Wolff BJ , Benitez AJ , Thurman KA , Diaz MH , Winchell JM , Schrag S . MMWR Morb Mortal Wkly Rep 2021 70 (14) 505-509 Psittacosis is typically a mild febrile respiratory illness caused by infection with the bacterium Chlamydia psittaci and usually transmitted to humans by infected birds (1). On average, 11 psittacosis cases per year were reported in the United States during 2000-2017. During August-October 2018, the largest U.S. psittacosis outbreak in 30 years (82 cases identified*) occurred in two poultry slaughter plants, one each in Virginia and Georgia, that shared source farms (2). CDC used C. psittaci real-time polymerase chain reaction (PCR) to test 54 human specimens from this outbreak. This was the largest number of human specimens from a single outbreak ever tested for C. psittaci using real-time PCR, which is faster and more sensitive than commercially available serologic tests. This represented a rare opportunity to assess the utility of multiple specimen types for real-time PCR detection of C. psittaci. C. psittaci was detected more frequently in lower respiratory specimens (59% [10 of 17]) and stool (four of five) than in upper respiratory specimens (7% [two of 28]). Among six patients with sputum and nasopharyngeal swabs tested, C. psittaci was detected only in sputum in five patients. Cycle threshold (Ct) values suggested bacterial load was higher in lower respiratory specimens than in nasopharyngeal swabs. These findings support prioritizing lower respiratory specimens for real-time PCR detection of C. psittaci. Stool specimens might also have utility for diagnosis of psittacosis. |
Antibody Responses after Classroom Exposure to Teacher with Coronavirus Disease, March 2020.
Brown NE , Bryant-Genevier J , Bandy U , Browning CA , Berns AL , Dott M , Gosciminski M , Lester SN , Link-Gelles R , Quilliam DN , Sejvar J , Thornburg NJ , Wolff BJ , Watson J . Emerg Infect Dis 2020 26 (9) 2263-5 After returning from Europe to the United States, on March 1, 2020, a symptomatic teacher received positive test results for severe acute respiratory syndrome coronavirus 2. Of the 21 students exposed to the teacher in the classroom, serologic results suggested past infection for 2. Classroom contact may result in virus transmission. |
Evaluation of Commercial Molecular Diagnostic Methods for the Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae .
Leal SMJr , Totten AH , Xiao L , Crabb DM , Ratliff A , Duffy LB , Fowler KB , Mixon E , Winchell JM , Diaz MH , Benitez AJ , Wolff BJ , Qin X , Tang YW , Gonzalez M , Selvarangan R , Hong T , Brooks E , Dallas S , Atkinson TP , Zheng X , Dien Bard J , Waites KB . J Clin Microbiol 2020 58 (6) We evaluated six commercial molecular tests targeting M. pneumoniae: the BioFire FilmArray Respiratory Panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex Respiratory Pathogen Panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB Research Use Only Polymerase Chain Reaction (PCR), and the SpeeDx Resistance Plus MP. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed-positives for M. pneumoniae The highest clinical sensitivities were found with the InGenius (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6%, 64.6%, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (p < 0.05). Specificities of all assays were 99.5 - 100%. The Resistance Plus MP detected macrolide resistance in 27/33 specimens resulting in a sensitivity of 81.8%. This study provides the first large scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of variable test performance on patient outcome. |
Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.
Diaz MH , Waller JL , Theodore MJ , Patel N , Wolff BJ , Benitez AJ , Morris T , Raghunathan PL , Breiman RF , Whitney CG , Blau DM , Winchell JM . Clin Infect Dis 2019 69 S311-s321 Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30-45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories. |
Epidemiology and Molecular Identification and Characterization of Mycoplasma pneumoniae, South Africa, 2012-2015.
Carrim M , Wolter N , Benitez AJ , Tempia S , du Plessis M , Walaza S , Moosa F , Diaz MH , Wolff BJ , Treurnicht FK , Hellferscee O , Dawood H , Variava E , Cohen C , Winchell JM , von Gottberg A . Emerg Infect Dis 2018 24 (3) 506-513 During 2012-2015, we tested respiratory specimens from patients with severe respiratory illness (SRI), patients with influenza-like illness (ILI), and controls in South Africa by real-time PCR for Mycoplasma pneumoniae, followed by culture and molecular characterization of positive samples. M. pneumoniae prevalence was 1.6% among SRI patients, 0.7% among ILI patients, and 0.2% among controls (p<0.001). Age <5 years (adjusted odd ratio 7.1; 95% CI 1.7-28.7) and HIV infection (adjusted odds ratio 23.8; 95% CI 4.1-138.2) among M. pneumonia-positive persons were associated with severe disease. The detection rate attributable to illness was 93.9% (95% CI 74.4%-98.5%) in SRI patients and 80.7% (95% CI 16.7%-95.6%) in ILI patients. The hospitalization rate was 28 cases/100,000 population. We observed the macrolide-susceptible M. pneumoniae genotype in all cases and found P1 types 1, 2, and a type 2 variant with multilocus variable number tandem repeat types 3/6/6/2, 3/5/6/2, and 4/5/7/2. |
Development of a multiplex TaqMan real-time PCR assay for the detection of Chlamydia psittaci and Chlamydia pneumoniae in human clinical specimens.
Wolff BJ , Morrison SS , Winchell JM . Diagn Microbiol Infect Dis 2017 90 (3) 167-170 Diagnosis of Chlamydia psittaci and Chlamydia pneumoniae infections has traditionally relied on serological assays. We developed a multiplex real-time PCR assay for detection of C. psittaci, C. pneumoniae and an internal control. Results of this assay demonstrated 100% concordance compared to results of previously tested human clinical specimens. |
Molecular characterization of Mycoplasma pneumoniae infections in two rural populations of Thailand from 2009-2012.
Whistler T , Sawatwong P , Diaz MH , Benitez AJ , Wolff BJ , Sapchookul P , Thamthitiwat S , Winchell JM . J Clin Microbiol 2017 55 (7) 2222-2233 Studies on Mycoplasma pneumoniae in Thailand have focused on urban centers and have not included the molecular characterization. In an attempt to provide a more comprehensive understanding of this organism, we conducted a systematic random sampling to identify 3000 nasopharyngeal swab specimens, collected from January 2009 through July 2012 during population-based surveillance for influenza-like illness in two rural provinces. M. pneumoniae was detected by real-time PCR in 175 (5.8%) specimens. Genotyping was performed using the major adhesion protein (P1) and multilocus variable-number tandem-repeat analysis (MLVA). Of the 157 specimens typed, 97 were P1 type 1 and 60 were P1 type 2. Six different MLVA profiles were identified in 149 specimens with 4/5/7/2 (40%) and 3/5/6/2 (26%) predominating. There was no discrete seasonality to M. pneumoniae infections. Examination of the 23S rRNA sequence for known polymorphisms conferring macrolide resistance revealed all 141 tested to possess the genotype associated with macrolide susceptibility. |
Comprehensive bioinformatics analysis of Mycoplasma pneumoniae genomes to investigate underlying population structure and type-specific determinants.
Diaz MH , Desai HP , Morrison SS , Benitez AJ , Wolff BJ , Caravas J , Read TD , Dean D , Winchell JM . PLoS One 2017 12 (4) e0174701 Mycoplasma pneumoniae is a significant cause of respiratory illness worldwide. Despite a minimal and highly conserved genome, genetic diversity within the species may impact disease. We performed whole genome sequencing (WGS) analysis of 107 M. pneumoniae isolates, including 67 newly sequenced using the Pacific BioSciences RS II and/or Illumina MiSeq sequencing platforms. Comparative genomic analysis of 107 genomes revealed >3,000 single nucleotide polymorphisms (SNPs) in total, including 520 type-specific SNPs. Population structure analysis supported the existence of six distinct subgroups, three within each type. We developed a predictive model to classify an isolate based on whole genome SNPs called against the reference genome into the identified subtypes, obviating the need for genome assembly. This study is the most comprehensive WGS analysis for M. pneumoniae to date, underscoring the power of combining complementary sequencing technologies to overcome difficult-to-sequence regions and highlighting potential differential genomic signatures in M. pneumoniae. |
Complete Genome Sequence of Mycoplasma pneumoniae Type 2 Reference Strain FH Using Single-Molecule Real-Time Sequencing Technology.
Desai HP , Morrison SS , Diaz MH , Benitez AJ , Wolff BJ , Winchell JM . Genome Announc 2017 5 (8) Mycoplasma pneumoniae type 2 strain FH was previously sequenced with Illumina (FH-Illumina) and 454 (FH-454) technologies according to Xiao et al. (2015) and Krishnakumar et al. (2010). Comparative analyses revealed differences in genomic content between these sequences, including a 6-kb region absent from the FH-454 submission. Here, we present a complete genome sequence of FH sequenced with the Pacific Biosciences RSII platform. |
Development of a multiplex taqMan real-time PCR assay for typing of Mycoplasma pneumoniae based on type-specific indels identified through whole genome sequencing.
Wolff BJ , Benitez AJ , Desai HP , Morrison SS , Diaz MH , Winchell JM . Diagn Microbiol Infect Dis 2016 87 (3) 203-206 We developed a multiplex real-time PCR assay for simultaneously detecting M. pneumoniae and typing into historically-defined P1 types. Typing was achieved based on the presence of short type-specific indels identified through whole genome sequencing. This assay was 100% specific compared to existing methods and may be useful during epidemiologic investigations. |
Improved detection of respiratory pathogens using high-quality sputum with TaqMan Array Card technology
Wolff BJ , Bramley AM , Thurman KA , Whitney CG , Whitaker B , Self WH , Arnold SR , Trabue C , Wunderink RG , McCullers J , Edwards KM , Jain S , Winchell JM . J Clin Microbiol 2016 55 (1) 110-121 New diagnostic platforms often use naso- or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multi-pathogen testing to high-quality sputum specimens to determine if more pathogens could be identified relative to NP/OP swabs. Children (<18 years old) and adults hospitalized with CAP were enrolled over 2.5 years through the Etiology of Pneumonia in the Community (EPIC) study. NP/OP specimens with matching high-quality sputum (defined as ≤10 epithelial cells/low power field [lpf] and ≥25 white blood cells/lpf or a q-score definition of 2+) were tested by TaqMan Array Card (TAC), a multi-pathogen real-time polymerase chain reaction (PCR) detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had ≥1 pathogen detected compared with NP/OP specimens in children (93% v. 68%, P<0.0001) and adults (88% v. 61%; P<0.0001); for each pathogen targeted, crossing threshold (Ct) values were earlier in sputum. Both bacterial (361 vs. 294) and viral detections (245 vs. 140) were more common in sputum versus NP/OP specimens, respectively, in both children and adults. When available, high-quality sputum may be useful for testing in hospitalized CAP patients. |
Ability of device to collect bacteria from cough aerosols generated by adults with cystic fibrosis
Ku DN , Ku SK , Helfman B , McCarty NA , Wolff BJ , Winchell JM , Anderson LJ . F1000Res 2016 5 1920 Background: Identifying lung pathogens and acute spikes in lung counts remain a challenge in the treatment of patients with cystic fibrosis (CF). Bacteria from the deep lung may be sampled from aerosols produced during coughing. Methods: A new device was used to collect and measure bacteria levels from cough aerosols of patients with CF. Sputum and oral specimens were also collected and measured for comparison. Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus mitis were detected in specimens using Real-Time Polymerase Chain Reaction (RT-PCR) molecular assays. Results: Twenty adult patients with CF and 10 healthy controls participated. CF related bacteria (CFRB) were detected in 13/20 (65%) cough specimens versus 15/15 (100%) sputum specimens. Commensal S. mitis was present in 0/17 (0%, p=0.0002) cough specimens and 13/14 (93%) sputum samples. In normal controls, no bacteria were collected in cough specimens but 4/10 (40%) oral specimens were positive for CFRB. Conclusions: Non-invasive cough aerosol collection may detect lower respiratory pathogens in CF patients, with similar specificity and sensitivity to rates detected by BAL, without contamination by oral CFRB or commensal bacteria. |
Isothermal detection of Mycoplasma pneumoniae directly from respiratory clinical specimens
Petrone BL , Wolff BJ , DeLaney AA , Diaz MH , Winchell JM . J Clin Microbiol 2015 53 (9) 2970-6 Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia (CAP) across patient populations of all ages. We have developed a loop-mediated isothermal amplification (LAMP) assay that enables rapid, low-cost detection of M. pneumoniae from nucleic acid extracts and directly from various respiratory specimen types. The assay implements calcein to facilitate simple visual readout of positive results in approximately one hour, making it ideal for use in primary care facilities and resource-poor settings. The analytical sensitivity of the assay was determined to be 100 fg by testing serial dilutions of target DNA ranging from 1 ng to 1 fg per reaction, and no cross-reactivity was observed against 17 other Mycoplasma species, 27 common respiratory agents, or human DNA. We demonstrate the utility of this assay by testing nucleic acid extracts (n=252) and un-extracted respiratory specimens (n=72) collected during M. pneumoniae outbreaks and sporadic cases occurring in the United States from February 2010 to January 2014. The sensitivity of the LAMP assay was 88.5% when tested on extracted nucleic acid and 82.1% when evaluated on un-extracted clinical specimens, compared to a validated real-time PCR test. Further optimization and improvements to this method may lead to a rapid, cost-efficient laboratory test for M. pneumoniae detection that is more widely available to primary care facilities, ultimately facilitating prompt detection and appropriate response to potential M. pneumoniae outbreaks and clusters within the community. |
Chlamydia psittaci Comparative Genomics Reveals Intraspecies Variations in the Putative Outer Membrane and Type III Secretion System Genes.
Wolff BJ , Morrison SS , Pesti D , Ganakammal SR , Srinivasamoorthy G , Changayil S , Weil MR , MacCannell D , Rowe L , Frace M , Ritchie BW , Dean D , Winchell J . Microbiology (Reading) 2015 161 (7) 1378-91 Chlamydia psittaci is an obligate intracellular bacterium that can cause significant disease among a broad range of hosts. In humans, this organism may cause psittacosis, a respiratory disease that can spread to involve multiple organs, and in rare untreated cases may be fatal. There are ten known genotypes based on sequencing the major outer membrane protein gene, ompA, of C. psittaci. Each genotype has overlapping host preferences and virulence characteristics. Recent studies have compared C. psittaci among other members of the Chlamydiaceae family and showed that this species frequently switches hosts and has undergone multiple genomic rearrangements. In this study, we sequenced five genomes of C. psittaci strains representing four genotypes, A, B, D and E. Due to the known association of the type III secretion system (T3SS) and polymorphic outer membrane proteins (pmps) with host tropism and virulence potential, we performed a comparative analysis of these elements among these five strains along with a representative genome from each of the remaining six genotypes previously sequenced. We found significant genetic variation in the pmps and T3SS genes that may partially explain differences noted in C. psittaci host infection and disease. |
Detection and characterization of Mycoplasma pneumoniae during an outbreak of respiratory illness at a university.
Waller JL , Diaz MH , Petrone B , Benitez AJ , Wolff BJ , Edison L , Tobin-D'Angelo M , Moore A , Martyn A , Dishman H , Drenzek CL , Turner K , Hicks LA , Winchell JM . J Clin Microbiol 2013 52 (3) 849-53 An outbreak at a university in Georgia was identified after eighty-three cases of probable pneumonia were reported among students. Respiratory specimens were obtained from 21 students for an outbreak investigation. The TaqMan Array Card (TAC), a qPCR-based multi-pathogen detection technology, was used to initially identify M. pneumoniae as the causative agent in this outbreak. TAC demonstrated 100% diagnostic specificity and sensitivity when compared to the multiplex qPCR assay for this agent. All M. pneumoniae specimens (n=12) and isolates (n=10) were found to be susceptible to macrolide antibiotics through genetic analysis. Strain diversity of M. pneumoniae associated with this outbreak setting was identified using a variety of molecular typing procedures, resulting in two P1 genotypes (types 1 (60%) and 2 (40%)) and seven different Multi-Locus Variable-Number Tandem-Repeat analysis (MLVA) profiles. Continued molecular typing of this organism, particularly during outbreaks, may enhance the current understanding of the epidemiology of M. pneumoniae and may ultimately lead to a more effective public health response. |
Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection.
Diaz MH , Waller JL , Napoliello RA , Islam MS , Wolff BJ , Burken DJ , Holden RL , Srinivasan V , Arvay M , McGee L , Oberste MS , Whitney CG , Schrag SJ , Winchell JM , Saha SK . PLoS One 2013 8 (6) e66183 Identification of etiology remains a significant challenge in the diagnosis of infectious diseases, particularly in resource-poor settings. Viral, bacterial, and fungal pathogens, as well as parasites, play a role for many syndromes, and optimizing a single diagnostic system to detect a range of pathogens is challenging. The TaqMan Array Card (TAC) is a multiple-pathogen detection method that has previously been identified as a valuable technique for determining etiology of infections and holds promise for expanded use in clinical microbiology laboratories and surveillance studies. We selected TAC for use in the Aetiology of Neonatal Infection in South Asia (ANISA) study for identifying etiologies of severe disease in neonates in Bangladesh, India, and Pakistan. Here we report optimization of TAC to improve pathogen detection and overcome technical challenges associated with use of this technology in a large-scale surveillance study. Specifically, we increased the number of assay replicates, implemented a more robust RT-qPCR enzyme formulation, and adopted a more efficient method for extraction of total nucleic acid from blood specimens. We also report the development and analytical validation of ten new assays for use in the ANISA study. Based on these data, we revised the study-specific TACs for detection of 22 pathogens in NP/OP swabs and 12 pathogens in blood specimens as well as two control reactions (internal positive control and human nucleic acid control) for each specimen type. The cumulative improvements realized through these optimization studies will benefit ANISA and perhaps other studies utilizing multiple-pathogen detection approaches. These lessons may also contribute to the expansion of TAC technology to the clinical setting. |
Multilocus variable-number tandem-repeat analysis of Mycoplasma pneumoniae clinical isolates from 1962 to the present: a retrospective study.
Benitez AJ , Diaz MH , Wolff BJ , Pimentel G , Njenga MK , Estevez A , Winchell JM . J Clin Microbiol 2012 50 (11) 3620-6 In this study we evaluated a recently developed multi-locus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The method is based on GeneScan analysis of five VNTR loci throughout the genome which define a specific genotype based on the number of tandem repeats within each locus. A retrospective analysis of 154 M. pneumoniae clinical isolates collected over the last 50 years and a limited (n=4) number of M. pneumoniae-positive primary specimens acquired by CDC was performed using MLVA. Eighteen distinct VNTR types were identified including two previously unidentified VNTR types. Isolates from several M. pneumoniae community outbreaks within the United States were also analyzed to examine clonality of a specific MLVA type. Observed in vitro variability of the Mpn1 VNTR locus prompted further analysis which showed multiple insertions or deletions of tandem repeats within this locus for a number of specimens and isolates. To our knowledge this is the first report showing variation within the Mpn 1 locus, thus affecting precise and reliable classification using the current MLVA typing system. The superior discriminatory capability of MLVA provides a powerful tool for greater resolution of M. pneumoniae strains and could be useful during outbreaks and epidemiological investigations. |
Rapid identification and discrimination of Brucella isolates using real-time PCR and high resolution melt analysis
Winchell JM , Wolff BJ , Tiller R , Bowen M , Hoffmaster A . J Clin Microbiol 2010 48 (3) 697-702 Definitive identification of Brucella species remains a challenge due to the high degree of genetic homology shared within the genus. We report the development of a molecular technique which utilizes real-time PCR followed by high resolution melt (HRM) curve analysis to reliably type members of this genus. Using a panel of seven primer sets, we have tested 153 Brucella spp. isolates with >99% accuracy when compared to traditional techniques. This assay provides a useful diagnostic tool that can rapidly type Brucella spp. isolates and has the potential to detect novel species. This approach may also prove helpful for clinical, epidemiological and veterinary investigations. |
Genotyping of Mycoplasma pneumoniae isolates using real-time PCR and high-resolution melt analysis
Schwartz SB , Thurman KA , Mitchell SL , Wolff BJ , Winchell JM . Clin Microbiol Infect 2009 15 (8) 756-62 Mycoplasma pneumoniae is an important respiratory pathogen, accounting for up to 25% of community-acquired pneumonia, and is a common cause of hospitalized pneumonia in otherwise healthy adults and children. Mycoplasma pneumoniae isolates can be classified into two main genomic groups (type 1 and type 2) based on sequence variation within the gene encoding the major adhesion molecule P1. Although numerous publications have described real-time PCR assays for the detection of M. pneumoniae, none has been able to discriminate the two genomic types. Here, a real-time PCR assay that can distinguish each type of M. pneumoniae utilizing high-resolution melt-curve analysis is reported. Using this method, 102 isolates obtained from patients from 1965 to the present, including those from recent outbreaks, were typed along with reference strains M129 (type 1) and FH (type 2). The results show that 55 isolates (54%) can be classified as type 1 and 47 isolates (46%) as type 2, and 100% correlation was demonstrated when compared with a standard PCR-restriction fragment length polymorphism typing procedure. Typing of isolates obtained from recent outbreaks in the USA has revealed the presence of both types. This assay provides a rapid, reliable and convenient method for typing M. pneumoniae isolates and may be useful for surveillance purposes and epidemiological investigations, and may provide insight into the biology of M. pneumoniae distribution within populations. |
Identification of P1 variants of Mycoplasma pneumoniae using High Resolution Melt Analysis
Schwartz SB , Mitchell SL , Thurman KA , Wolff BJ , Winchell JM . J Clin Microbiol 2009 47 (12) 4117-20 Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia. Although two genetically distinct types of M. pneumoniae are known, variants of each also exist. We used a real-time PCR HRM genotyping assay to identify clinical variants which may provide greater insight into the genetic distribution of M. pneumoniae strains. |
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