Bernard J. Wolff

Detection of Macrolide Resistance in Mycoplasma pneumoniae by Real-Time PCR and High-Resolution Melt Analysis

ABSTRACT Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides or azalides such as erythromycin or azithromycin. Recent studies have discovered the existence of macrolide-resistant strains within the population that have been mapped to mutations within the domain V region of the 23S rRNA gene. Currently, identification of these resistant strains relies on time-consuming and labor-intensive procedures such as restriction fragment length polymorphism, MIC studies, and sequence analysis. The current study reports two distinct real-time PCR assays that can detect the A2063G or A2064G base mutation (A2058G or A2059G by Escherichia coli numbering) conferring macrolide resistance. By subjecting the amplicon of the targeted domain V region of the 23S rRNA gene to a high-resolution melt curve analysis, macrolide-resistant strains can quickly be separated from susceptible strains. Utilizing this method, we screened 100 clinical isolates and found 5 strains to possess mutations conferring resistance. These findings were concordant with both sequencing and MIC data. This procedure was also used successfully to identify both susceptible and resistant genotypes in 23 patient specimens. These patient specimens tested positive for the presence of M. pneumoniae by a separate real-time PCR assay, although the bacteria could not be isolated by culture. This is the first report of a real-time PCR assay capable of detecting the dominant mutations that confer macrolide resistance on M. pneumoniae, and these assays may have utility in detecting resistant strains of other infectious agents. These assays may also allow for clinicians to select appropriate treatment options more rapidly and may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.

Genotyping of Mycoplasma pneumoniae isolates using real-time PCR and high-resolution melt analysis

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.

Genotyping of Chlamydophila psittaci by Real-Time PCR and High-Resolution Melt Analysis

ABSTRACT Human infection with Chlamydophila (Chlamydia) psittaci can lead to psittacosis, a disease that occasionally results in severe pneumonia and other medical complications. C. psittaci is currently grouped into seven avian genotypes: A through F and E/B. Serological testing, outer membrane protein A (ompA) gene sequencing, and restriction fragment length polymorphism analysis are currently used for distinguishing these genotypes. Although accurate, these methods are time-consuming and require multiple confirmatory tests. By targeting the ompA gene, a real-time PCR assay has been developed to rapidly detect and genotype C. psittaci by light-upon-extension chemistry and high-resolution melt analysis. Using this assay, we screened 169 animal specimens; 98 were positive for C. psittaci (71.4% genotype A, 3.1% genotype B, 4.1% genotype E, and 21.4% unable to be typed). This test may provide insight into the distribution of each genotype among specific hosts and provide epidemiological and epizootiological data in human and mammalian/avian cases. This diagnostic assay may also have veterinary applications during chlamydial outbreaks, particularly with respect to identifying the sources and tracking the movements of a particular genotype when multiple animal facilities are affected.

Association of Higher MERS-CoV Virus Load with Severe Disease and Death, Saudi Arabia, 2014.

More data are needed to determine whether modulation of virus load by therapeutic agents affects clinical outcomes.

Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection

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.

Identification of P1 Variants of Mycoplasma pneumoniae by Use of High-Resolution Melt Analysis

ABSTRACT 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 high-resolution melt genotyping assay to identify clinical variants which may provide greater insight into the genetic distribution of M. pneumoniae strains.

Detection and characterization of Mycoplasma pneumoniae during an outbreak of respiratory illness at a university

ABSTRACT An outbreak at a university in Georgia was identified after 83 cases of probable pneumonia were reported among students. Respiratory specimens were obtained from 21 students for the outbreak investigation. The TaqMan array card (TAC), a quantitative PCR (qPCR)-based multipathogen detection technology, was used to initially identify Mycoplasma pneumoniae as the causative agent in this outbreak. TAC demonstrated 100% diagnostic specificity and sensitivity compared to those of the multiplex qPCR assay for this agent. All M. pneumoniae specimens (n = 12) and isolates (n = 10) were found through genetic analysis to be susceptible to macrolide antibiotics. The 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 multilocus 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.

Chlamydia psittaci comparative genomics reveals intraspecies variations in the putative outer membrane and type III secretion system genes

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 tbl3SS genes that may partially explain differences noted in C. psittaci host infection and disease.

Dynamics of genome change among Legionella species

Legionella species inhabit freshwater and soil ecosystems where they parasitize protozoa. L. pneumonphila (LP) serogroup-1 (Lp1) is the major cause of Legionnaires’ Disease (LD), a life-threatening pulmonary infection that can spread systemically. The increased global frequency of LD caused by Lp and non-Lp species underscores the need to expand our knowledge of evolutionary forces underlying disease pathogenesis. Whole genome analyses of 43 strains, including all known Lp serogroups 1–17 and 17 emergent LD-causing Legionella species (of which 33 were sequenced in this study) in addition to 10 publicly available genomes, resolved the strains into four phylogenetic clades along host virulence demarcations. Clade-specific genes were distinct for genetic exchange and signal-transduction, indicating adaptation to specific cellular and/or environmental niches. CRISPR spacer comparisons hinted at larger pools of accessory DNA sequences in Lp than predicted by the pan-genome analyses. While recombination within Lp was frequent and has been reported previously, population structure analysis identified surprisingly few DNA admixture events between species. In summary, diverse Legionella LD–causing species share a conserved core-genome, are genetically isolated from each other, and selectively acquire genes with potential for enhanced virulence.

Molecular Characterization of Mycoplasma pneumoniae Infections in Two Rural Populations of Thailand from 2009 to 2012

ABSTRACT Studies on Mycoplasma pneumoniae in Thailand have focused on urban centers and have not included molecular characterization. In an attempt to provide a more comprehensive understanding of this organism, we conducted a systematic random sampling to identify 3,000 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 that all 141 tested to possess the genotype associated with macrolide susceptibility.