Molecular Testing of Serial Blood Specimens from Patients with Early Lyme Disease during Treatment Reveals Changing Coinfection with Mixtures of Borrelia burgdorferi Genotypes.

Borrelia burgdorferi is the etiological agent of Lyme disease. In the current study, we used direct-detection PCR and electrospray ionization mass spectrometry to monitor and genotype B. burgdorferi isolates from serially collected whole-blood specimens from patients clinically diagnosed with early Lyme disease before and during 21 days of antibiotic therapy. B. burgdorferi isolates were detected up to 3 weeks after the initiation of antibiotic treatment, with ratios of coinfecting B. burgdorferi genotypes changing over time.

Improved sensitivity for molecular detection of bacterial and Candida infections in blood.

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

Rapid PCR/ESI-MS-based molecular genotyping of Staphylococcus aureus from nasal swabs of emergency department patients.

BACKGROUND: A limitation of both culture-based and molecular methods of screening for staphylococcal infection is that current tests determine only the presence or absence of colonization with no information on the colonizing strain type. A technique that couples polymerase chain reaction to mass spectrometry (PCR/ESI-MS) has recently been developed and an assay validated to identify and genotype S. aureus and coagulase-negative staphylococci (CoNS). METHODS: This study was conducted to determine the rates, risk factors, and molecular genotypes of colonizing Staphylococcus aureus in adult patients presenting to an inner-city academic emergency department. Participants completed a structured questionnaire to assess hospital and community risks for infection with methicillin-resistant S. aureus (MRSA). Nasal swabs were analyzed by PCR/ESI-MS to identify and genotype S. aureus and CoNS. RESULTS: Of 200 patients evaluated, 59 were colonized with S. aureus; 27 of these were methicillin-resistant strains. Twenty-four of the 59 S. aureus carriers were co-colonized with a CoNS and 140 of the 200 patients were colonized exclusively with CoNS. The molecular genotypes of the 59 S. aureus strains were diverse; 21 unique molecular genotypes belonging to seven major clonal complexes were identified. Eighty-five of 200 patients carried strains with high-level mupirocin resistance. Of these eighty-five participants, 4 were colonized exclusively with S. aureus, 16 were co-colonized with S. aureus and CoNS, and 65 were colonized exclusively with CoNS. CONCLUSION: The prevalence of S. aureus and methicillin-resistant S. aureus colonization in a random sample of patients seeking care in Emergency Department was 29.5% and 13.5%, respectively. A substantial fraction of the S. aureus-colonized patients were co-colonized with CoNS and high-level mupirocin-resistant CoNS. Determining the molecular genotype of S. aureus during intake screening may prove valuable in the future if certain molecular genotypes become associated with increased infection risk.

Detection, identification, and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus PCR coupled with electrospray ionization/mass spectrometry.

As pulmonary fungal infections continue to increase due to an increasing number of immunocompromised patients, rapid detection and accurate identification of these fungal pathogens are critical. A broad fungal assay was developed by incorporating broad-range multilocus PCR amplification and electrospray ionization/mass spectrometry (PCR/ESI-MS) to detect and identify fungal organisms directly from clinical specimens. The aims of this study were to evaluate the performance of PCR/ESI-MS for detection, identification, and determination of the distribution of fungal organisms in bronchoalveolar lavage (BAL) fluid specimens. The BAL fluid specimens submitted for fungal culture at Vanderbilt University Medical Center between May 2005 and October 2011 were included. Cultures and identification were done using standard procedures. In addition, DNA was extracted from BAL fluid specimens, and fungal DNA amplification/identification were performed by PCR/ESI-MS. The results were compared with those of the standard cultures. A total of 691 nonduplicated BAL fluid specimens with sufficient leftover volume for molecular testing were evaluated using PCR/ESI-MS. Among them, 134 specimens (19.4%) were positive for fungi by both culture and PCR/ESI-MS testing. Of the dual-positive specimens, 125 (93.3%) were positive for Candida and Aspergillus species, with concordances between culture and PCR/ESI-MS results being 84 (67.2%) at the species level and 109 (87.2%) at the genus level. In addition, 243 (35.2%) and 30 (4.3%) specimens were positive only by PCR/ESI-MS or by culture, respectively (odds ratio [OR] = 11.95, 95% confidence interval [CI] = 7.90 to 18.17, P = 0.0000). Codetection of fungal organisms was noted in 23 (3.3%) specimens by PCR/ESI-MS, which was significantly higher than the 4 (0.6%) in which they were noted by culture (OR = 5.91, 95% CI = 1.93 to 20.27, P = 0.0002). Among 53 specimens in which cultures failed because of bacterial overgrowth, at least one fungus was identified in 26 specimens (47.3%) by PCR/ESI-MS. PCR/ESI-MS provides an advanced tool for rapid and sensitive detection, identification, and determination of the distribution of fungal organisms directly from BAL fluid specimens. Moreover, it detected fungal organisms in specimens in which cultures failed because of bacterial overgrowth. The clinical relevance of the significantly higher detection rate of fungal organisms by PCR/ESI-MS merits further investigation.

Broad-spectrum biosensor capable of detecting and identifying diverse bacterial and Candida species in blood.

We describe an assay which uses broad-spectrum, conserved-site PCR paired with mass spectrometry analysis of amplicons (PCR/electrospray ionization-mass spectrometry [ESI-MS]) to detect and identify diverse bacterial and Candida species in uncultured specimens. The performance of the assay was characterized using whole-blood samples spiked with low titers of 64 bacterial species and 6 Candida species representing the breadth of coverage of the assay. The assay had an average limit of detection of 100 CFU of bacteria or Candida per milliliter of blood, and all species tested yielded limits of detection between 20 and 500 CFU per milliliter. Over 99% of all detections yielded correct identifications, whether they were obtained at concentrations well above the limit of detection or at the lowest detectable concentrations. This study demonstrates the ability of broad-spectrum PCR/ESI-MS assays to detect and identify diverse organisms in complex natural matrices that contain high levels of background DNA.

PCR followed by electrospray ionization mass spectrometry for broad-range identification of fungal pathogens.

Invasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and to improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens. Using a database grounded by 60 ATCC reference strains, a total of 394 clinical fungal isolates (264 molds and 130 yeasts) were analyzed by PCR/ESI-MS; results were compared to phenotypic identification, and discrepant results were sequence confirmed. PCR/ESI-MS identified 81.4% of molds to either the genus or species level, with concordance rates of 89.7% and 87.4%, respectively, to phenotypic identification. Likewise, PCR/ESI-MS was able to identify 98.4% of yeasts to either the genus or species level, agreeing with 100% of phenotypic results at both the genus and species level. PCR/ESI-MS performed best with Aspergillus and Candida isolates, generating species-level identification in 94.4% and 99.2% of isolates, respectively. PCR/ESI-MS is a promising new technology for broad-range detection and identification of medically important fungal pathogens that cause invasive mycoses.

Rapid identification of Aspergillus terreus from bronchoalveolar lavage fluid by PCR and electrospray ionization with mass spectrometry.

We describe the application of PCR and electrospray-ionization with mass spectrometry (PCR/ESI-MS) to culture-negative bronchoalveolar lavage (BAL) fluid in order to identify septate hyphae noted by Gomori methenamine silver (GMS) staining of the fluid that was obtained from an immunocompromised woman with neutropenia following induction chemotherapy for treatment of acute myelogenous leukemia (AML). The patient was treated with empirical antifungal therapy, including intrathecal amphotericin B, while results of fungal cultures were pending. Ultimately, Aspergillus terreus, an amphotericin-resistant mold, was cultured from bilateral brain abscesses. PCR/ESI-MS correctly identified the mold.

Concurrent serotyping and genotyping of pneumococci by use of PCR and electrospray ionization mass spectrometry.

A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ionization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65- to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single- and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates.

Simultaneous identification of mycobacterial isolates to the species level and determination of tuberculosis drug resistance by PCR followed by electrospray ionization mass spectrometry.

Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs. The mechanisms of resistance for several drugs used to treat Mycobacterium tuberculosis are well understood and therefore should be amenable to determination by rapid molecular methods. We describe here the use of PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) in an assay that simultaneously determines INH and RIF resistance in Mycobacterium tuberculosis and identifies and determines the species of NTMs. The assay panel included 16 primer pairs in eight multiplexed reactions and was validated using a collection of 1,340 DNA samples from cultured specimens collected in the New York City area, the Republic of Georgia, and South Africa. Compared with phenotypic data, the PCR/ESI-MS assay had 89.3% sensitivity and 95.8% specificity in the determination of INH resistance and 96.3% sensitivity and 98.6% specificity in the determination of RIF resistance. Based on a set of 264 previously characterized liquid culture specimens, the PCR/ESI-MS method had 97.0% sensitivity and 99.9% specificity for determination of NTM identity. The assay also provides information on ethambutol, fluoroquinolone, and diarylquinoline resistance and lineage-specific polymorphisms, to yield highly discriminative digital signatures potentially suitable for epidemiology tracking.

Molecular characterization of drug-resistant Mycobacterium tuberculosis isolates circulating in China by multilocus PCR and electrospray ionization mass spectrometry.

We used multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to determine the genotype and drug resistance profiles for 96 Mycobacterium tuberculosis isolates circulating in regions of high and low tuberculosis (TB) endemicity in China. The dominant principal genetic group (PGG) circulating in China was PGG1, and drug-resistant gene mutations were more diversified in the region of low rather than high TB endemicity.

Evaluation of a PCR-electrospray ionization mass spectrometry platform for detection and identification of fungal pathogens directly from prospectively collected bronchoalveolar lavage specimens.

The incidence of invasive fungal infections is on the rise worldwide due to the growth of the immunocompromised population. We report here the use of a diagnostic assay that utilizes a universal extraction method, broad spectrum PCR amplification and analysis via electrospray ionization mass spectrometry (PCR/ESI-MS) to detect and identify more than 200 pathogenic fungi directly from bronchoalveolar lavage (BAL) specimens in less than 8 hours. In this study, we describe both analytical and clinical performance of the assay, when run with prospectively collected clinical BAL specimens. In 146 patients with probable and possible fungal infections defined by EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria, the PCR/ESI-MS assay demonstrated a sensitivity of 90.9% (95% CI: 76.4-96.9%) and a specificity of 82.3% (95% CI: 74.2-88.2%). This data demonstrates the utility of a non-culture based broad fungal targets molecular diagnostic tool for rapid and accurate diagnosis of invasive fungal infections in patients at risk of developing fungal diseases.

Occurrence, distribution, and origins of Streptococcus pneumoniae Serotype 6C, a recently recognized serotype.

The prevalence of Streptococcus pneumoniae serotype 6C, a recently recognized serotype that cross-reacts serologically with serotype 6A, was investigated. Isolates of serotype 6A in various collections were recovered, and serotype 6C was differentiated from 6A by multiplex PCR of DNA extracts by using appropriate primers. Antimicrobial susceptibility was performed by Clinical and Laboratory Standards Institute broth microdilution, and selected isolates were typed by pulsed-field gel electrophoresis, repetitive sequence-based PCR typing, and rapid multilocus sequence typing (MLST) by electrospray ionization mass spectrometry of PCR products. A total of 60 serotype 6C isolates were found: 30 of 122 Cleveland isolates collected from 1979 to 2007, 19 of 39 pediatric isolates collected nationwide in 2005 and 2006, and 11 pediatric isolates from Massachusetts collected in 2006 and 2007. Only four isolates were recovered prior to introduction of the conjugate pneumococcal vaccine in 2000; the earliest isolate was recovered in 1989. The sources of the isolates included blood (n = 5), the lower respiratory tract (n = 27), the sinus (n = 5), the ear (n = 2), and the nasopharynx (n = 18); isolates were recovered from 49 children and 11 adults. Pediatric isolates were found in all six major U.S. geographic regions. Antimicrobial susceptibility showed that 22 isolates were nonsusceptible to penicillin, macrolides, and trimethoprim-sulfamethoxazole, 8 had other resistance patterns, and 30 were fully susceptible. The three typing methods used showed similar clusters of up to eight isolates per cluster. MLST showed five clusters related to serotype 6A, two clusters related to serotype 6B, one cluster related to serotype 3, and one cluster related to serotype 34. This study documents the occurrence, nationwide distribution, diversity, likely origins, and increasing incidence after 2001 of this recently recognized serotype. Serotype 6C warrants consideration for addition to future conjugate pneumococcal vaccines.

Rapid determination of quinolone resistance in Acinetobacter spp.

In the treatment of serious bacterial infections, the rapid institution of appropriate antimicrobial chemotherapy may be lifesaving. Choosing the correct antibiotic or combination of antibiotics is becoming very important, as multidrug resistance is found in many pathogens. Using a collection of 75 well-characterized multidrug-resistant (MDR) Acinetobacter sp. isolates, we show that PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and base composition analysis of PCR amplification products can quickly and accurately identify quinolone resistance mediated by mutations in the quinolone resistance-determining regions of gyrA and parC, two essential housekeeping genes. Single point mutations detected by PCR/ESI-MS in parC (found in 55/75 of the isolates) and in gyrA (found in 66/75 of the isolates) correlated with susceptibility testing and sequencing. By targeting resistance determinants that are encoded by genes with highly conserved DNA sequences (e.g., gyrA and parC), we demonstrate that PCR/ESI-MS can provide critical information for resistance determinant identification and can inform therapeutic decision making in the treatment of Acinetobacter sp. infections.

Molecular genotyping of microbes by multilocus PCR and mass spectrometry: a new tool for hospital infection control and public health surveillance.

We describe a new technology for the molecular genotyping of microbes using a platform known commercially as the Ibis T5000. The technology couples multilocus polymerase chain reaction (PCR) to electrospray ionization/mass spectrometry (PCR/ESI-MS) and was developed to provide rapid, high-throughput, and precise digital analysis of either isolated colonies or original patient specimens on a platform suitable for use in hospital or reference diagnostic laboratories or public health settings. The PCR/ESI-MS method measures digital molecular signatures from microbes, enabling real-time epidemiological surveillance and outbreak investigation. This technology will facilitate understanding of the pathways by which infectious organisms spread and will enable appropriate interventions on a time frame not previously achievable.

Genotypic evolution of Acinetobacter baumannii strains in an outbreak associated with war trauma.

The Walter Reed Army Medical Center has experienced an influx of traumatically injured patients either infected or colonized with Acinetobacter baumannii. Using multilocus polymerase chain reaction (PCR) and mass spectrometry to genotype isolates, we found an atypical and evolving strain distribution, distinct from those found at nonmilitary hospitals in the United States.

Rapid identification of emerging infectious agents using PCR and electrospray ionization mass spectrometry.

Newly emergent infectious diseases are a global public health problem. The population dense regions of Southeast Asia are the epicenter of many emerging diseases, as evidenced by the outbreak of Nipah, SARS, avian influenza (H5N1), Dengue, and enterovirus 71 in this region in the past decade. Rapid identification, epidemiologic surveillance, and mitigation of transmission are major challenges in ensuring public health safety. Here we describe a powerful new approach for infectious disease surveillance that is based on polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry (ESI-MS) for accurate mass measurements of the PCR products, and base composition signature analysis to identify organisms in a sample. This approach is capable of automated analysis of more than 1,500 PCR reactions a day. It is applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens and will facilitate rapid characterization of known and emerging pathogens.

Recurrent structural RNA motifs, Isostericity Matrices and sequence alignments.

The occurrences of two recurrent motifs in ribosomal RNA sequences, the Kink-turn and the C-loop, are examined in crystal structures and systematically compared with sequence alignments of rRNAs from the three kingdoms of life in order to identify the range of the structural and sequence variations. Isostericity Matrices are used to analyze structurally the sequence variations of the characteristic non-Watson-Crick base pairs for each motif. We show that Isostericity Matrices for non-Watson-Crick base pairs provide important tools for deriving the sequence signatures of recurrent motifs, for scoring and refining sequence alignments, and for determining whether motifs are conserved throughout evolution. The systematic use of Isostericity Matrices identifies the positions of the insertion or deletion of one or more nucleotides relative to the structurally characterized examples of motifs and, most importantly, specifies whether these changes result in new motifs. Thus, comparative analysis coupled with Isostericity Matrices allows one to produce and refine structural sequence alignments. The analysis, based on both sequence and structure, permits therefore the evaluation of the conservation of motifs across phylogeny and the derivation of rules of equivalence between structural motifs. The conservations observed in Isostericity Matrices form a predictive basis for identifying motifs in sequences.

Broad-range survey of vector-borne pathogens and tick host identification of Ixodes ricinus from Southern Czech Republic.

Ixodes ricinus ticks are vectors of numerous human and animal pathogens. They are host generalists able to feed on more than 300 vertebrate species. The prevalence of tick-borne pathogens is influenced by host-vector-pathogen interactions that results in spatial distribution of infection risk. Broad-range polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was used to analyze 435 I. ricinus nymphs from four localities in the south of the Czech Republic for the species identification of tick-borne pathogens. Borrelia burgdorferi sensu lato spirochetes were the most common pathogen detected in the ticks; 21% of ticks were positive for a single genospecies and 2% were co-infected with two genospecies. Other tick-borne pathogens detected included Rickettsia helvetica (3.9%), R. monacensis (0.2%), Anaplasma phagocytophilum (2.8%), Babesia venatorum (0.9%), and Ba. microti (0.5%). The vertebrate host of the ticks was determined using PCR followed by reverse line blot hybridization from the tick's blood-meal remnants. The host was identified for 61% of ticks. DNA of two hosts was detected in 16% of samples with successful host identification. The majority of ticks had fed on artiodactyls (50.7%) followed by rodents (28.6%) and birds (7.8%). Other host species were wild boar, deer, squirrels, field mice and voles.

Fatal disseminated Rasamsonia infection in cystic fibrosis post-lung transplantation.

BACKGROUND: Disseminated fungal infections are a known serious complication in individuals with cystic fibrosis (CF) following orthotopic lung transplantation. Aspergillus fumigatus and Scedosporium species are among the more common causes of invasive fungal infection in this population. However, it is also important for clinicians to be aware of other emerging fungal species which may require markedly different antifungal therapies. CASE SUMMARY: We describe the first laboratory-documented case of a fatal disseminated fungal infection caused by Rasamsonia aegroticola in a 21-year-old female CF patient status post-bilateral lung transplantation, which was only identified post-mortem. Molecular analysis revealed the presence of the identical Rasamsonia strains in the patient's respiratory cultures preceding transplantation. DISCUSSION: We propose that the patient's disseminated fungal disease and death occurred as a result of recrudescence of Rasamsonia infection from her native respiratory system in the setting of profound immunosuppression post-operatively. Since Rasamsonia species have been increasingly recovered from the respiratory tract of CF patients, we further review the literature on these fungi and discuss their association with invasive fungal infections in the CF lung transplant host. CONCLUSION: Our report suggests Rasamsonia species may be important fungal pathogens that may have fatal consequences in immunosuppressed CF patients after solid organ transplantation.

The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

UNLABELLED: Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. DISCLAIMER: The IRIDICA BAC BSI Assay is not available in the United States.