Clinical chorioamnionitis at term X: microbiology, clinical signs, placental pathology, and neonatal bacteremia - implications for clinical care.

OBJECTIVES: Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5-12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia. METHODS: This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration ≥2.6 ng/mL. RESULTS: (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40-58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases. CONCLUSIONS: Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood.

Performance of PCR/Electrospray Ionization-Mass Spectrometry on Whole Blood for Detection of Bloodstream Microorganisms in Patients with Suspected Sepsis.

Blood culture (BC) often fails to detect bloodstream microorganisms in sepsis. However, molecular diagnostics hold great potential. The molecular method PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) can detect DNA from hundreds of different microorganisms in whole blood. The aim of the present study was to evaluate the performance of this method in a multicenter study including 16 teaching hospitals in the United States (n = 13) and Europe (n = 3). First, on testing of 2,754 contrived whole blood samples, with or without spiked microorganisms, PCR/ESI-MS produced 99.1% true-positive and 97.2% true-negative results. Second, among 1,460 patients with suspected sepsis (sepsis-2 definition), BC and PCR/ESI-MS on whole blood were positive in 14.6% and 25.6% of cases, respectively, with the following result combinations: BC positive and PCR/ESI-MS negative, 4.3%; BC positive and PCR/ESI-MS positive, 10.3%; BC negative and PCR/ESI-MS positive, 15.3%; and BC negative and PCR/ESI-MS negative, 70.1%. Compared with BC, PCR/ESI-MS showed the following sensitivities (coagulase-negative staphylococci not included): Gram-positive bacteria, 58%; Gram-negative bacteria, 78%; and Candida species, 83%. The specificities were >94% for all individual species. Patients who had received prior antimicrobial medications (n = 603) had significantly higher PCR/ESI-MS positivity rates than patients without prior antimicrobial treatment-31% versus 22% (P < 0.0001)-with pronounced differences for Gram-negative bacteria and Candida species. In conclusion, PCR/ESI-MS showed excellent performance on contrived samples. On clinical samples, it showed high specificities, moderately high sensitivities for Gram-negative bacteria and Candida species, and elevated positivity rates during antimicrobial treatment. These promising results encourage further development of molecular diagnostics to be used with whole blood for detection of bloodstream microorganisms in sepsis.

Limited diagnostic possibilities for bloodstream infections with broad-range methods: A promising PCR/electrospray ionization-mass spectrometry platform is no longer available.

Fast and accurate detection of causative agents of bloodstream infections remains a challenge of today's microbiology. We compared the performance of cutting-edge technology based on polymerase chain reaction coupled with electrospray ionization-mass spectrometry (PCR/ESI-MS) with that of conventional broad-range 16S rRNA PCR and blood culture to address the current diagnostic possibilities for bloodstream infections. Of 160 blood samples tested, PCR/ESI-MS revealed clinically meaningful microbiological agents in 47 samples that were missed by conventional diagnostic approaches (29.4% of all analyzed samples). Notably, PCR/ESI-MS shortened the time to positivity of the blood culture-positive samples by an average of 34 hr. PCR/ESI-MS technology substantially improved current diagnostic tools and represented an opportunity to make bloodstream infections diagnostics sensitive, accurate, and timely with a broad spectrum of microorganisms covered.

Molecular diagnosis of bloodstream infections in onco-haematology patients with PCR/ESI-MS technology.

OBJECTIVES: Onco-haematological patients are prone to develop infections, and antibiotic prophylaxis may lead to negative blood cultures. Thus, the microbiological diagnosis and subsequent administration of a targeted antimicrobial therapy is often difficult. The goal of this study was to evaluate the usefulness of IRIDICA (PCR/ESI-MS technology) for the molecular diagnosis of bloodstream infections in this patient group. METHODS: A total of 463 whole blood specimens from different sepsis episodes in 429 patients were analysed using the PCR/ESI-MS platform, comparing the results with those of blood culture and other clinically relevant information. RESULTS: The sensitivity of PCR/ESI-MS by specimen (excluding polymicrobial infections, n = 25) in comparison with blood culture was 64.3% overall, 69.0% in oncological patients, and 59.3% in haematological patients. When comparing with a clinical infection criterion, overall sensitivity rose to 74.7%, being higher in oncological patients (80.0%) than in haematological patients (67.7%). Thirty-one microorganisms isolated by culture were not detected by IRIDICA, whereas 42 clinically relevant pathogens not isolated by culture were detected moleculary. CONCLUSIONS: PCR/ESI-MS offers a reliable identification of pathogens directly from whole blood. While additional studies are needed to confirm our findings, the system showed a lower sensitivity in onco-haematological patients in comparison with previously reported results in patients from the Intensive Care Unit.

Direct detection of Anaplasma phagocytophilum by polymerase chain reaction followed by electrospray ionization mass spectrometry from human blood.

Bacterial pathogens not detectable via commercial blood culture assays represent an important challenge for infectious disease physicians, in particular if clinical symptoms of the illness are non-specific. In this report, Anaplasma phagocytophilum was detected directly in a peripheral blood sample from a febrile patient reporting a tick bite. This was done using a commercial system based on PCR followed by electrospray ionization mass spectrometry (ESI-MS). The diagnosis of a human granulocytic anaplasmosis infection was established using this diagnostic methodology for the first time. Human granulocytic anaplasmosis is a neglected zoonotic disease in Europe. Its seroprevalence is similar in North America and Europe, but in contrast to the USA, it is rarely diagnosed in the old world. PCR followed by ESI-MS is a novel, complex, but highly promising diagnostic methodology for the rapid assessment of rare or exotic pathogens, including intracellular bacteria.

Rapid detection and ruling out of neonatal sepsis by PCR coupled with Electrospray Ionization Mass Spectrometry (PCR/ESI-MS).

BACKGROUND: Sepsis is an important cause of morbidity and mortality in neonates and clinicians are typically required to administer empiric antibiotics while waiting for blood culture results. However, prolonged and inappropriate use of antibiotics is associated with various complications and adverse events. Better tools to rapidly rule out bacterial infections are therefore needed. AIMS: We aimed to assess the negative predictive value of PCR coupled with Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) compared to conventional blood cultures in neonatal sepsis. STUDY DESIGN: Prospective observational study. SUBJECTS: All consecutive neonates (<28days old) with clinical suspicion of sepsis. Samples for PCR/ESI-MS analysis were collected at the same time as samples for the blood culture, before the initiation of antibiotics. OUTCOME MEASURES: Our primary objective was to evaluate the negative predictive value of PCR/ESI-MS for the detection of bacteria in the bloodstream of newborns with suspected sepsis. Our secondary objective was the evaluation of the sensitivity, specificity and positive predictive value of the PCR/ESI-MS in such a neonatal population. RESULTS: We analysed 114 samples over 14months. The median age and weight were 32weeks+3days and 1840g, respectively. Two patients had negative PCR/ESI-MS results, but positive blood cultures. Overall, the negative predictive value was 98% (95%CI: 92% to 100%). CONCLUSIONS: Based on these results, PCR/ESI-MS analysis of blood samples of neonates with suspected sepsis appears to have a very good negative predictive value when compared to blood cultures as gold standard. This novel test might allow for early reassessment of the need for antibiotics.

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.

Emerging methodologies for pathogen identification in positive blood culture testing.

Bloodstream infections (BSIs) represent a major cause of death in developed countries and are associated with long-term loss of functions. Blood culture remains the gold standard for BSI diagnosis, as it is easy to perform and displays a good analytical sensitivity. However, its major drawback remains the long turnaround time, which can result in inappropriate therapy, fall of survival rate, emergence of antibiotic resistance and increase of medical costs. Over the last 10 years, molecular tools have been the alternative to blood cultures, allowing early identification of pathogens involved in sepsis, as well detection of critical antibiotic resistance genes. Besides, the advent of MALDI-TOF revolutionized practice in routine microbiology significantly reduced the time to result. Reviewed here are recent improvements in early BSI diagnosis and these authors' view for the future is presented, including innovative high-throughput technologies.

Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood.

When a bloodstream infection (BSI) is suspected, most of the laboratory results-biochemical and haematologic-are available within the first hours after hospital admission of the patient. This is not the case for diagnostic microbiology, which generally takes a longer time because blood culture, which is to date the reference standard for the documentation of the BSI microbial agents, relies on bacterial or fungal growth. The microbial diagnosis of BSI directly from blood has been proposed to speed the determination of the etiological agent but was limited by the very low number of circulating microbes during these paucibacterial infections. Thanks to recent advances in molecular biology, including the improvement of nucleic acid extraction and amplification, several PCR-based methods for the diagnosis of BSI directly from whole blood have emerged. In the present review, we discuss the advantages and limitations of these new molecular approaches, which at best complement the culture-based diagnosis of BSI.

[Mass spectrometry in bacteriology]. / Place de la spectrométrie de masse en bactériologie.

Recently, different bacteriological laboratory interventions that decrease reporting time have been developed. These promising new broad-based techniques have merit, based on their ability to identify rapidly many bacteria, organisms difficult to grow or newly emerging strains, as well as their capacity to track disease transmission. Maldi-TOF MS has been proven to be an accurate and reliable method for organism identification including bacteria, yeast, molds, and mycobacteria. It is rapid, with results often 24 hours earlier than traditional methods, and inexpensive. The range of applications of Maldi-TOF MS has been growing constantly, from rapid species identification to labor-intensive proteomic studies of bacterial physiology (bacterial resistance and virulence). The purpose of this review is to present the different solutions commercialized in France, summarize the place of this technology in microbiology lab and to analyze future perspectives in this field.

Salvage microbiology: opportunities and challenges in the detection of bacterial pathogens following initiation of antimicrobial treatment.

Broad-range 16S ribosomal RNA gene PCR coupled with Sanger sequencing was originally employed by soil scientists and was subsequently adapted for clinical applications. PCR coupled with electrospray ionization mass spectrometry has also progressed from initial applications in the detection of organisms from environmental samples into the clinical realm and has demonstrated promise in detection of pathogens in clinical specimens obtained from patients with suspected infection but negative cultures. We review studies of multiplex PCR, 16S ribosomal RNA gene PCR and sequencing and PCR coupled with electrospray ionization mass spectrometry for detection of bacteria in specimens that were obtained from patients during or after administration of antibiotic treatment, and examine the role of each for assisting in antimicrobial treatment and stewardship efforts. Following an exploration of the available data in this field, we discuss the opportunities that the preliminary investigations reveal, as well as the challenges faced with the implementation of these strategies in clinical practice.

Emerging commercial molecular tests for the diagnosis of bloodstream infection.

Bloodstream infection (BSI) by microorganisms can lead to sepsis. This condition has a high mortality rate, which rises significantly with delays in initiation of appropriate antimicrobial treatment. Current culture methods for diagnosing BSI have long turnaround times and poor clinical sensitivity. While clinicians wait for culture diagnosis, patients are treated empirically, which can result in inappropriate treatment, undesirable side effects and contribute to drug resistance development. Molecular diagnostics assays that target pathogen DNA can identify pathogens and resistance markers within hours. Early diagnosis improves antibiotic stewardship and is associated with favorable clinical outcomes. Nonetheless, limitations of current molecular diagnostic methods are substantial. This article reviews recent commercially available molecular methods that use pathogen DNA to diagnose BSI, either by testing positive blood cultures or directly testing patient blood. We critically assess these tests and their application in clinical microbiology. A view of future directions in BSI diagnosis is also provided.

Prospective comparison of RT-PCR/ESI-MS to Prodesse ProFlu Plus and Cepheid GenXpert for the detection of Influenza A and B viruses.

RT-PCR/ESI-MS has previously demonstrated the capability to detect and identify respiratory viral pathogens in nasopharyngeal swabs. This study expands on previous research by performing a prospective evaluation of RT-PCR/ESI-MS to detect and identify Influenza A and B viruses compared to Prodesse ProFlu Plus and combined ProFlu Plus and Cepheid Xpert Flu. ProFlu Plus was also used as a gold standard for comparison for respiratory syncytial virus detection. Using ProFlu Plus as a gold standard, RT-PCR/ESI-MS had sensitivity and specificity of 82.1% (23/28) and 100% (258/258), respectively, for Influenza A, 100% (16/16) and 99.6% (269/270), respectively for Influenza B, and 88.6% (39/44) and 99.6% (241/242) for any Influenza virus. Using matching results from ProFlu Plus and Xpert Flu as a gold standard, RT-PCR/ESI-MS had 85.2% (23/27) and 100% (259/259) sensitivity and specificity respectively for Influenza A, 100% (14/14) and 99.6% (270/272), respectively for Influenza B virus. Overall, RT-PCR/ESI-MS was not as sensitive as the combined gold standard of ProFlu Plus and Xpert Flu, although it has the capability of detecting other respiratory viruses.

A case of Mycobacterium sherrisii pneumonia diagnosed by PCR/ESI-MS method.

Mycobacterium sherrisii is an acid fast organism that is recently validated as a novel species. It is recognized to cause severe disease in immunosuppressed individuals especially in patients with HIV. We describe a case M. sherrisii pneumonia which was diagnosed using PCR coupled with electrospray ionization mass-spectrometry (PCR/ESI-MS) technology.

A case-control study evaluating RT-PCR/ESI-MS technology compared to direct fluorescent antibody and xTAG RVP PCR.

Waste nasopharyngeal swabs (N = 244) were evaluated by the reverse-transcriptase polymerase chain reaction/electrospray ionization mass spectrometry PLEX-ID Broad Respiratory Virus Surveillance Kit version 2.5 compared to direct fluorescent antibody and xTAG Respiratory Virus Panel for percent agreement, sensitivity, and specificity. Sensitivity and specificity were 91% (111/122) and 95.1% (116/122), respectively. Sensitivity by virus, except parainfluenza, was 92.9-100%, and specificity was 99-100%.

Detection and identification of viral pathogens in patients with hand, foot, and mouth disease by multilocus PCR, reverse-transcription PCR and electrospray ionization mass spectrometry.

BACKGROUND: Rapid detection and identification of viruses are important for early diagnosis and effective surveillance of hand, foot, and mouth disease (HFMD). We described a novel assay using multilocus PCR and reverse transcription-PCR coupled with electrospray ionization mass spectrometry (RT-PCR/ESI-MS) to simultaneously detect and identify human enterovirus A-D, adenovirus A-F, human herpesvirus 1-8, parvovirus B19 and polyomavirus. OBJECTIVES: To evaluate the accuracy and efficacy of the RT-PCR/ESI-MS method, to detect and type enterovirus from specimens of clinical diagnosed HFMD patients. STUDY DESIGN: In this study, 152 specimens of clinically diagnosed HFMD patients were studied by the assay using RT-PCR/ESI-MS method. The detection and typing of enterovirus by RT-PCR/ESI-MS were compared with results from reference molecular methods. RESULTS: The assay detected enteroviruses in 97 (63.8%) specimens, resulting in a sensitivity of 93.8% (95% CI: 91.8-95.7%) and a specificity of 87.5% (95% CI: 84.8-90.2%) compared with a reference clinical diagnostic test. Most enterovirus genotypes (65/84; 77%) determined by the platform were consistent with 5' UTR sequence analysis, and most misidentifications resulted from the virus library, which could be further improved by updating the enterovirus database. In addition to enteroviruses, herpesviruses, polyomaviruses, adenoviruses and human rhinoviruses were detected and identified in 55 (36%) HFMD specimens by RT-PCR/ESI-MS. CONCLUSION: With the capability of high throughput and detection and typing of multiple clinically relevant viruses simultaneously, RT-PCR/ESI-MS can be a rapid and robust laboratory tool for identifying viral pathogens.