Detection Of Microbial DNA Research Articles

DNA-sensing pathways in health, autoinflammatory and autoimmune diseases.

Detection of microbial DNA is a primary means of host defense. In mammalian cells, DNA-sensing pathways induce robust anti-microbial responses and initiation of adaptive immunity, leading to the eventual clearance of the infectious agent. However, while conferring the advantage of broad detection capability, the sequence-independent recognition mechanisms of most DNA sensors pose a significant challenge for mammalian cells to maintain ignorance to self-DNA under homeostatic conditions. In this Review, we summarize the fundamentals of DNA-sensing pathways and the intricate regulatory networks that keep these pathways in check. In addition, we describe how regulatory restraints can be defective and underlie human autoinflammatory and autoimmune diseases. Further, we discuss therapies in development that limit inflammation fueled by self-DNA or inappropriate activation of DNA-sensing pathways.

Bridging Flocculation of a Sterically Stabilized Cationic Latex as a Biosensor for the Detection of Microbial DNA after Amplification via PCR.

There is a high demand for rapid, sensitive, and accurate detection methods for pathogens. This paper demonstrates a method of detecting the presence of amplified DNA from a range of pathogens associated with serious infections including Gram-negative bacteria, Gram-positive bacteria, and viruses. DNA is amplified using a polymerase chain reaction (PCR) and consequently detected using a sterically stabilized, cationic polymer latex. The DNA induces flocculation of this cationic latex, which consequently leads to rapid sedimentation and a visible change from a milky-white dispersion to one with a transparent supernatant, presenting a clear visible change, indicating the presence of amplified DNA. Specifically, a number of different pathogens were amplified using conventional or qPCR, including Staphylococcus aureus, Escherichia coli, and Herpes Simplex Virus (HSV-2). This method was demonstrated to detect the presence of bacteria in suspension concentrations greater than 380 CFU mL-1 and diagnose the presence of specific genomes through primer selection, as exemplified using methicillin resistant and methicillin susceptible Staphylococcus aureus. The versatility of this methodology was further demonstrated by showing that false positive results do not occur when a PCR of fungal DNA from C. albicans is conducted using bacterial universal primers.

Interruption of post-Golgi STING trafficking activates tonic interferon signaling

Activation of the cGAS-STING pathway is traditionally considered a “trigger-release” mechanism where detection of microbial DNA or cyclic di-nucleotides sets off the type I interferon response. Whether this pathway can be activated without pathogenic ligand exposure is less well understood. Here we show that loss of Golgi-to-lysosome STING cofactors, but not ER-to-Golgi cofactors, selectively activates tonic interferon signalling. Impairment of post-Golgi trafficking extends STING Golgi-dwell time, resulting in elevated immune signalling and protection against infection. Mechanistically, trans-Golgi coiled coil protein GCC2 and several RAB GTPases act as key regulators of STING post-Golgi trafficking. Genomic deletion of these factors potently activates cGAS-STING signalling without instigating any pathogenic trigger for cGAS. Gcc2−/− mice develop STING-dependent serologic autoimmunity. Gcc2-deleted or Rab14-deleted cancer cells induce T-cell and IFN-dependent anti-tumour immunity and inhibit tumour growth in mice. In summary, we present a “basal flux” mechanism for tonic cGAS-STING signalling, regulated at the level of post-Golgi STING trafficking, which could be exploited for cancer immunotherapy.

Meningitis

The term aseptic meningitis encompasses all types of inflammations of the brain meninges other than that caused by pus producing organisms. It is usually a mild illness. Etiology of aseptic meningitis is very wide and includes many infections - both viral and non viral, drugs, malignancy and systemic illness. The most common cause is viral infection and enteroviruses - Coxsackie and ECHO viruses account for more than half of all cases. Clinical manifestations include headache, fever, malaise, photophobia and meningeal signs. Convulsions, neurological deficits and severe obtundation are rare except with certain non viral infectious meningitis. Diagnostic work up includes blood and cerebrospinal fluid (CSF) examination and serology for infectious meningitis. The polymerase chain reaction is a rapid and accurate method for detection of microbial DNA in CSF. Treatment is mainly supportive, except for the nonviral infectious etiology.

FP05.04 Robust Discrimination of Lung Cancer via Microbial DNA Detection and Machine Learning Classification

Human tissues, including tumors, are extensively colonized by taxonomically diverse microbes. Intra-tumoral microbial activity and events of cellular turnover and trafficking contribute to shedding microbial nucleic acids into the blood stream. Here we characterized microbial signatures present in primary-tumor tissue and in the blood of patients affected with different cancer types, with particular focus on lung cancer, and we demonstrated the discriminatory power of such microbial signatures for the identification and classification of lung cancer versus other cancer types.

Detection of Circulating Tumor Cells and Microbial DNA Fragments in Stage III Colorectal Cancer Patients under Three versus Six Months of Adjuvant Treatment.

Simple SummaryDuration of adjuvant therapy in stage III CRC is now under re-evaluation. The aim of the current study was the detection of microbial DNA that could be originated from the intestine (16S rRNA, E. coli, B. fragilis and C. albicans) and the detection of CTCs during treatment with FOLFOX or CAPOX, in stage III CRC patients. CTCs were significantly decreased after 3 months of treatment, whereas 6 months resulted to their increase again. A significant increase of CTCs was demonstrated in patients under FOLFOX for 6 months. A significant correlation was demonstrated following microbial DNAs and both CTCs detection at baseline and CTCs increase, between baseline and 3 months of treatment. The results provide additional evidence of non-inferiority of 3 over 6 months of treatment, mainly in patients under CAPOX.Oxaliplatin-fluoropyrimidine combination therapy is the gold standard treatment for patients with stage III colorectal cancer (CRC); however, treatment duration is now under re-evaluation. The aim of the study was the evaluation of the non-inferiority of three over six months treatment with FOLFOX or CAPOX, in stage III CRC patients. Peripheral blood samples from 121 patients were collected, at three time points during treatment and evaluated for circulating tumor cells (CTCs) and microbial DNA detection (16S rRNA, Escherichia coli, Bacteroides fragilis, Candida albicans). Of all patients, 41.3% and 58.7% were treated with FOLFOX and CAPOX, respectively. CTCs were significantly decreased and increased after three and six months of treatment, respectively. CAPOX tends to reduce the CTCs after 3 months, whereas there is a statistically significant increase of CTCs in patients under FOLFOX after 6 months. A significant correlation was demonstrated between microbial DNA detection and both CTCs detection at baseline and CTCs increase between baseline and three months of treatment. To conclude, the current study provides additional evidence of non-inferiority of three over 6 months of treatment, mainly in patients under CAPOX.

Detection of microbial DNA in the blood of colorectal cancer patients for the diagnosis of microbial translocation.

171 Background: Dysbiosis has been associated with diseases and is of a major public health importance. Can lead to the passage of viable bacteria, their products or their fragments from the intestinal lumen through the mesenteric lymph nodes and other sites, known as bacterial translocation. The aim of the study was to determine whether microbial translocation occurs in stage II/III-IV colorectal cancer (CRC) patients and evaluate the usefulness of blood PCR for diagnosis of such translocation. Also to correlate the presence of Toll-Like Receptor and Vitamin D Receptor polymorphisms with the detection of microbial DNA fragments in the blood of CRC patients. Methods: Peripheral blood was obtained from 397 CRC patients (adjuvant n = 202 and metastatic n = 195) and 32 healthy individuals. DNA from all subjects was analyzed using PCR for amplification of genomic DNA encoding 16S rRNA, β-galactosidase gene of E. coli, Glutamine synthase gene of B. fragilis and 5.8S rRNA found in C. albicans. Results: Significantly higher rates of 16S rRNA, β-galactosidase, Glutamine synthase and 5.8S rRNA detection was observed in the pool of CRC patients than the controls ( p< 0.001). All microbial DNA fragments detected were also significantly associated with the metastatic disease ( p< 0.001) leading to shorter survival rates ( p< 0.001). Moreover, individuals with the homozygous mutant alleles of either TLR or VDR gene polymorphisms had significantly higher detection rates of microbial DNA fragments. Conclusions: The detection of microbial DNA fragments in patients with CRC highlights the role of these microbes in cancer development, progression and therefore in patients’ survival.

Direct detection of Listeria monocytogenes DNA amplification products with quartz crystal microbalances at elevated temperatures

Current methods for identifying Listeria monocytogenes are both time and labor intensive. However, it is highly desirable to detect it rapidly and reliably to prevent and/or identify contamination of foodstuff. Herein we propose a mass-sensitive sensor operating at elevated temperatures, 50−55 °C, for that purpose. Sensitive and selective detection relies on distinguishing genes of genomic extract of L. monocytogenes. A thiol-modified ssDNA probe designed for virulence phosphatidylcholine-phospholipase C (plcB) immobilized on the Quartz Crystal Microbalance (QCM) serves as the recognition element. This hybridizes with synthetic Loop-mediated isothermal amplification (LAMP) products of target DNA on the active surface sensor. Discernible detection limits of approximately 3 × 10−1 to 3 × 100 CFU mL-1 of L. monocytogenes DMST 17303 gDNA were achieved. The QCMDNA sensor showed high sensitivity and selectivity for L. monocytogenes (100 %) with negligible interference by DNA of other foodborne pathogens, such as Salmonella Paratyphi A (24 %), Salmonella Weltevreden (24 %), Salmonella Typhi (16 %), Shigella boydii (22 %), and Shigella flexneri (13 %). The temperature covered is in the range of 50–55 °C for immobilizing DNA probe and DNA target hybridization. Hybridization response times were within 10−30 min, demonstrated by saturation of the respective sensor responses. It turned out that sensitivity of the hybridization response increases up to two times by co-immobilizing the probe and L-cysteine. The latter acts as a spacer to increase probe-probe distance. This work demonstrates the potential of the QCM sensor technique at elevated temperatures as a sensor platform for further development of sensitive, specific and rapid detection of microbial DNA.

Polymorphisms in interferon pathway genes and risk of Mycobacterium tuberculosis infection in contacts of tuberculosis cases in Brazil

BackgroundHost genetic polymorphisms may be important in determining susceptibility to Mycobacterium tuberculosis (Mtb) infection, but their role is not fully understood. Detection of microbial DNA and activation of type I interferon (IFN) pathways regulate macrophage responses to Mtb infection. MethodsWe examined whether seven candidate gene SNPs were associated with tuberculin skin test (TST) positivity in close contacts of microbiologically confirmed pulmonary TB patients in Brazil. Independent associations with TST positivity were tested using multivariable logistic regression (using genotypes and clinical variables) and genetic models. ResultsAmong 482 contacts of 145 TB index cases, 296 contacts were TST positive. Multivariable regression analysis adjusted for population admixture, age, family relatedness, sex and clinical variables related to increased TB risk demonstrated that SNPs in PYHIN1-IFI16-AIM2 rs1101998 (adjusted OR [aOR]: 3.72; 95%CI=1.15–12.0; p=0.028) and in PYHIN1-IFI16-AIM2 rs1633256 (aOR=24.84; 95%CI=2.26–272.95; p=0.009) were associated with TST positivity in a recessive model. Furthermore, an IRF7 polymorphism (rs11246213) was associated with reduced odds of TST positivity in a dominant model (aOR: 0.50, 95%CI: 0.26-0.93; p=0.029). ConclusionsPolymorphisms in PYHIN1-IFI16-AIM2 rs1633256, rs1101998 and in IRF7 rs11246213 were associated with altered susceptibility to Mtb infection in this Brazilian cohort.

Detection of Microbiota in Post Radiation Sinusitis.

A shift in the microbiota of chronic rhinosinusitis has been described after radiotherapy to the sinonasal cavity and skull base. There is a paucity of literature characterizing the bacteriology of post radiation sinusitis using next-generation gene sequencing techniques. This study aims to describe and compare the microbial flora of rhinosinusitis after radiotherapy using both culture and molecular techniques for microbial DNA detection. The medical records of patients treated with external beam radiation for sinonasal, nasopharyngeal, or skull base malignancy were reviewed at a tertiary care facility. Patients' sinonasal cavities were swabbed for routine culture or brushed for molecular gene sequencing. Swab specimens were processed for standard microbial culture, and brush specimens were sent for gene sequencing at Micro GenX Laboratory (Lubbock, Texas, USA). Twenty-two patients were diagnosed with chronic sinusitis after undergoing radiotherapy. Staphylococcus aureus was the most common organism identified by both culture and gene sequencing, followed by Pseudomonas aeruginosa. Several additional organisms were detected by gene sequencing that were not isolated by routine culture techniques. Gene sequencing identified pathogens differing from culture results in 50% of patients examined. The bacteriology of post radiation sinusitis appears to resemble the microorganisms responsible for chronic sinusitis in healthy adults. Next generation gene sequencing techniques may reveal additional organisms responsible for sinusitis and provide complementary results that may impact the medical treatment of post radiation sinusitis.

Use of a filter cartridge combined with intra‐cartridge bead‐beating improves detection of microbial DNA from water samples

Abstract Microbial communities play an important role in driving the dynamics of aquatic ecosystems. As difficulties in DNA sequencing faced by microbial ecologists are continuously being reduced, sample collection methods and DNA extraction protocols are becoming more critical to the outcome of any sequencing study. In the present study, I added a manual, intra‐cartridge, bead‐beating step in the protocol using a DNeasy® Blood & Tissue kit for DNA extraction from a filter cartridge without breaking the cartridge unit (“Beads” method) and compared its performance with those of two other protocols (“NoBeads” method, which was similar to the Beads method but without the bead‐beating step and “PowerSoil” method, which followed the manual of the DNeasy® PowerSoil kit after breaking apart the filter cartridge). Water samples were collected from lake, river, pond and coastal ecosystems in Japan and DNA was extracted using the three protocols. Then, the V4 region of prokaryotic 16S rRNA genes was amplified. In the library preparation process, internal standard DNAs were included to estimate the number of DNA copies. The DNA library was sequenced using Illumina MiSeq and sequences were analysed using the amplicon sequence variant (ASV) approach. I found that, (a) the total prokaryotic DNA yields were highest with the Beads method, (b) the number of ASVs (a proxy for species richness) was also highest with the Beads method, (c) overall community compositions were significantly different among the three methods and (d) the number of method‐specific ASVs was highest with the Beads method. These results were generally robust across samples from all aquatic ecosystems examined. In conclusion, the inclusion of a bead‐beating step performed inside the filter cartridge increased the DNA yield as well as the number of prokaryotic ASVs detected compared with the other two methods. Performing the bead‐beating step inside the filter cartridge causes no dramatic increase in either handling time or processing cost and it can reduce the potential contamination risk from the ambient air and/or other samples. Therefore, this method has the potential to become one of the major choices when one aims to extract aquatic microbial DNAs.

Phospholipid levels in blood during community-acquired pneumonia

Phospholipids, major constituents of bilayer cell membranes, are present in large amounts in pulmonary surfactant and play key roles in cell signaling. Here, we aim at finding clinically useful disease markers in community-acquired pneumonia (CAP) using comprehensive phospholipid profiling in blood and modeling of changes between sampling time points. Serum samples from 33 patients hospitalized with CAP were collected at admission, three hours after the start of intravenous antibiotics, Day 1 (at 12–24 h), Day 2 (at 36–48 h), and several weeks after recovery. A profile of 75 phospholipid species including quantification of the bioactive lysophosphatidylcholines (LPCs) was determined using liquid chromatography coupled to time-of-flight mass spectrometry. To control for possible enzymatic degradation of LPCs, serum autotaxin levels were examined. Twenty-two of the 33 patients with a clinical diagnosis of CAP received a laboratory-verified CAP diagnosis by microbial culture or microbial DNA detection by qPCR. All major phospholipid species, especially the LPCs, were pronouncedly decreased in the acute stage of illness. Total and individual LPC concentrations increased shortly after the initiation of antibiotic treatment, concentrations were at their lowest 3h after the initiation, and increased after Day 1. The total LPC concentration increased by a change ratio of 1.6–1.7 between acute illness and Day 2, and by a ratio of 3.7 between acute illness and full disease resolution. Autotaxin levels were low in acute illness and showed little changes over time, contradicting a hypothesis of enzymatic degradation causing the low levels of LPCs. In this sample of patients with CAP, the results demonstrate that LPC concentration changes in serum of patients with CAP closely mirrored the early transition from acute illness to recovery after the initiation of antibiotics. LPCs should be further explored as potential disease stage biomarkers in CAP and for their potential physiological role during recovery.

The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer.

Detection of microbial DNA is an evolutionarily conserved mechanism that alerts the host immune system to mount a defense response to microbial infections. However, this detection mechanism also poses a challenge to the host as to how to distinguish foreign DNA from abundant self-DNA. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP), which binds and activates the adaptor protein STING. However, cGAS can be activated by double-stranded DNA irrespective of the sequence, including self-DNA. Although how cGAS is normally kept inactive in cells is still not well understood, recent research has provided strong evidence that genomic DNA damage leads to cGAS activation to stimulate inflammatory responses. This review summarizes recent findings on how genomic instability and DNA damage trigger cGAS activation and how cGAS serves as a link from DNA damage to inflammation, cellular senescence, and cancer.

The detection of microbial DNA but not cultured bacteria is associated with increased mortality in patients with suspected sepsis—a prospective multi-centre European observational study

ObjectivesBlood culture results inadequately stratify the mortality risk in critically ill patients with sepsis. We sought to establish the prognostic significance of the presence of microbial DNA in the bloodstream of patients hospitalized with suspected sepsis. MethodsWe analysed the data collected during the Rapid Diagnosis of Infections in the Critically Ill (RADICAL) study, which compared a novel culture-independent PCR/electrospray ionization-mass spectrometry (ESI-MS) assay with standard microbiological testing. Patients were eligible for the study if they had suspected sepsis and were either hospitalized or were referred to one of nine intensive care units from six European countries. The blood specimen for PCR/ESI-MS assay was taken along with initial blood culture taken for clinical indications. ResultsOf the 616 patients recruited to the RADICAL study, 439 patients had data on outcome, results of the blood culture and PCR/ESI-MS assay available for analysis. Positive blood culture and PCR/ESI-MSI result was found in 13% (56/439) and 40% (177/439) of patients, respectively. Either a positive blood culture (p 0.01) or a positive PCR/ESI-MS (p 0.005) was associated with higher SOFA scores on enrolment to the study. There was no difference in 28-day mortality observed in patients who had either positive or negative blood cultures (35% versus 32%, p 0.74). However, in patients with a positive PCR/ESI-MS assay, mortality was significantly higher in comparison to those with a negative result (42% versus 26%, p 0.001). ConclusionsPresence of microbial DNA in patients with suspected sepsis might define a patient group at higher risk of death.

The utility of direct specimen detection by Sanger sequencing in hospitalized pediatric patients

Direct microbial DNA detection from clinical specimens by polymerase chain reaction and Sanger sequencing has been developed to address the innate limitations of traditional culture-based work-up. We report our institution's experience with direct specimen sequencing, its clinical utility, and barriers to effective clinical implementation.

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

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.

The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya

The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was little restriction of the parasites to particular zones.

Different presence of Chlamydia pneumoniae, herpes simplex virus type 1, human herpes virus 6, and Toxoplasma gondii in schizophrenia: meta-analysis and analytical study.

In the present study we have performed both a meta-analysis and an analytical study exploring the presence of Chlamydia pneumoniae, herpes simplex virus type 1, human herpes virus 6, and Toxoplasma gondii antibodies in a sample of 143 schizophrenic patients and 143 control subjects. The meta-analysis was performed on papers published up to April 2014. The presence of serum immunoglobulin G and immunoglobulin A was performed by enzyme-linked immunosorbent assay test. The detection of microbial DNA in total peripheral blood was performed by nested polymerase chain reaction. The meta-analysis showed that: 1) C. pneumoniae DNA in blood and brain are more common in schizophrenic patients; 2) there is association with parasitism by T. gondii, despite the existence of publication bias; and 3) herpes viruses were not more common in schizophrenic patients. In our sample only anti-Toxoplasma immunoglobulin G was more prevalent and may be a risk factor related to schizophrenia, with potential value for prevention.

Evolution, Multiple Acquisition, and Localization of Endosymbionts in Bat Flies (Diptera: Hippoboscoidea: Streblidae and Nycteribiidae)

Bat flies are a diverse clade of obligate ectoparasites on bats. Like most blood-feeding insects, they harbor endosymbiotic prokaryotes, but the origins and nature of these symbioses are still poorly understood. To expand the knowledge of bacterial associates in bat flies, the diversity and evolution of the dominant endosymbionts in six of eight nominal subfamilies of bat flies (Streblidae and Nycteribiidae) were studied. Furthermore, the localization of endosymbionts and their transmission across developmental stages within the family Streblidae were explored. The results show diverse microbial associates in bat flies, with at least four ancestral invasions of distantly related microbial lineages throughout bat fly evolution. Phylogenetic relationships support the presence of at least two novel symbiont lineages (here clades B and D), and extend the geographic and taxonomic range of a previously documented lineage ("Candidatus Aschnera chinzeii"; here clade A). Although these lineages show reciprocally monophyletic clusters with several bat fly host clades, their phylogenetic relationships generally do not reflect current bat fly taxonomy or phylogeny. However, within some endosymbiont clades, congruent patterns of symbiont-host divergence are apparent. Other sequences identified in this study fall into the widely distributed, highly invasive, insect-associated Arsenophonus lineage and may be the result of symbiont replacements and/or transient infections (here clade C). Vertical transmission of endosymbionts of clades B and D is supported by fluorescent signal (fluorescent in situ hybridization [FISH]) and microbial DNA detection across developmental stages. The fluorescent bacterial signal is consistently localized within structures resembling bacteriomes, although their anatomical position differs by host fly clade. In summary, the results suggest an obligate host-endosymbiont relationship for three of the four known symbiont clades associated with bat flies (clades A, B, and D).

A new workflow for the microbiological diagnosis of febrile neutropenia in patients with a central venous catheter

We aimed to improve the microbial diagnosis of first episodes of febrile neutropenia (FEFNs) since <30% of episodes are microbiologically documented. Consequently patients are usually treated by empirical antibiotic therapy. A prospective study evaluated a new workflow combining: (i) one 40 mL blood culture (BC) sampled from the central venous catheter; (ii) immediate incubation in an automated BC system on the ward; (iii) direct detection of microbial DNA in blood; and (iv) identification and susceptibility testing using rapid methods performed directly on positive BC bottles. Patients were also sampled for the standard workflow with two BC sets incubated in the central laboratory and assessed by classical procedures. One hundred and twenty consecutive FEFNs were included (February 2008-March 2009). The new workflow was as sensitive as the standard workflow, with BC positivity rates of 30% (36/120) and 28% (34/120), respectively (McNemar's χ(2) =0.67, P=0.41). Direct DNA detection was positive in nine episodes (7.5%) that were also positive in BC. The new workflow provided microbiological results significantly earlier than the standard workflow, with a shorter time to BC positivity (median 12 h 31 min, range 7 h 55 min-25 h 37 min versus median 13 h 01 min, range 9 h 31 min-43 h 33 min, P=0.004) and shorter turnaround times for identification and susceptibility testing, with most of the results obtained <24 h after BC sampling. We retrospectively estimated that the new workflow would lead to earlier adequacy of antimicrobial therapy in 30% of documented cases. Our new process improved the microbiological diagnosis in FEFNs. Cost effectiveness needs to be tested.