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Vaginal fluid analysis plays a crucial role in sexual assault investigations. However, vaginal fluid found at crime scenes is usually subject to a certain duration of exposure. This study thus aimed to assess the influence of different durations of exposure to indoor environment on the vaginal microbiota. The 16S rDNA high-throughput sequencing was used on vaginal fluid samples exposed for short-term (30 days) and long-term (240 days), respectively. Despite potential contamination from environmental microorganisms, particularly following long-term exposure, the results indicated that the vaginal microbiota after exposure was still dominated by Lactobacillus. Both in short-term and long-term exposure involving vaginal fluid, there were clusters with time-dependent characteristics, wherein the relative abundances of associated microbial genera showed a trend of increasing or decreasing over time. In addition, each bodily fluid presented with a unique array of dominant bacterial genera, enabling the differentiation of exposed vaginal fluid samples from other bodily fluids (semen, skin, saliva, feces) with a remarkable 98.75 % accuracy rate. Furthermore, the mean absolute error achieved by the long-term deposition time prediction model was 13.54 days. The mean absolute error for the short-term deposition time prediction model was notably lower, reaching just 2.05 days. In summary, this study investigates the variations in microbial communities within vaginal fluid subjected to different indoor exposure durations and explores their potential in body fluid identification and estimating the time since deposition, thereby contributing valuable supporting evidence in forensic investigations., Competing Interests: Declaration of Competing Interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The native vaginal microbiome plays a crucial role in maintaining vaginal health and disruption can have significant consequences for women during their lifetime. While the composition of the vaginal microbiome is important, current methods for monitoring this community are lacking. Clinically used techniques routinely rely on subjective analysis of vaginal fluid characteristics or time-consuming microorganism culturing. Surface-enhanced Raman spectroscopy (SERS) can aid in filling this gap in timely detection of alterations in the vaginal microbiome as it can discriminate between bacterial species in complex solutions including bacterial mixtures and biofluids. SERS has not previously been applied to study variations in vaginal Lactobacillus , the most common species found in the vaginal microbiome, in complex solutions. Herein, the SERS spectra of Lactobacillus crispatus ( L. crispatus ) and Lactobacillus iners ( L. iners ), two of the most common vaginal bacteria, was characterized at physiologically relevant concentrations. Subsequently, the ability of SERS to detect L. crispatus and L. iners in both pure mixtures and when mixed with a synthetic vaginal fluid mimicking solution was determined. In both pure and complex solutions, SERS coupled with partial least squares regression predicted the ratiometric bacterial content with less than 10% error and strong goodness of prediction ( Q 2 > 0.9). This developed method highlights the applicability of SERS to predict the dominant Lactobacillus in the vaginal micro-environment toward the monitoring of this community.

Rapid identification of pathogens in normally sterile body fluid (NSBF) is essential for appropriate patient management, specifically antimicrobial therapy. Limited sensitivity and increased time to detection of traditional culture prompted us to evaluate additional testing to contribute to the diagnosis of infection. The purpose of this study was to evaluate the GenMark Dx ePlex Blood Culture Identification (BCID) Panels on positive body fluids inoculated into blood culture bottles for the detection of microorganisms. A total of 88 positive body fluids from blood culture bottles were analyzed using a Gram-Positive, Gram-Negative, and/or Fungal pathogen BCID Panel based on the Gram stain result. Each result was compared to routine culture performed from the positive bottle. When using culture as a reference standard, we found the ePlex multiplex panel performed with a positive percent agreement of 96.5% and a negative percent agreement of 99.8%. The use of multiplex PCR may be a useful supplement to routine culture for NSBF in blood culture bottles., Importance: The identification of pathogens in normally sterile body fluid (NSBF) is performed using routine culture, the current gold standard. Limitations of this method include sensitivity and increased turnaround times which could potentially delay vital patient care, especially antimicrobial therapy. Adaptations of NSBF in blood culture bottles prompted us to consider the utility of additional methods to bridge the gap in diagnostic challenges for these life-threatening infections. Multiplex molecular panels have been manufactured for use with multiple specimen types including blood, cerebral spinal fluid, stool, and respiratory. Therefore, the purpose of this study was to evaluate the off-label use of ePlex Blood Culture Identification Panels on positive body fluids grown in blood culture bottles for the detection of microorganisms for research purposes., Competing Interests: E.A.P., J.E.M., M.V.P.-F.—Roche diagnostics provided reagents for this study.

Microbial communities present in body fluids can assist in distinguishing between types of body fluids. Metagenomic studies have reported bacterial genera which are core to specific body fluids and are greatly influenced by geographical location and ethnicity. Bacteria in body fluids could also be due to bacterial infection; hence, it would be worthwhile taking into consideration bacterial species associated with diseases. The present review reports bacterial species characteristic of diseased and healthy body fluids across geographical locations, and bacteria described in forensic studies, with the aim of collating a set of bacteria to serve as the core species-specific markers for forensic body fluid identification. The most widely reported saliva-specific bacterial species are Streptococcus salivarius, Prevotella melaninogenica, Neisseria flavescens, with Fusobacterium nucleatum associated with increased diseased state. Lactobacillus crispatus and Lactobacillus iners are frequently dominant in the vaginal microbiome of healthy women. Atopobium vaginae, Prevotella bivia, and Gardnerella vaginalis are more prevalent in women with bacterial vaginosis. Semen and urine-specific bacteria at species level have not been reported, and menstrual blood bacteria are indistinguishable from vaginal fluid. Targeting more than one bacterial species is recommended for accurate body fluid identification. Although metagenomic sequencing provides information of a broad microbial profile, the specific bacterial species could be used to design biosensors for rapid body fluid identification. Validation of microbial typing methods and its application in identifying body fluids in a mixed sample would allow regular use of microbial profiling in a forensic workflow., (© 2023. The Author(s).)

The analysis of biological fluids is crucial for the diagnosis and monitoring of diseases causing effusions and helps in the diagnosis of infectious diseases. The gold standard method for cell count in biological fluids is the manual method using counting chambers. The microbiological routine procedures consist of Direct Gram staining and culture on solid or liquid media. We evaluate the analytical performance of SYSMEX UF4000 (Sysmex, Kobe, Japan) and Sysmex XN10 (Sysmex, Kobe, Japan) in comparison with cytological and microbiological routine procedures. A total of 526 biological fluid samples were included in this study (42 ascitic, 31 pleural, 31 peritoneal, 125 cerebrospinal, 281 synovial, and 16 peritoneal dialysis fluids). All samples were analyzed by flow cytometry and subsequently processed following cytological and/or microbiological routine procedures. With regard to cell counts, UF4000 (Sysmex, Kobe, Japan) showed a performance that was at least equivalent to those of the reference methods and superior to those of XN10 (Sysmex, Kobe, Japan). Moreover, the bacterial count obtained with UF4000 (Sysmex, Kobe, Japan) was significantly higher among culture or Direct Gram stain positive samples. We established three optimal cutoff points to predict Direct Gram stain positive samples for peritoneal (465.0 bacteria/μL), synovial (1200.0 bacteria/μL), and cerebrospinal fluids (17.2 bacteria/μL) with maximum sensitivity and negative predictive values. Cell count and detection of bacteria by flow cytometry could be used upstream cytological and microbiological routine procedures to improve and accelerate the diagnosis of infection of biological fluid samples. IMPORTANCE The analysis of biological fluids is crucial for the diagnosis and monitoring of diseases causing effusions and helps in the diagnosis of infectious diseases. The possibility of carrying out cytological and microbiological analyses of biological fluid samples on the same automated machine would simplify the sample circuit (addressing the sample in a single laboratory, 24/7). It would also minimize the quantity of sample required. The performance of cytological and microbiological analysis by the flow cytometer UF 4000 (Sysmex, Kobe, Japan) has never been evaluated yet. This study has shown that bacterial count by flow cytometry using UF4000 (Sysmex, Kobe, Japan) could be used upstream of microbiological routine procedures to improve and to accelerate the diagnosis of infection of biological fluid samples.

Problem: Preterm birth (PTB) is a major cause of increased morbidity and mortality in newborns. The main cause of spontaneous PTB (sPTB) is the activation of an inflammatory response as a result of ascending genital tract infection. Despite various studies on the effects of the vaginal microbiome on PTB, a practical method for its clinical application has yet to be developed., Method of Study: In this case-control study, 94 Korean pregnant women with PTB (n = 38) and term birth (TB; n = 56) were enrolled. Their cervicovaginal fluid (CVF) was sampled, and a total of 10 bacteria were analyzed using multiplex quantitative real-time PCR (qPCR). The PTB and TB groups were compared, and a PTB prediction model was created using bacterial risk scores using machine learning techniques (decision tree and support vector machine). The predictive performance of the model was validated using random subsampling., Results: Bacterial risk scoring model showed significant differences (P < 0.001). The PTB risk was low when the Lactobacillus iners ratio was 0.812 or more. In groups with a ratio under 0.812, moderate and high risk was classified as a U. parvum ratio of 4.6 × 10 -3 . The sensitivity and specificity of the PTB prediction model using bacteria risk score were 71% and 59%, respectively, and 77% and 67%, respectively, when white blood cell (WBC) data were included., Conclusion: Using machine learning, the bacterial risk score in CVF can be used to predict PTB., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

High-throughput DNA sequencing technologies have facilitated the in silico forensic analysis of human microbiome. Specific microbial species or communities obtained from the crime scene provide evidence of human contacts and their body fluids. The microbial community is influenced by geographic, ethnic, lifestyle, and environmental factors such as urbanization. An understanding of the effects of these external stressors on the human microbiome and determination of stable and changing elements are important in selecting appropriate targets for investigation. In this study, the Forensic Microbiome Database (FMD) (http://www.fmd.jcvi.org) containing the microbiome data of various locations in the human body in 35 countries was used. We focused on skin, saliva, vaginal fluid, and stool and found that the microbiome distribution differed according to the body part as well as the geographic location. In the case of skin samples, Staphylococcus species were higher than Corynebacterium species among Asians compared with Americans. Holdemanella and Fusobacterium were specific in the saliva of Koreans and Japanese populations. Lactobacillus was found in the vaginal fluids of individuals in all countries, whereas Serratia and Enterobacter were endemic to Bolivia and Congo, respectively. This study is the first attempt to collate and describe the observed variation in microbiomes from the forensic microbiome database. As additional microbiome databases are reported by studies worldwide, the diversity of the applications may exceed and expand beyond the initial identification of the host., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cho and Eom.)

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a "stressed state" due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2021 Atheesha Singh and Tobias George Barnard.)

Background: Metagenomic next-generation sequencing (mNGS) of body fluids is an emerging approach to identify occult pathogens in undiagnosed patients. We hypothesized that metagenomic testing can be simultaneously used to detect malignant neoplasms in addition to infectious pathogens., Methods: From two independent studies (n = 205), we used human data generated from a metagenomic sequencing pipeline to simultaneously screen for malignancies by copy number variation (CNV) detection. In the first case-control study, we analyzed body fluid samples (n = 124) from patients with a clinical diagnosis of either malignancy (positive cases, n = 65) or infection (negative controls, n = 59). In a second verification cohort, we analyzed a series of consecutive cases (n = 81) sent to cytology for malignancy workup that included malignant positives (n = 32), negatives (n = 18), or cases with an unclear gold standard (n = 31)., Results: The overall CNV test sensitivity across all studies was 87% (55 of 63) in patients with malignancies confirmed by conventional cytology and/or flow cytometry testing and 68% (23 of 34) in patients who were ultimately diagnosed with cancer but negative by conventional testing. Specificity was 100% (95% CI 95-100%) with no false positives detected in 77 negative controls. In one example, a patient hospitalized with an unknown pulmonary illness had non-diagnostic lung biopsies, while CNVs implicating a malignancy were detectable from bronchoalveolar fluid., Conclusions: Metagenomic sequencing of body fluids can be used to identify undetected malignant neoplasms through copy number variation detection. This study illustrates the potential clinical utility of a single metagenomic test to uncover the cause of undiagnosed acute illnesses due to cancer or infection using the same specimen.

Optimisation of microbiological diagnostics in primarily sterile body fluids is required. Our objective was to apply EUCAST's RAST on primarily sterile body fluids in blood culture bottles with total lab automation (TLA) and to compare results to our reference method Vitek2 in order to report susceptibility results earlier. Positive blood culture bottles (BACTEC™ Aerobic/Anaerobic/PEDS) inoculated with primarily sterile body fluids were semi-automatically subcultured onto Columbia 5% SB agar, chocolate agar, MacConkey agar, Schaedler/KV agar and Mueller-Hinton agar. On latter, cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin were added. After 6 h, subcultures and RAST were imaged and MALDI-TOF MS was performed. Zone sizes were digitally measured and interpreted following RAST breakpoints for blood cultures. MIC values were determined using Vitek2 panels. During a 1-year period, 197 Staphylococcus aureus, 91 Enterococcus spp., 38 Escherichia coli, 11 Klebsiella pneumoniae and 8 Pseudomonas aeruginosa were found. Categorical agreement between RAST and MIC was 96.5%. Comparison showed no very major errors, 2/7 (28.6%) and 1/7 (14.3%) of major errors for P. aeruginosa and meropenem and ciprofloxacin, 1/9 (11.1%) for K. pneumoniae and ciprofloxacin, 4/69 (7.0%) and 3/43 (5.8%) for Enterococcus spp. and vancomycin and ampicillin, respectively. Minor errors for P. aeruginosa and meropenem (1/8; 12.8%) and for E. coli and ciprofloxacin (2/29; 6.5%) were found. 30/550 RAST measurements were within area of technical uncertainty. RAST is applicable and performs well for primarily sterile body fluids in blood culture bottles, partially better than blood-based RAST. Official EUCAST evaluation is needed.

Background: Clinicians commonly obtain endotracheal aspirate cultures (EACs) in the evaluation of suspected ventilator-associated infections. However, bacterial growth in EACs does not distinguish bacterial colonization from infection and may lead to overtreatment with antibiotics. We describe the development and impact of a clinical decision support algorithm to standardize the use of EACs from ventilated PICU patients., Methods: We monitored EAC use using a statistical process control chart. We compared the rate of EACs using Poisson regression and a quasi-experimental interrupted time series model and assessed clinical outcomes 1 year before and after introduction of the algorithm., Results: In the preintervention year, there were 557 EACs over 5092 ventilator days; after introduction of the algorithm, there were 234 EACs over 3654 ventilator days (an incident rate of 10.9 vs 6.5 per 100 ventilator days). There was a 41% decrease in the monthly rate of EACs (incidence rate ratio [IRR]: 0.59; 95% confidence interval [CI] 0.51-0.67; P < .001). The interrupted time series model revealed a preexisting 2% decline in the monthly culture rate (IRR: 0.98; 95% CI 0.97-1.0; P = .01), immediate 44% drop (IRR: 0.56; 95% CI 0.45-0.70; P = .02), and stable rate in the postintervention year (IRR: 1.03; 95% CI 0.99-1.07; P = .09). In-hospital mortality, hospital length of stay, 7-day readmissions, and All Patients Refined Diagnosis Related Group severity and mortality scores were stable. The estimated direct cost savings was $26 000 per year., Conclusions: A clinical decision support algorithm standardizing EAC obtainment from ventilated PICU patients was associated with a sustained decline in the rate of EACs, without changes in mortality, readmissions, or length of stay., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2021 by the American Academy of Pediatrics.)

Background: Acinetobacter species have been a leading cause of nosocomial infections, causing significant morbidity and mortality over the entire world including Ethiopia. The most important features of A. baumannii are its ability to persist in the hospital environment and rapidly develop resistance to a wide variety of antibiotics. This study aimed to determine trend of antimicrobial resistance in Acinetobacter species over a five years period., Method: A retrospective data regarding occurrence and antimicrobial resistance of Acinetobacter species recovered from clinical specimens referred to the national reference laboratory was extracted from microbiology laboratory data source covering a time range from 2014 to 2018. Socio-demographic characteristics and laboratory record data was analyzed using SPSS 20., Results: A total of 102 strains of Acinetobacter species were analyzed from various clinical specimens. Majority of them were from pus (33.3%) followed by blood (23.5%), urine (15.6%) and body fluid (11.7%). Significant ascending trends of antimicrobial resistance was shown for meropenem (12.5% to 60.7%), ceftazidime (82.1% to 100%), ciprofloxacin (59.4% to 74.4%), ceftriaxone (87.1% to 98.6%), cefepime (80.0% to 93.3%) and pipracillin- tazobactam (67.8% to 96.3%). However, there was descending trend of antimicrobial resistance for tobramycin (56.5% to 42.8%), amikacin (42.1% to 31.4%) and trimethoprim-sulfamethoxazole (79.0 to 68.2%). The overall rate of carbapenem non-susceptible and multidrug resistance rates in Acinetobacter species were 56.7% and 71.6%.respectively., Conclusion: A five year antimicrobial resistance trend analysis of Acinetobacter species showed increasing MDR and resistance to high potent antimicrobial agents posing therapeutic challenge in our Hospitals and health care settings. Continuous surveillance and appropriate infection prevention and control strategies need to be strengthened to circumvent the spread of multidrug resistant pathogens in health care facilities., Competing Interests: The authors have declared that no competing interests exist.

Rapid and direct observation of fungal spores or hyphae in clinical liquid specimens poses a challenge for the diagnosis of invasive fungal infection. To allow rapid detection of fungal pathogens, we designed a new method of fungal cell detection involving double fluorescence staining with calcium fluorescent white (CFW) and SYTOX green combined with single-cell real-time imaging flow cytometry (IFC). IFC allowed quick detection and analysis of detailed morphology of the spores and pseudohyphae of Candida albicans, and small hyphae and typical truncated large mycelia of Aspergillus fumigatus. Further, cell sorting based on fluorescence, the width-to-height ratio and bright-field parameters preferentially identified spores or hyphae with a typical cell wall. The specificity and overall coincidence rate of IFC for fungi detection in common clinical samples were 100% and 98.18%, respectively. Moreover, the detection rate by IFC (102/105, 97.14%) was significantly higher (P = 0.002) than that by wet mount method (89/105, 84.5%). Therefore, IFC is a reliable diagnostic method with a high potential for application for rapid diagnosis of fungal infection in the clinic.

We developed a metagenomic next-generation sequencing (mNGS) test using cell-free DNA from body fluids to identify pathogens. The performance of mNGS testing of 182 body fluids from 160 patients with acute illness was evaluated using two sequencing platforms in comparison to microbiological testing using culture, 16S bacterial PCR and/or 28S-internal transcribed ribosomal gene spacer (28S-ITS) fungal PCR. Test sensitivity and specificity of detection were 79 and 91% for bacteria and 91 and 89% for fungi, respectively, by Illumina sequencing; and 75 and 81% for bacteria and 91 and 100% for fungi, respectively, by nanopore sequencing. In a case series of 12 patients with culture/PCR-negative body fluids but for whom an infectious diagnosis was ultimately established, seven (58%) were mNGS positive. Real-time computational analysis enabled pathogen identification by nanopore sequencing in a median 50-min sequencing and 6-h sample-to-answer time. Rapid mNGS testing is a promising tool for diagnosis of unknown infections from body fluids.

Purpose: Analyze the diagnostic performance of suction drainage fluid culture for acute surgical site infection, which has not been specifically reported in spine surgery patients., Method: This was a retrospective single-center observational study including data from 363 patients who underwent aseptic instrumented spine surgery between 2015 and 2017. A suction drain was inserted in all cases. Data analyzed were patient age, gender, ASA score, indication for surgery (degenerative disease, tumor, trauma), spine level (cervical, thoracic, lumbar), procedure performed and spine level, operative time, body temperature, postoperative C-reactive protein time-curve, clinical aspect of surgical scar, bacteriology results of suction drainage fluid, and in case of revision surgery, lavage fluid. Major criteria for periprosthetic infection proposed by the Musculoskeletal Infection Society (MSIS) were accepted as the gold standard for the diagnosis of acute surgical site infection., Results: The overall rate of surgical site infection was 6.9% (5.76% for 1- or 2-level fusion, 5.81% for 3- or 4-level fusion, and 15.6% for 5-level fusion and above). The suction drain was withdrawn on the second postoperative day in 44.1% of cases and the third day in 39.1%. The sensitivity of suction drainage fluid culture for the diagnosis of surgical site infection was 20% [95%CI 6.8-40.7%] with a 96.2% [95%CI 93.2-97.9] specificity., Conclusion: The diagnostic performance of suction drainage fluid culture after aseptic instrumented spine surgery for acute surgical site infection is insufficient to warrant its use in routine practice.

Background: Information about the use of flow cytometry in the diagnosis of male urethritis is scarce. The current study aims to evaluate the performance of flow cytometry on first-voided urine in males with infectious urethritis (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis)., Methods: Male patients of the Andrology Centre (Tartu University Hospital, Estonia) were recruited during the period March 2015 -January 2018. Cases included 306 patients with infectious urethritis caused by Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and/or Trichomonas vaginalis. The control group consisted of 192 patients without uro-genital complaints, negative tests for C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis from first-voided urine and no inflammation in first-voided urine, mid-stream urine and urine after prostate massage. C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis were detected from first-voided urine using polymerase chain reaction (PCR) method. First-voided urine was analysed using urine particle analyzer Sysmex UF-500i., Results: The most prevalent infection was chlamydia (64.1%), followed by Mycoplasma genitalium (20.9%), gonorrhoea (7.8%) and trichomoniasis (1.6%). Gonorrhoea caused the highest flow-cytometric leucocyte/bacteria count, followed by chlamydia and Mycoplasma genitalium. Trichomonas vaginalis showed nearly absent inflammation in first-voided urine. Using an empiric flow-cytometry diagnostic threshold for urethritis in first-voided urine (leucocytes ≥ 15/μl and bacteria ≥ 20/μl) the total calculated sensitivity was over 90%. However, when applying such criteria for deciding whether to perform first-voided urine PCR for C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis or not, we could miss 23 cases with infectious urethritis that makes up 7,5% of all proven cases., Conclusions: Flow cytometry of first-voided urine can be considered as a rapid and objective screening method in case of suspected male infectious urethritis., Competing Interests: The authors have declared that no competing interests exist.

Secretory otitis media (SOM) is characterized by persistence of fluid in the middle ear, often following an episode of acute otitis media . Our hypothesis is that failure to eliminate bacterial or viral pathogens may result in persistent low-grade inflammation. In this study, we analyzed inflammatory mediators in middle ear fluids from 67 children with SOM. This was combined with determinations of viable bacteria by culture along with detection of bacterial and viral genetic material by real-time polymerase chain reaction (PCR). The inflammatory mediators found at the highest concentrations (>30 ng/mL) were stem cell growth factor-β (median 110 ng/mL), CXCL1, IL-16, IL-8, migration inhibitory factor, CXCL10, and CXCL9. Among bacterial pathogens, Moraxella catarrhalis and Haemophilus influenzae dominated, regardless of detection methods, while rhinovirus dominated among viral pathogens. Middle ear fluid levels of interleukin (IL)-1α, IL-17, IL-1β, fibroblast growth factor basic, and tumor necrosis factor correlated strongly with presence of bacteria detected either by culture or PCR, while IL-1RA, IL-3, IL-6, IL-8, CCL3, CCL4, and granulocyte-colony stimulating factor correlated significantly with real-time PCR values. CXCL10, CXCL9, CCL2, and TRAIL correlated significantly with viral nucleic acid levels. To conclude, persistence of viral and bacterial pathogens may fuel persistent inflammation in SOM. Bacteria caused a broad inflammatory response, while viruses chiefly elicited the interferon-induced chemokines CXCL9 and CXCL10.

Background: Microbiome and metagenomic studies have given rise to a new understanding of microbial colonization of various human tissues and their ability to impact our health. One human microbiome growing in notoriety, the vaginal microbiome, stands out given its importance for women's health, and is peculiar in terms of its relative bacterial composition, including its simplicity and typical domination by a small number of Lactobacillus species. The loss of Lactobacillus dominance is associated with disorders such as bacterial vaginosis, and efforts are now underway to understand the ability of Lactobacillus species to colonize the vaginal tract and adapt to this dynamic and acidic environment. Here, we investigate how various Lactobacillus species often isolated from the vaginal and intestinal cavities genomically and transcriptionally respond to iterative growth in simulated vaginal fluid., Results: We determined the genomes and transcriptomes of L. acidophilus, L. crispatus, L. fermentum, L. gasseri, and L. jensenii and compared profiles after 50, 100, 500, and 1000 generations of iterative passages in synthetic vaginal fluid. In general, we identified relatively few genetic changes consisting of single nucleotide polymorphisms, with higher counts occurring more frequently in non-vaginal isolated species. Transcriptional profiles were more impacted over time and tended to be more extensive for species that typically do not dominate the vaginal tract, reflecting a more extensive need to adapt to a less familiar environment., Conclusions: This study provides insights into how vaginal and non-vaginal Lactobacillus species respond and adapt to a simulated vaginal environment. Overall, trends indicate high genomic stability for all species involved, with more variability in the transcriptome especially for non-dominant species of the vaginal tract.

Nine Gram-stain-positive cocci, coccobacilli or short, rod-shaped strains recovered from clinical sources from patients located in two Canadian provinces and one environmental source were extensively studied. Clinical sources included blood cultures, cerebral spinal fluid, lymph node, lung biopsy and peritoneal fluid. Through 16S rRNA gene and whole genome sequencing analyses, the strains were found to cluster into three groups, closest to but distinguished from other genera in the family Propionibacteriaceae . The genomes from these bacteria had high G+C content, ranging from 67.8-69.56 mol%, and genome sizes of 3.02-4.52 Mb. Biochemical and chemotaxonomic properties including branched-chain cellular fatty acids, l-lysine diaminopimelic acid (ll-DAP) and cell-wall type A3γ (ll-DAP-gly) containing ll-DAP, alanine, glycine and glutamic acid were found and so the strains were therefore deemed to be consistent with other new genera in this family. Based on this investigation, we propose Enemella gen. nov., Enemella evansiae sp. nov., Enemella dayhoffiae sp. nov. and Parenemella sanctibonifatiensis gen. nov., sp. nov. for these taxa. Misidentified taxon ' Ponticoccus gilvus ' was found to be assignable to Enemella evansiae based on this study.

We studied the effects of IL-1β, IL-8, TNFα, and prostaglandin E2α in concentrations typically observed in health and during inflammation on the growth of vaginal microbiota and its resistance to factors inhibiting the synthesis of proteins, nucleic acids, and peptidoglycans. An increase in the cytokine levels, characteristic of inflammation, inhibits the growth of Lactobacillus population and improves its resistance to adverse factors. The growth of the population of opportunistic microorganisms (S. aureus, E. coli) is stimulated under these conditions, while their resistance to adverse factors decreases. Hence, it seems that the cytokines regulate the behavior of the host cells and of its bacterial symbionts.

Background: Blood-borne pathogen exposures (BBPEs) pose a risk to health care workers (HCWs). Needlestick injuries (NSIs) have declined overall, but not for surgical HCWs. There are limited data regarding BBPEs among medical students (MSs) in their clinical years. We aimed to quantify this risk for third- and fourth-year MSs., Methods: A literature review was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. The PUBMED database was searched to identify studies of third- and fourth-year MSs using the terms BBPE, NSI, and MS. Studies of other HCWs were excluded if MS data were not extractable. Additional studies were identified from references. Descriptive analysis was performed., Results: Seven of 171 articles published from 2002 to 2018 met study criteria. All used self-reported data from surveys/questionnaires. One-third of MSs reported BBPEs (n = 194/600, 32.3%) with a mean of 1 in 3.09 and a median of 1 in 3.53 (range: 1 in 1.9-8.3 students). Most events were NSIs (144/194, 74%) with a mean of 1 NSI per 4.05 MSs and median of 1 in 4.625 (range: 1 in 2.47-10.71). The remaining BBPEs reported included blood and bodily fluid splashes (n = 37, 19%), other mucocutaneous exposures (n = 7, 3.6%), and uncategorized injuries (n = 2, 1%)., Conclusions: One-third of senior MSs reported BBPEs during clinical rotations. Most BBPEs were NSIs. Quantifying this risk allows for anticipatory education and protocol development to protect students and other new HCWs. Educational efforts focused on NSI prevention before and during clinical rotations may help reduce BBPEs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Metagenomic next generation sequencing (mNGS) is becoming increasingly available for pathogen detection directly from clinical specimens. These tests use target-independent, shotgun sequencing to detect potentially unlimited organisms. The promise of this methodology to aid infection diagnosis is demonstrated through early case reports and clinical studies. However, the optimal role of mNGS in clinical microbiology remains uncertain., Content: We reviewed studies reporting clinical use of mNGS for pathogen detection from various specimen types, including cerebrospinal fluid, plasma, lower respiratory specimens, and others. Published clinical study data were critically evaluated and summarized to identify promising clinical indications for mNGS-based testing, to assess the clinical impact of mNGS for each indication, and to recognize test limitations. Based on these clinical studies, early testing recommendations are made to guide clinical utilization of mNGS for pathogen detection. Finally, current barriers to routine clinical laboratory implementation of mNGS tests are highlighted., Summary: The promise of direct-from-specimen mNGS to enable challenging infection diagnoses has been demonstrated through early clinical studies of patients with meningitis or encephalitis, invasive fungal infections, community acquired pneumonia, and other clinical indications. However, the proportion of patient cases with positive clinical impact due to mNGS testing is low in published studies and the cost of testing is high, emphasizing the importance of improving our understanding of 'when to test' and for which patients mNGS testing is appropriate., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Probiotic products have been shown to have beneficial effects on human hosts, but what happens in the gastrointestinal tract after its ingestion remains unclear. Our aim was to investigate the changes within the small intestines after a single intake of a fermented milk product containing a probiotic. We have periodically collected the small-intestinal fluids from the terminal ileum of seven healthy subjects for up to 7 h after ingestion by small-intestinal fluid perfusion using an endoscopic retrograde bowel insertion technique. The bacterial composition of the terminal ileum clearly revealed that the ingested probiotics ( Lactobacillus casei strain Shirota: LcS and Bifidobacterium breve strain Yakult: BbrY) occupied the ileal microbiota for several hours, temporarily representing over 90% of the ileal microbiota in several subjects. Cultivation of ileal fluids showed that under a dramatic pH changes before reaching the terminal ileum, a certain number of the ingested bacteria survived (8.2 ± 6.4% of LcS, 7.8 ± 11.0% of BbrY). This means that more than 1 billion LcS and BbrY cells reached the terminal ileum with their colony-forming ability intact. These results indicate that there is adequate opportunity for the ingested probiotics to continuously stimulate the host cells in the small intestines. Our data suggest that probiotic fermented milk intake affects intestinal microbes and the host, explaining part of the process from the intake of probiotics to the exertion of their beneficial effects on the host.

Background: Comparing the composition of microbial communities among groups of interest (e.g., patients vs healthy individuals) is a central aspect in microbiome research. It typically involves sequencing, data processing, statistical analysis and graphical display. Such an analysis is normally obtained by using a set of different applications that require specific expertise for installation, data processing and in some cases, programming skills., Results: Here, we present SHAMAN, an interactive web application we developed in order to facilitate the use of (i) a bioinformatic workflow for metataxonomic analysis, (ii) a reliable statistical modelling and (iii) to provide the largest panel of interactive visualizations among the applications that are currently available. SHAMAN is specifically designed for non-expert users. A strong benefit is to use an integrated version of the different analytic steps underlying a proper metagenomic analysis. The application is freely accessible at http://shaman.pasteur.fr/ , and may also work as a standalone application with a Docker container (aghozlane/shaman), conda and R. The source code is written in R and is available at https://github.com/aghozlane/shaman . Using two different datasets (a mock community sequencing and a published 16S rRNA metagenomic data), we illustrate the strengths of SHAMAN in quickly performing a complete metataxonomic analysis., Conclusions: With SHAMAN, we aim at providing the scientific community with a platform that simplifies reproducible quantitative analysis of metagenomic data.

Leptospirosis is a globally distributed disease associated with reproductive failures in livestock; however, its pathogenesis has not been fully elucidated. Results from the present study indicate there is a presence of Leptospira sp. in organs and fluids of fetuses from ewes slaughtered in the semiarid region of Brazil. Twenty-nine fetuses from 23 ewes determined to be Leptospira sp.-positive using PCR were sampled (14 and 15 in dry and rainy seasons, respectively). Fetal samples of blood, central nervous system (CNS), lung, liver, spleen, stomach contents, peritoneal fluid, kidney, bladder, urine and reproductive system were collected. Diagnostic methods included the microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Of the 29 fetuses, 24 (82.8 %) had at least one Leptospira sp.-positive organ or fluid, as determined using PCR, and of a total of 209 samples, 62 (29.7 %) contained leptospiral DNA. Of the 99 samples collected during the dry season, 42 (42.4 %) were positive, and of 110 samples collected during the rainy season, 20 (18.2 %) were positive (P = 0.0001). There was deoxyribonucleic acid (DNA) sequencing of three samples of kidney, CNS and liver, and in all of these, there was 99.3 % similarity with Leptospira interrogans. Leptospires were present in cultures of pooled samples from fetuses with deformities. Results indicate there is vertical (maternal-to-fetus) transmission which would represent an alternative transmission route for the spread of Leptospira sp. in ewes, suggesting molecular detection is essential in the investigation of leptospirosis in fetuses to identify animals that have been infected with this bacterium., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Development of a properly functioning gastrointestinal tract (GIT) at an early age is critical for the wellbeing and lifetime productivity of dairy cattle. The role of early microbial colonization on GIT development in neonatal cattle and the associated molecular changes remain largely unknown, particularly for the small intestine. In this study, we performed artificial dosing of exogenous rumen fluid during the early life of the calf, starting at birth through the weaning transition at 8 wk. Six calves were included in this study. At 8 wk of age, tissue from the ileum was collected and subjected to host transcriptome and microbial metatranscriptome analysis using RNA sequencing. A total of 333 genes showed significant differential expression (DE) (fold-change ≥2; adjusted P < 0.1, mean read-count ≥10) between the treated and control calves. Gene ontology analysis indicated that these DE genes are predominantly associated with processes related to the host immune response ( P < 0.0001). Association analysis between the host gene expression and the microbial genus abundance identified 57 genes as having significant correlation with the ileum microbial genera ( P < 0.0001). Of these, three genes showed significant association with six microbial genera: lysozyme 2 ( LYZ2 ), fatty acid binding protein 5 ( FABP5 ), and fucosyltransferase (FUT1 ). Specifically, the profound increase in expression of LYZ2 in treated calves suggests the initiation of antibacterial activity and innate response from the host. Despite the limitation of a relatively small sample size, this study sheds light on the potential impact of early introduction of microbes on the small intestine of calves.

Objectives: Pneumonia still remains a problem from the clinical and public health viewpoint because of the relevant epidemiological burden. The etiological diagnosis is important in the light of avoiding unnecessary antibiotic treatment and choosing the most appropriate therapeutical approach. This study is aimed at providing evidence on the proportion of microbiological ascertainment in pneumonia-related hospitalizations in one of the most important teaching hospitals in Rome., Methods: The study relied on the record linkage of two administrative databases of the same hospital: the electronic hospital discharge register and the microbiology laboratory surveillance database., Results: 2819 records were identified, where 46% had a microbiological ascertainment, significantly higher in males than in females (51% vs 40%) and in cases of pneumonia reported in secondary diagnosis instead of primary diagnosis (52% vs 42%). Medical patients had significantly lower proportion of ascertainment compared to surgical patients (43% vs 67%) whereas there were not differences between patients with emergency and elective admission. The overall mortality was 17%. Mortality was significantly higher: in surgical compared to medical patients (27% vs 15%), in ventilated compared to not ventilated patients (41% vs 11%), in cases with secondary diagnosis of pneumonia compared to a primary diagnosis (23% vs 11% ) and in hospitalized in intensive care unit-ICU- rather than in non-ICU (71% vs 12%)., Conclusion: The proportion of microbiological ascertaiment in pneumonia remains less than 50%. Albeit in line with other evidence, this result should call the attention on the impact of unknown etiological diagnosis on antibiotic treatment and resistance.

Background: Neisseria gonorrhoeae (gonococcus) is the etiologic agent for the sexually transmitted Infection gonorrhea, a disease with a significant global public health impact. The treatment regimen for gonorrhea has been changed frequently over the past few decades due to the organism's propensity for developing antibiotic resistance. This study investigated antimicrobial susceptibility patterns of quinolones, third-generation cephalosporin, and other relevant antimicrobials found in N. gonorrhoeae isolated from men presenting with urethral discharge at selected healthcare facilities in Addis Ababa, Ethiopia, with the aim of revising the national treatment regimen based on the information generated from this study., Methods: A total of 599 male patients presenting with urethral discharge were included in the current study. Urethral discharge specimens were cultured on Modified Thayer Martín media and suspected gonococcal colonies were confirmed using Oxidase and Superoxol tests followed by identification through a commercial kit (API-NHR). Antimicrobial susceptibility testing was performed by the Kirby-Bauer disc diffusion method using ciprofloxacin (5μg), ceftriaxone (30μg), cefixime (5μg), cefoxitin (30 μg), penicillin (10μg) and spectinomycin (100 μg) on enriched GC agar. Minimum Inhibitory Concentration (MIC) was also carried out using concentration gradient strips (E-tests) of the same antimicrobial agents., Results: The prevalence of gonococcal isolates in the current study was 69%. Out of the 361 gonococcal isolates, close to 68% were fluoroquinolone non-susceptible, with 60% resistant and 7% having an intermediate status. However, all tested isolates were susceptible to ceftriaxone. In addition, all of the isolates have shown reduced non-susceptibility to spectinomycin and cefoxitin., Conclusion: The prevalence of gonococcal isolates in men presenting with urethral discharge at selected healthcare facilities in Addis Ababa, Ethiopia was found to be high. The high level of fluoroquinolone resistance observed in gonococcal isolates recovered in this study necessitates revision of the national syndromic treatment guideline., Competing Interests: The authors have declared that no competing interests exist.

The lung microbiome is associated with host immune response and health outcomes in experimental models and patient cohorts. Lung microbiome research is increasing in volume and scope; however, there are no established guidelines for study design, conduct, and reporting of lung microbiome studies. Standardized approaches to yield reliable and reproducible data that can be synthesized across studies will ultimately improve the scientific rigor and impact of published work and greatly benefit microbiome research. In this review, we identify and address several key elements of microbiome research: conceptual modeling and hypothesis framing; study design; experimental methodology and pitfalls; data analysis; and reporting considerations. Finally, we explore possible future directions and research opportunities. Our goal is to aid investigators who are interested in this burgeoning research area and hopefully provide the foundation for formulating consensus approaches in lung microbiome research.

Purpose: To investigate the impact of early postoperative drainage fluid culture positivity on the development of clinically relevant postoperative pancreatic fistula (POPF) after pancreaticoduodenectomy (PD)., Methods: We assessed the positive prevalence, distribution, and drug sensitivity of microorganisms in drainage fluid collected on postoperative day (POD) 1 after PD from 465 patients., Results: Culture results were positive in pancreaticojejunostomy (PJ) drainage fluid from 26.0% of patients. Similar distributions of microorganisms were observed in the bile juice and PJ/hepaticojejunostomy (HJ) drainage fluid from these patients. PJ drain culture positivity was associated with an elevated drainage amylase level and with preoperative biliary drainage. No associations were seen between HJ drainage culture positivity and the drainage amylase and bilirubin levels. PJ drainage culture positivity was found to be an independent predictor of grade B/C POPF. According to the antibiogram, the bacteria identified were likely to be resistant to prophylactic antibiotics., Conclusions: PJ drainage culture positivity on POD 1 in combination with an elevated drainage amylase level is an early predictor of grade B/C POPF. PJ drainage culture positivity may be attributable to bile juice contamination caused by intraoperative spillage and early postoperative leakage from the PJ anastomotic sites.

As data on pediatric invasive candidiasis (IC) and the antifungal susceptibility pattern of associated isolates are scarce in Iran, this study aimed to determine species distribution and antifungal susceptibility profile of Candida species isolated from pediatric patients with suspected or documented IC. A total of 235 yeast strains recovered from normally sterile body fluids of patients admitted at the intensive care units of Children's Medical Centre, Tehran, Iran, were identified using CHROMagar Candida, molecular methods (ITS PCR-RFLP and sequencing), and MALDI-TOF. Susceptibility to amphotericin B, fluconazole, voriconazole, micafungin, and anidulafungin was determined according to the European on Antimicrobial Susceptibility testing reference microdilution method (EUCAST E.Def 7.3.1). Candida albicans (53.6%), C. parapsilosis (24.7%), and C. tropicalis (8.5%) were the most common species, followed by C. lusitaniae (4.3%), C. glabrata (3.0%), C. guilliermondii and C. orthopsilosis (each 1.7%), C. kefyr (1.3%), C. dubliniensis (0.8%), and C. intermedia (0.4%). Amphotericin B MICs were ≤1 mg/l for all Candida isolates. C. albicans isolates were susceptible to all five antifungal agents. All C. parapsilosis isolates categorised as intermediate to micafungin and anidulafungin, except two isolates that had the MICs >2 mg/l for micafungin. MIC50, MIC90, and MIC range for fluconazole were 0.25 mg/l, 1 mg/l, and 0.125 - ≥32 mg/l, respectively. Fluconazole and voriconazole showed 100% activity against the most prevalent Candida species. The low resistance rate, favorable safety profile and low cost of fluconazole make it a reasonable choice for treatment of candidemia/invasive candidemia in Iran., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: Real-time (RT) PCR is a rapid and accurate method that is widely used for the detection of Mycobacterium tuberculosis complex (MTB). The aim of this study was to evaluate and compare the performance of the Genedia MTB/NTM Detection Kit and the Anyplex plus MTB/NTM Detection kit in the detection of MTB and nontuberculous mycobacteria (NTM) in clinical specimens., Methods: From October 2017 to February 2018, 236 respiratory specimens and 137 non-respiratory specimens, from patients with suspected tuberculosis, were examined. AFB smear, culture, and RT-PCR using the Genedia MTB/NTM Detection kit (Green Cross Medical Science Corp.) and the Anyplex plus MTB/NTM Detection kit (Seegene) were applied. PCR performance in the detection of MTB and NTM was evaluated in relation to culture results and between the two assays., Results: Culture was positive for MTB in 30 (8.0%) of the 373 specimens and for NTM in 23 (6.2%). The sensitivity and specificity of MTB detection with the Genedia kit were 76.7% and 99.7%, respectively, whereas the Anyplex kit sensitivity and specificity for MTB detection were 86.7% and 97.5%, respectively. Both kits exhibited the same sensitivity (73.9%) for NTM detection, and the specificity was 100% and 99.4% for the Genedia and Anyplex kits, respectively., Conclusions: The Genedia and Anyplex kits demonstrated high sensitivity and specificity for the detection of MTB and NTM. Both kits have a high concordance rate and can be used more widely in clinical laboratories for the early detection of tuberculosis., (© 2019 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc.)

Transfaunation is supposed to stimulate normal rumen function and has been used as an ancillary treatment for indigestion. Although it is widely recommended, there are little research data on the efficacy and the necessary volume. The objective of the prospective clinical trial was the evaluation of the therapeutic efficacy of two different transfaunation volumes which can be obtained under practical conditions. Forty-five cattle suffering from indigestion were included in the study. A scoring system for the classification of rumen fluid was used. Scores were given in accordance with the importance of the parameter as an indication of microbial dysfunction. Animals with disturbed rumen fluid composition and activity were randomly assigned into 3 groups. Group 1 received 1 L of rumen fluid, group 2 received 5 L of rumen fluid and group 3 (control group) received 5 L of body temperature water. Rumen fluid analysis was repeated on days 1 and 4 after transfaunation. The feed intake of the animals was recorded. After the transfaunation of 1 L and 5 L, the rumen fluid score improved significantly from day 0 to days 1 and 4. Rumen fluid samples in the control group showed no significant improvement from day 0 to day 1. No significant differences were observed between the two treatment groups. But significant differences between the improvement of group 1 and the control group on days 1 and 4 and significant differences between group 2 and the control group on day 1 were detected. Small volumes of rumen fluid are easily obtainable by stomach tubes fitted with suction pumps. In summary, the transfaunation of as little as 1 L of rumen fluid caused significant improvement in the activity of rumen flora in cows suffering from indigestion., (© 2019 The Authors. Journal of Animal Physiology and Animal Nutrition published by Blackwell Verlag GmbH.)

Background: Acute otitis media (AOM) is a common ear infection caused by respiratory viruses and bacteria of the nasopharynx. The present study aimed to detect various respiratory viruses and bacteria in middle ear fluid (MEF) and nasopharyngeal aspirates (NPA) using polymerase chain reaction (PCR)., Methods: We collected MEF and NPA samples from 122 pediatric patients with AOM. Real-time PCR detected 11 types of respiratory viruses (respiratory syncytial virus A/B, parainfluenza virus 1/2/3, human metapneumovirus, influenza virus A/B, adenovirus, human bocavirus and rhino virus) and 7 types of bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Streptococcus pyogenes, Legionella pneumophila and Moraxella catarrhalis). MEF specimens were also examined using bacterial culture., Results: At least 1 respiratory viral or bacterial pathogen was detected in MEF of 120 cases (98%) by viral and bacterial PCR and of 93 cases (76%) by viral PCR and bacterial culture. Respiratory viruses were detected in NPA of 84 cases (69%) and MEF of 67 cases (55%). The most common virus detected in MEF was respiratory syncytial virus (21%), followed by parainfluenza virus (15%). All the viruses present in MEF were also detected in NPA specimens. Bacteria were detected by PCR in MEF of 109 cases (89%); H. influenzae was the most frequently detected (65%)., Conclusions: In many cases, pediatric AOM was found to constitute a respiratory polymicrobial infection. Multiplex PCR was useful to detect multiple respiratory viruses and bacteria in AOM. To understand intractable AOM, further studies regarding the clinical features of each viral and bacterial coinfection are required.

The success of fecal microbiota transplantation (FMT) as a treatment for Clostrioides difficile infection (CDI) has stirred excitement about the potential for microbiota transplantation as a therapy for a wide range of diseases and conditions. In this article, we discuss vaginal microbiota transplantation (VMT) as "the next frontier" in microbiota transplantation and identify the medical, regulatory, and ethical challenges related to this nascent field. We further discuss what we anticipate will be the first context for testing VMT in clinical trials, prevention of the recurrence of a condition referred to as bacterial vaginosis (BV). We also compare clinical aspects of VMT with FMT and comment on how VMT may be similar to or different from FMT in ways that may affect research design and regulatory decisions.

Several lines of evidence suggest that children born via Cesarean section (C-section) are at greater risk for adverse health outcomes including allergies, asthma and obesity. Vaginal seeding is a medical procedure in which infants born by C-section are swabbed immediately after birth with vaginal secretions from the mother. This procedure has been proposed as a way to transfer the mother's vaginal microbiome to the child, thereby restoring the natural exposure that occurs during vaginal birth that is interrupted in the case of babies born via C-section. Preliminary evidence indicates partial restoration of microbes. However, there is insufficient evidence to determine the health benefits of the procedure. Several studies, including trial, are currently underway. At the same time, in the clinic setting, doctors are increasingly being asked to by expectant mothers to have their babies seeded. This article reports on the current research on this procedure and the issues it raises for regulators, researchers, physicians, and patients.

Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis , Salmonella enterica , Aspergillus fumigatus , and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. The PCR cycle threshold ( C T ) was used to measure amplifiable cfDNA. In spiked samples, the median C T values for M. tuberculosis , S. enterica , and EBV cfDNA were significantly lower in blood collected in K 2 EDTA tubes than those in Streck and PAXgene blood collection tubes, and they were was significantly lower in urine preserved with EDTA (EDTA-urine) than in urine preserved with Streck reagent (Streck-urine). Blood and urine samples from TB patients preserved with K 2 EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosis C T values than with the Streck blood collection tube and Streck urine preservative. Processing delay increased the median pathogen C T values for Streck and PAXgene but not K 2 EDTA blood samples and for urine preserved with Streck reagent but not EDTA. Double-spin compared with single-spin plasma separation increased the median pathogen C T regardless of blood collection tube. No differences were observed between whole urine and supernatant and between fresh and thawed plasma and urine after 24 weeks at -80°C. Larger plasma and urine volumes in contrived and patient samples showed a significantly lower median M. tuberculosis C T These findings suggest that large-volume single-spin K 2 EDTA-plasma and EDTA-whole urine with up to a 24-h processing delay may optimize pcfDNA detection., (Copyright © 2019 Murugesan et al.)

Bronchoalveolar lavage (BAL) is recommended for diagnosing lung infiltrates (LI) in patients with hematologic malignancy (HM). Prospective data on the impact of BAL on survival are still lacking. We conducted a prospective observational study on patients who performed BAL for LI among 3055 HM patients hospitalized from January to September 2018. The BAL was performed in 145 out of 434 patients who developed LI, at a median time of four days from LI detection. The median age was 60 (1-83). Most patients had an acute myeloid leukemia/myelodisplastic syndrome (81), followed by lymphoma (41), acute lymphoblastic leukemia (27), and other types of HM (36). A putative causal agent was detected in 111 cases (76%), and in 89 cases (61%) the BAL results provided guidance to antimicrobial treatment. We observed a significantly improved outcome of LI at day +30 in patients who could receive a BAL-driven antimicrobial treatment (improvement/resolution rate: 71% vs 55%; P = .04). Moreover, we observed a significantly improved outcome in 120-day overall survival (120d-OS) (78% vs 59%; P = .009) and 120-day attributable mortality (120d-AM) (11% vs 30%; P = 0.003) for patients who could receive a BAL-driven treatment. The multivariate analysis showed that BAL-driven antimicrobial treatment was significantly associated with better 120d-OS and lower 120d-AM. We did not observe any severe adverse events. In conclusion BAL allows detection of a putative agent of LI in about 75% of cases, it is feasible and well tolerated in most cases, demonstrating that a BAL-driven antimicrobial treatment allows improvement of clinical outcome and survival., (© 2019 Wiley Periodicals, Inc.)

Objectives: Conventional microbiological procedures for the isolation of bacteria from biological fluids consist of culture on solid media and enrichment broth. However, these methods can delay the microbiological identification for up to 4 days. The aim of this study was to evaluate the analytical performance of Sysmex UF500i (Sysmex, Kobe, Japan) as a screening method for the detection of bacteria in different biological fluids in comparison with direct Gram staining and the conventional culture on solid media and enrichment broth., Methods: A total of 479 biological fluid samples were included in the study (180 ascitic, 131 amniotic, 56 synovial, 40 cerebrospinal, 36 pleural, 24 peritoneal, 9 bile and 3 pericardial fluids). All samples were processed by conventional culture methods and analyzed by flow cytometry. Direct Gram staining was performed in 339 samples. The amount of growth on culture was recorded for positive samples., Results: Bacterial and white blood cell count by flow cytometry was significantly higher among culture positive samples and samples with a positive direct Gram stain compared to culture negative samples. Bacterial count directly correlated with the amount of growth on culture (Kruskall-Wallis H χ2(3) = 11.577, p = 0.009). The best specificity (95%) for bacterial count to predict culture positivity was achieved applying a cut-off value of 240 bacteria/μL., Conclusions: Bacterial and white blood cell counts obtained with flow cytometry correlate with culture results in biological fluids. Bacterial count can be used as a complementary method along with the direct Gram stain to promptly detect positive samples and perform other diagnostic techniques in order to accelerate the bacterial detection and identification., Competing Interests: Sysmex España S.L. financially supported the study providing reagents and equipment. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Infections of the sterile body sites, when occurs typically have greater clinical urgency and these infections could be life threatening. For the appropriate management of patient, early detection and identification of organism is crucial. Hence, the present study was designed to evaluate the prevalence, organism profile and antimicrobial susceptibility pattern of isolates obtained from infection of body fluids., Methods: Laboratory and clinical records of all the sterile body fluid except blood and cerebrospinal fluid submitted to the Department of Microbiology, B. P. Koirala Institute of Health Sciences during the period of 5 years (January 2012 to December 2016) were analyzed., Results: Among 1835 samples, 196 fluids samples showed growth of organisms with an isolation rate of 10.68%. Escherichia coli (E. coli) was the most common organism followed by Staphylococcus aureus (S. aureus) and Acinetobacter calcoaceticus-baumannii complex. Antimicrobial susceptibility testing showed variable degree of resistance. Thirty percentage of organisms were multi-drug resistant, 10% were extensively-drug resistant, none was pan-drug resistant, 35% of E. coli and K. pneumoniae were extended spectrum ?-lactamase producer and 30% of S. aureus were methicillin-resistant S. aureus. The study showed increasing trends of multi-drug resistant, extensively-drug resistant, methicillin-resistant S. aureus and extended spectrum ?-lactamase over the years., Conclusions: The study showed infection rate of 10% among normally sterile body fluids. E.coli, S. aureus and Acinetobacter calcoaceticus-baumannii complex are the common organisms. The infections are associated with multi drug resistant organisms. Routine surveillance of multi-drug resistant in infection of body fluids is necessary to guide treatment.

Background: While all healthcare workers are exposed to occupational hazards, workers in sub-Saharan Africa have higher rates of occupational exposure to infectious diseases than workers in developed countries. Identifying prevalence and context of exposure to blood and bloodborne pathogens may help guide policies for prevention., Objective: This systematic review examined occupational exposure rates to blood and bloodborne pathogen among healthcare workers in sub-Saharan Africa., Methods: In November 2017, a comprehensive literature search was conducted to identify studies reporting exposure of health workers in African coutnries to blood and bodily fluids. Title, abstract and full text screening were used to narrow our search. Studies more than 10 years old, or published in non-English languages were excluded., Findings: Fifteen studies reported a variety of exposures. The lifetime prevalence of needlestick injury ranged from 22-95%, and one-year prevalence ranged from 39-91%. Studies included a range of descriptive statistics of knowledge, attitudes, practice and access factors related to exposures. Two studies reported 21-32% of respondents linked poor knowledge or training with prevention of needlestick injuries. Rates of recapping needles ranged from 12-57% in four studies. Attitudes were generally positive toward occupational safety procedures while access was poor., Conclusions: The high burden of blood and bloodborne pathogen exposures demonstrated here indicates a high risk for contracting bloodborne illnesses. Although the data are sparse, implementation of preventative policies based on current knowledge remains critical to minimize risk and reduce exposure. There remains a pressing need for high quality data on occupational hazards to identify the burden of exposures and inform preventive policies in Sub-Saharan Africa. Additional studies are needed to determine whether differential exposures exist between professions and the associations with knowledge, attitudes, practices, and access factors to create targeted strategies to diminish occupational hazards., Competing Interests: The authors have no competing interests to declare., (© 2019 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/.)

We report the design and operation of an integrated microfluidics system that uses cellulose ester dialysis membranes coupled with disposable carbon and copper electrodes for monitoring and concentration of microliter scale biofluid samples. Dialysis membranes are typically used for buffer exchange, but in this work, membranes with 100-500 Da MWCO were evaluated for feasibility in concentrating small volume samples. This is an alternative to the use of centrifugation, ultrafiltration, and evaporative methods, where quantitative inline monitoring of sample concentration is challenging. The impact of draw solution used, osmotic concentration gradient, pH, and temperature were studied for the optimized concentration of bodily fluids. A system using sucrose in the draw solution generated the best results, with water removal rates of 0.023 mL min -1 . PBS, urine, and saliva samples were concentrated up to 20-fold (PBS), 15-fold (urine), and 5-fold (saliva) in less than 3 h. The osmotic system further showed a 5-fold increase in the electrochemical signal for detecting pyocyanin, a biomarker for early diagnostics of the Pseudomonas aeruginosa pathogen in urine and saliva samples. Overall, the osmotic system can be easily integrated with point of care diagnostic systems for low cost improvement in signal amplification and limit of detection.

Liquid wastes from clinical biology automated systems are currently evacuated in the urban network after chemical treatment to eliminate a possible risk of infection. Since these wastes are ecotoxic because of the presence of numerous chemical reagents, we studied their intrinsic microbicidal power towards a selection of infectious agents widely found in clinical specimens. The objective was to determine if an additional anti-infectious treatment before elimination is necessary. Thus, we evaluated the bactericidal effect of liquid wastes of several automated systems towards four bacterial species (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis) and their virucidal activity against a non-enveloped virus, resistant in the environment (adenovirus). This effect was determined for different exposure times. Our results showed that the antibacterial activity was highly variable depending on the waste-bacteria pair considered (varying from no activity to complete sterilization of a strong bacterial inoculum). The liquid wastes were on the other hand globally inactive towards adenovirus.

Background: Although artificial insemination (AI) was developed as a means of controlling disease transmission, pathogens can still be transmitted to females in semen used for AI. In addition, bacteria can cause deterioration in sperm quality during storage. Semen becomes contaminated by the male's normal bacterial flora as it passes out of the reproductive tract but potential pathogens may also contaminate the semen. Therefore, semen samples from stallions to be used for AI are tested before the breeding season to minimize transmission of pathogens to inseminated mares. In Sweden, semen samples are tested at the National Veterinary Institute, Uppsala (SVA). For the present study, a retrospective analysis was made of potentially pathogenic bacteria isolated from samples submitted to the SVA from 2007 to 2017., Results: In our study, Taylorella equigenitalis was found infrequently (53 out of 25,512 samples), representing 11 out of 2308 stallions. If T. equigenitalis was detected, the stallions were treated with antibiotics and re-tested later in the same year. Klebsiella pneumoniae and beta haemolytic streptococci were the most commonly found potential pathogens, whereas Pseudomonas aeruginosa was also isolated occasionally. There were considerable differences in the number of species isolated each year., Conclusions: Potential pathogens were identified in relatively few of the samples submitted to SVA during this period, with T. equigenitalis not being identified since 2015. Of the other potential pathogens, K. pneumoniae and beta haemolytic streptococci were the most common. The information is relevant for determining guidelines on the testing and treatment of stallions before breeding.

Invasive fungal infections by opportunistic yeasts have increased concomitantly with the growth of an immunocompromised patient population. Misidentification of yeasts can lead to inappropriate antifungal treatment and complications. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy is a promising method for rapid and accurate identification of microorganisms. ATR-FTIR spectroscopy is a standalone, inexpensive, reagent-free technique that provides results within minutes after initial culture. In this study, a comprehensive spectral reference database of 65 clinically relevant yeast species was constructed and tested prospectively on spectra recorded (from colonies taken from culture plates) for 318 routine yeasts isolated from various body fluids and specimens received from 38 microbiology laboratories over a 4-month period in our clinical laboratory. ATR-FTIR spectroscopy attained comparable identification performance with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In a preliminary validation of the ATR-FTIR method, correct identification rates of 100% and 95.6% at the genus and species levels, respectively, were achieved, with 3.5% unidentified and 0.9% misidentified. By expanding the number of spectra in the spectral reference database for species for which isolates could not be identified or had been misidentified, we were able to improve identification at the species level to 99.7%. Thus, ATR-FTIR spectroscopy provides a new standalone method that can rival MALDI-TOF MS for the accurate identification of a broad range of medically important yeasts. The simplicity of the ATR-FTIR spectroscopy workflow favors its use in clinical laboratories for timely and low-cost identification of life-threatening yeast strains for appropriate treatment., (Copyright © 2019 American Society for Microbiology.)

In the present study, we investigated the potential of Bifidobacterium spp., isolated from ruminal fluid samples from buffaloes (Bubalus bubalis) for conjugated linoleic acid (CLA) production. A total of 294 isolates were obtained from 86 ruminal fluid samples using Bifidus Selective Medium (BSM) medium, and based on phospoketolase assay, 24 isolates were presumptively confirmed to be Bifidobacterium species. Further, the isolates were confirmed morphologically, biochemically and by PCR assays for genus specific (16s rDNA) and transaldolase genes. All 24 strains were positive for conversion of linoleic acid (LA) to CLA by spectrophotometric screening. Gas chromatographic analysis showed that the strains produced cis9, trans11 and tran10, cis12 CLA isomers in LA-supplemented deMan-Rogosa-Sharpe (MRS) broth. The strains were identified as B. thermophilum (n = 21) and B. pseudolongum (n = 3) based on 16 rDNA sequence analysis. The study shows that Bifidobacterium spp., present in the rumens of buffaloes produce CLA from LA and the strains may have the potential to be used as probiotics to enhance the nutraceutical value of ruminant food products., (Copyright © 2019. Published by Elsevier Ltd.)

Transformation is one of the mechanisms of acquisition of foreign genetic material leading to the emergence of multidrug resistant (MDR) bacteria. Recently, human serum albumin (HSA) was shown to specifically increase transformation frequency in the nosocomial pathogen Acinetobacter baumannii. To further assess the relevance of HSA as a possible modulator of A. baumannii transformation in host-pathogen interactions, in this work we examined the effect of different human fluids. We observed a significant increase in transformation frequencies in the presence of pleural fluid, whole blood cells and liquid ascites, and to a lesser extent with urine. The observed effects correlate with both HSA and bacterial content found in the assayed patient fluids. Taken together, these results are in agreement with our previous findings that highlight HSA as a possible host signal with the ability to trigger natural transformation in A. baumannii., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Three previously unidentified Gram-positive anaerobic coccoid bacteria, strains KhD-2 T , KHD4 T , and Kh-D5 T , isolated from a vaginal swab, were characterized using the taxonogenomics concept. The phylogenic analysis, phenotypic characteristics, and genotypic data presented in this report attest that these three bacteria are distinct from previously known bacterial species with standing in nomenclature and represent three new Peptoniphilus species. Strain KhD-2 T is most closely related to Peptoniphilus sp. DNF00840 and Peptoniphilus harei (99.7% and 98.2% identity, respectively); strain KHD4 T to Peptoniphilus lacrimalis (96%) and strain Kh-D5 T to Peptoniphilus coxii (97.2%). Strains KhD-2 T , KHD4 T , and Kh-D5 T DNA G+C contents are, respectively, 34.23%, 31.87%, and 49.38%; their major fatty acid was C 16:0 (41.6%, 32.0%, and 36.4%, respectively). We propose that strains KhD-2 T (=CSUR P0125 = DSM 101742), KHD4 T (=CSUR P0110 = CECT 9308), and Kh-D5 T (=CSUR P2271 = DSM 101839) be the type strains of the new species for which the names Peptoniphilus vaginalis sp. nov., Peptoniphilus raoultii sp. nov., and Peptoniphilu pacaensis sp. nov., are proposed, respectively., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)