Development and preliminary evaluation toward a new tuberculosis treatment monitoring tool: the PATHFAST TB LAM Ag assay.

The PATHFAST TB LAM Ag assay is based on a chemiluminescent enzyme immunoassay to quantify lipoarabinomannan (LAM) in sputum within 1 h, and was developed as an alternative to conventional culture methods for monitoring tuberculosis (TB) treatment. This study aimed to evaluate the analytical performance and initial clinical feasibility of using five Mycobacterium tuberculosis variants, 178 non-tuberculous mycobacteria (NTM), 34 upper respiratory and oral cavity microorganisms, 100 sputum specimens from untreated patients, and potential interfering substances, including 27 drugs. The results reveled a single-site repeatability coefficient of variation (CV) of 5.2%-7.0%, and a multi-site reproducibility CV of 7.1%-8.4%. The limit of blank, limit of detection, and limit of quantification were 3.03 pg/mL, 6.67 pg/mL, and 7.44 pg/mL, respectively. Linearity was observed over the analytical measurement range (10.0 pg/mL-50,000 pg/mL), and no hook effect was observed. The assay tended to cross-react with slow-growing NTMs, but not with common upper respiratory and oral cavity microorganisms, except Nocardia asteroides, Nocardia farcinica, and Tsukamurella paurometabola. No interference was observed in the presence of mucin, blood, or major anti-TB, anti-HIV, and anti-pneumonia drugs. Regarding clinical performance, the assay had a sensitivity of 88.8% (95% CI: 80.0%-94.0%) and specificity of 100.0% (95% CI: 83.9%-100.0%) using mycobacterial culture as the reference standard, and a correlation (Spearman's r = -0.770) was observed between LAM concentration and time to detection of culture. These findings show, for the first time, that the PATHFAST TB LAM Ag assay has potential value for monitoring TB treatment.

RNA-seq analysis revealed the pathogenicity of Vibrio vulnificus to American eel (Anguilla rostrata) and the strategy of host anti-V. vulnificus infection.

Vibrio vulnificus is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-V. vulnificus infection remains uncertain. In this study, American eels were infected with different dose of V. vulnificus to determine the LD50. Then, bacterial load in the liver and kidney histopathology were assessed post the LD50 of V. vulnificus infection. Additionally, gene expressions of 18 immune related genes in the liver, spleen and kidney were detected. Furthermore, transcriptome sequencing and enrichment of differentially expressed genes (DEGs) were analyzed in the eel spleens between pre-infection (Con_0), post-36 h (Vv_36), and post-60 h (Vv_60) infection. The results showed that LD50 of V. vulnificus to American eels was determined to be 5.0 × 105 cfu/g body weight, and the bacterial load peaked at 24 and 12 h post the infection (hpi) in the kidney and liver, respectively. The histopathology was highlighted by necrotic hepatocytes and splenic cells, congestion blood vessels in liver and spleen, atrophied glomeruli and vacuolization of renal tubular epithelial cells. The results of RT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated expression post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 16 DEGs play essential role to the immunosuppression in American eels, and the protein-protein interactions shed light on the widespread upregulation GEGs related to metabolism and immune response maintained the host cell homeostasis post the V. vulnificus infection, shedding new light on our understanding of the V. vulnificus pathogenesis towards understudied American eel and the host anti-V. vulnificus infection strategies in gene transcript.

Exploration of culturable bacterial associates of aphids and their interactions with entomopathogens.

Aphids shelter several bacteria that benefit them in various ways. The associates having an obligatory relationship are non-culturable, while a few of facultative associates are culturable in insect cell lines, axenic media or standard microbiology media. In the present investigation, isolation, and characterization of the culturable bacterial associates of various aphid species, viz., Rhopalosiphum maidis, Rhopalosiphum padi, Sitobion avenae, Schizaphis graminum, and Lipaphis erysimi pseudobrassicae were carried out. A total of 42 isolates were isolated using different growth media, followed by their morphological, biochemical, and molecular characterization. The isolated culturable bacterial associates were found to belong to the genera Acinetobacter, Bacillus, Brevundimonas, Cytobacillus, Fictibacillus, Planococcus, Priestia, Pseudomonas, Staphylococcus, Sutcliffiella, and Tumebacillus which were grouped under seven families of four different orders of phyla Bacillota (Firmicutes) and Pseudomonata (Proteobacteria). Symbiont-entomopathogen interaction study was also conducted, in which the quantification of colony forming units of culturable bacterial associates of entomopathogenic fungal-treated aphids led us to the assumption that the bacterial load in aphid body can be altered by the application of entomopathogens. Whereas, the mycelial growth of entomopathogens Akanthomyces lecanii and Metarhizium anisopliae was found uninhibited by the bacterial associates obtained from Sitobion avenae and Rhopalosiphum padi. Analyzing persistent aphid microflora and their interactions with entomopathogens enhances our understanding of aphid resistance. It also fosters the development of innovative solutions for agricultural pest management, highlighting the intricate dynamics of symbiotic relationships in pest management strategies.

Salmonid Rickettsial Septicemia (SRS) disease dynamics and Atlantic salmon immune response to Piscirickettsia salmonis LF-89 and EM-90 co-infection.

In Chile, Piscirickettsia salmonis contains two genetically isolated genogroups, LF-89 and EM-90. However, the impact of a potential co-infection with these two variants on Salmonid Rickettsial Septicemia (SRS) in Atlantic salmon (Salmo salar) remains largely unexplored. In our study, we evaluated the effect of P. salmonis LF-89-like and EM-90-like co-infection on post-smolt Atlantic salmon after an intraperitoneal challenge to compare changes in disease dynamics and host immune response. Co-infected fish had a significantly lower survival rate (24.1%) at 21 days post-challenge (dpc), compared with EM-90-like single-infected fish (40.3%). In contrast, all the LF-89-like single-infected fish survived. In addition, co-infected fish presented a higher presence of clinical lesions than any of the single-infected fish. The gene expression of salmon immune-related biomarkers evaluated in the head kidney, spleen, and liver showed that the EM-90-like isolate and the co-infection induced the up-regulation of cytokines (e.g., il-1ß, ifnγ, il8, il10), antimicrobial peptides (hepdicin) and pattern recognition receptors (PRRs), such as TLR5s. Furthermore, in serum samples from EM-90-like and co-infected fish, an increase in the total IgM level was observed. Interestingly, specific IgM against P. salmonis showed greater detection of EM-90-like antigens in LF-89-like infected fish serum (cross-reaction). These data provide evidence that P. salmonis LF-89-like and EM-90-like interactions can modulate SRS disease dynamics in Atlantic salmon, causing a synergistic effect that increases the severity of the disease and the mortality rate of the fish. Overall, this study contributes to achieving a better understanding of P. salmonis population dynamics.

Estimating bacterial load in S. aureus and E. coli bacteremia using bacterial growth graph from the continuous monitoring blood culture system.

BACKGROUND: We examined whether the time to positivity (TTP) and growth and detection plot graph (GDPG) created by the automated blood culture system can be used to determine the bacterial load in bacteremic patients and its potential association correlation with disease severity. METHODS: Known bacterial inocula were injected into the blood culture bottles. The GDPGs for the specific inocula were downloaded and plotted. A cohort of 30 consecutive clinical cultures positive for S. aureus and E. coli was identified. Bacterial load was determined by comparing the GDPG with the "standard" curves. Variables associated with disease severity were compared across 3 bacterial load categories (< 100, 100-1000, > 1000 CFU/mL). RESULTS: S. aureus growth was sensitive to the blood volume obtained whereas E. coli growth was less so. A 12-hour delay in sample transfer to the microbiology laboratory resulted in a decrease in TTP by 2-3 h. Mean TTP was 15 and 10 h for S. aureus and E. coli, respectively, which correlates with > 1000 CFU/mL and 500-1000 CFU/ml. For S. aureus, patients with a bacterial load > 100 CFU/mL had a higher mortality rate, (OR for death = 9.7, 95% CI 1.6-59, p = 0.01). Bacterial load > 1000 CFU/mL had an odds ratio of 6.4 (95% CI1.2-35, p = 0.03) to predict an endovascular source. For E. coli bacteremia, we did not find any correlations with disease severity. CONCLUSION: GDPG retrieved from the automated blood culture system can be used to estimate bacterial load. S.aureus bacterial load, but not E.coli, was associated with clinical outcome.

Transcriptome analysis of host anti-Aeromonas hydrophila infection revealed the pathogenicity of A. hydrophila to American eels (Anguilla rostrata).

Aeromonas hydrophila is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-A. hydrophila infection remains uncertain. In this study, LD50 of A. hydrophila to American eels was determined and bacterial load in the liver and kidney of eels was assessed post 2.56 doses of LD50 of A. hydrophila infection. The results showed that the LD50 of A. hydrophila to American eels was determined to be 3.9 × 105 cfu/g body weight (7.8 × 106 cfu/fish), and the bacterial load peaked at 36 h post the infection (hpi) in the liver. Then, the histopathology was highlighted by congestion in splenic blood vessels, atrophied glomeruli, and necrotic hepatocytes. Additionally, the results of qRT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 10 hub DEGs and 7 encoded proteins play essential role to the anti-A. hydrophila infection in American eels. Pathogenicity of A. hydrophila to American eels and RNA-seq of host anti-A. hydrophila infection were firstly reported in this study, shedding new light on our understanding of the A. hydrophila pathogenesis and the host immune response to the A. hydrophila infection strategies in gene transcript.

A threshold of 100 or more colony-forming units on the anesthesia machine predicts bacterial pathogen detection: a retrospective laboratory-based analysis.

PURPOSE: Preventing the spread of pathogens in the anesthesia work area reduces surgical site infections. Improved cleaning reduces the percentage of anesthesia machine samples with ≥ 100 colony-forming units (CFU) per surface area sampled. Targeting a threshold of < 100 CFU when cleaning anesthesia machines may be associated with a lower prevalence of bacterial pathogens. We hypothesized that anesthesia work area reservoir samples returning < 100 CFU would have a low (< 5%) prevalence of pathogens. METHODS: In this retrospective cohort study of bacterial count data from nine hospitals, obtained between 2017 and 2022, anesthesia attending and assistants' hands, patient skin sites (nares, axilla, and groin), and anesthesia machine (adjustable pressure-limiting valve and agent dials) reservoirs were sampled at case start and at case end. The patient intravenous stopcock set was sampled at case end. The isolation of ≥ 1 CFU of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Enterococcus, vancomycin-resistant Enterococcus, gram-negative (i.e., Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter spp.) or coagulase-negative Staphylococcus was compared for reservoir samples returning ≥ 100 CFU vs those returning < 100 CFU. RESULTS: Bacterial pathogens were isolated from 24% (7,601/31,783) of reservoir samples, 93% (98/105) of operating rooms, and 83% (2,170/2,616) of cases. The ratio of total pathogen isolates to total CFU was < 0.0003%. Anesthesia machine reservoirs returned ≥ 100 CFU for 44% (2,262/5,150) of cases. Twenty-three percent of samples returning ≥ 100 CFU were positive for ≥ 1 bacterial pathogen (521/2,262; 99% lower confidence limit, 22%) vs 3% of samples returning < 100 CFU (96/2,888; 99% upper limit, 4%). CONCLUSIONS: Anesthesia machine reservoir samples returning < 100 CFU were associated with negligible pathogen detection. This threshold can be used for assessment of terminal, routine, and between-case cleaning of the anesthesia machine and equipment. Such feedback may be useful because the 44% prevalence of ≥ 100 CFU was comparable to the 46% (25/54) reported earlier from an unrelated hospital.

RéSUMé: OBJECTIF: La prévention de la propagation des agents pathogènes dans la zone de travail de l'anesthésie réduit les infections du site opératoire. L'amélioration du nettoyage réduit le pourcentage d'échantillons de l'appareil d'anesthésie présentant ≥ 100 unités de formation de colonie (UFC) par surface échantillonnée. Le fait de cibler un seuil < 100 UFC lors du nettoyage des appareils d'anesthésie pourrait être associé à une prévalence plus faible d'agents pathogènes bactériens. Nous avons émis l'hypothèse que les échantillons des réservoirs de la zone de travail d'anesthésie < 100 UFC résulteraient en une faible prévalence (< 5 %) d'agents pathogènes. MéTHODE: Dans cette étude de cohorte rétrospective des données de décompte bactérien de neuf hôpitaux, obtenues entre 2017 et 2022, les mains des anesthésiologistes et des assistant·es en anesthésie, les sites cutanés des patient·es (narines, aisselles et aines) et les réservoirs de l'appareil d'anesthésie (soupape de réglage de limitation de pression et cadrans d'agent) ont été échantillonnés au début et à la fin de chaque cas. Les échantillons sur l'ensemble de robinets d'arrêt intraveineux des patient·es ont été prélevés à la fin de chaque cas. L'isolement de ≥ 1 UFC de staphylocoque doré, de staphylocoque doré résistant à la méthicilline, d'entérocoque, d'entérocoque résistant à la vancomycine, de staphylocoque à Gram négatif (c.-à-d. Klebsiella, Acinetobacter, Pseudomonas et Enterobacter spp.) ou à coagulase négative a été comparé pour les échantillons de réservoir retournant ≥ 100 UFC vs ceux qui comportaient < 100 UFC. RéSULTATS: Des bactéries pathogènes ont été isolées dans 24 % (7601/31 783) des échantillons de réservoir, 93 % (98/105) des salles d'opération et 83 % (2170/2616) des cas. Le rapport entre le nombre total d'isolats d'agents pathogènes et le nombre total d'UFC était de < 0,0003 %. Les échantillons pris sur les réservoirs d'appareils d'anesthésie ont retourné ≥ 100 UFC dans 44 % (2262/5150) des cas. Vingt-trois pour cent des échantillons retournés ≥ 100 UFC étaient positifs pour ≥ 1 agent pathogène bactérien (521/2262; limite de confiance inférieure à 99 %, 22 %) vs 3 % des échantillons retournant < 100 UFC (96/2888 ; 99 % de la limite supérieure, 4 %). CONCLUSION: Les échantillons pris sur les réservoirs de l'appareil d'anesthésie comportant < 100 UFC étaient associés à une détection négligeable d'agents pathogènes. Ce seuil peut être utilisé pour l'évaluation du nettoyage final, de routine et entre les cas de l'appareil et de l'équipement d'anesthésie. Une telle rétroaction peut être utile parce que la prévalence de 44 % de ≥ 100 UFC était comparable aux 46 % (25/54) rapportés précédemment dans un autre hôpital.

The efficacy of 2780 nm Er,Cr;YSGG and 940 nm Diode Laser in root canal disinfection: A randomized clinical trial.

OBJECTIVES: Effective disinfection of the root canals is the cornerstone of successful endodontic treatment. Diminishing the microbial load within the root canal system is crucial for healing in endodontically treated teeth. The aim of this study was to evaluate the effect of 2780 nm Er,Cr:YSGG and 940 nm diode lasers on the eradication of microorganisms from single-rooted teeth with asymptomatic apical periodontitis. MATERIALS AND METHODS: Thirty participants conforming to the inclusion criteria were randomly divided into 3 groups according to the disinfection protocol used; Conventional group: 2.5% Sodium Hypochlorite (NaOCl) and 17% EDTA solution NaOCl/EDTA, Dual laser group: 2780 nm Erbium, chromium: yttrium scandium-gallium-garnet (Er,Cr:YSGG) laser and 940 nm diode laser Er,CrYSGG/Diode, and Combined group: 17% EDTA and 940 nm diode laser EDTA/Diode. Bacterial samples were collected before and after intervention. The collected data were statistically analyzed using Friedman's test and Kruskal-Wallis test (P ≤ 0.05). RESULTS: The results of the study showed that both dual laser Er,CrYSGG/Diode and combined laser EDTA/Diode groups showed significantly less mean Log10 CFU/ml of aerobic and anaerobic bacterial counts than the conventional NaOCl/EDTA group. CONCLUSIONS: In this study we evaluated in vivo the bactericidal efficacy of three disinfection protocols for endodontic treatment of single-rooted teeth with apical periodontitis. The results indicated that both dual laser Er,CrYSGG/Diode and combined laser EDTA/Diode groups provide superior bactericidal effect compared to the conventional NaOCl/EDTA group. CLINICAL RELEVANCE: The integration of lasers into root canal disinfection protocols has demonstrated significant bacterial reduction which might promote healing and long-term success.

Clinical Manifestations and Stool Load of Atypical Enteropathogenic Escherichia coli Infections in United States Children and Adults.

BACKGROUND & AIMS: There is debate whether atypical enteropathogenic Escherichia coli (aEPEC) causes disease in adults. aEPEC is commonly detected in symptomatic and asymptomatic individuals. aEPEC, in contrast to typical EPEC, lacks bundle-forming pili, altering its pathogenicity. Here, we define for the first time the clinical manifestations of sporadic aEPEC infection in United States children and adults and determine whether EPEC load correlates with disease. METHODS: This is a retrospective case-control study of 380 inpatients/outpatients of all ages. EPEC load in stools was determined by quantitative polymerase chain reaction. RESULTS: Diarrhea, vomiting, abdominal pain, and fever were more prevalent in EPEC-positive cases than in EPEC-negative controls. aEPEC infection caused mostly acute, mild diarrhea lasting for 6 to 13 days. However, some had severe diarrhea with 10 to 40 bowel movements per day or had persistent/chronic diarrhea. Fever, vomiting, and abnormal serum sodium levels were more common in children. Adults more often reported abdominal pain and longer duration of diarrhea. Symptomatic aEPEC infection was associated with leukocytosis in 24% of patients. EPEC load >0.1% was associated with symptomatic infection; however, loads varied greatly. Co-infecting pathogens did not alter diarrhea severity or EPEC load. Longitudinal data reveal that some are colonized for months to years or are repeatedly infected. CONCLUSIONS: aEPEC is associated with a wide array of symptoms in adults, ranging from asymptomatic carriage to severe diarrhea. Higher EPEC loads are associated with presence of symptoms, but bacterial load does not predict disease or severity. Future studies are needed to understand bacterial and host factors that contribute to aEPEC pathogenicity to improve diagnostic tools and clinical care.

Factors affecting biofilm formation by bacteria on fabrics.

Fabrics act as fomites for microorganisms, thereby playing a significant role in infection transmission, especially in the healthcare and hospitality sectors. This study aimed to examine the biofilm formation ability of four nosocomial infection-causing bacteria (Acinetobacter calcoaceticus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) on cotton, polyester, polyester-cotton blend, silk, wool, viscose, and nylon, used frequently in the healthcare sector, by qualitative and quantitative methods. The impact of temperature, pH, and relative humidity (RH) on biofilm formation was also assessed. P. aeruginosa and S. aureus were strong biofilm producers, while E. coli produced weak biofilm. Wool (maximum roughness) showed the highest bacterial load, while silk (lowest roughness) showed the least. P. aeruginosa exhibited a higher load on all fabrics, than other test bacteria. Extracellular polymeric substances were characterized by infrared spectroscopy. Roughness of biofilms was assessed by atomic force microscopy. For biofilm formation, optimum temperature, pH, and RH were 30 °C, 7.0, and 62%, respectively. MgCl2 and CaCl2 were the most effective in removing bacterial biofilm. In conclusion, biofilm formation was observed to be influenced by the type of fabric, bacteria, and environmental conditions. Implementing recommended guidelines for the effective disinfection of fabrics is crucial to curb the risk of nosocomial infections. In addition, designing modified healthcare fabrics that inhibit pathogen load could be an effective method to mitigate the transmission of infections.

Inflammatory reaction and immune response of half-smooth tongue sole (Cynoglossus semilaevis) after infection with Vibrio anguillarum.

Frequently occurred bacterial diseases have seriously affected the aquaculture industry of half-smooth tongue sole (Cynoglossus semilaevis). Notably, vibriosis, with Vibrio anguillarum as one of the causative pathogens, is the most severe bacterial disease with severe inflammatory response of the host, leading to high mortality rates. In the present study, we explored the relationship between bacterial concentrations and host mortality, inflammatory reaction, and immune response in half-smooth tongue sole after infection with V. anguillarum at different concentrations (Treatment 1, 6.4 × 105 CFU/mL; Treatment 2, 6.4 × 106 CFU/mL). The mortality of Treatment 2 (77.5%) was significantly higher than that of Treatment 1 (10%), corresponding with bacterial concentrations. Although the number of deaths varies, intensive deaths were observed within 24 h post infection (hpi) in both bacterial concentration groups. Histopathological analyses revealed that fish tissues were most severely damaged at 24 or 48 hpi, and Treatment 2 was more severe than Treatment 1. A qRT-PCR-based detection method with virulence factor gene empA was established to quantify the bacterial loads in various tissues, and the bacterial loads were the highest at 24 hpi in Treatment 2, and at 48 hpi in Treatment 1. Additionally, the expression levels of complement genes (C5a, C3, C5, and C6), inflammatory factors (IL-1ß, TNF-α, and IL-10), and other immune-related genes (jak2, NF-κB1, stat3, and tlr3) were increased in various tissues after infection in both treatment groups, with most genes being most expressed at 24 or 48 hpi, and expression levels of inflammatory factors in Treatment 2 were higher than those in Treatment 1. Moreover, the expression of C5a was positively correlated with that of proinflammatory cytokines in both bacterial concentration groups. According to the results of this study, 24-48 hpi was a key node for early vibriosis detection and intervention. Compared with the low mortality of Treatment 1, the mass death of fish in Treatment 2 was suggested to be caused by uncontrolled excessive inflammatory reaction induced by the overactivation of complement system, especially C5a. We believe these results could provide theoretical basis for prevention, evaluation, and treatment of vibrio disease in tongue sole aquaculture, and lay a solid foundation for future functional analyses.

Pathogenicity of Edwardsiella anguillarum to American eels (Anguilla rostrata) and RNA-seq analysis of host immune response to the E. anguillarum infection.

Edwardsiella anguillarum is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-E. anguillarum infection remains uncertain. In this study, LD50 of E. anguillarum to American eels was determined and bacterial load in the liver and kidney of eels was assessed post the LD50 of E. anguillarum infection. The results showed that LD50 of E. anguillarum to American eels was determined to be 2.5 × 105 cfu/g body weight, and the bacterial load peaked at 36 and 72 h post the infection (hpi) in the kidney and liver, respectively. Then, the histopathology was highlighted by congestion in splenic blood vessels, atrophied glomeruli, and necrotic hepatocytes, as well as ultrastructural pathology in the kidney were charactered by acute nephritis, showing necrosis of the renal tubular epithelial cells, glomerular capillaries dilate, mitochondria swelling and ribosomes separate from the endoplasmic reticulum. Furthermore, the results of qRT-PCR revealed that 12 host immune-related genes showed significantly up or downregulated post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 6 hub DEGs play essential role to the anti-E. anguillarum infection in American eels. Pathogenicity of E. anguillarum to American eels and hub genes related host anti- E. anguillarum infection were firstly reported in this study, shedding new light on our understanding of the E. anguillarum pathogenesis and the host immune response to the E. anguillarum infection strategies in gene transcript.

Comparative evaluation of Duragen® and skim milk extenders to enhance ram semen quality and fertility: A promising alternative in ovine artificial insemination.

This study aimed to investigate the effectiveness of Duragen® and skimmed milk (SM) extenders on the quality parameters, bacterial load and fertilization ability of stored ram semen. A total of 50 ejaculates from Sardi rams (n = 5) aged 2.5-3 years, were collected and stored in Duragen® and SM at 15°C. The motilities and velocity parameters generated by the CASA system were then evaluated at 0, 8 and 24 h of storage. Afterward, bacterial loads of sperm extended in Duragen® and SM were determined at 0, 5 and 24 h of incubation. In addition, ewes (n = 100) aged 2 years, have been chosen in the same herd. The selected ewes were then synchronized and inseminated using semen extended in Duragen® and SM and stored for 5 h at 15°C. The results revealed that total and progressive motilities, straight velocity (VSL), straightness (SRT), lateral head displacement (ALH) and beat cross frequency (BCF) were not affected by the extender type after 24 h of storage (p > .05). However, curvilinear velocity (VCL), velocity average path (VAP), linearity (LIN) and wobble (WOB) showed higher values in Duragen® compared with SM extender after 24 h of storage (p < .05). Bacterial loads were observed mainly in sperm stored in SM at 5 h (183 UFC/mL) and at 24 h (357 UFC/mL) of incubation. However, the only case showing a bacterial load in Duragen® is when the storage time attains 24 h (199 UFC/mL). Concerning fertility, sperm diluted in both extenders resulting in high fertility rates which reaches 66% and 73% for Duragen® and SM, respectively, with no statistical difference (p > .05). In summary, Duragen® extender decreased bacterial load in stored semen and maintained high ram sperm quality and fertility. These findings suggest that Duragen® extender could be used as SM alternative in ovine artificial insemination (OAI).

Performance of Tuberculosis Molecular Bacterial Load Assay Compared to Alere TB-LAM in Urine of Pulmonary Tuberculosis Patients with HIV Co-Infections.

Alternative tools are needed to improve the detection of M. tuberculosis (M. tb) in HIV co-infections. We evaluated the utility of Tuberculosis Molecular Bacterial Load Assay (TB-MBLA) compared to lipoarabinomannan (LAM) to detect M. tb in urine. Sputum Xpert MTB/RIF-positive patients were consented to provide urine at baseline, weeks 2, 8, 16, and 24 of treatment for TB-MBLA, culture, and LAM. Results were compared with sputum cultures and microscopy. Initial M. tb. H37Rv spiking experiments were performed to validate the tests. A total of 63 urine samples from 47 patients were analyzed. The median age (IQR) was 38 (30-41) years; 25 (53.2%) were male, 3 (6.5%) had urine for all visits, 45 (95.7%) were HIV positive, of whom 18 (40%) had CD4 cell counts below 200 cells/µL, and 33 (73.3%) were on ART at enrollment. Overall urine LAM positivity was 14.3% compared to 4.8% with TB-MBLA. Culture and microscopy of their sputum counterparts were positive in 20.6% and 12.7% of patients, respectively. Of the three patients with urine and sputum at baseline, one (33.33%) had urine TB-MBLA and LAM positive compared to 100% with sputum MGIT culture positive. Spearman's rank correction coefficient (r) between TB-MBLA and MGIT was -0.85 and 0.89 with a solid culture, p > 0.05. TB-MBLA has the promising potential to improve M. tb detection in urine of HIV-co-infected patients and complement current TB diagnostics.

Point-of-care fluorescence imaging reveals extent of bacterial load in diabetic foot ulcers.

Elevated levels of bacteria, including biofilm, increase the risk of chronic wound infection and inhibit healing. Addressing asymptomatic high bacterial loads is challenged by a lack of clinical terminology and diagnostic tools. This post-hoc multicenter clinical trial analysis of 138 diabetic foot ulcers investigates fluorescence (FL)-imaging role in detecting biofilm-encased and planktonic bacteria in wounds at high loads. The sensitivity and specificity of clinical assessment and FL-imaging were compared across bacterial loads of concern (104 -109 CFU/g). Quantitative tissue culture confirmed the total loads. Bacterial presence was confirmed in 131/138 ulcers. Of these, 93.9% had loads >104 CFU/g. In those wounds, symptoms of infection were largely absent and did not correlate with, or increase proportionately with, bacterial loads at any threshold. FL-imaging increased sensitivity for the detection of bacteria across loads 104 -109 (P < .0001), peaking at 92.6% for >108 CFU/g. Imaging further showed that 84.2% of ulcers contained high loads in the periwound region. New terminology, chronic inhibitory bacterial load (CIBL), describes frequently asymptomatic, high bacterial loads in diabetic ulcers and periwound tissues, which require clinical intervention to prevent sequelae of infection. We anticipate this will spark a paradigm shift in assessment and management, enabling earlier intervention along the bacterial-infection continuum and supporting improved wound outcomes.

DNA extraction leads to bias in bacterial quantification by qPCR.

Quantitative PCR (qPCR) has become a widely used technique for bacterial quantification. The affordability, ease of experimental design, reproducibility, and robustness of qPCR experiments contribute to its success. The establishment of guidelines for minimum information for publication of qPCR experiments, now more than 10 years ago, aimed to mitigate the publication of contradictory data. Unfortunately, there are still a significant number of recent research articles that do not consider the main pitfalls of qPCR for quantification of biological samples, which undoubtedly leads to biased experimental conclusions. qPCR experiments have two main issues that need to be properly tackled: those related to the extraction and purification of genomic DNA and those related to the thermal amplification process. This mini-review provides an updated literature survey that critically analyzes the following key aspects of bacterial quantification by qPCR: (i) the normalization of qPCR results by using exogenous controls, (ii) the construction of adequate calibration curves, and (iii) the determination of qPCR reaction efficiency. It is primarily focused on original papers published last year, where qPCR was applied to quantify bacterial species in different types of biological samples, including multi-species biofilms, human fluids, and water and soil samples. KEY POINTS: • qPCR is a widely used technique used for absolute bacterial quantification. • Recently published papers lack proper qPCR methodologies. • Not including proper qPCR controls significantly affect experimental conclusions.

Cervical pessary and cerclage placement for preterm birth prevention and cervicovaginal microbiome changes.

INTRODUCTION: Our objective was to compare the vaginal microbiome in low-risk and high-risk pregnant women and to explore a potential association between vaginal microbiome and preterm birth. MATERIAL AND METHODS: A pilot, consecutive, longitudinal, multicenter study was conducted in pregnant women at 18-22 weeks of gestation. Participants were assigned to one of three groups: control (normal cervix), pessary (cervical length ≤25 mm) and cerclage (cervical length ≤25 mm or history of preterm birth). Analysis and comparison of vaginal microbiota as a primary outcome was performed at inclusion and at 30 weeks of gestation, along with a follow-up of pregnancy and perinatal outcomes. We assessed the vaginal microbiome of pregnant women presenting a short cervix with that of pregnant women having a normal cervix, and compared the vaginal microbiome of women with a short cervix before and after placement of a cervical pessary or a cervical cerclage. RESULTS: The microbiome of our control cohort was dominated by Lactobacillus crispatus and inners. Five community state types were identified and microbiome diversity did not change significantly over 10 weeks in controls. On the other hand, a short cervix was associated with a lower microbial load and higher microbial richness, and was not correlated with Lactobacillus relative abundance. After intervention, the cerclage group (n = 19) had a significant increase in microbial richness and a shift towards community state types driven by various bacterial species, including Lactobacillus mulieris, unidentified Bifidobacterium or Enterococcus. These changes were not significantly observed in the pessary (n = 26) and control (n = 35) groups. The cerclage group had more threatened preterm labor episodes and poorer outcomes than the control and pessary groups. CONCLUSIONS: These findings indicate that a short cervix is associated with an altered vaginal microbiome community structure. The use of a cerclage for preterm birth prevention, as compared with a pessary, was associated with a microbial community harboring a relatively low abundance of Lactobacillus, with more threatened preterm labor episodes, and with poorer clinical outcomes.

Survey on the current status of the indication and implementation protocols for bile replacement in patients with external biliary drainage with special reference to infection control.

PURPOSE: This survey of bile replacement (BR) was conducted on patients with external biliary drainage to assess the current status of indication and implementation protocol of BR with special reference to infection control. METHODS: A 12-item questionnaire regarding the performance of perioperative BR was sent to 124 institutions in Japan. RESULTS: BR was performed in 29 institutions, and the indication protocol was introduced in 19. BR was performed preoperatively in 11 institutions, pre- and postoperatively in 12, and postoperatively in 6. The methods used for BR administration included oral intake (n = 10), nasogastric tube (n = 1), enteral nutrition tube (n = 3), oral intake and enteral nutrition tube (n = 6), oral intake or nasogastric tube (n = 2), nasogastric tube and enteral nutrition tube (n = 2), and oral intake or nasogastric tube and enteral nutrition tube (n = 5). In 10 of 29 institutions, isolation of multidrug-resistant organisms and a high bacterial load were considered contraindications for the use of BR. Seven institutions experienced environmental contamination. CONCLUSIONS: Given the different implementation of BR among institutions, the appropriate indication and protocols for BR should be established for infection control.

What do positive and negative Cutibacterium culture results in periprosthetic shoulder infection mean? A multi-institutional control study.

BACKGROUND: Deep tissue culture specimens obtained at the time of revision shoulder arthroplasty are commonly positive for Cutibacterium. Clinical interpretation of positive cultures can be difficult. This was a multi-institutional study evaluating the accuracy of cultures for Cutibacterium using positive control (PC) and negative control (NC) samples. The relationship between time to culture positivity and strength of culture positivity was also studied. METHODS: Eleven different institutions were each sent 12 blinded samples (10 PC and 2 NC samples). The 10 PC samples included 2 sets of 5 different dilutions of a Cutibacterium isolate from a failed total shoulder arthroplasty with a probable periprosthetic infection. At each institution, the samples were handled as if they were received from the operating room. Specimen growth, time to culture positivity, and strength of culture positivity (based on semiquantitative assessment) were reported. RESULTS: A total of 110 PC samples and 22 NC samples were tested. One hundred percent of specimens at the 4 highest dilutions were positive for Cutibacterium. At the lowest dilution, 91% of samples showed positive findings. Cutibacterium grew in 14% of NC samples. Cutibacterium grew in PC samples at an average of 4.0 ± 1.3 days, and all of these samples showed growth within 7 days. The time to positivity was significantly shorter (P < .001) and the strength of positivity was significantly higher (P < .001) in true-positive cultures compared with false-positive cultures. CONCLUSIONS: This multi-institutional study suggests that different institutions may report highly consistent rates of culture positivity for revision shoulder arthroplasty samples with higher bacterial loads. In contrast, with lower bacterial loads, the results are somewhat less consistent. Clinicians should consider using a shorter time to positivity and a higher strength of positivity as adjuncts in determining whether a tissue culture sample is a true positive.

Antiseptic versus non-antiseptic solutions for preventing infection in acute traumatic wounds: a systematic review.

OBJECTIVE: To compare the effectiveness of antiseptic solutions to that of non-antiseptic solutions in reducing wound infection rate, reducing bacterial load and improving wound healing. METHOD: We searched PubMed MEDLINE, Cochrane Central Register of Controlled Trials (CENTRAL), ProQuest Medical Database and medRxiv for randomised controlled trials (RCTs) comparing antiseptic solutions with non-antiseptic solutions in simple, uncomplicated acute traumatic wounds. Qualitative data synthesis was employed. Risk of bias was assessed and GRADE assessment was used to evaluate quality of evidence. RESULTS: In this systematic review, four studies with a total of 875 participants were included, of which two studies showed a significant reduction of wound infection rate using povidone-iodine (p=0.001 and p=0.03). The use of non-antiseptic solutions significantly increased bacterial load on acute traumatic wounds (p=0.0001). The quality of evidence was very low. No studies reported on wound healing outcome. CONCLUSION: No robust conclusions can be implemented in clinical practice. Future studies are needed to compare the use of antiseptic and non-antiseptic solutions in acute traumatic wounds.