Culture-positive Samples Research Articles

BACTERIOLOGICAL STUDY OF VENTILATOR-ASSOCIATED PNEUMONIA AND ANTIBIOTIC SUSCEPTIBILITY OF ISOLATES

Objective: The present study determined the prevalence of various aerobic bacteria causing ventilator-associated pneumonia in adult patients. Initially the bacteria causing ventilator-associated pneumonia was isolated from ET samples and studied the antimicrobial susceptibility pattern of bacterial isolates.
 Methods: Total 250 endotracheal aspiration (ET) samples were collected from patients admitted in Medical, Respiratory and Surgical ICUs for 1 y period. ET aspirates were collected under aseptic precautions and processed as per standard operating procedure for the identification of microorganisms. The antibiotic susceptibility test was performed by using Kirby-Bauer disk diffusion method as per CLSI guidelines.
 Results: Out of the 250 samples processed, culture-positive were 34.8% (n=87) and culture-negative were 65.2% (n=163). Out of 87 culture-positive samples, polymicrobial growth was observed in 9.19% (n=8) and monomicrobial growth was observed in 90.8% (n=79). Gram negative bacilli 95.7% (n=91), and gram-positive cocci isolates are 4.2% (n=4). Among Gram-negative organisms isolated, A. baumannii is the most common isolate 33 (34.7%), followed by P. aeruginosa 28 (29.5%) and K. pneumoniae 20 (21.0%) E. coli 8 (8.4%) and E. cloacae 2 (2.1%). Out of 4 Gram-positive organisms isolated, 3 (3.1%) were MSSA, and 1(1.1%) was MRSA.
 Conclusion: VAP is increasingly associated with multidrug-resistant (MDR) pathogens due to the production of ESBL, Amp C b-lactamase, Metallo-b-lactamase. It is important to carry out aggressive surveillance to determine the prevalence of MDR organisms and to generate a local antibiogram periodically. Early and appropriate antibiotics in right doses followed by de-escalation based on microbiological culture results are essential to curtail the VAP rate. VAP bundle care shall be implemented correctly.

Study of antibiotic susceptibility pattern of the isolated organisms in otitis media

Background: Otitis media (OM) encompasses a spectrum of inflammatory conditions affecting the middle ear, contributing significantly to health-care visits and prescriptions. Complications arising from OM frequently result in avoidable hearing loss, particularly in developing nations. Aims and Objectives: This study aims to ascertain the bacterial profile and antimicrobial susceptibility pattern of ear infections characterized by ear discharge complaints. Materials and Methods: The present study was conducted in the microbiology department of a tertiary care hospital over a 2-year period. The study involved 581 samples diagnosed with OM. Trained nurses collected pertinent patient information, while both nurses and an ENT doctor collected samples during specimen collection, utilizing an otoscope and headlight. Thorough documentation of relevant history and physical examinations accompanied the meticulous collection of ear discharge. Results: Culture-positive samples accounted for 96.39% (560 samples), with no growth observed in 3.61% (21 samples). Gram staining revealed 570 positive samples. Of the 581 OM samples, aerobes were isolated from 73.67% and anaerobes from 51.64%. The total isolates numbered 845, with 61.54% being aerobic and 38.46% anaerobic. Among the bacterial isolates, gram-negative bacteria slightly exceeded gram-positive bacteria, constituting 60.57% and 39.42%, respectively. Conclusion: In conclusion, the isolated aerobes and anaerobes shed light on the prevalent organisms in our region causing OM. The antibiotic sensitivity analysis conducted in this study emphasizes the identification of drugs suitable for the earliest treatment of OM.

Defining a metagenomic threshold for detecting low abundances of Providencia alcalifaciens in canine faecal samples.

Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. Providencia alcalifaciens is suspected to play a role in the disease as it was commonly found in dogs suffering from AHDS during a Norwegian outbreak in 2019. The role of this bacterium as a constituent of the canine gut microbiota is unknown, hence this study set out to investigate its occurrence in healthy dogs using metagenomics. To decrease the likelihood of false detection, we established a metagenomic threshold for P. alcalifaciens by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for P. alcalifaciens was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for P. alcalifaciens. Finally, the metagenomic threshold was used to determine the occurrence of P. alcalifaciens in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project. The metagenomic assay and qPCR had a detection limit of 1.1x103 CFU P. alcalifaciens per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique k-mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, P. alcalifaciens was found in only 1.1% (95% CI [0.0, 6.8]) of the samples, and then in low relative abundances (median: 0.04%; range: 0.00 to 0.81%). The sensitivity of the qPCR and shotgun metagenomics assay was low, as only 40% of culture-positive samples were also positive by qPCR and metagenomics. Using our detection limit, the occurrence of P. alcalifaciens in faecal samples from healthy dogs was low. Given the low sensitivity of the metagenomic assay, these results do not rule out a significantly higher occurrence of this bacterium at a lower abundance.

The molecular detection of carbapenem markers with a two-levels amplification screening protocol: epidemiological and resistome insights.

Carbapenem-resistance is a challenging healthcare concern and require specific stewardship programs. Monitoring workflows include the identification from surveillance samples, such as rectal swabs. Although culture assays represent the gold standard, data report a significant effectiveness in detecting carbapenemases genes directly from rectal swabs. The aim of this study was to evaluate the REALQUALITY Carba-Screen kit (AB ANALITICA, Padova, Italy) in detecting carbapenemases genes directly from rectal swabs, also comparing its effectiveness to culture assays results. A next-generation sequencing (NGS) was performed to investigate the positive samples about resistance markers and sequence type (ST). A number of 136 rectal swabs were collected from the University Hospital Policlinico of Catania critical wards. The samples simultaneously underwent culture and molecular assays (REALQUALITY Carba-Screen kit). The molecular method included two-steps. The first step (1 h and 6 min) rapidly excluded negative samples, while the second one (1 h and 6 min) included only positive samples for a resistance confirmation. All the positive culture samples underwent NGS analysis. Statistical evaluations demonstrated high sensitivity (100%) and detection rates (92.6%) for the REALQUALITY Carba-Screen kit, which mostly correlated to the standard workflow. All the culture positive results matched the positive molecular results, which were mainly confirmed by the NGS resistome analysis. The identified ST appeared to be diversified and different from the clinically significative strains of the same setting, furnishing interesting epidemiological evidence. The molecular detection allowed a coordinate approach in a high-prevalence multi-drug-resistance area. The rapid identification with a multi-step procedure accelerated the infection control procedures, while the preliminary negative results reduced the overtreatment episodes. The molecular method efficacy was confirmed through the NGS. In conclusion, the molecular screening could initially lead to a more conservative approach, which may be reevaluated after a culture result about the microorganisms' identification and susceptibility profile.

Levels of Cytokines in Leptospirosis Patients with Different Serovars and rfb Locus.

Leptospirosis has a wide spectrum of clinical manifestations ranging from mild to severe disease. The cytokine response is considered one of the key drivers for this varying manifestation. The different cytokine response observed in patients with leptospirosis could be due to the variation of infecting serovars. Since the rfb locus codes for the lipopolysaccharide synthesis of the bacterial cell wall, which also determines the serovar, this locus may play a role in driving a specific cytokine response in the host. We investigated 12 commonly used cytokine profiles in serum samples of culture, microscopic agglutination test (MAT), or polymerase chain reaction (PCR)-positive patients with leptospirosis. The sequences of the rfb locus in culture-positive samples were generated from whole genome sequencing and serovar status was drawn from original data published. Isolated cultures were subjected to whole genome sequencing using the PacBio RS II system, and the resulting data were used to determine the species. The recovered genomic data were annotated with the Rapid Annotation using Subsystem Technology (RAST) subsystem, and the rfb locus was extracted. The cytokine analysis was carried out using the Qiagen human ELISA kit. Eighteen samples were found to be positive by culture, while the other 7 samples were positive by PCR or MAT. Infections from Leptospira interrogans serovar Autumnalis (5), Pyrogens (3), Icterohaemorrhagiae (1) Leptospira borgpetersenii (all 7 samples clustered in same clonal group with serovar status not determined), Leptospira weilii (1 with serovar status not determined), and Leptospira kirschneri serovar Grippotyphosa (1) were included in the analysis. Three patients [infected with Leptospira interrogansserovar Autumnalis (2) and Pyrogens (1)] and 2 MAT-positive patients (highest titer against serovar Bratislava of L.interrognas) were reported to have severe clinical manifestations, while the rest had mild to moderate symptoms. Although the serum cytokine concentration of patients with severe clinical manifestation was comparatively higher, a statistically significant difference was observed only for interleukin (IL)-1β (P < 0.05). IL-10/tumor necrosis factor-alpha (TNF-α) ratio was high in patients with severe complications. In general, patients infected with L. interrogans showed higher concentration of cytokines compared to L. borgpetersenii.

Intramedullary Positive Tissue Culture Increases the Risk of Reinfection Following One-Stage Septic Revision Total Knee Arthroplasty

BackgroundIntraoperative acquisition of representative tissue samples is essential during revision arthroplasty of the infected total knee arthroplasty (TKA). While the number of intraoperative tissue samples needed to identify the organism is well described in the literature, there is still a paucity of evidence regarding the location of positive intraoperative samples and their correlation to postoperative outcomes. MethodsThere were forty-two patients who had septic failure following one-stage revision TKA for periprosthetic joint infection who were identified between January 2009 and December 2017. They were matched to a control group of patients who had successful one-stage revision TKA without septic failure. The location of positive intraoperative tissue samples was categorized as: 1) soft tissue; 2) interface between bone and prosthesis; and 3) intramedullary (IM). Chi-square, Student’s t-, and Wilcoxon Mann-Whitney U-tests were used as appropriate. Univariate and multivariate logistic regression analyses were performed to evaluate predictors of septic failure. ResultsWeight > 100 kilograms (P = .033), higher Charlson Comorbidity Index (P < .001), and positive IM cultures (P < .001) were associated with a higher risk of reinfection after one-stage revision TKA. A positive IM sample carried a nearly five-fold increase in odds of reinfection (odds ratio 4.86, 95% confidence interval 1.85 to 12.78, P = .001). ConclusionsA positive IM culture sample is significantly associated with septic failure after one-stage exchange for periprosthetic joint infection of the knee. Patients who had positive IM cultures may benefit from longer postoperative antibiotic therapy for the treatment of one-stage exchange arthroplasty to minimize the risk of reinfection.

Molecular Amplification and Cell Culturing Efficiency for Enteroviruses' Detection in Cerebrospinal Fluids of Algerian Patients Suffering from Meningitis.

Enteroviruses (EVs) represent a major cause of viral meningitis, being responsible for nearly 1 billion infections each year worldwide. Several techniques were developed to obtain better diagnostic results of EV infections. Herein, we evaluated the efficiency of EV detection through isolation on both Rhabdomyosarcoma (RD) and Vero cell line cultures, conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. Thus, 50 cerebrospinal fluid (CSF) samples belonging to patients suspected to have viral meningitis in northern Algeria were collected, anonymously numbered from 1 to 50 and subjected to the above-mentioned techniques for EV detection. Using real-time RT-PCR, 34 CSF samples were revealed to be positive for viral origin of meningitis (68%). Thirteen of them were positive when the conventional RT-PCR was used (26%), and only three samples gave positive results when the cell culture technique was used (6%). Surprisingly, two cell culture-positive CSF samples, namely, 31 and 39, were negative using RT-PCR directly on the original samples. However, they turned to be positive when amplification was carried out on their corresponding cell culture supernatant. The cell-cultured viral isolates were then identified by sequencing their viral genome's VP1 regions. All of them were revealed to belong to the echovirus 27 strain. This investigation demonstrates that RT-PCR techniques are often more sensitive, accurate and much faster, providing reliable results within a clinically acceptable timeframe. However, viral isolation on cell cultures remains crucial to obtain enough viral load for serological tests or even to avoid the rare, but existing, false negative PCR.

Occurrence of Imipenem-Resistant Uropathogenic Escherichia coli in Pregnant Women: An Insight into Their Virulence Profile and Clonal Structure.

Uropathogenic Escherichia coli (UPEC) is the predominant pathogen in Urinary Tract Infection (UTI) in pregnant and non-pregnant women. Limited studies were initiated to explore UPEC from pregnant women with respect to imipenem resistance, pathogenicity, and their clonal lineage. In this study, imipenem resistance, phylogenetic background, virulence-associated genes, and clonal characteristics in UPECs isolated from pregnant and non-pregnant cohorts were investigated. E. coli was identified biochemically from urine culture-positive samples from pregnant and non-pregnant women. Carbapenem (meropenem, ertapenem, imipenem) susceptibility was determined by Kirby-Bauer disk diffusion test. The pathogenic determinants were identified by PCR. MEGA 11 was used to interpret clonal lineages from MLST. GraphPad Prism 8.0 and SPSS 26.0 were used for statistical interpretation. Results indicated highest resistance against imipenem compared to meropenem and ertapenem in UPECs isolated from pregnant (UPECp; 63.89%) and non-pregnant (UPECnp; 87.88%) women. Although phylogroup E was predominant in both imipenem-resistant isolates, acquisition of virulence factors was higher among UPECnp than UPECp. Akin to this observation, the presence of PAI III536 and PAI IV536 was statistically significant (p < 0.05) in the former. MLST analysis revealed similar clonal lineages between UPECnp and UPECp, which showed an overall occurrence of ST405 followed by ST101, ST410, ST131, and ST1195 in UPECnp and ST167 in UPECp, respectively, with frequent occurrence of CC131, CC405. Therefore, imipenem-resistant UPECp although discrete with respect to their virulence determinants when compared to UPECnp shared similar STs and CCs, which implied common evolutionary history. Thus, empiric treatment must be restricted in UTIs to especially protect maternal and fetal health.

Good Performance of Revised Scoring Systems in Predicting Clinical Outcomes of Aeromonas Bacteremia in the Emergency Department: A Retrospective Observational Study.

Aeromonas species, Gram-negative, non-sporulating, facultative, and anaerobic bacilli, widely distributed in aquatic environments, derive various infections, including bacteremia. Most of these infections were opportunistic and found in patients with predisposing conditions. Among the infections, bacteremia remains with notable mortality, reported from 15% to 45%. However, predicting systems for assessing the mortality risk of this disease have yet to be investigated. We aimed to validate the performance of specific predictive scoring systems to assess the clinical outcomes of Aeromonas bacteremia and applied the revised systems to predict mortality risk. A retrospective observational study reviewed patients with bacteremia caused by Aeromonas spp. based on at least one positive blood culture sample collected in the emergency department from January 2012 to December 2020. The outcome was in-hospital mortality. We used seven predictive scoring systems to predict the clinical outcome. According to the effectiveness in predicting mortality, we revised three of the seven predictive scoring systems by specific characteristics to refine their risk-predicting performances. We enrolled 165 patients with bacteremia caused by Aeromonas spp., including 121 males (73.3%) and 44 females (26.7%), with a mean age of 66.1 ± 14.9 years and an average length of hospital stay of 12.4 ± 10.9 days. The overall mortality rate was 32.7% (54/165). The non-survivors had significantly higher scores in MEDS (6.7 ± 4.2 vs. 12.2 ± 3.3, p < 0.001), NEWS (4.0 ± 2.8 vs. 5.3 ± 3.0, p = 0.008), and qSOFA (0.3 ± 0.6 vs. 0.6 ± 0.7, p = 0.007). Regarding mortality risk prediction, the MEDS demonstrated the best predictive power with AUC of ROC measured up to 0.834, followed by NEWS (0.626) and qSOFA (0.608). We revised the MEDS, NEWS, and qSOFA by hemoglobin and lactate. We found that the revised scores had better powerful performance, including 0.859, 0.767, and 0.691 of the AUC of ROC, if the revised MEDS ≥10, revised NEWS ≥8, and revised qSOFA ≥2, respectively. MEDS, NEWS, and qSOFA were good tools for predicting outcomes in patients with Aeromonas spp. bacteremia. The revised MEDS, NEWS, and qSOFA demonstrated more powerful predicting performance than the original scoring systems. We suggested that patients with higher scores in revised MEDS (≥10), revised NEWS (≥8), and revised qSOFA (≥2) received early goal-directed therapy and appropriate broad-spectrum antibiotic treatment as early as possible to reduce mortality.

PCR combined with lateral flow dipstick assay (PCR-LFD) for a rapid diagnosis of melioidosis.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. Septicemic melioidosis patients have a high mortality rate within 48 hours. To develop a polymerase chain reaction (PCR) combined with a lateral flow dipstick (LFD) assay for detection of B. pseudomallei in blood samples. The PCR with wcbG gene primers and a PCR-LFD test were developed. The specificity and sensitivity were determined using the B. pseudomallei and other bacterial DNAs. They were evaluated using 43 B. pseudomallei positive blood samples and another 43 blood samples positive for other microbial infections. The detection limit of the PCR-LFD test was 50 fg of bacterial gDNA or 1.0 CFU per 200 μl of blood. All B. pseudomallei were positive while B. thailandensis and selected gram-negative bacterial strains were negative. The PCR-LFD gave all positives with all 43 B. pseudomallei culture positive patient blood samples and all negative with 43 blood samples that were culture positive for K. pneumoniae, E. gallinarum, E. faecium, E. coli, S. aureus, A. baumannii, A. hydrophila, S. haemolyticus, S. pneumoniae, P. aeruginosa, E. cloacae, S. hominis, E. aerogenes, P. mirabilis, C. neoformans, C. albicans, A. caviae, E. faecalis and K. variicola. The developed PCR-LFD assay provided 100% sensitivity and 100% specificity compared to the conventional blood culture. The technique took only 1.5 hours that is easy and quick to perform compared to the 3-7 days of culture method. The new method of PCR with LFD could facilitate the detection to be a semi-point-of-care testing (POCT).

Bactericidal Effect of Triple Antibiotic Paste against Enterococcus faecalis in Dentinal Tubules: An Ex Vivo Study.

The aim of this study was to investigate the bactericidal effect of various concentrations of triple antibiotic paste (TAP) against Enterococcus faecalis (E. faecalis) in dentinal tubules using a bacterial culture assay and confocal laser scanning microscope (CLSM). Ninety human teeth were contaminated with E. faecalis (ATCC 29212) and randomly allocated into 5 groups; the negative control (without TAP), 1 mg/ml, 5 mg/ml, 7.5 mg/ml and 10 mg/ml TAP (n=18). After a 3-week TAP treatment, samples were collected from the root canal space, root dentin at 100-μm and 200-μm depth. The collected samples were subjected to a bacterial culture assay (n=10). Eight roots from each group underwent CLSM analysis to determine the live/dead bacterial cells. The bacterial culture assay results indicated that the negative control samples were all culturable. The number of culture-positive samples decreased after TAP treatment at 1, 5, 7.5 and 10 mg/ml, with 2, 2, 1 and 0 culturable samples, respectively. However, there was no significant difference among the TAP treatments. Surprisingly, the CLSM analysis demonstrated live bacteria in the dentinal tubules in all samples. The negative control had 52.36%+-3.24 live bacteria. TAP treatment at 10 mg/ml had the lowest percentage of live bacterial cells (40.58%+-5.40), followed by 7.5 mg/ml (44.14%+-6.03), 5 mg/ml (46.31%+-5.32) and 1 mg/ml (52.55%+-8.82). The percentage of live cells in the 10 mg/ml, 7.5 mg/ml and 5 mg/ml TAP groups were significantly lower than the 1 mg/ml TAP and negative control groups. TAP treatment significantly decreased the percentage of viable E. faecalis cells in the dentinal tubules and its bactericidal effect was dose-dependent.

Species Distribution and Antifungal Susceptibility of Candida spp among Superficial Candidiasis in Outpatients at RMCH

Background: The increase in the incidence of fungal infections, especially those caused by Candida albicans and non albicans Candida species, necessitates the understanding and treatment of Candida-associated infection. Due to frequent use of antifungal drugs, a shift is observed towards non-albicans Candida species and change in the pattern of susceptibility of antifungal drugs. The aim of this study was to isolate and identify the Candida species from different superficial candidial infections in adult patients and to observe their antifungal susceptibility patterns. Materials and Methods: A cross sectional study was performed over a period of one year in at tertiary care hospital that involved 180 patients. The candidial species were isolated, their antifungal susceptibility patterns were determined from vaginal swab, oral swab and skin appendages. Results: Commonest age group involved was of 30-39 years age (30%, 54/180). Among 67 culture positive samples,C. albicans was the most common isolates and found in 39/67(58.21%) cases. Non-candidal species likeC. tropicalis and C. kruseiwere isolated in16/67(23.88%), 09/67 (13.43%)cases respectively. Caspofungin was found to be the most sensitive and fluconazole was found to be the least sensitive antifungal drugs. Non-albicansCandida species showed more antifungal resistance than C. albicans. Conclusions: Candida albicans were predominant isolated from the superficial candidial infection but C. tropicalis was the most frequently isolated among non albicans species. They showed a wide range of susceptibility towards different antifungal agents and fluconazole was found to be less sensitive drugs. TAJ 2023; 36: No-2: 93-100

Diagnostic evaluation of viral versus bacterial tonsillopharyngitis using an artificial intelligence mobile application and symptom questionnaire

Objective: In this study, it was aimed to distinguish bacterial/viral tonsillopharyngitis (TP) by scoring the symptom and throat images of pediatric patients with artificial intelligence-based mobile application. &#x0D; &#x0D; Method: Fifty-one patients who applied to Sakarya University Training and Research Hospital, Department of Pediatrics and Diseases with acute tonsillopharyngitis were included. Samples were taken from patients and mouth/throat pictures were taken so that the tonsils and pharynx were clearly visible. In the microbiology laboratory, identification with culture/MALDI-TOF MS (Biomerieux, France) from the first samples, and nucleic acid isolation from the other for molecular tests were performed. Symptoms such as fatigue, sore throat, muscle pain, cough, sneezing, and runny nose were questioned from each patient on a scale of 1 to 5. By uploading the symptom results and throat pictures to the artificial intelligence application, it was aimed to distinguish bacterial/viral tonsillopharyngitis with the developed scoring system. &#x0D; &#x0D; Results: Of the 51 samples included in the study, 21 were culture positive and 30 were negative. The artificial intelligence application was defined as 20 out of 21 culture-positive samples, 3 out of 30 culture-negative samples as bacterial tonsillopharyngitis (Sensitivity: 95.2%, specificity: 90%). &#x0D; &#x0D; Conclusion: This study is one of the first to bring together the artificial intelligence application and microbiology. AI/scoring system may have a role to play in the diagnosis of bacterial vs viral TP, and in doing so may enable more appropriate antibiotic usage targeted to only bacterial TP infections. It is important to distinguish between bacterial and viral tonsillopharyngitis in the COVID-19 pandemic.

OVERVIEW OF BACTERIAL PATTERNS IN TABANAN GENERAL HOSPITAL

AbstractBackground: Resistant bacterial infections will affect the results of therapy, costs, spread of disease, and duration of illness. To minimize the losses incurred, it is necessary to monitor resistant bacterial and the use of antibiotics. Objective: To be able to identify, monitor, map and overcome antibiotic resistance and restrict bacterial transmission in hospitals, especially at the Tabanan General Hospital. Method: This research was conducted using a cross-sectional method using samples of inpatients and outpatients sent by clinicians to the Clinical Pathology Laboratory, Microbiology Sub Lab in period of January to December 2022. Results: Of the 590 positive culture samples in 2022, the majority were caused by Escherichia coli (15.25%) and 62.22% were ESBL. Apart from that, 8.31% were found to be Staphylococcus aureus and 53.06% were MRSA. Conclusion: The percentage of Escherichia coli bacteria that produce the ESBL enzyme is still high (62.22%). The percentage of Staphylococcus aureus bacteria that produce the MRSA enzyme is still high (53.06%). The antibiotics Meropenem, Amikacin, and Tigecycline are the antibiotics with the highest sensitivity to ESBL. The antibiotics Nitrofurantoin, Linezolid, and Quinupristin/Dalfopristin are the antibiotics with the highest sensitivity to MRSA. Keywords: bacterial patterns, description of bacterial distribution, antibiotic resistance

Prevalence, Associated Risk Factors and Major Bacterial Pathogens causing Bovine Mastitis on Selected Dairy Farms in and around Harar, Ethiopia

Aim Mastitis, an inflammation of the mammary gland, is a complex and costly disease in dairy herds. The main aim of this study was to estimate the prevalence of mastitis and associated risk factors for bacterial pathogens in lactating dairy cows. Methods A cross-sectional study was carried out. The study was conducted in Harar town and its district from November 2016 to April 2017. A total of 384 (264 crossbreds and 120 Holstein Frisian) milking cows were tested using the California mastitis test (CMT), direct diagnosis of obverse clinical mastitis, and bacterial culture in Harar City and its district. Results The result of the study indicated the prevalence of mastitis at the cow level was 58.60% (225/384), out of which 10.7% (41/384) and 47.9% (184/384) were clinical and subclinical, respectively. The quarter-level prevalence was 34.1% (513/1536); from this, the clinical and subclinical forms were 8.7% (131/1536) and 25.4% (382/1536), respectively, while 2% (31/1536) had blind teats. From 341 culture-positive samples, the following bacteria were isolated: Out of the total, Coagulase-negative Staphylococcus (CNS) (28.5%) was the most prevalent isolate, followed by Streptococcus species (27.5%), Staphylococcus aureus (21.4%), Escherichia coli species (14.4%), Micrococcus species (5.3%) and Corynebacterium species (2.9%). Age, parity, breed, production, and hygiene were significant associated risk factors, whereas management risk factors such as house types, milking systems, farming systems, and treatment history do not have a significant association with the prevalence of mastitis in the study area at this level of significance. Conclusion Based on the findings of this study, a thorough screening of high-producing and aged cows should be needed; optimum space should be required for those cows kept in intensive and closed houses to reduce the risk of contagious mastitis; attention should be paid to the informal use of drugs; and further research should be needed to view other risk factors for mastitis

Implementation of full-length 16S nanopore sequencing for bacterial identification in a clinical diagnostic setting

This study describes the implementation of 16S nanopore sequencing in a diagnostic lab for pathogen identification without prior enrichment. First, the universality of the test and taxonomic resolution was evaluated for 78 clinically relevant bacteria (69 known and 9 unknown bacterial cultures). Next, the diagnostic value of the test was evaluated based on clinical samples. It was shown that 16S sequencing can be used both for identification of unknown cultures and to find bacteria directly in the clinical sample without cultivation. All culture-positive samples (n=11) tested positive with 16S sequencing directly performed on the sample, but bacteria were found as well in 15/30 culture-negative samples. Pathogenic bacteria were found in a background of commensal flora, and even complex polymicrobial infections could be unraveled. This study demonstrates the feasibility of implementing 16S nanopore sequencing in a clinical diagnostic setting and demonstrates its value for the diagnosis of culture-negative and polymicrobial infections.

480. Duration of Infectious Virus Shedding by SARS-CoV-2 Omicron Variant among Immunocompromised Patients

Abstract Background Immunocompromised patients with coronavirus disease (COVID-19) shed SARS-CoV-2 RNA longer than usual, but the duration of viral shedding and criteria for nosocomial de-isolation in these patients remains unclear. Methods A prospective cohort study was performed at two tertiary medical centers in Japan during the Omicron epidemic waves from July 8, 2022, to January 30, 2023. Nasopharyngeal swab samples were serially collected from immunocompromised COVID-19 patients. We included populations with either hematological malignancies, solid tumors, autoimmune diseases, or human immunodeficiency virus infection. Patients were classified as severely or moderately immunocompromised based on the national COVID-19 guidelines in Japan. The relationship among patients' characteristics, immune status, quantitative reverse transcription PCR (qRT-PCR), and duration of infectious virus shedding between the two groups was assessed using Mann-Whitney U and Fisher's exact tests. Results Among 41 patients with 163 samples, nine patients and 47 samples were severely immunocompromised, and 32 patients and 116 samples were moderately immunocompromised. The median ages of the two groups were 70 (IQR: 56-71) and 70.5 (IQR: 60.75-75) years, respectively. In the severely immunocompromised group, 87.2% of the samples (41/47) were PCR-positive, with a median Cq value of 26.1 (IQR: 20.5-30.2), and 36.2% (17/47) were culture-positive. In the moderately immunocompromised group, 75.0% of the samples (87/116) were PCR-positive, with a median Cq value of 21.9 (IQR: 18.4-28.6), and 38.8% (45/116) were culture-positive (Table 1). Of the 62 culture-positive samples, five (8.1%) were culture-positive from day 20 onwards, all from severely immunocompromised patients (P = 0.001). In moderately immunocompromised patients, none were culture-positive on day 12 or later (Figure 1). Conclusion In COVID-19 patients, particularly those severely immunocompromised, the duration of viral shedding should be closely monitored. Further research is needed to establish a safe period for in-hospital de-isolation of immunocompromised patients. Disclosures All Authors: No reported disclosures

The effect of the transition to molecular diagnosis on the epidemiology and the clinical characteristics of bacterial gastroenteritis in Northern Israel

Background The transition to PCR-based diagnosis of bacterial gastroenteritis (BGE) can increase the sensitivity but might reduce the clinical specificity. The aims of this study were (1) to compare the effect of the change from culture to PCR-based diagnostics on the reported incidence and positivity rates of BGE due to Salmonella, Shigella and Campylobacter species and (2) to compare the demographics, medical background, clinical characteristics and pre-analytic variables between cases with PCR-positive, culture-negative samples to cases with PCR-positive, culture-positive samples. Methods The study was performed at the Emek Medical Centre that serves a population of 0.5 million people in Northern Israel. The study included two parts: (1) a retrospective cohort study, comparing the incidence and positivity rates of laboratory-diagnosed BGE from January 2016 until December 22nd, 2019 when culture was the sole method to January 2020 until April 2023 when PCR was used; (2) a prospective cohort study, conducted between November 2020 until April 2023 that compared the demographics and clinical characteristics of BGE cases that were diagnosed by PCR alone versus cases that were diagnosed by both PCR and culture. Results The incidence rate between-periods comparability ratio was only 113% since the incidence rate did not increase during 2020, the first year of the COVID-19 pandemic. The sample positivity rate increased since 2020, with between-periods comparability ratio of 159%. In the second period, the sample positivity rates of culture vs. PCR alone differed between the pathogens and were 90.2%, 63.8% and 54.2% for Salmonella, Campylobacter and Shigella species, respectively (p < 0.001). The following variables were identified as independent predictors of culture positivity: (1) Salmonella infection (O.R. = 10.6, 95% C.I. 3.6–31.1, p < 0.001); (2) Shigella infection (O.R. = 0.46, 95% C.I.0.23–0.93, p = 0.032); (3) time from sample submission to culture (O.R.=0.73, 95% C.I. 0.58–0.92, p = 0.008); (4) the presence of abdominal pain (O.R. = 1.98, 95% C.I. 1.04–3.79, p = 0.038) and the PCR mean Ct value (O.R. = 0.89, 95% C.I.0.85–0.94, p < 0.001). Conclusions The use of PCR had led to improved sensitivity, without noticeable decrease in the clinical specificity. This was especially important in the case of the more fastidious organisms.

Metagenomics for the microbiological diagnosis of hospital-acquired pneumonia and ventilator-associated pneumonia (HAP/VAP) in intensive care unit (ICU): a proof-of-concept study

BackgroundHospital-acquired and ventilator-associated-pneumonia (HAP/VAP) are one of the most prevalent health-care associated infections in the intensive care unit (ICU). Culture-independent methods were therefore developed to provide faster route to diagnosis and treatment. Among these, metagenomic next-generation sequencing (mNGS) has shown considerable promise.MethodsThis proof-of-concept study describes the technical feasibility and evaluates the clinical validity of the mNGS for the detection and characterization of the etiologic agents causing hospital-acquired and ventilator-associated pneumonia. We performed a prospective study of all patients with HAP/VAP hospitalized in our intensive care unit for whom a bronchoalveolar lavage (BAL) was performed between July 2017 and November 2018. We compared BAL fluid culture and mNGS results of these patients.ResultsA total of 32 BAL fluids were fully analyzed. Of these, 22 (69%) were positive by culture and all pathogens identified were also reported by mNGS. Among the culture-positive BAL samples, additional bacterial species were revealed by mNGS for 12 patients, raising the issue of their pathogenic role (colonization versus coinfection). Among BALF with culture-negative test, 5 were positive in mNGS test.ConclusionsThis study revealed concordant results for pneumonia panel pathogens between mNGS and culture-positive tests and identified additional pathogens potentially implicated in pneumonia without etiologic diagnosis by culture. mNGS has emerged as a promising methodology for infectious disease diagnoses to support conventional methods. Prospective studies with real-time mNGS are warranted to examine the impact on antimicrobial decision-making and clinical outcome.

Uterine microbial profiles in healthy postpartum dairy cows do not vary with sampling techniques or phases of estrous cycle

In this study, we aimed to compare uterine microbial profiles in postpartum dairy cows, determined by bacteriological culture and next-generation sequencing, using three uterine sampling techniques (swab, cytobrush, and lavage) and induced phases of the estrous cycle (estrus and diestrus). Fifteen healthy postpartum dairy cows at 53 ± 5 days postpartum were enrolled in the study. Uterine samples were collected during a fixed-time artificial insemination protocol. Viable bacteria were aerobically cultured from part of each sample, and bacterial isolates were identified through Sanger sequencing of the 16S rRNA gene. Total genomic DNA was extracted from the remainder of undiluted samples to quantify bacterial load using 16S rRNA qPCR and characterize the microbiome by metagenomic sequencing of the V1–V3 region of the 16S rRNA gene. Microbial profiles and composition were analyzed using the Shannon-Weaver diversity index and principal component analysis, respectively. Out of 87 samples, 88 % (77/87) were culture positive. The proportion of culture-positive uterine samples did not differ between sampling techniques (P = 0.39) or estrous cycle phases (P = 0.99). However, swab, cytobrush, and lavage techniques yielded 1.5, 9 and 9 times greater bacterial loads (P < 0.01), respectively, during diestrus than estrus phase. Moreover, during diestrus phase, the cytobrush method yielded 3 and 6 times more bacteria (P < 0.01) than both the lavage and swab methods. The most abundant bacterial genera identified from both bacteriological culture and metagenomic sequencing were Bacillus and Enterococcus, regardless of sampling technique or phases of the estrous cycle. Bacterial genera in moderate to low abundance through metagenomic sequencing included Streptococcus, Oscillospiraceae, and Lachnospiraceae. Notably, the uterine microbial profiles and composition, determined by metagenomic sequencing, did not differ by sampling techniques (P = 0.55 and P = 0.60, respectively) or estrous cycle phases (P = 0.34 and P = 0.17, respectively). In conclusion, our results suggest that any of the sampling techniques can be reliably used to study the uterine microbiome of healthy cows at random phases of the estrous cycle. However, it is important to consider potential differences in bacterial yield as a confounding factor.