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Abstract Background Candida species have emerged as a significant cause of opportunistic infections. Alongside the expression of various virulence factors, the rise of antifungal resistance among Candida species presents a considerable clinical challenge. Aim This study aimed to identify different Candida species isolated from clinical specimens, evaluate their antifungal sensitivity patterns, identify key genes regulating virulence mechanisms using multiplex PCR and to assess any correlation between their virulence profiles and antifungal resistance patterns. Method A total of 100 Candida spp. was isolated from 630 different clinical specimens and identified to the species level. Their antifungal susceptibility was phenotypically evaluated in accordance with CLSI guidelines using the Vitek-2 Compact System. Virulence markers, including biofilm formation capacity, protease production, melanin production, coagulase production and hemolysin production, were also phenotypically detected. The genetic determinants for biofilm formation and extracellular hydrolytic enzymes were assessed using a multiplex PCR assay. Results The prevalence of Candida spp. was 15.9%, with C. albicans (48%) and C. glabrata (16%) being the most common. C. albicans showed the highest virulence, with strong biofilm formation, and high proteinase and melanin production. Multiplex PCR revealed Hlp in 22.0%, Hwp in 80.0%, Als in 56.0%, and Sap genes in 56.0% of isolates. Virulence genes were more common in C. albicans than in non-albicans Candida (NAC). Resistance patterns significantly correlated with virulence profiles, with notable associations between flucytosine resistance and the presence of Hlp and Hwp genes. Conclusion The significant correlation between virulent markers such as germination, coagulase, hemolysin production and resistance patterns among different Candida isolates is crucial for predicting the severity and outcomes of Candida infections. This understanding aids in guiding tailored treatment strategies.

Background: Candida species have emerged as a significant cause of opportunistic infections. Alongside the expression of various virulence factors, the rise of antifungal resistance among Candida species presents a considerable clinical challenge. Aim: This study aimed to identify different Candida species isolated from clinical specimens, evaluate their antifungal sensitivity patterns, identify key genes regulating virulence mechanisms using multiplex PCR and to assess any correlation between their virulence profiles and antifungal resistance patterns. Method: A total of 100 Candida spp. was isolated from 630 different clinical specimens and identified to the species level. Their antifungal susceptibility was phenotypically evaluated in accordance with CLSI guidelines using the Vitek-2 Compact System. Virulence markers, including biofilm formation capacity, protease production, melanin production, coagulase production and hemolysin production, were also phenotypically detected. The genetic determinants for biofilm formation and extracellular hydrolytic enzymes were assessed using a multiplex PCR assay. Results: The prevalence of Candida spp. was 15.9%, with C. albicans (48%) and C. glabrata (16%) being the most common. C. albicans showed the highest virulence, with strong biofilm formation, and high proteinase and melanin production. Multiplex PCR revealed Hlp in 22.0%, Hwp in 80.0%, Als in 56.0%, and Sap genes in 56.0% of isolates. Virulence genes were more common in C. albicans than in non-albicans Candida (NAC). Resistance patterns significantly correlated with virulence profiles, with notable associations between flucytosine resistance and the presence of Hlp and Hwp genes. Conclusion: The significant correlation between virulent markers such as germination, coagulase, hemolysin production and resistance patterns among different Candida isolates is crucial for predicting the severity and outcomes of Candida infections. This understanding aids in guiding tailored treatment strategies. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli belongs to the Enterobacteriaceae family and is found in the digestive systems of humans and various animals as normal flora. Objective: This study aims to identify the optimal conditions necessary for the synthesis of hemolysin by E. coli isolated from a variety of clinical contexts. Methods: A total of 120 specimens were collected from different clinical sources in Baghdad Hospitals. Screening for hemolysin production was done by plate and Spectrophotometric methods. Furthermore, many conditions for producing hemolysin from clinical E. coli isolates were investigated including temperature, pH, incubation time, inoculum size, culture medium, and aeration. Results: Six isolates of E. coli exhibited hemolysin production. Optimal conditions for hemolysin production were determined: the temperature was 37 °C with an optical density of 0.931, the pH was 7 with an optical density of 0.976, the incubation period was 24 hours with an optical density of 0.885, 1% inoculum resulting in an optical density of 0.558, brain heart infusion as the best medium with an optical density of 1.697, and a shaker method showing superior results (optical density of 1.235) compared to static aeration (optical density of 0.988). Conclusions: This study identified the optimal conditions necessary for the synthesis of hemolysin by E. coli. By understanding these optimal conditions, researchers may be better equipped to develop strategies for managing and controlling the spread of these pathogenic bacteria. [ABSTRACT FROM AUTHOR]

It is difficult to differentiate between coronavirus disease 2019 (COVID-19) and influenza based on the symptoms. In the present study, a newly developed antigen rapid diagnostic test (Ag-RDT) called Panbio™ COVID-19/Flu A&B that can simultaneously detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/B virus was evaluated. Its accuracy was evaluated using 235 pairs of nasopharyngeal samples collected from patients with respiratory symptoms and fever (>37.5°C). Reverse transcription polymerase chain reaction was used as a reference method to evaluate the accuracy of the SARS-CoV-2 detection. We confirmed the accuracy of the developed Ag-RDT against the Omicron variant where the sensitivity and specificity were 94.8% and 100%, respectively. In addition, to identify the influenza A virus, a noninferiority test was conducted using a commercial Ag-RDT, which has a sensitivity and specificity in comparison with viral culture of 94.8% and 98.4%, respectively. The positive and negative predictive values for influenza A virus were 98.5% and 98.1%, respectively, for the Panbio COVID-19/Flu A&B test. The evaluation of this newly developed Ag-RDT using clinical samples suggests that it has a high efficacy in clinical settings. [ABSTRACT FROM AUTHOR]

Background: Monkeypox virus (MPXV) is spreading globally and nearly half of the infected people were human immunodeficiency virus (HIV) positive. Therefore, an in-depth understanding of the effects of HIV infection on the outcomes of MPXV infection is urgently needed. This study aimed to explore the clinical features, viral dynamics, and antibody response to MPXV infections in men who had sex with men (MSM) with and without HIV co-infection. Design or methods: MPXV-infected patients diagnosed by PCR were recruited in this study and were divided into MPXV and MPXV + HIV groups based on whether they were co-infected with HIV. Clinical data and samples were collected during of the hospital stay and follow up interviews. The symptoms and signs, laboratory examinations, viral shedding in various body fluids or swabs, antibody dynamics were tracked and compared between the two groups. Results: A total of 41 MPXV patients were recruited through June 2023 to September 2023 in Guangzhou. The MPXV group and MPXV + HIV group comprised 20 and 21 MSM, respectively. Patients in the two groups exhibited similar clinical characteristics except for pruritus and eschar, both were significantly fewer in MPXV + HIV group than in MPXV only group. Among the 355 clinical samples collected, MPXV DNA was detected in 100% of scabs, 97.4% of skin swabs, and 92.3% of exudate swabs from lesions, while the positive rate was 87.5% from oropharyngeal swabs, 59% from saliva, 51.3% from anal swabs, 50% from feces, 30.6% from urine samples, 37.5% of semen, and 28.2% from sera. Dynamics analysis revealed that viral DNA was undetectable in most patients 20 days after symptom onset. IgM and IgG antibodies to MPXV were detected in all patients with 3-5 days earlier in the MPXV group than in the MPXV + HIV group. Conclusion: This cohort analysis based on a large outbreak among MSM in Guangzhou indicated no obvious differences in clinical symptoms, viral DNA data, but antibody responses were 3-5 days later in mpox patients with HIV infection. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli belongs to the Enterobacteriaceae family and is found in the digestive systems of humans and various animals as normal flora. Objective: This study aims to identify the optimal conditions necessary for the synthesis of hemolysin by E. coli isolated from a variety of clinical contexts. Methods: A total of 120 specimens were collected from different clinical sources in Baghdad Hospitals. Screening for hemolysin production was done by plate and Spectrophotometric methods. Furthermore, many conditions for producing hemolysin from clinical E. coli isolates were investigated including temperature, pH, incubation time, inoculum size, culture medium, and aeration. Results: Six isolates of E. coli exhibited hemolysin production. Optimal conditions for hemolysin production were determined: the temperature was 37 °C with an optical density of 0.931, the pH was 7 with an optical density of 0.976, the incubation period was 24 hours with an optical density of 0.885, 1% inoculum resulting in an optical density of 0.558, brain heart infusion as the best medium with an optical density of 1.697, and a shaker method showing superior results (optical density of 1.235) compared to static aeration (optical density of 0.988). Conclusions: This study identified the optimal conditions necessary for the synthesis of hemolysin by E. coli. By understanding these optimal conditions, researchers may be better equipped to develop strategies for managing and controlling the spread of these pathogenic bacteria.

Abstract Background Antimicrobial resistance is one of the common global public health problems. The emergence of antimicrobial resistance is multifactorial, and tackling its development is challenging. Consequently, infections caused by resistant bacteria are unresponsive to conventional drugs, resulting in prolonged and severe illnesses, higher mortality rates, and considerable healthcare costs. Therefore, understanding the antimicrobial resistance profiles of bacterial pathogens is essential to optimize treatments and reduce the risks associated with infections. This study aimed to determine the antimicrobial resistance patterns of bacterial isolates from different clinical specimens at the Ethiopian Public Health Institute (EPHI). Materials and methods The retrospective cross-sectional study was conducted on the bacterial culture and antibiotic susceptibility reports of different clinical specimens referred to the Bacteriology Laboratory of EPHI from September 2015 to August 2019. Standard bacteriological techniques were used for the isolation and identification of the bacteria. Data were extracted from 840 patients’ records, which included the type of clinical sample cultured, the name of the bacteria, the representations of the antibiotics used for susceptibility testing, and the susceptibility results. Descriptive statistics were used to describe the bacterial isolates and the antimicrobial resistance profiles. Results Eight types of clinical specimens were analyzed for bacterial isolates and urine specimens were the most analyzed. Ten different genera of bacteria were identified by culture. Almost all the isolates were gram-negative bacteria, while only one species of gram-positive (Staphylococcus aureus) was reported. Antibiotic sensitivity patterns were tested on 840 culture isolates. Escherichia coli strains revealed more than 57% resistance to seventeen antibiotics. Klebsiella pneumoniae showed nearly 70% or greater resistance rates for 17 of the antibiotics used. The overall detected multidrug resistance (MDR) was 64.29%. The highest MDR was reported in Acinetobacter strains (84%) followed by K. pneumoniae (80%). Conclusions The multidrug resistance rates found in this study were alarming. Strengthening antimicrobial resistance surveillance at the national level is mandatory, and antimicrobial sensitivity testing should be accessible at local diagnostic centers.

Background: Antimicrobial resistance is one of the common global public health problems. The emergence of antimicrobial resistance is multifactorial, and tackling its development is challenging. Consequently, infections caused by resistant bacteria are unresponsive to conventional drugs, resulting in prolonged and severe illnesses, higher mortality rates, and considerable healthcare costs. Therefore, understanding the antimicrobial resistance profiles of bacterial pathogens is essential to optimize treatments and reduce the risks associated with infections. This study aimed to determine the antimicrobial resistance patterns of bacterial isolates from different clinical specimens at the Ethiopian Public Health Institute (EPHI). Materials and methods: The retrospective cross-sectional study was conducted on the bacterial culture and antibiotic susceptibility reports of different clinical specimens referred to the Bacteriology Laboratory of EPHI from September 2015 to August 2019. Standard bacteriological techniques were used for the isolation and identification of the bacteria. Data were extracted from 840 patients' records, which included the type of clinical sample cultured, the name of the bacteria, the representations of the antibiotics used for susceptibility testing, and the susceptibility results. Descriptive statistics were used to describe the bacterial isolates and the antimicrobial resistance profiles. Results: Eight types of clinical specimens were analyzed for bacterial isolates and urine specimens were the most analyzed. Ten different genera of bacteria were identified by culture. Almost all the isolates were gram-negative bacteria, while only one species of gram-positive (Staphylococcus aureus) was reported. Antibiotic sensitivity patterns were tested on 840 culture isolates. Escherichia coli strains revealed more than 57% resistance to seventeen antibiotics. Klebsiella pneumoniae showed nearly 70% or greater resistance rates for 17 of the antibiotics used. The overall detected multidrug resistance (MDR) was 64.29%. The highest MDR was reported in Acinetobacter strains (84%) followed by K. pneumoniae (80%). Conclusions: The multidrug resistance rates found in this study were alarming. Strengthening antimicrobial resistance surveillance at the national level is mandatory, and antimicrobial sensitivity testing should be accessible at local diagnostic centers. [ABSTRACT FROM AUTHOR]

We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma (p < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14–685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods. [ABSTRACT FROM AUTHOR]

Background: Biofilm-associated protein (Bap) in Acinetobacter baumannii is an essential factor in biofilm production and persistence in the hospital environment. Objectives: This study aimed to detect the bap gene in A. baumannii by real-time polymerase chain reaction (PCR) from clinical specimens in Khorramabad, Iran. Methods: This cross-sectional study was performed during April 2017 - April 2018 on 43 A. baumannii strains from clinical samples collected and identified by microbiological and biochemical tests. The expression of the bap gene was evaluated by real-time PCR. Data were analyzed with SPSS version 24. Results: Out of 43 A. baumannii strains, 23, 8, 3, 3, 3, and 3 samples were isolated from chest sputum, wounds, urine, tissues, blood, and, respectively. According to the PCR results, all isolates had the bap gene except one. Real-time PCR showed significant differences in the expression of the bap gene between A. baumannii isolates from diverse clinical samples. The highest expression of the bap gene was found in chest sputum and wound samples and had a significant difference with other samples (P < 0.0001). Conclusions:We observed the bap gene in most strains, with the high expression of this gene in chest sputum and wound samples. Therefore, further studies are recommended to find strategies to inhibit the expression of this gene and biofilm formation, which help treat infections caused by biofilm-forming A. baumannii strains. [ABSTRACT FROM AUTHOR]

Papillary thyroid cancer (PTC) is a common malignancy of the endocrine system, and its morbidity and mortality are increasing year by year. Traditional twodimensional culture of cell lines lacks tissue structure and is difficult to reflect the heterogeneity of tumors. The construction of mouse models is inefficient and time-consuming, which is difficult to be applied to individualized treatment on a large scale. Clinically relevant models that recapitulate the biology of their corresponding parental tumors are urgently needed. Based on clinical specimens of PTC, we have successfully established patient-derived organoids by exploring and optimizing the organoid culture system. These organoids have been cultured stably for more than 5 passages and successfully cryopreserved and retried. Histopathological and genome analysis revealed a high consistency of the histological architectures as well as mutational landscapes between the matched tumors and organoids. Here, we present a fully detailed method to derive PTC organoids from clinical specimens. Using this approach, we have developed PTC organoid lines from thyroid cancer samples with a success rate of 77.6% (38/49) until now. [ABSTRACT FROM AUTHOR]

Nucleic acid amplification testing (NAAT) for N. gonorrhoeae is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting N. gonorrhoeae . We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical urine specimens, and optimize assay kinetics. We collected urine specimens among men presenting with urethritis enrolling in a clinical trial at the Massachusetts General Hospital Sexual Health Clinic. We assessed the quantified DNA yield of detergent-based extractions with and without heat. We selected one detergent for extracting all specimens, paired with isothermal recombinase polymerase amplification for 90 minutes and lateral flow Cas13a detection, interpreted via pixel intensity analysis. We also trained a smartphone-based machine-learning model on 1,008 images to classify lateral flow results. We used the model to interpret lateral flow results from the clinical specimens. We also tested a modified amplification chemistry with a second forward primer lacking the T7-promoter to accelerate reaction kinetics. Extraction with 0.02% Triton X resulted in an average DNA yield of 2.6 × 10 6 copies/µL (SD ± 6.7 × 10 5 ). We treated 40 urine specimens ( n = 12 positive) with 0.02% Triton X, and using quantified pixel intensity analysis, the Cas13a-based assay correctly classified all specimens (100% agreement; 95% CI 91.2%-100%). The machine-learning model correctly classified 45/45 strips in the validation data set and all 40 lateral flow strips from clinical specimens. Including the second forward primer reduced incubation time to 60 minutes. Using point-of-care DNA extraction, our Cas13a-based lateral flow N. gonorrhoeae assay demonstrated promising performance among clinical urine specimens.IMPORTANCEUsing a CRISPR-based assay we previously developed for Neisseria gonorrhoeae detection, we developed new techniques to facilitate point-of-care use. We then demonstrated the promising performance of that assay in clinical specimens. Furthermore, we developed a smartphone-based machine learning application for assisting interpretation of lateral flow strip results. Such an assay has the potential to transform the care of sexually transmitted infections in low-resource settings where diagnostic tests are unavailable. A point-of-care pathogen-specific assay, paired with the connectivity offered by a smartphone application, can also support public health surveillance efforts in such areas.

The 1890s marked a significant milestone with the introduction of antibody-based agglutination and precipitation assays, revolutionizing the detection of bacterial pathogens in both animals and humans. This era also witnessed pivotal contributions to our understanding of humoral immunity, as researchers elucidated the structure and functions of antibody molecules, laying the groundwork for diagnostic applications. Among antibody isotypes, IgG is of paramount importance in diagnostic investigations given its definitive indication of infection or vaccination, coupled with its widespread presence and detectability across various specimen types, such as serum, colostrum, milk, oral fluids, urine, feces, and tissue exudate. Despite their resilience, immunoglobulins are susceptible to structural alterations induced by physicochemical and enzymatic processes, which can compromise the reliability of their detection. Here we review comprehensively the historical milestones, underlying mechanisms, and influencing factors (e.g., temperature, pH, storage) that shape the structural integrity and stability of IgG antibodies in aqueous solutions and various clinical specimens., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

ABSTRACT 16S rRNA gene sequencing is increasingly used in clinical practice for bacterial identification of clinical specimens. However, studies on its applicability to direct clinical specimens are limited. Here, we studied the diagnostic yield and impact of 16S rRNA gene sequencing from direct clinical specimens on antimicrobial management. Adult inpatients whose attending physician requested 16S rRNA gene sequencing and corresponding bacterial culture from a direct clinical specimen between January and December 2021 in a university hospital were prospectively included in this study. A total of 434 specimens from 374 patients were requested. Of these, 253 (58.3%) specimens were collected from patients whose final diagnosis indicated a bacterial infection, whereas 181 (41.7%) specimens were from nonbacterial infections. Using the final diagnosis as a “gold standard,” the sensitivity and specificity of 16S rRNA gene sequencing were 38.3% and 93.9%, respectively. Among the bacterial infection cases, the proportion of 16S rRNA gene sequencing-positive and culture-positive cases was 32.4%, and the proportion of sequencing-positive and culture-negative cases was 5.9%. The impact on antimicrobial management was evident in 10 (2.3%) specimens, which all resulted in the continuation of antibiotics. The impact on antimicrobial management was highest in skin and soft tissue infections, followed by bone and joint infections. In this study, the long turnaround time of 16S rRNA gene sequencing of clinical specimens was a limiting factor. In conclusion, the overall diagnostic yield of 16S rRNA gene sequencing in bacterial infection cases was fair, being useful in selected cases. Restrictions on test requests may improve test utilization in this setting. IMPORTANCE 16S rRNA gene sequencing has been increasingly used in clinical practice. Using the final diagnosis as a gold standard, the sensitivity of 16S rRNA gene sequencing was fair. In the setting with no 16S rRNA gene sequencing test ordering restrictions, only small percentages of the test results had an impact on antimicrobial management. Restrictions on test requests should be developed to maximize the benefit of the test.

Objective: To determine important errors in initial Gram staining of clinical specimens and evaluate the types of Gram-stain errors. Study Design: Cross-sectional study. Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Aug 2018 to Feb 2019. Methodology: Gram staining and culture inoculation of all specimens were performed per recommended guidelines. The Gram stain results on day-0 were compared with the results of the culture on day-2. There was no discrepancy if similar organisms were obtained on culture as on Gram stain. Nevertheless, if the Gram stain and culture results were not similar, it was termed a discrepancy. The consultant microbiologist reviewed all discrepant slides, and if not resolved, possible causes of error were sought, and the results documented. Results: Of the total 300 clinical specimens, errors were observed in the initial gram staining of 29 specimens (9.7%), whereas 271(90.3%) specimens were error-free. Upon evaluating these 29 errors, 11(38.0%) were observer errors which were resolved when reviewed by a consultant microbiologist. 14(48.0%) were technical errors, and 4(14.0%) results were discrepant due to the presence of anaerobic organisms, missed on initial aerobic cultures. Conclusion: The frequency of Gram stain errors in our study (9.7%) is not very high; nevertheless, it can have severe consequences in critical samples from seriously ill patients if wrong empirical antimicrobial treatment is begun based on a wrong initial Gram stain result.

We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma (p < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14–685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health concern, and it is responsible for both hospital and community associated infections globally. In this study, we aimed at determining the prevalence and pattern of resistance of MRSA to commonly used antibiotics in Anyigba, Nigeria. This was a hospital based, cross-sectional study conducted between August 2017 and February 2018. One hundred and fifty routine clinical specimens were collected from selected health facilities in Anyigba for laboratory analyses. Standard laboratory methods were employed in the identification of the isolates. Methicillin-resistant Staphylococcus aureus (MRSA) was confirmed using Oxacillin Resistance Screening Agar Base (ORSAB) supplemented with 2g/l of Oxacillin. Screening to determine the antibiotic resistance profiles of all confirmed MRSA isolates was by disc diffusion method. A total of 124 (82.7%) isolates of Staphylococcus aureus were recovered from clinical samples obtained. Of these isolates, 28 (22.6%) were methicillin resistant. The percentage distribution rate of MRSA was highest (50.0%) in urine samples. Majority of these isolates were resistant to at least four of the six antibiotics tested. Most of the isolates recovered from urine samples were resistant to Gentamicin (92.9%), Amoxicillin (100.0%) and Cotrimoxazole (85.7%). Our study showed a significant presence of MRSA isolates in the clinical specimens collected, with a relatively high rate to gentamicin, Amoxicillin and Cotrimoxazole antibiotics. This study highlights the need for monitoring of antimicrobial use considering the lack of innovation in the development of new antimicrobials which lessens efforts at combating infections caused by antibiotic-resistant pathogens.

Diagnosis of botulism caused by multiple serotypes of botulinum neurotoxin (BoNT) is still a challenge due to the lack of a reliable detection method. The present study develops a feasible laboratorial method based on an isotope dilution Immuno-Endopep-MS to detect BoNTs and determine their serotypes and activities in clinical samples. Eleven positive foodborne botulism cases out of a total of 17 suspected cases in China, 2019–2022, were determined by the established method. Blood, urine, vomitus, gastric mucosa samples, and food samples were employed and evidenced to be suitable for the detection. Results showed that, although single type A-intoxication was still the first cause among these foodborne botulism cases, other causes involving type E, type B, and their mixed types were also determined, providing a glimpse to the serotype profile of botulism happened in recent years in China. Furthermore, in order to provide insights into in vivo profiles of toxin serotypes, a comprehensive analysis of clinical specimens collected from one family of four patients was performed during a clinically and therapeutically relevant time frame. Serotypes and concentrations of BoNT in specimens revealed a good correlation with symptoms and progresses of disease. Additionally, serum was proved to be more suitable for detection of BoNT/A with a detection window up to 12 days. A urine sample, although rarely reported for foodborne botulism diagnosis, was validated to be suitable for testing BoNTs, with a longer detection window up to 25 days. To the best of our knowledge, this is the first comprehensive analytical research on in vivo profiles of serotypes A, B, and E in different types of specimens from mixed botulism cases. Our method and findings facilitate the toxin detection and identification by clinical diagnostic laboratories. [ABSTRACT FROM AUTHOR]

Between December 2020 and June 2021, 150 clinical samples (wounds, burns, sputum and bodily fluids) will be collected from individuals of all ages and genders. The sample selection process varies depending on clinical symptoms; (i.e. swelling, fever, abscesses, discomfort, fast breathing, discomfort at the infection site...etc.). and 65 environmental specimens (beds, tables, sinks, floors and instruments) were collected using swabs and transported in sterilized transport medium containers from seven hospitals in NaJaf (Sadr City Medical Hospital, Public Health Laboratory, Al-Hakim General Hospital, Al-Zahra Teaching Hospital, Al-Furat Middle Teaching Hospital, Central Blood Bank, and The Middle Euphrates Center for Tumors) to conduct research. All specimens were grown on blood agar and MaCconkey agar. Following bacteria growth, the isolates were identified by microscopic inspection and biochemical testing, including manual biochemical tests such as (oxidase, catalase, Simmon Citrate, Motility, Indole, Urease, Methyl Red, Kligler iron agar (KIA), Lactose fermentation and Voges-Proskauer). The VITEK-2 system was used to confirm the identification of K. pneumoniae. A total of 28(18%) of K. pneumoniae isolates were recognized. Other bacteria isolated were Escherichia coli, Pseudomonas aeruginosa and Streptococcus spp. in percentages of 37%, 22% and 11%, respectively. For molecular detection the kfu and magA risestenc gene. The genomic DNA of K. pneumoniae isolates was extracted using a wizard genomic DNA purification kit. The genomic DNA collected was examined. Using 1% agarose gel electrophoresis and the concentration and quality of the isolated genomic DNA were determined using a Nanodrop spectrophotometer device. In order to detect K. pneumoniae isolates using molecular methods, genomic DNA extraction of these isolates analyzed (2.5%) kfu. [ABSTRACT FROM AUTHOR]

Genetic testing using reverse transcriptase real-time polymerase chain reaction (rRT-PCR) is the mainstay of diagnosis of COVID-19. However, it has not been fully investigated whether infectious viruses are contained in SARS-CoV-2 genome-positive specimens examined using the rRT-PCR test. In this study, we examined the correlation between the threshold Cycle (Ct) value obtained from the rRT-PCR test and virus isolation in cultured cells, using 533 consecutive clinical specimens of COVID-19 patients. The virus was isolated from specimens with a Ct value of less than 30 cycles, and the lower the Ct value, the more efficient the isolation rate. A cytopathic effect due to herpes simplex virus type 1 contamination was observed in one sample with a Ct value of 35 cycles. In a comparison of VeroE6/TMPRSS2 cells and VeroE6 cells used for virus isolation, VeroE6/TMPRSS2 cells isolated the virus 1.7 times more efficiently than VeroE6 cells. There was no significant difference between the two cells in the mean Ct value of the detectable sample. In conclusion, Lower Ct values in the PCR test were associated with higher virus isolation rates, and VeroE6/TMPRSS2 cells were able to isolate viruses more efficiently than VeroE6 cells. [ABSTRACT FROM AUTHOR]

Diagnosis of botulism caused by multiple serotypes of botulinum neurotoxin (BoNT) is still a challenge due to the lack of a reliable detection method. The present study develops a feasible laboratorial method based on an isotope dilution Immuno-Endopep-MS to detect BoNTs and determine their serotypes and activities in clinical samples. Eleven positive foodborne botulism cases out of a total of 17 suspected cases in China, 2019–2022, were determined by the established method. Blood, urine, vomitus, gastric mucosa samples, and food samples were employed and evidenced to be suitable for the detection. Results showed that, although single type A-intoxication was still the first cause among these foodborne botulism cases, other causes involving type E, type B, and their mixed types were also determined, providing a glimpse to the serotype profile of botulism happened in recent years in China. Furthermore, in order to provide insights into in vivo profiles of toxin serotypes, a comprehensive analysis of clinical specimens collected from one family of four patients was performed during a clinically and therapeutically relevant time frame. Serotypes and concentrations of BoNT in specimens revealed a good correlation with symptoms and progresses of disease. Additionally, serum was proved to be more suitable for detection of BoNT/A with a detection window up to 12 days. A urine sample, although rarely reported for foodborne botulism diagnosis, was validated to be suitable for testing BoNTs, with a longer detection window up to 25 days. To the best of our knowledge, this is the first comprehensive analytical research on in vivo profiles of serotypes A, B, and E in different types of specimens from mixed botulism cases. Our method and findings facilitate the toxin detection and identification by clinical diagnostic laboratories.

The treatment of Klebsiella pneumoniae infections has become more difficult due to the emergence of multidrug-resistant strains. Fluoroquinolones have been widely used to treat respiratory tract infections and urinary tract infections caused by different bacteria. Therefore, this study aimed to evaluate fluoroquinolones resistance and detect ESBL producers in various clinical samples in Duhok Province, Iraq. K. pneumoniae was identified from different clinical samples by conventional microbiological tests. In addition, antibiotic susceptibility was detected by the Kirby-Bauer disc diffusion method. Out of 120 K. pneumoniae isolates, 73.3% were causing urinary tract infections, in most cases, 74.2% were among females and 25.8% among males, and among those 120 isolates, 76.7% of isolates were from outpatients, and 23.3% were inpatients. The resistance rate among all samples was 52.5%, 36.7%, 33.3%, 33.3%, ciprofloxacin, levofloxacin, norfloxacin, and ofloxacin, respectively in urine samples, the resistance to nalidixic acid was 22.5%. Among 120 samples, only 12 isolates were hypermucoviscosity. About 25% of ciprofloxacin and 17.5% of levofloxacin resistances were ESBL producers simultaneously. According to this study, K. pneumoniae is more susceptible to norfloxacin, ofloxacin, and nalidixic acid than ciprofloxacin and levofloxacin. Additionally, this study establishes a link between ESBL and fluoroquinolone resistance. [ABSTRACT FROM AUTHOR]

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex chronic condition affecting multiple body systems, with unknown cause, unclear pathogenesis mechanisms, and fluctuating symptoms which may lead to severe debilitation. It is frequently reported to have been triggered by an infection, but there are no clear differences in exposure to, or seroprevalence of, any particular viruses between people with ME/CFS and healthy individuals. However, herpes viruses have been repeatedly hypothesized to underlie the chronic relapsing/remitting form of MS/CFS due to their persistence in a latent form with periodic reactivation. It is possible that ME/CFS is associated with herpes virus reactivation, which has not been detectable previously due to insufficiently sensitive testing methods. Saliva samples were collected from 30 people living with ME/CFS at monthly intervals for 6 months and at times when they experienced symptom exacerbation, as well as from 14 healthy control individuals. The viral DNA load of the nine humanherpes viruses was determined by digital droplet PCR. Symptoms were assessed by questionnaire at each time point. Human herpesvirus (HHV) 6B, HHV-7, herpes simplex virus 1 and Epstein-Barr virus were detectable within the saliva samples, with higher HHV-6B and HHV-7 viral loads detected in people with ME/CFS than in healthy controls. Participants with ME/CFS could be broadly separated into two groups: one group displayed fluctuating patterns of herpesviruses detectable across the 6 months while the second group displayed more stable viral presentation. In the first group, there was positive correlation between HHV-6B and HHV-7 viral load and severity of symptom scores, including pain, neurocognition, and autonomic dysfunction. The results indicate that fluctuating viral DNA load correlates with ME/CFS symptoms: this is in accordance with the hypothesis that pathogenesis is related to herpesvirus reactivation state, and this should be formally tested. Herpesvirus reactivation might be a cause or consequence of dysregulated immune function seen in ME/CFS. The sampling strategy and molecular tools developed here permit such large-scale epidemiological investigations.

Aims: To reveal the sources of Aeromonas infection in Okinawa Prefecture of Japan, the species, virulence genes and clones of strains isolated from clinical specimens and well water were compared. Methods and Results: The properties of both isolates were investigated by sequencing of rpoD, detection of 10 virulence genes using PCR and genotyping with pulsed‐field gel electrophoresis. In all, 68 clinical and 146 well water strains of Aeromonas were isolated and the main species were A. caviae, A. dhakensis, A. hydrophila and A. veronii biovar sobria. Aeromonas dhakensis possessed various virulence genes; however, A. caviae possessed only fla. The same or similar clones were distributed in certain areas of Okinawa and one clone had survived several months in the biliary system of two patients, respectively. Conclusion: Although the same Aeromonas clone was not isolated from clinical and well water samples, our study revealed the detected patterns of virulence genes in both isolates, the distribution of identical/similar clones in the Okinawan environment and long‐time survival in patient's organs. Significance and Impact of the Study: We investigated the association between Aeromonas patients and well water exposure. This study provides the properties of species, virulence genes and clones of Aeromonas isolated from samples of these origins. [ABSTRACT FROM AUTHOR]

Klebsiella pneumonia is an opportunistic pathogen that has been implicated as one of commonest cause of hospital and community acquired infections. The capsule of Klebsiella pneumonia is an important virulence factor, involved in pathogenic mechanisms. So, this study aimed to isolate Klebsiella pneumonia from different clinical specimens from patients in AlDiwaniyah teaching hospital and determine some virulence factors (capsular genes)that used for serotyping of isolates. The study extended from August to November 2020.A total of 31 isolates from 80 different clinical specimens identified as Klebsiella pneumonia by traditional biochemical tests, Vitek system and 16SrRNA gene. The existence of three genes from7 tested capsular genes was detected by Polymerase chain reaction. The most common serotype were k2, k54, k57 where K2 detected in 4 (12.9 %), K54 in 12 (38.7 %),k57 in 4 (12.9).But, the other capsular polysaccaride genes k1, k3, k5,k20 not detected in all isolates of this study [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate human coronavirus NL63 (HCoV-NL63) prevalence among the other respiratory viruses such as parainfluenza, respiratory syncytial virus, and non-enteric adenoviruses in clinical specimens of Egyptian children and raw sewage samples. One hundred clinical specimens were collected from Egyptian children suffering from upper and lower respiratory viral infections in the years 2005–2006 to detect HCoV-NL63 genome using RT-PCR. All the specimens were negative for the virus. Also, a complete absence of HCoV-NL63 genome was observed in the twenty-four raw sewage samples collected from two wastewater treatment plants within Greater Cairo from February 2006 to January 2007. Using nested RT-PCR, parainfluenza virus type 1, respiratory syncytial virus type A, adenovirus type 4, and adenovirus type 7 were detected in 3%, 2%, 5%, and 2% of the clinical specimens, respectively. Of these viruses, only adenovirus type 4 was detected in 1/24 (4.17%) of the raw sewage samples, while a complete absence of the other investigated respiratory viruses was observed in the raw sewage samples. The low percentage of positivity in the clinical specimens, the concentration method of the raw sewage samples, and the indirect routes of transmission may be the reasons for the absence of respiratory viruses in raw sewage samples. On the other hand, enteric adenoviruses were detected in 21/24 (87.5%) of the raw sewage samples with a higher prevalence of adenovirus type 41 than adenovirus type 40. A direct route of transmission of enteric viruses to raw sewage may be the reason for the high positivity percentage of enteric adenoviruses in raw sewage samples. [ABSTRACT FROM AUTHOR]

The diversity of 61 Bacillus cereus strains isolated from different clinical specimens, food including raw milk and milk products, and water was evaluated. PFGE analysis could discriminate 61 distinct pulsotypes with similarity levels from 25 to 82%, which were divided into 13 clonal complexes. The similarity between clonal complexes was at least 40%. Clinical strains were divided into 10 clonal complexes, while the strains, isolated from milk, food and water were included in 9, 6 and 6 clonal complexes, respectively. Three clonal complexes were dominated by clinical isolates, while they were absent in two complexes. Bacterial isolates from food, being a probable source of alimentary toxoinfection, showed low similarity to isolates from stool specimens. The isolates from both sources were classified together in only 4 out of 13 clonal complexes. The large circular and linear plasmids with the sizes between 50 and 200 kb were detected in 24 (39.3%) and 14 (23%) B. cereus strains, respectively. Thirteen (21.3%) strains contained only one plasmid, two plasmids were found in 6 (9.8%) of strains, and three or more plasmids were obtained in 5 (8.2%) of tested strains. The plasmids were confirmed in 30.8% and 40% of isolates from clinical specimens and food and milk samples, respectively. No clear correlation between the PFGE profiles, the source as well as plasmid content among all tested strains was observed. [ABSTRACT FROM AUTHOR]

In recent years, the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus (AIV) gene recombination and reassortment. Until now, traditional RT-PCR, fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV, but these methods require high-level instruments and experimental conditions, not suitable for the rapid detection in field and farms. In order to develop a rapid, sensitive and practical method to detect and identify AIV subtypes, 4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification (RT-LAMP) method was established. Using this method, the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein, without cross reaction with other susceptible avian viruses. In addition, the detection limit of the common H1, H5, H7, and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU (plaque-forming unit), which was 10 times more sensitive than that using the routine RT-PCR. Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18% (14/335) comparing with 3.58% (12/335) from real-time RT-PCR. All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast, convenient and practical method for the clinic test and epidemiological investigation of AIV.

Listeria monocytogenes are Gram-positive bacilli cause listeriosis, associated with high rates of hospitalization and mortality. A total of 1362 samples were investigated included 647 clinical samples and 715 from food. This study investigated the differences in the seasonal occurrence of L. monocytogenes in Kurdistan region-Iraq. L. monocytogeneswas found in 48 samples (3.52%) out of 1362 samples. There were 7/642(1.09%) human clinical samples diagnosed with cold and moderate weather (November, December 2016, and January, February, April 2017). While 41/715(5.73%) of L. monocytogenesisolates were found among food samples, meat samples (n=37) and dairy samples (n=4). A total of 37/476 (7.77%) of the L. monocytogenes isolates in meat samplesweredetected during warm weather (from July through October 2016). In addition, L. monocytogenesin dairy products was found to be by 4/239 (1.68%), were isolated during the moderate season (October, November 2016, and April, May 2017). The study found that the incidence of meat contamination by L. monocytogenesincrease significantly during the warm season in comparison with other seasons. Furthermore, the cases of human Listeriosis caused correlated well with the seasonal levels of L. monocytogenesfound in dairy products. A statistically significant difference in the occurrence of L. monocytogenesisolates and seasons were identified in this study (P value=

Abstract Background The global emergence and spread of extended-spectrum beta-lactamases (ESBLs) producing Enterobacteriaceae have been threatening the ability to treat an infection. Hence, this study aimed to determine the prevalence of ESBL-producing and multi-drug resistance (MDR) Enterobacteriaceae (ESBLs-E) from different clinical specimens in Addis Ababa, Ethiopia. Methods A cross-sectional study was conducted from January 1 to May 30, 2017. A total of 426 Enterobacteriaceae isolates were identified from clinical specimens. The isolates were collected from four laboratories. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method on Muller Hinton agar (MHA). All Enterobacteriaceae were screened for ESBLs production using cefotaxime and ceftazidime as per CLSI guideline. Each ESBL screening positive Enterobacteriaceae were confirmed by a combination disk test (CDT). Data were entered and analyzed by using SPSS version-20. Result The most frequent Enterobacteriaceae were E. coli 228 (53.5%) and K. pneumoniae 103 (24.1%). The magnitude of ESBLs-E was 57.7% (246/426). The highest frequencies of ESBLs-E were observed in blood specimesns (84.4%) and the highest ESBLs production was observed in K. pneumoniae (85.4%). The highest resistance level was seen to sulfamethoxazole-trimethoprim (77.0%), amoxicillin with clavulanic acid (71.6%), cefotaxime (62.2%), cefepime (60.3%) and ceftazidime (60.8%). The overall magnitude of multi-drug resistance (MDR) level was 68.3%. Of ESBLs-E, 96.3% of them were MDR (P

Recently, increased frequencies of carbapenemase-producing Enterobacteriaceae have been reported worldwide. Among multiple genetic subtypes, oxacillinase (OXA)-48 β-lactamase-producing strains have been associated with inbound infection because they have been detected predominantly in patients who traveled outside of Japan. However, a recent case report of OXA-48 β-lactamase-producing Enterobacteriaceae suggested the latent spread of domestic infections. Due to a lack of specific inhibitors, culture-based detection of OXA-48 β-lactamase-producing bacteria is difficult. Thus, DNA-based detection methods, including PCR, direct sequencing and loop-mediated isothermal amplification (LAMP), have been employed. Among these methods, LAMP detection is more favorable than other methods because of its technical simplicity and low cost. We designed novel LAMP primers to detect OXA-48 β-lactamase-producing bacteria and investigated their possible clinical applications with bacterial genome-spiked human materials (cerebrospinal fluid, blood, feces, urine, and sputum). We evaluated the specificity of the LAMP primers using 37 bacterial strains: 8 standard, 9 reference, and 20 clinical Gram-negative strains. Our LAMP primers detected 10 copies of the OXA-48 type β-lactamase gene and exhibited no cross reactivity with other β-lactamase genes. Sensitivity was not influenced in any clinical sample, in contrast to PCR detection, which was strongly inhibited by substances in fecal samples. These results suggest the superior performance of LAMP compared with conventional PCR for detecting the OXA-48 type β-lactamase gene in various clinical samples. [ABSTRACT FROM AUTHOR]

Background: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. Methods: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). Results: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. Conclusions: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors. [ABSTRACT FROM AUTHOR]

Ninety three Klebsiella pneumoniae isolates were collected from clinical and water samples. Sixty- six (20.625%) clinical isolates were isolated from 320 clinical samples from different hospitals at Medical City Iraq, Baghdad. Twenty-seven (18%) water isolates were collected from 150 different water sources from different water samples of Baghdad city. All isolates were diagnosed by biochemical tests and by CHROM agar. The isolates were tested for their ability to beta-lactam antibiotics (Imipenem, Meropenem, Amoxiclave, Cetazidime, Ceftriaxone and Cefotaxime) resistance and Minimum Inhibitory Concentration (MIC) determined for each isolate by using agar dilution method. The results show that the clinical isolates were more resistance to beta lactam antibiotics than water isolates and both of them were (100 %) resist to Amoxiclav. The resistance percentage of the clinical isolates was (6.06%) Meropenem, (13.63%) Imipenem, (19.69%) Ceftazidime, (53.03%) Cefotaxime and (86.36%) Ceftriaxone, while the resistance percentage for the water isolates was zero for the Meropenem, Imipenem, Ceftazidime, Cefotaxime and Ceftriaxone. The most resistance clinical and water isolates for beta-lactam antibiotics were diagnosed by PCR technique and they were documented by the National Center for Biotechnology Information (NCBI) of American and registered for the first time in this study as a new K. pneumoniae strain; clinical isolates (GMH1 80, GMH2 80, GMH11 80 and GMH12 80), while water isolates (GMH3 80, GMH4 80, GMH5 80, GMH6 80, GMH7 80, GMH8 80, GMH9 80, and GMH10 80). [ABSTRACT FROM AUTHOR]

Rapid characterization of the causative agent(s) during a disease outbreak can aid in the implementation of effective control measures. However, isolation of the agent(s) from crude clinical samples can be challenging and time-consuming, hindering the establishment of countermeasures. In the present study, we used saliva specimens collected for the diagnosis of SARS-CoV-2-a good example of a practical target-and attempted to characterize the virus within the specimens without virus isolation. Thirty-four saliva samples from coronavirus disease 2019 patients were used to extract RNA and synthesize DNA amplicons by PCR. New primer sets were designed to generate DNA amplicons of the full-length spike (S) gene for subsequent use in a circular polymerase extension reaction (CPER), a simple method for deriving recombinant viral genomes. According to the S sequence, four clinical specimens were classified as BA. 1, BA.2, BA.5, and XBB.1 and were used for the de novo generation of recombinant viruses carrying the entire S gene. Additionally, chimeric viruses carrying the gene encoding GFP were generated to evaluate viral propagation using a plate reader. We successfully used the RNA purified directly from clinical saliva samples to generate chimeric viruses carrying the entire S gene by our updated CPER method. The chimeric viruses exhibited robust replication in cell cultures with similar properties. Using the recombinant GFP viruses, we also successfully characterized the efficacy of the licensed antiviral AZD7442. Our proof-of-concept demonstrates the novel utility of CPER to allow rapid characterization of viruses from clinical specimens., Importance: Characterization of the causative agent(s) for infectious diseases helps in implementing effective control measurements, especially in outbreaks. However, the isolation of the agent(s) from clinical specimens is often challenging and time-consuming. In this study, saliva samples from coronavirus disease 2019 patients were directly subjected to purifying viral RNA, synthesizing DNA amplicons for sequencing, and generating recombinant viruses. Utilizing an updated circular polymerase extension reaction method, we successfully generated chimeric SARS-CoV-2 viruses with sufficient in vitro replication capacity and antigenicity. Thus, the recombinant viruses generated in this study were applicable for evaluating the antivirals. Collectively, our developed method facilitates rapid characterization of specimens circulating in hosts, aiding in the establishment of control measurements. Additionally, this approach offers an advanced strategy for controlling other (re-)emerging viral infectious diseases., Competing Interests: The authors declare no conflict of interest.

Background: Monkeypox virus (MPXV) is spreading globally and nearly half of the infected people were human immunodeficiency virus (HIV) positive. Therefore, an in-depth understanding of the effects of HIV infection on the outcomes of MPXV infection is urgently needed. This study aimed to explore the clinical features, viral dynamics, and antibody response to MPXV infections in men who had sex with men (MSM) with and without HIV co-infection., Design or Methods: MPXV-infected patients diagnosed by PCR were recruited in this study and were divided into MPXV and MPXV + HIV groups based on whether they were co-infected with HIV. Clinical data and samples were collected during of the hospital stay and follow up interviews. The symptoms and signs, laboratory examinations, viral shedding in various body fluids or swabs, antibody dynamics were tracked and compared between the two groups., Results: A total of 41 MPXV patients were recruited through June 2023 to September 2023 in Guangzhou. The MPXV group and MPXV + HIV group comprised 20 and 21 MSM, respectively. Patients in the two groups exhibited similar clinical characteristics except for pruritus and eschar, both were significantly fewer in MPXV + HIV group than in MPXV only group. Among the 355 clinical samples collected, MPXV DNA was detected in 100% of scabs, 97.4% of skin swabs, and 92.3% of exudate swabs from lesions, while the positive rate was 87.5% from oropharyngeal swabs, 59% from saliva, 51.3% from anal swabs, 50% from feces, 30.6% from urine samples, 37.5% of semen, and 28.2% from sera. Dynamics analysis revealed that viral DNA was undetectable in most patients 20 days after symptom onset. IgM and IgG antibodies to MPXV were detected in all patients with 3-5 days earlier in the MPXV group than in the MPXV + HIV group., Conclusion: This cohort analysis based on a large outbreak among MSM in Guangzhou indicated no obvious differences in clinical symptoms, viral DNA data, but antibody responses were 3-5 days later in mpox patients with HIV infection., 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 © 2024 Yang, Xie, Zeng, Cao, Fan, Jiang, Lei, Wang, Li, Tang, Yu and Li.)

Abstract Objective Staphylococcus genus comprising both Staphylococcus aureus and coagulase negative staphylococci (CoNS) are widely distributed in nature and can infect diversity of hosts. Indeed, staphylococci are the major pathogens causing biofilm associated infections caused by contaminated hospital indwelling devices. These infections are persistent in nature being highly refractory to various stresses including antibiotics. Implementation of efficient diagnostic techniques for the biofilm production would help minimize the disease burden. Thus, early detection of pathogenic strains producing biofilms warrant the utmost importance in diagnostic laboratories especially in resource limited settings. Result Among 375 isolates collected from different clinical specimens, 214 (57%) were identified as coagulase negative staphylococci and 161 (43%) S. aureus. Detection of In-vitro biofilm formation in these isolates were carried out by three commonly used phenotypic assays and a genotypic assay. While evaluating the results, tissue-culture method with supplemented glucose and sucrose showed the best correlation with the results of genotypic assay.

To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID 50 per μL and copies per μL, and the newly established methods were tested in clinical specimens collected in recent years. The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID 50 per μL and 10 virus copies per μL, and for the complete VP1 gene was 1 CCID 50 per μL and 100 virus copies per μL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons. This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens. [ABSTRACT FROM AUTHOR]

Different isolates of eight species of dermatophytes: M. canis, T. interdigetale, T. rubrum, T. mentagrophytes, T. schoenleinii, T. simii, T. verrucosum and Epidermophyton floccosum, as well as Candida spp. Four species of dermatophytes were presented for different concentrations (25,50,100 ppm) of silver ions with plant in a mixture and under laboratory conditions. The colony diameter of the fungus was measured. The results showed that the inhibitory effect of silver ions was greater on the isolation of M. canis., T. interdigetale and T. mentagrophtes while the least affected was the T. rubrum cooperatively on the growth rate of the fungus. The concentration of 100 ppm silver nanoparticles gave the highest effect on fungus growth, The effect of nanoparticles depends on their concentrations and exposure time. [ABSTRACT FROM AUTHOR]

Among non-albicans Candida species, Candida glabrata is the leading cause of invasive infections in critically ill patients. It is intrinsically less susceptible to fluconazole/other azoles that limits therapeutic options. This study determined distribution of C. glabrata in clinical specimens and determined their susceptibility to fluconazole, caspofungin, and amphotericin B by E test. During 8-year period (2011–2018), 1,410 isolates were obtained from 1,410 patients including 600, 409, and 131 isolates from respiratory, urine, and bloodstream specimens, respectively. Proportion of C. glabrata isolates was nearly the same during the two 4-year periods. Demographic details were available from 731 patients and susceptibility data for 1,225 isolates. C. glabrata isolation from bloodstream, respiratory, and urine specimens was higher from elderly (>60 years) versus younger patients. More bloodstream and urine isolates were obtained from female patients, however, more respiratory isolates were recovered from male patients (p = <0.05). Resistance to all three drugs increased during 2015–2018 compared with 2011–2014 but was more pronounced for fluconazole (p = 0.001). More isolates with reduced susceptibility to fluconazole/amphotericin B were obtained from elderly patients versus younger subjects and urine versus respiratory samples (p = <0.05). Our data show increasing trends of reduced susceptibility to antifungals, particularly fluconazole, among clinical C. glabrata isolates in Kuwait. Most isolates with reduced susceptibility to fluconazole/amphotericin B were obtained from elderly patients and urine/respiratory samples with urinary tract appearing as the most favorable niche for antifungal drug resistance development. The study also highlights the need for continued surveillance and better antifungal drug stewardship to control resistance development in C. glabrata. [ABSTRACT FROM AUTHOR]

Introduction: Biomedical and translational research often relies on the evaluation of patients or specimens that meet specific clinical or laboratory criteria. The typical approach used to identify biospecimens is a manual, retrospective process that exists outside the clinical workflow. This often makes biospecimen collection cost prohibitive and prevents the collection of analytes with short stability times. Emerging data architectures offer novel approaches to enhance specimen-identification practices. To this end, we present a new tool that can be deployed in a real-time environment to automate the identification and notification of available biospecimens for biomedical research. Methods: Real-time clinical and laboratory data from Cloverleaf (Infor, NY, NY) were acquired within our computational health platform, which is built on open-source applications. Study-specific filters were developed in NiFi (Apache Software Foundation, Wakefield, MA, USA) to identify the study-appropriate specimens in real time. Specimen metadata were stored in Elasticsearch (Elastic N. V., Mountain View, CA, USA) for visualization and automated alerting. Results: Between June 2018 and December 2018, we identified 2992 unique specimens belonging to 2815 unique patients, split between two different use cases. Based on laboratory policy for specimen retention and study-specific stability requirements, secure E-mail notifications were sent to investigators to automatically notify of availability. The assessment of throughput on commodity hardware demonstrates the ability to scale to approximately 2000 results per second. Conclusion: This work demonstrates that real-world clinical data can be analyzed in real time to increase the efficiency of biospecimen identification with minimal overhead for the clinical laboratory. Future work will integrate additional data types, including the analysis of unstructured data, to enable more complex cases and biospecimen identification. [ABSTRACT FROM AUTHOR]

Pathogens causing sexually transmitted diseases (STDs) include viruses, bacteria, and parasites. The ability to rapidly and efficiently detect these pathogens in a single reaction still remains a health challenge. The aim of this study was to evaluate the clinical reliability and accuracy of the STD Direct Flow Chip Kit (Vitro, IVD-EC approved), which can simultaneously detect up to 9 different species of STD pathogens at once. This kit enables direct analysis—direct-PCR—of clinical specimens (urine, semen, endocervical, urethral, nasopharyngeal, and perianal swabs) without DNA purification for the following pathogens: Chlamydia trachomatis (serovars A-K and L1-L3), Haemophilus ducreyi, Herpes Simplex Virus (Types I and II), Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, and Ureaplasma. The Anyplex™ II STI-7 Detection Kit (Seegene, IVD-EC) was used as the reference's method. Existing discordances were resolved using either a third molecular assay or DNA sequencing. Clinical performance was evaluated at two different stages: (i) from purified DNA of three hundred and fifty-eight clinical specimens with a diagnostic sensitivity (SE) and specificity (SP) of 99.4% and 100%, respectively, and an agreement of 99% (kappa index, κ = 0.97) with the reference's method and; (ii) by direct-PCR from six hundred and thirty-three specimens rendering SE, SP, and agreement values of 98.4%, 99.9%, and 98.0% (κ = 0.95), respectively. The STD Direct Flow Chip Kit constitutes a promising alternative to routine procedures in diagnostic, allowing direct analysis of specimens and enabling the detection of a broad panel of pathogens. [ABSTRACT FROM AUTHOR]

The major study of this work was to evaluate the chemical composition and the inhibitory components of Parmotrema perlatum against the clinically isolated specimens of Staphylococcus, Streptococcus, Escherichia, Pseudomonas and Candida. The GC-FID-MS by using a Trace GC Ultra apparatus MS DSQ II detectors and FID-MS splitter under the operating conditions of a polar capillary column was employed to determine the chemical composition of Parmotrema perlatum essential oil. Amino acids viz (38.1%) and cymene (29.1%) were the most adequate constituents in this oil. The antibacterial activity of Parmotrema perlatum oil was tested against standard strains of bacteria and clinical strains isolated from patients with infections of the oral cavity, abdominal cavity, respiratory, genitourinary tracts, skin, and from the hospital environment. Kirby-Bauer disc diffusion technique was employed to determine the susceptibility of the organism towards the Parmotrema perlatum essential oil exhibited very strong antimicrobial activity to inhibit the growth of the clinical specimens. Application of natural antimicrobial based essential oil from Parmotrema perlatum can be potential source in the treatment and prevention of different types of infections as an alternative to the chemical - based antibiotics based on the investigation. [ABSTRACT FROM AUTHOR]

• The slope of residual γH2AX foci dose–response curve negatively correlates with local tumour control measured as the TCD 50 in seven HNSCC xenografts models. • The γH2AX assay is feasible in clinical HNSCC samples. • The inter-patient variability in slope might reflect different repair capacities, which could be used to individualise radiotherapy dose prescriptions according to tumour radiosensitivity. Predictive biomarkers can be instrumental to treatment individualisation of cancer patients and improve therapy outcome. Residual γH2AX foci represent a promising biomarker to predict tumour radiosensitivity. In this pre-clinical study, the slope of the dose–response curve was evaluated for its predictive relevance in head and neck squamous cell carcinoma xenografts (HNSCC). Additionally, the feasibility of the translated assay was tested in a clinical setting in patient derived HNSCC samples, and associations between residual γH2AX foci and clinical parameters were analysed. Seven HNSCC xenografts models (FaDu, SAS, SKX, UT-SCC-5, UT-SCC-14, UT-SCC-45, XF354) were used. Tumour bearing NMRI nude mice were randomly distributed to five treatment arms (0–8 Gy). Residual γH2AX foci (24 h post irradiation) were counted by visual scoring in a micromilieu dependent manner (assessed with BrdU and pimonidazole). The local tumour control values measured as TCD 50 (tumour control dose 50%) have previously been published. Patient derived HNSCC biopsies were cultivated ex vivo for 24 h including 4 h of pimonidazole and BrdU treatment, subsequently irradiated with 0–8 Gy and fixed after 24 h. In the pre-clinical study, the dose–response curve slopes negatively correlated with the tumour control dose after fractionated irradiation (TCD 50,fx , R 2 = 0.63, p = 0.032) and after single dose irradiation under homogeneous hypoxia (TCD 50,SD,clamp , R 2 = 0.66, p = 0.027). The γH2AX assay in clinical HNSCC samples showed a dose–response relationship, with the values of the slopes ranging from 0.099 Gy−1 to 0.920 Gy−1 (coefficient of variation = 52.8%). Slopes derived from patients were in the same ranges as the sensitive, moderate and resistant models of the pre-clinical study. Statistical analysis revealed a significant negative correlation between the slope and the patients' age (R 2 = 0.65, p = 0.001). These results further support the promise of the slope of the residual γH2AX foci dose–response as a biomarker for radiosensitivity. In the clinical samples, the variation in the slopes reveals patients' specific repair capacities, which could hold potential value for treatment individualisation. [ABSTRACT FROM AUTHOR]

Rapid spread of acquired Metallo-beta-lactamases (MBLs) among major Gram negative pathogens is an emerging threat and a matter of great concern worldwide. Especially increasing resistance to 3rd and 4th generation cephalosporin and carbapenems is an issue of high importance. Present study was aimed to determine the frequency and antimicrobial susceptibility patterns of MBL producing carbapenemresistant Gram-negative rods (GNRs) isolated from clinical specimens. A clinical descriptive study was conducted in Faisalabad, Pakistan from June 2016 to January 2017. A total of 152 clinical samples were processed according to standard microbiological methods. Isolated GNRs were subjected to susceptibility testing against various antibiotics as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Carbapenem-resistant isolates were subjected to detection of Metallo-beta-lactamase production by Double Disc Diffusion test (DDDT) and Modified Hodge's test. In 152 specimens, different species identified in order of their frequency were; Klebsiella pneumoniae (59; 38.8%), Pseudomonas aeruginosa (46; 30.3%), Escherichia coli (36; 23.7%), Klebsiella oxytoca (4; 2.6%), Serratia marcescens (3; 2%), Enterobacter agglomerans (2; 1.3%) and Enterobacter cloacae (2; 1.3%). Out of 103 carbapenemresistant isolates, 58 (55.1%) were positive by DDDT and 49 (46.7%) by MHT. Most of the carbapenems MBL resistant isolates were resistant towards drugs such as cephalosporin, ceftazidime but majority of them were sensitive to polymyxins, tazocin and fusidic acid. Proper screening of MBLs producing GNRs should be done from clinical specimens along with antimicrobial susceptibility testing for developing an effective treatment strategy and thus better control of infections caused by these bugs in future. [ABSTRACT FROM AUTHOR]

Background and Aim: In medical centers, after obtaining visceral clinical samples in suitable containers under certain circumstances, they are sent to mycology laboratory. Since sometimes it is impossible to test specimens immediately, therefore, they should be kept in the refrigerator. Thus, possibility of keeping samples, the confidently time of samples keeping in a refrigerator and the appropriate guidelines for the maintenance of visceral samples for clinical centers have a particular importance for practical purposes and are the aim of this study. Materials and Methods: At first, the specimen was examined by KOH direct microscopic examination for detection of fungal elements. After primary culture of visceral samples on the Sabouraud dextrose agar with chloramphenicol (SC) medium, the samples were sequentially placed in a refrigerator at temperature 2-8 ºc for 1, 2, 24 and 48 hs and each sample with the specific mentioned time culturing was performed on the Sabouraud dextrose agar (S) and SC media. Then the results of growth were recorded. Results: From 100 samples, 79 samples had grown, 20 samples with lack of growth and in 1 sample reduced growth were observed. Conclusion: After 48 hours of cold temperatures (2-8 ºc) visceral fungal clinical samples are able to grow in culture media. So samples which were not tested immediately, could be stored at temperatures 2-8 ºc in the refrigerator.

Aspergillus fumigatus is the most prevalent species that causes aspergillosis. A. fumigatus strains with tandem repeats in the cyp51A promoter have emerged in the environment. Aspergillus species other than A. fumigatus have also been recognized as causative agents of aspergillosis; however, they show lower susceptibility to antifungals compared with A. fumigatus. Therefore, it is important to precisely identify Aspergillus species and determine their antifungal susceptibility. Herein, we collected 119 mold strains isolated from clinical specimens collected at a hospital between November 2013 and December 2018. The collected strains were identified by sequencing several regions, including internal transcribed spacers, and determined their susceptibility to the antifungals itraconazole, voriconazole, and amphotericin B. Of 119 strains, 107 were Aspergillus species, which were identified as A. fumigatus (67), Aspergillus section Nigri (21), A. flavus (7), A. terreus (6), and A. nidulans (6). In Aspergillus section Nigri , the number of A. niger was less than the number of A. welwitschiae and A. tubingensis. Two azole-resistant A. fumigatus samples were included among the isolates. Four of the eight A. tubingensis isolates showed less susceptibility to voriconazole; however, all isolates of A. niger and A. welwitschiae were susceptible to itraconazole and voriconazole. Because of lack of susceptibility data for non-fumigatus Aspergillus and an increasing frequency of antifungal resistance among A. fumigatus , our data along with further surveillance may contribute to determining the frequency and susceptibility of Aspergillus spp. clinical isolates in Japan. [ABSTRACT FROM AUTHOR]

Introduction: Biomedical and translational research often relies on the evaluation of patients or specimens that meet specific clinical or laboratory criteria. The typical approach used to identify biospecimens is a manual, retrospective process that exists outside the clinical workflow. This often makes biospecimen collection cost prohibitive and prevents the collection of analytes with short stability times. Emerging data architectures offer novel approaches to enhance specimen-identification practices. To this end, we present a new tool that can be deployed in a real-time environment to automate the identification and notification of available biospecimens for biomedical research. Methods: Real-time clinical and laboratory data from Cloverleaf (Infor, NY, NY) were acquired within our computational health platform, which is built on open-source applications. Study-specific filters were developed in NiFi (Apache Software Foundation, Wakefield, MA, USA) to identify the study-appropriate specimens in real time. Specimen metadata were stored in Elasticsearch (Elastic N. V., Mountain View, CA, USA) for visualization and automated alerting. Results: Between June 2018 and December 2018, we identified 2992 unique specimens belonging to 2815 unique patients, split between two different use cases. Based on laboratory policy for specimen retention and study-specific stability requirements, secure E-mail notifications were sent to investigators to automatically notify of availability. The assessment of throughput on commodity hardware demonstrates the ability to scale to approximately 2000 results per second. Conclusion: This work demonstrates that real-world clinical data can be analyzed in real time to increase the efficiency of biospecimen identification with minimal overhead for the clinical laboratory. Future work will integrate additional data types, including the analysis of unstructured data, to enable more complex cases and biospecimen identification. [ABSTRACT FROM AUTHOR]

Abstract Optimal transportation of bacteria is important for accurate clinical interpretation, quantitative assays, and microbiome studies. A transport medium should ideally keep the bacteria alive without supporting growth or altering the relative proportions of the constituent species. We investigated the effect of nasal mucus and mucin on the growth and survival of two Staphylococcus aureus strains in liquid Amies transport medium at room temperature and 4 °C for 14 days. The study showed that the presence of nasal mucus in microbiological samples stimulated undesired S. aureus growth at room temperature in a dose-dependent manner. These findings underscore that microbiological samples from humans and animals should be stored at 4 °C until analysis to avoid undesired S. aureus growth. Highlights • S. aureus multiplies in nasal swabs in Amies medium during storage at room temperature. • Concentration of mucin influences the growth rate of S. aureus in nasal swabs. • The growth rate is dependent on the S. aureus strain. [ABSTRACT FROM AUTHOR]

Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2–12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected by rtPCR, and was most frequently found with P. multocida. The rtPCR assay enabled enhanced levels of detection over culture for all pathogens on both thermocycling platforms. The rotary-based thermocycler had significantly lower Cq cutoffs (35.2 vs. 39.7), which maximized agreement with gold standard culture or gel-based PCR results following receiver operating characteristic analysis to maximize sensitivity (Se) and specificity (Sp). However, overall assay Se and Sp were similar on both platforms (80.5% Se, 88.8% Sp vs. 80.1% Se, 88.3% Sp). Implementation of these tests could enhance the detection of these pathogens, and with high-throughput workflows could reduce assay time and provide more rapid results. The assays may be especially valuable in identifying coinfections, given that many more antemortem samples tested in our study were positive for 2 or more pathogens by rtPCR (n = 125) than were detected using culture alone (n = 25). [ABSTRACT FROM AUTHOR]