Multiplex Polymerase Chain Reaction Analysis Research Articles

Accuracy of RHC/c genotyping in Chinese Han population.

The RHCE gene plays an important role in the complex and polymorphic Rh blood group system. RHCE genotyping holds significant clinical and transfusion-related implications. The objective of this study was to evaluate the accuracy of RHC/c genotyping in the Chinese Han population. Blood samples were obtained from 653 Chinese Han blood donors. The serological RhD and RhCcEe types were determined using monoclonal antibodies. Subsequently, multiplex real-time polymerase chain reaction (PCR) analysis was performed for RHC and RHc genotyping. Additionally, exon 2 of RHCE and exon 1 of RHD were sequenced. The analysis in this study found 443 RhD-positive donors and 210 RhD-negative donors. Among the 653 total donors, discrepancies between the RHC genotyping results and the serological results were found in 37 individuals. Specifically, 6 false-positive RhC results in RhD-positive donors and 28 false-positive RhC results in RhD-negative donors were identified based on c.48C in RHCE exon 1. Additionally, 3 false-negative RhC results were observed in the RhD-positive donors due to a 109 bp insertion in RHCE intron 2. RHc typing demonstrated complete consistency between the real-time PCR and the serological results. In the Chinese Han population, RHC genotyping was reliable when consistent results were achieved by both c.48C-based and 109 bp insertion-based genotyping. Moreover, RHc genotyping based on c.203A and c.307C polymorphic loci demonstrated dependable performance.

Isolation and identification of probiotic lactobacilli from ruminant animals

Background Lactobacillus strains are a group of bacteria that provide health benefits to their hosts when consumed in proper amounts. They are which exhibit an important for intestinal microflora that constitutes a beneficial mechanism responsible for antimicrobial activity. Ruminant animals such as cattle, buffalo, goat, and sheep have formed a symbiotic relationship with ruminal microorganisms that synthesize fiber digesting enzymes. The microbial flora obtained from ruminal ingesta is an output of the composition, nature, and quality of the animals’ feed. It is found that the dominating flora are usually starch and complex polysaccharide degrading microbiota. Objective The aim of the present study was to isolate and identify probiotic lactobacilli present in some ruminant animals to investigate interspecies differences in probiotic Lactobacillus contents. Materials and methods Sixty samples were collected in triple manner under aseptic conditions from buffalo, cattle, sheep and goats including rectal, buccal, and nasal swabs. Following DNA extraction from the isolated bacteria, 16S rRNA multiplex polymerase chain reaction analysis was performed to recognize the obtained isolates. Results and conclusion A total of 38 isolates were identified as lactobacillus species including 7, 14, 8, and 9 isolates from buffalo, cattle, sheep, and goat, respectively. The buffalo samples displayed the lowest variability in lactobacilli with the identification of Lactobacillus delbrueckii only. Cattle, goat, and sheep samples showed the presence of Lactobacillus casei, Lactobacillus acidophillus, L. delbrueckii, Lactobacillus gasseri, and Lactobacillus rhamnosus. It is found that L. gasseri was the most frequently isolated species in cattle followed by L. rhamnosus, while L. delbrueckii was the most frequently isolated species in goat followed by L. casei. L. gasseri was the most frequently isolated strain in sheep followed by L. acidophillus and L. rhamnosus. These strains should be investigated in more detail, individually or in combination, for their potential health benefits. Understanding how these species interact with other microbiota community members in each host as well as how they interact with host cells, particularly immune cells, can provide valuable insight into their function both in health and disease.

Bacterial Flora and Treatment Strategies in Women With Escherichia coli Urinary Tract Infections.

Introduction This study explores the intricate relationship between bacterial flora and the occurrence of Escherichia coli (E. coli) infections in gynecological patients. It aims to provide insights into the various treatment strategies used to effectively manage bacterial pathogens, especially E. coli infections. By conducting a comprehensive analysis of the bacterial flora in gynecological patients, the study highlights the notable presence of E. coli, promptingfurther investigation into the factors that contribute to its colonization. The objective of the study is to comprehensively investigate and detect urinary tract infections (UTIs) specifically caused by E. coli among gynecological patients. The study aims to delve into bacterial flora prevalence, antibiotic resistance patterns, and potential virulence factors. Through this analysis, the study intends to identify effective strategies for rapid detection and diagnosis of UTIs caused by E. coliby utilizing advanced microbiological and molecular techniques. Furthermore, the study aims to formulate and propose a strategic treatment approach with a particular emphasis on selecting appropriate antibiotics to reduce the risk of severe infections and associated complications. Materials and methods The methodology employed in this study included the isolation and characterization of bacterial strains from clinical samples obtained from gynecological patients. A total of 52 urine specimens were collected from patients with complaints of infection in the urinary tract and infertility. These samples underwent both preliminary and confirmatory microbiological analysis, such as gram staining, biochemical confirmation test, and antibiotic susceptibility, and further proceeded with the multiplex polymerase chain reaction (PCR) technique. The results of PCR and antibiotic susceptibility revealed the specific gene involvement and resistant characteristics of E. coli. Results The findings revealed a total of 32 specimens positive for E. coli, of which 10 patients had infertility complaints and 22 patients had UTIs. The preliminary test, gram staining, showed the gram-negative bacilli E. coli, and the nutrient agar plate revealed smooth circular translucent colonies; MacConkey agar showed pink-colored lactose-fermented colonies; and the blood and chocolate agar plates showed grayish white moist gamma-hemolytic colonies. The biochemical confirmation of E. coli resulted in positive for indole and methyl red tests and negative for Voges-Proskauer and citrate utilization tests. The multiplex PCR analysis confirmed the E. coli strains with the presence of two target genes,stx2d and stx2e. Conclusion To summarize, this study offers valuable insights into the bacterial flora of gynecological patients impacted by E. coli infections, which provides a foundation for the development of precise and efficient treatment strategies. The results emphasize the importance of personalized treatment approaches that consider both the microbiological characteristics of the infection and the evolving landscape of antibiotic resistance. The implication of this research extends to enhancing clinical outcomes and alleviating the burden of E. coli infections in gynecological settings.

Clinical Implementation of β-Tubulin Gene-Based Aspergillus Polymerase Chain Reaction for Enhanced Aspergillus Diagnosis in Patients with Hematologic Diseases: A Prospective Observational Study.

The β-tubulin (benA) gene is a promising target for the identification of Aspergillus species. Assessment of the clinical implementation and performance of benA gene-based Aspergillus polymerase chain reaction (PCR) remains warranted. In this study, we assessed the analytical performance of the BenA probe PCR in comparison with the Aspergenius kit. We prospectively collected bronchoalveolar lavage (BAL) fluid via diagnostic bronchoscopy from adult patients with hematologic diseases. BenA gene-based multiplex real-time PCR and sequential melting temperature analysis were performed to detect the azole resistance of Aspergillus fumigatus. In total, 76 BAL fluids in 75 patients suspicious of invasive pulmonary aspergillosis (IPA) were collected. Before the application of PCR, the prevalence of proven and probable IPA was 32.9%. However, after implementing the benA gene-based PCR, 15.8% (12 out of 76) of potential IPA cases were reclassified as probable IPA. The analytical performance of the BenA probe PCR in BAL samples was comparable to that of the Aspergenius kit. The diagnostic performance was as follows: sensitivity, 52.0%; specificity, 64.7%; positive predictive value, 41.9%; negative predictive value, 73.3%; positive likelihood ratio, 1.473; and negative likelihood ratio, 0.741. Moreover, benA gene-based Aspergillus PCR discriminated all major sections of Aspergillus, including cryptic species such as Aspergillus tubingensis. Sequential melting temperature analysis successfully detected 2 isolates (15.4%) of A. fumigatus carrying resistant mutations. BenA gene-based Aspergillus PCR with melting temperature analysis enhances diagnostic accuracy and detects not only cryptic species but also resistant mutations of A. fumigatus. It shows promise for clinical applications in the diagnosis of IPA.

Pathological and molecular study of kidneys in apparently healthy cattle and sheep with special reference to Leptospira species in central and northern Jordan.

Renal pathological conditions can cause significant economic losses for livestock owners. This study investigated and described the gross pathology and histopathology of lesions found in the kidneys of sheep and cattle, with particular attention to the presence of Leptospira spp. Three hundred and sixty kidneys from apparently healthy sheep and cattle (208 and 152, respectively) were collected from slaughterhouses in Jordan for gross and histopathological examinations, multiplex polymerase chain reaction (PCR) analysis, and gene sequencing of Leptospira spp. Histopathological analysis of the samples revealed the following conditions: interstitial nephritis (4.44%), glomerulonephritis (4.16%), acute tubular necrosis (17.22%), and renal congestion (4.72%). In addition, in 26.9% of the samples, hyaline material was observed in the interstitium of the cortex and medulla. The PCR results revealed that 83 (23.1%) of the 360 samples were positive for Leptospira spp. 16S rRNA, 42/152 (28%) of cattle, and 41/208 (20%) of sheep. Four samples (two sheep and two cattle) were sequenced and deposited in GenBank with accession numbers from OL701310 to OL701313. Basic Local Alignment Search Tool search and nucleotide similarities between OL701311 and OL701313 resulted in the highest similarities with different Leptospira borgpetersenii strains, whereas OL701310 showed the highest nucleotide similarity (99.2%) with the Leptospira interrogans strain. Similarly, phylogenetic analysis revealed that OL701311 to OL701313 clustered together with different serovars of L. borgpetersenii, whereas OL701310 clustered with the L. interrogans clade. This is the first study to reveal a close association between pathogenic Leptospira spp. and kidney disorders in Jordanian cattle and sheep. These findings may help expand the current understanding of the causes and mechanisms of renal disease in cattle and sheep and contribute to developing more effective prevention and treatment programs.

Oxidative stress and genotoxicity in oral epithelial cells from subjects undergoing orthodontic treatment with fixed appliances.

The objective of this work was to evaluate the impact of fixed orthodontic appliances on oxidative stress (OS) and genotoxicity from oral epithelial cells. Samples of oral epithelial cells were obtained from fifty-one healthy voluntary subjects who had an indication for orthodontic treatment. The samples were obtained before treatment and after 6 and 9months of treatment. OS was evaluated by quantitating 8-hydroxy-2'deoxyguanosine (8-OHdG) and by performing relative gene expression with antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). DNA degradation and instability were evaluated by multiplex polymerase chain reaction (PCR) and fragment analysis for human identification. The quantitation results showed that 8-OHdG increased during treatment, although this increase was not statistically significant. SOD increased by 2.5- and 2.6-fold after 6 and 9months of treatment, respectively. CAT increased by threefold after 6months of treatment, while after 9months of treatment, the expression level decreased to a level similar to that before treatment. DNA degradation was found in 8% and 12% of DNA samples after 6 and 9months of treatment, respectively, while DNA instability was detected in only 2% and 8% of DNA samples after 6 and 9months of treatment, respectively. The results showed that OS and genotoxicity slightly changed after treatment with a fixed orthodontic appliance; in addition, a biological adaptation response to the treatment may occur after 6months. OS and genotoxicity in the buccal cavity are risk factors for oral and systemic diseases. This risk may be reduced through antioxidant supplementation, by using thermoplastic materials, or by reducing the orthodontic treatment time.

Cross-sectional analysis of Piroplasma species-infecting camel (Camelus dromedaries) in Egypt using a multipronged molecular diagnostic approach.

Camel piroplasmosis is a tick-borne disease (TBD) caused by hemoprotozoan parasites. Hereby, we describe a cross-sectional study aiming at identifying Piroplasma spp.-infecting camels in Egypt using a multipronged molecular diagnostic approach. A total of 531 blood samples from camels (Camelus dromedarius) were collected from slaughterhouses at different governorates in Egypt for analysis during the period from June 2018 to May 2019. Piroplasma spp. was identified using microscopical examination and several different and sequential polymerase chain reaction (PCR) assays targeting the 18S rRNA genes. The overall prevalence of Piroplasma spp. in microscopical and molecular analyses in the samples was 11% (58/531) and 38% (203/531), respectively. Further discriminative multiplex PCR analysis targeting the 18S rRNA gene applied on all Piroplasma spp.-positive samples allowed the detection of Theileria equi (41%), Babesia caballi (5.4%), Babesia bigemina (0.5%), and Babesia bovis (4%). Additionally, the blast analysis of nested (n) PCR, targeting the V4 region, amplicon sequences resulted in the identification of B. vulpes (22%), Babesia sp. (9%), and Theileria sp. (3%). Overall, the results of this study confirmed the high prevalence of TBDs caused by several types of piroplasm hemoparasites in camel and suggests the need for future interventions aimed at improving the control of these potentially debilitating diseases that may be t-hreatening important economic resources and food security in Egypt.

Minimally Required Personal Protective Equipment during Local Anesthesia Surgery in COVID-19: A Simulation Study.

Two mannequins were each connected to a test lung machine simulating a surgeon and patient with spontaneous breathing. An aerosol generator containing severe acute respiratory syndrome coronavirus 2 virion particle substitutes was connected to the patient mannequin. The sampling points for the target molecules were set at different distances from the patient mannequin and sent for multiplex quantitative polymerase chain reaction analysis. Three clinical scenarios were designed, which differed in terms of the operating room pressure and whether a fabric curtain barrier was installed between the mannequins. Analysis of the multiplex quantitative polymerase chain reaction results showed that the cycle threshold (Ct) value of the target molecule increased as the distance from the aerosol source increased. In the negative-pressure operating room, the Ct values were significantly increased at all sample points compared with the normal pressure room setting. The Ct value sampled at the surgeon mannequin wearing double face masks was significantly increased when a cloth curtain barrier was set up between the two mannequins. Double surgical masks provide elementary surgeon protection against COVID-19 in a negative pressure operating room, with a physical barrier in place between the surgeon and patient who is spontaneously breathing during local anesthesia or sedated surgery.

Evaluation of the Epidemiological, Clinical and Radiological Features of Pneumonia Cases Caused by Mycoplasma pneumoniae in Childhood

The current study aimed to investigate the clinical, laboratory and radiological findings of the pneumonia cases in children that were confirmed as M.pneumoniae by polymerase chain reaction (PCR) testing and to reveal the factors that can be decisive in the diagnosis. Seventy-seven children were included in this study. The median age of the patients was 31 months (1 month-17 years 4 months). The 63.6% of the patients were younger than five years of age, 53.2% were girls and 46.8% were boys. During the eight-year research period, the frequency of M.pneumoniae in the patients hospitalized with the diagnosis of pneumonia was found to be 3.1%. The rate of M.pneumoniae as the underlying factor of pneumonia was found to be statistically significantly lower in patients aged 0-60 months compared to the patients aged 61-216 months. In patients with M.pneumoniae accompanied by viruses, the age group was more likely to between 0-60 months. The most common symptoms were cough (96.1%) and fever (74%). Physical examinations revealed that 70.1% of the patients had rales, 63.6% had tachypnea, 45.5% had oropharyngeal hyperaemia, 35.1% had subcostal-intercostal retraction, 31.2% had long expiration period, 26% had rhonchus, 24.7% had decrease in breath sounds, 15.6% had cervical lymphadenopathy, 13% had tachycardia, 3.9% had otitis media, 3.9% had tonsil hypertrophy and 2.6% had a maculopapular rash. The rate of hypoxemia was found to be 42.2%. When the physical examination findings of patients with only M.pneumoniae detected in multiplex PCR analysis and those with accompanying viruses in M.pneumoniae were compared, tachypnea, oropharyngeal hyperemia and decreased breath sounds were found to be statistically significantly higher in patients with M.pneumoniae only. Retraction was detected more frequently in patients with accompanying viruses. When the laboratory results of the patients were evaluated according to age, leukocytosis was detected in only 18.2% of the patients, while the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were found to be high in 75% and 85.7% of the patients, respectively. In the multiplex PCR analysis, the CRP values of the patients with only M.pneumoniae were found to be higher than the patients with accompanying viruses. M.pneumoniae was accompanied by viruses at the rate of 40.3%. The most common accompanying viruses were rhinovirus, adenovirus, bocavirus and metapneumovirus. The 55.8% of the patients had lobar-segmental consolidation, 46.8% had parahilar-peribronchial thickening, 18.2% had atelectasis, 11.7% had pleural effusion, 9.1% had increase in reticulonodular density, 6.5% had lymphadenopathy whereas no abnormality was observed in 5.2% of them. No diffuse interstitial involvement was recorded. The CRP value of the patients who had lobar segmental consolidation which was detected through chest X-rays were statistically higher than those without consolidation. In multiplex PCR analysis, the rate of parahilar-peribronchial thickening detected in chest X-ray findings was found to be higher in patients with M.pneumoniae accompanied by viruses compared to those with only M.pneumoniae. The rate of the patients who were given empirical antibiotics against atypical agents was 45.5%. The rate of empirically administered antibiotic treatment for atypical agents after being hospitalization was higher in patients diagnosed with only M.pneumoniae compared to patients with M.pneumoniae and viruses. One patient (1.3%) died. As there are no typical clinical, laboratory or radiological findings specific to M.pneumoniae pneumonia, all of the findings should be assessed as a whole to establish a diagnosis. Besides, for the detection of M.pneumoniae, diagnostic tests which are cost effective, with rapid results and are capable of distinguishing colonisation from active infection should be developed.

Recent developments on the contribution of glutenin and puroindoline proteins to improve wheat grain quality

AbstractBackground and ObjectivesWheat is most staple food crop for making different end products. The major factors determining the wheat flour end‐use are glutenins and puroindolines. It is very important to identify the composition, variation, and functional characteristics of these key factors.FindingsThe glutenin subunits comprise two subgroups—high molecular weight glutenin subunits (HMW‐GS), and low molecular weight glutenin subunits (LMW‐GS). Puroindolines genes depict a wide diversity of both PINA and PINB allelic forms. The prevalence and relative proportions of these types may vary depending on several genetic and environmental factors.ConclusionCurrent review summarizes recently published research on the impact of glutenin genes governing gluten strength and puroindolines genes related to grain softness. It encompasses recent reports on the development of DNA‐based molecular markers utilized to identify novel alleles associated with HMW‐GS, LMW‐GS, and puroindoline genes in diverse wheat genotypes.Significance and NoveltySubstantial advancements have been made to identify associations between gene expression and grain quality and to develop improved wheat grain quality lines. Nevertheless, a novel marker system such as multiplex polymerase chain reaction and high throughput analysis is needed to screen large numbers of markers at low cost for rapid and precise analysis of germplasm, mapping, and marker‐assisted selection.

Using short tandem repeat profiling to validate cell lines in biobanks

Aim. To approve the COrDIS kit (Gordiz, Russia) for the authenticity of cell lines from the Bioresource Collection of the N.N. Blokhin National Medical Research Center of Oncology by the short tandem repeat (STR) profiling.Material and methods. The chosen method proved to be a reliable and reproducible option. With this approach, a number of polymorphic STR loci are amplified using commercially available primer sets. Polymerase chain reaction (PCR) products are analyzed simultaneously with size standards using automated fluorescent detection methods. The results are presented as a simple number code corresponding to the lengths of the PCR products amplified at each locus. By applying this method to cell lines, the laboratory can both authenticate commercial cell lines and build a database of their lines. In the work, we used the COrDIS EXPERT 26 kit (Gordiz, Russia), validated for molecular genetic identification of personality based on multiplex PCR analysis of 26 highly polymorphic loci of human genomic deoxyribonucleic acid. PCR results were analyzed by capillary electrophoresis using an automatic genetic analyzer with laser-induced fluorescence detection (Applied Biosystems 3500xL).Results. When testing the method, profiling of 37 cell lines was carried out, of which 18 were announced in international databases and 19 were unique, obtained at the N. N. Blokhin National Medical Research Center of Oncology, as well as a cell line mixture in order to determine the limits of contamination detection. The obtained results showed the correspondence of commercial cell lines with the data in international databases. Within the framework of this work, profiles of unique lines were obtained and the foundation of own genetic database was laid. Studies to identify the limit of contamination detection by another line have shown that even 4% of the contaminant culture in the total pool can be used to identify its individual alleles.Conclusion. The results obtained indicate the possibility of using the method to identify samples of the collection and detect intraspecific contamination.

Antimicrobial Sensitivity of Shiga toxin-producing Escherichia coli (STEC) and Virulence Genes of Representative Isolates in Port Harcourt, Nigeria

Aim: To assess the antimicrobial susceptibility, Shiga toxin-producing Escherichia coli (STEC) and virulence genes of representative isolates in some selected sites of Port Harcourt, Nigeria.
 Study Design: Case-controlled study.
 Place and Duration of Study: Selected places in Port Harcourt, Rivers State, Nigeria, between November, 2020 to November, 2021.
 Methodology: Three hundred and forty-nine (349) samples were analyzed, 80 meat and 63 waste waters from five abattoirs cited in the city, 46 meat samples from five selected roadside butchers sites in the city, 109 patients stool samples, 30 stool samples from food sellers, 20 stool samples from healthy subjects and 1 commercial bottled water which served as control samples. A combination of methods was employed: conventional culture using Tryptone soya broth as an enrichment media, chromagar STEC and serology with O157 latex reagents. Multiplex polymerase chain reaction analysis was used to screen for the presence of specific virulence genes in representative isolates and 16SrRNA sequence data was used to confirm the identity of isolates. GraphPad Prism 2.01 was used to perform the statistical analysis and p values < 0.05 were considered statistically significant.
 Results: The results showed that 20 (100%) of the selected isolates profiled for virulence gene harbored a combination of stx1 + stx2, while 45% carried the eaeA gene. The culture and molecular methods used in STEC detection showed a good level of agreement comparatively. The result of isolates sequenced for identification by targeting the 16S gene and compared with publicly available sequences on NCBI for phylogenetic analysis showed 100% identities to the ribosomal RNA gene of seven uncultured Escherichia coli strains with Gene Accession numbers (CP049290, MN208208, KY780343, CP058233 MK641327, CP082357, CP082129, MH346270). Isolates were resistant to Ceftazidime 98.9%, Cefuroxime 100%, Cefixime 100%, Augmentin 100% and sensitive to the quinolones and nitrofuratoin. The virulence gene combination pattern, the antibiogram pattern and the phylogenetic analysis result indicated genetic relatedness of isolates. 
 Conclusion: The representative isolates harbored stx1 and stx2 combination, only 45% harbored stx1, stx2 and eaeA genes combination. Phylogenetic analysis of the representative isolates confirmed the genetic similarity of isolates.

Direct detection of iro B, stn and hil A virulence genes in Salmonella enterica serovar typhimurium from non-ripened cheese

BackgroundMicroorganisms' survival is based on the importance of growth factors found in the microbial environment, as well as their overwhelming appetite for survival which is controlled by their genetic material. This study was designed to investigate the virulence patterns of Salmonella enterica serovar typhimurium associated with soft cheese. Two hundred and sixty (260) soft cheese samples were collected from vendors at Akure, Ikare and Owo in Ondo State, Nigeria. Molecular characterization of six (6) Salmonella enterica isolates for the possession of iro B, stn, hil A and spv C gene were revealed through multiplex polymerase chain reaction (PCR) analysis with DNA extraction, polymerase chain reaction (PCR) amplification, sequencing and genome blasting, respectively.ResultsFour (4) possess only iro B; two (2) possess only stn gene; and another three (3) possess hil A gene as none of the isolates possess spv C gene. This study revealed antibiotic-resistant Salmonella enterica serovar typhimurium is present in locally made soft cheese samples. It also revealed that virulence genes comprising hil A, stn and iro B as well as plasmids-mediating antibiotic resistance are present in S. enterica serovar typhimurium found in locally produced cheese.ConclusionsThe presence of molecularly elucidated virulence genes comprising Iro B, Stn and Hil A makes the soft cheese potentially viable for pathogenicity. This study recommends food-borne salmonellosis should be constantly monitored with appropriate cleaning of preparatory paraphernalia in cheese production. Constant sensitization of nomads to improve their awareness on milk-borne zoonosis and its associated risk factors is needed.

Prevalence, Antimicrobial Resistance, and Molecular Characterization of Escherichia coli Isolated from Food Contact Surfaces in Seafood Pre-Processing Plants (India)

Background: The survival of pathogens in biofilms poses a threat to food safety. The aim of this study was to determine prevalence, antimicrobial resistance, and molecular characterization of Escherichia coli strains.
 Methods: Swab samples (n=144) were collected from biofilm formed on food contact surfaces in seafood pre-processing plant in India. E. coli was isolated and identified using uid A gene by Polymerase Chain Reaction (PCR). The risk assessment of the isolates was carried out in terms of their drug resistance and the presence of virulence genes. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method. Phylogenetic grouping was done by quadruplex PCR. Molecular typing of the strains was performed by Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). Data were statistically analyzed using SPSS version 22.
 Results: Enteropathogenic E. coli (EPEC) strains were the most prevalent serotype. Multiplex PCR analysis revealed the presence of shiga toxin genes (stx1, stx2), intimin (eae), and enterohemolysin genes (hlyA). Shiga toxin gene stx2 showed the highest prevalence (83.33%). Among various phylogroups, B1 (45.56%) and B2 (30%) were the most prevalent phylogroups. Resistance to ampicillin (85.56%), piperacillin (84.44%), and cefpodoxime (85.56%) was widespread among the E. coli strains.
 Conclusion: The presence of genetically heterogeneous multi drug resistant E. coli strains with virulence potential showed a high risk in the seafood industry.

Microbial surveillance of Hajj tents: Bioaerosol sampling coupled with real-time multiplex PCR

Respiratory tract infections are common among pilgrims attending annual Hajj in Mecca, Saudi Arabia. Pilgrims typically spend most of the Hajj period inside ventilated tents, where microorganisms may be transmitted through bioaerosols and droplets. To perform microorganism surveillance inside Hajj tents and assess the similarities between microorganisms isolated from tent bioaerosol samples and nasopharyngeal swabs (NP) of tent occupants. Respiratory microorganisms in bioaerosols collected from Hajj tents over a 4-day period were compared with NP of tent occupants using real-time multiplex polymerase chain reaction analysis. A total of 152 samples were collected: 120 tent bioaerosol samples collected on days 9, 10, 11, and 12 of Dhu al-Hijjah, and 32 NP collected on day 12 of Dhu al-Hijjah (corresponding to 23/08/2018). Eighty-three (69.2%) bioaerosol samples tested positive for at least 1 microorganism, with the number of pathogens increasing over the 4 days of sampling. Twenty-seven (84.38%) NP swabs from tent occupants also tested positive. Microorganisms identified in pilgrim nasal carriage and tent bioaerosol samples were similar, and included K. pneumonia, S. aureus, S. pneumonia, human adenovirus, Moraxella, influenza A, and H. influenza. The data suggest that the Hajj tent environment may contribute to the spread of airborne infections during Hajj. This can have important ramifications for novel pathogens with pandemic potential.

Molecular typing and prevalence of antibiotic resistance and virulence genes in Streptococcus agalactiae isolated from Chinese dairy cows with clinical mastitis.

Bovine mastitis is a common disease occurring in dairy farms and can be caused by more than 150 species of pathogenic bacteria. One of the most common causative organisms is Streptococcus agalactiae, which is also potentially harmful to humans and aquatic animals. At present, research on S. agalactiae in China is mostly concentrated in the northern region, with limited research in the southeastern and southwestern regions. In this study, a total of 313 clinical mastitis samples from large-scale dairy farms in five regions of Sichuan were collected for isolation of S. agalactiae. The epidemiological distribution of S. agalactiae was inferred by serotyping isolates with multiplex polymerase chain reaction. Susceptibility testing and drug resistance genes were detected to guide the clinical use of antibiotics. Virulence genes were also detected to deduce the pathogenicity of S. agalactiae in Sichuan Province. One hundred and five strains of S. agalactiae (33.6%) were isolated according to phenotypic features, biochemical characteristics, and 16S rRNA sequencing. Serotype multiplex polymerase chain reaction analysis showed that all isolates were of type Ia. The isolates were up to 100% sensitive to aminoglycosides (kanamycin, gentamicin, neomycin, and tobramycin), and the resistance rate to β-lactams (penicillin, amoxicillin, ceftazidime, and piperacillin) was up to 98.1%. The TEM gene (β-lactam-resistant) was detected in all isolates, which was in accordance with a drug-resistant phenotype. Analysis of virulence genes showed that all isolates harbored the cfb, cylE, fbsA, fbsB, hylB, and α-enolase genes and none harbored bac or lmb. These data could aid in the prevention and control of mastitis and improve our understanding of epidemiological trends in dairy cows infected with S. agalactiae in Sichuan Province.

Simultaneous detection of SARS-CoV-2 and identification of spike D614G mutation using point-of-care real-time polymerase chain reaction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with high mortality and infectivity rates in humans since its emergence. Analysis using high-accuracy real-time polymerase chain reaction (PCR) is recommended for the detection of general respiratory viruses including SARS-CoV-2, but it takes a long time (e.g. ~ 6 h); moreover, on-site diagnosis is difficult owing to the need for skilled technicians and advanced laboratory facilities. Currently, the importance of point-of-care testing (POCT) is being emphasized for the rapid detection of SARS-CoV-2. Here, we developed a multiplex real-time reverse transcription PCR (rRT-PCR) analysis that not only detects SARS-CoV-2 but also D614G strains with higher contagiousness than wild types among SARS-CoV-2 mutants using probe-based rRT-PCR. Moreover, this method was applied to portable PCR equipment capable of POCT to confirm high detection sensitivity and specificity. Multiple assays were possible with fluorescence labeling of individual probes. Furthermore, using a microfluidic chip-based point-of-care testing rRT-PCR platform, detection time was reduced by more than half compared with the commonly used detection system. This demonstrates that our assay has 100% of high sensitivity and specificity and could thus aid in the rapid and simple screening of SARS-CoV-2 carrying the mutation. We present a rapid detection method for mutations in SARS-CoV-2.

Benign acute childhood myositis associated with influenza A/B in the paediatric emergency department and the efficacy of early-onset oseltamivir.

To investigate the association of benign acute childhood myositis (BACM) with respiratory viruses. Also, we aimed to assess the effect of antiviral treatment on the improvement and complications. This study was conducted at an urban-academic emergency department during four influenza-seasons (2016-2019), retrospectively. Demographics, clinical findings, laboratories, metabolic disease analyses and serological features were extracted from the medical records. Treatments, complications and outcomes were also recorded. A total of 114 children were included. The median age was 7.0 (min 1.25-max 17) years and 78.9% were male. The most common symptoms were leg pain (91.2%), anorexia (54.4%), fever (45.6%), sore throat (42.1%) and walking difficulty (32.5%). On admission, the median creatine phosphokinase level was 3332 IU/L (range, 1634-50 185), median aspartate aminotransferase 107 U/L (range, 38-1798). In the multiplex polymerase chain reaction analysis, 40.4% influenza B, 36.8% influenza A, 7.8% adenovirus, 7.8% parainfluenza virus, 5.3% rhinovirus, 5.3% respiratory syncytial virus and 1.8% Mycoplasma pneumoniae were detected. Rhabdomyolysis was developed in 6.7% and acute renal failure was seen in two patients. Oseltamivir was given in 44 (38.6%) patients who had influenza A/B. Metabolic disease screening tests were performed in 33.3% of patients and metabolic diseases were detected in 4 (3.5%) patients. The median recovery time was lower in patients with oseltamivir treatment (4 (min 2-max 5) - 5 (min 3-max 10) days) (P <0.001). Rhabdomyolysis is more common in BACM due to the influenza A virus. The early use of oseltamivir treatment was significantly associated with a shorter recovery time.

Development of a new multiplex quantitative real-time polymerase chain reaction method for Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum detection in cheese

Late blowing is a type of microbiological spoilage that causes unwanted changes in the taste, odour and texture of cheese due to the metabolic activities of Clostridium contaminating raw milk. This study aimed to develop a multiplex quantitative real-time polymerase chain reaction (PCR) analysis method for the rapid and simultaneous detection of Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum using specific primers and probes. The optimised method was determined to work with high sensitivity and specificity to quantify late-blowing Clostridium species in cheese samples. The lowest detectable gene copy number was 4.9 × 101, 7.8 × 101 and 8.5 × 101 for C. tyrobutyricum, C. butyricum and C. sporogenes, respectively. These results demonstrated that this method is a powerful tool for detecting and quantifying late-blowing agents in cheese. This is also the first multiplex qPCR study involving C. butyricum for detecting Clostridia, a late-blowing agent in cheese.

Multiplex Quantitative Real-Time Polymerase Chain Reaction and High-Resolution Melting Analysis for Identification of a Couple At-Risk of Having a Newborn with Severe Thalassemia

Many polymerase chain reaction (PCR)-based techniques have been used for routine diagnosis of α- and β-thalassemias. However, most require a multi step of post-PCR processes that are time-consuming and labor-intensive procedures. This study reported the successful use of multiplex quantitative real-time PCR (qPCR), with high-resolution melting (HRM) analysis for diagnosis of two common deletional α0-thalassemia (α0-thal) and 15 common β-thalassemia (β-thal) mutations, in order to identify a couple at-risk of having a newborn with severe thalassemia in the northern region of Thailand. With this approach, 22 (7.2%) of 306 couples were diagnosed as being at-risk for having a child with severe thalassemia, including three homozygous α0-thal, five homozygous β-thal and 14 Hb E (HBB: c.79G>A)/β0-thal disease. Our findings indicated that multiplex qPCR with HRM is applicable for routine molecular diagnosis in order to identify a couple at-risk of having a newborn with severe thalassemia, especially in an endemic region.