Results Of Multiplex Polymerase Chain Reaction Research Articles

Investigation of gut microbiota diversity according to infectious agent in pediatric infectious acute gastroenteritis in a Korean university hospital

BackgroundAcute gastroenteritis (AGE) is a common cause of pediatric morbidity and mortality worldwide. AGE can cause an imbalance in the intestinal microbiota. This study aimed to investigate the diversity of the gut microbiome in Korean children hospitalized for infectious AGE at a university hospital. MethodsA total of 23 stool samples from patients aged 5 months to 11 years with AGE were analyzed. Thirteen convalescent stool samples were collected 1 month after discharge. Multiplex polymerase chain reaction (PCR) for the five viruses and 16 bacteria-specific AGE pathogens (PowerChek Multiplex Real time PCR Kit, Seoul, Korea), and 16s rRNA sequencing (Illumina MiSeq Sequencing system, Illumina, USA) were performed. ResultsAccording to the results of multiplex PCR for causative pathogens, the microbiome taxonomic profile (MTP) of the gut microbiome in three groups of AGE, norovirus AGE (n = 11), Campylobacter AGE (n = 7) and Salmonella AGE (n = 5) was compared. The phylum Actinobacteria was significantly more abundant in the norovirus AGE (P = 0.011), whereas the phylum Proteobacteria was significantly more abundant in Salmonella AGE (P = 0.012). Alpha diversity, which indicates species richness and diversity, showed no statistical differences. However, beta diversity, representing the similarity in MTP between norovirus, Campylobacter, and Salmonella AGE, was significantly different (P = 0.007). In convalescence, compared with their corresponding AGE samples, the phylum Firmicutes; and the lower taxa Christensenellaceae (P = 0.0152) and Lachnospiraceae (P = 0.0327) were significantly increased. ConclusionsIn pediatric AGE, the type of infectious agent can affect the diversity and dominance of gut microbiota in pediatric patients. Furthermore, healthy gut bacteria increased during the period of 1 month after infection, allowing a return to a healthy state without causing long-term dysbiosis.

Presence of blaCTXM-1, blaCTXM-9, and blaTEM-1 Genes in Extended-spectrum β-lactamase-producing Escherichia coli Isolates from Hospital Wastewater

BACKGROUND: Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) are selectively proliferated in the human gut, excreted through feces, and deposited through wastewater lines, with hospital wastewater acting as a major reservoir of antibiotic resistance genes and resistant bacteria, thus pose adverse effects to human health. This study aimed to determine the presence of blaCTXM-1, blaCTXM-9, blaTEM-1, and blaSHV-1 genes in ESBL-EC in wastewater from selected hospitals in Manila and Quezon City, the Philippines.METHODS: Influent and effluent in twelve hospital wastewater treatment plants were collected, screened for cefotaxime-resistant E. coli, and examined for the ESBL production through phenotypic characterization using conventional bacterial identification, disk diffusion method, and VITEK® 2 Compact system and genotypic identification of ESBL-EC blaCTXM-1, blaCTXM-9, blaTEM-1, blaSHV-1 genes using multiplex polymerase chain reaction (PCR).RESULTS: Conventional bacterial identification methods and the VITEK® 2 Compact system results showed that both influent and effluent samples were positive for ESBL-EC at 33.3% and 16.7%, respectively. Multiplex PCR results revealed that various E. coli isolates were of ESBL-EC blaCTXM-1, blaCTXM-9, and blaTEM-1 genes. Multi-drug resistance was observed among all ESBL-EC isolates with resistance being highest against ampicillin, cefuroxime, ceftazidime, ceftriaxone, cefepime, piperacillin, and aztreonam.CONCLUSION: As the study revealed the presence of ESBL-producing bacteria, efforts must be made to ensure the prudent antimicrobial use with possible emphasis on antibiotic rotation accompanied by intensified infection prevention and control in hospital settings.KEYWORDS: antimicrobial resistance, beta-lactams, blaCTXM, blaTEM, extended-spectrum beta-lactamase, E. coli, hospital wastewater

Identificação molecular de genes de virulência de Escherichia coli e análise histopatológica em frangos condenados por aerossaculite

ABSTRACT: The present study identified virulence genes and pathological changes caused by Escherichia coli in chicken carcasses condemned for airsacculitis and assessed if the histopathological examination and polymerase chain reaction (PCR) were effective for studies like this. Trachea, liver, and lung were collected from 30 chickens with suspected airsacculitis that has been condemned in the inspection line. The samples were analyzed by PCR to simultaneously identify two virulence genes (iss and tsh genes) and for histopathological testing. PCR efficiently genotypically characterize the E. coli isolates, where the virulence genes iss and tsh were found in three birds simultaneously. The histopathological examination detected a predominance of heterophils and mononuclear cells in the trachea (100%), lung (90%), and liver (13.3%). The liver was the organ where practically no alteration was diagnosed. The results of multiplex PCR for the tsh and iss virulence genes indicate the great potential of the approach in the characterization of E. coli isolates. Unspecific identification did not occur, thus making it necessary to use technologies for the identification and prevention of this agent in aviaries and poultry abattoirs.

Molecular typing and antibiotic resistance patterns among clinical isolates of Acinetobacter baumannii recovered from burn patients in Tehran, Iran.

Acinetobacter baumannii (A. baumannii) is now considered a highly resistant pathogen to various types of antibiotics. Therefore, tracking the source of its prevalence and continuous control is crucial. This study aimed to determine antibiotic resistance and perform various molecular typing methods on clinical isolates of A. baumannii isolated from hospitalized burn patients in Shahid Motahari Burn Hospital, Tehran, Iran. Hospital isolates were confirmed by phenotypic and molecular methods. Then the sensitivity to different antibiotics was determined using the minimum inhibitory concentration (MIC) method. In order to perform molecular typing, three-locus dual assay multiplex polymerase chain reaction (PCR), multiple-locus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) methods were used. Among the 60 isolates collected, the frequencies of multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were 90 and 10%, respectively. The most effective antibiotics were colistin with 100% and tigecycline with 83.33% sensitivity. Isolates were 100% resistant to piperacillin/tazobactam and cephalosporins, and 68.3% were resistant to carbapenem. The results of multiplex PCR showed five groups that international clone I (IC I) and IC II were the most common. The MLVA method identified 34 MLVA types (MTs), 5 clusters, and 25 singletons. Multilocus sequence typing results for tigecycline-resistant isolates showed seven different sequence types (STs). Increasing antibiotic resistance in A. baumannii isolates requires careful management to control and prevent the occurrence of the pre-antibiotic era. The results of this study confirm that the population structure of A. baumannii isolates has a high diversity. More extensive studies are needed in Iran to better understand the epidemiology of A. baumannii.

Evaluating the utility of the A.F. Genital System test for pathogen diagnosis in acute male urethritis.

This study evaluated the effectiveness of the A.F. Genital System (Liofilchem® , Italy) in detecting pathogens compared with multiplex real-time polymerase chain reaction (PCR) in men with acute urethritis. Men diagnosed as having acute urethritis between 1 April 2021 and 31 December 2021 were included. Urethral swab samples were obtained for A.F. Genital System and PCR testing in a randomly determined order. The efficacy of the A.F. Genital System was analysed by comparing the results of the two tests. The study included 83 patients (mean age 34.1±11.3years). A urethritis pathogen was detected in 69 patients (83.1%) by PCR and only 15 patients (18.1%) with the A.F. Genital System. The sensitivity of the A.F. Genital System in detecting acute urethritis pathogens was 21.7% (95% confidence interval [CI]: 13.6-32.8), and the specificity was 100% (95% CI: 78.5-100). Its sensitivity was 20% (95% CI: 7.1-45.2) in the diagnosis of gonococcal urethritis and 19.1% (95% CI: 11.2-30.4) in the diagnosis of non-gonococcal urethritis. PCR detected two or more urethritis pathogens in 9 patients (13.0%), while no polymicrobial infection was detected with the A.F. Genital System. Based on the results of multiplex real-time PCR, the A.F. Genital System had very low sensitivity in the detection of pathogens in acute male urethritis. It should be kept in mind that using this test in patients with acute urethritis may result in a high missed diagnosis rate for urethritis pathogens.

Evaluation of Multiplex Real-time PCR and WHO Criteria for Diagnosing Childhood Bacterial Meningitis in a Tertiary Referral Hospital in Iran

Background: Childhood bacterial meningitis (BM) requires prompt and precise diagnosis to provide proper treatment and decline mortality and morbidity. Objectives: We aimed to evaluate the World Health Organization (WHO) criteria and polymerase chain reaction (PCR) for diagnosing BM in children admitted to a tertiary referral hospital in Shiraz, southern Iran. Materials: We included all 492 children aged one month to 17 years suspected of meningitis who had cerebrospinal fluid (CSF) leukocytosis admitted to Nemazi Hospital from August 2016 to September 2017. The CSF specimens were examined for routine analysis, Gram staining, and culture. A multiplex real-time PCR was used to identify Streptococcus pneumoniae, Haemophilus influenzae type b (Hib), and Neisseria meningitidis in the CSF samples. Seven viruses were also investigated using real-time PCR. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using the WHO criteria and the multiplex real-time PCR results. Results: Seventy-four CSF samples had leukocytosis. Nineteen (22.9%) patients had BM caused by S. pneumoniae (n = 14), Hib (n = 2), Salmonella enterica (n = 2), and N. meningitidis (n = 1). The PCR test detected all cases, except for two with Salmonella meningitis (sensitivity 89.4%, specificity 100%, PPV 100%, and NPV 96%). The WHO criteria detected all cases, except three who received antibiotics at least four days before performing lumbar puncture (sensitivity 84.2%, specificity 98.2%, PPV 94.1%, and NPV 94.7%). Enterovirus was the most common viral etiology (6.75%). Conclusions: The WHO criteria and the multiplex real-time PCR had high accuracy in our setting, and their use could decrease the antibiotic over-prescription in febrile children suspected of meningitis.

Multiplex Real-Time Polymerase Chain Reaction on Sputum for the Diagnosis of Pneumocystis jirovecii Pneumonia in Children: A Retrospective Study.

BackgroundPneumocystis jirovecii pneumonia (PCP) is a serious opportunistic infection in immunocompromised children. Real-time polymerase chain reaction (PCR) is widely used for the diagnosis of PCP due to its good accuracy. However, the diagnostic performance of multiplex real-time PCR on sputum in children with PCP has never been explored.MethodsMedical records of 63 consecutive pediatric patients were analyzed retrospectively, including 13 cases with PCP and 50 with non-PCP pneumonia. Pneumocystis jirovecii (P. jirovecii) and other co-pathogens detected by multiplex real-time PCR in sputum samples were summarized. Using clinical composite diagnosis as the reference standard, we further compared the diagnostic performance of multiplex real-time PCR to combined serological markers (1,3)-β-D-glucan plus lactate dehydrogenase. Additionally, modifications of antimicrobial treatment for pediatric PCP patients after the report of multiplex real-time PCR results were reviewed.ResultsIn children with PCP, nonproductive cough and shortness of breath were more common, lymphocyte count in peripheral blood was markedly lower, and serum levels of (1,3)-β-D-glucan and lactate dehydrogenase were much higher than non-PCP group. Multiplex real-time PCR reached a sensitivity of 100% in diagnosing PCP, which was better than serum (1,3)-β-D-glucan plus lactate dehydrogenase (76.9%). Its specificity (98.0%) significantly surpassed serum (1,3)-β-D-glucan plus lactate dehydrogenase (84.4%). Furthermore, multiplex real-time PCR showed a good performance in identifying co-pathogens in sputum of pediatric PCP patients. Cytomegalovirus, Epstein–Barr virus and Streptococcus pneumoniae were the most common co-pathogens in these patients. Initial antimicrobial treatment was modified in 76.9% of children with PCP after the report of PCR results.ConclusionMultiplex real-time PCR on sputum is a diagnostic tool with good performance for the identification of P. jirovecii as well as co-pathogens in children with PCP. Sputum may be an alternative to bronchoalveolar lavage fluid for PCR assay in children when bronchoscopic examination is not feasible.

Role of multiplex PCR in the early diagnosis of infective endocarditis

Infective endocarditis (IE) is a serious infection of the endocardium and heart valves that necessitate early diagnosis. The conventional blood culture has lots of false-negative results besides being time consuming. Polymerase chain reaction (PCR) is a rapid diagnostic tool that helps in saving the patients’ life. This study aimed to investigate the feasibility of multiplex PCR in early diagnosis of IE compared to the conventional blood culture, and to evaluate its impact on IE diagnosis in cases of negative blood cultures. The current study was conducted on 30 patients admitted to the Cardiology Department, Faculty of Medicine, Ain Shams Hospitals, Cairo, Egypt, which were diagnosed clinically as infective endocarditis according to the modified Duke’s criteria. After processing of the patient's blood samples, the blood cultures recorded positivity in 5 cases (16.7 %). The most common recovered bacteria were, Staphylococcus aureus 2(6.7 %), Staphylococcus epidermedis 1(3.3 %), Enterococcus faecalis 1(3.3 %), and Escherichia coli 1(3.3 %); however, no other pathogens were isolated. On the other hand, results of multiplex PCR showed positivity in 13 cases (43.3 %), mainly; Staphylococcus aureus 5(16.7 %), E. faecalis 3(10 %), Staphylococcus epidermidis 2(6.7 %), Pseudomonas aeruginosa 2(6.7 %), and E. coli 1(3.3 %). No other bacterial of fungal pathogens were detected by multiplex PCR. Finally, the multiplex PCR assay exhibited remarkable sensitivity and feasibility in IE diagnosis over blood culture, besides being a rapid and accurate diagnostic assay that enhances proper treatment.

Distribution of respiratory viruses: Evaluation of multiplex PCR results of 3074 patients.

Early and accurate diagnosis of acute respiratory infections is important because these diseases negatively affect public health and can lead to loss of workforce and an increase in health expenditures. In this study, we aimed to determine the respiration panel multiplex polymerase chain reaction (PCR) test results and seasonal distribution in our region. Three thousand and seventy-four patients samples multiplex PCR (Anatolia, Bosphore® Respiratory Pathogens Panel Kit v1) test results, which were sent to our laboratory, from 13 hospitals in our region between January 2018 and December 2018, were evaluated retrospectively. A total of 3074 patients samples, 1465 (48%) were positive and 1609 (52%) were negative test results. The most common factors were rhinovirus 30.2%, influenza A 23.1%, and respiratory syncytial virus (RSV) A/B 19.1%, respectively. When the distribution of these three most common viruses by months is examined, the most frequent months were determined as June for rhinovirus, November for influenza A, and February for RSV A/B. In the period between October and February, there was a significant increase in the positivity level of viral factors. The use of molecular methods in the diagnosis of respiratory infections will prevent unnecessary use of antibiotics and ensure correct and rapid treatment.

Diagnostic Utility of Biplex/Multiplex Polymerase Chain Reaction in Infectious Granulomatous Dermatitis in North Indian Population

A definite diagnosis of infectious granulomatous dermatitis (IGD) is difficult for both practicing dermatologists and dermatopathologists due to overlapping clinical and histomorphological features. We aimed to explore the role of multiplex polymerase chain reaction (PCR) for identifying a definite etiological agent for diagnosis and appropriate treatment in IGD in formalin-fixed paraffin-embedded tissue. Sixty-two cases of IGD were included, excluding leprosy. The histochemical stains including Ziehl-Neelsen, periodic acid-Schiff, and Giemsa were performed in all cases. A multiplex PCR was designed for detection of tuberculosis (TB) (IS6110 and mpt64), fungal infections (ITS1, ITS2; ZM1, and ZM3), and leishmaniasis (kDNA). The results of histomorphology, histochemical stains, and multiplex PCR were compared. Among 62 cases, the sensitivity rate of PCR detection for organisms was 16.7%, 0%, 100%, 72%, 75%, and 66.7% in patients with TB, suggestive of TB, leishmaniasis, fungal infections, and granulomatous dermatitis not otherwise specified and granulomatous dermatitis suggestive of fungus, respectively. The TB PCR using IS6110 primers was negative in all cases; however, PCR using mpt64 primers was positive in 33.33% cases of scrofuloderma. The histochemical stains including Ziehl-Neelsen for acid-fast bacilli, periodic acid-Schiff for fungus, and Giemsa for Leishman-Donovan bodies showed positivity in 11.3%, 43.5%, and 3.2%, respectively. A multiplex PCR (Mycobacterium tuberculosis, Leishmania, and panfungal) is highly recommended in all cases of IGD where an etiological agent is difficult to establish by skin biopsy and histochemical stains along with a clinicopathological correlation. This will augment in appropriate treatment and will reduce empirical treatment and morbidity in such patients.

High prevalence of blaCMY AmpC beta-lactamase in ESBL co-producing Escherichia coli and Klebsiella spp. clinical isolates in the northeast of Iran

ObjectiveThe production of β-lactamase enzymes such as AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) is among the main mechanisms for resistance to expanded-spectrum cephalosporins. The present study was conducted to investigate the prevalence and molecular epidemiology of plasmid-mediated AmpC beta (β)-lactamase in ESBL co-producing Escherichia coli (E. coli) and Klebsiella spp. (Klebsiella pneumoniae and Klebsiella oxytoca) clinical isolates in the northeast of Iran. MethodsA total of 602 E. coli and Klebsiella spp. clinical isolates were collected from three hospitals in Mashhad (northeast of Iran). A combination disk test (CDT) was performed for the phenotypic detection of ESBLs. Screening for the detection of AmpC β-lactamases was performed by a susceptibility test to a cefoxitin disc among ESBL producing isolates. A confirmatory test for AmpC β-lactamases was performed using the Mast® D68C test. Identification of plasmid-mediated AmpC cluster genes was done by multiplex polymerase chain reaction (PCR). ResultsAmong 336 ESBL-producing strains, 230 (68.4%) isolates were resistant to cefoxitin. Results of the Mast® D68C test showed that 30% (69/230) of cefoxitin-resistant isolates simultaneously exhibited ESBL and AmpC activity and 22% (51/230) of isolates probably showed multi-drug resistant (MDR) phenotype. Results of multiplex PCR among ESBL-positive isolates showed that, 16.7% (56/336) of isolates were positive for plasmid-borneampC cluster genes, and CMY (38%) was the most frequent genotype of plasmid mediated AmpC. ConclusionFindings of the study revealed that an increase in the prevalence of ESBL and AmpC co-producer in E. coli and Klebsiella spp. strains may become an important public health issue. Therefore, there is a vital need for surveillance of spread of these clinical isolates.

Isolation and characterization of Clostridium perfringens strains isolated from ostriches (Struthio camelus) in Vietnam.

Background and Aim:Clostridium perfringens can cause enteritis in ostriches. The toxin release is believed to play a major role in determining pathogenesis properties of these pathogenic bacteria. This study was conducted to isolate and characterize C. perfringens strains from ostriches in Vietnam for identifying if particular virulence factors of these pathogenic bacteria are associated with enteritis progress in ostriches.Materials and Methods:The prevalence of cpa, cpb, iA, etx, cpe, and cpb2 genes among C. perfringens isolates was determined by a multiplex polymerase chain reaction (PCR) method. The NetB toxin-encoding gene was detected by PCR and then sequenced to observe their variation. The expression of NetB toxin was checked by SDS-PAGE.Results:A total of 116 C. perfringens isolates were obtained from 318 fecal samples and 105 intestinal organs. Of 80 isolates from fecal samples, 33 isolates were from healthy and 47 isolates were from diseased ostriches. The results of multiplex PCR showed that all 116 C. perfringens strains from healthy and enteric disordered ostriches were positive for the alpha toxin-encoding gene (cpa). The cpe and cpb2 genes were found in only one and five diseased ostriches, respectively. The netB gene was detected in 1/33 (3.03%) C. perfringens isolates from healthy ostriches, in 8/47 (17.05%) isolates from feces, and in 7/36 (19.44%) intestinal contents of diseased ostriches. The full-length sequences of 5 out of 15 netB-positive isolates from diseased ostriches showed 100% identity to each other as well as to the netB sequences available in GenBank. All of these five isolates produced NetB toxin in vitro.Conclusion:Type A is the most prevalent among C. perfringens isolates from ostriches in Vietnam. Especially, the study provides data emphasizing the role of NetB toxin in causing necrotic enteritis by C. perfringens in ostriches.

Event-specific multiplex PCR method for four genetically modified cotton varieties, and its application

Multiplex polymerase chain reaction (PCR) methods have been developed and validated for screening, tracing, and regulating genetically modified (GM) crops in quarantine and environmental monitoring. In this study, we aimed to develop a method to simultaneously detect four GM cotton varieties in order to establish a screening system for cotton volunteers. Based on the sequence of DNA in the junction between introduced gene and flanking genomic DNA of four GM cotton events, herbicide-tolerant MON88701 and DAS-81910-7 and insect-resistant COT102 and T304-40, event-specific primers were designed and a multiplex detection method was developed. The simplex PCR results supported the multiplex PCR results; the amplification efficiency of the novel multiplex PCR method was increased compared with that of the Joint Research Centre (JRC) method. Based on the accuracy and efficiency, the method can be applied to detect and identify randomly mixed reference materials and suspected cotton volunteers. To apply this multiplex PCR method to living modified (LM) environmental monitoring samples, we performed additional PCR analysis to identify whether the volunteers were the four LM cotton varieties. As a result, 66 cotton volunteers were identified with stack event, comprising one or two of the four LM cotton events, and all stacks have been approved in South Korea for food, feed, and processing. These results indicated that our novel multiplex method is suitable for LMO identification.

Outcomes and clinical relevance of stool multiplex bacterial polymerase chain reaction in patients with acute diarrhea: single center experience.

Background/AimsDiagnostic stool multiplex polymerase chain reaction (PCR) testing has attracted considerable interest, because of its high sensitivity, short turnaround time, and ability to detect multiple organisms simultaneously. This study investigates the clinical usefulness of a stool multiplex bacterial PCR in patients with acute diarrhea.MethodsWe retrospectively evaluated the stool multiplex bacterial PCR results, clinical parameters, and clinical courses of patients hospitalized because of acute diarrhea between August 2014 and November 2016.ResultsA total of 725 patients (male, 372; mean age, 30.9 ± 29.3 years) underwent stool multiplex bacterial PCR. A total of 243 pathogens were detected in 226 patients. The detection rate of multiplex PCR testing was higher than that of stool culture (32.7% vs. 3.3%, p < 0.01). Severe symptoms of acute diarrhea (bloody diarrhea, frequent diarrhea) and prescribed empirical antibiotics were significantly more common in the positive multiplex PCR group (p = 0.02, p < 0.01, p < 0.01, respectively). However, mean durations of hospital stay were similar in the 2 groups according to the multiplex PCR results (p = 0.32). In addition, Campylobacter spp., which was the most commonly detected pathogen (97/243, 39.9%), was significantly associated with frequent diarrhea and prescribed empirical antibiotics (p < 0.01), but not with duration of hospital stay (p = 0.09).ConclusionsWe concluded that stool multiplex bacterial PCR might be a useful tool for identifying bacterial etiology in patients with acute diarrhea, especially in those with Campylobacter spp. infection.

Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay.

Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) and Gallinarum (S. Gallinarum) can result in pullorum disease and fowl typhoid in avian species, respectively, and cause considerable economic losses in poultry in many developing countries. Conventional Salmonella serotyping is a time-consuming, labor-intensive and expensive process, and the two biovars cannot be distinguished using the traditional serological method. In this study, we developed a rapid and reliable one-step multiplex polymerase chain reaction (PCR) assay to simultaneously identify and discriminate the biovars Pullorum and Gallinarum. The multiplex PCR method focused on three specific genes, stn, I137_08605 and ratA. Based on bioinformatics analysis, we found that gene I137_08605 was present only in S. Pullorum and S. Gallinarum, and a region of difference in ratA was deleted only in S. Pullorum after comparison with that of S. Gallinarum and other Salmonella serovars. Three pairs of primers specific for the three genes were designed for the multiplex PCR system and their selectivity and sensitivity were determined. The multiplex PCR results showed that S. Pullorum and S. Gallinarum could be identified and discriminated accurately from all tested strains including 124 strains of various Salmonella serovars and 42 strains of different non-Salmonella pathogens. In addition, this multiplex PCR assay could detect a minimum genomic DNA concentration of 67.4 pg/μL, and 100 colony forming units. The efficiency of the multiplex PCR was evaluated by detecting natural-occurring Salmonella isolates from a chicken farm. The results demonstrated that the established multiplex PCR was able to identify S. Gallinarum and S. Pullorum individually, with results being consistent with traditional serotyping and biochemical testing. These results demonstrated that a highly accurate and simple biovar-specific multiplex PCR assay could be performed for the rapid identification and discrimination of Salmonella biovars Gallinarum and Pullorum, which will be useful, particularly under massive screening situations.

Prevalence and seasonality of common viral respiratory pathogens, including Cytomegalovirus in children, between 0\u20135\xa0years of age in KwaZulu-Natal, an HIV endemic province in South Africa

BackgroundAcute respiratory tract infections contribute significantly to morbidity and mortality among young children in resource-poor countries. However, studies on the viral aetiology of acute respiratory infections, seasonality and the relative contributions of comorbidities such as immune deficiency states to viral respiratory tract infections in children in these countries are limited.MethodsA retrospective analysis of laboratory test results of upper or lower respiratory specimens of children between 0 and 5 years of age collected between 1st January 2011 and 31st July 2015 from hospitals in KwaZulu-Natal, South Africa. Respiratory specimens were tested for viral respiratory pathogens using multiplex polymerase chain reaction (PCR), HIV testing was performed either by serological or PCR methods. Cytomegalovirus (CMV) respiratory infection was determined using the CMV R-gene PCR kit.ResultsIn total 2172 specimens were analysed, of which 1175 (54.1%) were from males. The median age was 3.0 months (interquartile range [IQR] 1–7). Samples from the lower respiratory tract accounted for 1949 (89.7%) of all specimens. Respiratory multiplex PCR results were positive in 834 (45.7%) specimens. Respiratory syncytial virus (RSV) was the most commonly detected virus in 316 (32.1%) patients, followed by adenovirus (ADV) in 215 (21.8%), human rhinovirus (Hrhino) in 152 (15.4%) and influenza A (FluA) in 50 (5.1%). A seasonal time series pattern was observed for ADV (winter peak), enterovirus (EV) (autumn), human bocavirus (HBoV) (summer), and parainfluenza viruses 1 and 3 (PIV1 and 3) (spring). Stationary or untrended seasonal variation was observed for FluA (winter peak) and RSV (summer). HIV results were available for 1475 (67.9%) specimens; of these 348 (23.6%) were positive. CMV results were available for 714 (32.9%) specimens, of which 416 (58.3%) were positive. There was a statistically significant association between the coinfection of HIV and CMV with ADV.ConclusionsIn this study, we identified the most common respiratory viral pathogens detected among hospitalized children in KwaZulu-Natal. The coinfection between HIV and CMV was found to be associated with an increased risk of only adenovirus infection. Most viral pathogens showed a seasonal trend of occurrence. Our data has implications for the rational design of public health programmes.

English

This study aimed to identify some important virulence factors, including pap, fim, sfa, aer and hly genes, typical of uropathogenic Escherichia coli (UPEC) in isolates collected from diarrheic and urinary tract infected patients in Khartoum State by multiplex polymerase chain reaction (PCR) assay. A total of 100 clinical specimens (50 urine and 50 diarrhea) were collected. Samples were cultured and identified by conventional method. Most study population were females 57/100 (57%); 42 suffering from urinary tract infections (UTIs) and 15 from diarrhea, while males were 43/100 (43%); 8 suffering from UTIs and 35 from diarrhea. Among enrolled subjects, 83 were positive for one or more uropathogenic E. coli virulent genes, while 17 isolates were negative for all genes. The results of multiplex PCR revealed that thirty two (n=32) diarrheal samples and fourteen (n=14) urine samples were aer positive. Thirty three (n=33) urine samples and eight (n=8) diarrheal samples appeared as fim positive. The genes pap and hly were found in 24 and 14 urine samples, respectively and in 9 and 3 diarrheal samples, respectively, while sfa gene was detected only in 15 urine specimens. The study concluded that fim gene was highly prevalent among UTI patients while aer gene was highly prevalent among diarrhea patients. &nbsp; Key words: Uropathogenic Escherichia coli, fimH, aer, pap, sfa, hly, Sudan.

Genotypic detection of the virulence factors of uropathogenic Escherichia coli isolated from diarrheic and urinary tract infected patients in Khartoum State, Sudan

This study aimed to identify some important virulence factors, including pap, fim, sfa, aer and hly genes, typical of uropathogenic Escherichia coli (UPEC) in isolates collected from diarrheic and urinary tract infected patients in Khartoum State by multiplex polymerase chain reaction (PCR) assay. A total of 100 clinical specimens (50 urine and 50 diarrhea) were collected. Samples were cultured and identified by conventional method. Most study population were females 57/100 (57%); 42 suffering from urinary tract infections (UTIs) and 15 from diarrhea, while males were 43/100 (43%); 8 suffering from UTIs and 35 from diarrhea. Among enrolled subjects, 83 were positive for one or more uropathogenic E. coli virulent genes, while 17 isolates were negative for all genes. The results of multiplex PCR revealed that thirty two (n=32) diarrheal samples and fourteen (n=14) urine samples were aer positive. Thirty three (n=33) urine samples and eight (n=8) diarrheal samples appeared as fim positive. The genes pap and hly were found in 24 and 14 urine samples, respectively and in 9 and 3 diarrheal samples, respectively, while sfa gene was detected only in 15 urine specimens. The study concluded that fim gene was highly prevalent among UTI patients while aer gene was highly prevalent among diarrhea patients. Key words: Uropathogenic Escherichia coli, fimH, aer, pap, sfa, hly, Sudan.

Polymerase Chain Reaction and Goldmann-Witmer Coefficient to Examine the Role of Epstein-Barr Virus in Uveitis

ABSTRACTPurpose: To use polymerase chain reaction (PCR) and Goldmann-Witmer Coefficient (GWC) calculation to search for evidence that Epstein-Barr virus (EBV) causes uveitis.Methods: A prospective cross-sectional study where participants with positive multiplex EBV PCR results were further investigated by: 1) real-time PCR for EBV viral loads (VL) and 2) EBV GWC.Results: Eleven of 106 consecutive uveitis patients (10.4%) had positive multiplex PCR for EBV on aqueous humor sampling and 7/11 (63.6%) were HIV-positive. Only 4/10 (40%) cases had detectable intraocular EBV VLs which were always lower than the blood or plasma VL. EBV GWC was negative in all 10 cases tested. In 9/11 (81.8%) of these cases an alternative, more plausible cause of uveitis was identified.Conclusion: We found no evidence of active intraocular replication or antibody production to prove that EBV caused uveitis in these cases. In most cases an alternative treatable cause of uveitis was identified.

Prevalence of the microsporidian Nosema ceranae in honeybee (Apis mellifera) apiaries in Central Italy.

Nosema ceranae and Nosema apis are microsporidia which play an important role in the epidemiology of honeybee microsporidiosis worldwide. Nosemiasis reduces honeybee population size and causes significant losses in honey production. To the best of our knowledge, limited information is available about the prevalence of nosemiasis in Italy. In this research, we determined the occurrence of Nosema infection in Central Italy. Thirty-eight seemingly healthy apiaries (2 to 4 hives each) were randomly selected and screened from April to September 2014 (n = 11) or from May to September 2015 (n = 27). The apiaries were located in six areas of Central Italy, including Lucca (n = 11), Massa Carrara (n = 9), Pisa (n = 9), Leghorn (n = 7), Florence (n = 1), and Prato (n = 1) provinces. Light microscopy was carried out according to current OIE recommendations to screen the presence of microsporidiosis in adult worker honeybees. Since the morphological characteristics of N. ceranae and N. apis spores are similar and can hardly be distinguished by optical microscopy, all samples were also screened by multiplex polymerase chain reaction (M-PCR) assay based on 16S rRNA-gene-targeted species-specific primers to differentiate N. ceranae from N. apis. Furthermore, PCR-positive samples were also sequenced to confirm the species of amplified Nosema DNA. Notably, Nosema spores were detected in samples from 24 out of 38 (63.2%, 95% CI: 47.8–78.5%) apiaries. Positivity rates in single provinces were 10/11, 8/9, 3/9, 1/7, or 1/1 (n = 2). A full agreement (Cohen's Kappa = 1) was assessed between microscopy and M-PCR. Based on M-PCR and DNA sequencing results, only N. ceranae was found. Overall, our results highlighted that N. ceranae infection occurs frequently in the cohort of honeybee populations that was examined despite the lack of clinical signs. These findings suggest that colony disease outbreaks might result from environmental factors that lead to higher susceptibility of honeybees to this microsporidian.