Conventional Multiplex PCR Research Articles

A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis

BackgroundZoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp.MethodsWe compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites.ResultsThe protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile.ConclusionThe new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

Serotype Distribution, Antimicrobial Susceptibility, and Multilocus Sequencing Type (MLST) of Streptococcus pneumoniae From Adults of Three Hospitals in Shanghai, China.

Background: Streptococcus pneumoniae, a main causative agent associated with invasive and non-invasive infection in elderly population, is a major global health problem. After pneumococcal conjugate vaccines (PCV) and pneumococcal polysaccharide vaccines (PPV) were introduced, the distribution of S. pneumoniae serotypes has changed. There was currently limited data on epidemiology and status of antimicrobial resistance of S. pneumoniae in Shanghai.Objective: To determine the serotype distribution, antimicrobial susceptibility and molecular epidemiology of S. pneumoniae isolated from adults in Shanghai.Method: A total of 75 S. pneumoniae isolates consecutively collected from 2015 through 2017 were serotyped by conventional multiplex-PCR. The antimicrobial susceptibility was determined by broth microdilution method. The multilocus sequence type (MLST) was performed to estimate the molecular epidemiology.Results: The predominant serotypes among the isolates were 19F (20.00%), 3 (16.00%), 23F (9.33%), 14 (8.00%), and19A (5.33%). The prevalence of pneumococcal strains with serotypes targeted by vaccines PCV7, PCV10, PCV13, and PPV23 was 44, 45.33, 66.67, and 80%, respectively. Penicillin non-susceptible S. pneumoniae (PNSSP) accounted for 16% of the isolates examined and resistance to erythromycin, azithromycin, tetracycline, clindamycin, cefaclor and trimethoprim-sulfamethoxazole were found in 92.00, 90.67, 86.67, 81.33, 54.67, and 54.67% of isolates, with most isolates (78.67%) presenting multidrug-resistance. The top three sequence types (STs) were ST271 (17.33%), ST180 (9.33%), and ST81 (8.00%). The international resistance clone complexes Spain23F-1 (n = 4), Netherland3-31 (n = 8), and Taiwan19F-14 (n = 14) were identified.Conclusions: The S. pneumoniae isolates showed high genetic diversity in Shanghai and the prevalence of antimicrobial resistance was also high among S. pneumoniae isolates, most of which were multidrug-resistant. The spread of international resistance clones might contribute to the increase of resistant isolates. The PPV23 could protect against most pneumococcal capsular serotypes causing infection of adults in Shanghai.

Conventional culture diagnostics vs. multiplex PCR for the detection of causative agents of vascular graft infections - results of a single centre observational pilot study.

Background: Timely diagnosis of vascular graft infections is of major importance in vascular surgery. The detection of causative microorganisms is needed for specific medical treatment, but conventional culture is often slow, insensitive and inconclusive due to antibiotic pre-treatment. Detection of bacterial DNA by polymerase chain reaction (PCR) might bypass these problems. We hypothesised that multiplex PCR (mPCR) is feasible, fast and sensitive to detect causative microorganisms in vascular graft infections. Patients and methods: We performed a pilot observational prospective study comparing conventional culture and a commercial mPCR. Inclusion criteria were: confirmed graft infection, suspicious imaging, clinical suspicion, anastomotic aneurysm and repeated graft occlusion. Diagnostic methods were performed using identical samples. Time to result, microorganisms and antibiotic resistance in both groups were compared using Student's t-test or nonparametric tests. Results: 22 samples from 13 patients were assessed and 11 samples were negative for bacteria. Some showed multiple germs. In total, we found 15 different organisms. 13 samples matched, 9 had non-concordant results. Out of the mismatches 3 microorganisms identified in PCR were not detected by culture. Time to result with PCR was shorter (median 5h vs. 72h, p<0.001) than with culture. No resistance genes were detected by mPCR, but conventional culture allowed susceptibility testing and revealed resistance in 5 samples. Conclusions: mPCR seems to be a feasible and quick tool to detect causes of vascular graft infections within 24h and might be helpful in antibiotic pre-treated patients. The detection of antibiotic resistance with mPCR needs improvement for clinical practice.

Comparison of Gold Standard Genescan with NGS-Based TCR-Beta Clonality Analysis Using Oncomine TCR Beta-Short Read Assay

Introduction All mature T-cell-based lymphoid malignancies harbor identical (clonal) rearrangements of their T cell receptor (TCR) genes (van Dongen et al. Clinica Chimica Acta. 1991, 198, 1-92). The clonality assessment of the rearranged TCR gene is particularly important for the identification, characterization and monitoring of T-cell neoplasms since histology and immunophenotyping alone is not enough to make conclusive diagnosis in all cases. Diagnostic clonality testing is currently based on the parallel analysis of the rearranged TCR-gamma and TCR-beta chain genes. This is done using a multiplex PCR developed within the European BIOMED-2/EuroClonality consortium (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis of the resultant PCR products. Although, this method is considered the "gold standard", which is established worldwide in many molecular diagnostic laboratories, the resolution of this approach is limited, especially in cases with low percentage of clonally rearranged TCRs and a high mixture of non-clonally rearranged T-cells. Next generation sequencing (NGS) is a powerful tool to provide resolution at single gene level. This also holds true for NGS assays, which are able to detect all unique TCR rearrangements in a given sample with very high resolution and sensitivity. This approach can be employed for precise assessment of the immune repertoire, minimal residue disease and T-cell clonality. Detailed insights into the clonotypes provides a great potential for early diagnosis of T-cell neoplasms, and the identification of individual clones is essential for monitoring of the disease. Methods We investigated 19 formalin-fixed paraffin-embedded (FFPE) tissue samples from celiac disease patients (n = 14) and routine diagnostic cases suspicious of lymphoma (n = 5). After DNA extraction, we performed multiplex PCRs using the BIOMED-2 TCR-beta primer sets (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis. For TCR-beta NGS, the Oncomine™ TCR Beta-SR DNA Assay (Thermo Fisher Scientific) was used according to the manufacturer's instructions and sequencing was performed on the IonTorrent S5. Identification of individual clonotypes and bioinformatics analysis of the data was done with the help of the IonReporter software (Thermofisher Scientific). It is worth noting that the 14 celiac disease samples were previously analysed by a different TCR-beta NGS approach (Ritter et al. Gut. 2018, 67, 644-653) and utilized for the comparison of the NGS-data. Results 17 out of 19 cases analysed by TCR-beta multiplex PCR (BioMed-2) followed by capillary electrophoresis and by the Oncomine™ TCR Beta-SR assay displayed a very similar length distribution of the PCR products. This holds true for samples with a clonal appearance and for samples with an oligo-/polyclonal pattern. In two discrepant cases, the Primerset B of the BioMed-2 approach showed a dominant amplification product, which was not as clear by TCR-beta NGS in which both cases displayed an oligoclonal TCR-beta gene rearrangement pattern with a few dominant T-cell populations. Both samples were from celiac disease patients, which mainly present a pronounced oligoclonal TCR-beta gene rearrangement pattern. Strikingly, the NGS data from the Oncomine™ TCR Beta-SR DNA Assay delivered highly comparable results when compared to the sequencing data of Ritter et al. 2018 despite having completely different primer sets and a different NGS platform. Conclusion Our comparison of the conventional multiplex PCR (BioMed-2) and TCR-beta NGS (Oncomine™ TCR Beta-SR DNA Assay) demonstrated a very high concordance (17/19 cases) of the molecular data. The two discordant cases can be explained by an over-interpretation of dominant species BioMed-2 Primerset B amplifications, which often show up in cases with low T-cell content or oligoclonal T-cell counts. These T-cell clonotypes are detectable by TCR-beta NGS only at a low percentage because of combination of all type of rearrangements in one assay. The robustness and reliability of NGS-based TCR-beta clonality testing was demonstrated by comparison of two completely different assays, leading to very similar results for all celiac disease patients. We are thus very confident that NGS-based clonality testing will be the "gold standard" of the future. Disclosures Looney: Thermo Fisher Scientific: Employment. Allen:Thermo Fisher Scientific: Employment.

Development of a multiplex real-time PCR to differentiate the four major Listeria monocytogenes serotypes in isolates from meat processing plants

Listeria monocytogenes is an important foodborne pathogen, causative agent of listeriosis. The epidemiology and persistence of this bacterium in meat processing plants may be related to its serotype, so it is of utmost importance to carry out a correct differentiation of L. monocytogenes serotypes. The objective of this study was to develop a unique quadruplex real-time quantitative PCR (qPCR) method able to differentiate the four most predominant and worrying L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) in isolates from meat processing plants and ready-to-eat (RTE) dry-cured meat products. The design of specific primers and probes was based on the lmo0737, lmo0308, ORFC (locus genomically equivalent to gltA-gltB) and ORF2110 genes. A qPCR based on a fragment of the 16S rRNA gene was used to ensure the amplification of Listeria spp. genomic DNA. The standard curves showed efficiency values ranging between 92.3% and 105.8% and, R2 values > 0.98. The specificity of the method was also confirmed by the comparison of the results with those obtained by a previously reported conventional multiplex PCR. In addition, none of the strains which were not ascribed to L. monocytogenes amplified any of the target genes related to the four major serotypes of this pathogenic species. The qPCR, therefore, provides a sensitive, specific and rapid tool for identifying the L. monocytogenes serotypes 1/2a, 1/2b, 1/2c and 4b. This method could be very useful for identifying sources of L. monocytogenes contamination in the meat industry or for epidemiological monitoring of persistent strains throughout the processing of RTE meat products.

Multicountry Distribution and Characterization of Extended-spectrum β-Lactamase-associated Gram-negative Bacteria From Bloodstream Infections in Sub-Saharan Africa.

BackgroundAntimicrobial resistance (AMR) is a major global health concern, yet, there are noticeable gaps in AMR surveillance data in regions such as sub-Saharan Africa. We aimed to measure the prevalence of extended-spectrum β-lactamase (ESBL) producing Gram-negative bacteria in bloodstream infections from 12 sentinel sites in sub-Saharan Africa.MethodsData were generated during the Typhoid Fever Surveillance in Africa Program (TSAP), in which standardized blood cultures were performed on febrile patients attending 12 health facilities in 9 sub-Saharan African countries between 2010 and 2014. Pathogenic bloodstream isolates were identified at the sites and then subsequently confirmed at a central reference laboratory. Antimicrobial susceptibility testing, detection of ESBL production, and conventional multiplex polymerase chain reaction (PCR) testing for genes encoding for β-lactamase were performed on all pathogens.ResultsFive hundred and five pathogenic Gram-negative bloodstream isolates were isolated during the study period and available for further characterization. This included 423 Enterobacteriaceae. Phenotypically, 61 (12.1%) isolates exhibited ESBL activity, and genotypically, 47 (9.3%) yielded a PCR amplicon for at least one of the screened ESBL genes. Among specific Gram-negative isolates, 40 (45.5%) of 88 Klebsiella spp., 7 (5.7%) of 122 Escherichia coli, 6 (16.2%) of 37 Acinetobacter spp., and 2 (1.3%) of 159 of nontyphoidal Salmonella (NTS) showed phenotypic ESBL activity.ConclusionsOur findings confirm the presence of ESBL production among pathogens causing bloodstream infections in sub-Saharan Africa. With few alternatives for managing ESBL-producing pathogens in the African setting, measures to control the development and proliferation of AMR organisms are urgently needed.

Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths.

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin–ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

Differentiation of Brucella abortus and B.melitensis biovars using PCR-RFLP and REP-PCR.

Brucellosis is one of the most common zoonotic diseases of animal and human beings. This study aimed to differentiate the Brucella spp. and determines the patterns of biovars by using repetitive element palindromic (REP)-PCR and PCR restriction fragment length polymorphism (RFLP) methods. A total of 100 blood specimens suspected of harbouring brucellosis were collected. Conventional culture methods and multiplex PCR were used for the detection of Brucella genus and species; and REP-PCR was used for Brucella spp. differentiation and polymorphisms sequence analysis. In addition, to identify the biovar patterns of REP-PCR, PCR-RFLP was used. Eighty-three samples were identified as harbouring Brucella spp. by the implementation of multiplex PCR, 72 of which were detected as Brucella melitensis and 11 as B. abortus. Also, through analysing the results of PCR-RFLP, it was found that of 72 B. melitensis samples, 69 were B. melitensis biovar 1 and three species were from other biovars. In addition, the obtained patterns for all of the B. abortus samples were from biovars 3, 5, 6 and 9. This study also optimized a test for the detection of Brucella biovar with the REP-PCR method such that Brucella spp. and biovars could be separated in the shortest possible time.

Multiplex PCR assay for identification and quantification of bovine and equine in minced meats using novel specific nuclear DNA sequences

The 2013 horsemeat scandal demonstrated the importance and significance of developing analytical methods to accurately identify and quantify bovine and equine components in meats. In this study, we successfully screened the bovine- and equine-specific nuclear DNA sequences for detection of bovine and equine fractions in minced meats, respectively. Primers and probes designed from bovine- and equine-specific nuclear DNA sequences were critically analysed against 53 species, and then their specificity was evaluated against 23 species through PCR. The conservation of primers and probes binding regions was tested in 44 different individuals of bovine or equine. The limit of detection (LOD) is 0.05 ng in both conventional and real-time PCR, and the limit of quantification (LOQ) is down to 5%. Real-time PCR confirmed that bovine- and equine-specific nuclear DNA sequences are present as a fixed copy number in the genome. A multiplex conventional PCR method was established to effectively discriminate bovine (223 bp), equine (197 bp) and reference (129 bp) in one reaction. A multiplex real-time PCR was developed and confirmed in five binary mixtures of cattle and horse DNA, exhibiting adequate parameters of trueness (R.E. ≤ 9.35%) and precision (R.S.D. ≤ 8.88%). Furthermore, we confirmed that our method held good potential in the detection of meat products in real life. The method and strategy developed in this study will play an important role in combating adulteration and maintaining a fair market.

The rapid detection and differentiation of Mycobacterium tuberculosis complex members from cattle and water buffaloes in the delta area of Egypt, using a combination of real-time and conventional PCR.

Mycobacterium tuberculosis complex (MTBC) has the potential to cause infections in animals and human beings. The combination of real-time PCR targeting atpE or lpqT and RD1, and conventional PCR targeting regions of difference (RD) was rigorously evaluated as a descriptive molecular epidemiology tool. A total of 2100 cattle and buffaloes from the Menoufia, Sharkia, Gharbia, Dakahlia, Elbuhaira, and Cairo Governorates were tested by single intradermal comparative cervical tuberculin test (SICCT). The frequency was 74/2100 (3.5%); thereafter, on post-mortem examination (PM), 49/74 (66.21%) showed visible lesions, while only 25/74 (33.78%) were non-visible with a significant difference of (p < .0001). Real-time PCR using atpE or lpqT and RD1 similarly detected the frequency of infection, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, which represented 73/74 (98.65%), 98.65, 100, 100, 90.91, and 98.81%, respectively. Multiplex conventional PCR targeting RD1, 4, 9, and 12 confirmed that 49/74 (66.21%) were M.bovis, while the simplex conventional PCR targeting RD4 and RD9 confirmed mycobacteria in 71/74 (95.94%) samples, which included 61/74 (82.4%) M.bovis and 2/74 (2.7%) M.tuberculosis. Additionally, 8/74 (10.8%) exhibited mixed patterns of M.bovis and M.tuberculosis, and 3/74 (4.05%) were negative. There was a significant difference between the results of simplex and multiplex conventional PCR (p < .0001). Moreover, simplex conventional PCR targeting RD4 and RD9 proved higher sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, which were 95.95, 100, 100, 76.92, and 96.43%, respectively, when compared with the values of multiplex conventional PCR targeting RD1,4,9, and 12 which were 66.22, 100, 100, 28.57, and 70.24%, respectively. The repeatability results of real-time PCR using atpE or lpqT and RD1, and simplex conventional PCR targeting RD4 and RD9 were acceptable. In conclusion, a combination of real-time PCR using atpE or lpqT and RD1 as the first step with simplex conventional PCR targeting RD4 and RD9 as the second step was reliable as a diagnostic tool.

Molecular monitoring of the therapeutic effect of Albendazole on Ascaris lumbricoides and Ancylostoma duodenale infected children using conventional multiplex PCR

ABSTRACT Background: Globally, many individuals are infected with soil-transmitted helminthes (STHS)with majority occurring in tropical and subtropical regions of the world. Albendazole (ADZ) is a broad-spectrum anthelmintic used efficiently for STHs control. Upgrading the assessment of ADZ efficacy is important to confirm that drug resistance has not emerged. Objective: To assess the therapeutic efficiency of ADZ in both A.duodenale infections by conventional multiplex PCR. Subjects and Methods: Stool specimens from 191 boys and 123 girls, aged from 2to3 years were screened by saline wet mount and iodine stained smears and cellophane thick smears (Keto-Katz technique). All positive cases were given a single oral dose of 400 mg ADZ than 2 weeks later new stool samples were collected and subjected to multiplex PCR. Before medication, anemia assessment (figure prick capillary blood sample) was performed. Results: Prevalence of infection with A.lumbricoides and A.duodenale was 20.7% and 5.4%, respectively. Parasitic infections were more prevalent in age group 7-13 years (59%) than in 2-6 years (41%) respectively. Boys were slightly more affected than girls (52.6% versus 47.4%). Prevalence of parasitic infections among cases living in rural areas proved to higher than those living in urban areas (65.4% versus 34.6%). Examination for associated anemia showed that 35.9% of infected cases were anemic (Hb level < 11.5 mg/dl) versus 9.7% in non-infected cases. Diarrhea and colic were more prevalent in infected cases (39.7% and 62.8%, respectively) than in those that were non-infected (5.5% and 8.9%, respectively). The therapeutic efficacy of ADZ resulted in a cure rate of 93.8% and 88.2% for A.lumbricoides and A.duodenal infections, respectively; with an egg reduction rate (ERR)of 96.1% and 91.2%, respectively. The multiplex PCR sensitivity was 100% for detection of both worms; the specificity was 96.8% and 93.8%, respectively. With diagnostic accuracy of 96.9% and 94.1%, respectively. Conclusion: Multiplex PCR was useful for measurement of the therapeutic efficacy of ADZas an anthelminthic drug.

Real-time PCR followed by high-resolution melting analysis - a new robust approach to evaluate SCCmec typing of methicillin-resistant Staphylococcus aureus.

We designed a novel approach based on real-time PCR followed by high-resolution melting (HRM) analysis to determine the Staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureusstrains, which we compared against the results of conventional multiplex PCR SCCmec typing. Multiplex PCR (for ccr and mec gene complexes) was carried out as conventional method. The HRM analysis was then designed using standard strains of each SCCmec type. The M-PCR results included types III (33.33%), IV (43.33%) and V (23.33%). HRM analysis was able to distinguish all five types, which were used to set up the protocol with a sensitivity and specificity of 100% compared with the conventional method. This novel method for SCCmec typing has high specificity and sensitivity and can be conducted in a shorter period of time at lower costs.

GENETIC SURVEY OF INVASIVE S. PNEUMONIAE SEROTYPES IN BULGARIA FOR A 5-YEAR PERIOD

Streptococcus pneumoniae colonises the mucosal lining of the upper respiratory tract and is an important cause of invasive infections affecting young children, adults over 65 years of age, the immunocompromised and individuals with chronic diseases. Recent studies have shown variations in virulence based on the high rate of pneumococcal recombination. PCR-based molecular methods are highly sensitive, specific and are becoming the preferred tool for quick and accurate diagnosis of bacterial meningitis which is required to be defined within 2-3 hours.&#x0D; During the 5-year survey period (2013-2017), 202 materials received as cerebrospinal fluid samples and pneumococcal strains isolated from patients diagnosed with meningitis, were examined by Real-time PCR in the reference laboratory at NCIPD. Serotyping of S. pneumoniae-positive materials was performed with conventional multiplex PCR and Real-time PCR with primers for 41serotypes/serogroups.&#x0D; There is a high incidence of S. pneumoniae serotypes not covered by the pneumococcal conjugate vaccine (PCV10) currently used in Bulgaria. It was found that all cases of meningitis caused by S. pneumoniae vaccine serotypes occurred in patients that were not vaccinated.

2301. Streptococcus pneumoniae Serotyping: Assessing the Performance of a PCR- and Sequencing-Based Testing Algorithm

Background Streptococcus pneumoniae is a bacterium that causes significant morbidity and mortality worldwide. Its capsular polysaccharides have been used successfully as vaccine antigens, and to characterize S. pneumoniae into 92 different serotypes. Phenotypic (Quellung reaction) or genotopic (PCR or sequencing) methods can be used for serotype assignment, but the performance may vary between methods. This study compared the performance of the Quellung reaction, to an algorithm using PCR- and sequence-based serotyping technologies for vaccine-preventable or closely related serotypes.MethodsA panel of geographically diverse isolates of S. pneumoniae spanning 92 different serotypes was provided by various references laboratories worldwide. Each isolate was subjected to conventional multiplex PCR methods, using previously established methods. Sanger sequencing was performed using genetic signatures defined in the PneumoCaT database. When discrepant, Quellung reaction were repeated, and next-generation sequencing and comparative genomics was used to evaluate the sequence composition of the cps loci.ResultsAs expected, PCR was unable to assign serotype in some cases, and some serotype results were insufficiently discriminatory. Following sequencing, 86.3% (404/468) of isolates were concordant with the Quellung serotyping. Discrepant analyses are underway.ConclusionAn algorithm based on PCR and sequencing, or next-generation sequencing alone, shows much promise for serotyping of S. pneumoniae. However, discrepant results were noted, suggesting either our current understanding of genetic signatures conferring serotype-specificity might not be complete, or the Quellung reaction results were incorrect. Accurate methods for serotyping are essential to monitor the impact of pneumococcal vaccines, and understand the epidemiology of S. pneumoniae diseases.Disclosures S. A. McNeil, GSK: Grant Investigator, Research grant. Pfizer: Grant Investigator, Research grant. Merck: Collaborator and Consultant, Contract clinical trials and Speaker honorarium. Novartis: Collaborator, Contract clinical trials. Sanofi Pasteur: Collaborator, Contract clinical trials.

Occurrence and antimicrobial susceptibility of Campylobacter spp. on fresh and refrigerated chicken meat products in Central Italy.

This study investigated the presence and the level of Campylobacter spp. contamination in 41 thigh samples (with skin) and 37 skinless breast samples collected at the end of slaughter (T1) and after 10 day period at refrigeration temperature (4°C) (T2), corresponding to their commercial shelf life. The isolates were phenotypically classified as Campylobacter spp. and successively identified by conventional multiplex PCR. The antimicrobial susceptibility of the isolates from fresh thigh and breast samples was also determined via the microdilution method (MIC) in Eucamp microtitre plates with known scalar concentrations of: gentamicin (GEN), streptomycin (ST), ciprofloxacin (CIP), tetracycline (TET), erythromycin (ERY), and nalidixic acid (NA). A greater percentage of positivity for Campylobacter spp. (P<0.001) was observed in thighs and C.jejuni appeared to be the most common species identified at this level (P<0.001) followed from its association with C.coli. There was a global reduction of Campylobacter spp. in both thigh and breast samples at T2 (P<0.001) showing that the refrigeration was able to reduce Campylobacter count. The prevalence of resistance to CIP, TET, NA, and ERY was evidenced for C.jejuni and C. coli. The co (TET-NA, CIP-NA) and multiple resistant (CIP-TET-NA, CIP-TET- NA-ERY) isolates came from the thigh products. It should be highlighted the presence of Campylobacter spp. isolates resistant to ST occurred in breast samples, responsible for the ST-CIP co-resistance and ST-CIP-TE multi-resistance profiles, higher in breast than in thigh products (P>0.001). The presence of Campylobacter isolates resistant to ST can be further investigated since it is used for therapeutic treatment of several bacterial diseases in humans.

Dominance of International High-Risk Clones in Carbapenemase-Producing Pseudomonas aeruginosa: Multicentric Molecular Epidemiology Report from India

Background:Pseudomonas aeruginosa is one of the most common opportunistic pathogens that cause severe infections in humans. The burden of carbapenem resistance is particularly high and is on the rise. Very little information is available on the molecular mechanisms and its clonal types of carbapenem-resistant P. aeruginosa seen in Indian hospitals. This study was undertaken to monitor the β-lactamase profile and to investigate the genetic relatedness of the carbapenemase-producing (CP) P. aeruginosa collected across different hospitals from India. Materials and Methods: A total of 507 non-duplicate, carbapenem-resistant P. aeruginosa isolated from various clinical specimens collected during 2014–2017 across seven Indian hospitals were included. Conventional multiplex polymerase chain reaction for the genes encoding beta-lactamases such as extended-spectrum beta-lactamase (ESBL) and carbapenemase were screened. A subset of isolates (n = 133) of CP P. aeruginosa were genotyped by multilocus sequence typing (MLST) scheme. Results: Of the total 507 isolates, 15%, 40% and 20% were positive for genes encoding ESBLs, carbapenemases and ESBLs + carbapenemases, respectively, whilst 25% were negative for the β-lactamases screened. Amongst the ESBL genes, blaVEB is the most predominant, followed by blaPER and blaTEM, whilst blaVIM and blaNDM were the most predominant carbapenemases seen. However, regional differences were noted in the β-lactamases profile across the study sites. Genotyping by MLST revealed 54 different sequence types (STs). The most common are ST357, ST235, ST233 and ST244. Six clonal complexes were found (CC357, CC235, CC244, CC1047, CC664 and CC308). About 24% of total STs are of novel types and these were found to emerge from the high-risk clones. Conclusion: This is the first large study from India to report the baseline data on the molecular resistance mechanisms and its association with genetic relatedness of CP P. aeruginosa circulating in Indian hospitals. blaVIM- and blaNDM-producing P. aeruginosa is the most prevalent carbapenemase seen in India. Majority of the isolates belongs to the high-risk international clones ST235, ST357 and ST664 which is a concern.

Simultaneous detection of Theileria annulata and Theileria orientalis infections using recombinase polymerase amplification

Theileriosis is a disease of domesticated animals in tropical and subtropical countries and causes significant reductions in livestock productivity. The arid region of Punjab in Pakistan is notorious for the presence of the vector tick (Acari: Ixodidae) and tick-borne diseases, such as theileriosis and babesiosis. The distribution of Theileria annulata and T. orientalis in the Chakwal district of Punjab was determined by developing a multiplex recombinase polymerase amplification (RPA) assay as a scientific basis for formulating control strategies for bovine theileriosis. Specificity was evaluated using DNA from related piroplasm species, while analytical sensitivity was calculated using a long fragment of the enolase gene. A total of 188 blood samples were collected on FTA cards (Whatman®) from tick-infested asymptomatic breeds of cattle (Bos indicus, Bos taurus, and Bos indicus × Bos taurus) in the study district. Finally, infections with of T. annulata and T. orientalis were detected using the multiplex RPA and compared with the conventional multiplex polymerase chain reaction (PCR). The multiplex RPA specifically amplified 282-bp and 229-bp fragments of the enolase gene from T. annulata and T. orientalis and had no cross-reaction with other piroplasm species. It was determined that 45 (23.9%) and 5 (2.6%) out of 188 blood samples were positive for T. annulata and T. orientalis, respectively, when examined using RPA. Multiplex PCR detection indicated that 32 (17.0%) and 3 (1.6%) blood samples were positive for T. annulata and T. orientalis, respectively. In the present study, a specific RPA method was developed for simultaneous differentiation and detection of T. annulata and T. orientalis infections and used for the first time for the detection of the two bovine Theileria infections.

Elimination of experimentally induced bovine intramammary infection assessed by multiplex real-time PCR and bacterial culture

Diagnosis of bovine intramammary infection (IMI) has traditionally been based on bacterial culture, but currently IMI can also be detected with DNA based methods, such as multiplex real-time PCR. The aim of this study was to describe the elimination of bacteria in experimentally induced IMI on the quarter level, using conventional bacterial culture (BC) and multiplex real-time PCR. Two coagulase-negative staphylococcal species, Staphylococcus epidermidis and Staphylococcus simulans, were experimentally inoculated into 14 healthy quarters of 8 dairy cows during 4 consecutive study periods. Intramammary infections were followed with 20 milk samplings per each quarter. Milk somatic cell count was monitored to evaluate the inflammation process in the quarters. Four quarters cured spontaneously during the study period according to the culture. The PCR detected staphylococcal DNA from these quarters for several days after they were defined as cured in BC. Agreement between BC and PCR results varied from substantial to almost perfect agreement for the first 36 h postchallenge, decreasing to moderate levels toward the end of the sampling period. Based on this study, we recommend collecting possible follow-up samples to assess the bacteriological cure from IMI not until 2 to 3 wk after the onset of mastitis or after the quarter milk somatic cell count has normalized when PCR is used.

Development of Simple Multiplex Real-Time PCR Assays for Some Probiotics Detection

The main objective of this study to investigate the development of simple multiplex real time for some probioticsdetection. The probiotics defined as Live microorganisms when administered in adequate amounts confer a health benefiton the host. Most commonly used probiotics are Lactic acid bacteria (LAB) and bifido bacteria. There are other examplesof species used as probiotics (certain yeasts and bacilli). Probiotic supplements are popular now a day. From the beginningof 2000, research on them has increased remarkably. The beneficial effects of probiotics are now day’s widely studied intreatment of many prevailing diseases. In this study, we present the development of molecular methods. Conventionalmultiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers.

Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions

A novel genomic and plasmid target-based PCR platform was developed for the detection of Salmonella serovars Heidelberg, Dublin, Hadar, Kentucky, and Enteritidis. Unique genome loci were obtained through extensive genome mining of protein databases and comparative genomic analysis of these serovars. Assays targeting Salmonella serovars Hadar, Heidelberg, Kentucky, and Dublin had 100% specificity and sensitivity, whereas those for Salmonella Enteritidis had 97% specificity and 88% sensitivity. The limits of detection for Salmonella serovars Heidelberg, Kentucky, Hadar, Enteritidis, and Dublin were 12, 9, 40, 13, and 5,280 CFU, respectively. A sensitivity assay was also performed by using milk artificially inoculated with pooled Salmonella serovars, yielding a detection limit of 1 to10 CFU/25 mL of milk samples after enrichment. The minimum DNA detected using the multiplexed TaqMan assay was 75.8 fg (1.53 × 101 genomic equivalents [GE]) for Salmonella Heidelberg, 140.8 fg (2.8 × 101 GE) for Salmonella Enteritidis, and 3.48 pg (6.96 × 102 GE) for Salmonella Dublin. PCR efficiencies were 89.8% for Salmonella Heidelberg, 94.5% for Salmonella Enteritidis, and 75.5% for Salmonella Dublin. Four types of 30 pasteurized milk samples were tested negative by culture techniques and with a genus-specific Salmonella invA gene PCR assay. Among 30 chicken samples similarly tested, 12 (40%) were positive by both culture and the invA PCR. Testing of these 12 samples with the serovar-specific PCR assay detected single and mixed contamination with Salmonella Kentucky, Salmonella Enteritidis, and Salmonella Heidelberg. Five unique primers were designed and tested by multiplex conventional PCR in conjunction with the use of the multiplex TaqMan assay with three of the primers. The diagnostic assays developed in this study could be used as tools for routine detection of these five Salmonella serovars and for epidemiological investigations of foodborne disease outbreaks.