Rapid Molecular Method Research Articles

Multiplex PCR for simultaneous identification of the most common European Opisthorchiid and Heterophyid in fish or fish products

Among others, the families Opisthorchiidae and Heterophyidae includes several genera causing fish-borne zoonoses and distributed also in European Countries and that are included in the ParaFishControl (Advanced Tools and Research Strategies for Parasite Control in European farmed fish) H2020 EU project. Due to the small size of the metacercariae, the infective stage for human, these parasites cannot be detected visually in fish and monitoring requires expert application of time-consuming techniques. The aim of this was to develop a rapid and affordable molecular method based on multiplex PCR for simultaneous identification of metacercariae of the most common European Opisthorchiid and Heterophyid in fish or fish products.

Analysis on drug resistance-associated mutations of multi-drug resistant Mycobacterium tuberculosis based on whole-genome sequencing in China

Objective: To analyze the resistance mutational profiles of multi-drug resistant Mycobacterium tuberculosis in China and the correlation between major mutation types and genotypes based on the whole-genome sequencing data. Methods: Search and download of the genome-wide sequencing data of M. tuberculosis published in China by August 2019 on NCBI database were conducted. Mutation frequency of drug resistance-related gene loci based on whole-genome sequencing was used to predict the molecular susceptibility of strains, and the correlation between mutation types and genotypes was analyzed. Results: According to the results of molecular resistance and susceptibility profiles, 1 024 MDR strains were identified from 2 019 M. tuberculosis strains. The major mutation types of resistance-related genes to common drugs were katG S315T (73.2%, isoniazid), rpoB S450L (63.1%, rifampicin), rpsL K43R (70.0%, streptomycin), embB M306V (37.4%, ethambutol), pncA_promoter T (-11)C (7.9%, pyrazinamide), gyrA A90V (32.3%, fluoroquinolones), rrs A1401G (67.7%, second-line injection drugs), fabG1_promoter C (-15) T (87.0%, Ethionamide), folC I43T (30.4%, P-aminosalicylic acid). Among them, the frequencies of katG S315T, embB M306V, rpsL K43R, gyrA A90V in lineage 2 were significantly higher than those in lineage 4, and folC I43T was only found in lineage 2. The proportion of katG S315T was significantly higher in the ancient Beijing genotype compared to the modern genotype, in contrast, the proportion of rpsL K43R was significantly higher in modern Beijing genotype, the differences were significant (all P<0.05). Conclusions: The results showed the main mutation types of resistance-related genes of MDR strains to many commonly used anti-tuberculosis drugs in China based on whole-genome sequencing, providing a basis for the development of sensitive and specific rapid molecular detection methods. At the same time, it was also found that the major mutation types of MDR-related genes were related to the genotype of the strains.

Rickettsial Diseases: Not Uncommon Causes of Acute Febrile Illness in India.

Rickettsial diseases (RDs) are major under-diagnosed causes of arthropod borne acute febrile illness (AFI) presenting with a range of symptoms from mild self-limiting fever to fatal sepsis. The spotted fever group (SFG) and typhus group (TG) are major RDs, which are commonly caused by Rickettsia conorii and Rickettsia typhi, respectively. The limited availability and role of serological tests in the acute phase of illness warrants rapid reliable molecular methods for diagnosis and epidemiological studies. Two hundred patients with AFI in whom the routine fever diagnostics were negative, were enrolled over a period of two months (April 2019 to May 2019). DNA was extracted and in-house nested PCR using primers specific for both SPG and TG pathogens was used. The positive amplified products were sequenced for species identification and phylogenetic analysis was performed using MEGA 7.0.14 software (iGEM, Temple University, Philadelphia, PA 19122, USA). The demographic details of the RD cases were documented. The prevalence of RD among AFI cases was 7% (14/200); SFG and TG were identified as the cause in 4% and 3% of AFI cases, respectively. The median age of the RD cases was 22 years (range 2–65). The median duration of fever was 3 days (range 1–12). The RD cases presented with respiratory symptoms or signs (44.44%), jaundice (22.22%), abdominal pain (22.22%), diarrhea (22.22), vesicular rash (11.11%), vomiting (11.11%), loss of appetite (11.11%), headache (11.11%), leukocytosis (88.88% with mean count 22,750/mm3), and thrombocytopenia (33.33%). The cases were treated empirically with piperacillin-tazobactam (66.66%), clindamycin (44.44%), cefotaxime (33.33%), meropenem (33.33%), metronidazole (33.33%), doxycycline (22.22%), azithromycin (22.22%), ceftriaxone (11.11%), and amoxicillin-clavulanic acid (11.11%). The mortality among the RD cases was 11.11%. The present pilot study shows that RD is not an uncommon cause of AFI in north India. The febrile episodes are usually transient, not severe and associated with heterogenous clinical presentation without documented history of tick exposure in the hospitalized patients. The transient, non-severe, febrile illness could be due to transient rickettsemia resulting from empirical antimicrobial therapy as the rickettsial organisms are expected to be more susceptible to higher doses of β-lactam antibiotics. The study emphasizes the molecular method as a useful tool to identify rickettsial etiology in AFI.

Phenotypic and Genotypic Characterization of Streptomycin-Resistant Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates in Southern China.

Streptomycin (STR) is the first antibiotic used in the treatment of tuberculosis (TB) and the earliest antituberculosis drug with acquired resistance developed by Mycobacterium tuberculosis. The high prevalence of such resistance in many parts of the world limits its use for treating multidrug-resistant (MDR) TB. The aims of this study are to characterize of mutations in rpsL, rrs, and gidB genes in MDR M. tuberculosis isolates originating from southern China and to investigate possible relationship between mutations and strain genotypes for precise diagnosis and treatment. Sequences of rpsL, rrs, and gidB genes and the resistance profiles were analyzed for 218 MDR M. tuberculosis isolates. Our study showed that 68.35% of MDR M. tuberculosis isolates were resistant to STR and 89.91% of STR-resistant (STRR) isolates were Beijing lineage strains. Mutations were observed in STRR MDR M. tuberculosis isolates at the following rates: 72.48% in rpsL, 36.91% in rrs, and 15.44% in gidB. Compared with the phenotypic data, the combination of mutations in rpsL, rrs, and gidB has sensitivity and specificity of 96.64% and 100.00%, respectively. The most common mutations in STRR isolates were rpsL128,263 and rrs514,1401, of which rpsL128 showed association with Beijing lineage (p < 0.001). It is noteworthy that a1401g mutation was present in rrs, while MDR M. tuberculosis isolates were resistant to both STR and amikacin. Twenty two novel mutations were found in STRR isolates. These findings could be helpful to develop rapid molecular diagnostic methods and understand STR resistance in China for developing TB precision medicine and disturbance of drug-resistant TB transmission.

Mycobacterium tuberculosis kompleks ve Rifampisin Direncinin Saptanmasında Hızlı Moleküler Yöntemle Konvansiyonel Yöntemlerin Karşılaştırılması

Mycobacterium tuberculosis kompleks ve Rifampisin Direncinin Saptanmasında Hızlı Moleküler Yöntemle Konvansiyonel Yöntemlerin Karşılaştırılması

GeneXpert MTB-RIF assay as a rapid and effective molecular method for the diagnosis of tuberculosis in Erbil City

Background and objective: Tuberculosis is a major public health problem worldwide, especially in developing countries. The disease poses a massive threat to public health, causing increased morbidity and mortality. Therefore, early detection is essential to reduce the death rate and interrupt transmission. In this study, GeneXpert assay was used for the rapid identification of bacteria, and its sensitivity and specificity were compared with acid fast bacilli staining in both pulmonary and extrapulmonary clinical specimens. Methods: A total of 115 pulmonary and extrapulmonary samples were collected and diagnosed for tuberculosis detection. Acid fastbacilli, culture, and GeneXpert methods were used for the diagnosis of tuberculosis. Results: Out of 115 tuberculosis specimens, 70 were acid fast bacilli smear positive, while 97 samples were positive for Mycobacterium tuberculosis by GeneXpert assay. The results indicated a higher sensitivity of GeneXpert assay compared to the acid fastbacillistaining method. The sensitivity of GeneXpert was 91.6%, specificity 50%; positive and negative predictive values were 89.7% and 55.6%, respectively. No significant differences were detected between the Gene Xpert test results and the culture results (P = 0.815), and the total agreement between the tests was 84.3%. Conclusion: We concluded that the GeneXpert test results is sensitive as culture results for both pulmonary and extrapulmonary specimens. Although culture remains the gold standard for laboratory confirmation of tuberculosis disease, GeneXpert should become standard practice for patients suspected of tuberculosis, and all clinicians and public health tuberculosis programs should have access to molecular testing for tuberculosis to shorten the time of diagnosis.

Development and Validation of a Novel and Rapid Molecular Detection Method for High-Risk Human Papillomavirus in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

The most widely applied algorithm for human papillomavirus (HPV) detection in formalin-fixed, paraffin-embedded (FFPE) specimens of oropharyngeal head and neck squamous cell carcinoma (HNSCC) consists of p16INK4A immunostaining followed by PCR-based detection of high-risk HPV DNA on p16INK4A-immunopositive samples. However, in nonoropharyngeal HNSCC this algorithm fails, hampering correct interpretation of the prevalence and prognosis of HPV in these cases. In this study, we developed and validated a molecular HPV detection method for FFPE specimens of oropharyngeal and nonoropharyngeal HNSCC. Sectioning of FFPE blocks was circumvented by using punch biopsies from tumor-enriched regions of FFPE tissue blocks, and combined extraction was applied to obtain high-quality DNA and RNA from the punch biopsy. Next, PCR-based detection of HPV DNA was performed for 15 high-risk HPV types with subsequent detection of E6 mRNA for validation. The combined DNA/RNA FFPE test of tissue cores was assessed in well-characterized cohorts with known HPV status based on earlier work, that is, a cohort of oropharyngeal HNSCC (n = 80) and oral cavity HNSCC (n = 25), and reached an accuracy of 97% and 100%, respectively. In conclusion, our method is rapid, simple, and shows an excellent diagnostic performance for detection of HPV type 16. Ultimately, it can be applied for large cohort studies to determine the etiologic fraction and prognostic implication of HPV in nonoropharyngeal HNSCC.

New Species-Specific Primers for Molecular Diagnosis of Bactrocera minax and Bactrocera tsuneonis (Diptera: Tephritidae) in China Based on DNA Barcodes.

Tephritidae fruit flies (Diptera: Tephritidae) are regarded as important damage-causing species due to their ability to cause great economic losses in fruit and vegetable crops. Bactrocera minax and Bactrocera tsuneonis are two sibling species of the subgenus Tetradacus of Bactrocera that are distributed across a limited area of China, but have caused serious impacts. They share similar morphological characteristics. These characteristics can only be observed in the female adult individuals. The differences between them cannot be observed in preimaginal stages. Thus, it is difficult to distinguish them in preimaginal stages morphologically. In this study, we used molecular diagnostic methods based on cytochrome c oxidase subunit I and species-specific markers to identify these two species and improve upon the false-positive results of previous species-detection primers. DNA barcode sequences were obtained from 900 individuals of B. minax and 63 individuals of B. tsuneonis. Based on these 658 bp DNA barcode sequences of the cytochrome c oxidase subunit I gene, we successfully designed the species-specific primers for B. minax and B. tsuneonis. The size of the B. minax specific fragment was 422 bp and the size of the B. tsuneonis specific fragment was 456 bp. A series of PCR trials ensured the specificity of these two pairs of primers. Sensitivity assay results demonstrated that the detection limit for the DNA template concentration was 0.1~1 ng/μL for these two species. In this study, we established a more reliable, rapid, and low-cost molecular identification method for all life stages of B. minax and B. tsuneonis. Species-specific PCR can be applied in plant quarantine, monitoring and control of B. minax and B. tsuneonis.

Molecular and metabolic strategies for postharvest detection of heat-resistant fungus Neosartorya fischeri and its discrimination from Aspergillus fumigatus

Abstract Heat Resistant Fungi (HRF) and toxigenic fungi are considered as a serious problem both in the agricultural field and for human health, due to ascospores and mycotoxins production, which can contaminate fruit and, as a consequence, adversely affect the agricultural food chain. One strategy to identify these fungi is the use of modern molecular method, which include the analysis of DNA target regions for differentiation of the fungal species. However, previously developed methods included only the identification of pure strains but not the detection of Neosartorya fischeri in artificially contaminated food samples, such as fruit or juices. Therefore, the aim of presented study was to develop a detection method of Neosartorya fischeri in contaminated strawberry fruit and juice. The other strategy is the use of phenotypic assays to determine the metabolic profile of the fungi in order to facilitate a qualitative and quantitative detection of microorganisms based on specific substrates utilization and mycotoxins production. Accordingly, the study included an evaluation of the differences in phenotype profile between N. fischeri and the phylogenetically close fungus Aspergillus fumigatus, as a strategy of their differentiation and identification. PCR detection assay was developed that revealed the presence of N. fischeri DNA in all tested contaminated samples of fruit and juice. Therefore, it can be concluded that this rapid molecular method is an important tool for the evaluation of the postharvest quality of agricultural raw materials. Moreover, the results suggest that specific metabolic and mycotoxin patterns may be used as N. fischeri detection markers and strategy in discrimination of this fungus from A. fumigatus. The results indicated that N. fischeri and A. fumigatus had a different time period of carbon sources utilization, and particularly N. fischeri presented a more efficient carbon metabolism. Mycotoxins, verruculogen and fumitremorgin C, were detected after 4 days incubation of N. fischeri. Although metabolic assays are not such fast as molecular detection approach, they allow to deeper insight into the pathways activated by heat-resistant and toxigenic fungi. Therefore, both molecular and metabolic strategies of heat-resistant fungus detection and identification are complementary and can be used to measure postharvest quality of fruit and their products.

Evaluation of a multiplex gastrointestinal PCR panel for the aetiological diagnosis of infectious diarrhoea

Background: Infectious diarrhoea is a significant cause of morbidity worldwide. Culture and microscopy are time-consuming and have a low yield. New rapid molecular methods such as multiplex PCR, have been recently introduced for aetiological diagnosis. Aim of this study was to compare the diagnostic yield of the FilmArray gastrointestinal panel with that of standard culture for aetiological diagnosis of infectious diarrhoea.Methods: We performed a retrospective analysis of results of stool samples already processed as part of routine clinical care in the interval from March 2016 to March 2019.Results: One hundred and eighty-three stool samples from as many patients were both cultured and tested by FilmArray and the comparison of diagnostic accuracy between culture and FilmArray with respect to Campylobacter spp., Salmonella spp., Shigella spp., Yersinia enterocolitica and Shiga-like toxin producing E. coli O157 gave the following results: 100% (95% confidence interval (CI): 85–100%) sensitivity; 93.4% (95% CI: 87.9–96.6%) specificity; 74.3% (95% CI: 57.5–86.4%) positive predictive value; 100% (95% CI: 96.7–100%) negative predictive value; 2.9 (95% CI: 1.6–5.1) positive likelihood ratio; zero negative likelihood ratio. By means of FilmArray gastrointestinal (GI) panel, we could identify 34.5% more pathogens (p = .001). Bacteria were mostly detected in patients with 6 or more years of age (χ2=17.1; p = .009) during summer.Conclusions: FilmArray GI panel showed a very good diagnostic performance compared to culture for diagnosis of infectious diarrhoea and gave a more detailed picture of the spectrum of the pathogens involved.

Salmonella Serotyping; Comparison of the Traditional Method to a Microarray-Based Method and an in silico Platform Using Whole Genome Sequencing Data.

Salmonella is one of the most common causes of food-borne diseases worldwide. While Salmonella molecular subtyping by Whole Genome Sequencing (WGS) is increasingly used for outbreak and source tracking investigations, serotyping remains as a first-line characterization of Salmonella isolates. The traditional phenotypic method for serotyping is logistically challenging, as it requires the use of more than 150 specific antisera and well trained personnel to interpret the results. Consequently, it is not a routine method for the majority of laboratories. Several rapid molecular methods targeting O and H loci or surrogate genomic markers have been developed as alternative solutions. With the expansion of WGS, in silico Salmonella serotype prediction using WGS data is available. Here, we compared a microarray method using molecular markers, the Check and Trace Salmonella assay (CTS) and a WGS-based serotype prediction tool that targets molecular determinants of serotype (SeqSero) to the traditional phenotypic method using 100 strains representing 45 common and uncommon serotypes. Compared to the traditional method, the CTS assay correctly serotyped 97% of the strains, four strains gave a double serotype prediction. Among the inconclusive data, one strain was not predicted and two strains were incorrectly identified. SeqSero was evaluated with two versions (SeqSero 1 and the alpha test version of SeqSero 2). The correct antigenic formula was predicted by SeqSero 1 for 96 and 95% of strains using raw reads and assembly, respectively. However, 34 and 33% of these predictions included multiple serotypes by raw reads and assembly. With raw reads, one strain was not identified and three strains were discordant with phenotypic serotyping result. With assembly, three strains were not predicted and two strains were incorrectly predicted. While still under development, SeqSero 2 maintained the accuracy of antigenic formula prediction at 98% and reduced multiple serotype prediction rate to 13%. One strain had no prediction and one strain was incorrectly predicted. Our study indicates that the CTS assay is a good alternative for routine laboratories as it is an easy to use method with a short turn-around-time. SeqSero is a reliable replacement for phenotypic serotyping if WGS is routinely implemented.

A real-time PCR for rapid identification of Salmonella enterica Gaminara serovar

Salmonella is one of the leading causes of foodborne illnesses in the USA. When a Salmonella outbreak occurs, rapid identification of the causative serovar is important for tracing the source of contamination and for preventing the further spread of the illness. Each serovar is characterized by the presence of a group-specific somatic O-antigen(s) and an assortment of flagellar phase-1 and phase-2 antigens. As the traditional serotyping protocol is time consuming, labor intensive, and expensive, faster and less expensive molecular diagnostic methods are needed. This report outlines the development of a rapid multiplex real-time PCR procedure that facilitates the identification of Salmonella serogroup I and the serovars of the group. Using Salmonella Gaminara serovar (O16:d:1,7) as an example, first the gene(s) responsible for expression of the somatic O antigen, O16, and the nucleotide sequence of the variable-region of genes encoding the flagellar phase-1 (d) and phase-2 (1,7) antigens were identified. Then, a multiplex real-time PCR was designed that incorporated primers and probes specific for the three target genes and confirmed the specificity. The assay had 100% inclusivity for all three gene targets, detecting 2 genomic DNA copies of O16 and 1,7 gene targets and 10 copies of d gene target.Importance: Rapid molecular methods to identify Salmonella serovars should increase the precision of routine surveillance of clinically important serovars and promote public health.

EFFICACY OF GENE XPERT MTB/RIF ASSAY AND AFB CULTURE IN DIAGNOSIS OF MYCOBACTERIUM TB COMPLEX ON EBUS TBNA

SESSION TITLE: Tuesday Abstract Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM PURPOSE: Rationale – Tuberculosis and Sarcoidosis are two clinical entity that share same clinical, radiological and pathological features and differentiating between two might be an arduous task. As cultures for M. Tuberculosis in tissue specimens have prolonged turn-around times, rapid molecular methods such as MTB. Rif system has shown great promise in rapid diagnosis of tuberculosis through detection of DNA of M. Tuberculosis and concurrent identification of major mutation that confers rifampicin resistance. We aim to evaluate both efficacy and role of Xpert MTB / RIF Assay and Mycobacterium TB Culture for diagnosis of TB using EBUS TBNA samples in patients with intra-thoracic granulomatous lymphadenopathy. METHODS: This was a retrospective study of randomly selected 100 patients who underwent EBUS TBNA for intra-thoracic granulomatous lymphadenitis with Gene Xpert MTB / RIF Assay and diagnosed with either Tuberculosis or Sarcoidosis RESULTS: EBUS TBNA procedures were performed and a diagnosis of Sarcoidosis (n 17) or Tuberculosis (n 83) was made in 100 patients. Histological results with grades I to II were seen in 74 patients and grade III in 26 patients respectively. Necrotising granulomas were found in 26 / 83 (31.32%) while Non-necrotising granulomas were found in 57 / 83 (68.67%) patients diagnosed with Tuberculosis. Mycobacterium TB Culture was performed in 72 patients diagnosed with Tuberculosis and was positive in 11 patients – 09 / 26 Necrotising Granuloma vs 02 / 57 Non-necrotising Granuloma (34.61% vs 3.5%, P = 0.010). Gene Xpert MTB / RIF Assay were performed in 20 / 83 patients diagnosed with Tuberculosis and was positive in 4 patients – 01 / 07 Necrotising Granuloma vs 03 / 13 Non-necrotising Granuloma (14.30% vs 23.10%, P = 0.547). The Diagnostic Accuracy of Xpert MTB / RIF Assay in diagnosis of Tuberculosis was 70% (95% CI 45.72% to 88.10%), Sensitivity 40%, Specificity 100%, Disease Prevalence 50%, PPV 100%, NPV 62.50%. Of 2 patients finally diagnosed with Sarcoidosis also showed a positive result of Xpert MTB / RIF Assay which possibly suggests casual link between TB and Sarcoidosis CONCLUSIONS: Gene Xpert MTB / RIF Assay can be useful in patients with suspected Pulmonary TB either AFB smear negative or positive due to its rapid and simultaneous detection of Rifampicin resistance and especially beneficial in patients with MDR TB and HIV associated TB. Xpert MTB / RIF Assay has good sensitivity and PPV in context of culture positive intra-thoracic TB lymphadenopathy and can provide immediate diagnosis of likely drug resistance specially in highly endemic area like India CLINICAL IMPLICATIONS: Positive Xpert MTB / RIF Assay with very low MTB detection but culture negative results need to be read cautiously and should be well correlated with clinical and treatment history of patient since Gene Xpert Assay amplifies any DNA, of live or dead bacilli and required to avoid false positive result DISCLOSURES: No relevant relationships by UMANG SHAH, source=Web Response

Quantitative real-time PCR assay for the rapid identification of the intrinsically multidrug-resistant bacterial pathogen Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is emerging as an important cause of disease in nosocomial and community-acquired settings, including bloodstream, wound and catheter-associated infections. Cystic fibrosis (CF) airways also provide optimal growth conditions for various opportunistic pathogens with high antibiotic tolerance, including S. maltophilia . Currently, there is no rapid, cost-effective and accurate molecular method for detecting this potentially life-threatening pathogen, particularly in polymicrobial specimens, suggesting that its true prevalence is underestimated. Here, we used large-scale comparative genomics to identify a specific genetic target for S. maltophilia , with subsequent development and validation of a real-time PCR assay for its detection. Analysis of 167 Stenotrophomonas spp. genomes identified a conserved 4 kb region in S. maltophilia , which was targeted for Black Hole Quencher assay design. Our assay yielded the positive detection of 89 of 89 (100%) clinical S. maltophilia strains, and no amplification of 23 non- S. maltophilia clinical isolates. S. maltophilia was detected in 10 of 16 CF sputa, demonstrating the assay's utility for direct detection in respiratory specimens. The assay demonstrated good sensitivity, with limits of detection and quantitation on pure culture of ~10 and ~100 genome equivalents, respectively. Our assay provides a highly specific, sensitive and cost-effective method for the accurate identification of S. maltophilia , and will improve the diagnosis and treatment of this under-recognized pathogen by enabling its accurate and rapid detection from polymicrobial clinical and environmental samples.

Rapid Molecular Detection and Transmission of Bacterial Leaf Streak Pathogen, Xanthomonas Oryzae Pv. Oryzicola, in Rice Seeds

Xanthomonas oryzae pv. oryzicola (Xoc) is a seed-borne bacterial pathogen of rice. In this study, a rapid molecular detection method in seeds was developed to prevent unwanted movement of infected materials and greater yield loss. Crude DNA from ground seed extract supernatant was precipitated in ethanol and centrifuged. DNA pellets of homogenous subsamples were pooled and werepurified using SDS or a DNA extraction kit. Three pairs of primers previously designed on Xoc isolate BLS-256 genome sequence robustly amplify Xoc-specific DNA sequences. Primer T40 was found to efficiently detect Xoc but only in single primer reactions. While primers 3864 and 3866 can amplify Xoc DNA separately, however, their multiplex PCR reactions were unsuccessful, probably due to substrate competition in the reaction mixture. Optimized PCR mixture and conditions were able to detect as low as 10 cfu/mL Xoc cells and the direct extraction method and conditions were able to detect as low as 104 cfu/mL inoculated cells using primer T40. Lastly, Xoc transmission through seeds was observed only in plant samples which were heavily infected. Recovery from infection, at around 25-50% was also observed in some cultivars which also manifested good grain filling at 56 days after transplanting.

A Fully Integrated Real-Time Detection, Diagnosis, and Control of Community Diarrheal Disease Clusters and Outbreaks (the INTEGRATE Project): Protocol for an Enhanced Surveillance System.

BackgroundDiarrheal disease, which affects 1 in 4 people in the United Kingdom annually, is the most common cause of outbreaks in community and health care settings. Traditional surveillance methods tend to detect point-source outbreaks of diarrhea and vomiting; they are less effective at identifying low-level and intermittent food supply contamination. Furthermore, it can take up to 9 weeks for infections to be confirmed, reducing slow-burn outbreak recognition, potentially impacting hundreds or thousands of people over wide geographical areas. There is a need to address fundamental problems in traditional diarrheal disease surveillance because of underreporting and subsequent unconfirmed infection by patients and general practitioners (GPs); varying submission practices and selective testing of samples in laboratories; limitations in traditional microbiological diagnostics, meaning that the timeliness of sample testing and etiology of most cases remains unknown; and poorly integrated human and animal surveillance systems, meaning that identification of zoonoses is delayed or missed.ObjectiveThis study aims to detect anomalous patterns in the incidence of gastrointestinal disease in the (human) community; to target sampling; to test traditional diagnostic methods against rapid, modern, and sensitive molecular and genomic microbiology methods that identify and characterize responsible pathogens rapidly and more completely; and to determine the cost-effectiveness of rapid, modern, sensitive molecular and genomic microbiology methods.MethodsSyndromic surveillance will be used to aid identification of anomalous patterns in microbiological events based on temporal associations, demographic similarities among patients and animals, and changes in trends in acute gastroenteritis cases using a point process statistical model. Stool samples will be obtained from patients’ consulting GPs, to improve the timeliness of cluster detection and characterize the pathogens responsible, allowing health protection professionals to investigate and control outbreaks quickly, limiting their size and impact. The cost-effectiveness of the proposed system will be examined using formal cost-utility analysis to inform decisions on national implementation.ResultsThe project commenced on April 1, 2013. Favorable approval was obtained from the Research Ethics Committee on June 15, 2015, and the first patient was recruited on October 13, 2015, with 1407 patients recruited and samples processed using traditional laboratory techniques as of March 2017.ConclusionsThe overall aim of this study is to create a new One Health paradigm for detecting and investigating diarrhea and vomiting in the community in near-real time, shifting from passive human surveillance and management of laboratory-confirmed infection toward an integrated, interdisciplinary enhanced surveillance system including management of people with symptoms.International Registered Report Identifier (IRRID)DERR1-10.2196/13941

Recreational water monitoring: Nanofluidic qRT‐PCR chip for assessing beach water safety

AbstractImproved recreational water monitoring using rapid molecular genetic methods would decrease both public health risks and unnecessary beach closures. Hence, we developed a novel nanofluidic quantitative real‐time PCR (qPCR) in the OpenArray platform to (a) detect and quantify fecal indicator bacteria (FIBs; N = 2), (b) identify contaminant sources (microbial source tracking (MST); N = 7), and (c) detect human virulent pathogens (virulence gene markers; N = 15). Our water monitoring OpenArray plate reliably detects as few as two template copies/hole (OpenArray® well), with some marker sensitivities as low as single‐copy detection. The OpenArray plate showed high target sequence specificity and returned expected patterns of contaminants for fecal and sewage samples. We found Canada geese and seagulls were the leading causes of contamination at beaches that tested positive for coliforms. When we incorporated robotic DNA extraction, we were able to process samples from water to FIBs, MST, and waterborne pathogen detection and quantification within four hours. Our monitoring TaqMan qPCR assays in the OpenArray platform is uniquely valuable for regulatory agencies charged with beach water safety as well as for researchers interested in human health implications of aquatic microbial community structure.

Amplified fragment length polymorphism and whole genome sequencing: a comparison of methods in the investigation of a nosocomial outbreak with vancomycin resistant enterococci

BackgroundRecognition of nosocomial outbreaks with antimicrobial resistant (AMR) pathogens and appropriate infection prevention measures are essential to limit the consequences of AMR pathogens to patients in hospitals. Because unrelated, but genetically similar AMR pathogens may circulate simultaneously, rapid high-resolution molecular typing methods are needed for outbreak management. We compared amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) during a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VRE) that spanned 5 months.MethodsHierarchical clustering of AFLP profiles was performed using unweighted pair-grouping and similarity coefficients were calculated with Pearson correlation. For WGS-analysis, core single nucleotide polymorphisms (SNPs) were used to calculate the pairwise distance between isolates, construct a maximum likelihood phylogeny and establish a cut-off for relatedness of epidemiologically linked VRE isolates. SNP-variations in the vanB gene cluster were compared to increase the comparative resolution. Technical replicates of 2 isolates were sequenced to determine the number of core-SNPs derived from random sequencing errors.ResultsOf the 721 patients screened for VRE carriage, AFLP assigned isolates of 22 patients to the outbreak cluster. According to WGS, all 22 isolates belonged to ST117 but only 21 grouped in a tight phylogenetic cluster and carried vanB resistance gene clusters. Sequencing of technical replicates showed that 4–5 core-SNPs were derived by random sequencing errors. The cut-off for relatedness of epidemiologically linked VRE isolates was established at ≤7 core-SNPs. The discrepant isolate was separated from the index isolate by 61 core-SNPs and the vanB gene cluster was absent. In AFLP analysis this discrepant isolate was indistinguishable from the other outbreak isolates, forming a cluster with 92% similarity (cut-off for identical isolates ≥90%). The inclusion of the discrepant isolate in the outbreak resulted in the screening of 250 patients and quarantining of an entire ward.ConclusionAFLP was a rapid and affordable screening tool for characterising hospital VRE outbreaks. For in-depth understanding of the outbreak WGS was needed. Compared to AFLP, WGS provided higher resolution typing of VRE isolates with implications for outbreak management.

Liquefaction of pineapple peel: Pretreatment and process optimization

This study explored the optimization of pretreatment of pineapple peel for biogas generation. Pretreatments were carried out sulfuric acid and alkaline hydrogen peroxide prior to anaerobic digestion while the response surface methodology (RSM) was used for optimization of the pretreatment procedures. The physical, chemical, proximate and structural compositions of the peels were determined prior to and at the end of the pretreatment procedures. The dynamics of microorganisms in the reactors were also evaluated by rapid molecular methods while the Fourier Transform Infra-red (FTIR) spectroscopy was employed in the identification of the chemical changes as a result of pretreatments. The use of H2O2 pretreatment caused enormous lignin solubilization in the pineapple peel. In comparison, biogas production was 67% more in the alkaline pretreated pineapple peel than the biomass treated with acid and also 51% over the untreated samples. The total biogas volume produced from the acidic pretreated, alkaline pretreated, not sifted untreated and sifted untreated samples are 194.2 ± 3.0, 587.5 ± 5.2, 287.8 ± 2.1 and 245.4 ± 3.1 respectively. Thus, the alkaline pretreated experiment used lower retention time to achieve maximum gas production in this study. The use of alkaline H2O2 on lignocelluloses has remained unpopular prior to this study. However, its usage in this study yielded better result than all the conventional treatments in terms of lignin solubilization and improvement in methane yield. Economically, the use of H2O2 for pretreatment is adjudged feasible because the 1504 kWh t−1 TS thermal energy gain obtained from the biogas produced by the alkaline treated peel exceeded the 921 kWh t−1 TS used in the pretreatment. This gives a net thermal energy of 583 kWh t−1 TS. Whereas, the investment into acidic pretreatment of pineapple peel may not be economically justified because the total thermal energy gain of −200 kWh t−1 TS was far lower than the 1236 kWh t−1 TS thermal energy that was consumed during the pretreatment giving a net thermal energy of −1436 kWh t−1 TS. Therefore, the use of mild alkaline pretreatment is advocated in biogas generation from pineapple peel and also for biofertilizer production mostly in localities of mass production.

Use of nanotechnology for infectious disease diagnostics: application in drug resistant tuberculosis

BackgroundThe increased transmission of multidrug-resistant (MDR) tuberculosis (TB) poses a challenge to tuberculosis prevention and control in Sri Lanka. Isoniazid (INH) is a key element of the first line anti tuberculosis treatment regimen. Resistance to INH may lead to development of MDR TB. Therefore, early detection of INH resistance is important to curb spread of resistance. Due to the limited availability of rapid molecular methods for detection of drug resistance in Sri Lanka, this study was aimed at developing a simple and rapid gold nanoparticle (AuNP) based lateral flow strip for the simultaneous detection of the most common INH resistance mutation (katG S315 T, 78.6%) and Mycobacterium tuberculosis (MTb).MethodsLateral flow strip was designed on an inert plastic backing layer containing a sample pad, nitrocellulose membrane and an absorption pad. Biotin labeled 4 capture probes which separately conjugated with streptavidin were immobilized on the nitrocellulose. The test sample was prepared by multiplex PCR using primers to amplify codon 315 region of the katG gene and MTb specific IS6110 region. The two detection probes complementary to the 5′ end of each amplified fragment was conjugated with gold nanoparticles (20 nm) and coupled with the above amplified PCR products were applied on the sample pad. The hybridization of the amplified target regions to the respective capture probes takes place when the sample moves towards the absorption pad. Positive hybridization is indicated by red colour lines.ResultsThe three immobilized capture probes on the strip (for the detection of TB, katG wild type and mutation) were 100 and 96.6% specific and 100 and 92.1% sensitive respectively.ConclusionThe AuNP based lateral flow assay was capable of differentiating the specific mutation and the wild type along with MTb identification within 3 h.