Molecular genotyping methods and computerized analysis for the study of Salmonella enterica.

Phenotypic And genotypic characteristics of Salmonella enterica serotype Typhi have been determined for 29 isolates, from Baghdad in 2007. Conventional typing methods were performed by biochemical tests, and antimicrobial susceptibility test. Molecular typing performed by analysis plasmid DNA beside using the Random Amplified Polymorphic DNA (RAPD-PCR). For the latter, two universal primers that have selected for the high discriminatory power were used for RAPD analysis. All isolates were belong one biotype according to the differention by their ability to decarboxylat lysine, 29(100%) were lysine (+). All the isolates were susceptible to the Antibiotics used. However, all the strains free of plasmids. RAPD was capable of grouping the strains in 6 genotypic patterns using primer 784, in 4 genotypic patterns using primer 787. Conventional phenotypic typing methods, as well as the DNA plasmid analysis, presented non significant discriminatory power; however, RAPD-PCR analysis showed discriminatory power, reproducibility, easy interpretation and can be considered as a promising alternative typing method for S. Typhi.

The world has experienced an increased incidence and transboundary spread of emerging infectious diseases over the last four decades. We divided emerging infectious diseases into four categories, with subcategories in categories 1 and 4. The categorization was based on the nature and characteristics of pathogens or infectious agents causing the emerging infections, which are directly related to the mechanisms and patterns of infectious disease emergence. The factors or combinations of factors contributing to the emergence of these pathogens vary within each category. We also classified public health laboratories into three types based on function, namely, research, reference and analytical diagnostic laboratories, with the last category being subclassified into primary (community-based) public health and clinical (medical) analytical diagnostic laboratories. The frontline/leading and/or supportive roles to be adopted by each type of public health laboratory for optimal performance to establish the correct etiological agents causing the diseases or outbreaks vary with respect to each category of emerging infectious diseases. We emphasize the need, especially for an outbreak investigation, to establish a harmonized and coordinated national public health laboratory system that integrates different categories of public health laboratories within a country and that is closely linked to the national public health delivery system and regional and international high-end laboratories.

The recently observed increase in invasive Streptococcus pyogenes infections causes concern in Europe. However, conventional molecular typing methods lack discriminatory power to aid investigations of outbreaks caused by S. pyogenes. Therefore, there is an urgent need for high-resolution molecular typing methods to assess genetic relatedness between S. pyogenes isolates. In the current study, we aimed to develop a novel high-resolution core-genome multilocus sequence typing (cgMLST) scheme for S. pyogenes and compared its discriminatory power to conventional molecular typing methods. The cgMLST scheme was designed with the commercial Ridom SeqSphere+ software package. To define a cluster threshold, the scheme was evaluated using publicly available data from nine defined S. pyogenes outbreaks in the United Kingdom. The cgMLST scheme was then applied to 23 isolates from a suspected S. pyogenes outbreak and 117 S. pyogenes surveillance isolates both from the Netherlands. MLST and emm-typing results were used for comparison to cgMLST results. The allelic differences between isolates from defined outbreaks ranged between 6 and 31 for isolates with the same emm-type, resulting in a proposed cluster threshold of <5 allelic differences out of 1,095 target loci. Seven out of twenty-three (30%) isolates from the suspected outbreak had an allelic difference of <2, thereby identifying a potential cluster that could not be linked to other isolates. The proposed cgMLST scheme shows a higher discriminatory ability when compared to conventional typing methods. The rapid and simple analysis workflow allows for extended detection of clusters of potential outbreak isolates and surveillance and may facilitate the sharing of sequencing results between (inter)national laboratories.

Public health laboratories (PHLs) provide essential services in the diagnosis and surveillance of diseases of public health concern, such as tuberculosis. Maintaining access to high-quality laboratory testing is critical to continued disease detection and decline of tuberculosis cases in the United States. We investigated the practical experience of sharing tuberculosis testing services between PHLs through the Shared Services Project. The Shared Services Project was a 9-month-long project funded through the Association of Public Health Laboratories and the Centers for Disease Control and Prevention during 2012-2013 as a one-time funding opportunity to consortiums of PHLs that proposed collaborative approaches to sharing tuberculosis laboratory services. Submitting PHLs maintained testing while simultaneously sending specimens to reference laboratories to compare turnaround times. During the 9-month project period, 107 Mycobacterium tuberculosis complex submissions for growth-based drug susceptibility testing and molecular detection of drug resistance testing occurred among the 3 consortiums. The median transit time for all submissions was 1.0 day. Overall, median drug susceptibility testing turnaround time (date of receipt in submitting laboratory to result) for parallel testing performed in house by submitting laboratories was 31.0 days; it was 43.0 days for reference laboratories. The median turnaround time for molecular detection of drug resistance results was 1.0 day (mean = 2.8; range, 0-14) from specimen receipt at the reference laboratories. The shared services model holds promise for specialized tuberculosis testing. Sharing of services requires a balance among quality, timeliness, efficiency, communication, and fiscal costs.

Although the disease has been eliminated in Turkey malaria continues to be a threat due to increase in the number of people coming from or going to countries where the disease is endemic. In this study, we aimed to evaluate blood smears sent to the National Malaria Reference Laboratory within the malaria surveillance system. From March 2016 to July 2018 a retrospective study was conducted to compare the results of Malaria Reference Laboratory and Public Health Laboratories. A total of 16.827 blood stains were sent to our laboratory for approval. In Public Health Laboratories, 315 (1.88%) of the smears were positive, 16.510 (98.12%) were negative, and in the National Malaria Reference Laboratory 252 (1.50%) were positive, 16.466 were negative. In the Public Health Laboratories, one of the two samples considered to be malaria suspected was positive in the National Malaria Reference Laboratory and one was negative. In Public Health Laboratories 35.88% of smears were P. falciparum, 27.30% were Plasmodium spp., 20.96% were P. vivax, 14.92% were mixed infection, 0.63% were P. malariae, 0.31% were P. ovale, and in the Reference Laboratory 49.60% were Plasmodium spp., 29.37% were P. falciparum, 16.27% were P. vivax, 4.36% were mixed infection, 0.40% were P. malariae. In order to malaria surveillance system to be maintained in a healthy manner, preparation, staining, coding, packaging, transportation of blood slides is very important. Also if necessary, continuing training of laboratory staff working in malaria diagnosis is crucial.

Salmonella Enteritidis (SE) is a genetically homogenous serovar, which makes optimal subtype discrimination crucial for epidemiological research. This study describes the development and evaluation of an optimized multiple-locus variable number tandem-repeat assay (MLVA) for characterization of SE. The typeability and discriminatory power of this MLVA was determined on a selected collection of 60 SE isolates and compared with pulsed-field gel electrophoresis (PFGE) using restriction enzymes XbaI, NotI, or SfiI. In addition, the estimated Wallace coefficient (W) was calculated to assess the congruence of the typing methods. Selection of epidemiologically unrelated isolates and more related isolates (originating from layer farms) was also based on the given phage type (PT). When targeting six loci, MLVA generated 16 profiles, while PFGE produced 10, 9, and 16 pulsotypes using XbaI, NotI, and SfiI, respectively, for the entire strain collection. For the epidemiologically unrelated isolates, MLVA had the highest discriminatory power and showed good discrimination between isolates from different layer farms and among isolates from the same layer farm. MLVA performed together with PT showed higher discriminatory power compared to PFGE using one restriction enzyme together with PT. Results showed that combining PT with the optimized MLVA presented here provides a rapid typing tool with good discriminatory power for characterizing SE isolates of various origins and isolates originating from the same layer farm.

The rapid advancement of genome technologies holds great promise for improving the quality and speed of clinical and public health laboratory investigations and for decreasing their cost. The latest generation of genome DNA sequencers can provide highly detailed and robust information on disease-causing microbes, and in the near future these technologies will be suitable for routine use in national, regional, and global public health laboratories. With additional improvements in instrumentation, these next- or third-generation sequencers are likely to replace conventional culture-based and molecular typing methods to provide point-of-care clinical diagnosis and other essential information for quicker and better treatment of patients. Provided there is free-sharing of information by all clinical and public health laboratories, these genomic tools could spawn a global system of linked databases of pathogen genomes that would ensure more efficient detection, prevention, and control of endemic, emerging, and other infectious disease outbreaks worldwide.

Phenotypic and molecular typing of Vibrio cholerae O1 and O139 isolates from India

Genotypic characterisation by PFGE of Salmonella enterica serotype Enteritidis phage types 1, 4, 6, and 8 isolated from animal and human sources in three European countries

When considering methods of detecting Cryptosporidium in patient samples, clinical and public health laboratories have historically relied primarily on microscopy. However, microscopy is time intensive and requires trained personnel to accurately identify pathogens that are present. Even with skilled analysts, the parasitemia level has the potential to fall below the level of detection. In addition, public health laboratories do not always receive specimens in fixatives that are compatible with the desired microscopic method. Antigen-based and molecular methods have proven to be effective at identifying Cryptosporidium at low levels and require less training and hands-on time. Here, we have developed and validated a real-time polymerase chain reaction (RT-PCR) laboratory-developed test (LDT) that identifies Cryptosporidium hominis and Cryptosporidium parvum, and also includes detection at the genus level to identify additional species that occasionally cause disease in humans. Results of the molecular test were compared with those obtained from modified acid-fast microscopy, immunofluorescent microscopy, an antigen-based detection rapid test, and a commercial gastrointestinal panel (GI panel). Of 40 positive samples, microscopy and antigen-based methods were able to detect Cryptosporidium in only 20 and 21 samples, respectively. The GI panel detected 33 of the 40 positive samples, even though not all specimens were received in the recommended preservative. The LDT detected Cryptosporidium in all 40 positive samples. When comparing each method for the detection of Cryptosporidium, our results indicate the LDT is an accurate, reliable, and cost-effective method for a clinical public health reference laboratory.

Received signal strength (RSS) based location fingerprinting is a powerful wireless positioning technique. It estimates the target location by consulting a preliminary database and searching for the best matched RSS fingerprints. The construction and maintenance of a sufficient fingerprint database could be laborious and problematic. This paper proposes a new approach that utilizes the Kriging spatial interpolation algorithm to build complete fingerprint databases from sparsely collected measurements. The interpolation performance is analyzed over various extents of sparsity and number of measurements. The constructed fingerprint databases are utilized to locate a static target and the localization performances are analyzed. It is shown that the Kriging algorithm can be used to build RSS fingerprint databases of good accuracy based on sparsely collected measurements.

SUMMARYThe hygiene and marketing of fresh cream in England and Wales was investigated by a working party of the Public Health Laboratory Service (PHLS) between 1 October 1968 and 31 July 1969. Thirty-one cream-producing dairies were visited and observations made in the light of the Code of Practice published by the Milk and Milk Products Technical Advisory Committee of the Ministry of Agriculture, Fisheries and Food, and the Scottish Home and Health Department. Suggestions are made in this report to strengthen the code.A total of 5184 samples of fresh cream comprising 4385 heat-treated, 282 clotted and 517 untreated were examined. Details of production, age of the cream, distance and mode of distribution along the retail chain were available for most samples, and this history was, as far as possible, related to the laboratory findings. In the laboratory the samples were examined by the methylene blue test, colony count, conform andEscherichia coliI test and for pathogens.Staphylococcus aureuswas grown from 59 out of 3417 samples of cream; 54 of these were from untreated cream. Phage typing indicated that a proportion of these strains were of animal, presumably bovine, origin. Other human pathogens isolated included one each ofSalmonella typhimurium, Brucella abortus, E. colitype O126 andClostridium welchii.It appeared that heat-treated cream was much better, bacteriologically, than untreated; and that large dairies, in general, had better premises, more hygienic methods of preparation and the advantages of mechanical filling and capping of cartons; thus they offered the consumer a better product, bacteriologically, than some of the smaller dairies.A statistical analysis of the results of the tests used showed the methylene blue test, in spite of some anomalies, to be of more use as a screening test than any of the others. As this test is cheap and easy to carry out, the working party thought that it should be the test of choice. In view of the known anomalies, however, the working party recommends that the test should remain a screening or advisory test and have no penal function. Results should be reported as follows: cream not decolorizing the dye in 4 hr. at 37° C. (± 0·5°) after overnight incubation at 20° C. (± 0·5°) would be accepted as satisfactory; between ½ and 4 hr. at 37° C. (± 0·5°) as fairly satisfactory; but if the dye were decolorized at the end of the overnight incubation the sample would be provisionally classified as ‘unsatisfactory; requires further investigation’. A repeat sample would then be examined. If samples were repeatedly unsatisfactory, consultations would be arranged between the local authority, the laboratory and the dairy, with perhaps inspection of the dairy premises and retail storage conditions and further, more searching tests on samples made in an effort to eradicate production faults.

A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control.

Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of > or =97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of > or =99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

Background:Laboratory testing practices are an important part of sexually transmitted disease (STD) diagnosis and control. The goal of this article is to describe the volume and types of STD tests performed in public health laboratories (PHLs) in the United States in 2016.Methods:A total of 105 state and local PHLs who were members of the Association of Public Health Laboratories were invited to participate in a survey between May and August 2017. This web-based survey included questions on types of tests offered for different types of STDs, the number of tests offered in 2016, and number of samples that tested positive for each STD.Results:Eighty-one (77.1%) of 105 member PHLs responded. Overall in 2016, 2,242,728 Chlamydia trachomatis tests, 2,298,596 Neisseria gonorrhoeae (GC) tests, 1,235,037 Treponema pallidum (syphilis) tests, 177,571 Trichomonas vaginalis tests, 37,101 herpes simplex virus tests, and 8707 human papillomavirus tests were performed in 2016 in surveyed laboratories. Sixty-seven (82.7%) of PHLs offered C. trachomatis and GC nucleic acid amplification testing. Ninety percent of laboratories performed syphilis testing, 42% performed T. vaginalis testing, and 28.4% performed herpes simplex virus type–specific nucleic acid amplification testing. Few laboratories tested for human papillomavirus.Conclusions:This survey collected important information on PHL STD laboratory testing practices. Capacity for important reference laboratory testing, such as GC culture and antimicrobial susceptibility, are needed to support STD programs. Public health laboratories play a key role in STD screening, surveillance, and prevention by offering test methods that are not available at commercial laboratories. Funding constraints affect the resources available for STD screening and surveillance, and thus, it is important to continue to monitor testing practices and the capacity of PHLs to test for STDs.