Combining Multiple Assays Improves Detection and Serotyping of Foot-and-Mouth Disease Virus. A Practical Example with Field Samples from East Africa.

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012-2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVß6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%.

Utility of a multiplex real-time polymerase chain reaction for combined detection and serotyping of dengue virus in paediatric patients hospitalised with severe dengue: A report from Chennai.

Objective: Molecular detection and serotyping are rapid, sensitive and accurate techniques for early diagnosis of paediatric dengue. The present study evaluates multiplex real-time polymerase chain reaction (PCR) for diagnosis of dengue virus in children hospitalised with severe dengue (SD) and attempts to establish an association of clinical severity with specific serotypes. Methods: Four hundred and eighty-five samples were received from hospitalised paediatric patients with suspected dengue from March 2019 to February 2020. Multiplex real time PCR was employed for diagnosis. An in-house real-time PCR that combined diagnosis and serotyping was established. Non-structural protein 1 (NS1) assay and real-time PCR were assessed for their accuracy in diagnosing severe paediatric dengue. Results: Three hundred and twenty-five (67%) patients were positive for dengue RNA by real-time PCR. All four serotypes were identified throughout the year; dengue serotype 2 (DEN-2) was predominant (61%) followed by DEN-3, 20%. Compared to the commonly used NS1 testing, multiplex real-time PCR showed greater sensitivity in diagnosing SD. Conclusions: Compared to NS1, multiplex real-time PCR is a rapid and accurate diagnostic test for children hospitalised with SD. DEN-2 was the predominant serotype in severe cases. Continued surveillance of serotypes should be carried out year-round in endemic areas.

Highly Sensitive and Specific Detection and Serotyping of Five Prevalent Salmonella Serovars by Multiple Cross-Displacement Amplification.

Salmonella is a common cause of foodborne disease worldwide, including Australia. More than 85% of outbreaks of human salmonellosis in Australia were caused by five Salmonella serovars. Rapid, accurate, and sensitive identification of Salmonella serovars is vital for diagnosis and public health surveillance. Recently, an isothermal amplification technique, termed multiple cross-displacement amplification (MCDA), has been employed to detect Salmonella at the species level. Herein, seven MCDA assays were developed and evaluated for rapid detection and differentiation of the five most common Salmonella serovars in Australia: Typhimurium, Enteritidis, Virchow, Saintpaul, and Infantis. MCDA primer sets were designed by targeting seven serovar/lineage-specific gene markers identified through genomic comparisons. The sensitivity and specificity of the seven MCDA assays were evaluated using 79 target strains and 32 nontarget strains. The assays were all highly sensitive and specific to target serovars, with the sensitivity ranging from 92.9% to 100% and the specificity from 93.3% to 100%. The limit of detection of the seven MCDA assays was 50 fg per reaction (10 copies) from pure DNA, and positive results were detected in as little as 8 minutes. These seven MCDA assays offer a rapid, accurate, and sensitive serotyping method. With further validation in clinically relevant conditions, these assays could be used for culture-independent serotyping of common Salmonella serovars directly from clinical samples.

Comparison of six commercial systems for the detection of non-O157 STEC in meat and vegetables.

Shiga toxin-producing Escherichia coli (STEC) are important pathogens transmitted by food that may cause severe illness in human beings. Thus, systems for STEC detection in food should have increasingly higher sensitivity and specificity. Here we compared six commercial systems for non-O157 STEC detection in meat and vegetables and determined their sensitivity, specificity and repeatability. A total of 46 samples (meat n = 23; chard n = 23) were experimentally contaminated with strains O26:H11, O45:H-, O103:H2, O111:NM, O121:H19 and O145:NM isolated in Argentina. Strain detection was confirmed by isolation according to ISO 13136:2012. Detection of the stx and eae genes in meat samples was highly satisfactory with all commercial kits, but only five had 100% sensitivity and specificity in chard. Of four kits evaluated for serogroup detection, three had 100% sensitivity and specificity, and one had 93.7% sensitivity and 100% specificity. All kits were adequate to analyze meat but not vegetable samples, and were not therefore validated for the latter matrix. The challenge for microbiology laboratories is to identify the advantages and disadvantages of the available kits for STEC detection in food based on a clear knowledge of the particular needs of each laboratory.

Identification and capsular serotype sequetyping of Streptococcus pneumoniae strains.

PURPOSE: Correct serotype identification of Streptococcus pneumoniae (pneumococcus) is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. Furthermore, correct identification and differentiation of the pathogenic S. pneumoniae from closely related commensal species of the mitis group of the genus Streptococcus are essential for correct serotype identification. METHODOLOGY: A new protocol for determining the existing 98 serotypes of pneumococcus was developed, applying two PCR amplifications and amplicon sequencing, using newly designed internal primers. The new protocol was validated using S. pneumoniae genome sequences, reference strains with confirmed serotypes and clinical isolates, and comparing the results with those from the traditional Quellung reaction or antiserum panel gel precipitation, in addition to real-time PCR analysis. The taxonomic identifications of 422 publicly available (GenBank) genome sequences of S. pneumoniae, Streptococcus pseudopneumoniae and Streptococcus mitis were assessed by whole-genome sequence average nucleotide identity based on blast (ANIb) analysis. RESULTS: The proposed sequetyping protocol generates a 1017 bp whole cpsB region sequence, increasing resolution for serotype identification in pneumococcus isolates. The identifications of all GenBank genome sequences of S. pneumoniae were confirmed, whereas most of the S. pseudopneumoniae and almost all of the S. mitis genome sequences did not fulfil the ANIb thresholds for species-level identification. The housekeeping biomarker gene, groEL, correctly identified S. pneumoniae but often misclassified S. pseudopneumoniae and S. mitis as S. pneumoniae. CONCLUSIONS: These studies affirm the importance of applying reliable identification protocols for S. pneumoniae before serotyping; our protocols provide reliable diagnostic tools, as well as an improved workflow, for serotype identification of pneumococcus and differentiation of serogroup 6 types.

Accuracy of High-Throughput Nanofluidic PCR-Based Pneumococcal Serotyping and Quantification Assays Using Sputum Samples for Diagnosing Vaccine Serotype Pneumococcal Pneumonia: Analyses by Composite Diagnostic Standards and Bayesian Latent Class Models.

The lack of reliable diagnostic tests for detecting vaccine serotype pneumococcal pneumonia (VTPP) remains a challenging issue in pneumococcal vaccine studies. This study assessed the performances of high-throughput nanofluidic PCR-based pneumococcal serotyping and quantification assay methods using sputum samples (the nanofluidic sputum quantitative PCR [Sp-qPCR] assay) to diagnose 13-valent pneumococcal conjugate VTPP compared with the performance of the serotype-specific urinary antigen detection (UAD) assay using urine samples. Adult pneumonia patients from Japan were enrolled in this study between September 2012 and August 2014. Sputum samples were subjected to the nanofluidic Sp-qPCR assay, quantitatively cultured, and serotyped by the Quellung reaction (SpQt). Urine samples were tested by the UAD method. The diagnostic performances of these tests were assessed using composite reference standards and Bayesian latent class models (BLCMs). Among 244 total patients, 27 (11.1%) tested positive with the UAD assay, while 16 (6.6%) and 34 (13.9%) tested positive with the SpQt and nanofluidic Sp-qPCR assays, respectively, with a cutoff value of ≥104 DNA copies/ml, which showed the maximum value of the Youden index. Using BLCMs, the estimated prevalence for VTPP was 12.9%, and the nanofluidic Sp-qPCR assay demonstrated the best performance (sensitivity, 90.2%; specificity, 96.9%), followed by UAD (sensitivity, 75.6%; specificity, 97.9%) and SpQt (sensitivity, 45.8%; specificity, 99.5%). However, when a higher cutoff value of ≥107 DNA copies/ml was applied, the performance of UAD became comparable to that of Sp-qPCR. The vaccine serotype-specific pneumococcal DNA load in sputum among UAD-positive patients was 3 logs higher than that among UAD-negative patients (P = 0.036). The nanofluidic Sp-qPCR assay may be accurate and useful for detecting VTPP among adults.

Development and characterization of a synthetic DNA, NUversa, to be used as a standard in quantitative polymerase chain reactions for molecular pneumococcal serotyping.

Identification of Streptococcus pneumoniae and its more than 90 serotypes is routinely conducted by culture and Quellung reactions. Quantitative polymerase chain reactions (qPCRs) have been developed for molecular detection, including a pan-pneumococcus lytA assay, and assays targeting 79 serotypes. Reactions require genomic DNA from every target to prepare standards, which can be time consuming. In this study, we have developed a synthetic DNA molecule as a surrogate for genomic DNA and present new single-plex qPCR reactions to increase molecular detection to 94 pneumococcal serotypes. Specificity of these new reactions was confirmed with a limit of detection between 2 and 20 genome equivalents/reaction. A synthetic DNA (NUversa, ∼8.2 kb) was then engineered to contain all available qPCR targets for serotyping and lytA. NUversa was cloned into pUC57-Amp-modified to generate pNUversa (∼10.2 kb). Standards prepared from pNUversa and NUversa were compared against standards made out of genomic DNA. Linearity [NUversa (R2 > 0.982); pNUversa (R2 > 0.991)] and efficiency of qPCR reactions were similar to those utilizing chromosomal DNA (R2 > 0.981). Quantification with plasmid pNUversa was affected, however, whereas quantification with synthetic NUversa was comparable to that of genomic DNA. Therefore, NUversa may be utilized as DNA standard in single-plex assays of the currently known 94 pneumococcal serotypes.

Development of a Sensitive and Specific Polyclonal Antibody for Serological Detection of Clavibacter michiganensis subsp. sepedonicus.

The quarantine bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) causes bacterial ring rot (BRR) in potato but is difficult to detect, hampering the diagnosis of this disease. ELISA immunoassays have not been widely used to detect Cms because commercially available anti-Cms antibodies detect mainly EPS-producing bacteria and can fail to detect strains that do not produce EPS. In the current study, we developed a new type of polyclonal antibody that specifically detects Clavibacter michiganensis subsp. sepedonicus bacteria irrespective of their EPS level. We first found that the presence of bacterial EPS precluded quantitative measurement of bacteria by currently available immunoenzymatic methods, but that washing Cms cells with acidic and basic buffers to remove EPS before analysis successfully standardized ELISA results. We used a mix of three strains of Cms with diverse EPS levels to generate antigen for production of antibodies recognizing Cms cells with and without an EPS layer (IgG-EPS and IgG-N-EPS, respectively). The resulting IgG-N-EPS recognized almost all Cms strains tested in this work regardless of their mucoidal level. The availability of this new antibody renders immunological diagnostics of Cms more sensitive and reliable, as our newly developed antibodies can be used in many type of immunoassays. This work represents an important step forward in efforts to diagnose and prevent the spread of BRR, and the methods and solutions developed in this work are covered by six Polish, one European and one US patents.

Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa.

Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce CT values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region.

Mass Spectrometry-Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping.

BACKGROUND: Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS: On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS: Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 µg or 1.465 × 10(7) cells) level and close correlation (r(2) > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS: MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.

Cambios en la epidemiología de las gastroenteritis causadas por Salmonella durante 2005-2014 en Salamanca, España / Changes in the epidemiology of gastroenteritis caused by Salmonella during 2005-2014 in Salamanca, Spain

Introducción. En España no abundan estudios poblacionales actualizados sobre salmonelosis, a pesar de ser una de las etiologías de gastroenteritis agudas (GEAs) bacterianas más habituales en el mundo. El objetivo fue conocer los rasgos epidemiológicos más relevantes de las GEAs producidas por Salmonella spp. entre 2005-2014 en Salamanca (España). Métodos. Estudio descriptivo transversal realizado a partir del archivo informático del Servicio de Microbiología del Complejo Asistencial Universitario de Salamanca. El cultivo, aislamiento, identificación y serotipificación se realizaron según la metodología habitual. Resultados. Salmonella se aisló en 1.477 pacientes, representando el 47,7% del total de coprocultivos positivos y el 53,3% de todos los ingresos por GEA bacteriana. La prevalencia media fue de 42,1 casos/100.000 habitantes y año. La media de edad fue de 23 ± 28 años y la mediana 7 años. El 40,2% de todos los aislamientos se produjo en menores de 5 años, con una prevalencia media de 45,1 casos/10.000 habitantes y año. Globalmente, el serotipo aislado con más frecuencia fue S. Typhimurium con un 57%, seguido por S. Enteritidis con un 35,8%. Conclusiones. La prevalencia de Salmonella disminuyó a lo largo del tiempo. El grupo entre 0-4 años presentó la tasa más alta durante todo el periodo. Sin embargo, produjo el mayor porcentaje de hospitalizaciones por GEA bacteriana. El serotipo S. Typhimurium ha reemplazado en los últimos años al serotipo S. Enteritidis y predomina en pacientes de menor edad. Se aprecia una infranotificación de los casos de salmonelosis producidos en Salamanca a pesar de ser obligatoria su declaración desde 2007 (AU)

Background. In Spain there are not many updated population studies about salmonellosis, despite being one of the most common etiologies of acute gastroenteritis (AGEs) caused by bacteria in the world. The aim of the study was to know the most relevant epidemiological features of AGEs produced by Salmonella spp. between 2005 and 2014 in Salamanca (Spain). Methods. Descriptive cross-sectional study carried out through review of the clinical microbiologic records at Complejo Asistencial Universitario de Salamanca. Culture, isolation, identification and serotyping were performed according to standard methodology. Results. Salmonella was isolated in 1,477 patients, representing 47.7% of all positive stool cultures and 53.3% of all income bacterial AGE. The average prevalence was 42.1 cases/100,000 people per year. The mean age was 23 ± 28 years and the median 7 years. 40.2% of all isolates occurred in children under 5 years, with an average prevalence of 45.1 cases/ 10,000 people per year. Overall, the most frequently isolated serotype was S. Typhimurium with 57%, followed by S. Enteritidis with 35.8%. Conclusions. The prevalence of Salmonella decreased over time. The group aged 0-4 years had the highest rate throughout the period. However, Salmonella produced the highest percentage of hospitalizations for bacterial AGE. In recent years, S. Typhimurium serotype has replaced S. Enteritidis serotype and predominates in younger patients. It is observed under-reporting of cases of salmonellosis produced in Salamanca despite being mandatory notification of these since 2007 (AU)

Is the microagglutination test (MAT) good for predicting the infecting serogroup for leptospirosis in Brazil?

Leptospirosis is a zoonotic infection caused by pathogenic members of the genus Leptospira spp. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the disease. The aim of this study was to evaluate the ability of serology by the microscopic agglutination test (MAT) to predict the serogroups compared with results of identification of leptospires in São Paulo, Brazil. MAT correctly assigned the serogroup of the infecting isolate in 49/52 cases (94.23%). The serogroup Icterohaemorrhagiae was the predominant serogroup (88.46%). This study showed the usefulness of the MAT to correctly identify the infecting serogroup with a good overall agreement between the serologically-identified infecting serogroup and by identification of the isolate and can be used in epidemiological surveys in São Paulo. However, it should be complemented by the identification of Leptospira isolates.

Results of Salmonella enterica subsp. enterica serotype identification by Salmonella Check&Trace microarray in international External Quality Assurance Systems.

INTRODUCTION: Traditionally Salmonella enterica subsp. enterica serotypes are identified by slide agglutination with specific antisera for somatic, flagellar and sometimes capsular antigens. An alternative way is genoserotyping using for example a microarray, eg. commercially available test Check&Trace Salmonella. The goal of this study was to evaluate the Check&Trace Salmonella microarray for Salmonella enterica subsp. enterica serotype identification, using Salmonella strains provided by reference laboratories during External Quality Assurance Systems organized for national reference laboratories by ECDC and WHO GFN. MATERIAL AND METHODS: 80 Salmonella enterica subsp. enterica have been tested using Check & Trace Salmonella (Check-Points BC, Netherlands). Also classical slide agglutination was performed according to EN ISO 6579:2003/Al:2007 norm, used as reference method. RESULTS: In the group of 80 tested strains, 66% were identified correctly, 4% gave uncertain results and 29% showed "Salmonella, genovar" without a serotype, of which 69% were not included in the CTS list of serotypes. Finally one strain has been recognized incorrectly. DISCUSSION: Because of IVD certification lack, the CTS test could not be recommended to clinical laboratories. AOAC-RI and OIE certification for test cause, that CTS could be used in most food, environmental and veterinary laboratories with the condition, that all unrecognized strains should be sent to a reference laboratory, to type according to EN ISO 6579:2003/Al:2007 norm, by KWM serotyping or other equal alternative methods.

Molecular Serotyping and Pathogenic Potential of Listeria monocytogenes Isolated from Milk and Milk Products in Tamil Nadu, India.

Listeria monocytogenes, an important bacterial pathogen, is responsible for foodborne illnesses worldwide. Examination of food samples for the presence of L. monocytogenes and assessment of their pathogenicity is usually an effective strategy in the prevention of listeriosis. In the present study, we have tested 307 samples of milk and milk products from various places in Tamil Nadu, India for the presence of L. monocytogenes using ISO 11290 and U.S. Food and Drug Administration Bacteriological Analytical Manual methods. 16S rDNA sequencing and duplex polymerase chain reaction (PCR) analysis for prs and iap genes were used to identify L. monocytogenes at the species level. Fifteen of the 307 samples screen tested positive for L. monocytogenes. Molecular serotyping of the L. monocytogenes isolates by multiplex PCR revealed the predominance of the serogroups 1/2a and 4b. Fourteen of the 15 isolates contained all the virulence genes (inlA, inlB, hlyA, and plcA) screened for using multiplex PCR. Only one isolate of L. monocytogenes was negative for the plcA gene and in vitro phosphatidylinositol-phospholipase C activity. L. monocytogenes strains that belong to the serogroup 4b exhibited higher nematocidal activity against Caenorhabditis elegans than the serogroup 1/2a. Worms infected with L. monocytogenes were symptomatic with aberrant contraction of body muscles, loss of pharyngeal pumping, and decreased locomotion, which highlights the pathogenic potential of the L. monocytogenes isolates.

Identification of essential outstanding questions for an adequate European laboratory response to Ebolavirus Zaire West Africa 2014.

On August 8 2014, the World Health Organization (WHO) declared the outbreak of Ebola Virus Disease (EVD) evolving in West Africa since December 2013, a Public Health Emergency of International Concern (PHEIC). It is expected that the outbreak of Ebolavirus Disease (EVD) in West Africa will lead to increased testing of individuals in Europe for EVD. The severity of the situation in West Africa warranted a critical appraisal of the laboratory preparedness and response for EVD, with a focus on information needs for laboratories involved in diagnostics of rare viral diseases associated with the European Network for the Diagnostics of "Imported" Viral Diseases", ENIVD. Essential knowledge and knowledge gaps for an adequate laboratory response focusing on virus properties, infection kinetics, tests specifics and field performances were identified. An inventory of the laboratory capacity for EVD diagnostics among ENIVD laboratories was made.

Capsular Serotyping of Streptococcus pneumoniae by latex agglutination.

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the 'gold standard' Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. This method of production and quality control may also be suitable for other testing purposes.

[Determination of Salmonella serotypes by conventional and molecular methods]. / Salmonella Serotiplerinin Konvansiyonel ve Moleküler Yöntemler ile Belirlenmesi.

Determination of Salmonella enterica serotypes is crucial for epidemiological studies. Salmonella serotypes are defined on the basis of somatic (O) and flagellar (H) antigens, both of which are present in the cell wall of Salmonella. The aim of this study was to compare the results of molecular serotyping obtained by multiplex polymerase chain reaction (mPCR) with conventional serotyping results. Conventional serotyping has been performed in Ministry of Health Refik Saydam Hygiene Center as part of the National Laboratory of Enteric Pathogenes Surveillance Network (UEPLA). A total of 100 Salmonella strains, thay comprise 14 different serotypes by the reference laboratory have been investigated by using specific primers for Salmonella serogroups (A, B, C1, D and E) and Vi antigen gene clusters via mPCR method. Serotypes have been determined by applying four sequential mPCR targeting the fliC and fliB genes encoding the H1 antigens (H1: a, -b, -d, -g,m, -i, -r, -z10) and H2 antigen complexes (H2: 1,2, -1,5, -1,6, -1,7 and H: enx, enz15). The results of mPCR showed 100% consistency with the serogroups determined by the conventional method. Both sensitivity and specificity of mPCR according to each serogroups were found to be 100%. Results of serotyping that have been determined with the molecular antigenic formula showed accurate results for 2 (2%), probable results for 91 (91%) and incomplete formula for 7 (7%) isolates. Molecular serotyping results of the most common isolated Salmonella serotypes of which S.Enteritidis, S.Typhimurium and S.Paratyphi from clinical microbiology laboratories have been determined as probable results. Antigenic formula of these serotypes that detected using mPCR were considered to be consistent with the results of conventional serotyping when interpreted with epidemiologic data. The sensitivity of mPCR to identify S.Typhi which have been determined as accurate result with molecular serotyping was 100% for serogrouping and serotyping. Multiplex PCR is cheaper and faster for the serotyping of strains isolated in clinical laboratories, compared to the conventional methods. However since it is not possible to detect all serotypes by using molecular typing, this technique can not be currently considered as an alternative for conventional serotyping. Nevertheless molecular typing could be beneficial in providing the preliminary results earlier.

[Serologic study of experimental cholera polymer antigen diagnosticums].

AIM: Evaluation of quality indicators of constructed cholera antigen polymer diagnosticums by using a complex of specific anti-cholera sera. MATERIALS AND METHODS. Cell lysates of cholera vibrio strains Vibrio cholerae cholerae 1395, V. eltor Ogawa 2044, V. eltor Inaba 13020, V. cholerae O139 16064 were sensitins for experimental preparations. 3 sera from cholera patients, normal human sera, cholera O1 (Ogawa, Inaba) commercial horse, cholera O139 commercial rabbit and heterologic sera against shigella, salmonella, escherichia and yersinia as well as experimental cholera rabbit sera against O1 and O139 were used as control. RESULTS: The study established that diagnosticums based on V. cholerae cholerae 1395 and V. cholerae O139 16064 strain sensitins by quality indicators may be used in the future for construction of these diagnosticums. CONCLUSION: Antibody containing preparations--commercial horse O1 sera, rabbit experimental and commercial sera and MCA O139 demonstrating titers not lower than 1/5120-1/10240 may serve as a control of experimental diagnosticums in the absence of human sera from cholera patients.

Interlaboratory agreement of pulsed-field gel electrophoresis identification of Leptospira serovars.

Leptospirosis may be caused by > 250 Leptospira serovars. Serovar classification is a complex task that most laboratories cannot perform. We assessed the interlaboratory reproducibility of a pulsed-field gel electrophoresis (PFGE) identification technique developed by the Centers for Disease Control and Prevention (CDC). Blinded exchange of 93 Leptospiraceae strains occurred between San Antonio Military Medical Center (SAMMC) and the CDC. PFGE was performed and gel images were analyzed and compared with patterns present in each laboratory's database (CDC database: > 800 strain patterns; SAMMC database: > 300 strain patterns). Overall, 93.7% (74 of 79) of strains present in each receiving laboratory's database were correctly identified. Five isolates were misidentified, and two isolates did not match serovar PFGE patterns in the receiving laboratory's database. Patterns for these seven isolates were identical between laboratories; four serovars represented misidentified reference strains. The PFGE methodology studied showed excellent interlaboratory reproducibility, enabling standardization and data sharing between laboratories.

Multilaboratory validation of a luminex microbead-based suspension array for the identification of the 11 most clinically relevant Shiga toxin-producing Escherichia coli O serogroups.

Rapid and high-throughput identification and serotyping of Shiga toxin-producing Escherichia coli (STEC) O serogroups is important for detecting, investigating, and controlling STEC infection outbreaks and removing hazardous products from commerce. A Luminex microbead-based suspension array has been developed to identify the 11 most clinically relevant STEC serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. Here we present results of a blinded multilaboratory collaborative study involving 10 participants from nine laboratories using 55 unknown strains. From the total 495 analyses, two false-positive and three false-negative results were obtained, indicating the assay to be a rapid, high-throughput, and robust method for identifying clinically relevant STEC serogroups.