Cost of whole genome sequencing for non-typhoidal Salmonella enterica.

BACKGROUND: While whole genome sequencing (WGS) may be more expensive than traditional testing and polymerase chain reaction (PCR), simple cost comparisons ignore the potential for WGS to reduce the societal costs of non-typhoidal Salmonella enterica through public health action to prevent illness. METHODS: We determined how many cases the use of WGS data would need to prevent to be cost-equal to serotyping and MLVA, or culture independent testing based on PCR in Australia. We then examined the costs and cost-savings of current typing methods compared with WGS in outbreak scenarios. RESULTS: A median of 275 (90% CrI -55-775) or 1.9% (90% CrI -0.4%-5.4%) of notified serotyped Salmonella cases would need to be prevented for WGS to be cost-equal to current typing methods and 1,550 (90% CrI 820-2,725) or 9.6% of all notified Salmonella cases would need to be prevented to be cost-equal to PCR. WGS is likely to result in cost savings in prolonged outbreaks, where data can support earlier public health action. CONCLUSIONS: Despite currently having a higher cost per isolate, routine WGS of Salmonella was no more expensive than existing typing methods or PCR where >2% of illness was averted.

Herpes Simplex Virus Serotyping in Pregnant Women With a History of Genital Herpes and an Outbreak in the Third Trimester of Pregnancy: A Cost-Effectiveness Analysis.

OBJECTIVE: To estimate whether serotyping women with a history of genital herpes simplex virus (HSV) and an outbreak during the third trimester of pregnancy is cost effective compared with no serotyping. METHODS: We designed a decision-analytic model using TreeAge Pro software to assess an approach of routine HSV serotyping in a theoretical cohort of 63,582 women (an estimate of the number of women in the United States with a history of genital HSV and an outbreak during the third trimester of pregnancy). Outcomes included mild, moderate, and severe neonatal HSV, neonatal death, costs, and quality-adjusted life-years (QALYs) for both the woman and neonate. Probabilities, utilities, and costs were derived from the literature, and we used a willingness-to-pay threshold of $100,000 per QALY. Sensitivity analyses were performed to assess the robustness of the results. RESULTS: In our theoretical cohort, HSV serology screening resulted in 519, 8, and 15 cases of mild, moderate, and severe neonatal HSV, whereas no serology screening resulted in 745, 65, and 85 cases, respectively. Thus, HSV serology screening led to 226, 57, and 70 fewer cases of mild, moderate, and severe neonatal HSV, respectively, as well as 91 fewer neonatal deaths. Additionally, serology screening saved $61 million and gained 7,900 QALYs, making it a dominant strategy. Univariate sensitivity analysis demonstrated that serology screening was cost effective until the chance of progression from neonatal HSV infection to disease despite empiric antiviral treatment was greater than 23%. CONCLUSION: Serology screening in pregnant women with an outbreak in the third trimester of pregnancy and a history of genital HSV resulted in improved outcomes and decreased costs.

Molecular Typing of Different Salmonella Serotypes by Multiple-Locus Analysis of Tandem Repeats.

Salmonella enterica subspecies enterica causes salmonellosis in humans and animals and is an important cause of food infections worldwide. In recent years, the multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA), a fast molecular typing method with strong epidemiological discrimination, has facilitated the effective control of diverse infections. This study aimed at the typing of 28 human origined Salmonella enteritidis, Salmonella infantis, and Salmonella typhimurium strains by using a single MLVA protocol. Previously these strains have been identified by pulsed field gel electrophoresis (PFGE) method and it has been shown that each strain produced a distinct PFGE banding profile. One MLVA protocol was tested on 3 serotypes simultaneously and it produced three banding patterns specific to each of the three common Salmonella serotypes. MLVA also constitute a relatively more cost-effective and faster method than PFGE.

A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6.

Mannheimia haemolytica is an important respiratory pathogen of ruminants. Of the 12 capsular serovars identified, 1 and 6 are most frequently associated with disease in cattle, while 2 is largely a commensal. Comparative analysis of 24 M. haemolytica genomes was used to identify unique genes associated with capsular polysaccharide synthesis as amplification targets in a multiplex PCR assay to discriminate between serotype 1, 2, and 6 strains. The specificity of serotype specific gene targets was evaluated against 47 reference strains representing 12 known serovars of M. haemolytica and 101 field isolates identified through antisera agglutination as serotypes 1, 2, or 6. The results suggest this simple and cost-effective serotype specific PCR assay can be used as an alternative to agglutination based techniques to serotype the majority of M. haemolytica collected from bovines, thus averting the need to use animals and invest in expensive sera development for agglutination assays. In addition, the gene targets identified in this study can be used in silico to identify serotype 1, 2, and 6 strains in sequenced M. haemolytica isolates without the need for culture based analysis.

Utilization of a molecular serotyping method for Salmonella enterica in a routine laboratory in Alberta Canada.

Salmonella is one of the most common enteric pathogens related to foodborne illness. Alberta's Provincial Laboratory for Public Health (ProvLab) provides Outbreak and Surveillance support by performing serotyping. The Check&Trace Salmonella™ (CTS) assay (Check-Points, Netherlands), a commercial DNA microarray system, can determine the serotype designation of a Salmonella isolate with automated interpretation. Here we evaluate 1028 Salmonella isolates of human clinical or environmental sources in Alberta, Canada with the CTS assay. CTS was able to assign a serovar to 98.7% of the most frequently occurring human clinical strains in Alberta (82.5% overall), and 71.7% of isolates which were inconclusive by conventional methods. There was 99.7% concordance in environmental isolates. The CTS database has potential to expand to identify rare serovars. With the anticipated shift to molecular methods for identification, CTS provides an easy transition and demonstrates ease-of-use and reduces the turn-around-time of a reported result significantly compared to classical serotyping.

Quantum dots and duplex-specific nuclease enabled ultrasensitive detection and serotyping of Dengue viruses in one step in a single tube.

Leveraging on the enzymatic processing of Dengue virus (DV) RNA hybridized quantum dot-capped DNA capture probes (QD-CPs), an ultrasensitive assay for the detection and serotyping of DVs is described in the report. Briefly, DV-specific DNA CPs are first capped by QDs and then conjugated to magnetic beads. In a sample solution, strands of DV RNA form heteroduplexes with the QD-CPs on the magnetic beads. The CPs together with the QDs in the heteroduplexes are subsequently cleaved off the magnetic beads by a duplex-specific nuclease (DSN), releasing the QDs to the solution, freeing the target RNA strands, and availing them for another around of hybridization with the remaining QD-CPs. After removing the magnetic beads along with unreacted (uncleaved) QD-CPs by using a permanent magnet, ultrasensitive fluorescent detection of DV is realized through the cleaved QDs. Serotyping of DV is accomplished by a judicious design of the QD-CPs. The assay combines excellent signal generation by the highly fluorescent QDs and the effortlessness of utilizing magnetic beads in the removal of the unreacted QD-CPs. The highly efficient DSN cleavage in conjunction with its excellent mismatch discrimination ability permits serotyping of DVs in one tube with excellent sensitivity and selectivity.

Detection of co-colonization with Streptococcus pneumoniae by algorithmic use of conventional and molecular methods.

Detection of pneumococcal carriage by multiple co-colonizing serotypes is important in assessing the benefits of pneumococcal conjugate vaccine (PCV). Various methods differing in sensitivity, cost and technical complexity have been employed to detect multiple serotypes of pneumococcus in respiratory specimens. We have developed an algorithmic method to detect all known serotypes that preserves the relative abundance of specific serotypes by using Quellung-guided molecular techniques. The method involves culturing respiratory swabs followed by serotyping of 100 colonies by either capsular (10 colonies) or PCR (90 colonies) reactions on 96-well plates. The method was evaluated using 102 nasal swabs from children carrying pneumococcus. Multiple serotypes were detected in 22% of carriers, compared to 3% by World Health Organization (WHO)-recommended morphology-based selection of 1 to 3 colonies. Our method, with a processing cost of $87, could detect subdominant strains making up as low as 1% of the population. The method is affordable, practical, and capable of detecting all known serotypes without false positive reactions or change in the native distribution of multiple serotypes.

[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.

Comparison of dkgB-linked intergenic sequence ribotyping to DNA microarray hybridization for assigning serotype to Salmonella enterica.

Two DNA-based methods were compared for the ability to assign serotype to 139 isolates of Salmonella enterica ssp. I. Intergenic sequence ribotyping (ISR) evaluated single nucleotide polymorphisms occurring in a 5S ribosomal gene region and flanking sequences bordering the gene dkgB. A DNA microarray hybridization method that assessed the presence and the absence of sets of genes was the second method. Serotype was assigned for 128 (92.1%) of submissions by the two DNA methods. ISR detected mixtures of serotypes within single colonies and it cost substantially less than Kauffmann-White serotyping and DNA microarray hybridization. Decreasing the cost of serotyping S. enterica while maintaining reliability may encourage routine testing and research.

SALMATcor: microagglutination for Salmonella flagella serotyping.

Salmonella is a complex bacterial group with more than 2400 serovars widely distributed in nature; they are considered zoonotic because they can infect a variety of animals and be transmitted to humans. Usually, they cause alimentary acquired diseases such as gastroenteritis, typhoid fever, and others that can lead to severe complications and death. Serotyping is useful to differentiate among Salmonella, because it shows an important correlation with their clinical and epidemiological patterns; consequently, it is of high value for public health, animal health, agriculture, and industry. To characterize all known Kauffmann-White Salmonella serovars, over 250 antisera are required. Due to this and to high prices antisera, many laboratories worldwide have limitations in establishing Salmonella surveillance. Therefore, we developed and validated a Salmonella flagella microagglutination test (SALMATcor) that significantly reduces laboratory requirements of antisera. SALMATcor is based on scaling down, by fivefold, the antigen:antiserum volumes actually required for the reference method: flagella standard tube agglutination technique (STAT). Antigen preparation, temperatures, and incubation periods remained as established for STAT. The SALMATcor was validated according to ISO/DIS 16140:1999 protocol, which included 1187 comparisons of flagella determinations conducted by SALMATcor and STAT, on 141 Salmonella isolates of 12 common serotypes and the use of antiserum recommended for STAT. SALMATcor concordance was excellent (Cohen's kappa index 0.9982), obtaining relative accuracy >99.9% and relative specificity >99.9%. Additionally, SALMATcor has been used by CNRB-INCIENSA since 2004 to respond to all 40 Salmonella proficiency testing strains, provided by World Health Organization-Global Salmonella Surveillance Network, obtaining 100% concordance on serovar identification. On the basis of the results achieved with SALMATcor and considering that it also significantly reduces antiserum expenses, hand labor, glassware, and bench top and water bath space requirements (microtiter plates and micropipette tips are the only additional supplies), we envision that SALMATcor will contribute to establish a sustainable Salmonella serovar surveillance worldwide.

Antibody microarray typing, a novel technique for Streptococcus pneumoniae serotyping.

The use of antibody-based miniaturized devices for microbiological applications is a field poorly investigated in the era of more developed molecular amplification techniques. A novel antibody microarray for Streptococcus pneumoniae serotyping was developed, by printing nanolitre volumes of pneumococcal serotype-specific antibodies on multi-well slides. This microarray, which showed high specificity when tested against reference and clinical S. pneumoniae isolates, can be applicable as a faster, cost-effective and accurate serotyping technique for pneumococcal epidemiological studies.

Typing of Listeria monocytogenes strains isolated in Italy by inlA gene characterization and evaluation of a new cost-effective approach to antisera selection for serotyping.

AIMS: In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost-effective methodological approach based on preliminary PCRs screening was proposed. METHODS AND RESULTS: The isolates were analysed by conventional serotyping, multiplex-PCRs for serogroup and lineage identification and PCR-RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58.10%), II (22.85%), III (12.38%) and IV (6.67%). Among clinical strains, lineage I was more represented (68.75%) than lineage II; whereas, lineage II was more associated with food (90.24%) and environmental (85.72%) isolates. Most of food (89.02%) and environmental (85.71%) isolates were classified into truncated InlA profiles, whereas the 93.75% of clinical strains were associated with a complete form of the protein. CONCLUSION: Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs-based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost-effective approach for serotyping. It could help to improve a national surveillance network for L. monocytogenes infections in Italy.

[Development and validation of a multiplex polymerase chain reaction for molecular identification of Salmonella enterica serogroups B, C2, D and E]. / Desarrollo y validación de una reacción en cadena de la polimerasa multiple para la identificación de los serogrupos B, C2, D y E de Salmonella enterica.

INTRODUCTION: The scheme Kauffman-White (KW) for serotyping of Salmonella recognizes 46 O antigens, and 119 H antigens, thereby permitting the characterization of 2541 serotypes. The serotyping is a useful epidemiological tool in identifying circulating serotypes and to characterize outbreaks. However, the method presents technical limitations, difficulty in interpretation of results and high costs. OBJECTIVE: A multiplex polymerase chain reaction test (M-PCR) was developed as an alternative method for the identification of serogroups B, C2, D, and E of Salmonella enterica. MATERIALS AND METHODS: The M-PCR detected Salmonella genes rfbJ of serogroups B and C2 and wzx of serogroups D and E. To standardize the M-PCR, reference strains of Salmonella serogroups were compared. Amplification of invA gender-specific gene of Salmonella was included as internal control of amplification. To validate the test, a blind study was conducted to identify by M-PCR 400 isolates that had been previously characterized by serology. RESULTS: The M-PCR detected Salmonella serogroups with reproducible results (Kappa index = 0.95). The sensitivity of the test was between 98% to 100% and specificity between 96% to 100%. CONCLUSIONS: The polymorphisms in the Salmonella genes rfbJ and wzx permitted the development of a method for molecular typing of Salmonella serogroups that was sensitive, specific and reproducible.

Identification of serotype in culture negative pneumococcal meningitis using sequential multiplex PCR: implication for surveillance and vaccine design.

BACKGROUND: PCR-based serotyping of Streptococcus pneumoniae has been proposed as a simpler approach than conventional methods, but has not been applied to strains in Asia where serotypes are diverse and different from other part of the world. Furthermore, PCR has not been used to determine serotype distribution in culture-negative meningitis cases. METHODOLOGY: Thirty six serotype-specific primers, 7 newly designed and 29 previously published, were arranged in 7 multiplex PCR sets, each in new hierarchies designed for overall serotype distribution in Bangladesh, and specifically for meningitis and non-meningitis isolates. Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers. PCR-based serotyping of 367 strains of 56 known serotypes showed 100% concordance with quellung reaction test. The first 7 multiplex reactions revealed the serotype of 40% of all, and 31% and 48% non-meningitis and meningitis isolates, respectively. By redesigning the multiplex scheme specifically for non-meningitis or meningitis, the quellung reaction of 43% and 48% of respective isolates could be identified. Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases. CONCLUSIONS: This PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics.

Use of miniaturized protein arrays for Escherichia coli O serotyping.

Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross-reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O-serotyping methods.

A simple and low-cost paper-bridged method for Salmonella phase reversal.

We have developed a simple, rapid, and low-cost "paper-bridged" method for Salmonella phase reversal. More than 3500 isolates were tested in our laboratory, and the results indicated that this paper-bridged method is a useful alternative for phase reversal.