The maintaining of the active laboratory-based surveillance of the acute flaccid paralysis (AFP) cases in Romania in the framework of the strategic plan of the global polio eradication initiative

Until 2008 poliomyelitis was controlled in Romania by predominantly using Oral Poliovirus Vaccine Sabin (OPV); the alternative vaccination schedule (IPV formalin Inactivated Poliovirus Vaccine/OPV) will be implemented starting September 2008. The vaccination coverage with 4 doses of TOPV (trivalent oral polio vaccine) in the first 14 months of life has been > 90% since 1980. In Romania, the risk of the Vaccine-Associated Paralytic Poliomyelitis cases (VAPP) decreased from less than 2 VAPP cases/year in the 1995-2006 interval to 0 VAPP cases in 2007. The serological study was performed in 2006-2007 only in cases with pair serum samples from 28 acute flaccid paralysis (AFP) cases (age = 3 months - 14 years) and from 45 facial paralysis (FP) cases (age -6 months - 4 years 9 months). A high level of vaccinal coverage was shown for all poliovirus serotypes: >95% in AFP serum samples investigated; and for FP serum samples investigated the levels of antibodies against poliovirus (PV) serotypes were 98% for PV type 1; 87% for PV type 2: and 89% for PV type 3. If the European region is polio free since 2002, the risk of wild PV importation from endemic region remains present. The laboratory capacity for the fast detection and molecular investigations of the emergence of the new epidemic strains and a high level of population immunity must be maintained. A national seroprevalence study concerning all three PV serotypes must be performed.     

Multilocus sequence typing for global surveillance of meningococcal disease

The global surveillance of bacterial pathogens is particularly important for bacteria with diverse and dynamic populations that cause periodic epidemics or pandemics. The isolate characterization methods employed for surveillance should: (1) generate unambiguous data; (2) be readily implemented in a variety of scenarios and be reproducible among laboratories; (3) be scalable and preferably available in a high throughput format; and (4) be cost effective. Multilocus sequence typing (MLST) was designed to meet these criteria and has been implemented effectively for a wide range of microorganisms. The 'Impact of meningococcal epidemiology and population biology on public health in Europe (EU-MenNet)' project had amongst its objectives: (1) to disseminate meningococcal MLST and sequence-based typing throughout Europe by establishing a centre for training and data generation, and (2) to produce a comprehensive Europe-wide picture of meningococcal disease epidemiology for the first time. Data produced from the project have shown the distribution of a relatively small number of STs, clonal complexes and PorA types that account for a large proportion of the disease-associated isolates in Europe. The project demonstrates how molecular typing can be combined with epidemiological data via the Internet for global disease surveillance.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

In this study, we describe a detection system for the indirect detection of vaccinia virus by DNA analysis. The system uses quartz crystal microbalance (QCM) as the detection technique and polymerase chain reaction (PCR) for amplification. Different immobilization strategies for the capture probe on the quartz chip are studied. For the QCM detection of hybridisation, the influence of the structure and length of target DNA is analyzed. For the detection of DNA from an amplification product, an efficient denaturation procedure is developed. On the basis of these investigations, vaccinia virus DNA is detected with only a low number of amplification rounds and a short analysis time. Specificity can be clearly shown. To enhance the signal strength and to have a further proof of specificity, a gold nanoparticle-tagged enhancer sequence can be used.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

The qualitative and semiquantitative Western blotting technique enables the detection of separate proteins and the determination of subtypes and fragments by specific immunological reactions. Protein typing on immunoblots is restricted to antibody-specific determination, with the result of a specific banding pattern. For protein characterization, several antibodies that recognize different epitopes within the protein sequence are used. However, repeated or parallel gel runs are needed. Here we describe a sequential determination of prion proteins in healthy and pathological states that both consist of di-, mono-, and nonglycosylated isoforms using a single blot with two antibodies from two species that recognize one antigen with two epitopes. The band signals are visualized by using different chemiluminescent substrate reactions. This application can be used in the fields of diagnostics and public health to detect full-length and fragmented proteins and can also be used for characterization of overlaying proteins.     

The antimicrobial susceptibility of Escherichia coli isolated in eastern area of Romania. A surveillance study

To evaluate the resistance trends for Escherichia coli isolates during 1993-1999 period in Eastern Romania to: ampicillin (A), ampicillin-sulbactam (A/S), ceftazidime (CAZ), cefotaxime (CTA), ceftriaxone (CRO), aztreonam (AZT), ofloxacin (OF), ciprofloxacin (CIP), tetracycline (T) and chloramphenicol (C). We tested 2012 clinical isolates obtained from faeces and urine. MICs were determined by a dilution method in Mueller-Hinton agar (NCCLS guidelines). Resistance rates were analyzed using the NCCLS breakpoints for the fully susceptible category (moderately susceptible strains were classified as resistant). No significant differences were observed in susceptibility of E. coli to ampicillin and ampicillin/sulbactam in the last years. The high percentage of resistant isolates was observed in 1995 for ampicillin (89.7%). Higher incidences of resistance were detected for A, A/S, T; the addition of sulbactam restored A susceptibility only for a small percent. CAZ, CTA, CRO, AZT, OF and CIP resistance among E. coli isolates was progressively increased in the last period.     

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.     

A new approach to germinal mutation surveillance: pair-wise evaluation of component elements in unidentified multiple congenital abnormalities

In the Hungarian population-based surveillance of germinal mutations, 3 indicator conditions of offspring are being followed, namely 15 sentinel anomalies, Down syndrome and unidentified multiple congenital abnormality. The latter is discussed here as a possible indicator of germinal dominant gene and chromosomal mutations. The component congential abnormalities of unidentified multiple congenital abnormalities are classified into 45 groups. The component congenital abnormalities were reduced to pairs. A pair is a set of 2 independent component congenital abnormalities in index patients with 2 or more congenital abnormalities. Baseline figures of all component congenital abnormality pairs in 3722 unidentified multiple congenital abnormalities were determined in the study period 1973–1982. The observed data for 1983 were compared with expected occurrences based on baseline figures. This pair-wise evaluation of component elements within unidentified multiple congenital abnormalities seems to be an adequate surveillance method to detect any time cluster of congenital abnormality pairs due to environmental factors including germinal mutagens.     

Epidemiological and clinical features of 1,149 persons with Lyme disease identified by laboratory-based surveillance in Connecticut

Laboratory-based surveillance of Lyme disease in Connecticut during 1984 and 1985 identified 3,098 persons with suspected Lyme disease; 1,149 were defined as cases. Lyme disease incidence in Connecticut towns ranged from none to 1,407 cases per 100,000 population in 1985. A comparison of 1985 data with data from 1977 epidemiologic studies indicated that incidence increased by 129 percent to 453 percent in towns previously known to be endemic for Lyme disease and that Lyme disease had spread northward into towns thought to be free of Lyme disease in 1977. Children aged five to 14 years had the highest incidence. Of persons with Lyme disease, 83 percent had erythema migrans, 24 percent had arthritis, 8 percent had neurologic sequelae, and 2 percent had cardiac sequelae. The distribution of symptoms was age-dependent: case-persons less than 20 years old were almost twice as likely to have arthritis than older case-persons (35 percent versus 18 percent). Of persons with arthritis, 92 percent of those less than 20 years of age, compared to 68 percent of older persons, did not have antecedent erythema migrans. We conclude that Lyme disease is increasing in incidence and geographic distribution in Connecticut. Of those with Lyme disease, children may be more likely than adults to develop arthritis and have it as their first major disease manifestation.     

Epidemiological surveillance of syphilis patients in Colentina Hospital (Bucharest, Romania)

Syphilis remains a global health problem with an estimated 12 million people infected each year. In Romania a decrease in the syphilis prevalence can be observed. From 2002 (12,702 cases) and 2003 (9,698 cases) until 2006 (5,657 syphilis cases) the reduction can be explained through the intensified efforts of the Ministry of Pubic Health to fight STI. The decrease is probably not related to an improvement of the general health status and not a consequence of some epidemiological prevention and control measures but probably was done by the reorientation of the patients to the general practitioners and to the private practice medical offices and to the lack of reporting of the cases. In Colentina Hospital a similar abrupt decrease of new cases was registered from 2004 (259 cases) to 2006 (110 cases). General problems related to syphilis cases recorded at Colentina Hospital included the patient presentation for consultation in the advanced stages of the disease, the socio-economic and educational factors, proxenetism and the sexual aggression of minors. There is a need in strengthening of the public health component in the control and surveillance of HIV/AIDS and STI. This may need changes in the legal framework to improve reporting and to target vulnerable groups in prevention activities. Laboratory capacity needs to be increased in order to be able to properly diagnose STI and improve the control and patient management. The reporting needs to be improved and simplified as for reporting protocol, reporting forms, case definitions to be taken into account in the renewed STI surveillance.     

A blended approach to active learning in a physiology laboratory-based subject facilitated by an e-learning component

Learning via online activities (e-learning) was introduced to facilitate existing face-to-face teaching to encourage more effective student preparation and then informed participation in an undergraduate physiology laboratory-based course. Active learning was encouraged by hypothesis formation and predictions prior to classes, with opportunities for students to amend their e-learning submissions after classes. Automatic or tutor feedback was provided on student submissions. Evaluation of the course was conducted via student questionnaires, individual student interviews, and analysis of student marks in examinations and of the e-learning component. Student feedback on this entire subject in the university-wide quality of teaching survey was very high by University of Melbourne standards and most encouraging for the first implementation of such a curriculum modification. Results from further detailed surveys of student interactions and engagement and correlation analysis between student responses were also very supportive of the effectiveness of the course. There were no significant differences between examination marks in the new course with e-learning and the previous year without e-learning. However, there was a significant correlation between assessment of student e-learning work and their final examination mark. Correlation analysis between various survey responses helped interpret results and strengthened arguments for e-learning and suggested future improvements for student use of e-learning. This mode of e-learning used to support face-to-face learning activities in the laboratory can be adapted for other disciplines and may assist students in developing a greater appreciation and a deeper approach for learning from their practical class experiences.     

A novel approach for the construction of multiple shRNA expression vectors

The application of RNA interference (RNAi) as a research and therapeutic tool depends on its ability to silence genes in a sequence-specific manner. Recent studies have reported that the effective knockdown of genes can be achieved by multiple short hairpin RNAs (shRNAs) in a single vector. Moreover, this approach can depress several genes simultaneously. However, current methods for the construction of multiple shRNA vectors often suffer from vector instability and are time-consuming. Here, we describe a simple, quick and low-cost approach to construct a single vector expressing four shRNA sequences driven by four different promoters. Using this vector, we were able to improve the gene silencing efficiency and make it possible to silence four different genes simultaneously, further expanding the application spectrum of RNAi, both in functional studies and therapeutic strategies.     

A data-mining approach for multiple structural alignment of proteins

Comparing the 3D structures of proteins is an important but computationally hard problem in bioinformatics. In this paper, we propose studying the problem when much less information or assumptions are available. We model the structural alignment of proteins as a combinatorial problem. In the problem, each protein is simply a set of points in the 3D space, without sequence order information, and the objective is to discover all large enough alignments for any subset of the input. We propose a data-mining approach for this problem. We first perform geometric hashing of the structures such that points with similar locations in the 3D space are hashed into the same bin in the hash table. The novelty is that we consider each bin as a coincidence group and mine for frequent patterns, which is a well-studied technique in data mining. We observe that these frequent patterns are already potentially large alignments. Then a simple heuristic is used to extend the alignments if possible. We implemented the algorithm and tested it using real protein structures. The results were compared with existing tools. They showed that the algorithm is capable of finding conserved substructures that do not preserve sequence order, especially those existing in protein interfaces. The algorithm can also identify conserved substructures of functionally similar structures within a mixture with dissimilar ones. The running time of the program was smaller or comparable to that of the existing tools.     

A data-mining approach to spacer oligonucleotide typing of Mycobacterium tuberculosis

MOTIVATION: The Direct Repeat (DR) locus of Mycobacterium tuberculosis is a suitable model to study (i) molecular epidemiology and (ii) the evolutionary genetics of tuberculosis. This is achieved by a DNA analysis technique (genotyping), called sp acer oligo nucleotide typing (spoligotyping ). In this paper, we investigated data analysis methods to discover intelligible knowledge rules from spoligotyping, that has not yet been applied on such representation. This processing was achieved by applying the C4.5 induction algorithm and knowledge rules were produced. Finally, a Prototype Selection (PS) procedure was applied to eliminate noisy data. This both simplified decision rules, as well as the number of spacers to be tested to solve classification tasks. In the second part of this paper, the contribution of 25 new additional spacers and the knowledge rules inferred were studied from a machine learning point of view. From a statistical point of view, the correlations between spacers were analyzed and suggested that both negative and positive ones may be related to potential structural constraints within the DR locus that may shape its evolution directly or indirectly. RESULTS: By generating knowledge rules induced from decision trees, it was shown that not only the expert knowledge may be modeled but also improved and simplified to solve automatic classification tasks on unknown patterns. A practical consequence of this study may be a simplification of the spoligotyping technique, resulting in a reduction of the experimental constraints and an increase in the number of samples processed.     

A laboratory-based survey of Campylobacter infections in Prahova County

In developing countries, as well as in many western countries, members of the genus Campylobacter are recognized as one of the most common cause of acute bacterial enteritis. Campylobacter jejuni and Campylobacter coli isolation rates have been shown to be equal to, and sometimes higher than those of other enteric pathogens. The Microbiology Laboratory of the local Public Health Authority in Prahova County conducted a one and a half-year laboratory-based survey of Campylobacter infections in patients suffering from gastrointestinal symptoms. From a total of 3284 stool samples screened, the culture-positive ones confirmed the bacterial etiology for 551 diarrhea cases. Campylobacter was found in 345 specimens, being the most frequently isolated enteropathogen. C. jejuni outnumbered C. coli species (239 vs. 106 isolates). Salmonella isolates were the second local cause of diarrhea. The highest isolation rate of Campylobacter was found in children 5 years of age (262 strains). The prevalence of campylobacteriosis declined with age. The isolation rate of Campylobacter (10.5%), the unimodal age-specific distribution of cases, as well as the identification of polymicrobial infections among the screened population were epidemiological aspects resembling reports on campylobacteriosis in developing countries. The susceptibility of Campylobacter isolates to various antimicrobial agents, including macrolides and fluoroquinolones was also assessed. Among the screened isolates, Erythromycin retained a good activity, while an increased ciprofloxacin resistance was observed. The information gathered through this local study sustains the importance of Campylobacter in the etiology of autochthonous infectious diarrhea. A development of a national surveillance program regarding the most important foodborne pathogens would be beneficial for improving prevention and controlling measures.     

A multiple in silico program approach for the prediction of mutagenicity from chemical structure

We have conducted an evaluation of three of the most widely used commercial toxicity prediction programs, Toxicity Prediction by Komputer Assisted Technology (TOPKAT), Deductive Estimation of Risk from Existing Knowledge (DEREK) for Windows (DfW) and CASETOX. The three programs were evaluated for their ability to predict Ames test mutagenicity using 520 proprietary drug candidate (Test set 1) and 94 commercial (Test set 2) compounds. The study demonstrates that these three commercially available programs are useful, with limitations in their ability to predict mutagenicity over a wide range of chemical space, i.e. global predictivity. Individually, each of the programs performed at an acceptable level for overall accuracy, i.e. the ability to predict the correct outcome. However, analysis of the predictions indicates that the overall accuracy figure is heavily weighted by the ability of the programs to correctly predict non-mutagens, whereas none of the programs individually performed well in the prediction of novel mutagenic structures, i.e. Ames positive compounds. The performance of these programs' in predicting Ames positive mutagens appeared to be independent of the chemical utility of the compound, i.e. industrial, agricultural or pharmaceutical. The combination of program predictions provided some improvement in overall accuracy, sensitivity and specificity.     

A hierarchical model for analyzing multisite individual-level disease surveillance data from multiple systems

Passive surveillance systems are widely used to monitor diseases occurrence over wide spatial areas due to their cost-effectiveness and integration into broadly distributed healthcare systems. However, such systems are generally associated with imperfect ascertainment of disease cases and with heterogeneous capture probabilities arising from factors such as differential access to care. Augmenting passive surveillance systems with other surveillance efforts provides a way to estimate the true number of incident cases. We develop a hierarchical modeling framework for analyzing data from multiple surveillance systems that allows for individual-level covariate-dependent heterogeneous capture probabilities, and borrows information across surveillance sites to improve estimation of the true number of incident cases. Inference is carried out via a two-stage Bayesian procedure. Simulation studies illustrated superior performance of the proposed approach with respect to bias, root mean square error, and coverage compared to a model that does not borrow information across sites. We applied the proposed model to data from three surveillance systems reporting pulmonary tuberculosis (PTB) cases in a major center of ongoing transmission in China. The analysis yielded bias-corrected estimates of PTB cases from the passive system and led to the identification of risk factors associated with PTB rates, as well as factors influencing the operating characteristics of the implemented surveillance systems.     

Development and application of a multiple typing system for Clostridium difficile

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

A systematic approach for prioritizing multiple management actions for invasive species

The successful management and eradication of invasive species is often constrained by insufficient or inconsistent funding. Consequently, managers are usually forced to select a subset of infested areas to manage. Further, managers may be unaware of the most effective methods for identifying priority areas and so are unable to maximize the effectiveness of their limited resources. To address these issues, we present a spatially explicit decision method that can be used to identify actions to manage invasive species while minimizing costs and the likelihood of reinvasion. We apply the method to a real-world management scenario, aimed at managing an invasive aquatic macrophyte, olive hymenachne (Hymenachne amplexicaulis), which is one of the most threatening invasives in tropical Australia, affecting water quality, freshwater biodiversity, and fisheries.