[Molecular epidemiology--molecular typing of pathogenic bacteria using pulsed-field gel electrophoresis].
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Molecular epidemiology is a new field that has recently emerged from the integration of molecular biology into epidemiologic research. As yet, few countries have significant capabilities in molecular epidemiology due to a lack of trained molecular epidemiologists, and a shortage of necessary equipment and supplies. Thus, there is a great need for: 1) training in methods for integrating molecular biology and human genetics into epidemiology and public health, and 2) advanced biotechnology equipment, reagents and supplies for molecular analyses of potential genetic and environmental risk factors. The International Molecular Epidemiology Task Force (IMETAF) was established in 1993, and is actively contributing to the development of training programs, as well as effective technology transfer in several countries. This paper focuses on approaches for enhancing training opportunities in molecular epidemiology, many of which are being supported by IMETAF.
Molecular epidemiology supplies information on various stages of the multistep process of carcinogenesis and allows identification of biological markers that may indicate an increased risk of cancer. These markers have an established position in occupational and environmental epidemiology.
Molecular detection of Yam mosaic virus (YMV), the most important potyvirus for yam plants (Dioscorea spp.) has been limited by its high genetic diversity. According to recent phylogenetic data, suitable molecular tools were developed for detection and genotyping. A reliable and rapid molecular test is described using single-tube immunocapture (IC)-RT-PCR combining the use of a monoclonal antibody able to recognise all YMV serotypes and primers designed to take into account the whole variability of YMV. Phylogenetic data were then used to develop an uncoupled IC-RT-PCR procedure adapted to the genotyping of the three major phylogenetic groups and the two recombinants implicated in the YMV epidemics in the Caribbean and French Guiana. A third exploitation of phylogenetic data was carried out with the development of a typing test based on the analysis of short nucleotide sequences. The direct sequencing of a 450 nts fragment constituted a reliable complementary typing tool as the resulting information was quite similar to that obtained with sequences of the complete coat protein gene. The approach, consisting in a robust study of YMV variability followed by the use of phylogenetic data to develop reliable detection and genotyping tools, offers new perspectives for powerful molecular epidemiological studies.
Benzene is an organic solvent that has been used in industry for about 100 years throughout the world. Since 1973, a series of toxicological and molecular epidemiological studies on benzene were conducted by researchers at the Chinese Academy of Preventive Medicine (CAPM) (1973-1986) and subsequently by a collaboration between the CAPM and the National Cancer Institute (NCI) in the United States that began in 1986, which was joined by investigators from the University of California at Berkeley, the University of North Carolina at Chapel Hill, and New York University. The findings demonstrated that the risk of leukemia and lymphoma among benzene-exposed workers was significantly increased, with elevated risks for leukemia present not only at higher exposure but also among workers exposed to under 10 ppm. Therefore, the benzene permissible level was decreased to 1.8 ppm (6 mg/m(3)) and benzene-induced leukemia is treated as an occupational cancer in China. The benzene permissible level is 1.0 in the United States and in several other developed countries and it has been suggested to be decreased to 0.5 ppm (ACGIH). A number of potential biomarkers are related to benzene exposure and poisoning. Some of these are benzene oxide-protein adducts, chromosome aberration of lymphocytes, and GPA mutations in erythrocytes, a decrease in B cell and CD4(-)T cell counts in peripheral blood, and altered expression of CXCL16, ZNF331, JUN, and PF4 in lymphocytes. Variation in multiple benzene metabolizing genes may be associated with risk of benzene hematotoxicity, including CYP2E1, MPO, NQO1, and GSTT1.
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Molecular epidemiology represents an association between molecular biology and traditional epidemiologic research. Molecular epidemiology focuses on genetic and environmental risk factors at the molecular level that influences etiology and distribution of diseases oriented to early diagnosis and treatment. Since 1993 Mexico has been participating at the International Molecular Epidemiology Task Force (IMETAF) supported by the World Health Organization and Pan American Health Organization. Mexico was the first country in Latin American that established projects in molecular epidemiology. Presentations in the Symposium Projection of molecular epidemiology in medicine, expressed the future potential of genetic knowledge in identification of genes that contribute to susceptibility or resistance to diseases.
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Molecular epidemiology uses biomarkers and advanced technology to refine the investigation of the relationship between environmental exposures and diseases in humans. It requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples, and minimizing future research costs by use of banked samples. Many factors, such as tissue type, time of collection, containers used, preservatives and other additives, transport means and length of transit time, affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for cytogenetic, immunological and biochemical analyses. Given the multiple uses of the samples in molecular epidemiology studies, appropriate informed consent must be obtained from the study subjects prior to sample collection. Use of barcoding and electronic databases allow more efficient management of large sample banks. Development of standard operating procedures and quality control plans is a safeguard of the samples' quality and of the validity of the analyses results. Finally, specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples, as well as communication of study results.Here, we focus on the factors affecting the quality and the potential future use of biological samples and some of the provisions that must be made during collection, processing, and storage of samples, based on our experience in the Superfund Basic Research Program and Children's Environmental Health Center, at the University of California, Berkeley.
Progress in molecular biology and genetics is changing the practice of medicine and public health through the development of molecular diagnostics and targeted interventions for susceptible individuals. The ethical, legal and social issues that are becoming apparent as these important discoveries are introduced into practice will have an enormous impact on society. The accurate translation of this new genetic information from the laboratory to the community is an urgent need. Molecular epidemiology is at the foundation of this important link, and represents the scientific basis of public health for the 21st Century.
Molecular epidemiological studies of cancer generally require tumor tissue to evaluate somatic genetic alterations. Frequently this requires retrieval of fixed tissue blocks from hospital pathology archives. The availability of this material may be associated with disease severity, diagnostic practices, hospitals, or risk factors for disease. Tumor material is not available when the diagnosis is made clinically without histological confirmation. These characteristics create difficulties in defining the study base population. Incomplete access to tumor tissue has implications for description of the natural history of disease, estimates of the prevalence of mutation in the population, and evaluation of environmental exposures and critical target gene mutations. Differential diagnostic practices by age groups or across hospitals may create a biased population with respect to potential risk factors. However, this will not bias case-case comparisons unless the mutation of interest is associated both with the exposure of interest and the presence of a tumor block. When subjects with less severe disease are more likely to have biopsies, information regarding the natural history of the disease will be obscured. Investigation of the interaction of environmental agents and critical target gene mutations may be limited if, for example, an environmental agent is associated with a more aggressive form of the disease. Using an ongoing pancreatic cancer case-control study as an example, we discuss the potential for bias associated with differential availability of tumor blocks including consideration of tumor, patient, and hospital characteristics. Due to incomplete retrieval of tissue, the determinants of selection should be described in all studies using tumor tissue, and the implications for generalizability, power, and interpretation of findings in population-based studies should be considered.
The WHO DiaMond Molecular IDDM Epidemiology Sub-Project is testing the hypothesis that population variation in the frequency of high-risk HLA-DQ alleles is a primary determinant of the global patterns of IDDM incidence. Data are currently available for 16 populations, and reveal significant variations in the frequencies of HLA-DQA1 and DQB1 alleles among the case and the control groups. However, DQA1 x Arg-(52) and DQB1 x non-Asp-57 (ND) were consistent and independent markers of IDDM susceptibility in all populations, except Japan. Individuals who carried only DQA1 x R and DQB1 x ND alleles had an IDDM risk similar to that observed for first degree relatives of affected individuals (3%-5%). Such information is essential for the development of clinical strategies or disease prevention approaches for the general population or individuals at high-risk. Thus, the DiaMond Molecular Epidemiology Sub-Project provides an excellent model that can be followed to assess the impact of new genetic discoveries on medicine and public health practice for diabetes and other chronic diseases.
Molecular genotyping techniques developed during the past decade and conventional epidemiological methods have been used synergistically in studies of the transmission and pathogenesis of Mycobacterium tuberculosis. Research studies assessing contacts and outbreaks, risk factors for ongoing transmission, and exogenous reinfection with M. tuberculosis have advanced with applied molecular epidemiologic techniques. In addition, molecular epidemiologic approaches have enabled scientists to assess the impact of drug resistance on the transmission and pathogenesis of M. tuberculosis and to identify strains with broad temporal and spatial distributions. In the near future, the intersection of molecular epidemiology, bacterial population genetics, comparative genomics, immunology, and other disciplines will further our understanding of tuberculosis transmission and pathogenesis, contributing to the development of effective drugs and a vaccine against this important human pathogen.
HLA-DQA11 and DQB1 alleles coding for arginine (R) in position 52, and an amino acid other than aspartic acid (ND) in position 57, respectively, are strong genetic markers for IDDM in Caucasians. However, their contribution to the occurrence of the disease in Asian populations is less clear. As part of the WHO DiaMond Molecular Epidemiology Sub-Project, HLA-DQ molecular typing was performed for IDDM cases and non-diabetic controls from three populations in the Western Pacific Rim Region where incidence rates have been established (Hokkaido, Japan; Seoul, Korea; Auckland, New Zealand). DQA1*R homozygosity was significantly associated with IDDM in all areas. DQB1*ND homozygosity was also related to IDDM in Korea and New Zealand, but not in Japan. Individuals who were homozygous for DQA1*R and DQB1*ND were at highest IDDM risk in Korea and New Zealand, with the most striking findings in Auckland. In Japan, individuals carrying two DQA1*R, but only one DQB1*ND allele, were most likely to develop IDDM. These data revealed considerable genetic heterogeneity between Japan and Korea and suggest that DQA1*R and DQB1*ND alleles may explain a larger proportion of IDDM incidence in Caucasian compared to Asian populations.
Molecular epidemiology approaches provided for a new interpretation of the TB infection transmission dynamics, contributed to changing the focuses of attention and updated the monitoring practice. On the basis of 101 cases of isolates of Mycobacterium tuberculosis (MBT) complex sampled from 84 patients with pulmonary tuberculosis in the Siberian region, we proved that the independent methods of IS6110 RFLP genetic typing and VNTR-typing by five accurate repeat tandems of ETR A, B, C, D, and E bring about similar results and can be used in studying the MTB clonal structure population in the Siberian region for the purpose of defining the TB infection transmission dynamics. The most widespread genetic types were detected, i.e. Beeijing family strains, the S42 spoligotype, and the 31323 VNTR type, which account for 52.3% of all samples. The general parameters describing the epidemic process intensity were evaluated, i.e. those characterizing the strains (91.6%) and the transmission activity factor (72%). Consequently, each three of the four analyzed TB cases resulted from a recent transmission. However, there is a trend, within the analyzed samples, towards a higher percentage of clusterization in the age group ranging from 40 to 60. Such trend is typical of a prevalence of TB reactivation cases caused by MBT complex strains spread intensively in the discussed territory. As for the clusterized isolates, which are endemic for the territory, such data should be interpreted as a recent transmission only cautiously. 28.5% of the studied isolates are resistant to anti-TB drugs used in medical practice; and 35.7% of them are resistant to izoniazide and rifampicin, therefore, according to the WHO classification they are considered to be poly-antibiotics-resistant (PAR). No strict associations were found between the spectrum of antibiotics-resistance and any of genotypes, however, 30% of PAR strains are 32525 and 42525 types VNTR (spoligotype S1 or Beejing type).
'Molecular epidemiology' is defined as the integration of conventional epidemiological approaches with molecular techniques to track specific strains of pathogens in order to understand the distribution of disease in populations. It has become a very powerful tool in the study of Mycobacterium tuberculosis and human tuberculosis, where it has been exploited to provide 'added value' to conventional epidemiological approaches (contact tracing) and has often challenged accepted dogmas. It has been used to confirm epidemiologically suspected transmission, to detect epidemiologically unsuspected transmission, to identify risk factors and environments where transmission is occurring, to detect laboratory errors and to monitor the efficacy of tuberculosis control programmes. For Mycobacterium bovis and bovine tuberculosis, molecular epidemiology has a key role to play in providing more precise epidemiological data on the issues of interbovine transmission and the role of wildlife reservoirs in disease maintenance and transmission. M. bovis strains may also differ in key biological properties, such as virulence, transmissibility, stability and antigenic variation, which may help to explain field observations. There may be correlation between strain type and 'herd level' factors such as breakdown size etc. Molecular 'strain typing' studies have provided useful information in several countries, notably New Zealand, where strain typing data is used as an integral part of M. bovis control schemes, to influence the level of herd testing or wildlife control and to define the extent and spread of infected wildlife. This presentation will review the methods and approaches currently appropriate for M. bovis strain typing and will review selected applications as well as discussing future perspectives and challenges for the application of molecular epidemiology to bovine tuberculosis.
Molecular epidemiology is a term to describe the incorporation of molecular and other types of biomarkers into epidemiology. Molecular epidemiology uses the same paradigm as traditional epidemiology but the former represents the opportunity to use the enhances resolving power of molecular biology and contemporary biochemical science to assess exposure disease relationships. There are three types of biomarkers that can be used in this regard: They include markers of exposure, effect and susceptibility. These markers can be used as dependent and independent variables in most epidemiologic study designs. Critical in their use is that they are validated and field tested. This requires extensive collaboration between laboratory and public health scientist. Special attention also needs or be paid to the interpretation and communication of biomarker data and the ethical issues attendant to their use.
Molecular epidemiology (ME), a blend of molecular biology and epidemiology, is very useful to study the spread of tubercle bacilli in mini epidemics, outbreaks, to analyse the transmission dynamics of tuberculosis (TB) and to determine the risk factors for TB transmission in a community. ME has a great role in distinguishing between exogenous reinfection and endogenous reactivation. In the laboratory, molecular epidemiology can be used to identify cross contamination. Many new DNA typing methods have been introduced after the initial introduction of restriction fragment length polymorphism (RFLP) in 1993. An internationally accepted, standardized protocol for RFLP typing of the Mycobacterium tuberculosis complex using IS6110 was published in 1993 and is still used today. Most of the newer DNA typing methods are PCR based and microarray based methods are also available. This will enable individual strains of M. tuberculosis or clonal groups to be identified by specific phenotypic traits. ME will continue to be a useful tool in future to measure the impact of any public health intervention strategy for control of tuberculosis in the community.