Multiplex PCR for Differential Identification of Broad Tapeworms (Cestoda: Diphyllobothrium) Infecting Humans

The specific identification of broad tapeworms (genus Diphyllobothrium) infecting humans is very difficult to perform by morphological observation. Molecular analysis by PCR and sequencing represents the only reliable tool to date to identify these parasites to the species level. Due to the recent spread of human diphyllobothriosis in several countries, a correct diagnosis has become crucial to better understand the distribution and the life cycle of human-infecting species as well as to prevent the introduction of parasites to disease-free water systems. Nevertheless, PCR and sequencing, although highly precise, are too complicated, long, and expensive to be employed in medical laboratories for routine diagnostics. In the present study we optimized a cheap and rapid molecular test for the differential identification of the most common Diphyllobothrium species infecting humans (D. latum, D. dendriticum, D. nihonkaiense, and D. pacificum), based on a multiplex PCR with the cytochrome c oxidase subunit 1 gene of mitochondrial DNA.Human diphyllobothriosis is a widespread fish-borne zoonosis caused by tapeworms of the genus Diphyllobothrium Cobbold, 1858 (Cestoda: Diphyllobothriidea). Their life cycles are complex and involve two intermediate hosts (a copepod and a fish) and a definitive host (humans or other piscivorous mammals and aquatic birds). Infection takes place through the consumption of raw or undercooked fish harboring plerocercoid larvae and often remains unnoticed until the excretion of segments of the adult parasite (proglottids) in stools. Symptoms include various minor digestive problems occurring a few weeks after infection (mostly nausea, diarrhea, and abdominal pain); less commonly, prolonged or heavy infections can result in a pernicious anemia (7). About 14 species of Diphyllobothrium have been reported to infect humans. From a medical perspective, Diphyllobothrium latum, Diphyllobothrium nihonkaiense, Diphyllobothrium dendriticum, and Diphyllobothrium pacificum are the most important, because humans represent their preferred definitive hosts (17).The identification of Diphyllobothrium tapeworms by physicians and medical laboratories is generally based on the morphological observation of operculated eggs and segments of adult worms (7). Morphoanatomical criteria allow identification to the genus level but are not reliable to assess species identity, because the different taxa are extremely similar one another, and species differentiation relies on characteristics of the scolex or the genital apparatus observed on mature proglottids, which are often unavailable during human infections (20). To this end, molecular methods have been recommended (17). In the past, biochemical techniques (isoenzymatic assay or immunoelectrophoresis) have been used as alternatives to traditional tools for species identification (5, 6). Matsuura et al. (13) previously discriminated between D. latum and D. nihonkaiense by using restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA), as the profiles generated with three different restriction enzymes provided valuable species-specific markers. Since the late 1990s, the development of molecular biology based on the sequencing of nuclear and mitochondrial DNA (mtDNA) targets resulted in a better knowledge of the genus Diphyllobothrium. Notable results for the phylogenetic relationships among some taxa have been obtained by the sequencing of the 18S rRNA, cytochrome c oxidase subunit 1 (cox1 or COI), and NADH dehydrogenase subunit 3 (NADH3) genes and internal transcribed spacer 1 (ITS1) and ITS2 (19, 27, 29, 31).However, molecular methods are still rarely employed in routine laboratories due to economical (need of reagents and equipment such as a thermal cycler and sequencer) and technical (procedures are complicated and time-consuming and require trained personnel) reasons. Moreover, all human-infecting Diphyllobothrium species cause similar symptoms, and infections are successfully treated with praziquantel, which can lead one to think that a further, specific identification of the causative agent is useless for the practice.In contrast, the specific diagnostic identification of Diphyllobothrium parasites isolated from patients is of great importance from an epidemiological point of view. Human diphyllobothriosis has been estimated to affect 20 million people worldwide (4, 14), and recent studies indicate a recrudescence in some well-developed countries (26). Thanks to the use of molecular tools, locally acquired infections with allochtonous species are being reported more and more frequently. For instance, D. nihonkaiense, usually present in Japan and South Korea (8), has recently been detected in Switzerland (18, 23), France (15, 30), Finland (26), and North America (25). D. dendriticum, common in northern Europe, was found in a Swiss patient (24). A human case due to D. latum was reported in Taiwan (11). In Sao Paulo, Brazil, an outbreak with a significant economic and public health impact linked to the ingestion of raw salmon was proven to be caused by D. latum (16). In most of these cases the infective source consisted of imported fish; on the other hand, the origin of the intermediate host even brought into question the known geographical repartition of some species, i.e., D. nihonkaiense, harbored by Pacific salmons in North America (a hypothesis which is currently under verification). The molecular identification of broad tapeworms infecting humans can therefore contribute to map the present species distribution, to understand their life cycles (for example, the intermediate hosts of D. pacificum are still unknown), and to clarify which fish represent the most important sources of human infections.To date, the sequencing of some genomic DNA targets allowed the identification of many Diphyllobothrium species (17), and the use of RFLP enabled the differentiation between D. latum and D. nihonkaiense (13). Nevertheless, only a PCR-based method for routine discrimination between D. latum and D. nihonkaiense has been developed for use in diagnostic laboratories (9). In this study, we adapted an easy and rapid molecular technique (one that does not require the sequencing of PCR products) to be routinely used for the differential diagnosis of all principal Diphyllobothrium species infecting humans. The method is based on a multiplex PCR, which was successfully employed for cestode identification in previous studies (9, 28).     

Cancer detection and prevention

Studies were performed on cancer detection and prevention in the high cancer risk group. To improve the screening method in the diagnosis of unspecified cancer, a modified combination assay was devised using a combination of several tumor markers and adding risk factors of malignancies obtainable from blood samples. Five tumor markers, AFP, CEA, CA 19-9, DUPAN-2 and CA 125 were determined simultaneously in each serum sample obtained from 54 patients with various cancer diseases. A modified combination assay was also examined using the risk factors of malignancies, pepsinogen I/II ratio together with 5 tumor markers. Detection rate was elevated to 68.5% by a combination assay using 5 tumor markers. Detection rate by modified combination assay using the risk factor (pepsinogen I/II ratio) together with 5 tumor markers was further improved to 88.8% without decreasing the specificity. The modified combination assay is easy in practice and expected to contribute in the screening of unspecified cancer diseases.     

Ideology and violence prevention

Interpersonal violence is a major problem in US society in terms of the death and destruction it causes, the fear it generates, and the attention it receives. A recent trend has been to regard the problem of violence as an epidemic and to shape ideas of violence prevention according to public-health formulations. This process does not take into account the ideological nature of the proposed violence-prevention measures. Problems arise because this ideology is relevant to the potential effectiveness of violence prevention.     

艾滋病的预防与控制

艾滋病毒（HIV）于1982年随血液制品第Ⅷ因子从美国进入中国，1983年感染第一个中国公民。随后HIV通过静脉吸毒、性途径不断传入我国，并在国内继续传播，使HIV-1流行形势越来越严峻。已给我国人民生命造成严重危害并给社会和经济发展带来重大损失。为此必须加强防治。一个十分重要的问题是如何控制艾滋病的流行问题。根据国外的经验，特别是HIV严重流行国家如泰国、乌干达等国家做出了很好成绩。泰国从1990年开始，政府总理带头，协调各部门全民参与艾滋病的宣传教育，并采取各种有效干预措施，特别是在娱乐场所宣传100％使用安全套。结果，性病显著减少，艾滋病感染率也迅速下降。联合国艾滋病规划署认为，泰国开展积极的宣传教育和干预工作，成绩十分显著，到2004年减少了700万人免受艾滋病毒感染。其他国家如柬埔寨和乌干达也取得很好的成就。相反的，南非政府没有积极进行宣传教育和干预，艾滋病毒的感染率高达25％。有报告预测从2002-2010年，如果全球各国不积极进行宣传教育和干预，全球将有4800万HIV感染者。如果积极进行宣传教育和干预，就会减少到2900万人，即其中2／3可以避免HIV感染，由此可见宣传教育和干预的重要性。因此全面深入的开展宣传教育和干预是当前预防和控制艾滋病主要策略。特别是应该在农村、流动人口及青壮年中进行。     

Cardiovascular risk and atherosclerosis prevention

Until recently, coronary artery disease (CAD) was the leading cause of death in the developed countries. Its remarkable decline can be attributed to our knowledge of the major risk factors identified by several studies resulting in better prevention and treatment. Of the major risk factors, the ratio of apolipoprotein (apo) B/apo A1 followed by smoking, diabetes, and hypertension are the most important. A number of risk scores for men and women are now available to estimate the likelihood of development of CAD. However, because of the risk of CAD differs in various populations, some of the algorithms are more appropriate for some countries but not suitable for others. These risk assessment algorithms differ in the parameters they use. All the risk scores have some limitations such as different study populations; the age of the study is also different, and number of points awarded for age categories also differs among the various algorithms.In an effort to further improve the risk prediction, a number of biomarkers have been studied. In addition to plasma lipids, a lot of interest has focused on apo measurements; particularly of apo B. Another valuable biomarker is lipoprotein (a) [Lp(a)]. Lp(a) is not only atherogenic as low-density lipoprotein (LDL) but also prothrombotic, and several studies indicate that Lp(a) is an independent risk factor for CAD.The lipid profile provides a framework for appropriate management. This includes therapeutic lifestyle changes and medications. Lifestyle interventions are the cornerstone of CAD prevention strategies and are the first step in risk factor management. Of particular importance are smoking cessation, achievement and maintenance of ideal body weight, regular exercise, reduction in the intake of saturated fat and sugars, and decreasing level of stress. Of medications, lipid-lowering, anti-hypertensive, and anti-coagulant can be effectively used.The current strategies for risk assessment and prevention have been very successful contributing to the more than 50% decrease in CAD mortality over the last 20 years. Thus, in Canada, cardiovascular disease is no longer the leading cause of death.     

Vitamin E and cardiovascular prevention

The antioxidant properties of vitamin E are well established. In humans, they appear very clearly from the nutritional supplementation trials. There is a strong correlation between supplied doses (>= 50 mg/j), vitamin E content of LDL and antioxidant protection of LDL. The consumption studies mostly suggest that the cardiovascular disease risk is diminished by the vitamin E supplementation, this being not true for vitamin E supplied by food strictly. In spite of the fact that there is a coherence between these results due in particular to the highly atherogenic role of oxidized low density lipoprotein, it is not allowed to claim that only the increased protection of LDL against oxidation is responsible for the diminished risk. The cell-regulating properties of vitamin E that have been more recently discovered have also to be taken into account as regards the functions of platelets, monocytes-macrophages, endothelial cells and vascular smooth muscle cells. The LDL-vitamin E capacity at decreasing the superoxide anion production (involved in turn in the oxidation process of LDL) could also play a role in preventing cardiovascular risk. The nutritional intervention studies undertaken in secondary prevention indicate a beneficial effect in terms of cardiovascular morbidity, either for low dose (50 mg), or for higher dose (>= 270 mg/d) intake, but without effect in terms of mortality. A recent study presumably supports a beneficial effect at the dose intake of 300 mg/d only in terms of cardiovascular mortality. Only one intervention has been carried out in condition of primary prevention, leading to the absence of effect at the dose employed (50 mg/d). The studies on the mechanisms of action make plausible the beneficial effects observed in analytical or experimental epidemiology. However, the experimental epidemiology does not provide indisputable evidence for the efficacy of the secondary prevention of cardiovascular risk by vitamin E supplementation. There is no intervention study using doses higher than 50 mg/d in primary prevention. There is a need for such studies in the not too distant future. A period of several years will be necessary before having new data possibly resulting in a consensus achievement.