Status of diphtheria in Japan and in other countries
  In Japan diphtheria outbreaks occurred during certain periods before and after World War II. These epidemics have been brought down under control thanks to the efficacy of diphtheria immunization. In the former Soviet Union, supplies of the toxoid vaccine were delayed with the deterioration of sanitary conditions due to the economic failure in the 1990s. Approximately 110,000 people became infected and contracted the disease between 1992 and 1996, and around 4,000 died. The epidemic was brought down with emergency toxoid immunization under the assistance of the WHO and of UNICEF. This recent example has demonstrated again the efficacy of the toxoid vaccine. Given this background, the interest of the international community in the epidemiology, diagnosis, treatment and prevention of diphtheria rose, and the European Diphtheria Surveillance Network and the European Laboratory Working Group on Diphtheria (ELWGD) was established in 1993 with the support of the WHO, and this year, the 10th year since its inception, the scale of this organization was expanded to the whole world, and its name was changed to DIPNET. It is currently pursuing its activities primarily under the Health Protection Agency (HPA) of the UK. (Twelve main countries'members of the EU including UK and France, and 14 countries outside the European sphere.) Unfortunately since Japan did not participate in the network, having been informed of the Eighth International Conference in June 2004 in Copenhagen, Dr. Masaaki Iwaki was dispatched to this meeting so that his laboratory could be permanently involved in DIPNET activities, as a function of NIID. This article will give an outline of the situation regarding diphtheria prevalence in Japan and the issues concerning prophylactic vaccination, followed by a report on Diphtheria Surveillance Network (DIPNET).
Cases of diphtheria caused by Corynebacterium ulcerans (C.ulcerans) in Japan
  C.ulcerans was isolated and identified in two patients suspected of diphtheria, one in February 2001 and the other in November 2002 in a hospital in Chiba prefecture. Both of the two strains were toxigenic. These two cases of infection with toxigenic C.ulcerans were the first in this country. Several cases of infection due to contacts with infected cows or ingestion of contaminated raw milk are reported every year in Europe. However, the route of C.ulcerans infection is unknown in many cases, which is also true for these two cases in Japan. It was learned that one of these two patients was raising many cats. C. ulcerans is capable of acquireing the ability of toxin production through bacteris phage lysogenic conversion. The gene for diphtheria toxin is located on bacteriophages and upon lysogenization of the bacteriophage, the host bacterium becomes toxinogenic. The number of patients affected with diphtheria caused by Corynebacterium diphtheriaeiC. diphtheriaej has dramatically decreased in Japan with vaccination with diphtheria toxoid, and now cases have almost disappeared. However, a careful epidemiological survey on diphtheria toxin-producing C.ulcerans will be necessary in the years to come.
Issues regarding immunization with diphtheria toxoid vaccine
  Vaccination is recommended for travelers to areas with a high risk of diphtheria infection. In this case, the diphtheria toxoid for adults that is available in Japan may be used for protection against this disease. However, WHO is recommending immunization of adults with adsorbed purified pertussis-diphtheria-tetanus combined vaccine iDTaP jthat allows protection not only against diphtheria but also concurrently against tetanus and pertussis.
  Incidentally the inoculation of 0.1 ml of adsorbed diphtheria-tetanus combined toxoid (DT) is recommended as a booster dose at the age of 11~12 years under the current Prophylactic Vaccination Act, and adults are not vaccinated subsequently. Five-tenth ml of a Human Single Dose (1 HSD) is generally given to infants for the acquisition of basic immunity, and cases of inoculation of 0.5 ml mistakenly given as a booster dose at the age of 11~12 years have been reported on rare occasions. In foreign countries, DT vaccine in which the diphtheria toxoid component has been reduced (1 HSD: antigen volume 5 LfiLimit of Flocculationj) is used for schoolchildren and adults. The content of the diphtheria toxoid component in the DT and DTaP in Japan is generally 35 Lf and 25 Lf on average per 1 ml, and it is 5 Lf and 3 Lf respectively, for the tetanus toxoid. When comparing approximately the adjuvant content (aluminium content per ml) of the Japanese products with the foreign products, it is 1 mg in foreign countries versus 0.2 mg in Japan, or a difference of around five times. Since the route of inoculation is intramuscular in foreign countries, so far no data is provided on adverse reactions to compare foreign products with the Japanese products when used by Japanese injection route, i. e. subcutaneous injection. Nevertheless, strong adverse reactions compared to the Japanese preparations will be anticipated in a combination of high-concentration adjuvant and subcutaneous inoculation. It will be necessary to confirm in future through clinical studies whether it will be possible to vaccinate schoolchildren and adults with DTaP vaccine in Japan instead of DT vaccine used in other countries.
Participation in the First Meeting of the Diphtheria Surveillance Network (DIPNET) (WHO European Secretariat, Copenhagen)
  DIPNET is a meeting consisting of members from many countries from not only Europe but also all over the world. It is an expansion of the European Laboratory Working Group on Diphtheria (ELWGD) that was set up in response to the large-scale diphtheria epidemic that struck regions of the former Soviet Union in 1994. This year the ELWGD will meet for the 8th time and DIPNET will be convened for the 2nd time. It will also be the first meeting held under the WHO's official support.
  After attending this meeting, we felt that Japan should have participated earlier. There were many researchers on diphtheria renowned all over the world in various domains including epidemiology and fundamental research. We obtained a lot of valuable information notably on epidemiology from a practical point of view. The information regarding diphtheria epidemiology and research achievements communicated at this conference were focused on sero-epidemiology through determination of the serum antitoxin titer and on surveillance through the isolation of the pathogen. Geographically the meeting was broadly divided into reports from regions of the former Soviet Union, the so-called Newly Independent States (NIS) and the Russian Federation and from other regions (Europe, Middle East [Turkey], the American Continent [Canada], and Asia [Japan]). The different approaches made by the countries related to the epidemics and other countries were reflected during this conference, since the diphtheria large-scale outbreaks of the 1990s occurred in the NIS and in Russia.
  The communications made at the conference are outlined in the following in the order of the program.
Overview.... Recapitulation of the diphtheria large-scale epidemics in the 1990s in the NIS
  The number of reports on diphtheria in the NIS was 1,436 in 1990, 19,604 in 1993, 47,869 in 1994, and it peaked at 50,434 in 1995. This corresponds in fact to 88% of the global number of diphtheria case reports in 1995. The mortality at the beginning of the epidemic reached as much as 20% depending upon the country. This is due to the lack of availability of antitoxin and to inadequacies or delay in the treatment. It should also be noted that the proportion of adolescents and adults in the patient population was high. The epidemics were remarkable in urban areas with a high density of population. However outbreaks were also observed in non-urban areas in Central Asian countries. The majority of clinical isolates during the outbreaks were of the "gravis "biotype.
  Mass immunization campaigns targeted at adults were organized with the WHO's cooperation between 1992 and 1994 in the Russian Federation and in the NIS. The number of patients began to fall from 1996 in the Russian Federation and from 1997 in the NIS. Declines of 60% and more were recorded each year.
Session 1 - Diphtheria in the European Region and beyond (4 topics)
  This session was devoted to epidemiological reports focused on bacteriology of clinical isolates from the European region, Turkey and Canada. It was shown that, while diphtheria is not a problem requiring immediate attention in the European region and in North America, it is a disease that still requires an urgent and attentive response in Turkey, in the NIS and in Russia. It will be interesting to note that when observing over time C.diphtheriae isolated in Russia, two biotypes, namely gravis and mitis, are alternately predominant in 10~15 year cycles. This phenomenon of alternation is occasionally observed also with other pathogenic bacteria. However, this is a fact to be particularly taken into consideration in terms of epidemiology and control of C.diphtheriae. On the other hand, in the regions where diphtheria is well controlled such as North America, diphtheria caused by closely related species Corynebacterium ulcerans is becoming a problem than the disease due to C.diphtheriae. This fact will become clearer in the following sessions. Incidentally a communication on cutaneous diphtheria from England had been scheduled, but it was unfortunately canceled.
Session 2 - Diphtheria in the Newly Independent States of the former Soviet Union (5 topics)
  Reports on the epidemiology of diphtheria from the viewpoint of bacterial isolation in the NIS and in the Russian Federation were the focus of this session. A large number of NIS was freed from the aftermath of the large epidemics of the 1990s, and the incidence returned to the level observed before the outbreaks. However, Russia and Georgia have not yet fully recovered from the impact. It was reported that in spite of the strong will to bring diphtheria under control in those countries, the vaccination coverage had hit the ceiling since homeless people and vagabonds had not been immunized and the situation could not be controlled effectively. It was reported that the carriage rate for C.diphtheriae in schoolchildren between 1996 and 1999 was 8.1% in Georgia and that it fell to 0.8% from 2000 to 2001. A phenomenon of alternation of predominant strains is observed also in terms of indicators other than the biotype with C.diphtheriae isolated in the NIS. A more precise classification rather than the biotype is now possible, and a shift of Lyon type 'Otshakov type'St.Petersburg/Russia type is observed when looking at the ribotype or the Pulsed Field Gel Electrophoresis (PFGE) type.
Session 3 - Clinical, epidemiological and microbiological aspects of infection caused by C. diphtheriae and C. ulcerans (5 topics)
  A simulation study of the advantages and disadvantages in terms of public hygiene and costs, in laboratory diagnoses using pharynx swabs on persons in contact with diphtheria patients were introduced during this session. Since the results are contingent to how the secondary infection rate is set, further studies will reportedly be needed. Then cases of human infection due to C.ulcerans, an organism closely related to C.diphtheriae, were presented from France and Japan. It is now known that cows and cats may constitute a source of infection. The communication from France mentioned that dogs may also become a source of infection. Tests on dogs have been performed in Japan too, but C.ulcerans was not detected. The necessity of carrying out more detailed tests in future should be investigated. According to the WHO's new classification, the cases of C.ulcerans meet the criteria on diphtheria. However, since it is not clear whether C.ulcerans may elicit infection from humans to humans (for the time being there exist no known cases of human-to-human infection), a careful approach to whether C.ulcerans infection cases should be treated on the same basis as diphtheria should be taken in Japan while amassing cases for notification to the health authorities.
Session 4 - Molecular and genetic characterization of Corynebacterium diphtheriae (9 topics)
  During the first half of this session, A researcher@from the Sanger Centre (UK), who has recently finished the genome sequencing of C. diphtheriae, introduced a web site providing a convenient analytical technique for the genome. Then, from the US CDC, the application of real time PCR, which is more quantitative than the normal PCR, was presented. As the sample pretreatment required for this procedure is the same as that currently employed in Japan for the normal PCR, the real time PCR may be applicable in Japan in laboratories equipped with the apparatus. Real time PCR may become an indispensable tool for active surveillance in Japan. The second half of this session was devoted to the introduction of an attempt to elucidate the origin of NTTB (Non-Toxigenic Tox gene-Bearing) strains, which has been frequently isolated in recent years and poses a problem, by analyzing the structure of the bacteriophage attachment sites on the genome. The presenter pointed out that NTTB strains are isolated more frequently than the usual toxigenic strains in some countries.
Session 5 - Diphtheria immunity: strategies and sero-epidemiological studies (11 topics)
  Sero-epidemiology on diphtheria was a subject of an active discussion during this session. To begin with, a report was made on the standardization of the sero-epidemiological testing method in terms of antibody titer against diphtheria toxin, as a collaborative study among European countries. It will serve as an indicator of the efficacy of vaccines, since diphtheria toxin is the vaccine component. Concurrently the elevation of the blood antibody titer to diphtheria toxin may also be an indicator of infection. During this work done within the frame of the European Sero-epidemiology Network (ESEN-2) in the European region, sets of serum with a known antibody titer were distributed to each country. This project consisted of measuring the blood antibody titers to the diphtheria toxin with the various techniques used in each country, such as Vero cell neutralization test, double-antigen delayed time-resolved fluorescence assay (DA-DELFIA), double antigen ELISA (DA-ELISA), toxin binding inhibition test (ToBI), passive hemagglutination test and commercially available ELISA kits, and of comparing the results. The objective was to perform a statistical analysis, to calculate the level of correction that should be added to the results obtained with the respective methods and to make a cross comparison of the measured values possible. The conclusion obtained regarding the numerous measuring techniques was that cross comparison is possible by adding a correction to the measured values. However, a few exceptions were apparently noted.
  When determining the blood antibody titer against diphtheria toxin, the elevation of the antibody titer due to infection and that due to vaccination cannot be distinguished. Russia reported that this obstacle was overcome and that raise of antibacterial antibody titer due to C.diphtheriae infection was detected, by using a complex composed of a cell wall component and a peptide, both derived from of C.diphtheriae, as antigen. It was also reported that a clinical trial was completed in Russia with this antigen ten years ago under the name of Kodivac, and it has already been put to clinical application as a vaccine offering protection against infection caused by C.diphtheriae. If its efficacy is excellent, this vaccine could be used also for protection against NTTB. We keenly realized that we should have access to this vaccine and study it.