Four-year study of WHO virus reports on enteroviruses other than poliovirus

Bull. Org. mond. Sante 1972, 46, 329-336 Bull. Wld Hlth Org. Four-year study of WHO virus reports on enteroviruses other than poliovirus F. ASSAAD1 & W. CHAS. COCKBURN 2 In 1963 the World Health Organization established a system for collecting and distri- buting information on viruses. By 1970, 93 laboratories in 33 countries were participating. The present study is an analysis of the reports on coxsackieviruses A and B and echoviruses for the 4 years 1967-70. Among the coxsackieviruses A, type 9 was reportedmostfrequently, and the most frequently reported coxsackievirus B was type 3. Among the echoviruses, types 9, 6, and 30 were common. In the northern hemisphere the season of highest inci- dence for each of the three groups was June-October; in the southern hemisphere it was November-February. Most ofthe infections were in children and the clinical manifestations usually included aseptic meningitis, respiratory disease, skin eruptions, undifferentiated febrile illnesses, and gastroenteritis. The relative frequency of an association of a virus with a clinical syndrome differed not only between the three groups of viruses under study, but in a number ofinstances between the types within a group. As is well known there were a number of instances in which a specific clinical syndrome was linked to certain specific viruses-e.g., hand, foot, and mouth disease to certain types of coxsackievirus A, and myalgia (Bornholm disease) and cardiac conditions to coxsackieviruses B. There was also an apparent relation between age and symptoms-e.g., those due to the coxsackievirus B associated with Bornholm disease in persons over 15 years ofage. At the beginning of 1963 a system for collecting and distributing laboratory and epidemiological information on virus diseases was organized by WHO (World Health Organization, 1968). Under this system, the reference centres for enteric and respiratory viruses and suitable national virus labo- ratories complete standard record forms on infections diagnosed by virus isolation or serology. WHO then collates the forms and prepares quarterly and annual reports, which are distributed under confidential cover to the collaborating laboratories. By 1970, 93 laboratories in 33 countries were participating in this scheme. In some instances the reports cover the virus laboratory work of a whole country-e.g., Canada and the United Kingdom (although the data for Scotland are analysed sepa- rately). In others they cover only parts of a country -e.g., reports from California, USA, and from Melbourne, Australia. 1 Medical Officer, Virus Diseases, World Health Organi- zation, Geneva, Switzerland. 'Chief Medical Officer, Virus Diseases, World Health Organization, Geneva, Switzerland. There are many difficulties in comparing data from different laboratories: variations in the level of technical performance or in the facilities for the laboratory work; interest limited to certain groups of viruses; delays in reporting and in completion of tests (mainly the final typing of viruses); the empha- sis placed by public health authorities on the impor- tance of certain virus diseases in comparison with others, and so on. Notwithstanding these limitations, a comparison of data from different laboratories can be useful and the results may give valid indi- cations of time trends in viral infections on a global basis although a complete picture may not be de- fined. The present study is such an attempt and is limited to coxsackieviruses A and B and the echo- viruses. METHODS The study is restricted to the 4-year period for which the information has been stored on magnetic tape for computer analysis, 1967-70. The viruses are discussed separately by type. So far as the individual virus types in the northern hemi- 2808 - 329- F. ASSAAD & W. C. COCKBURN sphere are concerned, only laboratories providing more than 25 reports during any one of the reporting years under study are included. To provide a con- trast with the northern hemisphere, laboratories from the southern hemisphere-namely, Melbourne and South Africa, which report regularly-are included, even though less than 25 isolations were reported. Viruses reported during special surveys or special studies are excluded. FINDINGS Restricting the study of each virus type to the reports from laboratories that provided at least 25 re- ports in at least one reporting year has reduced the number of laboratories included in the study to 50 (see Annex). For the same reason only coxsackie- virus A types 2, 4, 5, 9, 10, and 16, coxsackievirus B types 1-5, and echoviruses types 1, 3, 4, 6, 7, 9, 11, 14, 17, 30, and 33 are included. The number of virus isolations varied from 2 233 for coxsackievirus A (ranging from 88 reports for coxsackievirus AIO to 1 016 reports for coxsackievirus A9) to 5 611 re- ports for coxsackievirus B (ranging from 650 for coxsackievirus BI to 1 619 for coxsackievirus B3), and to 8 621 reports for echovirus (ranging from 40 for echovirus 33 to 2 600 for echovirus 9). The most frequently reported types were, for coxsackievirus A, types A9 and A16; for coxsackie- virus B, types B3 and B2; and for echovirus, types 9, 6, and 30. With all virus types the number of reports fluctuated greatly from year to year. The yearly diffe- rences were more evident when considering individual countries or laboratories-e.g., of a total of 154 cases of coxsackievirus A5 reported by Japan, 146 were reported in 1967, 1 in 1968, 4 in 1969, and 3 in 1970. Similarly, of 308 cases of coxsackievirus A16, 14, 5, 55, and 234, respectively, occurred in the four years under study. England and Wales reported 425 cases of coxsackievirus Bi, 375 (88% of which occurred in 1970, and the Federal Republic of Germany reported 282 cases of coxsackievirus B2, 253 (90 %) of which occurred in 1968. The reports of echoviruses were also unevenly distributed; for example, England and Wales report- ed 163 cases of echovirus 3, 112 (69%) of which occurred in 1967, and California, USA, reported 247 cases of echovirus 30, 217 (88%) of which occurred in 1968. In any one country, one or more virus types predominated during a given year and Table 1. Number of reports of coxsackie and echoviruses by month of collection or month of receipt of specimen Coxsackievirus A a Coxsackievirus B Echovirus Month Northern hemisphere Northern hemisphere Southern hemisphere Northern hemisphere Southern hemisphere No. 9 No. % No. % No. % No. % January 26 1.5 49 1.3 36 15.2 112 1.8 36 9.8 February 35 2.0 28 0.7 27 11.4 103 1.7 36 9.8 March 39 2.2 43 1.1 22 9.3 62 1.0 30 8.2 April 34 1.9 60 1.6 12 5.1 61 1.0 30 8.2 May 132 7.5 151 3.9 7 3.0 224 3.6 27 7.4 June 285 16.1 326 8.3 6 2.5 483 7.8 12 3.3 July 368 20.8 636 16.5 8 3.4 1 046 16.9 9 2.5 August 291 16.4 999 26.0 8 3.4 1 646 26.6 12 3.3 September 192 10.8 683 17.8 9 3.8 1 162 18.8 9 2.5 October 155 8.8 532 13.8 16 6.8 827 13.4 40 10.9 November 152 8.6 228 5.9 40 16.9 339 5.5 80 21.9 December 62 3.5 108 2.8 46 19.4 118 1.9 45 12.3 Total 1 771 100.0 3 843 100.0 237 100.0 6183 100.0 366 100.0 a There were only a few reports from the southern hemisphere and they have not, therefore, been included. 330 ENTEROVIRUSES OTHER THAN POLIOVIRUS 331 Table 2. Number of reports on coxsackieviruses included in the study according to age a Coxsackievirus A, type: Coxsackie B, type: Age Total Total (years) 2 4 5 9 10 16 1 2 3 4 5 No. % No. % < 1 1 5 18 27 96 10 76 242 15.0 111 119 246 58 145 679 18.2 1- 4 77 15 121 197 46 318 774 47.8 153 236 327 146 251 1 113 29.8 5-14 16 2 26 240 14 74 372 23.0 153 267 292 150 268 1 130 30.3 all children 108 35 174 533 70 468 1 388 85.8 417 622 865 354 664 2 922 78.3 15-24 0 0 2 67 0 14 83 5.1 42 68 80 24 83 297 8.0 25-59 3 3 1 96 1 1 28 142 8.8 64 86 169 57 112 488 13.1 >60 0 0 0 5 0 0 5 0.3 7 4 10 0 5 26 0.7 all adults 3 3 3 168 11 42 230 14.2 113 158 259 81 200 811 21.8 total 111 38 177 701 81 510 1 618 100.0 530 780 1124 435 864 3 733 100.0 a Cases of unknown age are excluded. No breakdown by age is available for England, Wales, and Northern Ireland for 1967 and 1968. similar observations have been reported in the litera- ture (Brit. med. J., 1967; Keuth et al., 1970; Yamaoka, 1968). There is apparently no regular yearly pattern for the recurrence of specific types, although England and Wales reported coxsackievirus A9 in large num- bers in both 1969 and 1970 (212 and 204 cases, respectively) and in these two years it was reported to be epidemic in the United Kingdom (Brit. med. J., 1970a, 1971a). In the northern hemisphere, infections of all virus types were least common during the winter and spring months. They rose rapidly to peaks during the summer months and gradually declined each autumn (Table 1), although in the larger outbreaks cases sometimes continued into the winter. For example, in England and Wales an outbreak caused by coxsackievirus A16 occurred in 1969 and of the total of 112 cases reported 51 occurred in November and December. In the same areas in 1970 there was an outbreak caused by coxsackievirus A9 and the highest monthly number of cases was in November.' The great majority of the specimens of coxsackie- virus A came from children less than 5 years of age, except for type A9 where children less than 5 years of age accounted for less than 50% of the reports. 1 The two epidemics have been reported in more detail (Brit. med. J., 1970b, 1971a). With coxsackievirus B, irrespective of type, most of the reports related to children less than 14 years of age, the age group 0-4 years accounting for about 50% or less (Table 2). Most of the echoviruses were again isolated from children in the age group 0-14 years. The number of reports of cases of echovirus types 1, 3, 7, 11, 14, and 17 in the age group 0-4 years was greater than, or equal to, that for the age group 5-14 years. With the other types a larger number of isolations were obtained from children aged 5-14 years, by far ex- ceeding the numbers for the 0-4-year age group for types 6 and 30 (Table 3). Coxsackieviruses A were identified most frequently in cases of skin eruptions (in particular hand, foot, and mouth disease), aseptic meningitis, upper respi- ratory tract disease, undifferentiated febrile illness, and gastroenteritis (Table 4). Nearly 50% of the reported A9 infections were associated with disease of the central nervous system (CNS), mostly aseptic meningitis (see Brit. med. J., 1970b, for the outbreak in England and Wales in 1970; and Shingu et al., 1970, for the outbreak in Japan in 1968). Coxsackievirus B was most frequently associated with neurological conditions, mostly aseptic meningi- tis, respiratory illnesses, undifferentiated febrile dis- ease, and gastroenteritis (Table 4), but a considerable proportion of the reports were associated with myalgia (Bornholm disease) and cardiac conditions 332 F. ASSAAD & W. C. COCKBURN Table 3. Number of reports on echoviruses included in the study according to age a Echovirus, type: Age Total (years) 1 3 4 6 7 9 11 14 17 30 33 Total No. % < 1 24 35 12 133 78 213 76 30 25 64 3 693 11.3 1- 4 14 33 22 344 157 536 77 30 56 103 1 1 373 22.4 5-14 15 64 84 712 96 976 126 14 68 472 16 2 643 43.1 all children 53 132 118 1 189 331 1 725 279 74 149 639 20 4709 76.8 15-24 1 7 33 208 11 217 33 9 14 180 12 725 11.8 25-59 5 16 21 150 11 264 32 4 13 135 7 658 10.7 60 and over 1 0 0 14 1 14 6 1 1 1 0 39 0.6 all adults 7 23 54 372 23 495 71 14 28 316 19 1 422 23.2 total 60 155 172 1 561 354 2220 350 88 177 955 39 6131 100.0 a Cases of unknown age are excluded. No breakdown by age is available for England, Wales, and Northern Ireland for 1967 and 1968. Table 4. Number of reports on coxsackieviruses included in the study according to the main clinical manifestation a Coxsackievirus A, type: Coxsackievirus B, type: Main clinical signs or symptoms 2Total Total2 4 5 9 10 16 1 2 3 4 5 No. % No. % total respiratory 27 20 16 126 32 19 240 11.8 113 255 281 153 164 966 19.1(lower respiratory) (2) (3) (3) (33) (17) (6) (64) (3.1) (44) (60) (91) (49) (53) (297) (5.9) total CNS 26 7 12 425 13 6 489 23.9 145 548 569 210 436 1 908 37.6(paralytic CNS) (0) (1) (1) (8) (3) (0) (13) (0.6) (4) (8) (13) (9) (12) (46) (0.9) cardiac 1 5 0 5 1 0 12 0.6 17 35 51 14 50 167 3.3 muscular 1 2 0 11 0 1 15 0.7 57 62 57 27 32 235 4.6 gastrointestinal 16 12 20 87 7 3 145 7.1 57 181 204 98 68 608 12.0 skin/mucous membrane c 45 11 151 61 18 543 829 40.6 24 25 68 22 26 165 3.3 fever d 13 2 11 123 5 4 158 7.7 99 245 219 95 87 745 14.7 others 1 0 1 42 0 3 47 2.3 23 56 54 34 20 187 3.7 nil 7 32 7 29 1 1 21 107 5.2 3 30 38 14 4 89 11.8 total 137 91 218 909 87 600 2 042 100.0 538 1 437 1 541 667 887 5 070 100.0 a Cases with unknown clinical picture are excluded. b Includes Bornholm myalgia. c Includes exanthema and hand, foot, and mouth disease. d With no other localizing signs or symptoms. ENTEROVIRUSES OTHER THAN POLIOVIRUS 333 Table 5. Number of reports Qn echoviruses included in the study according to the main clinical manifestation a Echovirus, type: Main clinical signs/symptoms 1 3 4 6 7 9 11 14 17 30 33 Total No. % total respiratory 24 48 0 176 60 174 72 37 12 47 6 656 8.2 (lower respiratory) (8) (11) (0) (65) (11) (63) (22) (11) (2) (12) (3) (208) (2.6) total CNS 16 170 163 1 576 177 1 807 272 104 85 1 108 33 5511 69.1(paralytic CNS) (1) (1) (2) (8) (3) (13) (3) (5) (1) (2) (0) (39) (0.5) cardiac 2 0 1 17 1 11 7 0 3 7 0 49 0.6 muscularb 2 3 0 50 0 4 2 3 0 0 0 64 0.8 gastrointestinal 46 27 2 150 60 80 55 52 17 50 0 539 6.8 skin/mucous membrane C 0 6 2 31 40 109 14 9 3 6 0 220 2.8 fever d 9 23 2 144 93 160 61 20 42 52 0 606 7.6 others 2 13 0 43 9 43 10 16 0 7 0 143 1.8 nil 7 12 1 42 9 29 46 21 5 8 0 180 2.3 total 108 302 171 2229 449 2417 539 262 167 1 285 39 7968 100.0 a Cases with unknown clinical picture are excluded. b Includes Bornholm myalgia. c Includes exanthema. d With no other localizing signs or symptoms. m yocarditis and pericarditis). 1 The clinical picture showed only small differences among the five cox- sackievirus B types under study (Table 4). Table 5 shows that, with the exception of type 1, echovirus was identified most frequently from cases of CNS diseases, mostly aseptic meningitis.' Echo- virus was also associated with cases of respiratory illness, non-localizing fever, and gastroenteritis. Skin rashes were noted in cases of echovirus 9, as has been found previously (Brit. med. J., 1968). Irrespective of virus type, the largest number of cases of CNS disease were seen in children under 15 years of age (in particular in the age group 5-14 years), and the largest number of cases of respiratory illness in chil- dren under 5 years of age. In coxsackievirus A infections, skin eruptions, in- cluding hand, foot, and mouth disease, were encoun- tered mostly in children under 5 years of age. In contrast, in coxsackievirus B infections, myalgia 1 Coxsackievirus infections in Britain in 1970 are reported in detail in Brit. med. J. (1970c,1971b) and conform with the above observations. 2Some of the outbreaks reported to WHO have been described in greater detail in the literature-e.g., Brit. med. J. (1969a, 1969b, 1970d); National Communicable Disease Center (1969); Roggenkamp et al. (1970); Voorhoeve & Wilterdink (1969); Groh et al., (1971). was reported in disproportionately large numbers of adults, i.e., those over 15 years of age. DISCUSSION It is apparent that all types of enterovirus are worldwide in distribution, the limited number of virus types uncovered in individual studies being governed largely by the isolation technique used, by the number of types known at the time of study, and by the number of different typing sera available to, and by the interests of, the investigator. Variations in the virus types recovered in a given area from year to year were noted early in the study, and their tendency to fluctuate in prevalence has been well demonstrated in later longitudinal studies in- volving healthy children, hospitalized patients, and the serial sampling of sewage (Gelfand, 1961; Gel- fand et al., 1963; Brit. med. J., 1971b). In any one country, more than one type of entero- virus may become epidemic during the summer months. In some investigations (Gelfand, 1961; Moritsugu et al., 1970) the isolation of echovirus appeared to occur distinctly later than that of the coxsackieviruses. This was not apparent in the pre- sent study. F. ASSAAD & W. C. COCKBURN However, the results of the present study (Table 1) are in conformity with the observation made in tem- perate climates that infection goes on throughout the year, though at a much reduced rate in the winter. The present study unfortunately does not give information on isolations in tropical countries and thus it is not possible to see if there is a contrast in seasonal pattern. Studies on healthy subjects (Cole et al., 1951; Ramos-Alvarez & Sabin, 1956) indicate that young children constitute the most frequently infected group; older individuals are less frequently infected, pri- marily because they usually possess preexisting active immunity, but presumably also because of their more limited exposure to the virus. Investigations of patients with an enterovirus disease also show that the same inverse relationship exists between age and virus excretion. However, insufficient data are avail- able in the literature to determine accurately age- specific coxsackievirus attack rates; this is parti- cularly true for the infections caused by different types of coxsackievirus (Dalldorf & Melnick, 1965). In the case of echovirus, the literature shows a marked difference in the frequency of isolation of virus types by age (Wenner, 1962). It has to be remembered that both the age of the patients and the nature of their illness are likely to influence the submission of specimens. Also, in most cases entero- viruses are isolated from faeces and, since the entero- viruses can commonly be isolated from the faeces of healthy persons, the etiological significance of the isolation in individual cases may be questionable. Notwithstanding these reservations, there is in this study, as has been found by others (Sabin et al., 1958; Johnsson, 1954; McNair Scott, 1961), an apparent age-associated variation in the clinical reaction to in- fection-e.g., Bornholm disease or myalgia was more frequently reported in adults whereas respiratory dis- easewasmostlynotedinyoungerchildren. Noattempt is made in the present study to relate a clinical feature in certain age groups to the total number of persons investigated in this age group, since the clinical cases selected for laboratory examination were not neces- sarily representative of all infections with the virus. It is noted that while the majority of neurological cases were among children, in particular in the age group 5-14 years, none the less a disproportionately large number of neurological cases were reported in adults. The present study demonstrates for temperate areas the importance of enteroviruses other than poliovirus in the production of disease. The study also shows that persistent paralysis is not a common feature, particularly in neurological conditions, al- though it does sometimes occur. As the study was limited to 4 years no cyclic trends as have been reported, for example, in Britain (Brit. med. J., 1970b, 1971b) could be included. However, a follow-up study is contemplated, which may not only illustrate cyclic patterns within any one country but may also provide information on the spread of infection across national frontiers. ACKNOWLEDGEMENTS For the data used in this paper the authors were dependent on the virologists collaborating in the reporting scheme and they express their gratitude to them for their cooperation over the years. RESUME fTUDE, COUVRANT UNE PERIODE DE QUATRE ANS, DES RAPPORTS OMS RELATIFS AUX ENTEROVIRUS AUTRES QUE LES POLIOVIRUS En 1963, 1'OMS a mis sur pied un systeme de rassem- blement et de diffusion de renseignements sur les virus. Ce systeme a pris progressivement de l'extension et en 1970 93 laboratoires situes dans 33 pays y participaient et adressaient 'a 'OMS des rapports sur les infections virales diagnostiqu&es par isolement du virus ou examens serologiques. La presente etude analyse les rapports regus de 1967 a 1970 concernant les enterovirus autres que les poliovirus, c'est-a-dire les coxsackievirus A et B et les echovirus. Elle ne mentionne un type donne de virus que si un laboratoire signale l'avoir isol6 dans 25 au moins des rapports etablis au cours d'une des quatre annees consider&es. De ce fait, le nombre des laboratoires inclus dans l'etude est reduit a 50 et l'analyse est limitee aux 334 ENTEROVIRUSES OTHER THAN POLIOVIRUS 335 coxsackievirus des types 2, 4, 9, 10 et 16, aux coxsackie- virus B des types I a 5 et aux echovirus des types 1, 3, 4, 6, 7, 9, 11, 14, 17, 30 et 33. Parmi les coxsackievirus A, les types les plus frequem- ment signales ont e les types A9 et A16; parmi les coxsackievirus B, les types B3 et B2, et parmi les echovirus, les types 9, 6 et 30. Quel que soit le type, le nombre de mentions dans les rapports envoy6s par les laboratoires a varie considerablement d'une annee 'a 'autre. La periode de plus forte incidence, pour chacun des trois groupes, s'est etendue de juin a octobre dans l'hemisphere nord et de novembre a fevrier dans l'hemisphere sud. Les infections a coxsackievirus A ont atteint surtout les enfants de moins de 5 ans; seul le type A9 a e isole avec une frequence notable chez des sujets d'autres groupes d'age. L'incidence des infections a coxsackie- virus B a ete particulierement elev6e parmi les enfants de moins de 15 ans et 50% environ d'entre elles ont ete observees dans le groupe d'age 0-4 ans. Quant aux echovirus des types 1, 3 ,14 et 17, ils ont pour la plupart e isoles chez des enfants ag6s de 0 a 4 ans, les autres types etant aussi frequemment rencontres dans d'autres groupes d'age. Les manifestations cliniques des infections a coxsackie- virus et a echovirus ont consist6 notamment en m6ningite aseptique, atteinte de l'appareil respiratoire, eruption cutanee, affection febrile non differenciee et gastro- enterite. Une association entre l'isolement d'un virus et un syndrome clinique a et6 observee avec une frequence relative tres variable selon le groupe de virus et aussi, souvent, au sein d'un meme groupe, selon le type. On a releve une association entre des manifestations cliniques et certains virus: syndrome main-pied-bouche dans l'in- fection a coxsackievirus A, myalgie (maladie de Bomholm) et affections cardiaques dans l'infection a coxsackie- virus B. II semble exister une certaine relation entre l'age et les manifestations cliniques. Quel que soit le type de virus en cause, les atteintes du systeme nerveux central sont les plus frequentes chez les sujets de moins de 15 ans, et les maladies de l'appareil respiratoire chez les enfants de moins de 5 ans. Dans les infections a coxsackievirus A, les eruptions cutanees, et en particulier le syndrome main-pied-bouche, sont observees surtout chez les enfants de moins de 5 ans. En revanche, dans les infections a coxsackievirus B, ce sont essentiellement les adultes (sujets ages de plus de 15 ans) qui pr6sentent des atteintes de myalgie. La presente etude montre le role pathogene important imparti aux enterovirus autres que les poliovirus dans les regions temperees. Elle ne couvre qu'une periode de quatre annees, mais on envisage de la poursuivre afin de deceler d'eventuels aspects cycliques de ces infections et de recueillir des informations sur leur propagation d'un pays a d'autres. REFERENCES Brit. med. J., 1967, 4, 570 Brit. med. J., 1968, 1, 723 Brit. med. J., 1969a, 2, 703 Brit. med. J., 1969b, 3, 538 Brit. med. J., 1970a, 2, 674 Brit. med. J., 1970b, 4, 693 Brit. med. J., 1970c, 4, 811 Brit. med. J., 1970d, 3, 594 Brit. med. J., 1971a, 3, 650 Brit. med. J., 1971b, 2,178 Cole, R. M. et al. (1951) Amer. J. publ. Hith, 41, 1342-1358 Dalldorf, G. & Melnick, J. L. (1965) Coxsackie viruses. In: Horsfall & Tamm, Viral and rickettsial infections of man, 4th ed., Philadelphia, Lippincott, pp. 474-512 Gelfand, H. M. (1961) Progr. med. Virol, 3, 193-244 Gelfand, H. M. et al. (1963) Amer. J. Hyg., 78, 358-375 Groh, V. et al. (1971) Canad. med. Ass. J., 104, 297 Keuth, U. et al. (1970) Germ. med. Mth., 15, 254-261 Johnsson, T. (1954) Arch. ges. Virusforsch, 5, 384-400 McNair Scott, T. F. (1961) Advanc. Virus Res., 8, 165-197 Moritsugu, Y. et al. (1970) Japan J. med. Sci. Biol., 23, 161-175 National Communicable Disease Center (1969) Aseptic meningitis and non-polio enteroviruses: annual summary 1968, Atlanta, Ga., US Department of Health, Educa- tion, and Welfare, p.17 Ramos-Alvarez, M. & Sabin, A. B. (1956) Amer. J. publ. Hith, 46, 295-299 Roggenkamp, K. et al. (1970) Med. Klin., 15, 711-717 Sabin, E. R. et al. (1958) Amer. J. Dis. Child., 96, 197-219 Shingu, M. et al. (1970) Kurume med. J., 17, 205-210 Voorhoeve, Dr H. J. A. & Wilterdink, Dr J. B. (1969) Ned. T. Geneesk, 28, 1230 Wenner, H. A. (1962) Ann. N.Y. Acad. Sci., 101, 398-412 World Health Organization (1968) The work of WHO Reference Centres, Geneva Yamaoka, K. (1968) Jap. J. publ. Hlth., 15, 9-775 F. ASSAAD & W. C. COCKBURN Annex PLACES AND COUNTRIES INCLUDED IN THE STUDY * Melbourne, Australia Sofia, Bulgaria Belgium (6) Canada Copenhagen, Denmark England, Wales & Northern Ireland Federal Republic of Germany (21) Helsinki, Finland Lyon, France Utrecht, Netherlands Budapest, Hungary Tokyo, Japan Norway (5) Bucharest, Romania Stockholm, Sweden South Africa St Gall, Switzerland California State, USA New Haven, Conn., USA New York, N.Y., USA Atlanta, Ga., USA * The figures in parentheses show the number of laboratories reporting separately. 336