Virus excretion in smallpox

Bull. Org. mond. Sant) 1973, 48, 523-527Bull. Wld Hlth Org. Virus excretion in smallpox* 2. Excretion in the throats of household contacts J. K. SARKAR,1 A. C. MITRA,2 M. K. MUKHERJEE,3 & S. K. DE 4 Throat swabs of 34 of 328 family contacts of 52 smallpox cases, examined 4-8 days after the onset of the disease in the family, were positive for variola virus. The log titre of virusper swab rangedfrom 2 to 3.95. A higher proportion ofunvaccinated than ofvaccinated contacts excreted the virus. Only 4 of the virus-positive contacts developed clinical small- pox; this occurred 5-7 days after their swabs were examined. Excretion of virus in the throats of these contacts, a few of whom were in the incubation period of the disease, suggests the possibility that they could have spread the infection. This possibility, if kept in mind, may help in tracing the source of infection or in determining the incubation period in a few instances when difficulty is experienced. Smallpox is transferred direct from person to person in a continuing chain of transmission, and there is no recognized animal reservoir of the disease (WHO Expert Committee on Smallpox Eradication, 1972). It is generally believed that a patient is infectious to others from the time the disease is manifest. In tracing the source of an infection, therefore, it is usual to look for a smallpox patient from whom the new case might have caught the virus. The household contacts of smallpox cases are not usually thought of as possible sources of infection. In the study described here the virus content of the throats of household contacts was examined to determine if they could in any way be a potential threat in the spread of the disease. MATERIALS AND METHODS The word " contact " in this paper is used to denote a person belonging to the family of a viro- logically proved smallpox patient, living in intimate * Supported principally by a research grant from the World Health Organization. 1 Professor of Virology and Officer-in-Charge, WHO Research Project on Smallpox, Department of Virology, School of Tropical Medicine, Calcutta, India. 'Research Officer, WHO Research Project on Smallpox, Department of Virology, School of Tropical Medicine, Calcutta, India. " Demonstrator in Virology, School of Tropical Medi- cine, Calcutta, India. ' Assistant Director of Health Services (Smallpox), Government of West Bengal, Calcutta, India. contact with the case and often sleeping in the same room. The smallpox patients whose contacts were studied are designated " index cases "; they included patients admitted to the Infectious Disease Hospital, Calcutta, India, but the majority were cases en- countered during our visit to the infected localities. The presence of a vaccination scar was taken as evidence of primary vaccination. No attempt was made to ascertain the date of primary vaccination or revaccination, as the information offered was unreliable. Patients with no vaccination scar and those vaccinated 0-7 days before our visit were considered as unvaccinated. However, all the contacts, whether unvaccinated or vaccinated, were given a vaccination after an occurrence of the disease in the family. To denote the severity of the disease, the cases were divided broadly into " haemorrhagic ", " con- fluent", and " discrete" according to the criteria described in part 1 of this article (Sarkar et al., 1973). The date of onset was taken as the day of onset of fever. Specimens were collected from contacts once, except on 4 occasions when the collection was repeated. Because only contacts available on the day of visit were examined, not all the contacts of the index cases were included in the study. If any of the contacts under study later devel- oped smallpox, the cases were re-examined and further specimens were collected. The duration of contact was calculated from the day of onset of fever of the index cases. 3043 - 523 Table 1. Details of the virus-positive contacts of smallpox cases, the virus titre in their throats, and the clinical type of the index cases a Serial No. Age Primary Serial No. Duration Log titre of virus of contact (years) vaccination index case (days)b per swab -~~~~~~~~~~~~~~idxcs dy)t + + + + + + 1 (C) c 1 (C) 1 (C) 2 (H) d 2 (H) 3(C) 3 (C) 4 (C) 5 (C) 5 (C) 6 (C) 7 (C) 8(C) 8(C) 9 (C) 9 (C) 10(C) 10 (C) 11 (C) 12 (C) 12 (C) 13 (C) 13 (C) 14 (C) 14 (C) 15 (C) - 16(C) + 17 (C) - 17(C) - 18 (C) 19(C) + 20(C) _ 21 (H) + 22 (C) 7 7 7 4 4 5 5 4 4 4 4 4 4 4 5 5 7 7 7 6 6 6 6 7 7 6 7 4 4 7 5 8 7 8 2.60 2.0 2.0 3.00 2.44 2.44 2.00 2.09 2.0 2.44 2.09 2.0 2.44 2.0 2.0 2.0 3.0 3.0 2.0 Developed smallpox 5 days after swabbing 2.44 3.00 Developed smallpox 5 days after swabbing 3.00 2.44 2.95 2.0 3.95 (on 6th day) Developed smallpox 5 2.00 (on 11th day) days after swabbing. 0.00 (on 17th day) Revaccinated on 1st day of swabbing. 2.44 2.00 3.00 (on 4th day) 2.00 (on 11th day) 2.0 2.44 2.0 3.44 1.00 (on 13th day) 2.0 Developed smallpox 7 days after 1st swab- bing. Given primary vaccination on 1st day of swabbing. a All contacts were vaccinated after the index cases were detected. b From the onset of fever in the index case. c C = confluent. d H - haemorrhagic. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 5 19 7 11 24 58 62 17 11 9 5 3 12 14 7 9 52 46 45 6 5 23 1 % 10 5 55 27 28 29 30 31 32 33 34 14 36 5 months I 'h 6 10 12 37 SMALLPOX: 2 Before contacts' throats were swabbed, the cotton swabs were soaked in Hanks' basal salt solution (BSS) containing 0.5% bovine albumin and anti- biotics (penicillin and streptomycin). The specimens were immediately placed in ice containers and brought to the laboratory. The swabs were dipped in 1 ml of Hanks' BSS and squeezed; the fluid was then preserved at -20°C until inoculation. Egg inoculation and pock counts were performed as reported previously (Sarkar & Mitra, 1967). Identi- fications of variola virus were based on the character- istics of growth on egg chorioallantoic membrane (CAM). RESULTS Altogether, 328 contacts of 52 index cases were examined. Variola virus was present in the throats of 34, while the rest were negative. Data on the positive contacts and the log titres of virus in their throats are given in Table 1. There were 2 haemor- rhagic and 20 confluent cases among the index patients and the duration of contact with them varied from 4 to 8 days. Eighteen of the contacts were vaccinated and 16 were unvaccinated. Log virus titre in the throat ranged from 2 to 3.95 per swab. In 4 contacts (serial Nos. 21, 26, 29, and 33) the examination was repeated and virus titres were found to fall gradually. Four of the contacts (serial Nos. 19, 21, 26, and 29) developed smallpox; 3 of them had been vaccinated previously and one (serial No. 29) was vaccinated during the incubation period. The frequency of isolation of variola virus by age group from vaccinated and unvaccinated contacts is shown in Table 2, while Table 3 gives the frequency of isolation of virus among contacts exposed to haemorrhagic, confluent, and discrete cases of smallpox. DISCUSSION In this study 34 (10.36%) of the 328 contacts examined were found to have variola virus in their throats, but only 4 (8.8 %) of these 34 later developed the disease. Thus 30 contacts out of 328 were capable of excreting virus at some stage, although they never developed smallpox. Unfortunately it was not possible to perform serial throat cultures, and so the duration of these contacts' infectivity could not be assessed. For the same reason it is not known if any of the negative contacts excreted virus on any day before or after our visit. One contact (serial No. 33) who did not develop smallpox yielded throat swabs positive for virus on the 7th and 13th days of illness of his index case. It may be assumed that in the inter- vening period his throat carried the virus, although the virus titre was lower in the second sample. The quantum of virus excreted by a person has a definite bearing on infectivity. In all the contacts virus titre was more than 102 per swab, and in a number it was above 102. These concentrations of virus in the throat are hardly negligible, as similar concentrations were found in the throats of a number of smallpox cases (Sarkar et al., 1973). Because serial throat swabs were not taken we cannot say whether the virus titre in contacts' throats was ever higher than on the day of the single visit. Unfortuna- tely we do not yet know what is the minimum number of virus particles that must be implanted in the throat of a susceptible individual to produce disease in him. The potential ability of contacts to spread small- pox thus requires serious consideration, particularly since little restriction is put on their movements and any spread of infection by them, should it occur, will remain unnoticed. When searching for a source of infection epidemiologists are sometimes unable to establish that a new case has been in direct contact with a smallpox patient. In such a situation, the possible role of contacts in spreading the infection may merit consideration. There is at present no epidemiological support for the concept of spread of smallpox by household contacts. But when the origin of a new cases is traced to a visit to the house of an established case, it is difficult to say categorically that the visitor caught the infection from the case and not from a virus-positive household contact. Indeed, there is every chance of his coming into closer contact with members of the household than with the actual patient. Four of the contacts in this study (Nos. 19, 21, 26, and 29 in Table 1) developed smallpox 5-6 days after virus was detected in their throats. This raises the possibility of the spread of infection by a person while in the incubation period. The titre of virus in the 4 cases, particularly case 26, was fairly high, indicating that there was a fair chance of their spreading the disease to others. This finding, although not contradictory, is not consistent with the failure of McCallum et al. (1950) and Downie & McCarthy (1954) to isolate virus from the mouths of smallpox patients before rash appeared. The failure to isolate virus from the mouth washings of 5 smallpox cases during the first 2 days of the disease (Downie et al., 1961) might, as the authors pointed out, be due to 52.5 J. K. SARKAR ET AL. Table 2. Frequency of isolation of variola virus, by age group, from vaccinated and unvaccinated household contacts of smallpox cases Vaccinated contacts Unvaccinated contacts Age No. who No. whoea) No. from No. with later No. from No. with later(years whom cultures smallpox developed whom cultures smallpox developed obtained virus clinical obtained virus clinical smallpox smallpox < 1 14 0 0 17 1 (5.8%) 1 1-4 34 0 0 19 3 (15.7 %) 0 5-14 45 11 (24.4%) 1 8 7(87.5%) 0 > 15 176 7 (3.9 %) 2 15 5 (33.3 %) 0 totals 269 18 (6.7 %) 3 59 16 (27.1 %) 1 the fact that only a very small portion of the mouth washings was examined. It is difficult to reconcile the present findings with actual field experience, which indicates that smallpox transmission by drop- lets occurs mainly during the first week of the mani- fested disease (Rao et al., 1968). It is noteworthy that in cases 26, 29, and 33 the titre of virus in the throat fell to a certain extent after the disease became manifest. All 3 patients were vaccinated or revac- cinated during the incubation period, and it might be that vaccination played a part in the subsequent fall in virus titre. Two of the previously vaccinated contacts (Nos. 19 and 26) who developed smallpox were aged 45 and 55 years; it can be presumed that they had been vaccinated many years earlier. Case No. 21, aged 5 years, might have had his primary vaccination more than 4 years previously and case 29, a 5-month-old infant, was not vaccinated until the index case occurred in the family. Table 1 shows that 16 other unvaccinated contacts harbouring virus in their throats did not develop smallpox. In some of them, vaccination after exposure to the virus may have prevented the disease, and in others previous subclinical infection may have produced a level of immunity sufficient to arrest the onset of the disease process. Unfortunately the sera of the latter cases were not examined for antibody to test this hypothesis. In some patients the incubation period of the disease, calculated from the case history and apparent source of infection, does not fit the accepted incu- bation period. Here it is worth quoting the report of the United Kingdom Ministry of Health (1963) on the smallpox outbreak in Great Britain in 1961- 1962: " In other instances what might normally be regarded as the conjectured time of infection proves to fall when the patient and his supposed source of infection were not in contact. One has then to postulate a longer or shorter incubation period in order to continue to assume the relationship between Table 3. Frequency of isolation of variola viru s from household contacts exposed to haemorrhagic, confluent, and discrete cases of smallpox Vaccinated contacts Unvaccinated contacts Type No. who No. who of index No. from No. with later No. from No. with later case whom cultures smallpox developed whom cultures smallpox developed obtained virus clinical obtained virus clinical smallpox smallpox Haemorrhagic 18 1 (5.5%) 0 4 2(50%) 0 Confluent 216 17 (7.9 %) 3 40 14 (35 %) 1 Discrete 35 0 0 15 0 0 totals 269 18 (6.7 %) 3 59 16 (27.1 %) 1 526 SMALLPOX: 2 527 the two cases; or one must look elsewhere for a source on this occasion. Such a source might be a ' missed case ', and this suggests that persons other than contacts under observation have been placed at risk. The absence of other discovered cases in the general population weakens the ' missed case ' hypothesis and tends to throw one back on the assumption that, in these instances, the incubation period was in fact longer or shorter than usual ." The recognition that contacts of smallpox cases or patients during the incubation period can excrete the virus may explain some of the discrepancies observed in the incubation period. If a patient picks up the infection from a case before the disease is manifest, the incubation period will appear to be shorter than usual, while if he is infected by a contact of a smallpox case, the incubation period might appear to be longer. The data in Table 1 indicate that the age and vaccination status of contacts did not materially affect the virus content of their throats. Although there were only 2 haemorrhagic cases among the 22 index cases, their contacts had the same virus titre in their throats as the contacts of the 20 con- fluent cases. It is apparent from Table 3 that the haemorrhagic cases did not contribute more virus- positive contacts than the confluent cases. Virus- negative contacts were also associated with all 52 index cases studied. In other words, individual index cases had both virus-positive and virus- negative contacts. Regarding age or period of contact with the index cases, the virus-negative contacts did not differ materially from the virus-positive contacts. However, the two groups differed considerably in respect of vaccination status; 15.30% of the virus-negative contacts were unvaccinated, as against 47.05% in the virus-positive group. Of the total of 328 contacts studied, 16 of 59 (27.1 %) unvaccinated contacts, but only 18 of 269 (6.7%) vaccinated contacts, were virus-positive. This shows that previous vaccination had a marked influence on virus excretion in con- tacts' throats (Tables 2 and 3). The fact that only 10.36% of contacts were virus- positive indicates that their potential for spreading the infection is much less than that of smallpox patients, all of whom are capable of excreting virus in the throat (often at a higher titre) or the scabs, and many of whom excrete in the urine and con- junctiva (Sarkar et al., 1973). RISUM EXCRETION DE VIRUS AU COURS DE LA VARIOLE: 2. EXCRETION PAR LA GORGE CHEZ DES CONTACTS FAMILIAUX On a effectue des prelevements de secr6tions pharyn- gees chez 328 contacts appartenant a l'entourage familial de 52 varioleux 4 'a 8 jours apres la decouverte du premier cas. Chez 34 d'entre eux (10,36%), le virus 6tait pr6sent a des titres variant en valeur logarithmique de 2 a 3,95 selon le prelevement. Quatre de ces sujets positifs ont det6 atteints de variole 5-7 jours apres le prelevement, tandis qu'aucun cas ne s'est declar6 chez les contacts non excreteurs de virus. On comptait 47% de sujets non vaccines anterieurement parmi les excreteurs de virus et 15,3 Y. parmi les non excr6teurs, les deux groupes etant par ailleurs comparables sous le rapport de l'age et de la duree du contact avec le cas princeps. Une vaccination ant6rieure a fortement influence le taux d'ex- cretion de virus parmi les contacts: 27,1 % des contacts non vaccines et 6,7% des vaccines etaient positifs. Tous les contacts, d6ja' vaccines ou non, ont requ la vaccination antivariolique au moment du prelevement ou dans les jours qui l'ont pr6ced6. La presence du virus variolique dans les secretions pharyng6es de contacts, dont certains etaient en periode d'incubation de la maladie, donne h penser qu'ils ont pu transmettre l'infection. On doit tenir compte de cette possibilite lorsqu'il s'agit de d6terminer la source de l'infection ou la dur6e de la p6riode d'incubation dans des cas d'interpretation malaisee. REFERENCES Downie, A. W. & McCarthy, K. (1954) Pathogenesis of variola. In: Hartman, W. F. et al., ed., The dynamics of virus and rickettsial infections, New York, Blakiston, pp. 194-205 Downie, A. W. et al. (1961) Bull. Wld Hlth Org., 25, 49-53 MacCallum, F. 0. et al. (1950) Lancet, 259, 514-517 Rao, A. R. et al. (1968) Indian J. med. Res., 56, 1826-1854 Sarkar, J. K. & Mitra, A. C. (1967) Indian J. med. Res., 55, 13-20 Sarkar, J. K. et al. (1973) Bull. Wld Hlth Org., 48, 517 United Kingdom, Ministry of Health (1963) Smallpox 1961-62, London, Her Majesty's Stationery Office (Reports on Public Health and Medical Subjects, No. 109) WHO Expert Committee on Smallpox Eradication (1972) Wld Hlth Org. techn. Rep. Ser., No. 493