Paralytic poliomyelitis in Ontario: laboratory studies of two recent cases

Bull. Org. mond. Sante 1973, 49, 245-250Bull. Wid Hlth Org. Paralytic poliomyelitis in Ontario: laboratory studies of two recent cases T. P. SUBRAHMANYAN,l J. M. LESIAK,' F. APPLETON,2 & N. A. LABZOFFSKY1 Sporadic cases ofparalytic poliomyelitis are being reported with increasing frequency, particularly in unvaccinated persons, in several countries in which the disease had been absent for several years following adequate initial vaccination programmes. In Ontario, two paralytic cases occurred in unvaccinated children after several disease-free years. Detailed studies of the strains of poliovirus type 1 isolated from these patients showed that they were not vaccine strains. Contact surveillance in one case showed that 21 originally unvaccinated contacts were also excreting virulent virus. An intensive poliomyelitis vaccination programme using inactivated vaccine during 1955-57 led to a marked reduction in the incidence of the disease in Ontario (1), but it was not until the 1962 mass oral vaccination programme that paralytic poliomyelitis almost completely disappeared and the isolation of virulent strains of poliovirus became a rare event (7). This is well supported by the fact that between 1963 and 1972 poliovirus was isolated from paralytic cases only twice. The present paper describes the laboratory studies on these two isolates and on others obtained from several asymptomatic contacts of one of the patients. CASE HISTORIES Case I This occurred in 1969 in a small village with fewer than 20 families, situated in an isolated position and not frequented by outsiders. The 16-year-old boy, who developed paralytic poliomyelitis, had not been in any public assembly and mixed with only a few boys of his own age. His other contacts were very few, nearly all close relatives, and none of his family had been immunized against poliomyelitis. His main recreation was bathing in a nearby lake, usually with two companions, 12-year-old twins. On 15 July, he bathed with his companions and, not having his swimming trunks, he went into the water in his trousers. The evening turned cold and 1 Laboratory Services Branch, Ontario Ministry of Health, Toronto, Ont., Canada. 2 Medical Officer of Health, St Thomas-Elgin, Ont., Canada. he arrived home with pain in both lower limbs and shivering. The pain and his general condition became worse over the next few days and he was admitted to hospital on 20 July with a history of fever, gen- eral malaise, tightness and pain in the hamstrings, and aching in both quadriceps. On 21 July he devel- oped weakness of both quadriceps, especially the left one, which was followed by bilateral paralysis. Although he had no history of neck or back stiff- ness, his aunt noticed that he held his neck stiffly when he returned from the lake. By 22 July he was afebrile, and on 25 July his abdominal reflexes were present but both quadriceps were weak, especially the left, and his patellar, ankle, and plantar reflexes were difficult to obtain. His recovery then followed the usual course with con- tinued improvement. The whole village was offered immunization and the inhabitants were instructed in personal hygiene and sanitation. All the contacts in the village as well as in the hospital were kept under surveillance and stool specimens were obtained from each. Case 2 A 12-year-old boy was admitted to hospital on 15 October 1971, with a history of headache, loss of appetite for 3 days, and a sudden onset of weakness in the left leg on the day of admission. Examination on admission revealed a slightly elevated temperature, diminished breath sounds, localized rales at the right lung base, bilateral weak- ness in hand grip, marked weakness of the left leg, and slight weakness in flexion of the right hip. Tendon reflexes in the upper extremities, equal 3108 - 245 T. P. SUBRAHMANYAN ET AL. bilaterally, were 2+, ankle and patellar reflexes were absent on the left and 1+ to 2+ on the right. Plantar reflexes were down-going bilaterally. A diagnosis of radiculitis (Guillain-Barre syn- drome) and right lower lobe pneumonia was made. After treatment of the pneumonia with ampicillin, bed rest, and physiotherapy, the patient improved rapidly. This unimmunized boy had four brothers and sisters, only three of whom had been immunized. No contact or source of infection was determined. MATERIALS AND METHODS Specimens for virus isolation From patients. The specimens submitted from the two patients included stool, throat washings and cerebrospinal fluid (CSF). In the first case, stool specimens were collected on the 7th, 36th, 58th and 136th days after onset of the illness, and throat washings and cerebrospinal fluid specimen on the 7th day. In the second case, throat washings were collected on the 6th day, the specimens of CSF on the 6th and 17th days, and stool specimens on the 13th and 56th days of illness. From contacts. In connection with the first case only, 118 stool specimens were collected from 74 con- tacts belonging to 28 families. Roller tube cultures of primary African green monkey kidney cells (AGMK) and human fetal diploid cells (WI-38) were used for inoculation of the specimens. Specimens for serologic tests Serum samples from the first patient were collected on the 7th, 30th, and 58th days of illness, and from the second patient on the 7th and 22nd days. The neutralization test was employed for antibody titra- tion using 100 TCID50/0.1 ml of specific virus. Antigenic differentiation and marker tests Serodifferentiation between the strains of polio- virus was made by the McBride test (9) and by the Wecker test (17) as modified by Nakano & Gel- fand (15). Two monotypic antisera were used in these tests, one to the virulent Mahoney strain and the other to the attenuated Sabin strain. The tests were carried out in AGMK cultures grown in Falcon plastic bottles. Three genetic marker tests were employed in characterization of the virus isolates: (a) the ther- mal (T) or reproductive capacity temperature marker test (8); (b) the delayed (d) marker test (5); and (c) the monkey neurovirulence test. The T marker test was carried out in roller tube cultures of AGMK cells, and the d marker test in Falcon plastic bottle cultures. The results of both tests were interpreted according to Yoshioka et al. (18). The monkey neurovirulence test was kindly performed by the Virus Research Laboratory, Labo- ratory Centre for Disease Control, Ottawa. RESULTS Virus isolation and antibody studies The results of virus isolation from the two patients and their antibody responses are presented in Table 1. In the first case, poliovirus type 1 was isolated from the stool specimen on the 7th and 58th days after the onset of symptoms. Specimens of throat wash- ing and of CSF, both collected on the 7th day of illness, failed to yield the virus. Serum samples col- lected on the 7th and 36th days both had antibody titres of 1: 256 to poliovirus 1 and the specimen obtained on the 58th day had a titre of 1: 512. No antibodies to poliovirus type 2 or 3 were detected. In the second case, poliovirus type 1 was isolated from the throat washing specimen collected on the 6th day of illness and from a stool specimen obtained on the 13th day. The CSF specimens collected on the 6th and 17th days of illness both failed to yield the virus. Two serum samples obtained on the 6th and 22nd days of illness had antibody titres of 1: 256 to both poliovirus type 1 and type 2 but no antibodies to type 3. In a programme of contact surveillance in the first case, 118 stool specimens from 74 contacts belonging to 28 families were examined over a 12-week period and the results are given in Table 2. Poliovirus type 1 was isolated from 21 contacts belonging to 6 families. Virus was isolated more than once from 2 contacts and was circulating in the community for at least 5 weeks. 2 virus excretors belonged to the 0-5 age group, 11 to 5-20 years, and 8 were over 20 years of age. Characteristics of the virus isolates The isolates from the patients and from the con- tacts were tested for the T marker, while only the isolates from the patients were tested for the d marker and for the antigenic relationship to the standard virulent (Mahoney) and attenuated (Sabin) strains of poliovirus type 1. The results are presented in 246 LABORATORY STUDIES OF POLIOMYELITIS Table 1. Virus isolation and antibody response a Day of illness Case Test Specimen 7 36 | 58 |136 1 virus isolation stool polio 1 neg polio 1 neg CSF neg throat wash neg neutralization test serum polio type 1 1:256 1: 256 1: 512 polio type 2 < 1: 8 < 1:8 polio type 3 < 1:8 < 1 :8 6 13 |17 22 56 2 virus isolation stool polio 1 neg CSF neg neg throat wash polio I neutralization test serum polio type 1 1 :256 1:256 polio type 2 1:256 1:256 polio type 3 < 1:8 < 1:8 a neg = no virus isolated. Table 2. Isolation of poliovirus type 1 from contacts Number Number Positive virus isolation of families of persons . . Totalonce twice thrice 6 35 19 1 1 21 a 22 39 0 0 0 0 a Age distribution of virus excretors: 0-5 years = 2; 5-20 years = 11; >20 years = 8. Table 3. All the isolates from 1969 resembled the virulent Mahoney strain in the T marker test, while the virus from the 1971 case was an " intermediate " strain. In the d marker test, the isolates from both patients possessed the d+ marker, which is usually characteristic of virulent strains. Only the isolate from the 1969 patient was tested for monkey neuro- virulence and the estimated paralytic dose (PD50) of the virus was between 100 and 1000 PFU/ml. The results of antigenic differentiation tests by the McBride and modified Wecker tests are given in Table 4, and show that the relationship between the present isolates and the reference virulent and attenu- ated strains was not very close. Pilot serologic survey A pilot antibody survey showed that approxi- mately 7% of the sera received for virus serology lacked neutralizing antibodies to any of the three types of poliovirus, 11 % had detectable antibodies to one type only, while 61 % had antibodies to all three types of poliovirus. Antibodies to the two epidemiologically important types, 1 and 3, were lacking in approximately 15%. These results are presented in the accompanying tabulation. Total number of sera tested Number with antibody to all types Number with antibody to 2 types Types 1 & 2 Types 1 & 3 Types 2 & 3 Number with antibody to 1 type Type 1 Type 2 Type 3 Number with no polio antibodies 560 343 (61%) 118(21%) 26 43 49 62(11%) 24 19 19 37 (7%) DISCUSSION Mass vaccination programmes with trivalent vac- cine have dramatically reduced the incidence of 247 T. P. SUBRAHMANYAN ET AL. Table 3. Comparison of genetic markers of the virus isolates and of poliovirus type 1 reference strains Virus T marker d marker Monkey StrainSourceEOP at Character EOP under neurovirulenceStrain Source EOPt40 Character low NaHCO3b Character test Mahoney stock virus 1.0 T+ 0.3 d+ not done Sabin (L.Sc.) stock virus 4.5 T 3.0 d not done isolate 1 patient 1 1.0 T+ 0.6 d+ neurovirulent isolate 2 patient 2 3.0 "intermediate" 0.3 d+ not done isolates 3-23 contacts of patient 1 0.6-1.0 T+ not done not done a EOP at 400C = log (ITC:D6: at 470C) b KFlD ...r Inw N - InnPFU under normal NaHCO3 concentration\kPFU under low NaHCO3 concentration I paralytic poliomyelitis in many countries (3). With the introduction of the mass oral vaccination pro- gramme in 1962, the number of paralytic cases reported in Canada has shown an approximately hundred-fold decrease (4). Yet in Canada and else- where, an increasing number of sporadic paralytic cases in unvaccinated persons in several areas where the disease had been absent for several years indi- cates that the consolidation phase of poliomyelitis control may have been less than adequate. The two cases reported here as well as others in Quebec (16), Saskatchewan (2), and most recently in Connecticut (12), are all examples of unvaccinated persons, mostly between 5 and 20 years of age, contracting paralytic poliomyelitis. It is highly likely that in every case there were many others infected and probably shedding virulent virus for prolonged peri- ods, as for example the 21 unvaccinated contacts in this study. In Connecticut, polivirus type 1 was incriminated as the etiologic agent in at least 9 out of the 11 paralytic cases, which occurred in a school, and a contact surveillance study led to the isolation of type 1 virus from 6 asymptomatic contacts and also showed moderately high serum antibody levels in these and several other contacts (13). This indi- cates that the virus was disseminated more widely than the clinical cases alone would suggest. Another interesting observation in this apparently closed epidemic was that an illness survey in the school revealed a considerably higher than usual incidence of possible viral illnesses (40-61.5%), one atypical case yielding a virus that had not been identified at the time of the report (14). Even more disquieting is the evidence from sero- Table 4. Antigenic relationships among the virus isolates and poliovirus type 1 reference strains Virus McBride test (NK value a). Modified Wecker testVirus riets vau ) (mean % breakthrough). Antiserum to Antiserum to Strain Source Mahoney Sabin (L.Sc.) Mahoney Sabin (L.Sc.)strain strain strain strain Mahoney stock virus 100 62 50 100 Sabin (L.Sc.) stock virus 22 100 98 8 isolate 1 patient 1 50 82 64 not done isolate 2 patient 2 45 65 not done 20 a Normalized K value, which represents the rate at which the given antiserum neutralized the test strain compared to the rate at which it neutralized its homologous strain (considered as 100). 248 v cAur unaeri lowlMab - logV LABORATORY STUDIES OF POLIOMYELITIS 249 logic surveys. In Houston, Texas, antibody surveys in the immediate post-vaccination period showed a generally high level of immunity in the city's popula- tion against all three types of poliovirus (10). Para- lytic poliomyelitis then reappeared in Houston in 1968 and a serologic survey at that time revealed that 50% of all age groups lacked antibody to one or more poliovirus types. This was attributable to laxity in vaccinations at that time (11). The results of our pilot antibody studies, although limited and not confined to critical age groups, indicate that a similar situation might exist in Ontario. This is being investigated. Ing and McLeod (6) have recently reported that the vaccine currently in use in Ontario is quite efficacious so that any gap in immunity that exists would be due to persons who refuse to be vaccinated. The presence of such unvaccinated individuals in Ontario is shown by the first case in this study, which also shows that nonvaccine polio- virus strains of varying degrees of virulence are still in circulation thus posing a danger to unvaccinated persons. There is therefore clearly a need for longi- tudinal surveillance of seroimmunity at regular inter- vals to ensure adequacy of vaccination and to pre- vent an increase in the numbers of susceptible persons to levels sufficient to sustain outbreaks of the dis- ease. In addition, all suspected cases of poliomyelitis should be investigated virologically and epidemiologi- cally in detail. RtSUMt POLIOMYELITE PARALYTIQUE DANS L'ONTARIO: tTUDES DE LABORATOIRE CONCERNANT DEUX CAS RtCENTS Les programmes de vaccination de masse contre la poliomyelite ont entrain6 une reduction massive du nombre des cas paralytiques dans beaucoup de pays, dont le Canada, et l'isolement de souches sauvages de poliovirus y est devenu un fait exceptionnel. Cependant, il est apparu recemment que les succes initiaux de 1'era- dication de la maladie ne s'etaient pas accompagnes de mesures aussi efficaces destinees A consolider les resultats acquis. On signale de plus en plus frequemment des cas sporadiques de poliomyelite paralytique, notamment chez des sujets non vaccines, dans des regions qui 6taient restees pratiquement indemnes de l'affection depuis plusieurs annees. Deux cas recents survenus dans l'Ontario (Canada) chez des sujets non vaccines ont e etudies en detail. Chez chacun d'entre eux, on a isole un poliovirus de type 1 qui par les epreuves des marqueurs genetiques (carac- teres T et d) s'est revele apparente aux souches sauvages. Les tests de differenciation antigenique n'ont pu deceler de relation etroite entre ces virus et la souche vaccinale Sabin ou la souche virulente type (souche Mahoney) du poliovirus de type 1. Le virus isoIl chez le ler malade faisait preuve de neurovirulence pour le singe et plusieurs des contacts non vaccines ont excrete ce virus pendant de longues periodes. La decouverte au sein d'une collectivite considere, comme bien protegee contre la poliomy6lite d'un groupe de personnes non vaccin&es est preoccupante. En outre, une enquete serologique pilote a montre qu'il existait des lacunes dans l'immunite antipoliomyelitique globale de la population de l'Ontario. 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