Hong Kong influenza in the Sudan.

DISABILITY INDEX FOR LEPROSY PATIENTS 713 replace the detailed appraisal of each individual case but can provide an overall picture of the intensity of disabilities and the proportions of patients in each range of disability in a " mass " campaign or in a control programme. REFERENCES Martinez Dominguez, V., Bechelli, L. M. & Patwary, K. M. (1966) Int. J. Leprosy, 34, 244-254 World Health Organization Expert Committee on Lep- rosy (1960) Wid Hlth Org. techn. Rep. Ser., No. 189 Hong Kong Influenza in the Sudan by A. R. SALIM,1 Influenza-like diseases have been known to occur in the Sudan for a long time, but the true nature of these diseases has not been investigated. In 1957, there was an epidemic of an influenza-like disease which physicians attributed to the Asian influenza type of virus that was causing epidemics in other countries at that time. However, no virus isolations or serological studies were performed in the Sudan at that time. In the winters of 1968-69 and 1969-70, there were epidemics of an influenza-like disease throughout the country. Laboratory investigations were carried out to establish the nature of the causative organism(s) of these epidemics. The epidemic of 1968-69 During the summer months of 1968, there were no reports of influenza-like diseases in the country. Such diseases were first observed in December. In February 1969, there was a sudden increase in the number of patients with influenza-like disease and eventually a large proportion of the population in all age-groups was affected. 1 Head, Department of Microbiology and Parasitology, Faculty of Medicine, University of Khartoum, Khartoum, Sudan. In Khartoum, a town with a population of about 100 000 persons, all the out-patient departments in hospitals and health centres are attended by qualified medical officers and the incidence of influenza-like diseases in the period from January 1968 to Decem- ber 1969 is shown in Table 1. These figures represent the number of patients who were sufficiently ill to seek medical advice. The epidemic began to die out gradually in July 1969 The epidemic of 1969-70 Few cases of influenza-like diseases were reported during the period from June to September 1969. In December 1969, the number of cases began to rise and by January 1970, reports came from all parts of the country of an epidemic of an influenza-like disease. In Khartoum, the epidemic reached its maximum intensity in the second week of January 1970 and then began to subside gradually, until it died out at the end of May 1970 (see Fig. 1). The epidemic was more widespread and the cases were more severe than in the previous winter; the monthly incidence of influenza-like diseases reported in Khartoum town are shown in Table 1. There were very few complications from these two epidemics; this is indicated by the absence of a Table 1. Monthly incidence of influenza-like diseases in Khartoum, 1968-70 Year [ Jan. Feb. 1 March T April [ May 1 June [ July T Aug. Sept. T Oct. J Nov. Dec. 1968 13 10 73 103 98 140 128 0 0 0 0 12 1969 0 4 145 1 016 1 267 395 197 98 193 112 234 237 237 1970 6333 3139 616 227 174 157 120 2683E 714 NOTES 2000 1800- 1600- 1400- 1200 C), LL 1000 1 C) 800- 600- Fig. 1. Weekly incidence of influenza-like diseases in Khartoum from November 1969 to May 1970 significant rise in admissions of pneumonia cases in mycin were then added to the specimens to give the Khartoum hospitals and the absence of a final concentrations of 1000 IU/ml and 1000 ,tg/ml, significant rise in mortality from respiratory diseases. respectively. The specimens were left for 1 hour at 4°C and were either inoculated into embryonated Materials and methods eggs immediately or stored at -20°C until tested Laboratory investigations. During January 1970, later. at the peak of the epidemic, students at the Uni- Embryonated white Leghorn eggs, 13 days old, versity of Khartoum were selected for laboratory were inoculated by the amniotic-cavity route accord- investigations. Students were chosen because they ing to the method described by Busby, House & were more readily available for follow-up studies. Macdonald (1964, p. 170). At the end of the incuba- All students who reported to the University health tion period the amniotic fluids were collected centre were examined by qualified medical officers aseptically and tested for the presence of virus by the and each student was asked to report back after 3, haemagglutination test, employing human group 0 7, and 15 days. Those students who suffered from and fowl red blood cells (RBC). Blind passages an influenza-like disease were asked if they would were done only once for negative specimens. volunteer to take part in the investigation and only those willing to co-operate were included in these Haemagglutination (HA) test. Haemagglutination tests. tests were performed in plastic plates (WHO pattern) following the method of Busby, House &isolation of virus. Each patient was asked to gargle Macdonald (1964, p. 170). Doubling dilutions of 3 times, using sterile nutrient broth (about 10 ml). amniotic fluids were made in normal saline, starting The washings were collected in sterile bottles and at 1: 10 dilution. these were transported to the laboratory on ice in vacuum flasks. Allantoic cavity inoculation. When a haemag- Specimens were cultured on blood-agar plates, glutinating agent was obtained from the amniotic which were incubated either aerobically or anaerobi- fluids, 10-2 dilutions of the fluids were made in cally, and in nutrient broth. Penicillin and strepto- nutrient broth containing 1000 IU of penicillin and HONG KONG INFLUENZA IN THE SUDAN 715 1000 ,ug of streptomycin per ml. The mixtures were (3) titration of the antigen used; and then inoculated with a syringe into the allantoic (4) a positive convalescent serum from a patient cavities of 10-11-day-old embryonated eggs, using from whom an agent had been isolated. the method described by Busby, House & Macdonald (1964, p. 86, method 2). Volumes of 0.25 ml were Results used to inoculate each egg, 6 eggs being used for Virus isolations. From a total of 33 specimens each specimen. The eggs were incubated at 350C from throat gargles tested, 9 (27%) haemagglutinat- for 48 h and then chilled at 4°C overnight; the ing agents were recovered in embryonated eggs. allantoic fluids were then harvested and tested for the These agents always gave higher HA titres against presence of haemagglutinating agents by the HA human group 0 RBC than against fowl RBC when test described above, except that incubation during incubation was carried out at room temperature or the HA test was done at room temperature only and at 40C. Thus the isolates could be tentatively human group 0 RBC alone were used. identified as influenza type A viruses. Haemagglutination inhibition (HAI) test. Two Virus typing. Dr H. G. Pereira, Director, World samples of sera were obtained from patients suffering Influenza Centre, London, England, kindly agreed from influenza-like diseases; one sample was taken to type our virus isolates. He reported that all the at the acute stage and the second sample 15 days isolates were strains of influenza A2 virus and that later. Treatment of sera with trypsin and heat was they were indistinguishable from the A2/Hong found to be the best way of removing non-specific Kong/l/68 variant (Coleman et al., 1968). inhibitors from sera (Sampaio & Isaacs, 1953). The trypsin solution was prepared by dissolving 40 mg of Serological examinations. Two specimens of sera * ,s. . , , ,,, 1 , ~were obtained from only 25 of the 33 patientstrypsin (Difco 1: 250) in phosphate-buffered saline, examined with influenza-like disease. Sera were at pH 8.2; fresh trypsin solution was prepared for stored at -20'C until tested. When the typing of the each day's tests. Mixtures of equal volumes (0.2 ml) viruses was reported to us, these paired sera were of trypsin solution and sera were shaken and placed tested by the HAI test in order to reach a diagnosis inin a water-bath at 56C for 30 mmn; 1.2 ml of saline those cases from which no viruses had been isolated. were then added to give final serum dilutions of 1: 8. The infected allantoic fluids were titrated against the stri ientife as A ane wasseleced human group 0 RBC as described above for the for theatetn beci. HA test and the highest dilution of the allantoic agglutiation titre.Of the 25 paired sera examined, 18 showed 8-foldfluid that gave 50% haemagglutination was taken as . gor greater rises in titre (72%) and 7 were negative. I unit; 4 units of this antigen were used in the Thus, the number of cases diagnosed serologicallyHAI test. HAI test. was three times the number diagnosed by virusFirstly, twofold dilutions of the treated sera, isolation. Significant rises in titre were obtained in starting at 1: 8 dilution, were added to wells in 8 out of 9 cases from which viruses were isolated.haemagglutination plates in 0.2-mi volumes. Then, This shows that this strain of Hong Kong virus was4 HA units of the antigen, in a volume of 0.25 ml, the cause of the influenza-like disease. were added to all the wells containing the sera, and All the acute-phase sera, taken within 1-3 days of the two reagents were mixed throughly and weretet rehandere* onset of symptoms, contained antibody to this virus,allowed to stand at room temperature for 30 mm. A volume of 0.25 ml of 1.0% human group 0 RBC with ttres ranging from 1 8 to 1 128 was added to all the wells and was mixed thoroughly; Bacteriological examinations. Of the 33 throat the plates were kept at room temperature for 1 hour gargles only 3 samples showed the presence of and the test was read after the controls had settled. ,B haemolytic streptococci and only 2 showed co- The titre of the serum was taken as the reciprocal of agulase-positive staphylococci. the highest dilution that showed 50% inhibition of haemagglutination; 4-fold or greater rises in titre Discussion were taken as significant. The status of influenza in the Sudan has not been well understood in the past, owing mainly to the lack The controls for this test were: of laboratory facilities and perhaps also to the mild (1) 1: 8 serum dilution alone with RBC, nature of this disease in a country where major (2) RBC alone in saline, causes of fever are still rife, e.g., malaria, kala azar, 716 NOTES and typhoid. Certainly the epidemics of influenza- like diseases of 1957 and the last two epidemics of 1968-69 and 1969-70 were severe enough not to pass unnoticed. The first isolations of influenza viruses in the Sudan reported in this paper will no doubt add greatly to our knowledge of some of the influenza- like diseases that have occurred in this country in the past. ACKNOWLEDGEMENTS The author thanks Dr H. G. Pereira, Director, World Influenza Centre, National Institute for Medical Research, Mill Hill, London, England, for typing the viruses; Dr A. A. Ibrahim and Sister Fatima I. Musa of the University Students' Health Centre for invaluable help; and Dr S. El Sarag, Province Medical Officer of Health, Khartoum, for providing useful information about the epidemics. The technical assistance of A. Hassan is gratefully acknowledged. REFERENCES Busby, D. W. G., House, W. & Macdonald, J. R. (1964) Virological technique, London, J. & A. Churchill Ltd. Coleman, M. T., Dowdle, W. R., Pereira, H. G., Schild, G. C. & Chang, W. K. (1968) Lancet, 2, 1384-6 Sampaio, A. A. & Isaacs, A. (1953) Brit. J. exp. Path., 34, 152-158