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An epidemic of rotavirus diarrhoea in Manipur, India
521 TRANSACTIONS OF THE ROYALSOCIETY
OPTROPICALMEDICINE
An epidemic National
AND
of rotavirus
HYGIENE, VOL. 75, No. 4, 1981
diarrhoea
in Manipur,
India
I?. G. SENGUPTA, D. SEN, M. R. SAHA, S. NIYOGI, B. C. DEB, S. C. PAL Institute of Cholera and Enteric Diseases (I.C.M.R.), 3, Dr. M. Ishaque Road, Calcutta-700016, India IBETOMBI DEVI AND N. SUKUMAR SINGH
Regional Medical
College Hospital,
Imphal
and Directorate India
Summary Recurrent epidemics of acute diarrhoeal disease in young children have occurred during the winter months in Manipur (India) for many years. During November 1979, children admitted to hospital for acute diarrhoea were investigated and, in addition to pathogenic Escherichia coliand Shigella rotovirus was detected by ELISA in a number of stool samples, especially of those less than one year old. The importance of this finding and the need for further study are discussed. Introduction Recurrent epidemics of acutre diarrhoeas in children below two years of age have been reported from Manipur, a north-eastern state in India, since 1969. The epidemics started building up in an almost predictable manner from SeptemberOctober every year and usually subsided by January. The patients were mainly admitted to the Regional Medical College Hospital, Imphal, and durina the winter months about 80% of beds in the chyldren’s ward were occupied- by diarrhoeal children. The epidemics were investigated twice during 1974 (DEB et al., 1977) and 1976 when enteropathogenic Escherichia coli and Shigella were isolated from about 30% of diarrhoeal children. Facilities for detecting rotavirus were not available to the investigators. The present report describes the results of investigations of a similar epidemic of acute diarrhoeal diseases during November, 1979. Demographic features The state of Manipur comprises four hill districts and the central Imphal valley. 65% of the total population (1,073,OOO) live in the central valley and the population density was 48p er km2 (according to the 1971 census). The yearly rainfall amounts to 150-200 cm and the winter-is fairly severe. Parts of Imphal town have a supply of piped chlorinated water but this is intermittent and inadequate. The rest of the state depends on surface water for drinking and other domestic purposes. This surface water also becomes very scanty during winter months. The nightsoil disposal system is grossly inadequate in Imphal town and people use open land for defaecation.
of Health
Services, Government
of Manipur,
Methods Data for clinical and epidemiological investigations were collected from each case of gastroenteritis admitted at the Regional Medical College (R.M.C.) Hospital, Imphal and at Thoubal, Kakthing, Moirang and Bishenpur Primary Health Centres (l?.H.Cs). Microbiolorrical studies of 59 patients admitted to these hospitals during the period of investigation were included but 422 other in-patients in the hospitals could not be investigated microbiologically as their time of stay did not coincide with visits by the study team. Rectal swabs and catheter specimens of stools were collected in phosphate buffered saline from 51 cases of gastro-enteritis admitted to the paediatric ward of R.M.C. Hospital and eight children in hospital in Moirang and Thoubal P.H.C.s. Rectal swabs were also collected in Carry & Blair’s transport media and screened for the presence of Vibrio cholerae, V. parahaemolyticus, Shi,ella, Salmonella and E. -cola’ using standard laboratory techniques (Taint ICMR-GWB-WHO Cholera Studv Grouu. Calcutta, 1970; EDWARDS & EWING, 1972). -The s&i specimens collected in phosphate buffered saline UH 7.4 were kent at 4°C and examined bv Enzvme Linked Immunosorbent Assay (ELISAY within a week. The procedure followed was that of YOLKEN et al., 1977 with certain modifications as adopted by the N.I.H., USA (Yolken-personal communication). Each well of polyvinyl microtitration plate (Dvnatech Laboratories) was coated with 100 & of. diluted goat anti-rota serum (1 :lO,OOO in carbonate buffer DH 9 -6) and incubated at 4°C for 24 hours. The plate was then washed three times with phosphate buffered saline pH 7.4 containing ‘Tween 20’ at a concentration of O-5 ml/litre (PBS-Tween). 50 ~1 of a mixture of PBS-Tween containing 1 oA foetal calf serum (FCS) and 0.5% normal goat serum (GS) was added in each well and then 50 ~1 of stool suspension 2 to 10% diluted in PBS buker was added. The plate was incubated at 4°C overnight and washed three times with PBS-Tween. To each well 100 ~1 of guinea-pig anti-rota serum diluted 1:700 in PBS-Tweenz FCS-GS mixture was added and incubated at 37°C for one hour. The plate was washed three times with PBS-Tween and 100 ~1 of alkaline phosphatase labelled goat anti-guinea-pig serum diluted 1:400 in PBS-Tween-FCS-GS mixture
522
ROTAVIRUS
Table I-Distribution ality rates
of cases admitted
DIARRHOEA
EPIDEMIC
to hospital
IN
INDIA
during the two months with
their
case fat-
Number of casesadmitted to hospital Cumulative percentage
Deaths
Case fatality rate (%>
368
76.5
2
0.5
12-23
81
93.3
2
2.5
24-35
10
95.4
Nil
36-47
7
96.9
Nil
48-59
3
97.5
Nil
2 60”
12
100.0
Nil
481
100.0
4
Age groups (months) <12
Total
Admissions
-
0.8
*Between 5 and 13 years of age. Table II-Pattern of isolation during November, 1979 Enteropathogen isolate
of enteropathogens
in 59 paediatric
Number (N = 59)
diarrhoea
cases in Manipur Percentage
Rotavirus only Rotavirus + EPEC fo;avrus + Sh. flex2
45 7 Nil
76.3 11.9 1.7 -
Total
53
89.8
rSerotypes-044X74, aType 2.
086:B7,012:B16,0142:K86
was added, incubated at 37°C for one hour and was washed three times with PBS-Tween. Finally 100 ~(1 of p-nitrophenyl phosphate substrate (Sigma) at a concentration of 1 *O mg/ml in 10% diethanolamine buffer pH 9 08 was added and the plates incubated at room temperature until yellow colour appeared. The colour was matched with known weakly positive and negative controls. The positive controls consisted of Nebraska calf diarrhoea virus and the negative control was a rota-negative stool sample by electron microscopy. The unknown stool samples as well as the positive and negative controls were tested in duplicate. All positive samples were confirmed by blocking tests using bovine anti-rota serum. Reagents employed for ELISA, including blocking test and nositive control were kindly supplied by Dr. Kapikian of N.I.H., USA. Results 481 casesof acute diarrhoeas were admitted to the R.M.C. Hospital, Imphal, during OctoberNovember and four died, thereby giving an over-all casefatality rate of 0 ‘8% (Table I). In October 145 cases were admitted and 336 in November with
case fatality rates of 1.4% and 0*6%, respectively. As shown in Table I. 93.3% of these cases were less than two years old and, of these, 76.5% were aged below one year. The proportion of male to female cases was 1*9:1. Admissions of acute diarrhoea casesto R.M.C. Hospital, Imphal, started to increase from the last week of October and the peak incidence was noted during the week ending 13th November. Thereafter the epidemic gradually declined. No adult cases of gastro-enteritis were admitted to either the R.M.C. Hospital or to any of the Primary Health Centres during this period. The important clinical features included watery diarrhoeas (lOO*O%), vomiting (90x), fever 46.7 %), cervical lymphadenitis and pharyngeal erythema (43 -3%). Abdominal distension was noted in about 30.0% of cases. Clinically 95.0% of the children had mild to moderate degrees of dehydration, severe casesbeing only about 5 -0%. The mean duration of diarrhoea, as calculated from the onset of clinical symptoms till the stoppage of diarrhoea, was 68 hours, 93.3% of children were free from any clinical signs of malnutrition. 63 *3 0/0caseswere treated before admission with a wide range of anti-
P. G. SENGUPTA
biotics including streptomycin, tetracycline, chloramphenicol, neomycine and sulpha drugs either singly or in combination. All casesoccurred as a single casein their families. The percentage distribution of the affected families of 60 cases,studied by the team, showed that 46.7% of them used chlorinated tap water as their source of drinking water, compared to 40.0% and 13.3% who used pond and river water respectively. 45% of the families used open ground for defaecation and 33.3% used dug well, 16.7 sanitary and 5% service types of latrine. Most of the families (63.4%) had a monthly family income of below Rs.500/- per month. Rotavirus was detected in 53 of 59 stool samples examined by the ELISA technique thereby giving a detection rate of 89.8% (Table II). Enteropathogenic E. coli (EPEC) was isolated from seven stool samples which were also positive for rotavirus. A total of 29 strains of E. cali isolated from these specimens were tested for their enterotoxigenicity (both LT and ST) in rabbit ileal loop and suckling mice models. None of the strains were found to be toxigenic (ETEC). One sample yielded ShigeZZa flexneri type 2 in addition to rotavirus.
entities have been recurring every year in Manipur since 1969. Availability of a fresh group of susceptible population (children under one year) coupled with the favourable environmental factors that exist in the area during the winter months, probably sparked off recurrent epidemics every year. The exact cause of recurrence, the natural reservoir of rotavirus, the mode of transmission and the triggering environmental factors have yet to be determined. Cervical lymphadenitis and pharyngeal erythema with accompanying fever in a large proportion of cases may indicate involvement of the respiratory system as well. Acknowledgements
We thank both the Principal and the Superintendent of the Regional Medical College Hospital, Imphal for their active co-operation and support and Dr. I?. K. Singh, Director of Health, Government of Manipur, for his interest and advice. The excellent assistance offered by the Doctors in the Paediatric Ward of R.M.C. Hosoital and the laboratory staff of N.I.C.E.D., Cal&tta is also thankfully acknowledged.
Discussions
Rotaviruses have emerged as the most important viral agents aetiologically associatated with- severe diarrhoeal diseases in infants and children. In a number of hospital-based studies, carried out mostly in developed countries, rotaviruses have been detected in approximately 50% of infantile diarrhoea cases. However, there is little information about their importance and prevalence in the developing countries with tropical climates, where diarrhoeal diseasesrepresent one of the most important causes of morbidity and mortality among infants and younger children. Similarly, there is a lack of information regarding the natural history of the disease, e.g. the principal reservoir of human rotaviruses, their mode of transmission, seasonality, survival under different environmental conditions, etc. In the present, study rotaviruses have been detected in the faecal samples of 89 *8% of diarrhoeal children mostly under one year of age. It is most interesting to note that epidemics of similar clinical
523
et d.
References
Deb, B. C., De, S. I?., Singh, A., Singh, I?. K. & Pal, S. C. (1977). A massive outbreak of gastroenteritis amongst children in Manipur, 1973-74. Indian Journal of Medical Research, 65, 320-326.
Edwards, I?. R. & Ewing, W. H. (1972). Zdentificatian of Enterobacteriacea. (3rd. edit.) Minneapolis, USA. Burgess Publishing Co. Joint ICMR-GWB-WHO Cholera Study Group, Calcutta (1970). Cholera Carrier Studies in Calcutta, 1968. Bulletin of the World Health Organization,
43, 379-387.
Yolken, R. H., Kim, H. W., CIem, T., Wyatt, R. G., Kalica, A. R., Chanock, R. M. & Kapikian, A. Z. (1977a). Enzyme Linked Immunosorbent Assay (ELISA) for detection of human reovirus-like agent of infantile gastroenteritis. Lancet, ii, 263-266.
Accepted for publication
4th October, 1980.
OPTROPICALMEDICINE
An epidemic National
AND
of rotavirus
HYGIENE, VOL. 75, No. 4, 1981
diarrhoea
in Manipur,
India
I?. G. SENGUPTA, D. SEN, M. R. SAHA, S. NIYOGI, B. C. DEB, S. C. PAL Institute of Cholera and Enteric Diseases (I.C.M.R.), 3, Dr. M. Ishaque Road, Calcutta-700016, India IBETOMBI DEVI AND N. SUKUMAR SINGH
Regional Medical
College Hospital,
Imphal
and Directorate India
Summary Recurrent epidemics of acute diarrhoeal disease in young children have occurred during the winter months in Manipur (India) for many years. During November 1979, children admitted to hospital for acute diarrhoea were investigated and, in addition to pathogenic Escherichia coliand Shigella rotovirus was detected by ELISA in a number of stool samples, especially of those less than one year old. The importance of this finding and the need for further study are discussed. Introduction Recurrent epidemics of acutre diarrhoeas in children below two years of age have been reported from Manipur, a north-eastern state in India, since 1969. The epidemics started building up in an almost predictable manner from SeptemberOctober every year and usually subsided by January. The patients were mainly admitted to the Regional Medical College Hospital, Imphal, and durina the winter months about 80% of beds in the chyldren’s ward were occupied- by diarrhoeal children. The epidemics were investigated twice during 1974 (DEB et al., 1977) and 1976 when enteropathogenic Escherichia coli and Shigella were isolated from about 30% of diarrhoeal children. Facilities for detecting rotavirus were not available to the investigators. The present report describes the results of investigations of a similar epidemic of acute diarrhoeal diseases during November, 1979. Demographic features The state of Manipur comprises four hill districts and the central Imphal valley. 65% of the total population (1,073,OOO) live in the central valley and the population density was 48p er km2 (according to the 1971 census). The yearly rainfall amounts to 150-200 cm and the winter-is fairly severe. Parts of Imphal town have a supply of piped chlorinated water but this is intermittent and inadequate. The rest of the state depends on surface water for drinking and other domestic purposes. This surface water also becomes very scanty during winter months. The nightsoil disposal system is grossly inadequate in Imphal town and people use open land for defaecation.
of Health
Services, Government
of Manipur,
Methods Data for clinical and epidemiological investigations were collected from each case of gastroenteritis admitted at the Regional Medical College (R.M.C.) Hospital, Imphal and at Thoubal, Kakthing, Moirang and Bishenpur Primary Health Centres (l?.H.Cs). Microbiolorrical studies of 59 patients admitted to these hospitals during the period of investigation were included but 422 other in-patients in the hospitals could not be investigated microbiologically as their time of stay did not coincide with visits by the study team. Rectal swabs and catheter specimens of stools were collected in phosphate buffered saline from 51 cases of gastro-enteritis admitted to the paediatric ward of R.M.C. Hospital and eight children in hospital in Moirang and Thoubal P.H.C.s. Rectal swabs were also collected in Carry & Blair’s transport media and screened for the presence of Vibrio cholerae, V. parahaemolyticus, Shi,ella, Salmonella and E. -cola’ using standard laboratory techniques (Taint ICMR-GWB-WHO Cholera Studv Grouu. Calcutta, 1970; EDWARDS & EWING, 1972). -The s&i specimens collected in phosphate buffered saline UH 7.4 were kent at 4°C and examined bv Enzvme Linked Immunosorbent Assay (ELISAY within a week. The procedure followed was that of YOLKEN et al., 1977 with certain modifications as adopted by the N.I.H., USA (Yolken-personal communication). Each well of polyvinyl microtitration plate (Dvnatech Laboratories) was coated with 100 & of. diluted goat anti-rota serum (1 :lO,OOO in carbonate buffer DH 9 -6) and incubated at 4°C for 24 hours. The plate was then washed three times with phosphate buffered saline pH 7.4 containing ‘Tween 20’ at a concentration of O-5 ml/litre (PBS-Tween). 50 ~1 of a mixture of PBS-Tween containing 1 oA foetal calf serum (FCS) and 0.5% normal goat serum (GS) was added in each well and then 50 ~1 of stool suspension 2 to 10% diluted in PBS buker was added. The plate was incubated at 4°C overnight and washed three times with PBS-Tween. To each well 100 ~1 of guinea-pig anti-rota serum diluted 1:700 in PBS-Tweenz FCS-GS mixture was added and incubated at 37°C for one hour. The plate was washed three times with PBS-Tween and 100 ~1 of alkaline phosphatase labelled goat anti-guinea-pig serum diluted 1:400 in PBS-Tween-FCS-GS mixture
522
ROTAVIRUS
Table I-Distribution ality rates
of cases admitted
DIARRHOEA
EPIDEMIC
to hospital
IN
INDIA
during the two months with
their
case fat-
Number of casesadmitted to hospital Cumulative percentage
Deaths
Case fatality rate (%>
368
76.5
2
0.5
12-23
81
93.3
2
2.5
24-35
10
95.4
Nil
36-47
7
96.9
Nil
48-59
3
97.5
Nil
2 60”
12
100.0
Nil
481
100.0
4
Age groups (months) <12
Total
Admissions
-
0.8
*Between 5 and 13 years of age. Table II-Pattern of isolation during November, 1979 Enteropathogen isolate
of enteropathogens
in 59 paediatric
Number (N = 59)
diarrhoea
cases in Manipur Percentage
Rotavirus only Rotavirus + EPEC fo;avrus + Sh. flex2
45 7 Nil
76.3 11.9 1.7 -
Total
53
89.8
rSerotypes-044X74, aType 2.
086:B7,012:B16,0142:K86
was added, incubated at 37°C for one hour and was washed three times with PBS-Tween. Finally 100 ~(1 of p-nitrophenyl phosphate substrate (Sigma) at a concentration of 1 *O mg/ml in 10% diethanolamine buffer pH 9 08 was added and the plates incubated at room temperature until yellow colour appeared. The colour was matched with known weakly positive and negative controls. The positive controls consisted of Nebraska calf diarrhoea virus and the negative control was a rota-negative stool sample by electron microscopy. The unknown stool samples as well as the positive and negative controls were tested in duplicate. All positive samples were confirmed by blocking tests using bovine anti-rota serum. Reagents employed for ELISA, including blocking test and nositive control were kindly supplied by Dr. Kapikian of N.I.H., USA. Results 481 casesof acute diarrhoeas were admitted to the R.M.C. Hospital, Imphal, during OctoberNovember and four died, thereby giving an over-all casefatality rate of 0 ‘8% (Table I). In October 145 cases were admitted and 336 in November with
case fatality rates of 1.4% and 0*6%, respectively. As shown in Table I. 93.3% of these cases were less than two years old and, of these, 76.5% were aged below one year. The proportion of male to female cases was 1*9:1. Admissions of acute diarrhoea casesto R.M.C. Hospital, Imphal, started to increase from the last week of October and the peak incidence was noted during the week ending 13th November. Thereafter the epidemic gradually declined. No adult cases of gastro-enteritis were admitted to either the R.M.C. Hospital or to any of the Primary Health Centres during this period. The important clinical features included watery diarrhoeas (lOO*O%), vomiting (90x), fever 46.7 %), cervical lymphadenitis and pharyngeal erythema (43 -3%). Abdominal distension was noted in about 30.0% of cases. Clinically 95.0% of the children had mild to moderate degrees of dehydration, severe casesbeing only about 5 -0%. The mean duration of diarrhoea, as calculated from the onset of clinical symptoms till the stoppage of diarrhoea, was 68 hours, 93.3% of children were free from any clinical signs of malnutrition. 63 *3 0/0caseswere treated before admission with a wide range of anti-
P. G. SENGUPTA
biotics including streptomycin, tetracycline, chloramphenicol, neomycine and sulpha drugs either singly or in combination. All casesoccurred as a single casein their families. The percentage distribution of the affected families of 60 cases,studied by the team, showed that 46.7% of them used chlorinated tap water as their source of drinking water, compared to 40.0% and 13.3% who used pond and river water respectively. 45% of the families used open ground for defaecation and 33.3% used dug well, 16.7 sanitary and 5% service types of latrine. Most of the families (63.4%) had a monthly family income of below Rs.500/- per month. Rotavirus was detected in 53 of 59 stool samples examined by the ELISA technique thereby giving a detection rate of 89.8% (Table II). Enteropathogenic E. coli (EPEC) was isolated from seven stool samples which were also positive for rotavirus. A total of 29 strains of E. cali isolated from these specimens were tested for their enterotoxigenicity (both LT and ST) in rabbit ileal loop and suckling mice models. None of the strains were found to be toxigenic (ETEC). One sample yielded ShigeZZa flexneri type 2 in addition to rotavirus.
entities have been recurring every year in Manipur since 1969. Availability of a fresh group of susceptible population (children under one year) coupled with the favourable environmental factors that exist in the area during the winter months, probably sparked off recurrent epidemics every year. The exact cause of recurrence, the natural reservoir of rotavirus, the mode of transmission and the triggering environmental factors have yet to be determined. Cervical lymphadenitis and pharyngeal erythema with accompanying fever in a large proportion of cases may indicate involvement of the respiratory system as well. Acknowledgements
We thank both the Principal and the Superintendent of the Regional Medical College Hospital, Imphal for their active co-operation and support and Dr. I?. K. Singh, Director of Health, Government of Manipur, for his interest and advice. The excellent assistance offered by the Doctors in the Paediatric Ward of R.M.C. Hosoital and the laboratory staff of N.I.C.E.D., Cal&tta is also thankfully acknowledged.
Discussions
Rotaviruses have emerged as the most important viral agents aetiologically associatated with- severe diarrhoeal diseases in infants and children. In a number of hospital-based studies, carried out mostly in developed countries, rotaviruses have been detected in approximately 50% of infantile diarrhoea cases. However, there is little information about their importance and prevalence in the developing countries with tropical climates, where diarrhoeal diseasesrepresent one of the most important causes of morbidity and mortality among infants and younger children. Similarly, there is a lack of information regarding the natural history of the disease, e.g. the principal reservoir of human rotaviruses, their mode of transmission, seasonality, survival under different environmental conditions, etc. In the present, study rotaviruses have been detected in the faecal samples of 89 *8% of diarrhoeal children mostly under one year of age. It is most interesting to note that epidemics of similar clinical
523
et d.
References
Deb, B. C., De, S. I?., Singh, A., Singh, I?. K. & Pal, S. C. (1977). A massive outbreak of gastroenteritis amongst children in Manipur, 1973-74. Indian Journal of Medical Research, 65, 320-326.
Edwards, I?. R. & Ewing, W. H. (1972). Zdentificatian of Enterobacteriacea. (3rd. edit.) Minneapolis, USA. Burgess Publishing Co. Joint ICMR-GWB-WHO Cholera Study Group, Calcutta (1970). Cholera Carrier Studies in Calcutta, 1968. Bulletin of the World Health Organization,
43, 379-387.
Yolken, R. H., Kim, H. W., CIem, T., Wyatt, R. G., Kalica, A. R., Chanock, R. M. & Kapikian, A. Z. (1977a). Enzyme Linked Immunosorbent Assay (ELISA) for detection of human reovirus-like agent of infantile gastroenteritis. Lancet, ii, 263-266.
Accepted for publication
4th October, 1980.