A planned comparison of the sensitivity and specificity of ELISA and RT–PCR for VP6 resulted in the identification of a high rate of asymptomatic infections in the birth cohort under follow-up, during the period May–June 2003. The mean age of the cohort at this time was 6 months (s.d.=3·8 months). Of the 371 children who contributed to the period May–June 2003, 118 (31·8%) had been previously infected with rotavirus. Of a total of 1191 non-diarrhoeal samples from 371 children collected over the 2-month period of May–June 2003, 22 (1·9%) were positive by ELISA and of these, 362 samples were tested by RT–PCR and 147 (40·6%) were positive (). This peak in PCR detection in asymptomatic samples was confined to a 2-week period at the end of May and in early June, since subsequent samples from the same children were negative. During this 2-week period, there was a significantly higher detection of positive samples (127 PCR positives/236 PCR tested, 53·8%) (P<0·001) than during the period of May–June 2003, excluding the 2-week period (20 PCR positive/126 PCR tested, 15·9%). The detection rate by PCR was also higher (P<0·001) than in the previous follow-up period from the beginning of enrolment of the birth cohort for samples tested by PCR (119 PCR positive/585 PCR tested, 20·3%, from 1 April 2002 to 26 May 2003).
Fig. 1 Testing of non-diarrhoeal samples for rotavirus by reverse transcription–polymerase chain reaction (RT–PCR) for the VP6 gene in a birth cohort under surveillance (——). The numbers of samples negative (□) and positive (more ...)
Genotyping of the VP7 gene was attempted for 50 samples in order to determine whether this was an outbreak due to a single strain, and VP7 types could determined for 29 samples. In 19 surveillance samples, a single G type was identified and in 10, there were multiple G types indicating mixed infection. The G types identified were G1 (n=25), G2 (n=7), G4 (n=1), G9 (n=4) and G10 (n=7). The VP4 types were P and P and P, the latter was found only in association with G10 strains. The proportion of multiple infections was significantly higher (P<0·001) in this 2-week period than in the period up to 26 May 2003, during when 66 of the 119 samples positive by VP6 PCR had G or P type determined and only 1·5% had multiple G or P types.
A total of 6/28 (21·4%) of surveillance samples tested for noroviruses during the period 27 May to 10 June 2003 were positive. Of these, two samples were also positive for rotaviruses. Other enteric pathogens were not tested in the surveillance samples.
During May–June 2003, there were 166 diarrhoeal samples of which three (1·8%) were positive for rotavirus antigen by EIA, and 34 (20·5%) were positive by VP6 PCR, this represented no difference in the 20·3% positivity by PCR that had been seen in diarrhoeal samples from the community tested from the start of follow-up for the birth cohort, where one of 10 diarrhoeal samples examined for noroviruses was positive. Bacteria identified in diarrhoeal samples included Salmonella C1 (n=1), Vibrio cholerae O1 (n=1), non-O1 vibrios (n=3), and Aeromonas (n=6).
Of the 70 diarrhoeal samples from the period 27 May to 10 June 2003, mixed enteric infections were found in 6·3% and of the 670 diarrhoeal samples from the period of follow-up prior to 27 May 2003, 3·5% had mixed infections (P=0·5).
There was a marked increase in the number of Cryptosporidium
-associated cases of diarrhoea in the cohort in the period June–August 2003, with 14 infections during this period, whereas only six infections were documented in the period March 2002–May 2003 and seven infections during September 2003–March 2004. Earlier hospital-based data do not indicate any seasonality of cryptosporidial (data not shown) or rotaviral [14
] infections in Vellore.
When the age of infected children in the cohort was studied, it was found that during the period of study, the median age of children with diarrhoea was 3·6 months, while the median age of children providing surveillance or asymptomatic samples was 6·3 months (Mann–Whitney U test P=0·0002). A similar pattern was seen in children infected with rotavirus where rotavirus diarrhoea was seen at a median age of 3·5 months but asymptomatic rotavirus infections were seen at a median age of 5·5 months (Mann–Whitney U test P=0·0004) as shown in .
Fig. 2 The distribution of samples from asymptomatic (- - -) and symptomatic (——) children of different ages in a birth cohort tested for rotavirus by reverse transcription–polymerase chain reaction for the VP6 gene during the period (more ...)
In order to identify a possible cause for the increased incidence of enteric pathogen shedding by the children, the water supply to the area was investigated. Most people in the area utilized the municipal water for drinking, with other water sources reserved for washing. The municipal water supply to the study families was from two main overhead tanks, Ramnaickanpalayam and Kaspa, which supply domestic and public stand pipes. Water supply is intermittent () and during the 92-day period from May to July 2003, water was supplied a total of five times. Since the increased enteric pathogen positivity began after the third week of May 2003, it is interesting to note that water was pumped to the two areas on 21 and 24 May 2003.
Drinking water supply to study areas
Additional data on disease due to other potential waterborne pathogens was collected at the referral hospital and showed no increase in numbers or proportion positive among samples tested for cholera during this time compared to the same months in the previous 3 years, but there were increases in cases of Cryptosporidium diarrhoea during June–August 2003 and hepatitis E cases during the following month from Vellore town (individual data not shown).