Incidence and risk factors of paediatric rotavirus ... - Wiley Online Library

Tropical Medicine and International Health volume 8 no 9 pp 840–846 september 2003

Incidence and risk factors of paediatric rotavirus diarrhoea in northern Ghana The Navrongo Rotavirus Research Group1 Navrongo Health Research Centre, Navrongo, Ghana

Summary

We measured the type-specific incidence of paediatric rotavirus diarrhoea in an area of northern Ghana. Over 1 year, diarrhoea 1717 episodes were identified, of which 677 (39%) were positive for rotavirus. Risk factors for rotavirus infection included old age, wasting, high Vesikari score and the episode occurring in the dry season. Rotavirus-positive episodes tended to be more acute, causing vomiting and greater dehydration, and were more likely to require hospitalization. The incidence was 0.089 episodes per personyear for all diarrhoea, and 0.035 for rotavirus diarrhoea. The observed incidence decreased markedly with distance from the nearest health centre, suggesting a large unobserved burden. G2P[6], G3P[4] and G9P[8] made up more than half the genotypes detected, but the remainder were diverse. There is a large burden of rotavirus diarrhoea, but the effectiveness of future vaccines could be diluted by the high polymorphism of the virus, and the difficulty of reaching remote populations. keywords rotavirus, diarrhoea, incidence, risk factors, burden, Ghana

Introduction Diarrhoea is a major cause of paediatric morbidity and mortality, causing approximately 3 million deaths per year (Bern et al. 1992; Murray & Lopez 1997), around 20% of which are caused by rotavirus (de Zoysa & Feachem 1985). Rotaviruses are members of the Reoviridae and have a genome that consists of 11 segments of dsRNA (Estes 2001). They are triple-layered particles with the middle and outer layers comprising VP6, VP7 and VP4, respectively. They are classified into groups, subgroups and G and P types according to reactivities of epitopes on the VP6, VP7 and VP4 proteins or nucleic acid sequence variability within the genes encoding these proteins (Kapikian et al. 2001). Both VP7 and VP4 elicit neutralizing antibodies and may confer limited cross-protection against other G and P types (Kapikian et al. 2001). The diversity of rotaviruses is associated with their ability to re-assort upon dual infection of a single cell (Ramig 1997). This results in diverse rotavirus types, including re-assortants derived from animal and human rotavirus strains, cocirculating in the population at any one time within the same geographical area (Iturriza-Go´mara et al. 2001). Studies determining the distribution of rotavirus G and P types circulating within Africa indicated both a diverse population of cocirculating types and co-infection with more than one type, the prerequisites for re-assort1

Authors listed at end of paper.

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ment. Rotavirus G1P (Steele 2000) was the most common type found overall but G3P (Ramig 1997) was the most common type in West Africa (Steele 2000). In addition, the range of different G and P types indicated that novel viruses may have originated through re-assortment in the past. Vaccines have the potential for greatly reducing this burden of disease, despite the recent withdrawal of the rhesus re-assortment tetravalent vaccine (RRTV) (de Zoysa & Feachem 1985; Bresee et al. 1999; Coffin 2000; Murphy et al. 2001). Parallel to an immunogenicity trial of this vaccine in northern Ghana, a surveillance study was conducted to estimate the incidence and risk factors for rotavirus diarrhoea, in order to assess the potential benefits of vaccination, and guide the design of efficacy trials. Materials and methods The project was approved by the ethics committees of the London School of Hygiene and Tropical Medicine and the Ministry of Health, Ghana. Study site The study was conducted in Kassena-Nankana District (KND) of northern Ghana (Figure 1). This is one of the 110 districts in Ghana and lies in the Guinea Savannah belt between 1030¢ and 1100¢ N, and 1.00¢ and 1030¢ W. It borders Burkina Faso to the north. KND has an area of about 1675 km2 and a population of 140 000, living in

ª 2003 Blackwell Publishing Ltd

Tropical Medicine and International Health

volume 8 no 9 pp 840–846 september 2003

The Navrongo Rotavirus Research Group Rotavirus diarrhoea in northern Ghana

N • Paga health centre • KNE health centre • War Memorial Hospital, ˚ health centre Navrongo Chiana ˚ Biu health centre Roads Health facility in study Other health facility Boundary of KassenaNankana district

• ˚

10

0

10

centres (KNE), which caused sample collection to be halted from 3 to 8 August 1999 inclusive. A standard form was used to collect information on histories of illness and vaccination, and on anthropometry. Height was measured using a length board, and weight using an electronic weighing scale (Soehnle, CMS Weighing Equipment Ltd, London, UK). To permit calculation of the Vesikari score as a measure of diarrhoea severity (Ruuska & Vesikari 1990), information was collected on: duration of diarrhoea, peak number of stools passed per day, duration of vomiting, peak frequency of vomiting per day, degree of fever, presence and severity of dehydration and treatment.

20 km

Figure 1 Map of the study area.

roughly 13 000 dispersed compounds, plus the town of Navrongo which has a population of about 20 000. The Navrongo Demographic Surveillance System (NDSS) monitors population dynamics in the area (Binka et al. 1999). Each compound is visited by an NDSS fieldworker once every 90 days to register events such as pregnancies, births, deaths, and in- and out-migrations. The main occupation of the people is subsistence farming, predominantly of millet and livestock. KND has a mean monthly temperature range of 20–45 C and rainfall averages 800–1000 mm per annum, occurring mainly between May and October. Diarrhoea surveillance The study was based at three health facilities: the Paga and Kassena-Nankana East (KNE) health centres, and War Memorial Hospital in Navrongo (Figure 1). Trained field staff attached to these three health facilities screened mothers and other carers by asking whether their children had diarrhoea and, if so, the number of stools. Children were eligible for inclusion in the study if they were aged no more than 24 months and had passed three or more loose stools per day, on any of the previous 3 days. Most such children are resident in the district, although some are brought from outside, including Burkina Faso. Children whose carers reported them as living in KND were traced to find their identifying number in the NDSS (Binka et al. 1999). Treatment given included oral rehydration salts (either administered under observation at the clinic, or given to the mother for home use), antibiotics and antipyretics. If necessary, children were referred to the district hospital for possible admission. Children were not followed up for a mortality endpoint. Fieldwork lasted from August 1998 to July 2000. There was a short outbreak of cholera in the vicinity of one of the health


Laboratory procedures A stool sample was taken if possible, otherwise a rectal swab was performed. These were transported on ice to Accra for virus identification. For detection of rotavirus by enzyme-linked immunosorbent assay (ELISA), a 10% suspension of stool was made in phosphate-buffered saline (PBS) pH 7.2 and used for group A antigen detection, using the DAKO IDEIA rotavirus ELISA kit (Dako Diagnostics Ltd, Cambridgeshire, UK) as per the manufacturer’s instructions. Rectal swabs were immersed in 1 ml PBS, allowed to soak for 30 min and suspensions were expressed from the cotton swab. This was repeated and the two washings were pooled for the assay. The tests were read both visually and spectroscopically at a wavelength of 450 nm. Each plate included a negative and positive control and all tests were performed in duplicate. The dsRNA genome was extracted from all ELISApositive specimens by the phenol/chloroform method and layered on a 10% polyacrylamide vertical slab gel followed by electrophoresis overnight at 100 V using the discontinuous buffer system. Gels were silver stained and a sample was regarded as rotavirus RNA-positive if patterns typical of rotavirus electropherotypes were seen. All polyacrylamide gel electrophoresis (PAGE)-positive samples with sufficient viral RNA were subjected to VP7 (G-) and VP4 (P-) typing by RT-PCR. For rotavirus genotyping, RNA was extracted from 100 ll of 10% faecal suspensions in PBS with guanidinium isothiocyanate and silica (Boom et al. 1990) and eluted in 26 ll of RNase-free sterile distilled water containing 40 units of ribonuclease inhibitor (RNAsin, Promega, Madison, WI, USA). The extracted RNA was used to generate cDNA for subsequent amplification after a reverse transcription assay with random primers (hexamers) or by specific priming using MuLV reverse transcriptase (Life Technologies, Gaitherburg, MD, USA) (IturrizaGo´mara et al. 1999). 841




G-typing was performed using a semi-nested PCR and adapted from the method of Gouvea et al. (1990). The VP7 was amplified by PCR with the Beg9 and End9 primers. The second round typing PCR was a multiplex PCR using standard protocols (WHO Rotavirus Workshop Manual, Pretoria, South Africa, 1999). This incorporated the primers RVG9, and aBT1, aCT2, aET3, aDT4, aAT8 and aFT9, which are specific for G-types 1, 2, 3, 4, 8 and 9, respectively. All amplified products were examined by gel electrophoresis in 2% agarose gels (Seakem, Flowgen, Leicestershire, UK) containing 4 mg/ml ethidium bromide under standard conditions. P-typing was performed using a semi-nested PCR adapted from the method of Gentsch et al. (1992). The first round PCR amplified an 876 bp fragment of the gene 4 of group A rotaviruses, using the consensus primers Con2 and Con3. The second round typing PCR incorporated Con3 as well as 1T-1, 2T-1, 3T-1, 4T-1 and 5T-1, which are specific for types P (Steele 2000), P (Estes 2001), P (Ramig 1997), P (Bresee et al. 1999) and P (Coffin 2000), respectively. The PCR reaction mix for the first and second round amplification was the same as that for the G-typing except for the primer concentrations (1 lm Con2 and Con3 for the first round, and 2 lm of each for the second). Forty PCR cycles were performed with annealing performed at 50 C for 1 min. The second round PCR cycle was reduced to 25 cycles. PCR products were examined as described above. Data management and statistical analysis The forms were double-entered into a FoxPro database in Navrongo. Logistic regression was used to analyse factors associated with rotavirus infection. As some children had more than one episode, the Huber–White method was used to adjust the standard errors to allow for the lack of independence of such episodes (Carlin et al. 1999). However, as the number of episodes per child was only 1.2, the results were similar to those from an unadjusted logistic regression. Analysis was performed with STATA version 7 (Stata Corporation, College Station, TX, USA) and S-PLUS version 3.3 (Statistical Sciences Ltd, Oxford, UK). Results A total of 2085 diarrhoea episodes were recorded at the three centres, of which 1717 were experienced by children in the NDSS database. Analysis will be restricted to these 1717 episodes, which occurred in 1448 children as shown in Table 1. The distribution of diarrhoea episodes, and the proportion rotavirus-positive, are shown in Table 2. Overall, 677 of the 1717 episodes (39%) were positive for group A 842

Table 1 Diarrhoea episodes in children 24 months of age in the Kassena-Nankana District (1998–1999)

Diarrhoea episodes

Detected cases* (total cases ¼ 1448) [no. (%)]

1 2 3 4 5 6

1218 198 27 4 0 1

(84.1) (13.6) (1.9) (0.2) (0.0) (0.0)

* There were 1717 episodes in 1448 children.

rotavirus by ELISA. The number of diarrhoea episodes increased with age within the first year [(87 of 1717), or 5%, of episodes were in 0–2-month olds, but 19% (331 of 1717) in 9–11-month olds), then decreased in the second year [41% (701 of 1717) for the whole 12–24-month range]. There was a tendency for the proportion positive for rotavirus to increase with age. There were fewer episodes in females than males, but the proportions of these, positive for rotavirus were similar between the sexes. More than a quarter (28%) of episodes were in children who were wasted (more than two standard deviations below the NCHS median weight for height), and these were more likely to be positive for rotavirus. More than half (54%) of the diarrhoea episodes were moderate or severe (Vesikari score 9 or more), and severity was associated with the presence of rotavirus. Multiple regression showed that this relationship was stronger in the dry season than in the wet season. Rotaviruses were detected less frequently from stools of children with diarrhoea brought in from more than 5 km. There was a very strong association between detection of rotavirus in stools of children with diarrhoea and season, with more than 50% of the dry season diarrhoea episodes being positive for rotaviruses compared with )2) Wasted (Z < )2) Mild (0–8 points) Moderate (9–14 points) Severe (>15 points) 5 Dry

658 432 1109

278 148 592

42 34 53

1.10 (0.88–1.37) 0.78 (0.61–1.01) 1.0

1.07 (0.84–1.37) 0.69 (0.53–0.92)

608

85

14

0.14 (0.1–0.18)

0.20 (0.14–0.29)à

Wet

Multivariable

(0.59–1.60) (0.71–1.93) (0.77–2.07) (0.78–2.00)

1.04 1.33 1.80 1.53

(0.61–1.77) (0.78–2.26) (1.05–3.07) (0.92–2.53)

(0.80–1.18)

1.01 (0.81–1.26)

(1.12–1.71)

1.33 (1.03–1.71)

(1.51–2.27) (1.58–3.82)

1.87 (1.45–2.41) 2.61 (1.51–4.51)

The relationship between Vesikari grade and ELISA differed between wet and dry seasons. The figures quoted in the table are for the dry season. In the wet season, the odds ratios (and 95% confidence interval) were 0.93 (0.58–1.49) for moderate grade (relative to mild), and 0.28 (0.04–2.14) for severe. à As noted above, there was an interaction between season and Vesikari grade. The odds ratio in the table is for mild Vesikari grade. For moderate grade, the odds ratio (95% confidence interval) was 0.10 (0.07–0.15) comparing wet vs. dry season. For severe, it was 0.02 (0.0003–0.17).

distance from the nearest health facility. Figure 3 shows that attendance with diarrhoea (whether all-cause or with rotavirus) declined strongly with distance. This suggests that a large number of diarrhoea cases are not seen by health personnel. Could this unobserved burden of rotavirus disease potentially be counteracted by a rotavirus vaccine delivered through the EPI? To try to address this question, Figure 3 also shows the number of children who had received all three doses of DPT (diphtheria, pertussis and tetanus) vaccine, as a proportion of eligible clinic attendees. Vaccine coverage also generally decreases with distance, although much less sharply than rotavirus incidence, and it seems reasonable to assume that the population coverage is less than this. Therefore, even a totally efficacious vaccine would still leave many children unprotected in remote locations. Table 4 shows the distribution of group A rotavirus strains, as defined by their G- and P-types, and how this changes over time. More than one strain was detected in samples on four occasions. Thirteen different G–P combinations were identified. G2P (Ramig 1997), G3P (Estes 2001) and G9P (Steele 2000) strains constituted 54% of all typed rotaviruses. In the first rotavirus peak in the study –


corresponding to the fourth quarter of 1998 and the first of 1999 – G2P6 and G3P4 constituted 65% of strains. In the second peak, the predominant strain switched to G9P8, which constituted 40% of strains. Discussion As found elsewhere (Cunliffe et al. 1998; Van Man et al. 2001), a large proportion (39%) of diarrhoea episodes seen at a health facility were rotavirus-positive. This proportion increased from birth to 1 year of age, levelling off in the second year. The episodes with more severe Vesikari score were more likely to be rotavirus positive. As found elsewhere (Cunliffe et al. 1998), rotavirus occurred mainly in the dry season, when it was present in most episodes. Peaks of diarrhoea incidence from other causes did occur outside the dry seasons. Previous data from West Africa indicated that G3P (Ramig 1997) was the predominant strain circulating between 1996 and 1999 (Steele 2000). In Ghana, the incidence of infection with G3P (Ramig 1997) declined throughout the study and G9P (Steele 2000) was the predominant type in 1999 and 2000. This mirrors the 843




Table 3 Association between rotavirus status and the components of the Vesikari score Rotavirus positive

Diarrhoea duration in days (points)

Maximum number of stools/24 h (points)

Duration of vomiting in days (points)

Maximum number of vomiting episodes/24 h (points)

Temperature in C (points)

Dehydration (points)

Treatment (points)

No.

+

%

Odds ratio for being enzyme linked immunosorbent assaypositive (95% confidence interval)

7.5

Distance from nearest health centre (km)

Figure 3 Diarrhoea incidence as a function of distance (in kilometres) from the nearest health centre, using the Navrongo Demographic Surveillance System to obtain the population denominator. Also shown is diphtheria, pertussis and tetanus vaccine coverage, using clinic attendees as a denominator.





Table 4 Rotavirus genotypes (Kassena Nakana District, 1998–1999)

Rotavirus G/P type

1998

Number detected

Q3

Q4

G1p[6] G1p[8] G2p[4] G2p[6] G2p[8] G3p[4] G3p[6] G3p[8] G8p[6] G8P[8] G9p[6] G9p[8] G9p[10] Mixed G/P type G)P/GP[)]à Untypable

– – – – – – – – – – – – – – – –

– – – – – 10 2 – – – – – – – –

Total

2

12

Q1

2000

Q2

Q3

2 31 2 17 3 – 5 – – – – 2 4 7

– – – 1 – – – – 1 – – – – – – 1

– – – – – – – – – – – – – – – –

5 4 49 2 2 4 20

77

3

–

99

4 –

Q4 2 3 –

Q1

Q2

Q3 – – – –

– – 14 5

– – – – – – – – – – – – – – – –

– – – – – – – – – – –

7 3 2 43 9 27 6 1 6 5 8 58 2 4 22 35

45

–

–

238

1 – –

3 3 – 1 1 –

8 4 – – – – – 4 9

Total

Multiple G-types and multiple P-types were detected in the same sample: each of these was counted as two genotypes. à Typed for either the G- or the P-type, but nothing detected for the other.

types and the potential for new strains to emerge through re-assortment. The significant numbers of incompletely or untyped strains may indicate genetic drift or shift, leading to the generation of strains untypable through the accumulation of point mutations or the lack of specific oligonucleotide primers for amplifying novel strains (Adah et al. 1997; Iturriza-Go´mara et al. 2000b). The study demonstrated the great diversity of rotavirus strains circulating in rural Ghana. Although three strains – G2P6, G3P4 and G9P8 – constituted 53% of strains typed, 13 distinct strains were identified on the basis of G–P combination. A further 24% of rotavirus positive specimens were untypeable or could only be incompletely typed. This is in line with other studies in rural Africa (Cunliffe et al. 1998). Only 41% of strains identified contained G types 1–4 which constitute the RRTV vaccine. This is in marked contrast to the studies in developed countries where more that 95% of strains contains G type 1–4 (Gentsch et al. 1992; Iturriza-Go´mara et al. 2000a). This may have implications for developing an appropriate vaccine strategy to protect against this diversity of strains. Heterotypic protection is generated following rotavirus infection, but the precise correlate of protection and the scope of heterotypic protection is not fully defined (Jiang et al. 1999) and should be a priority for future research (Cunliffe et al. 2002). The high incidence of rotavirus disease highlights the potential health benefit of an effective rotavirus vaccine. However, our data suggest that it may be difficult to achieve a high coverage in areas distant from health facilities.


Acknowledgements We are grateful to M. Adjuik and F. Kondayire for assistance with the geographical information system (GIS). Financial support was provided by the United Kingdom Department for International Development, project number R7232. References Adah MI, Rohwedder A, Olaleyle OD & Werchau H (1997) Nigerian rotavirus serotype G8 could not be typed by PCR due to nucleotide mutation at the 3¢ end of the primer binding site. Archives of Virology 142, 1881–1887. Bern C, Martines J, de Zoysa I & Glass RI (1992) The magnitude of the global problem of diarrhoeal disease: a ten-year update. Bulletin of the World Health Organization 70, 705–714. Binka FN, Ngom P, Phillips JF, Adazu K & MacLeod BB (1999) Assessing population dynamics in a rural African society: the Navrongo Demographic Surveillance System. Journal of Biosocial Sciences 31, 375–391. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM & van der Noordaa J (1990) Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28, 495–503. Bresee JS, Glass RI, Ivanoff B & Gentsch JR (1999) Current status and future priorities for rotavirus vaccine development, evaluation and implementation in developing countries. Vaccine 17, 2207–2222.

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Authors Professor Fred N. Binka, School of Public Health, University of Ghana, PO Box 13, Legon, Ghana. E-mail: [email protected] (corresponding author). Mr Francis K. Anto, Dr Abraham R. Oduro, Ms Elizabeth A. Awini and Dr Alex K. Nazzar, Navrongo Health Research Centre, PO Box 114, Navrongo, Upper East Region, Ghana. E-mail: [email protected], [email protected], [email protected], [email protected] Dr George E. Armah and Mr Richard H. Asmah, Noguchi Memorial Institute for Medical Research, PO Box 25, Legon, Ghana. E-mail: [email protected], [email protected] Prof. Andrew J. Hall, Prof. Felicity Cutts and Dr Neal Alexander, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK. E-mail: [email protected], [email protected], [email protected] Dr David Brown and Dr Jon Green, Central Public Health Laboratory, 61 Colindale Avenue, London, NW9 5HT, UK. E-mail: [email protected], [email protected] Dr Jim Gray and Dr Miren Iturriza-Go´mara, Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QW, UK. E-mail: [email protected], [email protected]

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