Group A rotavirus-associated diarrhea in children seeking treatment in ...

Journal of Clinical Virology 40 (2007) 289–294

Group A rotavirus-associated diarrhea in children seeking treatment in Indonesia夽 Shannon D. Putnam a,∗ , Endang R. Sedyaningsih b , Erlin Listiyaningsih a , Sri Pandam Pulungsih c , Komalarini d , Yati Soenarto e , Octavianus Ch. Salim f , Decy Subekti a , Mark S. Riddle g , Timothy H. Burgess a , Patrick J. Blair a a

b

U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia National Institute of Health Research and Development, Indonesian Ministry of Health, Jakarta, Indonesia c Infectious Diseases Hospital Prof. Dr. Sulianti Saroso, Jakarta, Indonesia d Sumber Waras Hospital, Jakarta, Indonesia e Medical Faculty, Gadjah Mada University, Yogyakarta, Indonesia f Medical Faculty, Trisakti University, Jakarta, Indonesia g U.S. Naval Medical Research Center, Silver Springs, MD, USA Received 17 December 2006; received in revised form 11 September 2007; accepted 15 September 2007

Abstract Background: Globally, group A rotavirus causes significant morbidity and mortality among children. Limited data exist on the epidemiology of rotavirus disease among Indonesian children. Objectives: We describe the epidemiology of rotavirus-associated diarrhea among Indonesian children 0.8b p = 0.001d

Age (months) 0–5 6–11 12–23 24–35 36–47 48–60

108 (13.5%) 336 (42.0%) 232 (31.5%) 66 (8.2%) 21 (2.6%) 18 (2.3%)

205 (16.8%) 355 (29.1%) 421 (34.5%) 134 (11.0%) 63 (5.2%) 44 (3.6%)

p < 0.0001b

Hours of diarrhea pre-visit Hospitalized Hospitalization-days

48 (48–96) 654 (81.3%) 4 (3–6)

48 (48–72) 886 (72.2%) 5 (4–6)

p = 0.03d p < 0.0001a p = 0.1d

Dehydration Mild Moderate Severe

128 (16.2%) 369 (46.8%) 292 (37.0%)

484 (39.9%) 454 (37.5%) 272 (22.6%)

p < 0.0001b

Overall death Diarrhea-related deaths Vomit Fever Nausea Fatigue Chills Abdominal pain Bloody stool

0 (0.0%) 0 (0.0%) 576 (71.6%) 635 (79.1%) 355 (44.3%) 515 (64.1%) 39 (3.7%) 321 (26.2%) 10 (1.2%)

6 (0.6%) 3 (0.2%) 591 (48.2%) 907 (74.0%) 373 (30.4%) 606 (49.5%) 21 (3.9%) 285 (35.5%) 60 (4.9%)

p = 0.03e p = 0.2e 2.7 (2.2–3.2)f 1.3 (1.1–1.6)f 1.8 (1.5–2.2)f 1.8 (1.5–2.2)d 0.9 (0.5–1.5)f 1.6 (1.3–1.9)f 0.2 (0.1–0.5)f

a b c d e f

Either antigen or RNA detection. Pearson Chi-square test. IQR = interquartile range. Kruskal–Wallis test. Fisher’s exact test. Odds ratio (95% confidence interval).

fied DNA was stored at −70 ◦ C in storage buffer for future studies. Rotavirus positivity was defined as a positive test by either Rotaclone or rotavirus PCR positive. 2.4. Statistical analysis All data were double-data entered into MS Access (Microsoft Inc., Redmond WA). Categorical data were analyzed by the use of either Chi-square (expected cell frequency >5) or Fisher’s exact test (expected cell frequency ≤5). Continuous data were assessed for normality and if normally distributed, a parametric statistic was used (Student’s T-test, ANOVA). If the data were non-normally distributed, then non-parametric testing was used (Kruskal–Wallis). Data was imported into SAS v8.2 (SAS, Cary, NC), which was used for all statistical analyses. All statistical tests were two-tailed and significance was defined as p < 0.05.

3. Results Between February 2004 and 2005, 1660 study subjects ≤60 months of age provided a stool sample that was tested

for rotavirus. The median age of all study subjects was 11 months (interquartile range (IQR) = 7–18) and 59.9% of the participants were males. The overall rotavirus positivity was 45.5% (n = 755). There was a downward trend in rotavirus prevalence with increasing age: 13.5% among 0–≤6 months; 42.0% among 6–11 months; 31.5% among 12–23 months; 8.2% among 24–35; 2.6% among 36–47 and 2.3% among 48–60 months of age (test of trend, β = −0.13, p = 0.0007). Compared to children with non-rotavirus-associated diarrhea, children with rotavirus-associated diarrhea were younger (p = 0.01) and more likely to be hospitalized (p < 0.0001) (Table 1). Length of hospitalization and time of symptom onset prior to evaluation were similar between the two groups. Symptoms more commonly associated with rotavirus-associated diarrhea included vomiting (OR = 2.4, 95%CI = 1.9–3.0), fever (OR = 1.4, 95%CI = 1.1–1.8), nausea (OR = 1.4, 95%CI = 1.2–1.8), fatigue (OR = 1.5, 95%CI 1.2–1.8) and dehydration (p < 0.0001; test for trend, p < 0.0001). Presence of bloody stool was significantly less common in children with rotavirus-associated diarrhea (OR = 0.2, 95%CI = 0.09–0.4). Six children with diarrhea died, with three diarrhea-related deaths. However, no deaths

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S.D. Putnam et al. / Journal of Clinical Virology 40 (2007) 289–294

Table 2 Distribution of VP7 (G-type) and VP4 (P-type) genotypes including mixed infections from Indonesian children—2003–2004

G-type G1 G1 G2 G1 G4 G2 G2 G3 G2 G4 G3 G3 G4 G4 G4 G9 G8 G9 G[negative] G[non-typeable] P-type P[4] P[4] P[6] P[4] P[8] P[6] P[6] P[8] P[6] P[11] P[8] P[8] P[11] P[9] P[10] P[11] P[Negative] P[non-typeable]

Rotaclone positive (N = 748)

Rotaclone negative (N = 182)

Number

Percent

Number

84 4 1 130 1 2 12 2 17 346 1 69 26 53

11.2 0.5 0.1 17.4 0.1 0.3 1.6 0.3 2.3 46.3 0.1 9.2 3.5 7.1

120

16.2

2 84 1 2 183 22 5 10 4 155 153

0.3 11.3 0.1 0.3 24.7 3.0 0.7 1.4 0.5 20.9 20.7

Percent

4

2.2

4 1

2.2 0.6

13 136 24

7.1 74.7 13.2

1

0.6

1 1

0.6 0.6

160 8

93.6 4.7

occurred among the children with rotavirus-associated diarrhea. To assess the insensitivity of the antigen-based assay, we conducted RT-PCR on a random sample of Rotaclone negative specimens. We found that out of 182 samples, 46 (25.3%) were RT-PCR positive for a G-type and 11 (6.0%) were positive for a P-type (Table 2). P[8] was the most common P-genotype (19.6%) (Table 3) identified. Mixed infections were common within and across both G- and P-genotypes (Table 3), with the combination of G9P[8] the most common dual genotype identified (n = 151). Overall, the most common G-genotype identified in our study was G9 (45.8%) (Table 3). The identification of rotavirus over the course of the study demonstrated a biomodal seasonality, peaking near the end of the rainy season and again near the end of the dry season (Fig. 1).

4. Discussion This study found that rotavirus is a major cause of diarrhea among Indonesian children and provides epidemiological baseline data for rotavirus-associated diarrhea among

Table 3 G-types and P-types combinations among rotavirus positive specimens G-type

P-type

Frequency

Percent

G1 G1 G1 G1 G1 G1 G2 G2 G2 G2 G2 G2 G2 G3 G4 G4 G4 G4 G2 G9 G9 G9 G9 G9 G9 G9 G9 G4 G9 G4 G9 G4 G9 G4 G9 G4 G9 G4 G9 G4 G9 G4 GN GN GN GN GNT GNT GNT GNT GNT GNT

PN PNT P[10] P[4] P[6] P[8] PN PNT P[10] P[4] P[6] P[8] PN P[4] PN PNT P[8] PN PN PNT P[11] P[8] P[4] P[6] P[8] P[8] P[4] PN PNT P[11] P[11] P[8] P[4] P[6] P[8] P[9] PN PNT P[4] P[6] PN PNT P[10] P[4] P[6] P[8]

5 8 2 9 46 5 19 18 4 66 13 9 3 8 9 6 5 2 12 23 4 4 5 28 2 80 91 2 17 13 12 124 3 139 3 8 2 35 11 3 10 4 11

1.64 0.88 0.22 0.98 5.03 0.55 2.08 1.97 0.44 7.22 1.42 0.98 0.33 0.88 0.98 0.66 0.55 0.22 1.31 2.52 0.44 0.44 0.55 3.06 0.22 8.75 9.96 0.22 1.86 1.42 1.31 13.57 0.33 15.21 0.33 0.88 0.22 3.83 1.20 0.33 1.09 0.44 1.20

NT = not-typeable; N = negative. A single isolated was identified with the following genotypes: G1 P[11], G1 P[6] P[8], G1 G4 PNT, G2 P[11] P[6], G2 G1 PN, G2 G1 P[4], G2 G1 P[6], G2 G1 P[8], G3 P[10], G3 G2 P[4], G4 P[6], G4 G3 PN, G4 G3 PNT, G8 P[6], G9 P[9], G9 G4 P[11] P[6], GN P[11] P[8], GN P[8], GNT P[11], GNT P[9].

Indonesian children requiring healthcare. A bimodal seasonality pattern was discernable with the identification of rotavirus, but the reason behind the bimodal distribution remains unknown. The most common G- and P-genotypes identified were G9 and P[8]. Together, these genotypes composed the largest genotype group. The deployment and implementation of interventional programs to reduce morbidity and mortality associated with rotavirus infections in the developing world requires baseline clinical and epidemiological data to evaluate the efficacy of any intervention. In addition, such studies can provide an estimate of the burden


Fig. 1. Percent of monthly pediatric diarreha samples positive for rotavirus. Note: Rainy season (November–April); dry season (May–October).

of diarrheal disease, associated healthcare costs, and provide accurate estimates of pathogen-specific disease. Currently, there are two rotavirus vaccines available, a monovalent vaccine composed of G1 P[8] and a pentavalent vaccine composed of G1–G4 and P[8]. While historical data indicate that G1–G4 had accounted for up to 85% of all rotavirus infections, recent reports indicate that G9 is rapidly becoming predominant in many parts of the world (Kirkwood et al., 2004). In contrast, a study conducted in 2000–2001 at Gajah Mada hospital in Yogyakarta, Indonesia reported the most common genotypes as G1–G4 (Dr. Yati Soenarto, Personal Communication, 2005). A recently reported Australian outbreak documented a temporal displacement of rotavirus G1–G4 by the G9 genotype. A potential implication of this displacement could be the continued spread of the G9 genotype due to the limited immunity among the general population and the low efficacy of the recently introduced rotavirus vaccines against this genotype. Our data support this genotype displacement due to the large number of G9 rotavirus identified. In addition, G9 was commonly associated with G4 and P[8]. If there is a significant degree of cross-protection, then there should be an overall reduction in the morbidity and mortality associated with rotavirus disease. If however, there is no crossprotection, genotype-specific vaccines may actually facilitate the spread of G9, and other non-vaccine genotypes (Cubitt et al., 2000; Iturriza-Gomara et al., 2000; Linhares et al., 2006). 4.1. Study limitations A major limitation of this study was the cross-sectional design, in which we were not able to estimate the incidence of disease. Although we had a full year of data, we were only able to estimate the prevalence of rotavirus and specific genotypes to that time frame, addition years of data will provide a broader picture of rotavirus dynamics. We were unable to

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conduct any follow-up on any of the study subjects, possibly underestimated mortality since very ill children may have been removed from the hospital by their parents. This may account for the failure to detect any rotavirus-associated diarrhea deaths in our study. Moreover, some symptoms are hard to measure in infants and young children and must be provided by a caretaker. Another limitation of this study was the use of a rotavirus antigen detection immunoassay to screen all stool samples for the presence of rotavirus. It is likely that the antigen detection method underestimated the true prevalence of rotavirus due to insensitivity. Studies have shown that RTPCR is a much more sensitive detection method, but may not be cost-effective for clinical laboratories. However, RT-PCR, especially when stool is the source specimen, can be affected by unknown factors that interfere with amplification. We addressed the possible underestimation of the antigen detection issue by conducting RT-PCR testing on a random sample of Rotaclone-negative specimens, in which several positive results were noted. This may reflect asymptomatic shedding of low copy number of virus or rotavirus with a VP6 antigen that cannot be detected by Rotaclone. Another possibility is continual asymptomatic shedding of rotavirus in the population and within our sample, possibility due to the advanced age of the control group. This issue requires further study to elucidate the exact mechanisms of asymptomatic shedding of rotavirus.

Conﬂict of interest No conflicts of interest exist among any of the authors.

Acknowledgments The authors wish to thank Dr. Joe Bresee (U.S. CDC, Atlanta, GA) and Dr. Thomas Wierzba for their detailed review and editing of this manuscript. In addition, the authors wish to thank all hospital and laboratory staff, both at NAMRU2 and GOI-NIHRD, who contributed to the execution of this study.

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