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Nitric oxide production in acute gastroenteritis in Indian children
Transactions of the Royal Society of Tropical Medicine and Hygiene (2009) 103, 849—851
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/trstmh
SHORT COMMUNICATION
Nitric oxide production in acute gastroenteritis in Indian children Thuppal Varadachari Sowmyanarayanan a, Sathish Kumar Natarajan a, Anup Ramachandran a, Rajiv Sarkar a, Prabhakar Devarajan Moses b, Anna Simon b, Indira Agarwal b, Solomon Christopher c, Gagandeep Kang a,∗ a
Department of Gastrointestinal Sciences, Christian Medical College, Vellore 632004, Tamil Nadu, India Departments of Child Health Units I, II and III, Christian Medical College, Vellore 632004, Tamil Nadu, India c Department of Biostatistics, Christian Medical College, Vellore 632002, Tamil Nadu, India b
Received 7 April 2009; received in revised form 7 May 2009; accepted 7 May 2009 Available online 23 June 2009
KEYWORDS Acute gastroenteritis; Nitric oxide; Rotavirus; Intestinal permeability; Children; India
Summary This study compared nitric oxide (NO) levels in 110 children with, and 110 children without, infectious gastroenteritis. Post-infection intestinal function was assessed in a subset. At least one pathogen was identified in 47.2% of cases. The most common diarrhoeal pathogens were rotavirus (22.7%) and norovirus genogroup II (11.8%). The levels of NO measured by median urinary nitrite:creatinine ratio were significantly higher in children with diarrhoea [23.6; interquartile range (IQR) 12.3—46.7] than without diarrhoea (7.8; IQR 4.1—13.2), P < 0.001. The ratio was not significantly different between diarrhoeal cases with and without pathogens (P = 0.148). Six of twelve children tested had intestinal dysfunction. © 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Nitric oxide (NO) is an inflammatory mediator synthesized through the L-arginine-NO pathway by the NO synthase enzymes. The inducible isoform (iNOS) is activated during acute infection and inflammation. A number of recent studies have documented increased levels of NO during infectious diarrhoea, particularly with rotavirus and
Cryptosporidium, suggesting a pathophysiological role for NO during acute infection.1,2 We investigated paediatric diarrhoeal illness presenting to the hospital of the Christian Medical College in southern India, to identify the aetiology of diarrhoea and measure the levels of NO in children with infectious gastroenteritis and controls, particularly with regard to severity of disease.
2. Materials and methods ∗ Corresponding author. Tel.: +91 416 228 2052; fax: +91 416 228 2486. E-mail address: [email protected] (G. Kang).
Children <5 years presenting with acute gastroenteritis requiring hospitalization for rehydration were enrolled from January 2006 to December 2007. Disease severity
0035-9203/$ — see front matter © 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2009.05.003
850
T.V. Sowmyanarayanan et al.
was assessed using a 20-point scoring system.3 For each child with diarrhoea, an age-matched healthy control was enrolled from an urban slum in the same town. Stool was tested for ova and parasites by microscopy; culture for Salmonella, Shigella, Campylobacter, Aeromonas and enteropathogenic Escherichia coli; ELISA for group A rotavirus and PCR for norovirus genogroup II. NO was measured in the first urine post-admission from cases and early morning urine from controls using a copper-cadmium alloy coupled with Griess reaction.4 Urine was collected in isopropanol to restrict the degradation of urinary nitrates by bacteria. NO levels were expressed as a function of urinary nitrite and creatinine (NO:Cr). Nitrite levels were estimated in the water from paediatric wards and the community where the controls were selected. D-xylose absorption tests were done 4 weeks post-infection to study intestinal permeability in a subset of 12 children with acute gastroenteritis for whom specific pathogens were identified in stool. The Mann-Whitney U test was performed to determine the significance of differences observed between cases and controls.
3. Results During the study, 757 children were admitted with diarrhoea, 110 of whom were enrolled and age matched with 110 controls recruited from an urban slum. Stool pathogens were identified in 52 cases (47.2%), with rotavirus (22.7%), norovirus genogroup II (11.8%) and Shigella spp. (4.5%) being the most common pathogens. The median NO:Cr ratio in children with diarrhoea [23.6; interquartile range (IQR) 12.3—46.7] was significantly higher than children without diarrhoea (7.8; IQR 4.1—13.2) (P < 0.001) (Figure 1). Levels of nitrites in the water from the paediatric wards and the community showed minimal and similar amounts of nitrites, thus not affecting measurements. There was no significant difference in the levels of NO in children in whom a pathogen was identified (22.5; IQR 10.1—35.4) (Table 1) and those with no pathogen (26.9; IQR 15.7—51.1) (P = 0.148). Disease severity score showed very severe dehydration in 6, severe dehydration in 48 and moderate dehydration in 56 children. Comparison of NO:Cr levels in children with very severe or severe dehydration and moderate dehydration showed no significant difference (P = 0.413).
Table 1 Nitric oxide levels measured as urinary nitrite and creatinine (NO:Cr) ratios for different pathogens Pathogen
No. of cases
Nitric oxide (NO:Cr ratio)
Aeromonas Ascariasis Cryptosporidium Enteropathogenic Escherichia coli Giardia Norovirus genogroup II Rotavirus Shigella spp.
1 1 2 2
22.67 27.47 15.89 (median) 24.05 (median)
1 13 25 5
104.66 22.67 (median) 20.5 (median) 12.3 (median)
Figure 1 Box-and-whisker plot of urinary nitrite and creatinine (NO:Cr) ratio measured in cases and controls. The bars indicate the median values (P < 0.001).
Abnormal intestinal function with D-xylose values less than the cut-off was seen in six children, with five positive for rotavirus with a mean (SD) value of 13.7 (2.5) and one for Aeromonas spp. Of the six children with normal Dxylose values, mean (SD) of 36.3 (13.3), three had rotavirus diarrhoea.
4. Discussion The study documents significantly elevated levels of NO in children with diarrhoea, not related to the severity of the illness. The lack of correlation may possibly be due to an early activation and expression of iNOS in the gut, unrelated to the level of replication of the pathogen, which has previously been shown to be associated with disease.5 Among the three most common pathogens, NO levels were significantly higher among children with viral pathogens than with Shigella spp. (P < 0.01) infection, although inflammation is expected to be greater in the latter. This is consistent with an early activation of iNOS, unrelated to the severity of disease and possibly the degree of inflammation. High NO (104.66) was seen in one child with giardiasis; however, the child also had Micrococcus septicaemia, which could result in high NO levels. Although differences in dietary habits may affect NO measurements, all children in this study were <5 years of age and resident in and around Vellore, India, and therefore expected to have similar diets. Most cases were only on intravenous fluids during the first few hours of admission. As the first urine passed post-admission was collected from all children for testing, the interference of diet on NO values was expected to be minimal.
Nitric oxide in acute gastroenteritis Although the number of samples was small, it was interesting to note that five of six children with abnormal D-xylose absorption tests and three of six children with normal D-xylose absorption tests had prior rotaviral infections. A larger study of intestinal function would permit analysis of pathogen-specific association and a correlation with NO levels. Authors’ contributions: All authors were involved in designing the study protocol and analysing and interpreting the data; PDM, AS and IA clinically assessed the children; GK did the microbial analysis of the stool samples; AR and SKN performed and interpreted the urine analysis for NO and urine D-xylose; RS and SC did the statistical analysis; TVS collected the data. All authors contributed to the preparation of the manuscript and read and approved the final version. GK and TVS are guarantors of the paper. Acknowledgements: We would like to thank the people of Ramnaickanpalayam, Chinnallapuram and Kaspa for their co-operation. We are thankful to the Departments of Child Health I, II and III and Department of Gastrointestinal Sciences, Christian Medical College, Vellore, India. Funding: The work was funded by the CMC Fluid Research Fund, Christian Medical College, Vellore, India.
851 Conflicts of interest: None declared. Ethical approval: The study protocol was approved by the Institutional Research Board, Christian Medical College, Vellore, India and signed informed consent was obtained from the parent(s)/guardian(s) of the study children.
References 1. Rodriguez-Diaz J, Banasaz M, Istrate C, Buesa J, Lundgren O, Espinoza F, et al. Role of nitric oxide during rotavirus infection. J Med Virol 2006;78:979—85. 2. Kukuruzovic R, Robins-Browne RM, Anstey NM, Brewster DR. Enteric pathogens, intestinal permeability and nitric oxide production in acute gastroenteritis. Pediatr Infect Dis J 2002;21:730—9. 3. Ruuska T, Vesikari T. Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes. Scand J Infect Dis 1990;22:259—67. 4. Sastry KV, Moudgal RP, Mohan J, Tyagi JS, Rao GS. Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy. Anal Biochem 2002;306:79—82. 5. Kang G, Iturriza-Gomara M, Wheeler JG, Crystal P, Monica B, Ramani S, et al. Quantitation of group A rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in South India. J Med Virol 2004;73:118—22.
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/trstmh
SHORT COMMUNICATION
Nitric oxide production in acute gastroenteritis in Indian children Thuppal Varadachari Sowmyanarayanan a, Sathish Kumar Natarajan a, Anup Ramachandran a, Rajiv Sarkar a, Prabhakar Devarajan Moses b, Anna Simon b, Indira Agarwal b, Solomon Christopher c, Gagandeep Kang a,∗ a
Department of Gastrointestinal Sciences, Christian Medical College, Vellore 632004, Tamil Nadu, India Departments of Child Health Units I, II and III, Christian Medical College, Vellore 632004, Tamil Nadu, India c Department of Biostatistics, Christian Medical College, Vellore 632002, Tamil Nadu, India b
Received 7 April 2009; received in revised form 7 May 2009; accepted 7 May 2009 Available online 23 June 2009
KEYWORDS Acute gastroenteritis; Nitric oxide; Rotavirus; Intestinal permeability; Children; India
Summary This study compared nitric oxide (NO) levels in 110 children with, and 110 children without, infectious gastroenteritis. Post-infection intestinal function was assessed in a subset. At least one pathogen was identified in 47.2% of cases. The most common diarrhoeal pathogens were rotavirus (22.7%) and norovirus genogroup II (11.8%). The levels of NO measured by median urinary nitrite:creatinine ratio were significantly higher in children with diarrhoea [23.6; interquartile range (IQR) 12.3—46.7] than without diarrhoea (7.8; IQR 4.1—13.2), P < 0.001. The ratio was not significantly different between diarrhoeal cases with and without pathogens (P = 0.148). Six of twelve children tested had intestinal dysfunction. © 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Nitric oxide (NO) is an inflammatory mediator synthesized through the L-arginine-NO pathway by the NO synthase enzymes. The inducible isoform (iNOS) is activated during acute infection and inflammation. A number of recent studies have documented increased levels of NO during infectious diarrhoea, particularly with rotavirus and
Cryptosporidium, suggesting a pathophysiological role for NO during acute infection.1,2 We investigated paediatric diarrhoeal illness presenting to the hospital of the Christian Medical College in southern India, to identify the aetiology of diarrhoea and measure the levels of NO in children with infectious gastroenteritis and controls, particularly with regard to severity of disease.
2. Materials and methods ∗ Corresponding author. Tel.: +91 416 228 2052; fax: +91 416 228 2486. E-mail address: [email protected] (G. Kang).
Children <5 years presenting with acute gastroenteritis requiring hospitalization for rehydration were enrolled from January 2006 to December 2007. Disease severity
0035-9203/$ — see front matter © 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2009.05.003
850
T.V. Sowmyanarayanan et al.
was assessed using a 20-point scoring system.3 For each child with diarrhoea, an age-matched healthy control was enrolled from an urban slum in the same town. Stool was tested for ova and parasites by microscopy; culture for Salmonella, Shigella, Campylobacter, Aeromonas and enteropathogenic Escherichia coli; ELISA for group A rotavirus and PCR for norovirus genogroup II. NO was measured in the first urine post-admission from cases and early morning urine from controls using a copper-cadmium alloy coupled with Griess reaction.4 Urine was collected in isopropanol to restrict the degradation of urinary nitrates by bacteria. NO levels were expressed as a function of urinary nitrite and creatinine (NO:Cr). Nitrite levels were estimated in the water from paediatric wards and the community where the controls were selected. D-xylose absorption tests were done 4 weeks post-infection to study intestinal permeability in a subset of 12 children with acute gastroenteritis for whom specific pathogens were identified in stool. The Mann-Whitney U test was performed to determine the significance of differences observed between cases and controls.
3. Results During the study, 757 children were admitted with diarrhoea, 110 of whom were enrolled and age matched with 110 controls recruited from an urban slum. Stool pathogens were identified in 52 cases (47.2%), with rotavirus (22.7%), norovirus genogroup II (11.8%) and Shigella spp. (4.5%) being the most common pathogens. The median NO:Cr ratio in children with diarrhoea [23.6; interquartile range (IQR) 12.3—46.7] was significantly higher than children without diarrhoea (7.8; IQR 4.1—13.2) (P < 0.001) (Figure 1). Levels of nitrites in the water from the paediatric wards and the community showed minimal and similar amounts of nitrites, thus not affecting measurements. There was no significant difference in the levels of NO in children in whom a pathogen was identified (22.5; IQR 10.1—35.4) (Table 1) and those with no pathogen (26.9; IQR 15.7—51.1) (P = 0.148). Disease severity score showed very severe dehydration in 6, severe dehydration in 48 and moderate dehydration in 56 children. Comparison of NO:Cr levels in children with very severe or severe dehydration and moderate dehydration showed no significant difference (P = 0.413).
Table 1 Nitric oxide levels measured as urinary nitrite and creatinine (NO:Cr) ratios for different pathogens Pathogen
No. of cases
Nitric oxide (NO:Cr ratio)
Aeromonas Ascariasis Cryptosporidium Enteropathogenic Escherichia coli Giardia Norovirus genogroup II Rotavirus Shigella spp.
1 1 2 2
22.67 27.47 15.89 (median) 24.05 (median)
1 13 25 5
104.66 22.67 (median) 20.5 (median) 12.3 (median)
Figure 1 Box-and-whisker plot of urinary nitrite and creatinine (NO:Cr) ratio measured in cases and controls. The bars indicate the median values (P < 0.001).
Abnormal intestinal function with D-xylose values less than the cut-off was seen in six children, with five positive for rotavirus with a mean (SD) value of 13.7 (2.5) and one for Aeromonas spp. Of the six children with normal Dxylose values, mean (SD) of 36.3 (13.3), three had rotavirus diarrhoea.
4. Discussion The study documents significantly elevated levels of NO in children with diarrhoea, not related to the severity of the illness. The lack of correlation may possibly be due to an early activation and expression of iNOS in the gut, unrelated to the level of replication of the pathogen, which has previously been shown to be associated with disease.5 Among the three most common pathogens, NO levels were significantly higher among children with viral pathogens than with Shigella spp. (P < 0.01) infection, although inflammation is expected to be greater in the latter. This is consistent with an early activation of iNOS, unrelated to the severity of disease and possibly the degree of inflammation. High NO (104.66) was seen in one child with giardiasis; however, the child also had Micrococcus septicaemia, which could result in high NO levels. Although differences in dietary habits may affect NO measurements, all children in this study were <5 years of age and resident in and around Vellore, India, and therefore expected to have similar diets. Most cases were only on intravenous fluids during the first few hours of admission. As the first urine passed post-admission was collected from all children for testing, the interference of diet on NO values was expected to be minimal.
Nitric oxide in acute gastroenteritis Although the number of samples was small, it was interesting to note that five of six children with abnormal D-xylose absorption tests and three of six children with normal D-xylose absorption tests had prior rotaviral infections. A larger study of intestinal function would permit analysis of pathogen-specific association and a correlation with NO levels. Authors’ contributions: All authors were involved in designing the study protocol and analysing and interpreting the data; PDM, AS and IA clinically assessed the children; GK did the microbial analysis of the stool samples; AR and SKN performed and interpreted the urine analysis for NO and urine D-xylose; RS and SC did the statistical analysis; TVS collected the data. All authors contributed to the preparation of the manuscript and read and approved the final version. GK and TVS are guarantors of the paper. Acknowledgements: We would like to thank the people of Ramnaickanpalayam, Chinnallapuram and Kaspa for their co-operation. We are thankful to the Departments of Child Health I, II and III and Department of Gastrointestinal Sciences, Christian Medical College, Vellore, India. Funding: The work was funded by the CMC Fluid Research Fund, Christian Medical College, Vellore, India.
851 Conflicts of interest: None declared. Ethical approval: The study protocol was approved by the Institutional Research Board, Christian Medical College, Vellore, India and signed informed consent was obtained from the parent(s)/guardian(s) of the study children.
References 1. Rodriguez-Diaz J, Banasaz M, Istrate C, Buesa J, Lundgren O, Espinoza F, et al. Role of nitric oxide during rotavirus infection. J Med Virol 2006;78:979—85. 2. Kukuruzovic R, Robins-Browne RM, Anstey NM, Brewster DR. Enteric pathogens, intestinal permeability and nitric oxide production in acute gastroenteritis. Pediatr Infect Dis J 2002;21:730—9. 3. Ruuska T, Vesikari T. Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes. Scand J Infect Dis 1990;22:259—67. 4. Sastry KV, Moudgal RP, Mohan J, Tyagi JS, Rao GS. Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy. Anal Biochem 2002;306:79—82. 5. Kang G, Iturriza-Gomara M, Wheeler JG, Crystal P, Monica B, Ramani S, et al. Quantitation of group A rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in South India. J Med Virol 2004;73:118—22.