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Correlation between genotype of Giardiaduodenalis and diarrhoea
International Journal for Parasitology 32 (2002) 229–231 www.parasitology-online.com
Letter to the Editor Correlation between genotype of Giardia duodenalis and diarrhoea
Infection with the protozoan parasite Giardia duodenalis (syn. Giardia intestinalis) is emerging as the leading nonviral cause of infectious diarrhoea in children attending daycare centres (Thompson, 2000). Conditions in day-care centres are ideal for the transmission of this parasite with potentially susceptible children concentrated in an area where it is difficult to maintain adequate hygiene standards. The symptoms of Giardia infection are highly variable and some individuals may shed infectious cysts in their faeces without showing any overt clinical signs (Farthing, 1997). It is not fully understood why some individuals develop clinical giardiasis while others remain asymptomatic; however, host factors and ‘strain’ variation of the parasite are both likely to be involved (Thompson, 2000). Although morphologically identical isolates of G. duodenalis have been shown to be phenotypically and genetically heterogeneous (Thompson et al., 2000), the majority of G. duodenalis isolates from humans and some species of domestic animals can be grouped into one of two distinct genetic Assemblages, A and B (Homan et al., 1992; Mayrhofer et al., 1995; Thompson et al., 2000). Isolates belonging to these Assemblages are found throughout the world, although Assemblage B isolates appear to be less widespread and restricted to localised endemic foci (Thompson and Meloni, 1993; Meloni et al., 1995). Isolates of G. duodenalis have been shown to differ in their pathogenicity, virulence and other biological characteristics. While there are biological characteristics that are capable of distinguishing between isolates of different genotypes, to date there is only one study that has been able to correlate a genotypic marker with phenotype (Homan and Mank, 2001). This study, carried out in the Netherlands, involved genotyping Giardia isolates obtained from patients between 8 and 60 years of age who presented themselves to their general practitioner with diarrhoeal symptoms. The patients were split into two symptomatic groups based on the severity of the diarrhoea. A strong correlation was found between Assemblage A and what was classed as intermittent diarrhoea, and Assemblage B and persistent diarrhoea. Based on these observations the authors hypothesised that Assemblage A isolates would be more prevalent in asymptomatic infected individuals (Homan and Mank, 2001). Here we report the results from the genotyping of Giardia isolates that were
obtained from children participating in a longitudinal study of day-care centres in Western Australia. Monthly faecal samples were obtained from children under 5 years of age attending 10 day-care centres in the period February 1998 to May 2000. The consistency of the faecal samples was recorded as normal or diarrhoeal. Samples were initially screened by microscopy for the presence of Giardia cysts using routine zinc sulphate flotation. Cysts were purified using standard saturated salt and glucose gradients, and DNA extracted as previously described (Hopkins et al., 1997). Genotyping was carried out using a nested PCR procedure, amplifying a region of the SSU-rDNA using the primers RH11 and RH4 as previously described (Hopkins et al., 1997) and the newly designed primer pair GiarF: GAC GCT CTC CCC AAG GAC and GiarR: CTG CGT CAC GCT GCT CG for the secondary PCR. Amplification and sequencing of the amplification products were carried out using conditions described by Hopkins et al. (1997). Isolates were grouped into the genetic Assemblages A or B based on analysis of the resulting 130 bp sequence. This region has previously been shown to be reliable in differentiating between genotypes (Hopkins et al., 1997). Isolates of genetic Assemblage A have bases G, T and C at positions 44, 62 and 72, respectively, compared to isolates of genetic Assemblage B that have bases C, G and G at positions 44, 62 and 72, respectively and an insertion, A, at position 43 of the SSU-rDNA gene (Hopkins et al., 1997; Monis et al., 1999). A total of 1306 samples were screened for Giardia from 353 children over the course of the study and 7.6% (27) of the children sampled were positive for Giardia at least once. Of these positive children, 35% were positive more than once over the period of the study. Sequence information was obtained for 36 isolates of Giardia from 23 different children. Thirty percent (7/23) of these children were infected with isolates of G. duodenalis that were genotyped as Assemblage A, and 70% (16/23) had isolates that were genotyped as Assemblage B (Fig. 1). Children found to be infected more than once were always infected with organisms of the same assemblage although it is not clear if this represents continuous infection or reinfection by other organisms belonging to the same assemblage. Statistical analysis revealed that children infected with Giardia isolates from Assemblage A were 26 times more likely to have diarrhoea than children with Assemblage B organisms (2.2, 304.7 95% confidence interval, P , 0:005). Our study has shown that Giardia infection in children is not necessarily accompanied by diarrhoea. Thus surveys
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(01)00340-X
230
Letter to the Editor
Fig. 1. Association of Giardia duodenalis genetic Assemblages A and B with symptomatic and asymptomatic infection in children.
which are based on sampling only individuals with diarrhoea may underestimate the true prevalence of G. duodenalis and, in particular, the epidemiological role played by Assemblage B isolates. Our results are in contrast to those of Homan and Mank (2001) who found a strong correlation between Assemblage B and severe, persistent diarrhoea. However, it must be stressed that their study involved patients who were all symptomatic and the observed correlation was between the genotype and the degree of severity of the symptoms rather than between genotype and presence or absence of symptoms (Thompson, 2001). It should also be noted that this degree of severity was based on an evaluation of questionnaires completed by patients and is thus dependent on a subjective self-assessment of their diarrhoeal complaints. In contrast, we found a clear correlation between the presence of a particular genotype and the occurrence or absence of symptoms. The same children were monitored over the course of the study and none of the children who were positive and asymptomatic were found to be symptomatic at any time during the sampling period. However, when comparing the results of the two studies the difference in age of the individuals sampled should be taken into account. In the Homan and Mank study the patients ranged from 8 to 60 years of age, whereas in our study the children were under 5 years of age. This raises interesting questions as to whether the infectivity and pathogenesis of Giardia genotypes may vary in hosts of different age. From our own results we can hypothesise that Assemblage B genotypes are more prevalent in asymptomatic children than those of Assemblage A. Thus the exclusion policies of day-care centres may be ineffective in limiting
the transmission of Giardia since asymptomatic children harbouring Assemblage B isolates will still attend the centres even though they may be shedding infectious cysts. In addition, staff caring for seemingly healthy individuals may be more complacent with hygiene standards thus leading to increased potential for transmission within the day-care centre and the community. Similarly, the high prevalence of infection in some communities may, in part, be due to the lack of obvious symptoms and therefore the failure to treat infected people. This may explain why, in our experience, infections with isolates of G. duodenalis from Assemblage B are more common in community settings such as isolated Aboriginal communities and day-care centres. Correlation between the presence of diarrhoea and Giardia genotype does not necessarily imply that a particular genotype is more virulent than isolates that are less commonly associated with diarrhoea. Chronic infections with Giardia, particularly those in children in community settings, are not always associated with the presence of diarrhoea. It has been suggested that such persistent, chronic infections are more likely to be associated with nutritional disorders and failure to thrive (AstiazaranGarcia et al., 2000; Thompson and Lymbery, 1996). This raises the question of what symptoms constitute ‘virulence’, and it may be that in such circumstances, an isolate of Giardia associated with an acute episode of diarrhoea is in fact less virulent than an isolate associated with a chronic infection.
References Astiazaran-Garcia, H., Espinosa-Cantellano, M., Castanon, G., ChavezMunguia, B., Martinez-Palomo, A., 2000. Giardia lamblia: effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus). Exp. Parasitol. 95, 128–35. Farthing, M.J., 1997. The molecular pathogenesis of giardiasis. J. Pediatr. Gastroenterol. Nutr. 24, 79–88. Homan, W.L., Mank, T.G., 2001. Human giardiasis: genotype linked differences in clinical symptomatology. Int. J. Parasitol. 31, 822–6. Homan, W.L., van Enckevort, F.H., Limper, L., van Eys, G.J., Schoone, G.J., Kasprzak, W., Majewska, A.C., 1992. Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes. Parasitol. Res. 78, 316–23. Hopkins, R.M., Meloni, B.P., Groth, D.M., Wetherall, J.D., Reynoldson, J.A., Thompson, R.C.A., 1997. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J. Parasitol. 83, 44–51. Mayrhofer, G., Andrews, R.H., Ey, P.L., Chilton, N.B., 1995. Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris. Parasitology 111, 11–17. Meloni, B.P., Lymbery, A.J., Thompson, R.C.A., 1995. Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: implications for reproductive biology, population structure, taxonomy, and epidemiology. J. Parasitol. 81, 368–83. Monis, P.T., Andrews, R.H., Mayrhofer, G., Ey, P.L., 1999. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol. Biol. Evol. 16, 1135–44.
Letter to the Editor Thompson, R.C.A., 2000. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int. J. Parasitol. 30, 1259–67. Thompson, R.C.A., 2001. Human giardiasis: genotype-linked differences in clinical symptomatology. Trends Parasitol. 17, 465. Thompson, R.C.A., Lymbery, A.J., 1996. Genetic variability in parasites and host-parasite interactions. Parasitology 112, S7–S22. Thompson, R.C.A., Meloni, B.P., 1993. Molecular variation in Giardia. Acta Trop. 53, 167–84. Thompson, R.C.A., Hopkins, R.M., Homan, W.L., 2000. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol. Today 16, 210–3.
231
C. Read, J. Walters, I.D. Robertson, R.C.A. Thompson* Division of Veterinary and Biomedical Sciences, World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, Murdoch University, South Street, Murdoch, WA 6150, Australia * Corresponding author. Tel.: 161-8-9360-2466; fax: 161-8-9310-4144. E-mail address: [email protected] (R. C. A. Thompson)
Letter to the Editor Correlation between genotype of Giardia duodenalis and diarrhoea
Infection with the protozoan parasite Giardia duodenalis (syn. Giardia intestinalis) is emerging as the leading nonviral cause of infectious diarrhoea in children attending daycare centres (Thompson, 2000). Conditions in day-care centres are ideal for the transmission of this parasite with potentially susceptible children concentrated in an area where it is difficult to maintain adequate hygiene standards. The symptoms of Giardia infection are highly variable and some individuals may shed infectious cysts in their faeces without showing any overt clinical signs (Farthing, 1997). It is not fully understood why some individuals develop clinical giardiasis while others remain asymptomatic; however, host factors and ‘strain’ variation of the parasite are both likely to be involved (Thompson, 2000). Although morphologically identical isolates of G. duodenalis have been shown to be phenotypically and genetically heterogeneous (Thompson et al., 2000), the majority of G. duodenalis isolates from humans and some species of domestic animals can be grouped into one of two distinct genetic Assemblages, A and B (Homan et al., 1992; Mayrhofer et al., 1995; Thompson et al., 2000). Isolates belonging to these Assemblages are found throughout the world, although Assemblage B isolates appear to be less widespread and restricted to localised endemic foci (Thompson and Meloni, 1993; Meloni et al., 1995). Isolates of G. duodenalis have been shown to differ in their pathogenicity, virulence and other biological characteristics. While there are biological characteristics that are capable of distinguishing between isolates of different genotypes, to date there is only one study that has been able to correlate a genotypic marker with phenotype (Homan and Mank, 2001). This study, carried out in the Netherlands, involved genotyping Giardia isolates obtained from patients between 8 and 60 years of age who presented themselves to their general practitioner with diarrhoeal symptoms. The patients were split into two symptomatic groups based on the severity of the diarrhoea. A strong correlation was found between Assemblage A and what was classed as intermittent diarrhoea, and Assemblage B and persistent diarrhoea. Based on these observations the authors hypothesised that Assemblage A isolates would be more prevalent in asymptomatic infected individuals (Homan and Mank, 2001). Here we report the results from the genotyping of Giardia isolates that were
obtained from children participating in a longitudinal study of day-care centres in Western Australia. Monthly faecal samples were obtained from children under 5 years of age attending 10 day-care centres in the period February 1998 to May 2000. The consistency of the faecal samples was recorded as normal or diarrhoeal. Samples were initially screened by microscopy for the presence of Giardia cysts using routine zinc sulphate flotation. Cysts were purified using standard saturated salt and glucose gradients, and DNA extracted as previously described (Hopkins et al., 1997). Genotyping was carried out using a nested PCR procedure, amplifying a region of the SSU-rDNA using the primers RH11 and RH4 as previously described (Hopkins et al., 1997) and the newly designed primer pair GiarF: GAC GCT CTC CCC AAG GAC and GiarR: CTG CGT CAC GCT GCT CG for the secondary PCR. Amplification and sequencing of the amplification products were carried out using conditions described by Hopkins et al. (1997). Isolates were grouped into the genetic Assemblages A or B based on analysis of the resulting 130 bp sequence. This region has previously been shown to be reliable in differentiating between genotypes (Hopkins et al., 1997). Isolates of genetic Assemblage A have bases G, T and C at positions 44, 62 and 72, respectively, compared to isolates of genetic Assemblage B that have bases C, G and G at positions 44, 62 and 72, respectively and an insertion, A, at position 43 of the SSU-rDNA gene (Hopkins et al., 1997; Monis et al., 1999). A total of 1306 samples were screened for Giardia from 353 children over the course of the study and 7.6% (27) of the children sampled were positive for Giardia at least once. Of these positive children, 35% were positive more than once over the period of the study. Sequence information was obtained for 36 isolates of Giardia from 23 different children. Thirty percent (7/23) of these children were infected with isolates of G. duodenalis that were genotyped as Assemblage A, and 70% (16/23) had isolates that were genotyped as Assemblage B (Fig. 1). Children found to be infected more than once were always infected with organisms of the same assemblage although it is not clear if this represents continuous infection or reinfection by other organisms belonging to the same assemblage. Statistical analysis revealed that children infected with Giardia isolates from Assemblage A were 26 times more likely to have diarrhoea than children with Assemblage B organisms (2.2, 304.7 95% confidence interval, P , 0:005). Our study has shown that Giardia infection in children is not necessarily accompanied by diarrhoea. Thus surveys
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(01)00340-X
230
Letter to the Editor
Fig. 1. Association of Giardia duodenalis genetic Assemblages A and B with symptomatic and asymptomatic infection in children.
which are based on sampling only individuals with diarrhoea may underestimate the true prevalence of G. duodenalis and, in particular, the epidemiological role played by Assemblage B isolates. Our results are in contrast to those of Homan and Mank (2001) who found a strong correlation between Assemblage B and severe, persistent diarrhoea. However, it must be stressed that their study involved patients who were all symptomatic and the observed correlation was between the genotype and the degree of severity of the symptoms rather than between genotype and presence or absence of symptoms (Thompson, 2001). It should also be noted that this degree of severity was based on an evaluation of questionnaires completed by patients and is thus dependent on a subjective self-assessment of their diarrhoeal complaints. In contrast, we found a clear correlation between the presence of a particular genotype and the occurrence or absence of symptoms. The same children were monitored over the course of the study and none of the children who were positive and asymptomatic were found to be symptomatic at any time during the sampling period. However, when comparing the results of the two studies the difference in age of the individuals sampled should be taken into account. In the Homan and Mank study the patients ranged from 8 to 60 years of age, whereas in our study the children were under 5 years of age. This raises interesting questions as to whether the infectivity and pathogenesis of Giardia genotypes may vary in hosts of different age. From our own results we can hypothesise that Assemblage B genotypes are more prevalent in asymptomatic children than those of Assemblage A. Thus the exclusion policies of day-care centres may be ineffective in limiting
the transmission of Giardia since asymptomatic children harbouring Assemblage B isolates will still attend the centres even though they may be shedding infectious cysts. In addition, staff caring for seemingly healthy individuals may be more complacent with hygiene standards thus leading to increased potential for transmission within the day-care centre and the community. Similarly, the high prevalence of infection in some communities may, in part, be due to the lack of obvious symptoms and therefore the failure to treat infected people. This may explain why, in our experience, infections with isolates of G. duodenalis from Assemblage B are more common in community settings such as isolated Aboriginal communities and day-care centres. Correlation between the presence of diarrhoea and Giardia genotype does not necessarily imply that a particular genotype is more virulent than isolates that are less commonly associated with diarrhoea. Chronic infections with Giardia, particularly those in children in community settings, are not always associated with the presence of diarrhoea. It has been suggested that such persistent, chronic infections are more likely to be associated with nutritional disorders and failure to thrive (AstiazaranGarcia et al., 2000; Thompson and Lymbery, 1996). This raises the question of what symptoms constitute ‘virulence’, and it may be that in such circumstances, an isolate of Giardia associated with an acute episode of diarrhoea is in fact less virulent than an isolate associated with a chronic infection.
References Astiazaran-Garcia, H., Espinosa-Cantellano, M., Castanon, G., ChavezMunguia, B., Martinez-Palomo, A., 2000. Giardia lamblia: effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus). Exp. Parasitol. 95, 128–35. Farthing, M.J., 1997. The molecular pathogenesis of giardiasis. J. Pediatr. Gastroenterol. Nutr. 24, 79–88. Homan, W.L., Mank, T.G., 2001. Human giardiasis: genotype linked differences in clinical symptomatology. Int. J. Parasitol. 31, 822–6. Homan, W.L., van Enckevort, F.H., Limper, L., van Eys, G.J., Schoone, G.J., Kasprzak, W., Majewska, A.C., 1992. Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes. Parasitol. Res. 78, 316–23. Hopkins, R.M., Meloni, B.P., Groth, D.M., Wetherall, J.D., Reynoldson, J.A., Thompson, R.C.A., 1997. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J. Parasitol. 83, 44–51. Mayrhofer, G., Andrews, R.H., Ey, P.L., Chilton, N.B., 1995. Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris. Parasitology 111, 11–17. Meloni, B.P., Lymbery, A.J., Thompson, R.C.A., 1995. Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: implications for reproductive biology, population structure, taxonomy, and epidemiology. J. Parasitol. 81, 368–83. Monis, P.T., Andrews, R.H., Mayrhofer, G., Ey, P.L., 1999. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol. Biol. Evol. 16, 1135–44.
Letter to the Editor Thompson, R.C.A., 2000. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int. J. Parasitol. 30, 1259–67. Thompson, R.C.A., 2001. Human giardiasis: genotype-linked differences in clinical symptomatology. Trends Parasitol. 17, 465. Thompson, R.C.A., Lymbery, A.J., 1996. Genetic variability in parasites and host-parasite interactions. Parasitology 112, S7–S22. Thompson, R.C.A., Meloni, B.P., 1993. Molecular variation in Giardia. Acta Trop. 53, 167–84. Thompson, R.C.A., Hopkins, R.M., Homan, W.L., 2000. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol. Today 16, 210–3.
231
C. Read, J. Walters, I.D. Robertson, R.C.A. Thompson* Division of Veterinary and Biomedical Sciences, World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, Murdoch University, South Street, Murdoch, WA 6150, Australia * Corresponding author. Tel.: 161-8-9360-2466; fax: 161-8-9310-4144. E-mail address: [email protected] (R. C. A. Thompson)