![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
*
M. R. C. Dunn Nutritional Laboratory, Downham's Lane, Milton Road, Cambridge CB4 1XJ, United Kingdom, and Keneba, The Gambia, and
Human Nutrition Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, United Kingdom
1To whom correspondence should be addressed.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1-antichymotrypsin and total IgM, IgA
and IgG immunoglobulins. However, infant growth over the whole 6-mo
period (i.e., between 2 and 8 mo of age) was not related to mean
Giardia-specific antibody titers, nor the time of first
exposure to the parasite. The data suggest that giardiasis in these
very young breast-fed children occurs as a mild, acute disease, and its
presence could not explain the marked, long-term growth faltering
observed in many of the subjects.
KEY WORDS: • giardiasis • intestinal parasite • Giardia antibodies • infant growth • The Gambia
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). While undoubtedly infection
with this parasite can at times result in severe diarrhea and
dehydration requiring hospitalization, such cases are rare and
infection commonly occurs without overt clinical symptoms (Wolfe 1990
). Nevertheless, because giardiasis is associated with
malabsorption of fat and reduced mucosal disaccharidase activity in the
small intestine (Buret et al. 1992
, Farthing 1993
, Katelaris and Farthing 1992
), there is a
general acceptance that infection with this parasite is likely to
result in failure to thrive and poor growth in infants and children
(Islam 1990
, Thompson et al. 1993
).
Evidence in the literature for such an effect is, however, rather
sparse. Gupta and Urrutia (1982)
reported increased
growth in weight and height of Guatemalan children in whom giardiasis
had been virtually eliminated by twice-monthly doses of metronidazole.
However metronidazole is a wide-spectrum antibiotic, and the
improvement noted cannot be unequivocally assigned to its anti-giardial
properties. In another Guatemalan study (Farthing et al. 1986
), Giardia-positive children were found to have
poorer weight gain than those without this infection (P = 0.03) but only in the second year of life; no effect was seen in the
first or third years. Similar small effects on growth were reported
from studies in The Gambia (Cole and Parkin 1977
) and in
Zimbabwe (Loewenson et al. 1986
, Mason and Patterson 1987
). However, these data do not give a clear guide
of the importance of G. intestinalis infection as a cause of
childhood growth faltering and malnutrition in developing countries.
Poorer than expected growth during the first year of life is a frequent
occurrence in infants living in the rural West Kiang area of The Gambia
(Lunn et al. 1991
). Dietary deficiencies seem unable to
explain the poor growth (Prentice 1993
), but close
associations between gastrointestinal disease and both height and
weight growth were described. In particular, repeated intestinal
permeability tests indicated the presence of a persistent enteropathy
in the small intestine of slow-growing infants (Lunn et al. 1991
). Giardia intestinalis causes persistent damage
to the mucosa of the small intestine (Farthing 1993
),
and previous work showed the parasite to be common in this part of The
Gambia (Cole and Parkin 1977
). The aim of this
investigation was to determine to what extent G.
intestinalis infection was associated with the etiology of the
observed mucosal enteropathy and the consequence of such involvement on
infant growth faltering in this area. In this study, the presence of
infection was assessed serologically by the measurement of
Giardia-specific immunoglobulins (Goka et al. 1986
).
|
SUBJECTS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anti-Giardia antibodies.
Giardia-specific IgA, IgM and IgG antibodies
(GS-IgA,2
GS-IgM and GS-IgG) in plasma were measured by ELISA as described by
Goka et al. (1986)
. Giardia antigen was
prepared as described from Portland 1 strain trophozoites grown in
axenic culture (Keister 1983
). Plasma was assayed at
1/200 dilution of the sample in phosphate buffered saline (pH 7.2)
containing 10 g/L bovine serum albumin. The assay differed from that
described by Goka et al. (1986)
only in the substitution
of o-phenylenediamine as substrate for the peroxidase
reaction. Optical density was read at 492 nm on a Labsystems Multiscan
MCC/340 plate reader (Basingstoke, Hants, UK).
Serial blood samples were taken monthly from the age of 2 mo, i.e., at
a time when the infants were fully breast-fed and unlikely to have been
exposed to the parasite. GS antibody titers at this age would be
expected to be low and give a baseline value. In subsequent samples an
optical density reading greater than 0.53 (at 492 nm) for GS-IgM was
taken as an indication of Giardia infection. This value
represents the mean value of the initial, i.e., 8-wk titer plus 1
SD (Al-Tukhi et al. 1993a
)
Plasma proteins.
Plasma 1-antichymotrypsin, albumin and total
immunoglobulins IgA, IgM and IgG concentrations were measured by
immunoturbidometric techniques using a Cobas Bio (Roche, Welwyn Garden
City, Herts. UK) centrifugal analyzer. The
1-antichymotrypsin was determined according to
Calvin and Price (1986)
using Dako (High Wycome, Bucks,
UK) antibodies and a Serotec (Kidlington, Oxon, UK) calibrator. The
albumin and immunoglobulin assays also used Dako antibodies but were
calibrated against Dako standards.
Intestinal permeability test.
This is a noninvasive technique which provides information about the
function and integrity of the mucosa of the small intestine. In this
test, a low lactulose/mannitol ratio suggests mucosal normality,
whereas a raised level indicates loss of absorptive capacity, breaches
of the mucosal barrier, or both (Lunn et al. 1991
).
The test solution contained 200 g of lactulose (as Duphalac,
Duphar Labs., Southampton, UK) and 50 g of mannitol (Sigma, Poole,
Dorset, UK) per L of water, and infants were given approximately 2 mL
(accurately measured) of solution per kg body weight. Urine was
collected over the next 5 h in a urine bag fitted with a drainage
tube ("Hollister U-bag," Abbot Labs, Queensborough, Kent, UK).
Urine bags were inspected regularly and any urine produced was drained
into collecting bottles containing a few drops of chlorhexidene
gluconate (2 g/L) as a bacteriostat. The total 5-h volume was recorded
and an aliquot taken and stored at -20°C prior to analysis in
Cambridge. Infants were not allowed food for 2 h after the test
dose but were encouraged to drink water. Urinary concentrations of
lactulose and mannitol were measured by automated enzymatic procedures
on a centrifugal analyzer (Cobas Bio) (Northrop et al. 1990
, Lunn and Northrop-Clewes 1992
). Intestinal
permeability (expressed as the lactulose/mannitol, L/M, ratio) was
calculated as urinary lactulose concentration divided by urinary
mannitol concentration. Urinary lactose was measured by a similar
technique (Northrop et al. 1990
) and the 5 h
excretion rate calculated.
Ethical approval for the study was obtained from the Medical Research Council-Gambian Government Ethical Committee. The study was part of a larger investigation into many aspects of growth, nutrition and infection in Gambian infants. Parents of the subjects were invited to give their consent after the purpose and requirements of the studies had been explained to them and they were free to withdraw their children at any time.
Statistical analyses were performed using SPSS for Windows, version 8.0
(SPSS, Chicago, IL). Repeated observations from individual infants were
analyzed by analysis of covariance to assess within-subject
correlations (Bland and Altman 1995
). Other data were
assessed by ANOVA, linear regression or chi-square analysis as
indicated. Growth data were corrected for age by regression. Intestinal
permeability was log transformed to correct for a skewed distribution.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
),but the largest increase was seen in GS-IgM. Titers of GS-IgM and
GS-IgA in 2-mo-old children were close to United Kingdom control
values. In contrast, GS-IgG levels were frequently elevated, probably
as a result of absorption of maternal IgG antibodies from breast milk.
GS-IgA titers varied in a similar way to GS-IgM, but appeared to be
less sensitive: the latter were invariably increased when IgA
antibodies rose, but GS-IgA was elevated only on 67% of the occasions
when GS-IgM was high. Nevertheless there was a good correlation between
titers of all three classes of antibody in individual samples, (GS-IgM
vs. GS-IGA, r = 0.44; GS-IgM vs. GS-IgG,
r = 0.50; GS-IgA vs. GS-IgM, r = 0.41;
P < 0.001 at all times).
|
shows the age distribution of this occurrence together with the
cumulative total of infants having had at least one positive titer.
Clearly the data show that infants are exposed to Giardia
from an early age with peak seroconversion occurring at 3–4 mo. By 6
mo of age, more than 80% of the subjects had experienced at least one
GS-IgM positive titer, and all but three infants gave a positive titer
before the end of the study, when the infants were 8-mo old. The months
of April, May and June represented the most likely time for the first
appearance of positive titers (Fig. 2
A)and GS-IgM titers were also at their highest during May and June (Fig. 2B)
. GS-IgG and GS-IgA titers also peaked during these 3 mo (data not
shown).
|
|
Elevated GS-IgM titers were associated with poor weight gain during the
2-wk period before the sample was obtained, (Fig. 3
A).Moreover, the higher the antibody titer, the more severe the weight
deficit. In contrast, in the 2 wk following elevated antibody titers
(Fig. 3B)
, weight gain increased, the greatest gain being in infants
who had exhibited the highest GS-IgM antibody titers. Weight-for age
Z-scores showed similar relationships and statistical significance.
|
)and with decreased mannitol absorption. No correlation was observed
with lactulose uptake, but urinary lactose excretion rose with
increased titers. High antibody titers were not associated with
episodes of diarrhea or vomiting in these children. Infants (18.6%)
had an episode of diarrhea and 6% vomited in the 2-wk period before a
positive titer was found, but this did not differ significantly from
corresponding figures of 20.3 and 7.1% before a negative titer.
|
1-antichymotrypsin concentrations became elevated
with increasing GS-IgM titers (Table 2)
, suggesting that production of
GS antibodies was associated with a systemic inflammatory response.
Such a mechanism may explain part of the poor weight gain seen in
infants with high GS-IgM titers as high concentrations of plasma
1-antichymotrypsin were overall related to poor weight
gain (r = 0.34, P < 0.001). No changes
occurred in plasma albumin concentrations during infection. Close relationships were also observed between plasma GS Ig titers of each class and the respective total Ig concentration: for GS-IgM and total IgM, r = 0.784; for GS-IgA and total IgA, r = 0.603; and for GS-IgG and total IgG, r = 0.420; P < 0.001 for each, (n = 60).
Predictive value of raised anti-Giardia antibody titers on long-term growth.
Long-term effects of raised anti-Giardia antibodies on the
growth of individual infants were examined by taking the mean of each
measurement made over the whole of the 6-mo study period for each
child. The mean monthly weight gain over this period was 375 ±
13 g (SEM) with a range from 143 to 552 g.
Corresponding values for length growth were a mean of 1.43 ± 0.07
cm and a range from 0.44 to 2.65 cm. Comparing the mean growth rate for
each child with its mean GS-IgM titer showed no long-term relationship
with either weight or length gain, (r = 0.13 and 0.07;
P = 0.3 and 0.6, respectively). Similarly, no
significant relationship was seen with GS-IgA or GS-IgG. Weak
associations between mean GS-IgA titer and
1-antichymotrypsin (r = 0.26,
P < 0.05) and intestinal lactulose uptake
(r = 0.28, P < 0.05) were present, but
a correlation no longer existed between antibody titer and intestinal
permeability. Also no relationship existed between any of these
parameters and the age at which infants first showed a positive GS-IgM
titer.
The association between GS antibodies and their respective total Ig
type was still strong when the data was analyzed in this way. The
regression coefficient for GS-IgM and total IgM, r =
0.780, indicated that the GS antibody titer could predict greater than
60% of the variation in total IgM (Fig. 4
).Corresponding coefficients for IgA and IgG were r =
0.740 and 0.543, respectively, (n = 60,
P < 0.001 in all cases).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), the infants are
gradually introduced to weaning foods from 2–3 mo coinciding with the
onset of growth faltering. In this study, 3–4 mo was the peak age for
the appearance of GS IgM antibodies in the plasma of the infants,
suggesting that exposure to G. intestinalis occurred at
about the same time or soon after weaning foods were first given.
Exposure occurring so early in life demonstrates not only the high
endemicity of the parasite in this area of The Gambia, but also a high
prevalence of fecal contamination of the infants' food or water.
Elevated GS-IgM concentrations in the blood were associated with poor
weight gain of infants; previous to the blood sample, the higher the
GS-IgM titer the poorer the weight gain. The raised antibody titer was
also associated with an elevated intestinal permeability due to reduced
mannitol uptake and a small but significant increase in the plasma
concentration of the acute-phase protein,
1-antichymotrypsin. Although increased
Giardia antibodies were not associated with diarrhea and
vomiting, the reduced weight gain, raised intestinal permeability and
small systemic inflammatory response are in keeping with a mild episode
of giardiasis. Maldigestion of lactose occurs in giardiasis
(Farthing 1993
) and could raise urinary excretion of
this sugar with increasing titers. The impact of the infection on
growth however seems to be short-lived. In the weeks following high
titers, weight gain was greater than average with infants making up
much of the previous weight loss. The effectiveness of recovery was
such that when weight or height growth over the whole 6 mo of life was
considered, no relationship with mean GS antibody titers could be
demonstrated. Giardiasis in these very young children thus appears to
be an acute illness that soon resolves.
The failure to demonstrate any long-term association between elevated
GS antibody titers and growth makes it unlikely that this infection
could account for a significant part of the progressive growth
faltering observed in these infants. Given the high potential for
infection and re-infection in the region, the pathogenic nature of the
parasite and the relatively immature immune system of infants of this
age, this result is rather surprising. It is however in keeping with
other reports of the impact of Giardia on this age group
(Farthing et al. 1986
), and probably the infection may
have been limited by protective agents present in breast milk. Human
milk was shown to have both cytotoxic (Gillin 1987
) and
antiadherent activity (Crouch et al. 1991
), and GS
secretory IgA was demonstrated (Walterspiel et al. 1994
). In field studies, Morrow et al. (1992)
reported a greater incidence of diarrhea and vomiting in infants and
young children who were not receiving breast milk. However their
studies also showed that there were no differences in the prevalence of
G. intestinalis cysts in the feces, i.e., the number of
children carrying the infection did not differ between breast-fed and
nonbreast-fed children. The lack of symptoms observed in the present
study is clearly in agreement with such data.
The relevance of the close correlation between the titers of GS-Ig
classes, and respective total plasma Ig is not clear. Statistically,
the 6-mo mean GS-IgM titer could predict greater than 60% of the 6-mo
total IgM mean. Corresponding figures for IgA and IgG were 54 and 29%.
One interpretation is that this represents the proportion of respective
total Ig that is GS. However a more likely explanation is that mucosal
damage associated with the parasite allowed translocation of various
antigenic macromolecules which elicited a polyclonal Ig response
(Suskind et al. 1976
).
Several groups used the presence of GS Ig in plasma to diagnose
infection with G. intestinalis. Goka et al. (1986)
reported a good correlation between specific
anti-Giardia IgM titers and the presence of giardiasis
assessed by standard techniques. GS IgG appeared less useful as such
antibodies persisted long after the infection was over in contrast to
IgM antibodies which fell sharply after the infection was cleared. More
recently the use of raised antibody titers as a reliable indicator of
G. intestinalis infection was confirmed in studies of
Gambian children (Sullivan et al. 1991
) and in Saudi
Arabian subjects (Al-Tukhi et al. 1993b
). In both the
latter studies, the specificity of the technique was greater than 93%
though sensitivity was a little lower. In the current study, the
availability of serial samples minimizes the likelihood of false
positive and negative values. The first samples were taken at the age
of 2 mo, a time when the infants were fully breast fed and thus
unlikely to have become infected, and were found to have low titers of
GS IgM and IgA. Increased titers, signifying the first G.
intestinalis infection, could be clearly observed as a sharp
increase from this early, baseline value.
The results do not suggest that giardiasis is of little importance in
the etiology of malnutrition and growth faltering of children in
general. The episodes seen in these particular infants were very mild;
they were not associated with diarrhea or vomiting and resolved
quickly. In older children or those receiving little or no breast milk,
the disease appears to be more severe and consequently may compromise
growth (Farthing 1993
). However, further studies in such
children are required to determine the full impact of this parasite on
growth.
|
FOOTNOTES |
|---|
Manuscript received July 28, 1998. Initial review completed November 25, 1998. Revision accepted December 10, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. Al-Tukhi M. H., Ackers J. P., Al-Ahdal M. N., Peters W. Enzyme-linked immunosorbent assay for the detection of anti-Giardia specific immunoglobulin G in filter paper blood samples. Trans. R. Soc. Trop. Med. Hyg. 1993;87:36-38[Medline]
2. Al-Tukhi M. H., Ackers J. P., Al-Ahdal M. N., Taha M. A., Peters W. ELISA for detection of anti-Giardia specific IgM-response in serum. J. Trop. Med. and Hyg. 1993;96:333-336
3.
Bland J. M., Altman D. G. Calculating correlation coefficients with repeated observations: Part 1—correlation within subjects. Br. Med. J. 1995;310:446
4. Buret A., Hardin J. A., Olson M. E., Gall D. G. Pathophysiology of small intestinal malabsorption in gerbils infected with Giardia lamblia. Gastroenterol 1992;103:506-513[Medline]
5.
Calvin J., Price C. P. Measurement of serum 1-antichymotrypsin by immunoturbidimetry. Ann. Clin. Biochem. 1986;23:206-209
6. Cole T. J., Parkin J. M. Infection and its effect on the growth of young children. Trans. R. Soc. Trop. Med. Hyg. 1977;76:196-198
7. Crouch A. A., Seow W. K., Whitman L. M., Thong Y. H. Effect of human milk and infant milk formulae on adherence of Giardia intestinalis. Trans. R. Soc. Trop. Med. Hyg. 1991;85:617-619[Medline]
8. Farthing M. J. G. Pathogenesis of Giardiasis. Trans. R. Soc. Trop. Med. Hyg. 1993;87, suppl. 3:17-21
9. Farthing M. J. G., Mata L., Urrutia J. J., Kronmal R. A. Natural history of Giardia infection of infants and children in rural Guatemala and its impact on physical growth. Am. J. Clin. Nutr. 1986;43:393-403
10. Gillin F. D. Giardia lamblia: the role of conjugated and unconjugated bile salts in killing by human milk. Exptl. Parasitol. 1987;63:74-83
11. Goka A. K. J., Rolston D. D. K., Mathan V. I., Farthing M. J. G. Diagnosis of Giardiasis by specific IgM antibody enzyme-linked immunosorbent assay. Lancet 1986;i:184-186
12.
Gupta M. C., Urrutia J. J. Effect of periodic antiascaris and antigiardial treatment on nutritional status of pre-school children. Am. J. Clin. Nutr. 1982;36:79-86
13. Islam A. Giardiasis in developing countries. Meyer E. A. eds. Giardiasis 1990:235-266 Elsevier Amsterdam.
14.
Katelaris P. H., Farthing M. J. G. Diarrhea and malabsorption in Giardiasis: A multifactorial process. Gut 1992;33:295-297
15. Keister D. B. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans. R. Soc. Trop. Med. Hyg. 1983;33:487-488
16. Loewenson R., Mason P. R., Patterson B. A. Giardiasis and the nutritional status of Zimbabwean schoolchildren. Ann. Trop. Paediatr. 1986;6:73-78[Medline]
17. Lunn P. G., Northrop-Clewes C. A. Intestinal permeability: Update on the enzymatic assay of mannitol. Clin. Chim. Acta 1992;205:151-152[Medline]
18. Lunn P. G., Northrop-Clewes C. A., Downes R. M. Intestinal permeability, mucosal injury and growth faltering in Gambian infants. Lancet 1991;338:907-910[Medline]
19. Mason P. R., Patterson B. A. Epidemiology of Giardia lamblia infection in children: Cross-sectional and longitudinal studies in urban and rural communities in Zimbabwe. Am. J. Trop. Med. Hyg. 1987;37:277-282
20. Meyer E. A. Giardiasis 1990 Elsevier Amsterdam.
21. Morrow A. L., Reves R. R., West M. S., Guerrero M. L., Ruizpalacios G. M., Pickering L. K. Protection against infection with Giardia lamblia by breast-feeding in a cohort of Mexican infants. J. Pediatr. 1992;121:363-370[Medline]
22. Northrop C. A., Lunn P. G., Behrens R. H. Automated enzymatic assays for the determination of intestinal permeability probes in urine: 1. Lactulose and lactose. Clin. Chim. Acta 1990;187:79-88[Medline]
23. Prentice A. Nutrient requirements for growth, pregnancy and lactation: The Keneba experience. S. Afr. J. Clin. Nutr. 1993;6:33-38
24. Prentice A., Paul A. A. Contribution of breast milk to nutrition during prolonged breast-feeding. Atkinson S. Hanson L. Chandra R. eds. Human lactation 4. Breast-feeding, nutrition, infection and infant growth in developed and emerging countries 1990:88-101 ARTS Biomedical Publishers St. Johns, Newfoundland.
25. Sullivan P. B., Neale G., Cevallos A. M., Farthing M. J. G. Evaluation of specific serum anti-Giardia IgM antibody response in diagnosis of Giardiasis in children. Trans. R. Soc. Trop. Med. Hyg. 1991;85:748-749[Medline]
26.
Suskind R., Sirishinha S., Vitayasai V., Edelman R., Damrongsak D., Charupatana C., Olson R. E. Immunoglobulins and antibody response in children with protein-calorie malnutrition. Am. J. Clin. Nutr. 1976;29:836-841
27. Thompson R. C. A., Reynoldson J. A., Mendis H. W. Giardia and Giardiasis. Adv. Parasitol. 1993;32:71-160[Medline]
28.
Walterspiel J. N., Morrow A. L., Guerrero M. L., Ruizpalacios G. M., Pickering L. K. Secretory anti-Giardia lamblia antibodies in human milk-protective effect against diarrhea. Pediatrics 1994;93:28-31
29. Wolfe M. S. Clinical symptoms and diagnosis by traditional methods. Meyer E. A. eds. Giardiasis 1990:175-185 Elsevier Amsterdam.
This article has been cited by other articles:
|
|
 |
|
  M.-G. Hollm-Delgado, R. H. Gilman, C. Bern, L. Cabrera, C. R. Sterling, R. E. Black, and W. Checkley Lack of an Adverse Effect of Giardia intestinalis Infection on the Health of Peruvian Children Am. J. Epidemiol., September 15, 2008; 168(6): 647 - 655. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
