Context: Giardia lambia is a common intestinal protozoon with a higher prevalence rate in tropical countries as compared to the Western world. Histological changes occur in duodenal mucosa in cases of chronic giardiasis that may be similar to celiac disease or tropical sprue. Aim: As the prevalence of giardiasis is high in our country, the present study was undertaken to study the changes in duodenal mucosa of patients with asymptomatic giardiasis and endoscopically normal duodenum. Study Design: The study included 47 duodenal biopsies out of 372 total cases of endoscopic biopsies examined, in which trophozoites of Giardia were identified. A detailed histo-morphological study of these cases was carried out and the histological findings were evaluated. The major histopathological features as observed in cases of giardiasisi.e inflammation, increased intra epithelial lymphocytes (IELs) and villous abnormalities were compared between patients without giardiasis and those with giardiasis. Statistical Analysis Used: P-value using chi-square test was calculated. Results: P-value was found to be significant for inflammation and villous abnormalities whereas increased IEL’s were found to be an insignificant histological parameter. Conclusion: Although the histological features associated with giadiasis were non-specific, a higher incidence of villous changes with increased IEL’s was found. This study highlighted the importance of identification of Giardia trophozoites in duodenal biopsies to avoid misdiagnosis of celiac disease or tropical sprue which may have similar clinical and histological features but have different management strategies.
Keywords: Celiac disease, duodenal biopsy, Giardiasis, trophozoites, villous abnormalities
|How to cite this article:
Varma K, Misra V, Misra SP, Dwivedi M. Histomorphometric study of changes in duodenal mucosa of patients presenting with Giardiasis. Ann Trop Med Public Health 2015;8:50-4
|How to cite this URL:
Varma K, Misra V, Misra SP, Dwivedi M. Histomorphometric study of changes in duodenal mucosa of patients presenting with Giardiasis. Ann Trop Med Public Health [serial online] 2015 [cited 2020 Oct 25];8:50-4. Available from: https://www.atmph.org/text.asp?2015/8/3/50/157628
Giardia lamblia , the cause of human giardiasis, is among the most common intestinal protozoa worldwide. It has a higher prevalence rate in tropical countries like the Indian subcontinent in comparison to the western countries like the U.S.  In India, prevalence rates of giardia infection in patients with diarrhea range from 0.4 % to as high as 70% in low socioeconomic groups.  Also a large majority (35-70%) of individuals infected with giardiasis remain asymptomatic. 
A wide range of histological changes have been reported in duodenal biopsies of these patients but none have been found to be specific.  Diagnosis of giardiasis in a duodenal biopsy specimen requires recognition of trophozoites in mucus adjacent to the epithelial surface.  As the prevalence of Giardiasis is high in our country; the present study was undertaken to study the changes in duodenal mucosa of asymptomatic patients with Giardiasis and endoscopically normal duodenum.
|Materials and Methods|
Three hundred and seventy-two asymptomatic subjects were included in the study. After informed consent, full history and clinical details were noted. A thorough upper gastrointestinal endoscopy was done and two biopsies were taken from the normal appearing duodenum. Out of 372 cases, 350 (94%) were studied in detail, and 22 (6%) cases were not included owing to insufficient material or improper orientation of the sections. 3-5 micron thick sections from paraffin embedded blocks were cut and stained with Haematoxylin and eosin, modified Giemsa, and Loeffler’s methylene blue stain  and PAS as and when required. Histological details of duodenal biopsy were studied according to Whitehead et al.  Changes in villous height and architecture, changes in surface epithelial lining, grade of inflammation in lamina propia and presence of any parasite were noted.
Duodenal biopsies showing normal villi in >75% area of all the sections examined with at least 4-6 normal villi in a row were considered normal [Figure 1]. Any properly oriented section showing branching and fusion of villi in more than 75% area of the section were recorded as villous abnormalities [Figure 2].
|Figure 1: Duodenal biopsy showing normal villi with normal Villous: Crypt ratio. (H and E ×100)
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|Figure 2: Duodenal biopsy showing mainly branched, fused and partially flattened villi. (H and E ×100)
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Increased inflammation in the lamina propria was observed subjectively on the basis of increased mononuclear cell density in the lamina propria in comparison to a normal duodenal biopsy along with lymphoid aggregates and increasing number of neutrophils [Table 1]. 
|Table 1: Distribution of significant histopathological findings in all patients (n = 350)
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Giardia trophozoites were identified by their definite shape (Pear shaped or sickle shaped depending upon the plane of section) along with nuclei in hematoxylin and eosin (H&E;) stained sections [Figure 3]. Modified Giemsa and Loeffler’s methylene blue stains were used in sections with low density of parasite to differentiate it from artifacts like mucus tags [Figure 4].
|Figure 3: Duodenal biopsy showing presence of Giardia trophozoites-pear shaped (arrow) and sickle shaped (arrowhead). Lining epithelium shows increased IELs (double arrows). (H and E ×400)
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|Figure 4: Duodenal biopsy showing sparse presence of pear shaped Giardia trophozoites. (Loeffler’s Methylene Blue ×400)
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Height of villous and depth of crypt were measured in 10 consecutive HPF/section and mean villous: Crypt (V:C) ratio was calculated using image analysis software (Image pro-plus, media cybernetics v 6). Grades of villous atrophy was graded as follows: Grade 1, normal (V:C ratio >1); Grade 2, mild (V:C 1:1); Grade 3, moderate (V:C- <1); and Grade 4, severe (V:C- 0). 
The number of IEL/100 epithelial cells in the surface epithelial lining as well as in crypts [Figure 2] were counted by reducing the field area, using a piece of paper with central pin hole (pin hole method) in the eyepiece of the microscope.
Mean (SD) IEL count in normal duodenal biopsies was calculated. Biopsies showing IEL count >2SD of normal values were counted as number (%) of biopsies showing increased IEL count It was 25.56 (3.6) [Table 1] and [Table 3].
A Chi-square test with and without Yates’ correction was used to find out any significance in histological features of biopsies with and without giardiasis. P value ≤0.05 was taken as critical level of significance. This research protocol was approved by the institutional ethics committee and the participants had given written informed consent.
Trophozoites of Giardia were identified in duodenal biopsies of 47/350 (13.4%) patients. These cases were studied in detail. None of the patients presented with diarrhea or any other specific symptoms. On endoscopy, duodenum was normal in all cases (100%). The age of patients with giardiasis ranged from 8 to 65 years with a mean age of 28.5 years. The male:female ratio was 2.4: 1. As seen in [Table 2] most common histological finding in duodenal biopsy was inflammation in 17 (36.2%) cases followed by increased IEL’s in 15 (31.9%). Abnormal villous architecture was present in 8 (17%) cases [Figure 1]. Out of 8 cases, 2 cases showed partial villous atrophy (grade 3) and 6 showed fusions of apex and branching of villi (grade 2) [Table 2].
|Table 2: Significant histological findings in cases of Giardiasis
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|Table 3: Comparison of histological parameters between patients with and without Giardiasis
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The major histopathological features as observed in cases of giardiasis i.e. inflammation, increased IEL’s and villous abnormalities was compared between patients without giardiasis and those with giardiasis. The difference in inflammation and villous abnormalities was statistically significant (P < 0.001) whereas the difference in the number (%) of biopsies showing increased IEL’s was not statistically significant [Table 3].
Giardia lamblia is a worldwide pathogen and is the most commonly diagnosed enteric protozoal infection, found in the United States.  In India, prevalence rates of giardia infection in patients with diarrhea range from 0.4 % to as high as 70% in low socioeconomic groups.  Also a large majority (35-70%) of individuals infected with giardiasis remain asymptomatic. 
There is even a higher prevalence rate in developing nations (40%) in comparison to 2-7% in the US.  In India prevalence is high but due to lack of screening modalities this disease is underdiagnosed. Prevalence rate in our cohort was 13.42% (47 cases) which is a high. Most of these cases were chronic carriers. In present study prevalence of Giardia was higher in males as compared to females. Similar findings have also been reported in a large series of enteric giardiasis by Oberhuber et al.  Duodenum has been documented as the most common site of intestinal Giardiasis. Lebwohl et al. found it to be the site of giardiasis in 77.2% cases. 
Presence of giardiasis in 13.4% of endoscopically normal appearing duodenal biopsies from asymptomatic patients suggests that giardia infestation may not always present with classical symptoms and may be missed. These asymptomatic patients may play an important role in spreading the parasite. These findings have been substantiated by earlier reports. Lopez et al, Casemore et al. and Walker et al. found that 60-80% and 81.8% of infected cases of giardiasis to be asymptomatic. ,, Chronic giardiasis typically presents as malaise, abdominal bloating and discomfort i.e. non-specific symptoms.  Persistent infection and chronic disease in some patients may be explained by immune evasion due to antigenic variation in variant specific surface protein (VSP), regulated by a mechanism similar to RNA interference. 
Oberhuber postulated that since Giardia inhabits the upper duodenum and proximal intestine, hence non-specific upper gastrointestinal symptoms are a more common presentation than diarrhea in cases of giardiasis.  The above findings also emphasize the fact that endoscopic biopsies must be performed routinely even in asymptomatic cases.
A thorough literature search revealed that very few studies have focused on the histological changes in giardiasis hence a detailed histomorphological study of all cases with giardiasis was done in the present study. Villous changes were seen in 8 (17%) of our cases and this finding was significantly higher as compared with non-giardiasis patients. In a large study of 567 cases, Oberhuber et al. found duodenitis with mild villous flattening in 18 subjects (3.7%) cases which is less compared to our study. As most of our cases were asymptomatic and chronic carriers, repeated infection led to a higher incidence of villous abnormality as compared with Western data. 
Another study by Arevalo et al. showed a much higher incidence of villous atrophy than reported by other authors.  Thirty cases were included. Atrophic villous architecture was seen in 61.2%, increase in number of intraepithelial lymphocytes in 63.3% and the occurrence of lymphoid follicles in 43.3%. Cases with atrophy showed greater frequency of increase in the number of intraepithelial lymphocytes than cases without atrophy.  Also partial villous changes with increased IEL’s characteristic of celiac disease has been found in giardiasis, but they can be differentiated from the former on the basis of identification of giardia trophozoites.  Nonspecific duodenitis associated with giardiasis was the most common histological findings (36.91%), whereas raised IEL’s were found in 31.91% cases but the difference was not significant when compared to non-giardiasis cases. Oberhuber et al. also did not find a substantial increase in intraepithelial lymphocytes and postulated that increased IEL’s in symptomatic giardiasis could be the result of bacterial super infection which could provoke immigration of intraepithelial lymphocytes.  Crypt hyperplasia with increased intraepithelial lymphocytes have been reported in erlier studies. Recent studies using athymic mice have found that epithelial injury are T-cell dependant. Further research indicates that loss of brush border surface area, reduced dissacharides activities and decreased crypt to villous ratio are mediated by CD 8 + T cells whereas both CD 8 and CD 4 small mesentric lymph node T cells regulate the influx of intra-epithelial lymphocytes. 
Although the histological features associated with giardiasis were non-specific, a higher incidence of villous changes with increased IEL’s was found. This study highlights the importance of identification of giardia trophozoites in duodenal biopsies to avoid misdiagnosis of celiac disease or tropical sprue which may have similar clinical and histological features but different management strategies.
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Source of Support: None, Conflict of Interest: None
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