| Abstract|| |
Objective: Direct microscopic visualization is the most specific method for detecting intestinal parasites and is commonly achieved by stool examination or mucosal biopsy. However, postfixation, the intestinal biopsy fragment is often curled, and the entire surface of the biopsied mucosa is seldom viewed microscopically. Tissue processing further distorts morphology of the organisms and causes diagnostic difficulties. Examining multiple sections for parasite detection is time-consuming and often requires aid of special stains and/or immunohistochemistry. To overcome these disadvantages, we hypothesized that the fixative in which biopsies are transferred may provide a valid representation of the biopsied mucosal surface and therefore aid in the identification of mucosal surface parasites.Materials and Methods: Formalin in which biopsies were transferred was retained, stored at 4°C and processed with a cytocentrifuge. Totally, 120 consequent duodenal biopsy fixatives were processed in this way and the cytocentrifuged smears visualized after May-Grunwald-Giemsa staining. Findings of these smears were correlated with their corresponding formalin fixed paraffin embedded tissue sections.Results: Cytocentrifuged formalin preparations were found to be representative of the mucosal surface contents. Giardia trophozoites were visualized in 10/120 preparations with distinct morphological characteristics which were seldom appreciable in tissue sections, eliminating the need for special stains. Furthermore, two of the corresponding histology sections did not demonstrate the parasites despite step sections, while in one case few parasites could be identified in the step sections. Conclusions: Cytocentrifuged fixative preparation is a simple and cost-effective technique which can be routinely employed for intestinal parasite characterization.
Keywords: Cytocentrifugation, fixative, formalin, Giardia, intestinal parasites
|How to cite this article:|
Joshi P, Das P, Iyer V, Gupta SD. Cytocentrifuged biopsy fixative preparation: A simple cost-effective technique facilitating microscopic diagnosis of lumen-dwelling intestinal parasites. Indian J Pathol Microbiol 2017;60:202-5
|How to cite this URL:|
Joshi P, Das P, Iyer V, Gupta SD. Cytocentrifuged biopsy fixative preparation: A simple cost-effective technique facilitating microscopic diagnosis of lumen-dwelling intestinal parasites. Indian J Pathol Microbiol [serial online] 2017 [cited 2020 Feb 24];60:202-5. Available from: http://www.ijpmonline.org/text.asp?2017/60/2/202/208390
| Introduction|| |
Parasitic infections comprise a significant proportion of the spectrum of gastrointestinal diseases, especially in the developing countries. They are of considerable importance as they reflect the status of hygiene and sanitation in a population and cause a wide spectrum of clinical manifestations. A multitude of parasites are known to cause gastrointestinal diseases in humans, and accurate characterization of the organism is essential to determine both clinical relevance and duration of therapy. Current diagnostic options available include a broad array of techniques ranging from microscopic examination to immunological and molecular assays. The choice of diagnostics utilized varies depending on the degree of clinical suspicion.
Although a range of symptoms can be directly ascribed to intestinal parasites, in many cases they present as superadded infections over conditions such as celiac disease or inflammatory bowel disease, modifying their clinical course, or persist as silent/subclinical infections. The symptoms in many cases are also nonspecific. Specific targeted and expensive investigations are unlikely to be considered up front in these situations, and therefore it is imperative that a simple screening tool, done routinely, can identify and characterize the parasites with as high sensitivity and specificity as possible.
The most common of such screening tools employed is stool examination by microscopy, three separate samples of which can identify over 90% of the parasitic infections. The ideal number of samples to be examined however is debatable and varies with the prevalence of infections in the population along with the preparation technique and screener expertise.,, Concentration techniques and ELISA assays further improve the sensitivity, but overall these techniques are labor intensive and require high technical expertise. Further, in infections like Giardia, stool examination also has the disadvantage that the trophozoites disintegrate rapidly and cyst passage is extremely variable and not related to the clinical symptoms. Mucosal biopsies, though invasive, are commonly done during evaluation of various gastrointestinal diseases. The sensitivity of biopsies in the detection of Giardia is, however, variable and in general considered to be lesser than that of stool examination. This may be due to the focal/patchy nature of the infection, fixation, and tissue processing artefacts as well as due to the fact that the entire mucosal surface biopsied is seldom examined microscopically. Nevertheless, mucosal biopsies are often the last resort for establishing the diagnosis in cases where stool examinations have been negative.
Being concerned about the sensitivity of biopsies in identifying mucosal parasites and to offer unbiased screening and conclusive diagnoses to patients who have undergone endoscopic biopsies, we hypothesized that the fixative in which biopsy fragments are transferred is likely to be representative of the mucosal surface contents and therefore serve as an easy source for targeted screening of intestinal parasites.
| Materials and Methods|| |
Cytocentrifugation of fixative
A total of 120 sequential samples of duodenal biopsies, received in the Department of Pathology, were collected without any prior suspicion of parasitic infection or any other inclusion criteria. Instead of discarding the fixative (10% neutral buffered formalin) in which biopsies are transferred, the tissue pieces were picked from the containers using clean forceps and the remaining fixative and container were stored at 4°C for a variable period ranging from 1 day to 1 week. No mechanical agitation was applied while taking out the biopsy fragments. The entire volume of formalin, ranging from 5 to 8 ml, was then centrifuged at 2000 rpm for 5 min and the supernatant was decanted. The sediment was processed in a cytocentrifuge (Cyto-Tek, Japan) at 2000 rpm for 2 min, depositing contents in the sediment on a 14 mm × 14 mm area on the slide. The slides were then air dried, stained with May-Grunwald-Giemsa (MGG) and visualized in a light microscope by two pathologists independently. Water used during MGG staining was also centrifuged and used as negative control. As a protocol, the duodenal biopsies are collected in new disposable containers at our institute. Institutional Ethical Clearance and written informed consent from patients were obtained.
Corresponding formalin fixed paraffin embedded (FFPE) histology slides of the biopsy samples collected were retrieved and screened for parasites by two pathologists without any bias, blinded of the findings of cytocentrifuged smears. Subsequently, if the cytocentrifuged sample revealed parasites and biopsy slides did not, then further step sections (varying from 20 to 30) were taken and reviewed.
| Results|| |
Cytocentrifuged fixative is representative of the biopsied mucosal surface contents
The villous surface of duodenal mucosal biopsies showed the presence of mucin, desquamated and degenerated epithelial cells, bacterial colonies, nondescript granular material, red blood cells, and a few inflammatory cells. These were all represented in the cytocentrifuged samples and were not seen as efficiently in the FFPE slides [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d,[Figure 1]e,[Figure 1]f. Some of the slides, in addition, showed the presence of long fibers, likely derived from the tissue paper pieces on which biopsied mucosal fragments were placed for preserving orientation.
|Figure 1: Cytocentrifuged fixative is representative of the biopsied mucosal surface contents. Red blood cells (a; MGG, ×200), bacilli (b; MGG, ×600), degenerated epithelial cells (c; MGG, ×600), and mucin (d; MGG, ×600) are also observed in the cytocentrifuged preparations, similar to the mucosal surface contents seen in formalin fixed paraffin embedded sections (e; H and E, ×400 and f; H and E, ×200)|
Click here to view
Diagnosis of intestinal parasites
Trophozoites of Giardia lamblia could be identified in 10/120 (8.3%) of the cytocentrifuged samples. The pear shaped outlines of the parasites could be discerned in most cases at ×10 objective [Figure 2]a, and on further magnification (×40 objective) four pairs of flagella, axoneme, and two symmetrically arranged nuclei were clearly visible [Figure 2]b and [Figure 2]c. The identification of these features was aided by the predominant face-on deposition of these parasites on the slide as opposed to the side-on view (sickle-shaped) in many tissue sections. Extracellular mucin and desquamated cells, which are the closest mimics of the parasite in tissue sections [Figure 2]d, could be easily differentiated, even from parasites which showed mild degenerative changes [Figure 2]e and [Figure 2]f. No other known intestinal parasites could be identified in the 120 samples studied.
|Figure 2: Trophozoites of Giardia lamblia in cytocentrifuged formalin preparations. Giardia trophozoites in cytocentrifuged preparations (a; MGG, ×100) with easily appreciable flagella, axoneme and two nuclei (b and c; MGG, ×1000) in comparison to a formalin fixed paraffin embedded section (d; H and E, ×1000). Trophozoites with degenerative changes (e and f; MGG, ×600) and differentiation from mucin globules (arrows in e; MGG, ×600)|
Click here to view
Of the ten positive cytology samples, only seven corresponding histology sections showed the presence of the parasites. Of the remaining three cases, in one case, a few parasites could be identified on further step sections. FFPE sections which were marked suspicious for parasites showed typical trophozoite morphology on cytology preparation, thus eliminating the need for further step sections or special stains [Figure 3]a,[Figure 3]b,[Figure 3]c,[Figure 3]d,[Figure 3]e,[Figure 3]f. The numbers of parasites detected in a slide on histology and cytology were comparable. There was no case where parasites were identified in histology but absent in cytology preparations.
|Figure 3: Advantages of cytocentrifuged preparations over formalin fixed paraffin embedded sections in the identification of Giradia trophozoites. Suspicious profiles of Giardia in formalin fixed paraffin embedded sections (a and b; H and E, ×600) resolved in corresponding cytocentrifuged preparation (c; MGG, ×600). Case where G. trophozoites were not found in one slide (d and e; H and E, ×600) but could be identified in subsequent serial sections (f; H and E, ×600)|
Click here to view
Cytocentrifuged preparations of some cases, in addition, revealed the presence of crescentic and thin branching filamentous structures [Figure 4]. In one case, a ciliated organism was identified [Figure 4]a, while in another a structure resembling spermatozoa was present [Figure 4]b. However, none of these could be identified in the corresponding tissue sections, and they could not be characterized further.
|Figure 4: Miscellaneous findings in cytocentrifuged formalin preparations. Ciliated organism (a; MGG, ×1000), spermatozoa and cyst-like structure (b and c; MGG, ×600), branching hyphal forms (d; MGG, ×600), and crescentic structures (e and f; MGG, ×600) observed in cytocentrifuged preparations which could not be further characterized|
Click here to view
| Discussion|| |
Gastrointestinal parasitic infections are common in India with a prevalence rate ranging from 46.5% to 97.4%, showing wide variations depending on geographical and socioeconomic status. The worldwide prevalence ranges from 1% to 65%. These infections account not only for a variety of gastrointestinal symptoms but are also associated with a range of extraintestinal manifestations such as arthritis, ocular pathologies, and allergies.
In addition to primary infections, the rate of these infections occurring in preexisting conditions such as celiac disease and inflammatory bowel disease is also high., In these cases, superadded infections can worsen the symptoms  and also mimic refractory celiac disease unresponsive to gluten free diet. Identification of Giardia in the context of celiac disease is also important as the parasites can themselves cause a variable degree of villous atrophy and an increase in intraepithelial lymphocytes.
The technique described by us is likely to be most useful in these patients who undergo repeated endoscopic biopsies in the course of their primary disease and complement the process of parasite detection in biopsies done after repeated stool examinations have been negative. Our technique is of relevance for histopathologists, as cytocentrifuged fixative preparation along with routine FFPE slide examination can provide better diagnostic yield. This is especially important as the wide variability in the prevalence of intestinal parasitic infestations may not only be related to geographical variations but also may be related to the diligence of histopathologists in picking up the luminal parasites. This technique can be performed even at very low cost community set-up.
Stool examination has the inherent advantage in being noninvasive and representing the entire length of gastrointestinal tract (GIT) and is the investigation of choice when gastrointestinal infections are suspected. However, a targeted mucosal biopsy is often the primary modality identifying infections in cases with atypical presentations (without diarrhea) and also likely to yield more positive results in patients with a lower load of infection. When these biopsies have been done, our technique can ease the process of screening at minimal extra cost and effort. As the complete, intact parasite is being visualized in cytology, it facilitates finer structures of the organism to be appreciated, reducing dependency on step cuts, special stains, rarely immunohistochemistry  and electron microscopy, for diagnostic confirmation and thus may be very useful in resource-strapped health care centers and also improve turn-around time of reporting. In principle, our technique is similar to duodenal aspirate examination and endoscopic brush cytology, which have been reported to have a higher parasite detection rate, as compared to endoscopic biopsy., However, in comparison to a brush cytology smear, a cytocentrifuged preparation is much easier to screen and does not need a separate sample to be collected. A collection of gastric or duodenal aspirates also requires accessories such as endoscopic suction cannula and a special preservation technique for sample collection, which may further increase the cost of routine diagnostic screening.
Giardia is the most common parasite infecting GIT worldwide, and our findings reflect the same. The prevalence of 5.5% to 11% has been reported from North India, as also observed in our study (8.3%). Other lumen-dwelling parasites which are likely to be detected by this technique would be Entamoeba species, Cryptosporidium, Strongyloides, Isospora, Cyclospora, and Microsporidia. However, given the rarity of the later infections, much larger sample size needs to be screened to evaluate the sensitivity of this technique in their detection. For Entamoeba species, similar screening with colonic biopsies may be performed, which was not done in our study.
The advantages of cytocentrifuged fixative preparations are thus focused parasite screening, better and faster visualization, reduced dependency on ancillary techniques for confirmation and reduced turn-around time at minimal extra cost. It can also serve as a readily accessible sample for molecular analysis in research settings, circumventing the need for dissection/isolation from FFPE tissue sections and collection of separate aspirate/biopsy/stool samples. A disadvantage is that at least a cytocentrifuge is needed to implement this procedure, along with the routine histology set up.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Senay H, MacPherson D. Parasitology: Diagnostic yield of stool examination. CMAJ 1989;140:1329-31.
Cartwright CP. Utility of multiple-stool-specimen ova and parasite examinations in a high-prevalence setting. J Clin Microbiol 1999;37:2408-11.
Branda JA, Lin TY, Rosenberg ES, Halpern EF, Ferraro MJ. A rational approach to the stool ova and parasite examination. Clin Infect Dis 2006;42:972-8.
Wahnschaffe U, Ignatius R, Loddenkemper C, Liesenfeld O, Muehlen M, Jelinek T, et al.
Diagnostic value of endoscopy for the diagnosis of giardiasis and other intestinal diseases in patients with persistent diarrhea from tropical or subtropical areas. Scand J Gastroenterol 2007;42:391-6.
Kaur R, Rawat D, Kakkar M, Uppal B, Sharma VK. Intestinal parasites in children with diarrhea in Delhi, India. Southeast Asian J Trop Med Public Health 2002;33:725-9.
Kang G, Mathew MS, Rajan DP, Daniel JD, Mathan MM, Mathan VI, et al
. Prevalence of intestinal parasites in rural Southern Indians. Trop Med Int Health 1998;3:70-5.
Fletcher SM, Stark D, Harkness J, Ellis J. Enteric protozoa in the developed world: A public health perspective. Clin Microbiol Rev 2012;25:420-49.
Halliez MC, Buret AG. Extra-intestinal and long term consequences of Giardia duodenalis
infections. World J Gastroenterol 2013;19:8974-85.
Rostami Nejad M, Ishaq S, Al Dulaimi D, Zali MR, Rostami K. The role of infectious mediators and gut microbiome in the pathogenesis of celiac disease. Arch Iran Med 2015;18:244-9.
Yamamoto-Furusho JK, Torijano-Carrera E. Intestinal protozoa infections among patients with ulcerative colitis: Prevalence and impact on clinical disease course. Digestion 2010;82:18-23.
Iyer VH, Augustine J, Pulimood AB, Ajjampur SS, Ramakrishna BS. Correlation between coinfection with parasites, cytomegalovirus, and Clostridium difficile
and disease severity in patients with ulcerative colitis. Indian J Gastroenterol 2013;32:115-8.
Korkmaz U, Duman AE, Gurkan B, Sirin G, Topcu Y, Dindar G, et al.
Nonresponsive celiac disease due to Strongyloides stercoralis
infestation. Intern Med 2012;51:881-3.
Edling L, Rathsman S, Eriksson S, Bohr J. Celiac disease and giardiasis: A case report. Eur J Gastroenterol Hepatol 2012;24:984-7.
Hopper AD, Cross SS, McAlindon ME, Sanders DS. Symptomatic giardiasis without diarrhea: Further evidence to support the routine duodenal biopsy? Gastrointest Endosc 2003;58:120-2.
Sinelnikov I, Sion-Vardy N, Shaco-Levy R. C-kit (CD117) immunostain is useful for the diagnosis of Giardia lamblia
in duodenal biopsies. Hum Pathol 2009;40:323-5.
Klima M, Gyorkey P, Min KW, Gyorkey F. Electron microscopy in the diagnosis of giardiasis. Arch Pathol Lab Med 1977;101:133-5.
Patwari AK, Anand VK, Malhotra V, Balani B, Gangil A, Jain A, et al.
Brush cytology: An adjunct to diagnostic upper GI endoscopy. Indian J Pediatr 2001;68:515-8.
Zieglauer H, Höpfl-Kreiner I. The value of the “impression smear” in detecting Giardia lamblia
infection (author's transl). Padiatr Padol 1978;13:165-73.
Dib HH, Lu SQ, Wen SF. Prevalence of Giardia lamblia
with or without diarrhea in South East, South East Asia and the Far East. Parasitol Res 2008;103:239-51.
Department of Pathology, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]