Screening of Auramine-Stained Smears of All Fecal Samples Is a Rapid and Inexpensive Way to Increase the Detection of Coccidial Infections

International Journal of Infectious Diseases (2008) 12, 47—50

http://intl.elsevierhealth.com/journals/ijid

Screening of auramine-stained smears of all fecal samples is a rapid and inexpensive way to increase the detection of coccidial infections

T. Hanscheid a,b,*, J. Melo Cristino a,b, M.J. Salgado b a Unidade de Microbiologia Molecular e Infecc¸a˜o, Instituto de Medicina Molecular, Faculdade de Medicina, Hospital Universita´rio de Santa Maria, Av. Prof. Egas Moniz, P-1649-028 Lisbon, Portugal b Laborato´rio de Microbiologia, Hospital de Santa Maria, Lisbon, Portugal

Received 25 July 2006; received in revised form 22 February 2007; accepted 13 April 2007 Corresponding Editor: J. Peter Donnelly, Nijmegen, The Netherlands

KEYWORDS Summary Coccidia; Introduction: Coccidia are important causes of diarrhea that is often indistinguishable from other Auramine; forms of community-acquired diarrhea. However, the detection of oocysts is often only per- Rapid screening; formed when explicitly requested, as part of the ova and parasite (O&P) examination. Reappraisal Cost-effective; and understanding of the accurate staining characteristics of auramine O (AuO), which stains Feces nucleic acids, may permit the inexpensive and reliable identiﬁcation of coccidian oocysts at routine workup of all fecal samples. Methods: AuO-stained smears were prepared from all stool samples received for stool culture in transport medium (SC) and from concentrated stools received for the ova and parasite (O&P) examination. Results: A total of 3732 samples for stool cultures and 3132 samples for O&P examinations were included. Ninety-one samples (1.3%) from 52 patients yielded Coccidia (45 Cryptosporidium spp and 7 Isospora belli). In seven cases oocysts were only detected in samples sent for stool culture in transport medium. The oocysts showed a typical staining pattern and were easy to recognize. The observation of one smear took only around 30 seconds, and the reagents and glass slide for one smear did not exceed US$ 0.03. Conclusions: The screening of all fecal samples with AuO-stained smears is a rapid and inexpensive way to increase the detection of coccidial infections, which in most laboratories can be incorporated into the microscopic workup for mycobacteria. # 2007 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Introduction

* Corresponding author. Tel.: +351 217999458; Coccidia are an important cause of diarrhea, and diarrhea fax: +351 217999459. caused by Coccidia is clinically very difﬁcult to distinguish from E-mail address: [email protected] (T. Hanscheid). community-acquired diarrhea caused by other pathogens,

Table 1 Distribution of positive samples from patients with coccidial infection

Sample type received Only SC Only O&P SC and O&P Positive sample Only SC Only O&P Only SC Only O&P SC and O&P Cryptosporidium spp 41117 22 Isospora belli 2202 1 SC, stool culture; O&P,ova and parasite examination. In six cases only samples for stool culture were received and in 13 cases only samples for ova and parasite examination. such as bacteria. Detection is typically based on microscopic In mycobacteriology, auramine O (AuO) has widely observation of smears, exploiting the acid-fast properties of replaced carbolfuchsin, especially because of the ease of the organisms.1 However, using carbolfuchsin-stained smears observing smears at low power (200Â). Interestingly, AuO has often requires the use of high-power microscopy (1000Â), been reported to yield ambiguous results when used to screen which is cumbersome and time-consuming. Other detection for Coccidia.6 However, reappraisal of the often unknown methods appear to be more sensitive, such as antigen detec- nucleic acid staining characteristics of AuO shows that AuO tion assays, immunoﬂuorescent staining (IF),2 and nucleic acid produces very characteristic images of coccidian oocysts. ampliﬁcation methods including real-time PCR.3 However, The aim of this study was to investigate if AuO staining is these methods are expensive and just the cost for reagents a feasible and inexpensive way to screen for Coccidia in all can be in the order of US$ 2—8.4 Possibly as a result of this, fecal specimens submitted for O&P examination as well as diagnostic guidelines such as the Cumitech 30A recommend fecal specimens sent in a transport medium for the detection the screening for Coccidia only if explicitly requested, as part of bacterial pathogens. of the ova and parasite (O&P) examination.1 Furthermore, leading guidelines from the Infectious Diseases Society of Materials and methods America suggest only bacterial cultures in the work-up of community-acquired diarrhea.5 Following these recommenda- All fecal samples submitted to the laboratory for the detec- tions, it is possible that coccidial infections may be missed or tion of bacterial pathogens (stool cultures, SC) or examina- diagnosed with a considerable delay. tion for O&P were screened for the presence of coccidian

Figure 1 Images of Cryptosporidium (A, B) and Isospora belli (C, D) oocysts. Auramine O stained smears prepared from unconcentrated stools submitted in transport medium. Typical auramine O staining patterns due to the nucleic acid staining inside the sporozoites (C) and sporocysts (D) (see text for detailed description). Magnification: top row, 200Â, bottom row, 1000Â. (Microscope: Leica DM2500, Wetzlar, Germany; filterset L5, blue; excitation: bandpass filter 460—500 nm, dichroic mirror 505 nm; emission: bandpass filter 512—542 nm). Screening of feces for Coccidia using auramine 49 oocysts. Smears from SC samples were prepared using only in the case of prolonged diarrhea (>7 days).5 Applying 10—20 ml of the stool—ETMTM transport medium mixture such a policy, coccidial infections may be missed or diagnosed (AlphaTec, Vancouver, WA, USA). Samples for O&P exam- with a considerable delay. inationwerefirstconcentratedusingthestandardformol— Some studies have investigated the screening of all fecal ethyl acetate procedure,1 before using 10—20 ml to prepare samples for Coccidia, although samples were usually concen- smears. Smears were dried and stained according to the trated as part of the O&P examination.8,9 Also, in some phenol auramine O protocol commonly used for staining countries, such as the UK, guidelines recommend the screen- mycobacteria.7 After air drying, the whole area of the ing for coccidial infections in all symptomatic indivi- smear was examined with a 10Â objective using a fluor- duals.10,11 In this study, the routine examination of SC lead escent microscope (Zeiss, Go¨ttingen, Germany), with to the detection of seven coccidial infections, while in 23 of the L09 longpass filterset (excitation: bandpass filter the 33 cases (69%) the oocysts were also easily detectable in 450—490 nm, dichroic mirror 510 nm; emission: longpass the unconcentrated SC samples, despite the dilution effect of filter 515 nm). the transport medium. Many laboratories use carbolfuchsin-stained smears, Results which require prolonged microscopic examination, with reported observation times of up to seven minutes per smear.4 Lower observation times may decrease the sensitiv- A total of 3732 samples for stool cultures and 3132 samples ity. Thus, it is not too surprising that AuO-stained smears for O&P examinations were included. Ninety-one samples seem to have a higher yield than carbolfuchsin, only sur- (1.3%) from 52 patients yielded Coccidia (45 Cryptosporidium passed by indirect immunofluorescent staining.8,12 Yet, AuO, spp and 7 Isospora belli). Of the 52 patients, 38 were adults often used in combination with rhodamine B, has not been and 14 children, while 42 were HIV-infected. The distribution generally accepted. This is possibly because it is felt that of positive samples from patients with coccidial infections is coccidial oocysts are difficult to distinguish from other summarized in Table 1. fluorescent particles, with a high yield of ambiguous Detection and identification of the coccidian oocysts was results.6 This may be due to the wide-held belief that AuO easy and observation of each smear took only approximately stains the wall of acid-fast organisms,6,13 as endorsed by 30 seconds (Figure 1). No staining of the cyst wall of either authoritative textbooks.14 In fact, AuO has been reported to Cryptosporidium or Isospora was observed. The typical pat- stain the cyst wall of Coccidia,6 and stained Cryptosporidium tern of Cryptosporidium oocysts consisted of a heterogeneous oocysts have simply been described as fluorescent round intracellular staining, which at higher amplification can be bodies.4,8 recognized as localizing in the sporozoites (Figure 1, B). However, a reappraisal of the AuO staining characteristics Oocysts of Isospora belli showed either a heterogeneous seems overdue, as already back in 1951 it was reported that staining of the interior of the cyst (Figure 1, D1 and D2) or AuO seems to be a nucleic acid stain.15 Supporting this finding staining was localized to the sporocyst (Figure 1, D3 and D4). are recent reports on the fluorescent enhancement of AuO on Contrary to this, artifacts were recognizable by their often binding to DNA/RNA and confocal microscopy images of irregular shape and homogeneous staining without any dis- mycobacteria, showing a heterogeneous intracellular stain- cernable interior staining pattern, in the majority of cases ing pattern.16 Cryptosporidium oocysts can show the pre- easily visible at low power (100Â—200Â). viously reported ‘erythrocyte’ staining appearance, however In the 23 cases where samples for stool cultures and for at only a very low magnification (100Â). Closer observations O&P examination were positive, it was noted that the fecal of the oocysts with 200Â or higher magnification reveals the concentration for the parasite examination increased the typical heterogeneous staining property of an intracellular number of oocysts observed by a factor of 3—10. However, nucleic acid stain (Figure 1): in Cryptosporidium oocysts the in seven cases oocysts were only detected in samples sent for AuO stains the nucleic acids located within the sporozoites, SC in transport medium (Table 1). which become visible (Figure 1, B, insert). In the case of Based on the average observation time of 30 seconds per Isospora belli, the sporocyst(s) are easily recognizable as smear, the total time for observing the 3732 smears prepared brightly fluorescent round bodies, located within the egg- from SC samples was 31.1 hours (an average of 1 hour per shaped non-fluorescent oocyst wall (Figure 1, D3 and D4). positive case), while the total time for the 3132 O&P samples However, in some cases the sporocysts are absent and the was 26.1 hours (an average of 0.6 hours per positive case). fluorescence is heterogeneously distributed within the Iso- The total cost for reagents and glass slides for all smears did spora oocysts (Figure 1, D1 and D2), possibly reflecting a not exceed US$ 250. heterogeneous distribution of nucleic acids. However, to obtain best results, laboratories may want Discussion to consider three important aspects: (1) Screening with which objective? Although screening can be done using Screening of AuO-stained slides of fecal smears is a rapid and the 10Â objective, suspicious images should be checked inexpensive way to detect Coccidia. Coccidial infections with the 20Â,40Â, or if indicated the 100Â oil immersion often present with symptoms of acute gastroenteritis and objective. (2) Bandpass or longpass filter? Auramine O and physicians may only send stools for bacterial culture. In fact, rhodamine B can be excited with light at a wavelength of guidelines on the diagnosis and treatment of infectious around 460—480 nm (blue—green) and emit across a wide diarrhea from the Infectious Diseases Society of America spectrum, beyond 700 nm, with emission peaks in the low recommend this as the initial approach in patients with 500 nm region for AuO and high 500 nm region for rhoda- community-acquired diarrhea and reserve O&P examinations mine B (http://omlc.ogi.edu/spectra/PhotochemCAD/ 50 T. Hanscheid et al. html/index.html). Most fluorescent microscopes use band- References pass filters of around 460—480 nm for excitation and longpass (LP) emission barrier filters around 510 nm. Thus, 1. Gracia LS. Cumitech 30A. Selection and use of laboratory all wavelengths of fluorochromes emitting above 510 nm procedures for the diagnosis of parasitic infections of the (green, orange, and red) are visible. Consequently, when gastrointestinal tract. Washington D.C.: American Society for stainingwithAuO(andrhodamineB)theuseofsuchanLP Microbiology; 2003. barrier filter can make an object fluoresce so brightly that it 2. Garcia LS. Diagnostic medical parasitology. In: Truant AL, editor. is difficult to discern internal structures. Thus, some micro- Manual of commercial methods in clinical microbiology. Washing- ton D.C.: American Society for Microbiology; 2002. p. 274—305. scopists may find the images clearer by using a bandpass 3. Verweij JJ, Blange RA, Templeton K, Schinkel J, Brienen EA, van barrier filter (BP) that only allows green light to pass within Rooyen MA, et al. Simultaneous detection of Entamoeba histo- a range of 30—40 nm (Figure 1). (3) AuO alone or AuO/ lytica, Giardia lamblia, and Cryptosporidium parvum in fecal rhodamine B mixture? Concerning the use of rhodamine B, it samples by using multiplex real-time PCR. J Clin Microbiol should be noted that it is not known to stain nucleic acids 2004;42:1220—2. and it seems very likely that rhodamine B causes a rather 4. MacPherson DW, McQueen R. Cryptosporidiosis: multiattribute nonspecific staining of the oocysts. This makes it difficult to evaluation of six diagnostic methods. J Clin Microbiol 1993;31: distinguish oocysts from artifacts, which would explain the 198—202. findings reported in several studies.4,6,10 Thus rhodamine is 5. Guerrant RL, van Gilder T, Steiner TS, Thielman NM, Slutsker L, not only unnecessary, but may render the observation more Tauxe RV, et al. Practice guidelines for the management of infectious diarrhea. Clin Infect Dis 2001;32:331—51. difficult. 4 6. Baron EJ, Schenone C, Tanenbaum B. Comparison of three Contrary to previous reports, we found that AuO methods for detection of Cryptosporidium oocysts in a low- reagents are very inexpensive and easy to prepare in-house prevalence population. J Clin Microbiol 1989;27:223—4. amounting to no more than US$ 0.03 per sample. Often, 7. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Wa- many more smears are stained to screen for mycobacteria shington Jr CW. Color atlas and textbook of diagnostic micro- and thus the additional staining of a few fecal smears per biology. Philadelphia: Lippincott; 1997. p. 1337—9. day adds very little additional technician time for the 8. Casemore DP, Armstrong M, Sands RL. Laboratory diagnosis of staining. 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Laboratory ascertainment of Cryptosporidium and ing of all fecal samples for Coccidia as part of the O&P local authority policies for investigating sporadic cases of cryp- examination. Furthermore, it may also be useful to extend tosporidiosis in two regions of the United Kingdom. Commun Dis this screening to fecal samples submitted for bacterial cul- Public Health 2002;5:114—8. ture. Both approaches may reduce the number of clinically 12. Arrowood MJ, Sterling CR. Comparison of conventional staining late diagnosed or even otherwise undiagnosed cases of coc- methods and monoclonal antibody-based methods for Cryptos- poridium oocyst detection. J Clin Microbiol 1989;27:1490—5. cidial infections. Furthermore, it may contribute to the 13. Fukunaga HT, Murakami T, Gondo KS, Ishihara T. Sensitivity of control of this infection with the (earlier) availability of acid-fast staining for Mycobacterium tuberculosis in formalin- results for public health professionals and a structured sur- fixed tissue. Am J Respir Crit Care Med 2002;166:994—7. veillance system. 14. Chapin KC, Lauderdale T. Reagents, stains and media: bacter- iology. In: Murray PR, Baron EJ, Pfaller MA, Jorgensen JH, Yolken Acknowledgments RH, editors. Manual of clinical microbiology. 8th ed. Washington D.C.: American Society for Microbiology; 2003. p. 365. 15. Oster MG. [Fluorescence of auramine O in the presence of We are indebted to Dr Howard M. Shapiro from The Center for nucleic acid] (article in French). C R Hebd Seances Acad Sci Microbial Cytometry (West Newton, MA 02465-0031, USA) for 1951;232:1708—10. sharing with us his research and knowledge on the staining 16. Hanscheid T, Ribeiro CM, Shapiro HM, Perlmutter NG. 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