Comparison of Three Collection-Preservation Methods for

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1981, p /81/ $02.00/0 Vol. 14, No. 6 Comparison of Three Collection-Preservation Methods for Detection of Intestinal Parasites DONALD L. PRICE Multidisciplinary Services and Research Institute, The University of Sarasota, Sarasota, Florida Received 11 August 1980/Accepted 30 June 1981 A study was designed to compare the effectiveness and time efficiency of three collection-preservation methods individually and grouped to form systems. Employees and members of their families of an agricultural grower in Hillsborough County, Fla., provided all fecal specimens used in the study. The 160 individuals participating were members of 64 families and represented a cross section of all job activity levels including management. Specimens were collected in Merthiolate/iodine/Formalin, Formalin (10% neutral buffered solution), and polyvinyl alcohol fixative. Parasites were found in the specimens from 39 (24.4%) of the 160 individuals participating, which represented 29.7% of the 64 families. No single method was effective in recovering all of the parasites found. When the methods were grouped to form diagnostic systems, the Merthiolate/iodine/Formalin system appeared to be more effective for parasite recovery as well as more time efficient than the Formalin/polyvinyl alcohol fixative system. Intestinal parasites cause untold discomfort and debility among much of the world's population and are widely distributed in the United States. The Centers for Disease Control have reported that 15.6% of 414,820 fecal specimens examined at state and territorial public health laboratories during 1976 contained one or more pathogenic or nonpathogenic parasites and that 18.3% of 363,567 did so in 1977 (2, 3). Although these results do not provide sufficient data to determine a true prevalence rate, they do indicate that major sanitation problems exist in many localities and suggest that potential public health problems may also exist. Unpublished reports from the Florida State Regional Laboratory at Tampa indicate a particularly high prevalence of parasitic infections in rural agricultural communities. A study, initiated in southern Hilsborough County, Fla., was designed to test the effectiveness and efficiency of three methods currently in use for collecting and preserving fecal specimens as a part of the process of recovering and identifying intestinal parasites. A second objective of the study was to determine the prevalence of the parasites in a population with a known structure living and working in a rural agricultural environment. MATERIALS AND METHODS Employees and their families from Speedling, Inc., an agricultural grower in southern Hillsborough County, Fla., supplied single fecal specimens used in the study. The participants in the study represented most employment areas of the company including management, office, engineering, greenhouse, and transportation personnel. Fecal cartons were distributed to employees by management personnel with instructions to return them to a collection point on their way to work in the morning. Laboratory personnel were present to process, fix, and preserve fecal specimens as thev were brought to the collection point. For collection-preservation of fecal specimens, three preservatives were used: MIF (Merthiolate/iodine/ Formalin) (7, 8), Formalin (10S% neutral buffered solution) (5), and PVA (polyvinyl alcohol fixative) (5). MIF-preserved specimens were processed and examined by direct smear as described by Price (7) and after a modified Formalin-ether sedimentation (6, 7). After standing undisturbed for an hour or more, an interface layer usually formed in the MIF-preserved specimen vial. Samples for examination were drawn from the base of the interface layer using a 5-mmdiameter, straight, glass pipette. Where interface layers were not formed, the was drawn from onethird the distance from the base of the vial to the surface of the solids layer. For Formalin-ether sedimentation, the MIF-preserved specimen was shaken, poured into a 15-ml conical centrifuge tube, and mixed with applicator sticks to break up any stubborn particles. All but 1.5 ml of the specimen was returned to the original vial, and the concentration procedure was performed on the in the centrifuge tube (6). Wet preparations to be examined were ringed with Vaspar (equal parts of Vaseline and paraffin) so that they could be reviewed if there was a question regarding identification. All prepared slides were examined using a 1Ox objective and a 1Ox ocular, with confirmation of identifi- 656

2 VOL. 14, 1981 cation made using the same ocular and the 100x oil immersion objective. Formalin-preserved specimens were examined directly using Dobell and O'Connor's iodine to aid in finding and identifying parasites (5). A Formalin-ether sedimentation, as described for MIF-preserved specimens, was performed on each Formalin-preserved. For examination, a few drops of Dobell and O'Connor's iodine was added to the sediment, and wet preparations were ringed with Vaspar and examined in the same manner as were the MIF-preserved specimens. PVA-preserved specimens were centrifuged at 2,000 rpm for 2 min, the supernatant fluid was poured off, and a portion of the sediment was placed on blotting paper. When the edges of the specimen began to dry, a small portion of the specimen was carefully spread on each of several slides. More even distribution of the heavier cysts in the specimen has been observed using a lower centrifuge speed and a shorter time, a modification of the technique developed by the California State Health Department and described by Melvin and Brooke (5). Slides were dried for a minimum of 2 h, but usually overnight, and when thoroughly dry, they were stained with Wheatley's modified trichrome stain (5). Stained slides were covered and scanned using a lox ocular and a 10x objective, and then 150 or more microscopic fields were examined using the same ocular and a 100x oil immersion objective. Measurements of the average time required for each procedure included all processing relative to the procedure after the specimen fixed in one of the preservatives had reached the laboratory. For the Formalinand MIF-fixed specimens, the time including apparatus setup was essentially the same and was easily measured since the entire processing could be completed without a break. Since the PVA-fixed specimens require a special setup and had several breaks in time when other work could be performed, a special method was employed to determine the time. Preparation of solutions and activities related to the maintenance of the staining setup were prorated over the number of specimens processed. Although actual drying time for prepared slides was not included, the extra handling time was taken into account, as was the setup time for mounting cover glasses. Without considering all such details, an accurate measurement of time would not have been possible. The average number of fecal specimens per week for laboratories is approximately 10. Two separate calculations were made, one for laboratories processing 1 or 2 slides daily, and one for those able to process 10 or more slides in a batch. The time used for actual slide handling was calculated by subtracting all time unrelated to the staining and processing from the total time elapsing for preparing slides, staining slides, and mounting cover glasses. To insure that no method was favored, only one slide was examined from each specimen processed by each method, making a total of five slides examined for each specimen collected. A limit of 15 to 20 min was allowed for the examination of each slide, simulating the time usually allocated in a clinical laboratory environment. All slides were examined for at least the INTESTINAL PARASITES: METHODS COMPARISON 657 minimum 15 min. In cases where technical personnel required confirmation of an identification and it could not be done immediately, the slides were marked indicating the area of the parasite on the slide, and confirmation was made later, usually within 2 days. Time for confirmation was not included in the time analysis study. RESULTS Parasites, representing seven species, were found in fecal specimens of 39 (24.4%) of the 160 individuals participating in the study (Table 1). Those participating were from ail levels of activity of Speedling, Inc., including management, and were representative of 64 families. Nonpathogenic or pathogenic parasites were found in one or more members of 19 (29.7%) families. Both cysts and trophozoites of protozoa were found in some specimens but, where trophozoites were found, cysts of the same species were also present in the specimen. The three collection-preservation methods used produced varied results in relation to the numbers and species of parasites recovered (Table 2). The Formalin-ether sedimentation method applied to the MIF-preserved specimen yielded the largest number of parasites found by a single method, recovering 40 of the 57 parasites found by all methods combined (Tables 2 and 3). Direct examination of the Formalin-preserved specimen and the trichrome-stained, PVA-preserved specimen yielded the smallest number of parasites, recovering 23 and 25, respectively, of the 57 parasites found (Tables 2 and 3). Formalin and PVA are often used as a "system" consisting of examination of the Formalinfixed directly and after Formalin-ether sedimentation, and examination of a trichromestained slide prepared from the PVA-preserved specimen. Using this system, 38 of the 57 para- TABLE 1. Number offecal specimens examined, representing 160 individuals, in which each species ofparasite was found No. of Parasite specimens with % with parasites parasites Protozoa Pathogens: Giardia lamblia Nonpathogens Entamoeba coli Entamoeba hartmanni Iodamoeba butschlii Endolimax nana Helminths Nematodes (roundworms): Trichuris trichiura Cestodes (tapeworms): Hymenolepis nana

3 658 PRICE J. CLIN. MICROI3IOL. TABLE 2. Numbers of individuals having positive feval s for each parasite species and the number detected by each method No. having positive fecal Method Enta- Enta- Iodamoeba Endolunioax Giar-dia Hvmenole- Trichuris rnoeba nioeba coli harinanni butschlii nana lam blia pis nana trichiura All MIF direct Formalin-ether on MIF l Direct + Formalin-ether on MIF' Formalin direct Formalin-ether on Formalin Direct + Formalin-ether on Formalin Trichrome on PVA Direct + Formalin-ether on Formalin + trichrome on PVA< " Either of the combinations of direct and sedimentation on the MIF or direct and sedimentation on the Formalin + trichrome on the PVA constitutes a diagnostic system for detection of intestinal parasitic infections. TABLE 3. Comparison of methods and systems for nonpathogens and pathogens Methods and systems Nonpatho- Pathogens gens Total parasites found MIF direct Formalin-ether on MIF Formalin direct 6 17 Formalin-ether on Formalin Trichrome on PVA 9 16 Formalin/PVA system MIF system 25 2:3 sites were recovered, whereas the MIF system, consisting of examination of the MIF-preserved specimen directly and after Formalin-ether sedimentation, recovered 48 of the 57 parasites found by a combination of all methods (Table 3). The average time required for all processing associated with each method is presented in Table 4. In many laboratories, a sufficient number of specimens are submitted daily to allow batch processing. The average times required for batch processing of 10 specimens is given in Table 5, demonstrating a lesser time for the MIF system than for the Formalin/PVA system. DISCUSSION The percentage (24.4%) of individuals providing fecal specimens in which parasites were found was about as expected. State Health Laboratories in Florida for similar populations and periods of time reported a prevalence of 15.8%. In an unpublished study conducted in Sarasota TABLE 4. Methods and systems Time comparison for individual methods and systems Time required (min)` Slide prepn Slide examination MIF direct 2 15 Formalin-ether on 6 15 MIF Formalin direct 2 15 Formalin-ether on 6 15 Formalin PVA 6 NA" Trichrome staining of slides Formalin/PVA 74 (3 slides) 50 system MIF system 8 (2 slides) 30 ` Includes all time involved after receiving a specimen in the fixative for completing the examination. h NA, Not applicable. County in which 150 individuals representing various economic strata, including 29 migrant agricultural workers, provided fecal specimens, a prevalence of 39.3% was reported. Parasites were found in the fecal specimens of 39 of the employees of Speedling, Inc., 21 specimens having pathogens (Table 1). Giardia lamblia was the predominant parasitic infection in the study and in the State of Florida laboratory reports. The 160 individuals participating represented 64 families, but not all family members participated in the study. Since all parasites found, either pathogenic or nonpathogenic, are transmitted through contamination by the oral route, family members not participating may

4 VOL. 14, 1981 TABLE 5. Time comparison for systerns with batch processing of 10 specimens Systems Time required (min) Prepn tion Total Formalin/PVA system Formalin PVA slide 30 NA" 30 Trichrome staining Total time, 10 s 614 Total time, each 61 MIF system Total time, 10 s Total time, each 32 Time difference favoring 29 MIF system, each "NA, Not applicable. have been infected also. Many of the participants and nonparticipants were taking medication for intestinal problems which may have affected the numbers of parasites present in specimens and, to some extent, the number of individuals found infected. Only a single fecal specimen was obtained from each individual. Since there are great variations in the numbers of parasites present in fecal specimens from day to day, a somewhat higher prevalence might be expected. On the other hand, individuals having intestinal symptoms are more likely to participate in a voluntary study. Work conducted after the study was completed suggests a balancing of these two factors, making the prevalence figures in the studies relatively accurate. Finding the parasites posed a greater problem than identifying those found, as indicated by the results. It follows that the most efficient and effective method or methods should be used when possible. None of the methods used independently were totally effective (Table 3). When two or more methods are used in combination, they constitute a system. The system should be effective for recovery of trophozoites and cysts of protozoa, eggs of helminths, and juvenile nematodes. Each procedure has its advantages and disadvantages. The three collection-preservation methods meet these requirements as follows. (i) Formalin. Formalin fixes and preserves cysts of protozoa and eggs of helminths. Juvenile nematodes are usually but not always fixed adequately for identification. Trophozoites of protozoa are often inadequately fixed. Formalinpreserved specimens can be concentrated by the Formalin-ether sedimentation method. INTESTINAL PARASITES: METHODS COMPARISON 659 (ài) PVA. PVA fixes and preserves protozoan trophozoites and cysts, although some cysts may be distorted. Eggs of helminths and juvenile nematodes are also preserved but are not usually diagnosed on permanent slides. PVA-preserved specimens are usually smeared on slides and stained with hematoxylin or trichrome stains. The method is considered excellent for protozoa but not for helminths, and PVA-preserved specimens do not concentrate well in commonly used concentration methods. Concentration methods, by definition, should increase the number of parasites present in a given volume of feces. Since a permanent smear from PVA-preserved specimens is equivalent to a direct smear in volume, any concentration method performed on the PVA-preserved specimen should increase the number of parasites found on the slide prepared from the concentrate. Garcia et al. (4) conducted a study comparing trichrome-stained slides and Formalinether sedimentation on PVA-preserved fecal specimens from 13,194 outpatients. The paper reported that far greater numbers of protozoa were found on examination of the stained direct smears than in the concentrate from the Formalin-ether sedimentation. I have experienced similar results. (iii) MIF. MIF fixes, stains, and preserves trophozoites and cysts of protozoa, eggs of helminths, and juvenile nematodes. MIF specimens are readily concentrated by the MIFC method (1) or by a modified Formalin-ether sedimentation method (6). Formalin-ether sedimentation will concentrate only those stages of parasites well preserved and is generally considered suitable for cysts of protozoa and eggs of helminths. It can be used on Formalin- and MIF-preserved specimens. Either the Formalin/PVA system or the MIF system as described is satisfactory for the recovery of the stages of intestinal parasites. A comparative effectiveness of each method individually and when combined in the two systems is given in Tables 2 and 3. Some parasites were recovered by each system that were not recovered by the other. Of the total of 57 parasites recovered, the Formalin/PVA system recovered 66.7%, whereas the MIF system recovered 84.2%. The Formalin/PVA system requires the examination of three prepared slides, which accounts in part for the longer time, whereas the MIF system requires only two, and results were obtained indicating a greater effectiveness. Examination of each fecal specimen was performed as it would be in a clinical laboratory environment. There is little doubt that by re-

5 660 PRICE peated examination of the specimens prepared by each method, most of the parasites missed could eventually be found. Some differences in parasite recovery by the two systems may be attributed to slight modifications in procedures. I consider the modifications employed to be improvements in procedures. The efficiency as well as the effectiveness of the procedures and systems was being tested. For this aspect of the study, repeated examination of the specimen was not considered feasible. In Table 4, the average time required for each method performed on a single specimen is presented. Although the time for examination of the prepared slides was essentially the same for each method, the preparation time for stained smears from the PVA-preserved specimens took considerably longer. By batching specimens in groups of 10, the time required per specimen for maintenance of the staining setup and the time for preparation can be reduced. It takes approximately the same time to conduct all steps necessary to stain slides with trichrome, including setup, placing slides in racks, and transferring them between solutions whether there are 2 or 25 slides; but the greater the number of slides, the greater the time required for preparing the smears and mounting cover glasses on the stained smears. There is not, therefore, a direct reduction in time required by increasing the number of specimens processed. J. CLIN. MICROBIOL. Table 5 presents the differences in time required for the two systems. The MIF system was more time efficient in that less time was required for preparation and examination of the specimen. Under the conditions and within the procedures used in this study, the MIF system appeared to be more effective and efficient than the Formalin/PVA system. ACKNOWLEDGMENTS I thank the staff of the Hillsborough County Migrant Health Center, Fla., for coordinating the study, and the staff of Speedling, Inc., for their cooperation and participation. Studies were supported, in part, by Marion Laboratories, Inc., Kansas City, Mo. Some equipment and materials used were furnished by Scientific Products, Ocala, Fla. LITERATURE CITED 1. Blagg, W., E. L. Schloegel, N. S. Mansour, and G. I. Khalaf A new concentration technic for the demonstration of protozoa and helminth eggs in feces. Am. J. Trop. Med. Hyg. 4: Center for Disease Control Intestinal parasite surveillance, annual sumrnary, Center for Disease Control, Atlanta, Ga. 3. Center for Disease Control Intestinal parasite surveillance, annual summary, Center for Disease Control, Atlanta, Ga. 4. Garcia, L. S., T. C. Brewer, and D. A. Bruckner A comparison of the formalin-ether concentration and trichrome-stained smear methods for the recovery and identification of intestinal protozoa. Am. J. Med. Technol. 45: Melvin, D. M., and M. M. Brooke Laboratory procedures for the diagnosis of intestinal parasites, p Publication no. (CDC) Center for Disease Control, Atlanta, Ga.. 6. Price, D. L In Hepatic, intestinal, and pulmonary trematodes. In CRC handbook series in clinical laboratory science, sect. E: Clinical microbiology 2: Price, D. L Culturette brand MIF procedure kit. Marion Scientific Corp., Kansas City, Mo. 8. Sapero, J. J., and D. K. Lawless The "MIF" stain-preservation technic for the identification of intestinal protozoa. Am. J. Trop. Med. Hyg. 2: