Abstract
Objectives
Diagnosis of Platynosomum fastosum Kossack 1910, a liver fluke of cats, can be challenging, with infections potentially not identified depending on the fecal analysis method used. The aim of this study was to compare three methods for identification of P fastosum eggs in feces.
Methods
Feces from 50 cats with unknown infection status from a location endemic for P fastosum were analyzed. The methods used included double centrifugation with Sheather’s sugar flotation solution (specific gravity 1.28); the Mini-Parasep (Apacor) flotation kit with ZnSO4 (specific gravity 1.20); and the Mini-Parasep sedimentation kit with Triton X/ethyl acetate solution.
Results
Thirty-four cats were identified as positive for P fastosum. Of these cats, 33 were identified with the double centrifugation method, 16 with the Mini-Parasep flotation kit and 11 with the Mini-Parasep sedimentation kit. Using the total number of samples identified as positive by at least one method as the true positive, the sensitivity of the methods was 97.1% for double centrifugation, 47.1% for the Mini-Parasep flotation kit and 32.4% for the Mini-Parasep sedimentation kit. Other parasite eggs (eg, Ancylostoma tubaeforme, Trichuris species and Toxocara cati) also were detected more frequently with the double centrifugation method.
Conclusions and relevance
Of the three methods tested, double centrifugation with Sheather’s sugar flotation solution was the most effective in identifying cats with P fastosum and other helminth eggs in their feces. The lower specific gravity of the flotation solution in the Mini-Parasep flotation kit likely influenced the results. A primary drawback of the Mini-Parasep sedimentation kit was the amount of sediment. While more sediment was examined than recommended in the manufacturer’s instructions, this was still only a small portion of the total sediment. In addition, reading the slides took longer than with the other methods.
Introduction
Platynosomum fastosum Kossack 1910 (Subclass Digenea, Family Dicrocoeliidae; synonyms Platynosomum concinnum, Platynosomum illiciens and Platynosomum planicipitus) infects the liver, gall bladder and bile ducts of cats.1,2 Cats become infected by consuming the second intermediate host (isopods; Oniscidea species) or paratenic hosts (lizards; Anolis species, Hemidactylus species) containing infective metacercariae.1,3 It is most commonly found in tropical and subtropical regions worldwide, with reports also in cats imported from endemic regions. Most cats are asymptomatic but clinical signs (eg, progressive anorexia, lethargy and icterus) and even death can occur with chronic infections and high-intensity infections, which can cause hepatic insufficiency and extrahepatic biliary duct obstruction.1,3 Most standard deworming protocols that use praziquatel at a dose of 5 mg/kg body weight are ineffective against P fastosum; 1 therefore, diagnosis of infections is critical so that the appropriate treatment (20 mg/kg body weight) can be implemented.
The least invasive of the diagnostic methods for P fastosum is fecal examination. The standard fecal diagnostic method for many years has been formalin-ether sedimentation. 4 However, formalin-ether sedimentation is not a routine fecal analysis method in most veterinary diagnostic laboratories or in clinical settings. A potential alternative to formalin-ether sedimentation is the Mini-Parasep (Apacor) sedimentation kit (MPS), which has been used for the diagnosis of the liver fluke Opisthorchis viverrini in humans and helminth infections in cats and dogs.5,6 The kit, available worldwide, is a single-use device that includes a mixing chamber, filter and sedimentation chamber.
The purpose of this study was to compare the performance of double centrifugation with Sheather’s sugar flotation solution (DCFS) to the MPS and the Mini-Parasep (Apacor) flotation kit (MPF) for diagnosing P fastosum infection in naturally infected cats. DCSF was selected as a comparison to the MPS owing to its high sensitivity for many eggs found in feces, particularly those with higher specific gravity (SPG). 7 MPF was included as an alternative flotation method. As with the MPS, it is a single-use device that includes a mixing chamber, filter and flotation chamber.
Materials and methods
From April to August 2017, fecal samples were collected from the litter boxes of individually housed cats (25 male and 25 female) from a population known to be endemic for P fastosum. 8 All cats were estimated to be >6 months of age and enrolled in the Feral Cat Program (FCP) at Ross University School of Veterinary Medicine (RUSVM). The FCP, including housing of the cats, and fecal collections were performed under RUSVM Institutional Animal Care and Use Committee-approved protocols. Feces were refrigerated (4–8°C) until analysis, which was within 7 days of collection. Feces were mixed thoroughly by hand prior to removing aliquots for the analysis and egg identification was based on morphology with Zajac and Conboy (2012) used as a reference. 9
The standard operating procedure at the RUSVM diagnostic laboratory was used for the DCFS. The SPG of the Sheather’s sugar flotation solution was confirmed each day prior to use and ranged from 1.27 to 1.28. One gram of feces was mixed with water, strained and then centrifuged for 5 mins at 500 g in a 15 ml test tube. The supernatant was poured off and the sediment thoroughly mixed with the flotation solution. Flotation solution was added to form a positive meniscus and then a coverslip (22 × 22 mm) was placed on the tubes prior to centrifugation (5 mins; 500 g; full-swing bucket centrifuge [LW Scientific Universal Centrifuge]). After centrifugation, the samples sat for approximately 10–15 mins to allow additional eggs to float as described by Dryden et al; 7 all parasite diagnostic stages seen under the coverslip were counted.
The Mini-Parasep kits were used as per the manufacturer’s instructions with three exceptions: the exact amount of feces used was weighed; more feces than suggested by the manufacturer (a pea-sized scoop) was used; and, for the MPF, the minimum flotation time was longer (10 mins vs 5 mins). For the MPF, 1 g feces was used, thoroughly mixed with ZnSO4 (SPG 1.20; supplied by the kit manufacturer) and centrifuged once (5 mins; 500 g). After centrifugation, additional flotation solution was added to form a positive meniscus and a 22 × 22 mm coverslip was placed on top and allowed to sit for 10–15 mins; all parasite diagnostic stages seen under the coverslip were counted. For the MPS, 0.5 g feces was used, mixed with Triton X/ethyl acetate solution and centrifuged once (3 mins; 1200 g). Approximately 200 µl sediment was examined with all parasite diagnostic stages seen recorded.
Sensitivity and specificity were calculated using MedCalc Software bvba (https://www.medcalc.org/). For the calculations, all samples negative by all three methods were considered true negatives and any sample positive by at least one method was considered a true positive.
Results
A total of 34 cats were identified as positive for P fastosum with at least one of the methods. Thirty-three of these were identified with the DCSF method with the number of eggs per gram of feces (EPG) ranging from 1 to 106 (mean 14.2) (Table 1). Sixteen cats were identified as positive using the MPF method, one of which was not identified with the DCSF method; the number of eggs detected per gram of feces was slightly lower (1–6; mean 1.7). All samples identified as positive with the MPS method also were positive with one of the other methods; EPG was not calculated with sedimentation. The comparative sensitivity of the DCSF, MPF and MPS methods for P fastosum were 97.1%, 47.1% and 32.4%, respectively. Other parasites seen in the fecal samples and the corresponding sensitivity are presented in Table 1.
Comparative performance of the double-centrifugation sugar flotation and flotation and sedimentation Mini-Parasep (Apacor) protocols in diagnosing Platynosomum fastosum eggs and other common parasites in 50 domestic cats
Other (number positive Sheather’s, ZnSO4, sedimentation): coccidian (10, 4, 3), capillarids (1, 1, 0), metastrongyloid larvae (1, 0, 1), Physaloptera species (1, 0, 0), taeniid (7, 0, 1), Dipylidium caninum (1, 1, 0)
SPG = specific gravity; CI = confidence interval
Discussion
Based on the results of this study, DCSF is the more sensitive method for detecting P fastosum eggs in feces. These results corroborate with the findings of Krecek et al and Rocha et al.8,10 In the study by Rocha et al, 10 5/40 samples from cats with unknown infection status were found to be positive for P fastosum using single centrifugation with Sheather’s sugar flotation solution, whereas no samples were identified as positive with formalin-ether sedimentation. In a study on parasite prevalence in cats on St Kitts, double centrifugation with Sheather’s sugar flotation solution also identified the most cats with P fastosum. 8
In comparison to DCSF, MPF was not as sensitive. The lower SPG of the flotation solution, which comes with the kit, could have contributed to this lower sensitivity. Results from a study by Souza-Dantas et al were similar to that found in this study with a ZnSO4 (SPG 1.2) method identifying 3/5 positive cats with necropsy counts used to confirm infection. 11 In contrast, Ramos et al found that ZnSO4 with a SPG of 1.35 was effective in diagnosing P fastosum using the FLOTAC technique, which is designed to have a detection threshold of 1 EPG. 12 While samples from only three cats were used in the work by Ramos et al, 12 it does suggest that if a higher SPG solution was used with MPF, sensitivity might be improved.
While MPS has been used successfully for liver fluke egg detection in human feces, a challenge with the cat samples was the quantity of sediment. While the filter system removes large particles, heavy small particles in the cat feces results in large quantities of sediment (approximately 300 µl). Only 0.5 g feces could be analyzed, given the amount of sediment, biasing results against the method. Modifications, including sieving through cheesecloth prior to placing in the kit tubes, might decrease the sedimentation issues, although some eggs might be missed in this step. Ramos et al, 12 Rocha et al 10 and Krecek et al 8 also found that sedimentation methods were less sensitive than flotation methods with centrifugation. In contrast to these studies, Leal et al, 13 in a study with 12 positive cats (based on necropsy) found that serial analysis of fecal samples with formalin-ether sedimentation with centrifugation was more sensitive than centrifugation with saturated sugar flotation solution. The operator experience, time spent examining the material and the volume examined might play a role in the difference seen in these studies with false-negative results occurring as a result of the difficulty in identifying eggs when there is a large quantity of debris. While more time spent sifting through the material would likely make it feasible to identify the eggs, in a clinical setting the time required would likely be a drawback compared with the other methods.
An advantage to identifying a flotation method sensitive for P fastosum eggs is that infection can be detected during routine fecal analysis and potentially enable detection prior to the appearance of clinical signs. In this study, all three methods enabled identification of other helminth eggs and larvae in feces with DCSF being overall the most effective method.
An inherent limitation in comparing fecal diagnostic methods is the uneven distribution of eggs within fecal material. This could account for some of the difference in sensitivity of the methods, particularly with the cestode and Physaloptera species eggs, which had low egg counts. This highlights the importance of thoroughly homogenizing the fecal material prior to taking samples for analysis. Even in this study, with mixing of the feces prior to removing aliquots, there were some differences in egg detection that were likely due to differences in distribution. This is highlighted in one sample in which one egg was detected with MPF, three eggs with MPS and none with DCSF, suggesting that at low numbers, even with mixing, eggs can be missed regardless of the method used when only 1 g feces is analyzed. Another limitation in this study is that true infection, based on necropsy counts, was not known. Therefore, sensitivity could be overestimated for all of the methods.
Conclusions
Flotation methods, specifically double centrifugation with Sheather’s sugar flotation solution and potentially ones with ZnSO4 with higher SPG, can be sensitive alternatives to formalin-ether sedimentation, enabling detection of P fastosum infections during routine fecal diagnostics. With regard to the three methods tested, DCFS had higher sensitivity for the detection of not only P fastosum, but also Trichuris species, Ancylostoma tubaeforme and Toxocara cati.
Footnotes
Acknowledgements
The authors thank the Ross University School of Veterinary Medicine’s Feral Cat Program members for assistance with fecal collection and Apacor, UK, for donating test kits.
Conflict of interest
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Ethical approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed with the cats (The Feral Cat Program, the housing of cats within the program and the collection of feces for this study) were in accordance with the ethical standards of Ross University School of Veterinary Medicine (RUSVM) and performed under RUSVM Institutional Animal Care and Use Committee approved protocols.
