A simple, robust and semi-automated parasite egg isolation protocol.
Mes Ted H M,Eysker Maarten,Ploeger Harm W
Large-scale parasite quantification is required for improving our understanding of the epidemiology and genetics of host-parasite interactions. We describe a protocol that uses a low-density salt solution for flotation and centrifugation of nematode eggs. Subsequently, sucrose flotation and precipitation are used to obtain clear egg preparations. Most traditional quantification protocols such as the McMaster technique are unsuited for the standardized processing of large numbers of samples and the analysis of large amounts of feces per sample. Consequently, they are suited only for small-scale surveys. Our protocol, which can be used to analyze up to 6 g of feces, results in clear egg preparations that are concentrated in wells of a microtiter plate and that are suited for digital recording and automated counting. Starting from a fecal suspension in the first flotation solution to a digital recording requires approximately 40 min per 24 samples.
Determination of the specific gravity of certain helminth eggs using sucrose density gradient centrifugation.
David E D,Lindquist W D
The Journal of parasitology
The specific gravities of ten species of helminth eggs were determined using sucrose density gradient centrifugation. Fecal or egg concentrate was layered over a 3 to 54% sucrose density gradient. The gradient was then centrifuged at 800 g for 20 min, allowing 5 min for acceleration and 5 for deceleration. Bands formed were identified and measured. Refractive index was measured at the middle of narrow bands, or at the level at which the concentration of eggs was highest, in the case of wide bands or when no band was formed. The specific gravity corresponding to this refractive index was taken as the specific gravity of the eggs. The ten species of helminth eggs studied and specific gravities measured on three or four gradients were: Toxascaris leonina, 1.0559; Ancylostoma caninum, 1.0559; Toxocara canis, 1.0900; Parascaris equorum, 1.0969; Toxocara cati (embryonated), 1.1005; Ascaris suum, 1.1299; Trichuris suis, 1.1299; Trichuris vulpis, 1.1453; Taenia sp., 1.2251; and Physaloptera sp., 1.2376. These determinations agree with or approximate those of previous workers. The specific gravities of P. equorum, T. suis, Taenia sp., and Physaloptera sp., are reported for the first time.
Sensitivity and efficiency of selected coproscopical methods-sedimentation, combined zinc sulfate sedimentation-flotation, and McMaster method.
Becker Ann-Christin,Kraemer Amelie,Epe Christian,Strube Christina
Coproscopical methods used in veterinary-parasitological diagnostics were validated according to their sensitivity (Se) and egg recovery rate [efficiency (Ef)]. Validation of the combined sedimentation-flotation method and the modified McMaster method was performed by using feces spiked with eggs of Ancylostoma caninum, Uncinaria stenocephala, Cooperia oncophora, cyathostomins, Ascaris suum, Toxascaris leonina, Toxocara canis, Trichuris vulpis, Moniezia expansa, and Anoplocephala perfoliata. For validation of the sedimentation method, Fasciola hepatica eggs were used. With the combined sedimentation-flotation method using ZnSO4 as flotation medium [specific gravity (SG) 1.30], 5 g fecal samples of all tested parasite species (concentration levels 1, 5, 10, 20, 40, 60, and 80 epg) were reproducibly detected "positive" (100 % Se) as of 80 epg. The Ef of the combined sedimentation-flotation method, defined as percentage of rediscovered eggs, revealed clear differences between parasites and showed the highest value for cyathostomins and the lowest for U. stenocephala and T. leonina eggs. The average Ef for all parasite species at 80 epg was 1.50 %. With the McMaster method (concentration levels 1, 30, 50, 80, 100, 500, and 1000 epg), all tested parasite species were detected reliably positive as of 500 epg with a mean Ef of 46.4 %. When evaluating the sedimentation method (concentration levels 1, 5, 10, 15, 20, 25, and 30 epg), F. hepatica eggs were reproducibly found in 5 g fecal samples as of 20 epg with 20.0 % Ef. The result that the combined zinc sulfate sedimentation-flotation method (SG 1.30) as flotation medium provides diagnostic certainty only as of 80 epg has to be considered at preventing zoonoses. If pet owners wish to prevent any zoonotic infection ("zero tolerance"), a monthly anthelminthic treatment should be advised instead of monthly fecal examinations.
Modified sugar centrifugal flotation technique for recovering Echinococcus multilocularis eggs from soil.
Matsuo Kayoko,Kamiya Haruo
The Journal of parasitology
Among soil-transmitted parasitic diseases, alveolar hydatidosis due to the ingestion of Echinococcus multilocularis eggs is becoming a serious problem in Hokkaido, the northern most island of Japan. Dissemination of the infection far from the endemic areas can occur if motor vehicles transmit soil contaminated with eggs. No appropriate and validated method for recovering the taeniid eggs from soil is available. A modified sugar centrifugal flotation technique, using a sucrose solution of specific gravity 1.27 and 0.05% Tween-80, was evaluated as a method to successfully recover eggs from soil. Contamination levels as low as 10 eggs per gram could be detected. This method may be useful to determine the prevalence of E. multilocularis, its transmission, and the potential for by monitoring soil contamination with eggs.
Specific gravity of Opisthorchis viverrini eggs.
Harnnoi T,Wijit A,Morakote N,Pipitgool V,Maleewong W
Journal of helminthology
The specific gravity of the eggs of the liver fluke Opisthorchisviverrini was determined using a sucrose gradient centrifugation and found to range from 1.2713 to 1.3043. The peak egg count was located at the sucrose fraction with a specific gravity of 1.2814. An attempt to float eggs in saturated sodium nitrate solution, sp.gr. 1.4, failed. Examination of human stool specimens for Oviverrini eggs by simple flotation in saturated sodium nitrate solution and the formol-ether sedimentation technique revealed that the flotation technique was not as efficient as the sedimentation technique. It was suggested that the flotation techniques were inappropriate for the detection of Oviverrini eggs in faeces or contaminated soil.
Flotation techniques (FLOTAC and mini-FLOTAC) for detecting gastrointestinal parasites in howler monkeys.
Alvarado-Villalobos Mayra Alejandra,Cringoli Giuseppe,Maurelli Maria Paola,Cambou Aurelie,Rinaldi Laura,Barbachano-Guerrero Arturo,Guevara Roger,Chapman Colin A,Serio-Silva Juan Carlos
Parasites & vectors
BACKGROUND:Analyses of environmental correlates of the composition of gastrointestinal parasite communities in black howler monkeys (Alouatta pigra) have been hindered by inadequate calibration techniques of detection and quantification methods of the parasites. Here we calibrate samples and compare the likelihood of parasite detection using two flotation techniques, FLOTAC and Mini-FLOTAC, and compare flotation solution, preservation method and dilution ratio for egg detection and counts of the most common parasites (Controrchis spp. and Trypanoxyuris spp.) in howler monkeys. RESULTS:For samples preserved in 5% formalin, the Mini-FLOTAC technique was the best option for qualitative and quantitative copro-microscopic analysis. This technique displays an 83.3% and 100% detection of Controrchis spp. and Trypanoxyuris spp. infections, respectively. For the trematode Controrchis spp., more eggs per gram of feces (EPG) were recorded with the flotation solution (FS) #7 (zinc sulfate; specific gravity SG = 1.35) at 1:20 and 1:25 dilution than other methods. By contrast, for the nematode Trypanoxyuris spp., the best results were recorded with FS1 (sucrose and formaldehyde; SG = 1.20) at 1:10 dilution. CONCLUSIONS:We recommend the Mini-FLOTAC technique for general use with parasite analysis on frugivore/folivores like the howler monkey, especially if many samples are analyzed. The technique has a high detection rate and the best EPG counts, allowing the qualitative and quantitative analysis of parasite load among the species or populations without the need for specialized equipment.
[Concordance between the zinc sulphate flotation and centrifugal sedimentation methods for the diagnosis of intestinal parasites].
Inês Elizabete De Jesus,Pacheco Flavia Thamiris Figueiredo,Pinto Milena Carneiro,Mendes Patrícia Silva de Almeida,Da Costa-Ribeiro Hugo,Soares Neci Matos,Teixeira Márcia Cristina Aquino
Biomedica : revista del Instituto Nacional de Salud
INTRODUCTION:The diagnosis of intestinal parasitic infections depends on the parasite load, the specific gravity density of the parasite eggs, oocysts or cysts, and the density and viscosity of flotation or sedimentation medium where faeces are processed. OBJECTIVE:To evaluate the concordance between zinc sulphate flotation and centrifugal sedimentation in the recovery of parasites in faecal samples of children. MATERIALS AND METHODS:Faecal samples of 330 children from day care centers were evaluated by zinc sulphate flotation and centrifugal sedimentation techniques. The frequencies of detection of parasites by each method were determined and the agreement between the diagnostic techniques was evaluated using the kappa index, with 95% confidence intervals. RESULTS:The faecal flotation in zinc sulphate diagnosed significantly more cases of Trichuris trichiura infection when compared to centrifugal sedimentation (39/330; 11.8% vs. 13/330; 3.9%, p<0.001), with low diagnostic concordance between methods (kappa=0.264; 95% CI: 0.102-0.427). Moreover, all positive samples for Enterobius vermicularis eggs (n=5) and Strongyloides stercoralis larvae (n=3) were diagnosed only by zinc sulphate. No statistical differences were observed between methods for protozoa identification. CONCLUSIONS:The results showed that centrifugal flotation in zinc sulphate solution was significantly more likely to detect light helminths eggs such as those of T. trichiura and E. vermicularis in faeces than the centrifugal sedimentation process.