Laura Rinaldi and Giuseppe Cringoli Dept. Pathology and Animal Health, University of Naples Federico II, Naples, Italy
Parasitological Diagnosis for STH and other parasites Copromicroscopic techniques
COPROMICROSCOPIC TECHNIQUES AND PARASITIC ELEMENTS DETECTABLE Techniques Eggs Parasitic elements Oocysts Cysts Larvae Direct Smear O O O O Simple Flotation in tube O O O Flotation in centrifuge (Wisconsin) O McMaster O O Mes method O Sedimentation O Baermann O Kato Katz O O O Ether/ethyl acetate concentration O O O FLOTAC O O O O Quantitative techniques = O Qualitative techniques = O
Helminths - Protozoa broncopolmonari COPROLOGICAL examination Gastro-intestinal tract - Liver, lungs Sensu stricto EGGS LARVAE OOCYSTS CYSTS Helminths and protozoa which inhabit the GI tract and lungs of the hosts eliminate their eggs/larvae with the faeces of hosts Ab Sensu lato DNA
STH Ascaris lumbricoides Trichuris trichiura Ancylostoma duodenale and Necator americanus
Faecal Egg Count (FEC) techniques are considered relatively straightforward and protocols such as the Kato-Katz technique and the ether-based concentration method in the human field have been available for many years.
Kato-Katz technique Advantages - Simplicity and low cost Disadvantages -Small amount of feces exam. (usually 41.7 mg) and thus low analytic sensitivity (24 EPG) - Fresh samples Low sensitivity and accuracy Ether-based concentration Advantages - Stool samples preserved - Diagnosis of both helminths and protozoa Disadvantages - Eggs can be broken or altered - Reference laboratory - Qualitative
Multivalent techniques characterized by high sensitivity, specificity, accuracy
FLOTAC
FLOTAC BASIC PRINCIPLES Flotation
FLOTAC BASIC PRINCIPLES Flotation Translation Centrifugation - If flotation takes place in a centrifuge, all the parasitic elements can float to the top, - if the top portion of the flotation suspension is cut transversally (i.e. translated), all the parasitic elements can be collected and observed under the microscope
Flotation in centrifuge Translation the debris sinks to the bottom, and the parasitic elements float to the top
flotation FLOTAC Centrifugation translation It has two flotation chambers
FLOTAC
FLOTAC READING DISC COMPONENTS TRANSLATION DISC BASE
5 ml Flotation chambers 5 ml Tot 10 ml
FLOTAC Ruled grids READING DISC
Base 100 X Base 400 X
2 - FLOTAC
FS recipes
11
10 g of feces fixed 1:4 10 g of feces + 30 ml of 1) Formalin 5% 2) Formalin 10% 3) SAF
Dilution ratio 1:10 1:25 humans Add 90 ml of tap water
The faecal suspension is homogenized using a house-hold mixer
The suspension is poured through a wire mesh screen having an aperture of 250 m
The two flotation chambers of the FLOTAC need 10 ml, 1 ml more is to work easily 11 ml
The tubes are centrifuged The tube is centrifuged
The tubes are centrifuged for 3 minutes at 1,500 r.p.m. The tubes are centrifuged The tube is centrifuged for 3 minutes at 1,500 rpm (170 g)
pellet
The supernatant is poured off and discarded
pellet
The tube is then filled with the chosen FS to the previous 11 ml level
The pellet and the FS are well mixed
The two flotation chambers of the FLOTAC are filled using a Pasteur pipette
75 mm
The FLOTAC centrifuge adaptor has been specially designed for microtitre centrifuge rotor
The Flotac centrifuge adaptor has been specially designed for microtitre centrifuges rotor
The FLOTAC in a centrifuge with a microtiter rotor
The FLOTAC is centrifuged for 5 minutes at 1,000 rpm
Centrifugation can take place also in a manual centrifuge Manual centrifuge Hettich
Centrifugation can take place also in a manual centrifuge FLOTAC Manual centrifugation Hettich
Centrifugation can take place also in a manual centrifuge FLOTAC Manual centrifugation Hettich
FLOTAC Manual centrifugation Hettish Centrifugation can take place also in a manual centrifuge
The FLOTAC can also be manually centrifuged
Details of a manual centrifuge system for the FLOTAC
Details of a manual centrifuge system for the FLOTAC
Manual centrifuge system for the FLOTAC
3 m 1.80 m Special Manual centrifuge system for the FLOTAC
Transparent microscope adaptor under the microscope
The FLOTAC mounted on the transparent microscope adaptor under the microscope
Flotac techniques 1 - Flotac basic technique 2 - Flotac dual technique 3 - Flotac double technique 4a,b,c - Flotac pellet techniques 5 - Faecal egg count calibration Herbivores Dogs Humans Herbivores Dogs Humans Herbivores Dogs Humans Dogs Humans Herbivores Dogs Humans and animals
Flotac basic technique Analytic sensitivity 1 FS
Flotac dual technique 2 FS
Flotac double technique Sample 1 Sample 2 1 FS Sample 1 Sample 2
Example of Calibration Ancylostoma caninum in dogs
Calibration studies for STH and other helminths: Italy Switzerland China Kyrgyzstan Cote D Ivoire Zanzibar Faecal? Preservation Methods Flotation Solutions Faecal dlituion Washing with Ether
Jurg Utzinger from Swiss Tropical and Public Health Institute, Basel, Switzerland
Marco Albonico PHL, Pemba, Zanzibar
Calibration studies for STH and other helminths: Faecal Preservation - Formalin 5% (better than formalin 10% or SAF, do not freeze) - Vacuum Flotation Solutions FS2 (NaCl) for FLOTAC Basic Technique FS2 (NaCl) and FS7 (ZnSO4) for FLOTAC Dual Technique STH, cestoda, protozoa Trematoda, protozoa Faecal dlituion 1:25 Washing with Ether NO!
Calibration studies for STH and other helminths
Stool sample collection
Fill FLOTAC -Fixative -Vacuum-packed
FLOTAC FLOTAC Advantages Disadvantages -Stool samples fresh or preserved -Clarity of reading -Diagnosis of both helminths and protozoa - More complicated techniques -Equipped laboratories - More extensive training of laboratory workers -High sensitivity and accuracy
Empty Clarity of the reading Full
Clarity of the reading Note that the ruled lines are transparent, allowing for the counting of eggs under them Grid without faecal suspension seen under a microscope FLOTAC - Empty Grid with faecal suspension seen under a microscope FLOTAC - Full
Clarity of the reading Horse -GI strongyles 100 x
Dog - Eggs of Trichuris 400 x
Clarity of the reading Sheep F. hepatica ghost (200x)
Clarity of the reading Sheep - Lungworm larva (100x)
Validation Studies on animals Many comparative studies of the FLOTAC techniques and other copromicroscopic techniques. In general, the FLOTAC technique produce: - number of negative results lower than those produced by other techniques - Mean EPG which are greater than those produced by the other techniques - Variances (coefficient of variation) of EPG which are lower than those produced by the other techniques, thus indicating high reliability
Campbell, C., Cringoli, G., Coles, G.C. Ivermectin resistance in cyathostomins in UK horses. XXI Congress of the WAAVP, Ghent, Belgium, August, 2007; Davies, P., Wells, L., Cringoli, G., Coles, G.C. The prevalence of Anoplocephala perfoliata in abattoir and stabled horses. XXI Congress of the WAAVP, Ghent, Belgium August, 2007.
Routine diagnosis Centro Regionale per il Monitoraggio Parassitosi
2009
Parasitol Res. 2010 Aug;107(3):555-60. Epub 2010 May 26. Comparison of the FLOTAC technique with the McMaster method and the Baermann technique to determine counts of Dictyocaulus eckerti L1 and strongylid eggs in faeces of red deer (Cervus elaphus). Bauer BU, Pomroy WE, Gueydon J, Gannac S, Scott I, Pfister K. Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand. Abstract The FLOTAC flotation technique has been introduced as a new diagnostic tool to detect parasitic elements from faeces. Samples from naturally infected young deer were used for counting Dictyocaulus larvae and strongylid eggs. The FLOTAC technique, using 11 different flotation solutions with specific gravities (sg) between 1.20 and 1.45, was compared with the Baermann technique and the saturated sodium chloride (sg 1.20)-based McMaster method. In addition, a comparison was made between the FLOTAC technique with magnesium sulphate (sg 1.28) and the Baermann technique for larval recovery from faeces that were examined on the day of collection or after 7 days storage at 4 degrees C. On the whole egg counts between the FLOTAC using different flotation solutions and the McMaster were unremarkable. In contrast, variations of larval counts were detected between different flotation solutions as well as with the Baermann technique. Most flotation solutions with a specific gravity of 1.20 floated significantly fewer lungworm larvae (p < 0.05) compared to flotation solutions with a higher specific gravity. Magnesium sulphate (sg 1.28) consistently produced the highest mean larval counts in all conducted experiments. Larval counts using magnesium sulphate (sg 1.28) were higher than with the Baermann technique both on the day of collection and after 7 days. Overall, the use of magnesium sulphate (sg 1.28) with FLOTAC for larval counts resulted in higher counts than the Baermann recovery technique and was the better choice of those flotation solutions examined. Furthermore, magnesium sulphate (sg 1.28) was also reliable for strongylid egg detection with the FLOTAC apparatus.
Flotac - human
Kato-Katz technique China Ether concentration technique FLOTAC technique
Soil-transmitted helminths, Schistosoma
2008
School children n 102 COTE D IVOIRE
2009
2010
2010
2010
FLOTAC in the World 113 countries
FLOTAC to Universities - 27 Vet schools Shools of Medicine
Fondazione Ivo De Carneri ZANZIBAR Tanzania ottobre 2009
FLOTAC hosts to Naples - 86 Vet schools Shools of Medicine
Costa d Avorio Agosto 2009
Costa d Avorio Agosto 2009
1 st International Course on FLOTAC techniques Naples, June 22-25, 2009 2 nd International Course on FLOTAC techniques Naples, February 16-19, 2010 3 rd International Course on FLOTAC techniques Naples, November 15-19, 2010
FLOTAC at WHO 2008
Regional Center for Monitoring Parasites of Immigrants FLOTAC for diagnosi of human parasitic infections - Naples
Naples, Immigrants and Parasites October 2008 May 2010 The movements of people, including immigrants, rural to urban migration, refugees and tourists, introduce and mix their cultural preferences, customs, and behavioral patterns. The concomitant changes in the environment, climate, technology, land use, human behavior, and demographics converge to favour the emergence of infectious and parasitic diseases
Migration to the European Union has increased exponentially, with 1.9 million new registered immigrants in 2008. Italy has overcome the European average, with a value of 7.2% and now it is third after Spain (11.7%) and Germany (8.2%). For both geographical and historical reasons, the city of Naples in the Campania region is receiving an increasing number of immigrants and with 61,169 immigrants in 2008 is the first recipient city in southern Italy.
Naples, Immigrants and Parasites October 2008 May 2010 Center for Immigrants Health Protection situated at the Ascalesi Hospital Regional Center for Monitoring Parasites of Immigrants 721 Protozoa Helminths Section of Veterinary Parasitology Faculty of Veterinary Medicine University of Naples Federico II, Italy
Naples, Immigrants and Parasites Study population = immigrants (n = 721) which were visited at the Ascalesi Hospital between October 2008 and March 2010 for general health problems. The immigrants were asked to provide a single faecal sample at morning and to bring it at the Ascalesi Hospital within two hours from collection. Laboratory Procedures Flotac dual tecnique (analytic sensitivity = 2 eggs/larvae/(oo)cysts per gram of faeces 2 Flotation solutions FS 4 - Sodium nitrate (1.200) FS 7 - Zinc Sulphate (1.350) FS 3 - Zinc Sulphate (1.200) for amoebae identification
FLOTAC MULTIVALENT TECHNIQUES Flotac dual technique Humans N PARASITES 1 Ancylostoma duodenalis 2 Ascaris lumbricoides 3 Tricostrongylus spp. 4 Trichuris trichura 5 Strongyloides stercoralis 6 Enterobius vermicularis 7 Schistosoma mansoni 8 Dicrocoelium dendriticum 9 Hymenolepis nana 10 Balantidium coli 11 Dientamoeba fragilis 12 Blastocystis hominis 13 Entamoeba hystolitica 14 Entamoeba hartmanni 15 Entamoeba coli 16 Endolimax nana 17 Giardia duodenalis 18 Enteromonas spp.
Naples, Immigrants and Parasites - RESULTS Nematoda Trematoda Cestoda Protozoa Number of examined = 721 PARASITE POSITIVES % Trichuris trichiura 31 4.30 Hookworms 25 3.47 Ascaris lumbricoides 8 1.11 Trichostrongylus spp. 4 0.55 Strongyloides stercoralis 2 0.28 Enterobius vermicularis 3 0.42 Schistosoma mansoni 10 1.39 Dicrocoelium dendriticum 5 0.69 Hymenolepis nana 13 1.80 Taenia spp. 2 0.28 Balantidium coli 1 0.14 Dientamoeba fragilis 4 0.55 Blastocystis spp. 315 43.67 Entamoeba hartmanni 40 5.55 Entamoeba coli 134 18.59 Entamoeba hystolitica/dispar 72 9.99 Endolimax nana 107 14.84 Giardia duodenalis 35 4.85 Enteromonas spp. 1 0.14 Chilomastix spp. 6 0.83 Iodamoeba butschlii 3 0.42 % POSITIVES 398 55.2
Geo-referenced parasitological results of the immigrants tested for intestinal parasites in Naples, southern Italy
Country No. Immigrants No. positives FLOTAC No. Positive Ether concentr. Algeria 20 10 8 Apolide 1 0 0 Bangladesh 102 56 43 Benin 1 0 0 Bielorussia 2 0 0 Brasile 1 1 0 Bulgaria 7 4 3 Burkina Faso 40 25 21 Camerun 1 0 0 Capo Verde 1 1 1 Cina 9 2 2 Colombia 1 1 0 Congo 1 1 0 Costa d'avorio 21 14 13 Ecuador 1 1 1 Egitto 2 2 2 Eritrea 1 0 0 Gambia 2 2 2 Georgia 3 1 1 Ghana 4 2 2 Guinea 13 9 8 No. immigrants No. Positives FLOTAC Country No. Immigrants No. positives FLOTAC No. Positive Ether concentr. India 8 6 4 Kirghizistan 6 2 1 Liberia 3 0 0 Mali 4 2 3 Marocco 30 16 15 Mauritania 1 0 0 Nigeria 44 22 19 Pakistan 62 34 26 Peru 1 0 0 Polonia 1 0 0 Rep. Dominicana 2 1 1 Romania 8 4 4 Russia 9 3 5 Sierra Leone 1 0 0 Somalia 5 2 2 Sri Lanka 10 4 2 Senegal 258 156 135 Tanzania 5 2 2 Togo 2 0 0 Tunisia 11 5 5 Ucraina 15 7 8 Yemen 1 0 0 No. Positives Ether concentration OVERALL 721 398 339
Trichuris trichiura r COUNTRY Bangladesh Burkina Faso Pakistan Sri Lanka Sénégal positive negative Examined Positives % Immigrants 721 31 4,3 4.3%
Ancylostoma duodenale r COUNTRY Bangladesh Burkina faso Liberia Nigeria Pakistan Sénégal positive negative Examined Positives % Immigrants 721 25 3,5 3.5%
Ascaris lumbricoides r COUNTRY Bangladesh Guinea Nigeria Sénégal positive negative Examined Positives % Immigrants 721 8 1,1 1.1%
FLOTAC strategy for DOLF (STH) -Fill-FLOTAC and FLOTAC apparatus FREE -Set up a strategy for spatial sampling by geo-referencing villages (epidemiological unit) and examine a representative number of stool samples -Set up Reference Laboratories in each country
Spatial sampling methods Cross-sectional SURVEYS a) selection of the study area b) selection of the study population and calculation of the sample size c) selection of the sample in the study area (proportional allocation, grid sampling, transect sampling, etc.) d) parasitological survey e) geo-referencing of the study units f) drawing maps by Geographical Information Systems
GRID SAMPLING
The Campania region of southern Italy Systematic grid sampling Distribution of parasites in sheep and goat farms
The Campania region of southern Italy Systematic grid sampling 10 x 10 Km Distribution of parasites in sheep and goat farms
The Campania region of southern Italy Systematic grid sampling 10 x 10 Km centroid Distribution of parasites in sheep and goat farms
FLOTAC strategy for DOLF (STH) Standardized Procedures -Collect stool samples and preserve in formalin 5% or vacuum-packed -Flotation Solutions (NaCl) -Training (2-3 days) for Technicians (on site) -20/30 Samples per day per person - External and Internal Quality control Results of any copromicroscopic technique could be different due to either technician error or inherent limitations to the technique
Need of better diagnosis and cure of parasitic infections with standardized procedures It is our responsibility here, there and everywhere! Unguja, Zanzibar, 2009 Pemba, Zanzibar, 2009
Korea, 2008 Pemba, 2009 Perù, 2009 Italia, 2005 Zanzibar, 2009 Cina, 2007 Thank you
DISEASE MAPPING Selection of the study area
DISEASE MAPPING Selection of study population and calculation of sample size N = (N x n)/(n + n) n = 1.96 P EXP (1 - P EXP ) d 2 (Thrusfield, 1995)
DISEASE MAPPING Distribution of the sample in the study area Random sampling Systematic sampling Proportional allocation Use of grids
Parasitological survey DISEASE MAPPING
DISEASE MAPPING Georeferencing the study units (e.g., farms, municipalities, regions, etc.)
The Nazioni current di provenienza era of di global 522 immigrati warming, a Napoli, global parassitologicamente movement and monitorati presso il laboratorio di parassitologia dell AOC globalization, pathogens can move further, faster and in greater numbers than ever before
Faculty of Veterinary Medicine Department of Pathology and Animal Health Section of Parasitology and Parasitic Diseases