Laura Rinaldi and Giuseppe Cringoli. Dept. Pathology and Animal Health, University of Naples Federico II, Naples, Italy

Transcription

1 Laura Rinaldi and Giuseppe Cringoli Dept. Pathology and Animal Health, University of Naples Federico II, Naples, Italy

2 Parasitological Diagnosis for STH and other parasites Copromicroscopic techniques

3 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

4 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

5 STH Ascaris lumbricoides Trichuris trichiura Ancylostoma duodenale and Necator americanus

6 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.

7 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

8 Multivalent techniques characterized by high sensitivity, specificity, accuracy

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12 FLOTAC

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14 FLOTAC BASIC PRINCIPLES Flotation

15 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

16 Flotation in centrifuge Translation the debris sinks to the bottom, and the parasitic elements float to the top

17 flotation FLOTAC Centrifugation translation It has two flotation chambers

18 FLOTAC

19 FLOTAC READING DISC COMPONENTS TRANSLATION DISC BASE

20 5 ml Flotation chambers 5 ml Tot 10 ml

21 FLOTAC Ruled grids READING DISC

22 Base 100 X Base 400 X

23 2 - FLOTAC

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29 FS recipes

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32 10 g of feces fixed 1:4 10 g of feces + 30 ml of 1) Formalin 5% 2) Formalin 10% 3) SAF

33 Dilution ratio 1:10 1:25 humans Add 90 ml of tap water

34 The faecal suspension is homogenized using a house-hold mixer

35 The suspension is poured through a wire mesh screen having an aperture of 250 m

36 The two flotation chambers of the FLOTAC need 10 ml, 1 ml more is to work easily 11 ml

37 The tubes are centrifuged The tube is centrifuged

38 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)

39 pellet

40 The supernatant is poured off and discarded

41 pellet

42 The tube is then filled with the chosen FS to the previous 11 ml level

43 The pellet and the FS are well mixed

44 The two flotation chambers of the FLOTAC are filled using a Pasteur pipette

45 75 mm

46 The FLOTAC centrifuge adaptor has been specially designed for microtitre centrifuge rotor

47 The Flotac centrifuge adaptor has been specially designed for microtitre centrifuges rotor

48 The FLOTAC in a centrifuge with a microtiter rotor

49 The FLOTAC is centrifuged for 5 minutes at 1,000 rpm

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52 Centrifugation can take place also in a manual centrifuge Manual centrifuge Hettich

53 Centrifugation can take place also in a manual centrifuge FLOTAC Manual centrifugation Hettich

54 Centrifugation can take place also in a manual centrifuge FLOTAC Manual centrifugation Hettich

55 FLOTAC Manual centrifugation Hettish Centrifugation can take place also in a manual centrifuge

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59 The FLOTAC can also be manually centrifuged

60 Details of a manual centrifuge system for the FLOTAC

61 Details of a manual centrifuge system for the FLOTAC

62 Manual centrifuge system for the FLOTAC

63 3 m 1.80 m Special Manual centrifuge system for the FLOTAC

64 Transparent microscope adaptor under the microscope

65 The FLOTAC mounted on the transparent microscope adaptor under the microscope

66 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

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68 Flotac basic technique Analytic sensitivity 1 FS

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70 Flotac dual technique 2 FS

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72 Flotac double technique Sample 1 Sample 2 1 FS Sample 1 Sample 2

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79 Example of Calibration Ancylostoma caninum in dogs

80 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

81 Jurg Utzinger from Swiss Tropical and Public Health Institute, Basel, Switzerland

82 Marco Albonico PHL, Pemba, Zanzibar

83 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!

84 Calibration studies for STH and other helminths

85 Stool sample collection

86 Fill FLOTAC -Fixative -Vacuum-packed

87 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

88 Empty Clarity of the reading Full

89 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

90 Clarity of the reading Horse -GI strongyles 100 x

91 Dog - Eggs of Trichuris 400 x

92 Clarity of the reading Sheep F. hepatica ghost (200x)

93 Clarity of the reading Sheep - Lungworm larva (100x)

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95 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

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97 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.

98 Routine diagnosis Centro Regionale per il Monitoraggio Parassitosi

99 2009

100 Parasitol Res Aug;107(3): 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.

101 Flotac - human

102 Kato-Katz technique China Ether concentration technique FLOTAC technique

103 Soil-transmitted helminths, Schistosoma

104 2008

105 School children n 102 COTE D IVOIRE

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108 2010

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112 2010

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114 FLOTAC in the World 113 countries

115 FLOTAC to Universities - 27 Vet schools Shools of Medicine

116 Fondazione Ivo De Carneri ZANZIBAR Tanzania ottobre 2009

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118 FLOTAC hosts to Naples - 86 Vet schools Shools of Medicine

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121 Costa d Avorio Agosto 2009

122 Costa d Avorio Agosto 2009

123 1 st International Course on FLOTAC techniques Naples, June 22-25, nd International Course on FLOTAC techniques Naples, February 16-19, rd International Course on FLOTAC techniques Naples, November 15-19, 2010

124 FLOTAC at WHO 2008

125 Regional Center for Monitoring Parasites of Immigrants FLOTAC for diagnosi of human parasitic infections - Naples

126 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

127 Migration to the European Union has increased exponentially, with 1.9 million new registered immigrants in 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.

128 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

129 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

130 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.

131 Naples, Immigrants and Parasites - RESULTS Nematoda Trematoda Cestoda Protozoa Number of examined = 721 PARASITE POSITIVES % Trichuris trichiura Hookworms Ascaris lumbricoides Trichostrongylus spp Strongyloides stercoralis Enterobius vermicularis Schistosoma mansoni Dicrocoelium dendriticum Hymenolepis nana Taenia spp Balantidium coli Dientamoeba fragilis Blastocystis spp Entamoeba hartmanni Entamoeba coli Entamoeba hystolitica/dispar Endolimax nana Giardia duodenalis Enteromonas spp Chilomastix spp Iodamoeba butschlii % POSITIVES

132 Geo-referenced parasitological results of the immigrants tested for intestinal parasites in Naples, southern Italy

133 Country No. Immigrants No. positives FLOTAC No. Positive Ether concentr. Algeria Apolide Bangladesh Benin Bielorussia Brasile Bulgaria Burkina Faso Camerun Capo Verde Cina Colombia Congo Costa d'avorio Ecuador Egitto Eritrea Gambia Georgia Ghana Guinea No. immigrants No. Positives FLOTAC Country No. Immigrants No. positives FLOTAC No. Positive Ether concentr. India Kirghizistan Liberia Mali Marocco Mauritania Nigeria Pakistan Peru Polonia Rep. Dominicana Romania Russia Sierra Leone Somalia Sri Lanka Senegal Tanzania Togo Tunisia Ucraina Yemen No. Positives Ether concentration OVERALL

134 Trichuris trichiura r COUNTRY Bangladesh Burkina Faso Pakistan Sri Lanka Sénégal positive negative Examined Positives % Immigrants ,3 4.3%

135 Ancylostoma duodenale r COUNTRY Bangladesh Burkina faso Liberia Nigeria Pakistan Sénégal positive negative Examined Positives % Immigrants ,5 3.5%

136 Ascaris lumbricoides r COUNTRY Bangladesh Guinea Nigeria Sénégal positive negative Examined Positives % Immigrants ,1 1.1%

137 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

138 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

139 GRID SAMPLING

140 The Campania region of southern Italy Systematic grid sampling Distribution of parasites in sheep and goat farms

141 The Campania region of southern Italy Systematic grid sampling 10 x 10 Km Distribution of parasites in sheep and goat farms

142 The Campania region of southern Italy Systematic grid sampling 10 x 10 Km centroid Distribution of parasites in sheep and goat farms

143 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

144 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

145 Korea, 2008 Pemba, 2009 Perù, 2009 Italia, 2005 Zanzibar, 2009 Cina, 2007 Thank you

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147 DISEASE MAPPING Selection of the study area

148 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)

149 DISEASE MAPPING Distribution of the sample in the study area Random sampling Systematic sampling Proportional allocation Use of grids

150 Parasitological survey DISEASE MAPPING

151 DISEASE MAPPING Georeferencing the study units (e.g., farms, municipalities, regions, etc.)

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153 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

154 Faculty of Veterinary Medicine Department of Pathology and Animal Health Section of Parasitology and Parasitic Diseases

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