laura rinaldi and giuseppe cringoli - biostatistics · laura rinaldi and giuseppe cringoli dept....

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 Parasitic elements

Eggs Oocysts CystsLarvaeDirect Smear O O O OSimple Flotation in tube O O OFlotation in centrifuge (Wisconsin) OMcMaster O OMes method OSedimentation OBaermann OKato Katz O O OEther/ethyl acetate concentration O O OFLOTAC O O O O

Quantitative techniques = O Qualitative techniques = O

Helminths -

Protozoa

broncopolmonari

Ab DNA

EGGS

LARVAE

OOCYSTS

CYSTS

COPROLOGICAL

examination

Sensu stricto

Sensu latoHelminths and protozoa which inhabit the GI tract and lungs of the hosts eliminate their

eggs/larvae with the faeces of hosts

Gastro-intestinal tract - Liver, lungs

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

Ether-based

concentrationAdvantages

- Stool samples preserved

- Diagnosis of both

helminths and protozoa

Disadvantages

- Eggs can be broken or altered

- Reference laboratory

- Qualitative

Low sensitivity and accuracy

Multivalent techniques

characterized by high

sensitivity, specificity, accuracy

FLOTAC®

FlotationFLOTAC® BASIC PRINCIPLES

Flo

tati

on

Centrifugation

Translation

- 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

FLOTAC® BASIC PRINCIPLES

Flo

tati

on

in

cen

trif

ug

e

Translation

the debris sinks to the bottom, and the

parasitic elements float to the top

Centrifugation

translation

flo

tati

on

FLOTAC®

It has two flotation chambers

FLOTAC

FLOTAC

COMPONENTS

BASE

TRANSLATION DISC

READING DISC

5 ml 5 ml

Tot 10 ml

Flotation chambers

FLOTAC

Ruled

grids

READING DISC

Base 100 X Base 400 X

2 - FLOTAC

FS recipes

10 g of feces fixed 1:4

10 g of feces + 30 ml of

1) Formalin 5%

2) Formalin 10%

3) SAF

Add 90

ml of tap

waterDilution ratio 1:10

1:25

humans

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

11 ml

The two flotation chambers of the

FLOTAC need 10 ml,

1 ml more is to work easily

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

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

Manual centrifugeHettich

Centrifugation can take place also in a manual centrifuge

FLOTAC Manual centrifugationHettich


FLOTAC Manual centrifugationHettish


The FLOTAC can also bemanuallycentrifuged

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

1 FS

Analytic sensitivity

Flotac dual technique

2 FS

1 FS

Sample 1 Sample 2

Flotac double technique

Sample 2

Sample 1

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

Faecal dlituion 1:25

Washing with Ether NO!

Flotation Solutions FS2 (NaCl) for FLOTAC Basic Technique

FS2 (NaCl) and FS7 (ZnSO4) for FLOTAC Dual Technique

STH, cestoda,

protozoa

Trematoda,

protozoa

Calibration studies for STH and other helminths

Stool sample collection

-Fixative

-Vacuum-packed

Fill FLOTAC

FLOTAC- More complicated

techniques

-Equipped laboratories

- More extensive training

of laboratory workers

Advantages

-Stool samples fresh or

preserved

-Clarity of reading

-Diagnosis of both

helminths and protozoa

-High sensitivity and

accuracy

Disadvantages

FLOTAC

Clarity of the reading

FullEmpty

FLOTAC - Empty FLOTAC - Full

Grid without faecal suspension seen

under a microscope

Grid with faecal suspension seen

under a microscope

Note that the ruled lines are transparent, allowing for the counting of eggs

under them


Horse -GI strongyles 100 x


Dog - Eggs of Trichuris 400 x

Sheep – F. hepatica “ghost” (200x)


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.

Centro Regionale per il Monitoraggio Parassitosi

Routine diagnosis

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

China

Kato-Katz technique

Ether concentration techniqueFLOTAC technique

http://www.who.int/en/

Soil-transmitted helminths, Schistosoma

School children n° 102

COTE D’IVOIRE

FLOTAC in the World – 113 countries

FLOTAC to Universities - 27

Vet schools

Shools of Medicine

ZANZIBARTanzania

Fondazione Ivo De Carneri

ottobre 2009

FLOTAC hosts to Naples - 86

Vet schools

Shools of Medicine

Costa d’Avorio

Agosto 2009

1st International Course on FLOTAC techniques

Naples, June 22-25, 2009

2nd International Course on FLOTAC techniques

Naples, February 16-19, 2010

3rd 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

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

Naples, Immigrants and ParasitesOctober 2008 – May 2010

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.

Center for Immigrants’

Health Protection situated

at the Ascalesi Hospital

Regional Center for Monitoring

Parasites of Immigrants

Section of Veterinary Parasitology

Faculty of Veterinary Medicine

Naples, Immigrants and ParasitesOctober 2008 – May 2010

University of Naples Federico II, Italy

721 Protozoa Helminths

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.

2 Flotation solutions

FS 4 - Sodium nitrate (1.200)

FS 7 - Zinc Sulphate (1.350)

Flotac dual tecnique

(analytic sensitivity = 2 eggs/larvae/(oo)cysts per gram of faeces

Laboratory Procedures

FS 3 - Zinc Sulphate (1.200) for

amoebae identification

Naples, Immigrants and Parasites

FLOTAC – MULTIVALENT TECHNIQUES

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.

Flotac dual technique

Humans

Number of examined = 721PARASITE 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

Naples, Immigrants and Parasites - RESULTS

Nematoda

Trematoda

Cestoda

Protozoa

Geo-referenced parasitological results of the immigrants tested for

intestinal parasites in Naples, southern Italy

CountryNo.

ImmigrantsNo. positives

FLOTACNo. Positive

Ether concentr.Algeria 20 10 8Apolide 1 0 0Bangladesh 102 56 43Benin 1 0 0Bielorussia 2 0 0Brasile 1 1 0Bulgaria 7 4 3Burkina Faso 40 25 21Camerun 1 0 0Capo Verde 1 1 1Cina 9 2 2Colombia 1 1 0Congo 1 1 0Costa d'Avorio 21 14 13

Ecuador 1 1 1Egitto 2 2 2Eritrea 1 0 0Gambia 2 2 2Georgia 3 1 1Ghana 4 2 2Guinea 13 9 8

CountryNo.

ImmigrantsNo. positives

FLOTACNo. 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 0Rep. 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 0No.

immigrantsNo. Positives

FLOTACNo. Positives

Ether concentration

OVERALL 721 398 339

rTrichuris trichiura

COUNTRY

Bangladesh

Burkina Faso

Pakistan

Sri Lanka

Sénégal

Examined Positives %

Immigrants 721 31 4,3

positive

negative

4.3%

rAncylostoma duodenale



COUNTRY

Bangladesh

Burkina faso

Liberia

Nigeria

Pakistan

Sénégal

positive

negative

3.5%

rAscaris lumbricoides

COUNTRY

Bangladesh

Guinea

Nigeria

Sénégal



positive

negative

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

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

Spatial sampling methods

Cross-sectional SURVEYS

GRID SAMPLING

The Campania region of southern Italy

Systematic grid sampling

Distribution of parasites

in sheep and goat farms

10 x 10 Km





10 x 10 Km

centroid





FLOTAC strategy for DOLF (STH)

-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

Standardized Procedures

Results of any copromicroscopic

technique could be different due to either

technician error or inherent limitations to

the technique

Unguja, Zanzibar, 2009 Pemba, Zanzibar, 2009

Need of better diagnosis and cure of parasitic

infections with standardized procedures

It is our responsibility…

…here, there and everywhere!

Thank you

Korea, 2008

Zanzibar, 2009 Cina, 2007

Perù, 2009Pemba, 2009

Italia, 2005

DISEASE MAPPING

• Selection of the study area

• Selection of study population and

calculation of sample size

N° = (N x n)/(N + n)

n = 1.96 PEXP (1 - PEXP)

d2

(Thrusfield, 1995)

DISEASE MAPPING

• Distribution of the sample in the study area

•Random sampling

•Systematic sampling

•Proportional allocation

•Use of grids

DISEASE MAPPING

• Parasitological survey

DISEASE MAPPING

• Georeferencing the study units (e.g., farms, municipalities,

regions, etc.)

DISEASE MAPPING

Nazioni di provenienza di 522 immigrati a Napoli, parassitologicamentemonitorati presso il laboratorio di parassitologia dell’AOCThe current era of global warming, global movement and

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

laura rinaldi and giuseppe cringoli - biostatistics · laura rinaldi and giuseppe cringoli dept....

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