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Epidemiology en diagnostics

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Background information

Application of diagnostic PCRs
Drawbacks of microscopy
Overview and codes of available PCRs:
- Intestinal Protozoa
- Stool/urine Helminths
- Tissue/blood parasites
- Multiplex format
Molecular diagnostic protocols

Application of diagnostic PCRs

The (multiplex) real-time PCRs developed by our group have been employed to map the prevalence and distribution of parasitic infections in both the Netherlands and several low income countries. We deployed our protocols on faecal samples collected in a broad range of countries, including Ethiopia (amoebiasis, microspori¬diosis), Ghana (intestinal nematodes, amoebiasis), Malawi (diarrhoea causing protozoa, hookworm), Senegal (schistosomiasis), Bangladesh and India (diarrhoea causing protozoa), Mozambique (intestinal parasites, Strongyloides), Ecuador (intestinal parasites, Taenia) and Peru (strongyloidiasis). In addition, urine samples from Ghana and Kenya have been used for PCR detection of Schistosoma DNA.

There are several arguments supporting the use of real-time PCR in epidemiological surveys:

Species-specific detection and quantification of parasite DNA is a highly sensitive and specific diagnostic procedure compared to the more classical methods, such as microscopy (see also below). Real-time PCR may potentially serve as a new gold-standard for detecting active infection of some parasites.
The simultaneous detection of a variety of parasites within a single clinical sample using a multiplex real-time PCR turns this approach into a powerful diagnostic tool. Several of our multiplex assays are listed below.
The simple procedure for faecal sample collection without the need for a cold chain and the high throughput potential of the protocol provide additional value for epidemio¬logical research. When mixed with ethanol, stool sampling can take place in rural and remote areas in the tropics without the need of cool storage or transportation.
The multiplex real-time PCR has thus far proven to be a promising diagnostic alternative for the evaluation of intervention programmes or for species-specific monitoring of drug trials in geographic regions where these parasites are most common.
An increasing number of research centres situated in developing countries offer real-time PCR applications. After collection in the field, samples can be simply transferred to such a centre for further analysis.
Isolated DNA may eventually be used to identify molecular markers of resistance.

At the LUMC we incorporated several of these PCRs in the routine clinical diagnostics of parasitic infections as well, with some of them even replacing microscopy.

Application form (pdf) for parasitological diagnostic research. (for a word-file please send an email to Dr. L. van Lieshout)

Drawbacks of microscopy

Classically, diagnosis of intestinal protozoa and helminths is performed by microscopic examination of faecal samples. However, microscopy has several important disadvantages, which reduces its efficacy in large scale population based studies:

Correct identification greatly depends on the experience and skills of the microscopist i.e. microscopy often lacks standardisation and possibilities for quality control are limited,
Sensitivity is low, therefore examination of multiple samples is often needed,
It is very difficult, sometimes even impossible, to differentiate pathogenic protozoa from non-pathogenic species,
Some protozoa require complicated staining procedures; often these are the opportunistic species which may cause life threatening diarrhoea in AIDS-patients.

PCR based methods for species-specific detection of parasite DNA in faecal samples have been shown, by our group and others, to be a highly sensitive and specific technique to diagnose numerous parasitic infections. The recent introduction of real-time PCR, in particular the multiplex real-time PCR, opened the way to develop specific diagnostic panels, applicable for defined populations at risk.

Besides faecal samples, parasite specific DNA can also be detected in other clinical material. For example real-time PCR can be performed on tissue samples (Leishmania, Toxoplasma), blood samples (Plasmodium species) and urine samples (Schistosoma)

Overview and codes of available PCRs

All PCRs are real-time and include PhHV target as an internal control.

Intestinal Protozoa

His (H)	-	Entamoeba histolytica
Dis	-	Entamoeba dispar
C	-	Cryptosporidium parvum/hominis
G	-	Giardia lamblia
Ib	-	Isospora belli
Cc	-	Cyclospora cayeteanensis
D	-	Dientamoeba fragilis
Eb(M)	-	Enterocytozoon bieneusi
Ei(M)	-	Encephalitozoon spp.

Stool/urine Helminths

S	-	Strongyloides stercoralis
A	-	Ancylostoma duodenale
N	-	Necator americanus
O	-	Oesophagostomum bifurcum
Al	-	Ascaris lumbricoides
Sch	-	Schistosoma spp.
Sm	-	Schistosoma mansoni
Sh	-	Schistosoma haematobium
Tsag	-	Taenia saginata
Tsol	-	Taenia solium

Tissue/blood parasites

Lsh	-	Leishmania spp.
Tox	-	Toxoplasma gondii
Pf	-	Plasmodium falciparum
Pv	-	Plasmodium vivax
Po	-	Plasmodium ovale
Pm	-	Plasmodium malariae

Multiplex format

His/Dis	-	Entamoeba histolytica / Entamoeba dispar
HGC	-	Entamoeba histolytica / Giardia / Cryptosporidium
DGC	-	Dientamoeba / Giardia / Cryptosporidium
BDGC	-	Blastocystis / Dientamoeba / Giardia / Cryptosporidium
MSp	-	Enterocytozoon bieneusi / Encephalitozoon spp.
Sm/Sh	-	Schistosoma mansoni / Schistosoma haematobium
ANOS	-	Ancylostoma / Necator / Oesophagostomum / Strongyloides
ANAS	-	Ancylostoma / Necator / Ascaris / Strongyloides
TTH	-	Taenia saginata / Taenia solium / Hymenolepis
Malaria	-	P. falciparum /P. vivax / P. ovale / P. malariae