The microscopic detection of parasites is generally accepted as the gold standard, although it has also been widely acknowledged that it falls short on effectiveness in both patient care and epidemiology.
Each year approximately 320.000 Dutch patients with gastro-intestinal complaints consult their general practitioner (Van Duynhoven et al., 2005). Guidelines for general practitioners to request stools were created by the Dutch Society of General practitioners (NHG) (Brühl et al., 2007) to improve daily practice for managing patients with gastro-intestinal complaints and can be summarized as follows:
The guidelines for the diagnostic standard for acute diarrhoea recommends additional diagnostic analysis for intestinal protozoa when the diarrhoea continues for more than 10 days. The GP should ask for a fresh stool sample for it to be analysed within hours after production. When the outcome is negative, two more stool samples should be requested, produced with an interval of a few days. If available, preference should be given to the TFT procedure. If no intestinal parasites were discovered in three samples and protozoan infection is still suspected, the laboratory could be consulted to perform a copro-antigen test for
G. lamblia detection. Detection of E. histolytica with PCR and
Cryptosporidium with specific staining methods have to be explicitly asked for as these techniques are usually not performed on a routine basis. (Translated from Dutch by R. ten Hove).
Although the diagnostic standard procedure for general practitioners as described above is fairly straightforward, many additional patients visiting their general practitioner were diagnosed with G. lamblia- and Cryptosporidium-infection by the molecular diagnostic approach (chapter 2 and chapter 3). Detection of these cases with the conventional diagnostic approach had failed because cases did not meet the criteria for parasite examination or, even if cases answered the criteria for parasite examination, specific parasite infections were not suspected by the general practitioners. Last but not least, supported by the semi-quantitative results, real-time PCR has the ability to detect very low loads of intestinal parasites that would most likely be missed with microscopic and antigen detection methods.
The design of real-time PCR panels for specific patient groups could improve the standard diagnostic procedures and patient diagnostics (Verweij, 2004). The advantages of such real-time PCR panels can further be supported by the results of
chapters 2, 3 and 4. Table 11.4 gives examples of real-time PCR panels that could be used in different patient groups. In children, G. lamblia and Cryptosporidium are two important pathogens. Although D. fragilis has been suggested to be also a potential pathogen in children, more studies are needed to support this statement. Some of the microbiological diagnostic laboratories in The Netherlands have replaced microscopic examination of SAF-preserved stool samples with a real-time PCR panel including a D. fragilis-specificPCR which can help to elucidate the pathogenic role of this parasite (Bruijnesteijn van Coppenraet et al., n.d.).
Table 11.4. Real-time PCR assays that were developed and evaluated summarized as components of different patient groups. Several targets have also been combined and evaluated as multiplex real-time PCR assays.
Adults Children Entamoeba histolytica Giardia lamblia Cryptosporidium Dientamoeba fragilis Giardia lamblia Cryptosporidium
Travellers Immune compromised
Entamoeba histolytica Giardia lamblia Cryptosporidium Strongyloides stercoralis Schistosoma b Cyclospora cayetanensis b Isospora belli b E. bieneusi c Strongyloides stercoralis a Cryptosporidium Isospora belli Enterocytozoon bieneusi Encephalitozoon spp.
a: Recommended when corticosteroid treatment is scheduled. b: Optional for those returning from high-risk areas.
c: Actual prevalence among travellers needs further investigation.
In immunocompromised patients the most important opportunistic intestinal parasites can be related to the conditions of the immune suppression. For example,
S. stercoralis hyperinfection is associated with corticosteroid treatment and HTLV infection but not with HIV infection. Travellers comprise a very diverse group, including immigrants, adoption children, tourists, businessmen, expatriates, army personnel, etc. who may have been exposed to a wide spectrum of infectious micro- organisms. Many studies have assessed the relative frequency of intestinal parasitic diseases to characterize demographic and travel-related predictors of infections (Bottieau et al., 2006, 2007; Van De Winkel et al., 2007; Cobelens et al., 1998; Muennig et al., 1999; Von Sonnenburg et al., 2000; Whitty et al., 2000; Saiman et al.,
2001; Okhuysen, 2001, 2007; Ansart et al., 2005; Steffen, 2005; Bailey et al., 2006; Thors et al., 2006; Freedman et al., 2006; Seybolt et al., 2006; Caruana et al., 2006; Wilson et al., 2007; Sudarshi et al., 2003; Boggild et al., 2006; Brouwer et al., 1999). To reach a consensus on which diagnostic procedures to perform for this patient group remains very complicated. In chapter 4 the difference in the number of detected cases using conventional- and the molecular-diagnostic approach was small. However, in the setting of a travel clinic the full arsenal of diagnostic tests are used to cover most of the intestinal pathogenic parasite species. Profit can be gained by simplifying these diagnostic procedures. An extensively automated diagnostic screening system for the most important parasites can be more cost-efficient. Nevertheless, it is still difficult to decide which parasite targets would be needed in a standard screening panel. Real-time PCR showed to be more effective in detecting all four targeted parasite species than conventional diagnostic methods. Moreover, only few additional parasite species were diagnosed with microscopy, while probably many more unexplained causes of gastro-intestinal complaints might be resolved by the application of additional real-time PCR targets.