Tag Archive for Parasite

EU funds project to reduce parasite impact on fishery products

Click on the flag for more information about Spain SPAIN
Thursday, February 07, 2013, 22:30 (GMT + 9)

Scientists at Sea Technology Centre (Cetmar) from Spain are involved in a community project to mitigate the impact of parasites in fishery products of the European Union (EU) and imported ones, a situation that harms consumers and the industry.

The aim of the study is to reduce this impact through new technological solutions and management tools.

The Parasite project is funded by the Seventh Framework Programme of the European Union for R&D.

During the next three years, Cetmar Foundation will work in this project led by Santiago Pascual from the Institute of Marine Research under the Spanish National Research Council (CSIC), the Galician regional government reported.

Eleven EC research institutions, three Asian ones, and six EU SMEs participate in this multidisciplinary consortium.

The project will seek to make progress in three lines:

  • Improving food security;
  • Strengthening competitiveness of European fisheries products ‘from boat to plate’;
  • The potential improvement of European food safety policy.

The initial meeting will take place between 7 and 8 February at CSIC headquarters.


Original article

By Analia Murias

Thesis: Molecular Detection of Intestinal Parasites for Clinical Diagnosis and Epidemiology



The detection of intestinal parasitic infections for routine diagnosis and for epidemiological research still depends mainly on microscopical examination of stool samples for the identification of helminth eggs and protozoan trophozoites and cysts. Because microscopy has several limitations, additional diagnostic methods (e.g. culture, antigen/antibody detection) have been accessed to surpass obstacles in detection and characterization of intestinal parasites. Although such additional methods increases sensitivity, the amount of hands-on time accumulates substantially.

During the last years remarkable progress has been made on another diagnostic methods that are based on Polymerase Chain Reaction (PCR) technique. DNA isolation from stool can be processed in a semi- or fully-automated system where after specific DNA of multiple targets can be simultaneously amplified, visualized and semi-quantified in a closed tube system with multiplex real-time PCR. The molecular diagnostic approach was merged with an alternative diagnostic strategy where clusters of patients with shared characteristics are routinely screened for a selected number of parasites species. This new diagnostic strategy was assessed for the routine diagnosis and epidemiology of intestinal parasites in patients consulting general practitioners.

Samples of a general practice patient group were processed on a real-time PCR panel for the detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium (HGC PCR). The retrospective analysis with HGC PCR was compared with the results obtained with those of routinely performed microscopy. The results revealed that significant numbers of G. lamblia and Cryptosporidium infections remained undetected with microscopy. On the other hand, the parasites that have been detected with microscopy but not with real-time PCR consisted mainly of non-pathogenic parasites and Dientamoeba fragilis, although the pathogenicity of the latter is disputed. The results showed that, compared to the molecular approach, microscopy provided limited additional value in routine diagnosis of general practice patients with gastro-intestinal complaints, even with the use of multiple sampling procedure of faecal specimens in combination with fixatives (also referred to as the triple faeces test (TFT) procedure). The introduction of the TFT method in Dutch laboratories resulted of D. fragilis being increasingly diagnosed. Although D. fragilis has been suggested to be a potential pathogen in children, more studies are needed to support this statement. The introduction of a D. fragilis real-time PCR in routine diagnostics can help to elucidate the pathogenicity of this parasite. Cases of clinical E. histolytica infections (amoebiasis) in The Netherlands are very rare and can be associated to travel in areas where the parasite is endemic. Still, in several cases the source of infection could not be explained. Unselected screening for this parasite can be appreciated because of its major clinical importance for the patient and its potential to spread among household members.

Many intestinal parasites species, such as hookworms or Cyclospora cayetanensis, have adapted their transmission pattern and life cycle to specific environmental factors. Being bound to these factors, these parasites appear only occasionally in The Netherlands and surrounding countries; usually they are taken along by travellers returning from countries where the parasites are endemic. Real time PCRs for more exotic parasites can be used as extensions of basic molecular assays in clinical diagnostic settings. The relative frequency of a wide variety of intestinal parasitic diseases has been assessed in a population of travellers (such as tourists, immigrants, expats, etc.) using two different diagnostic approaches. The “overall view” of the conventional approach (i.e. microscopy combined with antigen tests) was compared with the molecular diagnostic approach, where only those species that are targeted in the assay are detected. The molecular diagnostic method showed more effective in detecting the targeted parasites than the conventional diagnostic approach, in particular E. histolytica and Strongyloides stercoralis for which additional diagnostic techniques are needed op top of microscopy. Only few additional parasite species have been detected with microscopy, while many more unexplained causes of gastro-intestinal complaints might be resolved by the application of additional real-time PCR targets.

Using the molecular hight throughput screening system in combination with a simple collection procedure for stool specimens, valuable data has been produced that has yielded new insights in the epidemiology of several parasitic diseases.

The prevalence of Cryptosporidium in Dutch patients with gastro-intestinal complaints attending their general practitioner has so far exceeded the figures in previous studies. The highest infection rate has been detected among children aged under five years with a peak in the month of September; almost one-third of them had been infected with Cryptosporidium. Microscopic examination for Cryptosporidium, which requires an additional staining procedure of the faecal smear, was specifically requested by the general practitioner twenty-one times and was found positive in 13 cases once, whereas with HGC PCR 80 cases were detected. The lack of request for additional diagnostic procedures is not the only reason for missing infections. Basic microscopic stool examination has often not been requested, leaving a substantial number of gastro-intestinal parasitic infections undiagnosed.

Symptoms of G. lamblia infections are highly variable, and can range from asymptomatic to the presence of severe gastro-intestinal complaints. Data in this thesis showed that, although the G. lamblia Cycle-threshold (Ct)-values correlated with the number of specified gastro-intestinal complaints, the parasite was still detected in 4.7% of persons who did not have complaints. The reasons for the clinical heterogeneity of G. lamblia infections are not fully understood. Although the epidemiological role of G. lamblia assemblages (i.e. group of genotypes) had been suggested as an important factor associated with gastro-intestinal complaints, this could not be proven for adults. Nevertheless, in children the assemblages might still be relevant for the clinical presentation of the patient.

In this thesis, additional real-time PCRs have been evaluated to cover the most important intestinal parasite species for epidemiological studies and to be used as extensions of basic molecular diagnostic assays in clinical settings. Isospora belli (recently renamed as Cystoisospora belli), Encephalitozoon intestinalis and Enterocytozoon bieneusi are opportunistic pathogens that can cause life-threatening diarrhoea and malabsorption, in immuno-compromised patients. With increased awareness, infections with E. bieneusi are diagnosed not only in immune-compromised patients, but also showed presence in asymptomatic persons. Furthermore, the high prevalence of weak infections or low shedding of spores (high Ct-values) have been observed among populations in sub-Saharan- and East-African countries. The phylogenetic study on microsporidia infections in persons with different clinical backgrounds indicated a dynamic evolutionary process between genotypes of E. bieneusi. One specific genotype was restricted to transplantation patients receiving immuno-supressives and another genotype showed its preferential habitat in patients living with HIV/AIDS, which further emphasizes the predisposition for specific hosts by different E. bieneusi isolates.

Last but not least, real-time PCRs were developed for the detection of Schistosoma mansoni and Schistosoma haematobium. Both Schistosoma species could successfully be detected in stool samples with Ct-values correlating with the results of quantitative microscopy. S. haematobium could also be detected in DNA isolated both from urine and from cervico-vaginal lavages. Results indicated that real-time PCR may potentially serve as a gold standard to determine the prevalence and intensity of Schistosoma infections in surveys. The real-time PCR may even be provided as a diagnostic tool for urine, stool and other clinical samples. This might prove helpful in particular for the difficult to diagnose female genital schistosomiasis. The semi-quantitative outcome of the PCR might be used as a predictor in the disease pathology and help to determine if observed lesions are caused by the parasite eggs.

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Chapter 11 – General Discussion

Molecular diagnosis of intestinal parasites

Real-time PCR has been established as a sensitive and specific qualitative and quantitative technique within the fields of routine diagnostic virology and bacteriology (Read et al., 2000; Niesters, 2002; Klein, 2002; Mackay, 2004; Iijima et al., 2004; Espy et al., 2006; Schuurman et al., 2007a)⁠. Several real-time PCRs for the detection of intestinal parasites have been developed showing excellent specificity and sensitivity and accepted as objective tools for case confirmation and gold standard in the development of new diagnostic tools. Implementation of molecular methods in routine diagnostics moved a step forward with the development of the first multiplex real-time PCR for the simultaneous detection of the three most important diarrhoea causing protozoa (Verweij, 2004)⁠. Recent developments in improved and simplified DNA isolation and automatizing of these methods and the broader availability of real-time PCR in diagnostic microbiology laboratories will allow these methods to be used as an alternative tool for the routine diagnosis of intestinal parasitic infections (Verweij, 2004; Espy et al., 2006)⁠. The high specificity of DNA-based methods is one of major advantages and at the same time one of the major constraints as these methods will only detect the specific DNA targets for which primers and detection probes are designed. This is in contrast with the “overall view” of conventional diagnosis of intestinal parasites using microscopy. This thesis describes a number of studies on the implementation of real-time PCR for the detection and quantification of intestinal parasites to validate and optimize it’s use in different diagnostic settings and in epidemiological surveys.

Sample collection

In industrialized countries it is common practice that stool samples are sent to the laboratory by regular mail. This delay between stool production and analysis has little or no effect on the microscopic detection of parasite eggs and cysts. The same applies for the detection of parasitic DNA with real-time PCR; in a study on the DNA detection of Dientamoeba fragilis, a parasite which lacks a cyst stage, isolation and the outcome of the PCR was reproducible even after 8 weeks of storage at 4°C (Verweij et al., 2007b)⁠.

For epidemiological studies, the storage of stool samples might nevertheless be a major problem. It is not exceptional that these studies are conducted in remote areas with a (sub)tropical climate, where power cuts are a daily phenomenon. To avoid degradation of collected samples, faecal specimens can be suspended in ethanol which will preserve the samples for at least 3 months even at tropical temperatures. This is illustrated by the detection of Schistosoma haematobium DNA in stool samples collected from 10 infected persons (figure 11.1). Although S. haematobium eggs are normally excreted via the urine, in chapter 9 S. haematobium DNA detection in stool is shown to be feasible. Figure 11.1a shows similar DNA amplifications of isolated S. haematobium DNA from stool samples that were stored either frozen or suspended in ethanol at room temperature. Storage and transport of non-fixated stool samples at room temperature as a control was not feasible under the tropical conditions. For urine, S. haematobium DNA was detected in most samples stored at different temperatures (figure 11.1b). However, the temperature at which the urine samples were stored does influence the quantitative results.

Chapter 10 describes a sensitive method for the detection of S. haematobium DNA from vaginal washings. These samples were collected under difficult conditions and at that time the researchers postulated that a more simple collection procedure and diagnostics are necessary. The use of vaginal swabs already proved to be a simple and sensitive method for the detection of several female genital infections (Whiley et al., 2005; Caliendo et al., 2005; Jaton et al., 2006)⁠. It is therefore worthwhile to investigate if vaginal swabs could also be applied for the diagnosis of female genital schistosomiasis. For epidemiological studies, reliable storage methods which preferably allow the intensity of infection to be (semi-)quantified, still need to be tested both for urine samples and vaginal swabs.


Figure 11.1. Detected Schistosoma haematobium Cycle threshold (Ct)-values of DNA isolated from stool samples (a) collected from 10 patients living in an endemic area with S. haematonium infection determined by microscopic egg counts in urine. Samples of stool were stored in frozen condition or as ethanol suspension at room temperature over a period of two weeks. Additionally, urine samples (b) were collected and divided in three parts, with each part stored at a different temperature for a period of two weeks. No ethanol or other fixatives were added to the urine. S. haematobium Ct-values were detected from DNA isolated from the urine samples. Urine and stool samples were collected and kindly provided by Dr. Akim Adegnika in collaboration with the medical research unit of the Albert Schweitzer Hospital in Lambaréné, Gabon.

11.1a 11_1b


Sample analysis

The isolation of parasitic DNA is one of the most important steps for the implementation of DNA-based methods. It is therefore essential to check for each new target if the isolation procedure is capable of extracting the DNA from the faecal sample. For example, a Trichuris trichiura-specific PCR was successful for the detection of T. trichiura-DNA extracted from an adult worm, however, isolation of DNA from the T. trichiura eggs in faeces failed. Several pernicious treatments (e.g. sonification and microwaving) have been used in vain to release the DNA from the eggs. DNA isolation from T. trichiura eggs remains a challenge, even more so if considered that the method should be feasible for routine sample preparation.

Faecal contaminants can cause inhibition of the DNA amplification. An internal control is therefore essential to monitor the efficiency of the amplification process. One of the targets of a multiplex assay amplifies and detects Phocin Herpes virus (PhHV) (Niesters, 2002)⁠, of which a fixed amount is added to one of the isolation buffers. A higher Ct-value than expected or the absence of the signal indicates a less efficient amplification process which can result in a false negative outcome of one of the other targets. PhHV is preferred because of simplicity and standardization: the same buffer with the internal control is also used for all multiplex assays and for other samples, such as tissue or urine samples. Detection of PhHV amplification does not necessarily mean that faecal DNA has been isolated. DNA amplification from normal flora found in the gastro-intestinal track has been suggested as control for faecal DNA isolation. However, the amount of normal gut flora fluctuates between samples resulting in different Ct-values and as a consequence (partial) inhibition would not be recognized. Another suggestion is to include one positive faecal sample for each target within a series of samples prepared for DNA isolation. This procedure would need sufficient supply of positive material and it still can not assure that DNA isolation in other samples has succeeded.

Real-time PCR offers the ability to amplify and detect multiple DNA targets in a single reaction. Important advantages of a multiplex assay include reduced reagent costs and a high throughput potential. However, during the DNA amplification, inhibition can occur due to the simultaneous amplification of multiple targets in one assay. Part of designing a multiplex real-time PCR involves a background test, in which the amplification of one target is measured in the presence and absence of a second target. The background test shows the performance of the assay to detect a small number of DNA copies of one target in the presence of more abundant copy numbers of another parasite species. Combining of the real-time PCR for the detection of Isospora belli detection (Ten Hove et al., 2008)⁠ with the microsporidia real-time PCR (Verweij et al., 2007c)⁠ into one assay failed at this step. Amplification of multiple targets was not possible without the inhibitive effect on the amplification of the microsporidia DNA. It is not clear yet if the performance of this multiplex assay will improve by using another type of real-time PCR apparatus. Otherwise, a new design of these assays is needed.

Several real-time PCRs are already effectively employed in routine diagnosis and epidemiology. Because of the high specificity of real-time PCR, an assay could fail to detect genetically related species or strains (e.g. Cryptosporidium species and E. histolytica strains) due to variations in the DNA sequence of the primer and probe regions. Also, rare parasite species will be missed with standard real-time PCR panels. In patients with continuing gastro-intestinal complaints in which the initial real-time PCR screening does not reveal the cause of the complaints, microscopy is still required as an additional tool. The challenge for the future will be to maintain a profound knowledge on the morphological characteristics of in these more difficult academic cases.

In routine diagnostics and in epidemiology, often large numbers of samples are processed. With increasing numbers of samples, the amount of hands-on time for sample preparation and analysis can become a major bottleneck and human errors might occur more easily. Microscopy can not be subjected to automated process whereas molecular diagnostics can be incorporated into a high throughput robotic workstation. Several automated nucleic acid extraction platforms are available for various specimens and applications (Jongerius et al., 2000; Kessler et al., 2001; Espy et al., 2001; Knepp et al., 2003)⁠. Beside the reduction on hands-on time, increased consistency and reproducibility of DNA-extraction and -analysis will improve standard operation procedures in the laboratory.

Giardia lamblia

Giardia lamblia is worldwide one of the most common intestinal parasites causing gastro-intestinal complaints. Real-time PCR for this parasite is therefore considered as the first choice as a target in a diagnostic panel. Technical evaluation of the PCR already demonstrated 100% specificity and a higher sensitivity as compared to microscopy and antigen tests (Verweij et al., 2003b, 2004a)⁠. Table 11.1 gives the results of the diagnostic tests for G. lamblia in a routine academic setting (Clinical Laboratory for Microbiology, Leiden University Medical Centre). It is clear that real-time PCR shows a higher sensitivity for the detection of G. lamblia. In this thesis, G. lamblia real-time PCR was evaluated as part of a routine diagnosis for intestinal parasites in a western European setting. In previous studies, G. lamblia has been reported to be a common pathogen in The Netherlands with a prevalence of 5% among general practice patients with gastro-intestinal complaints (De Wit et al., 2001c)⁠. A comparable prevalence is reported in chapter 2 after routine screening with microscopy of general practice patients. When real-time PCR was applied on the same samples, the prevalence increased to 9.3%. In cases for which no microscopical examination for intestinal parasites was requested by the general practitioners, G. lamblia was detected in 6.5% of the samples. This finding concurs with those from two other studies (Mank et al., 1995a; Van den Brandhof et al., 2006)⁠. Mank et al. reported that more than a third of detected pathogenic protozoa, mainly G. lamblia, would have been missed if the laboratory would have complied with the general practitioners’ requests and Van den Brandhof et al. concluded that test request by general practitioners do not always comply with existing knowledge of the etiology of gastro-enteritis. Another reason for missed infections mentioned in the latter study, is the general restrictive policy in The Netherlands with regard to requesting tests.

Table 11.1. Test results (- = negative and + = positive) retrieved from the validation report for implementation of real-time PCR in routine diagnosis of Giardia lamblia at the clinical laboratory for microbiology at Leiden University Medical Centre (Unpublished data from Dr. J.J. Verweij).


Antigentest a

Real-time PCR

Number of samples

Ct range

Median Ct






20,1 – 38,0





27,3 – 36,7




30,7 – 42,8



a Alexon-Trend ProSpecT EIA for the detection of G. lamblia.

b These samples include: 10 samples from four patients where in successive samples G. lamblia was also detected by microscopy and/or antigen test; 11 samples from six patients where G. lamblia infection was found in a family member; 3 samples from three patients in which only one out of a series of three was positive with PCR.


Although G. lamblia is regarded as a pathogenic parasite, in case-control studies it was also found in controls (Whitty et al., 2000; De Wit et al., 2001c; Hörman et al., 2004; Amar et al., 2007)⁠. In chapter 4, half of all detected cases of G. lamblia originated from returned travellers without gastro-intestinal complaints. The G. lamblia positive samples were further characterized in chapter 5 to analyse the role of G. lamblia assemblages (i.e. defined group of genotypes) in symptomatic and asymptomatic returned travellers. The data showed that increasing G. lamblia Ct-values is in concordance with decreasing gastro-intestinal complaints, translated as symptoms-score. However, the assemblages of G. lamblia revealed no association with the presence or absence of any clinical symptoms in this group of travellers. Previous studies suggested the epidemiological role of G. lamblia assemblages in children as an important factor associated with gastro-intestinal symptoms (Read et al., 2002; Haque et al., 2005; Sahagun et al., 2008; Kohli et al., 2008; Peréz Cordón et al., 2008)⁠. In adults, like most subjects in chapter 4 and 5, the association of assemblages with gastro-intestinal symptoms is less evident and can even give contradicting results (Homan & Mank, 2001; Gelanew et al., 2007; Sahagun et al., 2008)⁠. It is suggested that the presence of symptoms in adults more likely to be related with underlying conditions such as the degree of host adaptation, nutritional and immunological status. The results of studies on the association of G. lamblia assemblages with gastro-intestinal complaints are summarized in table 11.2.


Table 11.2. Summary of studies on the association of Giardia lamblia assemblages with clinical symptoms.


Clinical symptoms

Reference Study area Study group Gene target

Assemblage A

Assemblage B

Homan et al., 2001 Netherlands n=18; 8-60yrs GDH

Mild, intermittent diarrhoea

Severe, persistent diarrhoea

Read et al., 2002 Australia n=23; <5yrs SSU-rDNA



Haque et al., 2005 Bangladesh n=211; <10yrs SSU-rDNA



Gelanew et al., 2007 Ethiopia n=80; all ages β-giardin

Less symptomatic

More symptomatic

Sahagun et al., 2008 Spain n=72; <5yrs TPI

More symptomatic

Less symptomatic

n=65; 5-72yrs TPI

No difference

Peréz et al., 2008 Peru n=201; 0-9yrs GDH


High shedding, non diarrhoeic

Kohli et al., 2008 Brazil n=189; children SSU-rRNA

No difference

Ten Hove et al., 2008 Worldwide

N=156; mostly adults


No difference


Future studies on transmission patterns in households in combinations with detailed description of underlying conditions could give more inside on the role of G. lamblia assemblages on the clinical presentation.


Cryptosporidium infections were studied in both the general practice patients and returned travellers. Microscopic detection of Cryptosporidium requires additional staining methods to visualize the parasite in faecal samples. This additional diagnostic procedure is not always included in routine stool analysis or requested by the health care providers, probably because cryptosporidiosis is usually associated with immune-depression, in particular HIV/AIDS. Given the sporadic requests from the health care providers for additional Cryptosporidium detection, prevalence of cryptosporidiosis is highly underestimated (chapter 2) (Van Gool et al., 2003; Jones et al., 2004)⁠. The prevalence of Cryptosporidium in patients with diarrhoea as described in chapter 2 and 3 has so far exceeded the figures provided in other studies (Petry, 1998; Banffer & Duifhuis, 1989; Rodriguez-Hernandez et al., 1996; De Wit et al., 2001a; Vandenberg et al., 2006a). In the month of September 2005, Cryptosporidium (mainly C. hominis) was detected in up to 30% of all children aged under five with gastro-intestinal complaints. The reason for the high prevalence described in these chapters could be due to the unselected screening of patients by highly sensitive real-time PCR analysis in combination with sample collection during a seasonal peak of cryptosporidiosis (Van Asperen et al., 1996; Wielinga et al., 2007)⁠. A common source of infection has not been identified but cases did not show any geographic clustering (unpublished observations). A low prevalence of Cryptosporidium was shown in the group of mainly adult travellers, even among those who returned from ‘high risk areas’.

Entamoeba histolytica

The very low prevalence of Entamoeba histolytica infections in the general practice patient population of chapter 2 could suggest that the E. histolytica assay is redundant in the diagnostic real-time PCR panel. Although infection with E. histolytica is rare in The Netherlands, the assay was included because of its major clinical importance for the patient and its potential to spread among household members (Stanley, 2003)⁠. Even though a clinical E. histolytica infection is normally associated with tropical areas, this is not always evident and can therefore delay the diagnosis (Vreden et al., 2000; Edeling et al., 2004; Veneman et al., 2006)⁠. Such a case emerged during screening for Cryptosporidium with the E. histolytica-G. lamblia-Cryptosporidium PCR (HGC-PCR) of an additional 1000 general practice patients as described in chapter 3. The E. histolytica real-time PCR positive individual concerned a young male with severe diarrhoea and an unknown travel history. Despite several microscopic analyses over a period of more than 3 years, still no pathogen was detected that could explain the gastro-intestinal complaints. Eventually the patient was treated as a clinical amoebic infection (T. Schuurman, personal communication). As described in the introduction, the classical method for detection and diagnosis of E. histolytica infection is not that straightforward and it is essential that physicians are familiar with epidemiology, clinical syndromes, management of infections and available diagnostic tests. The introduction of the HGC-PCR, in particular for travellers, greatly facilitates the difficult diagnosis of E. histolytica infections.

Strongyloides stercoralis

The diagnosis of a Strongyloides stercoralis infection by microscopy involves multiple sampling and concentration methods. The Baermann concentration method is widely used, but the sensitivity of the test relies on the number of samples examined. Furthermore, there are several reasons why this S. stercoralis detection procedure is omitted, like in chapter 4 because the samples were too old, did not have the right consistency, or only a small amount of faeces was available. Moreover, it was shown that physicians often don’t consider strongyloidiasis in their differential diagnosis, which could place patients at iatrogenic risk (Loutfy et al., 2002; Boulware et al., 2007)⁠. In the setting described in chapter 4, the screening for S. stercoralis infection with real-time PCR proved rewarding, but may also be an important method to avoid hyper-infection in patients in need of immune-suppressive treatment.

Isospora belli and microsporidia

In the epidemiology of opportunistic intestinal parasitic infections in immune-suppressed and immune-incompetent patients, real-time PCR proved to be a useful tool for the detection of Isospora belli (chapter 6) and microsporidia (chapter 7) infections. In Blantyre, Malawi, the real-time PCRs were used for a study on the aetiology of diarrhoea in hospitalized patients (figure 11.2). Stool samples were collected from HIV/AIDS positive- (n = 226) and HIV-negative- (n = 46) patients. I. belli and Enterocytozoon bieneusi were detected by real-time PCR in 175 (64%) and 197 (73%) of all cases, respectively. Preliminary data analyses show that the intensities of the parasitic infections (reflected in Ct-values) were associated with the severity of diarrhoea (Beadsworth, unpublished data). Moreover, I. belli was more common in patients with 100-200 CD4-cells/mm3, while E. bieneusi was mostly detected in patients with <100 CD4 cells/mm3 (figure 11.2).


Figure 11.2. Percentage of detected Isospora belli positive cases and Enterocytozoon bieneusi cases with detected Ct-values below 35, in HIV-negative (n=46) and -positive (n = 226) patients categorized by their HIV status (reflected by CD4 counts). Samples were collected from patients in Blantyre, Malawi (Unpublished data from L. van Lieshout, M.B. Beadsworth et al.).


The decrease in the number of I. belli infections in the last patient group is not necessarily the result of the lower CD4 counts (Kartalija & Sande, 1999; Certad et al., 2003)⁠. It could also be due to the in vivo competition between the two parasitic infections. Further data analysis, including the results of Cryptosporidium detection is still pending (Beadsworth et al., in preparation).

Because of higher awareness of microsporidiosis together with the availability of the highly sensitive and specific diagnostic techniques based on PCR, microsporidia infections are increasingly diagnosed in transplant recipients, children, seniors and travellers. The detection of microsporidia infections in these patient populations in western settings is mostly clear cut. However, in the study in Malawi, but also in unpublished observations in Ethiopia, a very high prevalence of E. bieneusi infections is observed. In half of the detected cases Ct-values were above 35 reflecting a very low DNA-load. The clinical relevance of these findings is not clear. Possible explanations for these cases could be just an infection with a low parasite load, the presence of asymptomatic carriers or the results of an ubiquity of spores in tropical environments with high HIV/AIDS prevalence among the population (Van Gool et al., 1997; Cegielski et al., 1999; Tumwine et al., 2002; Siński, 2003; Nkinin et al., 2007; Samie et al., 2007; Raccurt et al., 2008)⁠. To elucidate the association of microsporidia infections with different clinical backgrounds, E. bieneusi genotypes were characterised in several patient cohorts. The phylogenetic study in chapter 8 describes a clustering in different human populations for some of the genotypes of E. bieneusi. Using the same techniques for detection and characterization, future studies could attempt to follow transmission patterns involving (asymptomatic) household members, domestic animals and the environment to determine the potential of E. bieneusi as a zoonotic and water-borne pathogen.

Schistosoma spp.

In chapter 9 a multiplex real-time PCR approach on the cytochrome c oxidase subunit 1 (cox1) gene was compared with egg counts determined by Kato-smears on preselected series of faecal samples. These were collected from subjects living in an area endemic for both Schistosoma mansoni and Schistosoma haematobium. The multiplex real-time PCR showed 100% specificity using an extensive number and variety of controls and 100% sensitivity in subjects with more than 100 eggs per gram faeces, with few false negatives in patients excreting lower numbers of eggs. Although eggs of S. haematobium were not observed in stool samples, its DNA could successfully be detected. As expected the sensitivity of S. haematobium DNA detection in faeces is lower than the sensitivity achieved by microscopic examination of urine samples. Preliminary results using real-time PCR on DNA isolated from urine samples, collected in a S. haematobium-endemic area in Gabon showed promising results (figure 11.1). For further improvement on the sensitivity of the molecular test, another real-time PCR was developed targeting the internal transcribed spacer 2 (ITS-2) (Obeng et al., 2008)⁠. The ITS-2 PCR improved Schistosoma detection considerably, in particular for the detection of S. mansoni (table 11.3). Unlike the cox1 gene, the real-time PCR assay on ITS-2 does not discriminate between DNA of S. mansoni and DNA of S. haematobium.

Table 11.3. Amplification of a specified amount of Schistosoma mansoni DNA and S. haematobium DNA isolated from adult worms, with the species specific cox1 real-time PCR assay and the genus specific ITS-2 real-time PCR assay reported in detected Ct-values.



Schistosoma mansoni


Schistosoma haematobium

Target DNA


cox1 PCR



cox1 PCR


100 ng





10 ng





1 ng





100 pg





10 pg





1 pg





100 fg





10 fg






The ITS-2 PCR was further evaluated using urine samples collected in Ghana and compared with CCA strips and microscopy (Obeng et al., 2008)⁠. The results indicate that real-time PCR may potentially serve as a gold standard to determine the prevalence and intensity of Schistosoma infections in surveys. Moreover, these results and the findings described in chapter 9 suggest the versatility of real-time PCR as a diagnostic tool for urine, stool and other clinical samples. In chapter 10 the performance of Schistosoma real-time PCR was evaluated for the diagnosis of female genital schistosomiasis on DNA isolated from vaginal lavages. This provisional investigation showed that Schistosoma real-time PCR is a valuable tool for diagnosis in research and evaluation projects.

multiplex real-time PCR for routine diagnostics

The implementation of real-time PCR in routine diagnostics has encountered several paradigms in the medical community on assumptions, concepts, practices and even values that constitute the way on how parasites should be diagnosed. 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 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 met 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 real-time PCR panel including D. fragilis-specific PCR which can help to elucidate the pathogenic role of this parasite (Bruijnesteijn van Coppenraet et al., n.d.)⁠.

In immune-compromised patients the most important opportunistic intestinal parasites can be related to the conditions of the immune suppression. For example, S. stercoralis hyper infection is associated with corticosteroid treatment and HTLV infection but not with HIV infection.

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.

AdultsEntamoeba histolytica

Giardia lamblia


ChildrenDientamoeba fragilis

Giardia lamblia


TravellersEntamoeba histolytica

Giardia lamblia


Strongyloides stercoralis

Schistosoma b

Cyclospora cayetanensis b

Isospora belli b

E. bieneusi c

Immune compromisedStrongyloides stercoralis a


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.


Travellers comprise a very diverse group, including immigrants, adoption children, tourists, businessmen, expats, army personal, etc. that may have been exposed to a wide spectrum of infections. 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 overall 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 in simplifying these diagnostic procedures. An automated diagnostic screening system for the most important parasites can be more cost-efficient. Nevertheless, it is still difficult to decide which parasite targets are needed in the standard screening panel. Real-time PCR showed more effective in detecting all four targeted parasite species than with conventional diagnostic methods. Moreover, only few additional parasite species were diagnosed with microscopy, while many more unexplained causes of gastro-intestinal complaints might be resolved by the application of additional real-time PCR targets.

concluding remarks

Real-time PCR has opened new perspectives on diagnosis of intestinal parasites. Nevertheless, the process of improving diagnostic methods is still continuing. It is evident that in the future the throughput of real-time PCR platforms will improve substantially by increasing the number of targets, speeding up sample processing time and introducing plates with a larger number of wells. Already a real-time PCR system has been assessed for the detection of five viral targets on 384 well format (Molenkamp et al., 2007)⁠. Moreover, diagnostic technologies, such as biosensors, micro-DNA arrays and microfluid technologies are advancing (Wang et al., 2004; Van Merkerk, 2005; Taguchi et al., 2005; Stein et al., 2006)⁠. At the same time, new parasitic diseases can emerge while others disappear. Therefore, any diagnostic method, new or old, should only be applied when it has proven to provide meaningful analysis. The present thesis shows that real-time PCR provides a feasible and effective alternative method in the routine diagnosis of intestinal parasites in patient care and epidemiology.

The initial implementation of real-time PCR in a laboratory requires a large investment. On the other hand prices of the equipment and consumables continue to decrease and become more competitive compared with the high costs of staff in a laboratory. Cost-effectiveness of real-time PCR could increase when diagnostic assays are extended with additional parasitic, bacterial, fungal and/or viral targets. Nevertheless, it needs to be taken into consideration that although cost efficiency on consumables and labour-time might favour one diagnostic methodology, the amount of reimbursement for a laboratory can still vary extensively due to different declaration settlements in health care systems.

A major challenge that remains is the access to sensitive and specific diagnostic tools in low income countries. Although the real-time PCR method brings forward new exciting prospects on the epidemiology of intestinal parasites in remote and endemic areas, it has little value for clinical diagnostics in most of these settings. Real-time PCR can perform a prominent role as a gold standard in quality control for the evaluation of new inexpensive rapid tests for field diagnostics. Even more, field applicable PCR formats can be developed with instruments being further miniaturized, integrated into all sample processing and made less expensive (Gerdes et al., 2008; Yager et al., 2008)⁠. A giant leap in public health can be made if medical practices in resource poor settings could lean on such advances.

Chapter 1 – General Introduction


Since the late nineteenth century a vast amount of scientific data has been compiled on the causal relationship between parasites and diseases in man. It was anticipated that as a consequence of the overall medical advances and global initiatives in eradication programs, infectious parasitic diseases would eventually become a thing of the past. Malaria, sleeping sickness, visceral leishmaniasis, Chagas’ disease, river blindness and guinea worms are some examples of diseases on which international cooperative intervention programs have focused in the 20th century (Hopkins et al., 2005, 2008; Fèvre et al., 2006; Yamagata & Nakagawa, 2006; Alvar et al., 2006)⁠. In developed countries, many parasitic diseases have been eradicated or their prevalence has declined significantly. However, despite enormous efforts in eradication programs, it is striking to observe that ‘classical’ parasitic diseases continue to re-emerge in other parts of the world (Molyneux, 2004; Hotez et al., 2007; Stratton et al., 2008)⁠. Not only do diseases re-emerge, other parasitic infections (e.g. microsporidia and Cryptosporidium) have been described for the first time only recently (Lashley & Durham, 2007; Topazian & Bia, 1994)⁠. Factors that have contributed to the decline or the emergence of human pathogenic parasites include changes in sociocultural patterns and human behaviour as well as the overall expansion of human activities and their impact on ecosystems. Nevertheless, the exact reasons are often complex and interrelated (Cohen & Larson, 1996; Gubler, 1998; Lashley, 2003)⁠.

The human intestinal tract may be a habitat to a variety of parasites, ranging from microscopic small microsporidia to meters long tapeworms. Observations on the most common and larger intestinal worms were already described in the earliest civilizations (Cox, 2002)⁠. Smaller intestinal parasites that were not visible by eye could only be observed after the invention of the microscope. In the seventeenth century Antonie van Leeuwenhoeck was the first to observe these little animals (“dierkens”), which were later described as Giardia lamblia. The invention of the microscope caused a major breakthrough in parasitology and has ever since been the classical tool for identifying parasites. Only a small portion of all intestinal parasite species may cause serious health problem, but most live in the gastro-intestinal tract without causing significant harm to its host. In some way they may even have a positive effect on the host’s immunological system (Yazdanbakhsh et al., 2002)⁠.

Despite that our understanding of parasites has improved enormously over the last decades, the tools that are used for parasite detection have remained largely the same. Laboratory diagnosis of intestinal parasite infections still depends mainly on microscopical examination of stool samples for the identification of helminth eggs and protozoan trophozoites and cysts. Nonetheless, the use of microscopy in diagnostic laboratories has several important disadvantages. Some parasite species cannot be differentiated based on microscopy only, while detection of other species may need well trained and experienced technicians. The overall diagnostic sensitivity of microscopy is low and in settings with relatively large numbers of negative results, microscopy can be tedious with relatively high costs for each detected case. Some alternatives for microscopic detection of intestinal parasites have been developed. Those based on parasite antigen detection in stool and antibody detection in serum are sensitive and clinically relevant for diagnosing specific infections but still have their limitations. More promising developments in parasite diagnostics can be found in the field of molecular parasitology. Shortly after the development of polymerase chain reaction (PCR) in 1988 (Saiki et al., 1988)⁠, De Bruijn (De Bruijn, 1988)⁠ predicted this technique to become a valuable way of diagnosing parasitic diseases. Recently, a range of DNA based methods for the detection of intestinal parasites has been described and postulations have been made on the tremendous impact of the implementation of automated DNA isolation and combination of multiplex real-time PCR assays for the detection of parasites, viruses, and bacteria on the differential laboratory diagnosis of diarrhoeal diseases (Morgan & Thompson, 1998a; Mackay, 2004; Verweij, 2004; Monis et al., 2005; Espy et al., 2006)⁠. In this thesis, strategies for the most effective application of these techniques in patient care and epidemiology are evaluated and additional assays for parasitic targets that are currently missing have been developed and validated.

Challenges of microscopic diagnosis

In Western European laboratories the diagnosis of intestinal parasites by microscopy is facing several important methodological issues that concern the reliability of the analysis. Because so many parasite species are present in faecal specimens in low quantities only, infections are often missed in a direct smear examination. Therefore, concentration methods which increase the recovery of protozoa cysts and helminth eggs, such as the formol-ether sedimentation method, have become a routine procedure in clinical diagnostic laboratories (Ridley & Hawgood, 1956; Allen & Ridley, 1970; anonymous, 1977; Polderman, 2004)⁠. For further improvement of the sensitivity, it has been recommended to perform stool examination on samples obtained on different days at intervals of 2 to 3 days (Nazer et al., 1993; Branda et al., 2006)⁠. On the other hand sensitivity can be affected if microscopy can not be performed within one hour after defecation, due to the rapid disintegration of trophozoites. To overcome this problem a preservative, such as sodium acetate acetic acid formalin (SAF), can be added to the stool sample immediately after production. This increases the chance of detecting protozoan parasites, in particular Dientamoeba fragilis (Mank et al., 1995b)⁠.

During the last decade an increasing number of laboratories in The Netherlands have implemented a Triple Faeces Test (TFT)-protocol in order to solve some of these sensitivity limiting factors. In this TFT procedure the patient collects faeces on three consecutive days in two tubes already containing SAF (Van Gool et al., 2003)⁠. The SAF preserved specimens are in particular suitable for the detection of trophozoites and for making permanent stains (e.g. with chlorazol black dye (CB) or Iron Haematoxylin-Kinyoun (IHK)). The unpreserved specimen is used for formol-ether concentration of protozoan cysts and helminth eggs, and the detection of Strongyloides stercoralis larvae. Although the TFT-protocol has been received as a valuable adaptation of conventional microscopic analysis, one has to consider that the TFT procedure is a more time-consuming approach and needs specific training of the microscopist. Studies in which the recovery of intestinal parasites have been compared using TFT protocol versus the conventional diagnostic method (ether-sedimentation of one fresh stool sample) are limited. Data of two such studies showed that the majority of additional gain in the TFT-protocol are non-pathogens while for the pathogens the largest profit goes mainly to Giardia lamblia and Dientamoeba fragilis (although the clinical relevance of the latter is still disputed) (Van Gool et al., 2003; Vandenberg et al., 2006a)⁠. For some intestinal parasite species, neither examination of a formal-ether concentrated fresh stool sample nor a TFT-procedure is sufficient. Additional procedures are required for the specific detection of Strongyloides stercoralis (e.g. Baermann method) and microsporidia (e.g. optical white staining). For the detection of coccidia an acid fast staining procedure is essential, which may be included if the IHK permanent staining is used, but is not covered in the CB staining. Examination with UV fluorescence microscopy is needed for the detection of Cyclospora cayetanensis (Polderman, 2004)⁠.

Not surprisingly, all these methodological issues are hardly subjects of discussion in laboratories in less developed countries. The application of diagnostic tools does not only depend on the stated objectives of a clinical diagnostic laboratory, but also on the practical limitations when working under basic conditions. More extensive procedures for parasite detection, such as complicated staining techniques or the use of fluorescent light microscopy or even a centrifuge, are often not at hand. Standard routine diagnostic stool examination in resource-poor settings is generally limited to direct faecal smear examination. Additional diagnostic methods for the detection of a specific parasite species are applied mostly when diagnosis is part of major intervention studies, such as the Kato method for monitoring Schistosoma infections in high endemic areas (Katz et al., 1972)⁠.

Last but not least, the last methodological aspect affecting the reliability of microscopy is the performance of the microscopist. Proper identification of the parasite depends highly on her or his skills and experience. Keeping up with high standards and performance in clinical diagnostic laboratories requires continued in service-training of the technicians accompanied by indispensable internal- and external proficiency testing (Bartlett et al., 1994)⁠.

There are also factors, which are beyond the techniques used and the performance of the technicians, explaining why parasites may remain undetected. As stated, certain parasite species such as S. stercoralis or C. cayetanensis need specific diagnostic procedures in order to be detected microscopically in the most sensitive and specific way. Because many of these procedures are time-consuming and laborious, they are often not included in routine stool examination, certainly not when the parasite is not very common in the region. Instead many laboratories perform these additional procedures only for individual patients, either based on a special request from the health care provider or justified by information provided about the patient such as travel history or immune disorders. Thus, depending on the stringency of the selection criteria used and the, often limited, information provided by the health care taker, a relevant proportion of parasite infections may be left undiscovered (Whitty et al., 2000; Jones et al., 2004)⁠.

Alternative techniques

A more provoking approach would appear to be the design of a diagnostic strategy where clusters of patients with some general characteristics are routinely screened for a selected number of parasite species. Such an approach can have a major impact on cost-efficiency in diagnostic laboratories with less demand on highly specialized human resources and labour-intensive microscopy. An initial step in this direction has been the development of immunoassay’s (e.g., direct immunofluorescence assays [IFA] or enzyme immuno assays [ELISA]) for antigen detection of Giardia lamblia, Cryptosporidium and Entamoeba histolytica, which give highly reproducible results which are less dependent on the skills of a lab-technician. High sensitivity and specificity was established with the ELISA for Giardia lamblia in comparison with microscopy (Mank et al., 1997)⁠, but less consistent results were seen with Cryptosporidium antigen detection (Doing et al., 1999; Katanik et al., 2001; anonymous, 2004; Weitzel et al., 2006)⁠ whereas the sensitivity and specificity of Entamoeba histolytica stool antigen detection assays decreases substantially unless samples are examined or frozen shortly after production (Tanyuksel & Petri, 2003; Visser et al., 2006)⁠. For the diagnosis of schistosomiasis in epidemiological research, circulating anodic antigen and circulating cathodic antigen (CCA) detection in serum and urine antibodies have been proposed as an alternative method (Van Lieshout, 1996)⁠. In an effort to improve field based diagnosis of schistosomiasi, antigen capture dipsticks that detects CCA in urine have been developed (Van Dam et al., 2004; Stothard et al., 2006)⁠. Sensitivity and specificity of the antigen capture dipsticks still needs to be improved for low endemic areas and so far the test has no proven value for the diagnosis of Schistosoma haematobium infections (Stothard et al., 2006; Legesse & Erko, 2008)⁠. Nevertheless, CCA dipsticks have the important advantage to be able to semi-quantify Schistosoma mansoni infections and not having to rely on a well equipped laboratory.

During the last years, remarkable progress has been made in developing diagnostic methods that are based on the Polymerase Chain Reaction (PCR) technique, particularly since major drawbacks with this technique were readdressed. Initially, DNA isolation from faecal specimens was hindered by time-consuming methods and the presence of inhibitory substances in such samples. PCR was also known as a laborious and expensive technique and inefficient when large number of samples had to be screened for multiple diagnostic targets. Conventional methods of DNA amplification of large number of samples poses a risk to cross-contamination. However, newly developed DNA isolation- and PCR-methods have greatly reduced these obstacles. DNA isolation from stool can be processed in a semi- or fully-automated system with reduced chance of cross-contamination, while removing most of the inhibitory components (Verweij, 2004)⁠. After isolation, specific DNA can be amplified and visualized with real-time PCR in closed tubes using fluorescent molecules, which minimizes the risk of cross contamination. Hence, the PCR system simultaneously amplifies and determines the level of amplified DNA products (figure 1.1). Oligonucleotides have been combined with various fluorescent labels that emit light at different wavelengths. In this way, the real-time PCR system has the potential to detect several targets simultaneously in a multiplex real-time PCR assay. An important advantage of a multiplex assay is the reduction in reagent costs and labour time. Furthermore, one of the targets can be assigned to an internal control that can demonstrate inhibitory factors during the amplification process. Several real-time PCR assays have already been developed for clinical diagnosis but because only a limited number of targets can be assigned to one assay, the targets of choice need careful evaluation (Mackay, 2004; Espy et al., 2006)⁠.

pcr curves

Figure 1.1. Detection of parasite DNA from faecal suspension containing 1, 10, 100 or 250 oocysts of a parasite. The continuous curved lines show increased fluorescent signal of the parasite specific probes and the dotted curved lines are from corresponding internal controls. Samples with higher concentration of initial DNA templates will cross the fluorescent threshold (horizontal arrow) at a lower amplification cycle number.

As mentioned before, in developed countries parasitic infections are limited to only a few species and some of those can be associated with specific clinical conditions or patient’s background. Patient characteristics could therefore be used as a prognostic tool for the design of a new diagnostic decision tree by taking into account the clinical and demographic characteristics of the patients, such as age, travel history, immune-status, etc. With a decision-tree strategy, the most advantageous diagnostic panel could be determined for conducting real-time PCR analyses.

PCR has been successfully applied in another area of work, which is epidemiological research in endemic areas. It has been shown that field based diagnosis could be replaced by PCR analysis in a central laboratory (Verweij et al., 2003a). Stool specimens are collected, suspended in alcohol and can then be stored at room temperature for a period of several months. Upon arrival at a laboratory with PCR facilities, samples can be further processed for the detection of parasite infections. Using automated DNA isolation methods and a real-time PCR system, samples can be processed even more rapidly with little chance of contamination. A sensitive high-throughput system could bring major improvements in following-up, monitoring or evaluating parasite intervention studies (Mabey et al., 2004; Urdea et al., 2006)⁠. Furthermore, the (semi-quantitative) numeric outcome of real-time PCR analysis greatly facilitates the processing, interpretation and reporting of collected data (Verweij et al., 2007a)⁠.

In the following paragraphs, specific diagnostic techniques and diagnostic challenges for the most important intestinal parasitic infections will be discussed for each species separately. Although S. mansoni and S. haematobium are in actual fact blood-flukes, one paragraph will elaborate on the detection of Schistosoma species in stool-, urine- and genital-specimens.

Entamoeba histolytica.

The perception of the worldwide and regional epidemiology of E. histolytica infection has changed after the formal acceptance that the organism called E. histolytica in fact consists two genetically distinct species, now termed as Entamoeba histolytica (the pathogen) and Entamoeba dispar (a commensal) (WHO/PAHO/UNESCO, 1997; Stanley, 2003)⁠. Prevalence of E. histolytica would therefore be grossly overestimated when the non-pathogenic Entamoeba dispar is consistently recognized as an E. histolytica infection (Kebede et al., 2003)⁠. Epidemiological surveys that used techniques for specific E. histolytica detection, have reported prevalence as high as 21% in asymptomatic individuals (Gathiram & Jackson, 1987)⁠. In countries where adequate measures are taken for human waste disposal and food- and water-safety, cases of E. histolytica infections are sporadic as the parasite is transmitted faecal-orally. In developed countries those still at risk are mainly immigrants and travellers returning from high risk areas, as well as their close relatives (Jelinek et al., 1996; Vreden et al., 2000; Edeling et al., 2004; Boggild et al., 2006)⁠. Recognition of E. histolytica is of major importance in the clinical laboratory because of its potential of developing into a life-threatening disease. E. histolytica infection can result in colitis, dysentery and abscess in organs, most often the liver, with an estimated 40.000 – 100.000 deaths annually (Walsh, 1986; Stanley, 2003)⁠. Specific detection of E. histolytica cannot be achieved with microscopy alone as cysts and (small) trophozoites of E. histolytica and E. dispar are morphologically indistinguishable. Therefore, additional methods such as ELISA’s for E. histolytica antigen detection in faeces are employed, although their performance highly depends on the state of the stool samples (Fotedar et al., 2007)⁠. Furthermore, an indirect immunofluorescence assay or a enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies against E. histolytica is a highly sensitive and specific tool to establish the diagnosis of invasive amoebiasis or liver abscess (Jackson et al., 1985; Stanley, 2003; Verweij, 2004)⁠. Disadvantages with serology on E. histolytica / E. dispar carriers are that in early E. histolytica infections the test give false-negative results and in those from endemic areas the test could give false-positive results (Stanley, 2003; Visser et al., 2006)⁠⁠⁠. Real-time PCR for the detection of both Entamoeba species in stool samples has been recognized as a highly valuable alternative and has become the preferred method in patient diagnostics (Blessmann et al., 2002; Verweij et al., 2003a; Visser et al., 2006)⁠.

Giardia lamblia

Giardia lamblia (synonyms: G. duodenalis and G. intestinalis), a flagellated intestinal protozoan parasite, is a common cause of gastroenteritis worldwide (Guerrant et al., 1990)⁠. Many people with Giardia infection remain asymptomatic and therefore it took many years until the parasite was classified as a pathogen (Rendtorff, 1954; Thompson, 2000)⁠. Diagnosis of G. lamblia is usually performed by microscopic examination of stool samples for the presence of cysts and/or trophozoites. The excretion of the parasite can be highly variable and therefore analyses of multiple stool samples and concentration techniques are recommended to increase sensitivity (Danciger & Lopez, 1975; Marti & Koella, 1993; Mank et al., 1995b; Hiatt et al., 1995; Polderman, 2004)⁠. Other suggested procedures for the detection of G. lamblia are concentration techniques and examination of freshly preserved stool samples for the recovery of vegetative stages (Mank et al., 1995b; Polderman, 2004)⁠.

The alternatives for microscopic diagnosis of G. lamblia include immunoassays for direct immunofluorescence (DFA) and G. lamblia antigen detection (Garcia & Shimizu, 1997; Aldeen et al., 1998; Maraha & Buiting, 2000)⁠. Compared to microscopy, immunoassay tests have shown increased sensitivity with reduced number of samples required for examination (Mank et al., 1997; Mank & Zaat, 2001) but still multiple stool sample analysis is needed for optimal sensitivity (Hanson & Cartwright, 2001)⁠. The second diagnostic alternative is G. lamblia DNA detection in stool by real-time PCR, which showed a higher sensitivity than microscopy and immunoassay analyses and can further reduce the required number of stool samples for analysis (Verweij et al., 2003b)⁠.

G. lamblia has also been extensively studied with DNA-based methods for classification of subgroups. From humans and various animals, G. lamblia strains have been isolated and characterised as different genotypes. The genotypes that can infect humans are clustered in assemblage A and B (Monis et al., 1996; Homan et al., 1998)⁠ and several studies have associated assemblages with differences in clinical symptoms. The results of these studies are difficult to compare and sometimes are even conflicting. This subject will be further discussed in chapters 5 and 11.

Cryptosporidium hominis / C. parvum

Cryptosporidium spp. are coccidian protozoan parasites that infect various vertebrate and invertebrate hosts. At least seven Cryptosporidium species have been associated with gastro-intestinal disease in humans: C. hominis, C. parvum, C. meleagridis, C felis, C. canis, C. suis and C. muris  (Xiao & Ryan, 2004; Caccio et al., 2005)⁠. Of these, C. hominis and C. parvum are the two species found most often in humans. Infections with other Cryptosporidium species are sporadic and are associated with a deficient immune system (Pieniazek et al., 1999; Xiao et al., 2000; Gatei et al., 2002)⁠. Among immunocompromised individuals, especially those living with AIDS, Cryptosporidium is recognised as a potentially life-threatening opportunistic parasite and prevalence is often high in areas affected by the HIV/AIDS pandemic (Guerrant, 1997)⁠. C. hominis /C. parvum may show seasonal distribution patterns and has been recognised as the cause of several water-borne outbreaks (Rose et al., 2002; Karanis et al., 2007; Semenza & Nichols, 2007; Wielinga et al., 2007)⁠. Last, C. parvum is more associated with zoonotic transmission of infected cattle (Huetink et al., 2001; Lake et al., 2007)⁠.

Microscopic detection of Cryptosporidium infection usually includes a concentration method in combination with a modified acid fast staining. Microscopic examination can be time-consuming and is highly dependent on technical expertise in a clinical laboratory. As an alternative for microscopy, a variety of commercial tests (IFA and ELISA) have been evaluated for Cryptosporidium detection in stool specimens, that have the advantages of improved sensitivity and rapid turnover with little hands-on work (Garcia & Shimizu, 1997; Katanik et al., 2001; Weitzel et al., 2006)⁠. However, the applicability of copro-antigen tests in diagnostic laboratories needs to be interpreted with some caution because of conflicting reports on the specificity of the antigens and to the sensitivity of immunodetection methods over microscopy (anonymous, 1999; Doing et al., 1999)⁠.

Cryptosporidium-detection methods based on PCR has been an important instrument for studying the taxonomy and transmission of the parasite (Laxer et al., 1991; Jiang & Xiao, 2003; Caccio et al., 2005)⁠. More recently Cryptosporidium PCR assays were also developed with focus on use in routine clinical diagnostics (Morgan et al., 1998; Morgan & Thompson, 1998b; Verweij et al., 2004a)⁠.

Isospora belli

Infection with the coccidian Isospora belli (recently renamed as Cystoisospora belli (Barta et al., 2005; Samarasinghe et al., 2008)⁠) is associated with chronic and severe diarrhoea, in particular in persons living with AIDS and in other immunocompromised individuals (Ferreira, 2000; Lewthwaite et al., 2005; Atambay et al., 2007)⁠. Infections are also seen in children and travellers to tropical regions (Okhuysen, 2001; Goodgame, 2003; Jongwutiwes et al., 2007)⁠. An I. belli infection is usually diagnosed by microscopic detection of the parasite oocysts in stool samples. The oocysts have a thin transparent shell that makes detection of the oocysts in unstained direct smears difficult and additional microscopic, concentration, and/or staining methods are needed to improve sensitivity (Franzen et al., 1996; Lainson & da Silva, 1999; Bialek et al., 2002)⁠. The oocysts can be stained with modified Ziehl-Neelson method and also show auto-fluorescence using a microscope with ultra-violet (UV) light source. A nested PCR method with Southern blot hybridization was described by Muller et al. (Müller et al., 2001)⁠ as a helpful technique for the detection of very mild I. belli infections. However, these are very laborious procedures and therefore, not efficient for use in epidemiological studies or in routine diagnostic laboratories. Instead, a real-time PCR was developed for the specific detection of I. belli DNA in faecal samples which will be further discussed in chapter 6.

Cyclospora cayetanensis

Cyclospora cayetanensis, a coccidian intestinal protozoon, is reported to be in the USA the cause of several outbreaks of diarrhoeal illness after import of contaminated fruits and vegetables (Herwaldt, 2000)⁠. Infections have also been associated with travellers returning from endemic areas, in particular South-East Asia or South-America (Puente et al., 2006; Kansouzidou et al., 2004; Gascon et al., 1995; Blans et al., 2005)⁠. The parasite oocysts are easily missed in faecal wet-mount preparations and staining with modified Ziehl-Neelson gives variable results. However, the thin oocysts wall can well be distinguished by auto-fluorescence using UV-microscopy. Real-time PCR detection for C. cayetanensis has been described as a highly sensitive and specific alternative for microscopy (Varma et al., 2003; Verweij et al., 2003c)⁠.

Dientamoeba fragilis

Dientamoeba fragilis is a gastro-intestinal flagellate with high prevalence worldwide and is still subject to debate whether the parasite can be considered as a cause of gastro-intestinal illness (De Wit et al., 2001b; Okhuysen, 2001; Johnson et al., 2004; Stark et al., 2006; Vandenberg et al., 2006b; Farthing, 2006)⁠. Microscopic diagnosis of the parasite is hindered by its quick decomposition and thus relies on the analysis of fresh stool samples or stool samples fixated immediately after production. Despite the improved diagnosis of D. fragilis by the introduction of the TFT protocol the clinical value of this procedure still needs to be evaluated as the protozoa are largely present in asymptomatic persons(De Wit et al., 2001c)⁠. Johnson (Johnson et al., 2004)⁠ described in his review a number of studies incriminating D. fragilis as a legitimate enteric pathogen. Still, despite of the clinical improvement following the elimination of D. fragilis from symptomatic patients, it cannot be ruled out that a substantial number of these patients were suffering from a pathogen that remained undetected with the conventional diagnostic methods used. The introduction of highly sensitive molecular diagnostic methods for intestinal parasites, including the recent developed real-time PCR for the diagnosis of D. fragilis (Verweij et al., 2007b)⁠, will most likely further clarify the importance of routine diagnosis of D. fragilis.


Microsporidia are ubiquitously present in nature worldwide. It is a diverse group that represents more than 1200 species with a wide variety of hosts. Enterocytozoon bieneusi is the most common  species known to cause disease in man. The genus Encephalitozoon has three species identified as human pathogens: E. cuniculi, E. hellem and E. intestinalis. E. intestinalis is the second most prevalent species infecting humans. Before onset of the AIDS pandemic, microsporidial infections had only been described in 10 cases (Weber et al., 1994)⁠, but by the expansion of the pandemic, microsporidia came increasingly under attention. The parasites have become important opportunistic pathogens causing several clinical syndromes, most often life-threatening diarrhoea and malabsorption. Apart from its occurrence among AIDS patients, microsporidiosis is nowadays increasingly reported in transplant recipients, children, elderly people and travellers (Fournier et al., 1998; Guerard et al., 1999; Lopez-Velez et al., 1999; Müller et al., 2001; Tumwine et al., 2002; Lores et al., 2002; Leelayoova et al., 2005; Samie et al., 2007)⁠. Evidence that E. bieneusi is also present in asymptomatic carriers has been accumulating over the last decade (Mathis et al., 2005; Nkinin et al., 2007)⁠. This suggests that E. bieneusi is a very common intestinal parasite, while the severity of the disease is associated with the immune status of the person. It is also possible that the virulence of E. bieneusi is related to different genotypes as recent finding indicate that at least some genotypes show host specificity (Liguory et al., 2001; Sulaiman et al., 2003b, 2004)⁠. Although progress is made in increasing the repertoire of techniques for microscopic detection of microsporidia (Garcia, 2002)⁠, the interpretation of slides can be very difficult because of the small size of the spores and the variability in the quality of the staining. Moreover, light-microscopy does not allow accurate species identification which it is important as E. intestinalis can effectively be treated with albendazole (Sobottka et al., 1995)⁠, whereas no established treatment is available for Enterocytozoon bieneusi (Conteas et al., 2000)⁠. Several PCR assays have been developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis, although their application in routine diagnosis is still limited (Katzwinkel-Wladarsch et al., 1996; Kock et al., 1997; Franzen & Müller, 1999; Notermans et al., 2005)⁠. In chapter 7 a multiplex real-time PCR for the simultaneous detection of E. bieneusi and Encephalitozoon spp. in faecal samples in routine diagnosis is described.

Strongyloides stercoralis

Strongyloides stercoralis is a soil-transmitted helminth and those living or travelling in (sub)tropical regions are at risk for acquiring an infection. The S. stercoralis infection can be perpetuated through a low-grade auto-infection cycle for many decades while most individuals remain asymptomatic (Concha et al., 2005)⁠. Under certain conditions, however, an asymptomatic S. stercoralis infection can transform into a fulminant fatal hyperinfection with mortality rates of up to 87% (Siddiqui & Berk, 2001; Marcos et al., 2008)⁠. Development into a hyperinfection is attributed to a decrease in host resistance caused by debilitating disease, malnutrition or immunosupressive drugs, in particular corticosteroids (Keiser & Nutman, 2004; Fardet et al., 2007)⁠. Because of the poor prognosis of a hyperinfection, individuals scheduled for immunosupressive therapy are usually screened for S. stercoralis infection. In patients with a chronic infection, diagnosis of S. stercoralis is known to be problematic as the number of larvae in a stool sample can be very low. Multiple sampling and concentration methods such as Baermann and copro-culture technique are essential to increase the detection rates (Steinmann et al., 2007)⁠.

The success of applying immunodiagnostic assays for the detection of specific antibodies depends on the purity of the antigen used, the antibody isotypes selected for measurement, as well as the population on which the assay is applied on (Polderman et al., 1999; Sudarshi et al., 2003; Van Doorn et al., 2007)⁠. Recent infections may result in false-negative results, while persons with a history of residency in tropical regions may prove false-positive as many different assay platforms can not distinguish past from current infection. Antibody detection against a crude extract of S. stercoralis filariform larvae can also show cross-reactivity with other helminth infections such as filariasis or schistosomiasis. A positive result with serology may therefore be indicative for S. stercoralis infection upon which further searches for the parasite may be required (Grove, 1996)⁠. In chapter 4 and in a study from Verweij et al (Verweij et al., 2009)⁠   results indicate that a recent developed S. stercoralis real-time PCR assay may provide a valuable alternative as a test for diagnostics and for epidemiological studies.


Hookworms are parasitic nematodes that can be found in most of the world’s tropical and subtropical countries. The adult worms live in the jejunum and duodenum, attached to the mucosa and submucosa. The two most important species infecting man are Ancylostoma duodenale and Necator americanus. Light infections often go unnoticed, while heavy infections can cause hypochrome anaemia; those most at risk are children and pregnant women (Bethony et al., 2006; Calis et al., 2008)⁠. Hookworm infections are also occasionally found among persons returning from endemic areas (Ansart et al., 2005; Bailey et al., 2006)⁠. Infections can be diagnosed by the microscopic detection of eggs in direct smears or by the examination of concentrated stool samples. Based on the egg morphology, species differentiation is not possible. For the morphological species identification, a copro-culture technique is required to allow eggs to develop and hatch to release the filariform larvae that carry species-specific characteristics. Nevertheless, reliable identification requires time and skilled personnel. Although the differentiation has no clinical importance, it may be a valuable element in epidemiological studies. A recent developed multiplex real-time PCR assay can therefore be regarded as a highly valuable alternative for specification and also semi-quantitative detection of both hookworm species (Verweij et al., 2007a)⁠.

Schistosoma haematobium / S. mansoni

Schistosoma spp are blood dwelling fluke worms causing schistosomiasis or bilharzia (Gryseels et al., 2006)⁠. Worldwide an estimated 200 million people are infected with an approximately 85% of those living on the African continent (Chitsulo et al., 2000)⁠. The two most common species infecting humans are Schistosoma mansoni and S. haematobium. In schistosomiasis, the pathology is largely determined by the deposition of eggs in the tissues and the extent of granulomatous reactions and fibrosis in the affected organs. Most studies on the pathology of Schistosoma infections have focused on the hepatosplenic, bladder and kidney pathology. More recently, attention is drawn to a neglected, but socially important form of pathology of S. haematobium infections: female genital schistosomiasis (Poggensee & Feldmeier, 2001; Talaat et al., 2004; Kjetland et al., 2005)⁠. What is more indeed, recent studies suggest that as a result of induced inflammation in the semen-producing pelvic organs and in the uterine cervix, both male- and female genital schistosomiasis might constitute a risk factor for HIV transmission (Feldmeier et al., 1994; Leutscher et al., 2005; Kjetland et al., 2006; Secor & Sundstrom, 2007). In developed countries the diagnosis is mainly focused on travellers returning from endemic areas and a routine diagnostic laboratory usually selects methods with the highest sensitivity, specificity and predictive value which include antibody detection in serum and concentration methods for microscopic egg detection in faeces and urine (Lademann et al., 2000; Van Lieshout et al., 2000; Bottieau et al., 2006, 2007)⁠. For studies conducted in endemic areas, important criteria for selection of the diagnostic technique include cost of equipment and adequacy of a method both for the population studied and the involved field-workers. Urine filtration technique for S. haematobium (Peters et al., 1976)⁠ and Kato thick smear for S. mansoni (Katz et al., 1972)⁠ are commonly used in control programs and epidemiological studies. These methods allow quantification by egg counts, which generally correlate with worm burdens and morbidity. However, due to a substantial variation in egg excretion light infections are easily missed and examination of stool or urine needs to be repeated on several days (Engels et al., 1996)⁠. The use of CCA dipsticks may provide a valuable alternative technique for field diagnosis of intestinal schistosomiasis, although less so for urinary schistosomiasis (Van Dam et al., 2004; Stothard et al., 2006; Legesse & Erko, 2008)⁠. So far, no acceptable method has been found for diagnosing genital schistosomiasis and for measuring the morbidity of the disease (Kjetland et al., 2005)⁠.

To date, conventional PCR methods for the detection of Schistosoma DNA in human samples have been published (Pontes et al., 2002, 2003; Sandoval et al., 2006)⁠. More recently, a real-time PCR using SYBR Green dye for the detection of S. mansoni has been published (Gomes et al., 2006)⁠. Although high sensitivity on control DNA was achieved, this real-time PCR only detected S. mansoni DNA. A more desirable assay would detect both S. mansoni and S. haematobium, combined with an internal control. Such a multiplex real-time PCR is described in chapter 9.

Other nematodes

Finally, two more soil-transmitted nematodes with worldwide distribution are discussed below. The most prevalent one, Ascaris lumbricoides, resides in the small intestines of approximately 1.2 billion people worldwide (Bethony et al., 2006; Lammie et al., 2006)⁠. Infection with A. lumbricoides is often accompanied by Trichuris trichiura, an inhabitant of the large intestines of about 800 million people worldwide (Bethony et al., 2006)⁠. Although most infections with these nematodes remain unnoticed by the patient, high worm loads, especially in children, may cause severe complications such as dietary deficiencies and delayed physical and cognitive development (Hotez et al., 2007)⁠. To control the high morbidity due to these soil transmitted nematodes, including also hookworms, preventive chemotherapy was endorsed through regular administration of antihelminthic drugs in national control programs (Crompton, 2006)⁠. However, a recent review and meta-analysis suggested that the efficacy of available anthelminthic drugs is highly overestimated (Keiser & Utzinger, 2008)⁠. Diagnosis of A. lumbridoides and T. trichiura infections is relatively easy using microscopy for detection of the distinctive eggs in stool samples. A real-time PCR for A. lumbricoides targeting the rDNA has been described by Pecson (Pecson et al., 2006)⁠ for use in both research and routine diagnosis. The diagnosis of T. trichiura infection still relies on microscopic examination of stool samples.

The strategy for real-time PCR in parasite diagnostics

Based on the given summary of various diagnostic methods for intestinal parasites, the choice of a certain diagnostic technique clearly depends on the objectives and constraints of the laboratory. Choices can be made on the basis of the number of samples, the diagnostic targets and the accuracy of the tests, whereas restrictions may depend on the costs per detection. For the detection of parasites in areas with limited laboratory facilities, several practical constraints can be added. Depending on the diagnostic strategy, real-time PCR might be a cost-efficient diagnostic tool: different real-time PCR detection panels can be designed specifically targeting the most important parasites for the specific patient populations.

Two most important causes of protozoan diarrhoea worldwide are Giardia and Cryptosporidium (Caccio et al., 2005)⁠. For The Netherlands, prevalence of Giardia is estimated to be around 5% in patients with gastroenteritis, followed by Cryptosporidium with 2% based on microscopy of a single stool sample (De Wit et al., 2001a)⁠. Recently, a multiplex real-time PCR was developed for simultaneous detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum / C. hominis (HGC-PCR) (Verweij et al., 2004a)⁠. Although infection with E. histolytica is rare in the Dutch population, the target was included in the HGC-PCR because of its potential of developing into a life-threatening disease. In chapter 2 the HGC-PCR is evaluated for use in routine diagnostic laboratories as an alternative to the diagnosis procedure normally preferred. This study evaluates whether the molecular approach could eliminate unnecessary testing by directing the initial examination to parasites that are most prevalent in the target population, unless clinical considerations dictate otherwise. For the same patient group, results of Cryptosporidium detection with the HGC-PCR are discussed in more detail in chapter 3. In contrast to local patients attending their general practitioner, returned travellers are at risk of harbouring a larger variety of intestinal parasites. For this group of individuals the most effective diagnostic approach is investigated in chapter 4 by comparing diagnostic care as usual (i.e. microscopy and copro-antigen detection) with and an adapted real-time PCR panel. Also patient characteristics and clinical data were recorded to develop a diagnostic strategy for implementation of molecular diagnostics methods in the routine diagnosis of intestinal parasitic infection in returning travellers and immigrants. With the detailed clinical and demographic data available, the assemblages of G. lamblia are investigated in chapter 5 for their association with the presence and severity of gastro-intestinal symptoms. Isospora belli is a protozoan intestinal parasite which is responsible for diarrhoea with morbidity directly related to the degree of immunodepression. Chapter 6 describes the development of a real-time PCR assay for the detection of I. belli in stool samples. The assay also has potential to be part of a PCR panel for the immune-compromised patient group when combined in a panel with a multiplex real-time PCR assay for the detection of Enterocytozoon bieneusi and Encephalytozoon spp. (chapter 7). Although E. bieneusi is recognised as an opportunistic parasite of HIV/AIDS patients, the parasite is increasingly described also in transplantation patients, travellers, elderly people and children. To elucidate the dynamics of microsporidia infections in different human populations, chapter 8 describes several identified genotypes obtained from those living in a region with high HIV prevalence (Malawi) and The Netherlands, comparing immune-competent and patients with different types of acquired immune deficiencies.

Obviously, in low income countries real-time PCR has little value for day-to-day diagnosis of intestinal parasites because of high costs. On the other hand, prices of the necessary equipment and consumables are becoming more attractive and new developments in molecular parasitology have made it feasible to introduce real-time PCR in field research as an alternative for conventional microscopic diagnosis. In the field, research is often conducted under basic conditions and can be very strenuous when good storage facilities and appropriate diagnostic equipment are not available. Proper microscopic analysis also depends on the availability of well-trained and supervised technicians. The option for multiplex real-time PCR system was therefore evaluated in chapter 9 as a new method for the detection and quantification of Schistosoma mansoni and S. haematobium in stool samples collected in an area endemic for both species. Finally, this molecular diagnostic approach was evaluated as an indicator for clinical manifestations of genital Schistosoma haematobium infection in rural Zimbabwean women (chapter 10).