2.

3 Intestinal Parasites
2.3.1 Protozoa
Entamoeba histolytica is the cause of amoebic colitis, amoebic dysentery and
amoebic liver abscess and is responsible for an estimated 100,000 deaths per year.
Until about 25 years ago, differences in clinical symptoms were explained by the
existence of pathogenic and non-pathogenic strains of E. histolytica. Biochemical,
immunological and genetic differences between these “strains”, however, are so
large that it is now generally accepted that they are in fact two different species,
named E. histolytica and Entamoeba dispar. Because the potentially invasive
E. histolytica is morphologically indistinguishable from the non-invasive E. dispar,
microscopic examination of faecal samples only, is not suitable for a definitive
diagnosis of E. histolytica infection and additional methods, such as the detection of
specific antigens or specific DNA sequences are necessary. Although antigen
detection is specific, its sensitivity is too low, when compared with PCR, for use in
patient care.
Giardia Lamblia infections are very common, occur globally and are seen as one
of the most important non-viral causes of diarrhoea in industrialized countries.
Since the time of Anthony van Leeuwenhoek, the diagnosis has been based on
microscopic investigation of (multiple) faecal samples using concentration techniques.
The detection of Giardia antigens by ELISA has been accepted in recent
years as a cost-saving alternative. PCR targeting the SSU rDNA has excellent
sensitivity and specificity when compared to microscopic and antigen based
detection strategies. Giardia can be divided into various genotypes (assemblages)
on the basis of Restriction Fragment Length Polymorphism (RFLP) analysis and
DNA sequence analysis of several (household) genes. However, the clinical significance
of the various assemblages is still unclear and therefore, these genotyping
methods are used in epidemiological and basic research settings only.
Cryptosporidium-associated diarrhoea is best known for the severe symptoms in
AIDS patients during the HIV/AIDS epidemic in the 1980s and 1990s.
Subsequently, improved diagnostic methods have been developed. Probably due to
this awareness, Cryptosporidium is now also known as the cause of large water- and
food-borne outbreaks of gastroenteritis in immunocompetent individuals. Modified
acid-fast staining techniques are often used for the detection of Cryptosporidium
oocysts in stool. However, the sensitivity and specificity appear to be low and
proper identification is very dependent on the experience and skills of the microscopist.
Although immunological detection methods are available, these are not as well accepted as in the diagnosis of
Giardia infections due to low sensitivity and
specificity. PCR based techniques have been proven to be a sensitive and specific
alternative for the detection of Cryptosporidium in faecal samples. In a study
including 950 patients from the Groningen region in the Netherlands, who visited
their family doctor with gastrointestinal symptoms, Cryptosporidium was demonstrated
using real-time PCR in 20% of children under five.
PCR based techniques have also been shown to have a high sensitivity for the
detection of other coccidia-like infections, such as Cystoisospora belli (Isospora
belli) and Cyclospora cayetenensis. Another example in which PCR is a worthwhile
alternative in the diagnosis of opportunistic pathogens is microsporidiosis.
Microscopic detection of the very small spores of different microsporidium species
in faecal samples, even after additional staining techniques, has proven to be
extremely difficult and time-consuming. Amplification of microsporidium-specific
DNA has been shown to be a sensitive and specific tool in the diagnosis of
microsporidium infections. Probably due to the increased attention on
microsporidiosis during the AIDS epidemic and the improvement of diagnostic
techniques, such infections are now being diagnosed more frequently in transplant
patients, children, the elderly and in travellers.
Dientamoeba fragilis was first described in 1918 as a commensal living amoeba
of the gastro-intestinal tract of humans. Subsequently, through antigen based and
ultrastructure studies and through the analysis of the 16S ribosomal RNA, the
organism was classified as a flagellate, though a flagellum is missing. Since its
discovery, the pathogenicity of this organism has remained controversial. In recent
years, several authors have published on the clinical significance of D. fragilis as a
cause of gastrointestinal distress, unfortunately, a consensus on the pathogenicity is
lacking. One of the issues preventing progress is that many D. fragilis infections
remain without symptoms. Until recently, a cyst stage of D. fragilis was not recognized.
Therefore, the fragile trophozoites were detected and identified by
microscopic examination in fresh stool or by the use of fixatives and permanent
staining techniques. The sensitivity of a single microscopic examination is not high,
because the day-to-day variation of D. fragilis trophozoites in the faeces seems even
more irregular than observed in other intestinal protozoan infections, such as G.
lamblia and E. histolytica. Immunological techniques for the detection of D. fragilis
are not available. Real-time PCR has been proven to be a highly sensitive and
specific alternative to the diagnosis of D. fragilis, in which the influence of the
day-to-day variation of the results is greatly reduced. Dientamoeba DNA can be
detected successfully with qPCR, even after storage of the faecal samples for up to
eight weeks at 4 °C, without loss of sensitivity. Genetic variations of the small
subunit ribosomal RNA (SSU rRNA) gene and of the internal transcribed spacer 1
(ITS1) of D. fragilis have not been associated with the occurrence of clinical
symptoms. Furthermore, in a recent multicentre case-control study in the
Netherlands, D. fragilis was detected more often in controls as compared to diarrhoeal
cases, which has led many laboratories to remove D. fragilis from their
primary screening panel. Blastocystis is the most common intestinal parasite in the faeces of both patients
with gastrointestinal complaints and in asymptomatic individuals. It is thus, even
more so than with D. fragilis, unclear whether it is necessary to detect this parasite.
PCR-based techniques display a higher sensitivity when compared to microscopy
and culture and provide the possibility of performing sub-typing by means of DNA
sequence analysis. Factors that seem to be associated with the clinical significance
of Blastocystis are the parasite load, the genotype or subtype, and the use of
antimicrobial treatment (Fig. 2.1).