Tag Archive for intestinal

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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Predictors of parasitic and bacterial causes of diarrhoea in travellers.

Trans R Soc Trop Med Hyg. 2012 Sep;106(9):549-53. doi: 10.1016/j.trstmh.2012.04.008. Epub 2012 Jul 20.
Geographic, symptomatic and laboratory predictors of parasitic and bacterial causes of diarrhoea in travellers.
McGregor AC, Whitty CJ, Wright SG.

Hospital for Tropical Diseases, Capper Street, London WC1E 6AU, UK. alastairmcgregor@yahoo.co.uk

An observational study of patients presenting with diarrhoea to a walk-in service for returning travellers was conducted with the aim of identifying features that would help predict whether pathogens were bacterial or parasitic. In total, 509 cases were included, of which a bacterial aetiology was found in 55/440 (12.5%) and a parasitic cause in 51/428 (11.9%). Patients with symptoms of ≤14 days were significantly more likely to have a bacterial diagnosis than those with longer symptoms (p<0.001), whereas parasitic causes of diarrhoea were not associated with length of symptoms and became proportionately more likely with time. Raised CRP, faecal white cells and fever were all predictive of positive bacterial culture (p<0.001, p=0.001 and p=0.001, respectively) but did not predict parasitic infection. Travellers to South and Southeast Asia were more likely to have parasites detected in their stool than travellers to other tropical areas (OR=1.96; p=0.041). Gender, ethnicity, reason for travel and length of stay abroad were not significantly associated with the faecal pathogen identified.

Note from Robert ten Hove

The study on bacterial and parasitic infections among returning travelers was well performed. Interesting to see such a difference in travelers destinations. The majority of patients at the British clinic returned from India / South-East Asia whereas in The Netherlands and Belgium the majority of travelers return from the African continent. Nonetheless, nothing remarkable about percentages. It was good to read that Dientamoeba fragilis is not considered as a pathogen.

The most important message of this article is the one that is not mentioned. The writers do suggest that the absence of a good gold standard makes estimating the sensitivity of a stool microscopy difficult… In several countries (at least in The Netherlands and Belgium), Molecular diagnostics is now considered to be the gold standard, both for the detection of parasites as for several bacteria species. Why still dabbling through the poo with culture, ELISAs, Ridley’s, Ziehl-Neelson staining, fluorescend microscopy, and so on? When you got molecular diagnostics!