Taking the battle to the molecular level
1 Feb 2006 by Evoluted New Media
Molecular detection of parasite DNA is proving to be a useful weapon in the battle against parasitic enteric disease
Molecular detection of parasite DNA is proving to be a useful weapon in the battle against parasitic enteric disease
A parasite is an animal or vegetable organism that lives within or upon another, deriving nourishment from its host. The smallest animal parasites are the Protozoa - single-celled organisms, some of which are pathogenic. Malaria - a febrile disease caused by Plasmodium - is perhaps one of the better-known protozoan diseases. However, this protozoan parasite is also responsible for a wide range of other illnesses, including diarrhoea and other enteric diseases.
Here, Chris Waghorn and Wemmie Elsenga of Oxiod talk to parasitology expert, Dr Lisette van Lieshout about some of the most common protozoan parasites known to cause enteric disease in man and the usefulness of available methods for detecting these organisms.
Giardia and Cryptosporidium
Giardia and Cryptosporidium are major causes of protozoal diarrhoea in humans worldwide1.
Dr van Lieshout expands, “Giardia and Cryptosporidium are important waterborne parasitic infections, responsible for a number of outbreaks of gastrointestinal disease around the world every year. Giardiasis is also considered to be an important travel related disease. Often, imported Giardia infections seem to cause more severe intestinal complaints, but other factors such as immune status, nutritional status and age of the host also seem to be of importance.”
In the Netherlands, the general prevalence of Giardia is around 5% and Cryptosporidium is around 2%7, 8, depending on the study population. In a review of disease outbreaks associated with recreational water in the United States between 1971 and 2000, the most common cause identified was Cryptosporidium (15%). Giardia was responsible for 6% and other causes were bacterial. Bacterial disease was associated with fresh, untreated water such as lakes, ponds and rivers, whereas parasite infections were predominantly associated with treated water such as swimming and wading pools9. Similarly, a single outbreak of cryptosporidiosis in Sweden in 2002 affected 800-1000 people and was linked to a public swimming pool10. Transmission of these organisms has also been associated with drinking water, due to their ability to survive for long periods outside the body and to survive normal amounts of chlorine used to purify community water supplies.
Infants and young children are particularly susceptible to protozoan parasites, like Giardia and Cryptosporidium. This is a particular problem in the developing world, where current estimations suggest that giardiasis can be present in up to 30% of young children in some countries4.
“There are indications that intestinal protozoa are much more common in developing countries, where diarrhoea is a major cause of morbidity and mortality in HIV-positive individuals and young children,” continues Dr van Lieshout. “Accurate prevalence in these countries is difficult to assess, however, due to inadequate diagnostic procedures.”
Entamoeba histolytica
Entamoeba histolytica (causing amoebiasis) is another of the most important diarrhoea-causing protozoan parasites in the world, causing mild symptoms to severe disease, including amoebic dysentery. Every year 40-50 million people develop clinical amoebiasis, which is responsible for some 100,000 deaths11.
Amoebiasis is most common in developing countries, in immigrants from developing countries and in people who have travelled to developing countries12. It is endemic in parts of Africa, South America, India and South East Asia 13. In industrialised countries, human to human transmission of E. histolytica is rare, but not unheard of. Even with Western hygiene, persistent cyst passage has resulted in transmission to household contacts14.
In 1997, the species then known as Entamoeba histolytica was officially recognized as being actually two separate species – the potentially invasive E. histolytica and the non-invasive commensal E. dispar15. These two species are morphologically identical in the cyst and small trophozoite stages and so species-specific diagnosis of invasive amoebiasis could not be made microscopically unless the larger red blood cell-engulfing trophozoites were found15.
The team at Leiden University have been involved in research to differentiate E. histolytica from E. dispar by molecular means.
For example, a high prevalence of intestinal amoebiasis in Ethiopia was reported by microscopy. However, upon analysis of 108 microscopy-positive samples by PCR, only one sample was confirmed to contain invasive E. histolytica, 77 samples were found to be non-invasive E. dispar and 30 were negative for E. histolytica or E. dispar15.
This possible over-reporting of E. histolytica infection was confirmed in a larger study involving the analysis of 772 samples from primary school children and prisoners in Ethiopia. 213 samples were Entamoeba-positive by microscopy, of which 91.5% were the non-invasive E. dispar and none were confirmed as E. histolytica16.
Similarly, in Northern Ghana, a high prevalence of intestinal parasites - E. histolytica/E. dispar (39.8% determined by microscopy) and Giardia (21.5%) - was reported. PCR analysis found that only one case of Entamoeba infection was E. histolytica and 82.8% were E. dispar17. This confirmed the low sensitivity and specificity of microscopy on the analysis of a single stool sample. Since many amoebic infections are incorrectly attributed to E. histolytica, they are treated unnecessarily with amoebicidal drugs15.
Importance of detection
Gastrointestinal parasites, along with other infections, can be a significant problem for immunocompromised individuals, especially in resource-poor settings18. The three major parasitic causes of gastroenteritis discussed in this article often have similar clinical presentations. To complicate matters further, symptoms may also be similar to those caused by other gastrointestinal disease, such as bacterial and viral gastroenteritis. Sensitive and specific diagnosis of the cause of infection is important for improving patient management and infection control6. There is, therefore, a need for accessible diagnostic tests.
Dr van Lieshout comments, “As a parasitologist, I would say it is always important to identify the correct parasite, certainly for the clinically relevant parasites for which proper treatment is available, such as Giardia and E. histolytica. But, to the same extent, it is of equal importance to rule out - with a high level of certainty - the presence of a parasitic infection, so that one can look for other explanations for the clinical complaints and prevent unnecessary treatment and diagnostic delay. However, optimal microscopy is a challenge within routine diagnostics. The sensitivity is often limited for numerous reasons. Sensitivity may be increased by analysing multiple stool samples, but still the individual quality of the microscopist remains the most crucial aspect. Furthermore, as illustrated by our findings in Ethiopia and Ghana, the specificity of microscopy may be lower than expected, leading to over diagnosis of conditions such as amoebiasis.”
Microscopy has been the Gold Standard for the detection of protozoan parasites for many years. However more sensitive and specific detection can now be achieved using other methods.
Microscopy
Microscopic diagnosis of intestinal protozoan disease involves the morphological identification of the parasites within stool samples. This depends entirely on the skill, patience and experience of the microscopist.
Microscopy has proven to be unsatisfactory for the reliable differentiation between red blood cell-engulfing amoeba and non-invasive amoeba in wet smears in the routine diagnostic setting15.
Molecular methods
Dr van Lieshout’s laboratory has been involved in the development of molecular methods for the identification of intestinal parasites.
“During the last decade our section has been very active in the development of molecular diagnostic tools, in particular the detection of parasite DNA in stool samples,” she explains. “We started this line of research with the differentiation of Entamoeba histolytica and Entamoeba dispar. Now we have developed a range of molecular tests, including several multiplex real time PCR's for the detection of Giardia, Cryptosporidium, Entamoeba histolytica, microsporidia, hookworm species and Strongyloides.
“Compared to microscopy, molecular detection of parasite DNA has proven to be a highly specific and sensitive approach to diagnose infection. So far, molecular techniques have been mainly used for small epidemiological surveys and in reference laboratories. However, due to more simple procedures, reduced risks of contamination, the introduction of internal controls and reduced costs, it is expected that these techniques become increasingly applicable both for routine diagnostics and for large-scale epidemiological studies.”
Immunological methods
Some identification techniques rely on the detection of specific target antigen using monoclonal antibody technology. Some of these methods, such as the ProSpecT and Xpect assays available from Oxoid, have the advantage of being completely portable. They can be performed quickly and conveniently without the need for capital equipment, making them ideal for routine screening in laboratories that do not have the facilities or expertise for molecular techniques. 
Fast and convenient
The ProSpecT range of ELISA assays for the direct detection of enteric pathogens from faecal specimens include assays for each of the most important intestinal parasites, i.e. Giardia, Cryptosporidium, Entamoeba histolytica and a Cryptosporidium/Giardia dual assay. The tests are not dependent on the shedding of live organisms but detect antigen produced by the parasites within the gut. Figure 1: ProSpecT Microplate Assays
The microplates (Figure 1) can be incubated at room temperature and results, which are available within 2 hours, can be read visually or spectrophotometrically. Each assay in the ProSpecT range, including those for bacterial enteric pathogens, uses the same procedure and share common reagents, allowing several strips for different pathogens to be set up and run simultaneously. This is particularly useful in situations where the symptoms and location alone are unable to guide investigators as to the possible cause of disease.
Direct detection of Cryptosporidium and Giardia from faecal specimens is also possible via a range of rapid lateral flow tests – Xpect Giardia (Figure 2), Xpect Cryptosporidium and the dual assay, Xpect Giardia/Cryptosporidium. Diluted sample is simply added to the sample window of the test unit and within just 15 minutes the result can be read. A line at the control level demonstrates that the test has been performed correctly. An additional line at the result level indicates that the test is positive for the target organism. The absence of this line indicates that the test is negative.
Figure 2: Interpretation of results with Xpect Giardia
“We use the ProSpecT Giardia test for routine diagnostics,” concludes Dr van Lieshout. “We chose this test because of its relative high sensitivity compared to microscopy, its high specificity and because Giardia is the most common parasite in our patient population. Also ProSpecT is easy to use and gives a uniform interpretation19.”
“Early in 2004 we did a comparison with another type of Giardia copro-antigen test and found the other test to be less sensitive. In total we analysed almost 150 stool samples. About one third of the ProSpecT positive samples showed a negative result in the compared test. This reduced sensitivity could be simply explained by the fact the samples were not fresh or stored under frozen conditions, but stored for a longer period in formalin at 4oC. Still, for logistical reasons we would always prefer a copro-antigen test that would perform equally well on formalin or SAF-fixed stool samples. The ProspecT test showed similar results when retesting stool samples that were stored in formalin for a period considerably longer than 3 months.
“We have also applied the ProSpecT Giardia test in a small epidemiological study. Collection of only a single stool sample from each participant was feasible, so it was no great surprise we found the copro-antigen test to detect significantly more cases compared to microscopy. For those researchers who have no access to molecular techniques, copro-antigen detection is a sensitive and specific diagnostic tool and may be of value in epidemiological Giardia research.”
By Chris Waghorn and Wemmie Elsenga, Oxoid
Characteristics of giardiasis:
• Giardia live in the intestine and are passed in stools2. The parasites attach to the lining of the small intestine where they sabotage the body’s absorption of fats and carbohydrates from digested foods3.
• Symptoms include diarrhoea, flatulence, greasy floating stools, stomach cramps, upset stomach, nausea. Severe disease can lead to weight loss and dehydration 2, 3.
• Those most at risk of infection include, children in day care, childcare workers, parents of infected children, international travellers, people who drink untreated water, swimmers who swallow infected water and drinkers of contaminated water2.
• As few as 10 parasites in a glass of water are enough to cause severe giardiasis in humans3.
• It has a 1-2 week incubation period and symptoms may persist for 2-6 weeks. In chronic giardiasis, symptoms are recurrent and may persist for months2.
• Several prescription drugs are available to treat giardiasis including metronidazole and tinidazole 2, 4.
Characteristics of cryptosporidiosis:
• Cryptosporidium lives in the intestine of its host and is shed in stools.
• Symptoms can be mild to severe, including watery diarrhoea, stomach cramps, fever, nausea, vomiting, dehydration and weight loss 4, 5.
• Those most at risk of serious disease are young children, pregnant women and the immunocompromised5. It has particularly severe manifestations in AIDS patients6.
• Infection has a 2-10 day incubation period and usually lasts for 1-2 weeks if the patient is otherwise healthy5.
• The disease is self-limiting in most patients and, until recently, there have been no effective drugs for the treatment of cryptosporidiosis4. A new drug, nitazoxanide, has been approved for the treatment of diarrhoea caused by Cryptosporidium in those with healthy immune systems5.
Characteristics of Entamoeba histolytica infection:
• Symptoms can be mild (loose stools, stomach pain, stomach cramps) to severe (severe diarrhoea, stomach pain, bloody stools filled with mucous, fever, amoebic dysentery)12, 13.
• It can invade the liver and cause abscesses12.
• Amoebiasis is contagious and can spread very quickly, especially in regions where drinking water becomes contaminated13.
• Infection has an incubation period of up to 4 weeks12, 13 and, if treated, may last for around 3 weeks13.
• Antiamoebic drugs are available, including metronidazole, tinidazole, secnidazole and ornidazole. Asymptomatic carriers of E. histolytica can be treated with iodoquinol or paromomycin4.
References:
1. Cacciò SM, Thompson RCA, McLauchlin J and Smith HV (2005) Trend Parasitol 21(9): 430-437
2. CDC Fact Sheet – Giardia infection
3. KidsHealth.org – Giardiasis
4. Gupta Y, Gupta M, Aneja S and Kohli K (2004) Ind J Paed 71(1):55-58
5. CDC Fact Sheet – Cryptosporidium infection
6. Verweij JJ, Blangé RA, Templton et al (2004) J Clin Microbiol 42(3):1220-1223
7. de Wit MA, Koopmans MP, Kortbeek LM, Wannet WJ, Vinje J, van Leusden F, Bartelds AI, van Duynhoven YT (2001) Am J Epidemiol 154(7):666-74
8. Mank TG, Zaat JO (2001) Expert Opin Investig Drugs 10(8):1513-9
9. Craun GF, Calderon RL and Craun MF (2005) Int J Environ Health Res 15(4): 243-262
10. Insulander M, Lebbad M, Stenstrom TA and Svemingsson B (2005) Scan J Infect Dis 37(5): 354-360
11. Verweij JJ, Laeijendecker D, Brienen EAT et al (2003) J Clin Micro 41(11): 5041-5045
12. CDC Fact Sheet – Amebiasis
13. KidsHealth.org – Amebiasis
14. Vreden SGS, Visser LG, Verweij JJ et al (2000) Clin Infect Dis 31(4):1101-1104
15. Kebede A, Verweij JJ, Petros B and Polderman AM (2004) Trop Med Int Health 9(5): 651
16. Kebede A, Verweij JJ, Endeshaw T et al (2004) Annal Trop Med Parasitol 98(1): 43-48
17. Verweij JJ, Oostrogel F, Brienen EAT et al (2003) Trop Med Int Health 8(12): 1153
18. Lewthwaite P, Geoffrey G, Hart A et al (2005) Curr Opin Infect Dis 18(5): 427-435
19. Mank TG, Zaat JO, Deelder AM, van Eijk JT, Polderman AM. (1997) Eur J Clin Microbiol Infect Dis 16(8):615-9
