Lecture 2: Diagnostic challenges in parasitology

Question 1

What are trophozoites?

Answer 1

Trophozoites are the stage where the intestinal protozoan parasite is moving around. It is very lively, it is vegetative, it is feeding, it is reproducing asexually. As soon as this trophozoite leaves the human via defecation, it disintegrates. However, within the human body, the trophozoite is the stage causing morbidity.

Question 2

What are cysts?

Answer 2

Some trophozoites become cysts. Cysts have a decreased metabolism, a rounded, thick external cell wall. It does not feed itself, it is not motile, it does not reproduce. However, it is very resistant to the environment and can be transmitted both directly via faeces and contaminated water and food. Therefore, it is the infective stage of an intestinal protozoa.

Question 3

How do helminths multiply?

Answer 3

Adults (male&female or hermaphrodite) produce eggs or larvae

Question 4

What happens to the eggs and larvae produced by helminths?

Answer 4

They leave the host. This means there is a stable parasite population.

Question 5

What is very characteristic of helminth infections?

Answer 5

They very often cause chronic disease. The damage caused by the parasites is caused by the number of worms the patient is infected with. The disease is chronic because worms can live for years and sometimes even decades.

Question 6

What are the three most clinically relevant intestinal protozoa?

Answer 6

Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum/ Cryptosporidium hominis

Question 7

Which group are most likely to be infected with Entamoeba histolytica in the Netherlands?

Answer 7

Travellers from tropical countries and their partners and family members.

Question 8

What are the clinical symptoms caused by infection with Entamoeba histolytica?

Answer 8

The trophozoite stage can cause ulcers in the intestines, which can cause bloody diarrhoea (dysentery). The parasite can even migrate further into the human body, in particular to the liver where it can cause abscesses. These abscesses are potentially life threatening.

Question 9

What are the clinical symptoms caused by infection with Entamoeba histolytica?

Answer 9

The trophozoite stage can cause ulcers in the intestines, which can cause bloody diarrhoea (dysentery). The parasite can even migrate further into the human body, in particular to the liver where it can cause abscesses. These abscesses are potentially life threatening.

Question 10

Which group of people are most likely to be infected with Giardia lamblia in the Netherlands?

Answer 10

It is endemic in the Netherlands, especially in children (in particular in the late summer). Travellers from countries with low hygiene are also likely to be infected.

Question 11

What are the clinical symptoms caused by infection with Giardia lamblia?

Answer 11

The trophozoite stage causes the morbidity. It causes diarrhoea, other gastro-intestinal complaints and malabsorption as the trophozoites stick to the wall of the intestine. Chronic diarrhoea which continues for a couple of weeks is very characteristic for Giardia lamblia infection.

Question 12

What are the clinical symptoms caused by infection with Cryptosporidium parvum/ Cryptosporidium hominis? .

Answer 12

It causes gastro-intestinal complaints of quite severe chronic diarrhoea. The diarrhoea is self-limiting within a couple of weeks in healthy individuals, but can be severe and even life threatening in the immune-compromised

Question 13

Which group of people are most likely to be infected with Cryptosporidium parvum/ Cryptosporidium hominis in the Netherlands?

Answer 13

It is endemic in the Netherlands and is seen relatively more in children and in the late summer. Outbreaks are related to water (both recreational and drinking).

Question 14

What is a diagnostic challenge for Cryptosporidium parvum/ Cryptosporidium hominis?

Answer 14

When the parasite is excreted as an oocyst in in the stool, the oocysts can’t be easily visualised with microscopy. To visualise them, an acid-fast staining is needed.

Question 15

How is Cryptosporidium parvum/ Cryptosporidium hominis transmitted?

Answer 15

Water contaminated with faeces of an infected person gets ingested orally

Question 16

Which stages of the parasite are used to diagnose with a microscope from stool samples?

Answer 16

The (oo)cysts or the trophozoites. For Entamoeba histolytica and Giardia lamblia both cysts and trophozoites can be used, but for Cryptosporidium parvum/ Cryptosporidium hominis only the oocysts can be used.

Question 17

What is the problem with looking at trophozoites for diagnosis?

Answer 17

They disintegrate very quickly: within an hour after defaecation. This could be fixed (haha) by asking the patient to mix their faeces after defaecating with a fixative like formalin.

Question 18

What is the diagnostic challenge for diagnosing Entamoeba histolytica?

Answer 18

The cysts and trophozoites are morphologically the same as the non-pathogenic Entamoeba dispar. The only difference is that the small trophozoites of Entamoeba histolytica can develop into large trophozoites which can ingest human red blood cells (hematophagous trophozoites).

If you only look at the cysts, 90% of the cysts are Entamoeba dispar.

Question 19

What are the advantages and disadvantages of microscopy for diagnosis of intestinal protozoa?

Answer 19

Advantages are that it is available, affordable, and specific (but not for Entamoeba histolytica). Disadvantages are the low sensitivity, low specificity for Entamoeba histolytica and additional staining procedures are needed for Cryptosporidium. Three separate stool samples need to be collected on three consecutive days need to be examined to do a proper examination, as there is quite some day-to-day variation.

Question 20

What is serology?

Answer 20

Antibody detection

Question 21

What are the advantages and disadvantages serology for diagnosis of intestinal protozoa?

Answer 21

Advantages: fast, sensitive. Disadvantages: Only for invasive species (so not for Giardia lamblia), tests are done per parasite (so if the test is not ordered for the right parasite, it will be missed), it is aspecific (for most tests and parasites, people remain positive with serology after they are cured. It can take months to years for the antibodies to disappear), they can crossreact with other species, it is not always sensitive (early in infection).

Question 22

What are the advantages and disadvantages of antigen detection for diagnosis of intestinal protozoa?

Answer 22

Advantages: fast, more sensitive than microscopy, less training required, reproducible. Disadvantages: sensitivity not optimal, test is for one parasite (so other parasites are missed), high costs

Question 23

What are the advantages and disadvantages of molecular diagnostics (q-PCR) for diagnosis of intestinal protozoa?

Answer 23

Advantages: fast, sensitive & specific, multiplex setting, reproducible, (semi-)quantitative, high throughput, QC options. Disadvantages: requires particular setup of laboratory.

Question 24

How many Plasmodium species can infect humans and which one is the most important?

Answer 24

Five. Plasmodium falciparum is the most important.

Question 25

Where does transmission of plasmodium mostly take place and why?

Answer 25

Most of the transmission takes place in the tropics. This is due to the distribution of the Anopheles mosquito.

Question 26

How many people die yearly of malaria?

Answer 26

Nearly 400.000. Most of the deaths are in sub-Saharan Africa

Question 27

Who is most at risk of dying from malaria?

Answer 27

People infected with Plasmodium falciparum, children under 5, pregnant women, travellers (non-immune people)

Question 28

What is crucial to control malaria and prevent the serious consequences of the disease?

Answer 28

Early diagnosis and treatment. In particular Plasmodium falciparum can be life threatening days after the start of the first symptoms.

Question 29

How is malaria diagnosed?

Answer 29

By microscope with two different procedures: thick smear and thin smear.

Question 30

What is a thick blood smear?

Answer 30

A thick layer of blood on a glass slide. Normally this would cause the layer of red blood cells to be too thick, but for a thick blood smear, the slide is dipped into water. This causes haemolysis of the white and red blood cells. Then the staining will be performed. Because of the haemolysis, the content of the cells is the only thing sticking to the slide. This means that you will see the content of the white (nuclei) and red (possibly Plasmodium parasites) blood cells.

Question 31

What are the advantages of a thick smear?

Answer 31

You can analyse a large amount of blood

Question 32

What is a thin blood smear?

Answer 32

A tiny drop of blood spread out over the surface of a glass slide. Before the blood is stained, it is fixed by heating or by dipping it into ethanol. Because of this fixation, all the cells stay intact. This means that you can study the parasite’s morphology in a more natural way.

Question 33

What is the advantage of a thin smear?

Answer 33

It is very useful for differentiating the different species of Plasmodium

Question 34

What is the difference in practice for microscopy, especially in low income countries?

Answer 34

Blood smears made differ a lot in quality, which means that the diagnosis also differs in quality. The detection limit can range, depending on performance.

Question 35

What are the advantages and disadvantages of microscopy for diagnosis of malaria?

Answer 35

Advantages: high sensitivity (thick smear 1-200 p/uL), species differentiation (thin smear), quantification, fast. Disadvantages: fast enough, laborious, observer dependent, (training) costs.

Question 36

When can serology for diagnosis of malaria be used?

Answer 36

Not in a clinical setting: developing antibodies takes valuable time (so patients infected with Plasmodium falciparum could already be dead before the test is finished). It can be used to screen for exposure in a population. This gives an idea about the prevalence of malaria in that population and can be used for public health purposes and research.

Question 37

When are antigens detectable in malaria?

Answer 37

When the parasite is in the red blood cell stage. The most famous is the histidine-rich protein, which is excreted into the circulation. Another one is Plasmodium lactate dehydrogenase.

Question 38

What is possible with the newest antigen tests for malaria?

Answer 38

They can not only show if the patient has malaria or not, but they can also differentiate between the major Plasmodium species.

Question 39

What are the advantages and disadvantages of antigen tests for malaria diagnosis?

Answer 39

Advantages: fast (15 minutes), less training required, sensitive for P. falciparum (100 p/ul, not as sensitive as microscopy, but okay), high availability, low costs. Disadvantages: sensitivity not optimal for non-P. falciparum, not quantitative, hot-spots are still missed (need for ultra-sensitive tests), asymptomatic people often missed, deletions in target antigen have been described (which do not react to the test anymore, causes false-negatives).

Question 40

What are the advantages and disadvantages of molecular diagnostics (q-PCR) for malaria diagnosis?

Answer 40

Advantages: super sensitive (50 p/mL-5 p/uL, way more sensitive than microscopy), high specificity, quantitative, capable to process large sample numbers, reproducible. Disadvantages: high cost, slow (can take days), not very available (but this is changing! This is because there are more kits). It can be very useful as quality control for microscopy.

Question 41

What is a LAMP test?

Answer 41

A simplified form of a PCR. The disadvantage is that it can’t differentiate between different Plasmodium species yet, but it is very sensitive and fast. It can be used as a first line of screening, which is more useful in non-endemic countries. If the LAMP is negative, the patient does not have malaria. If it is positive, additional testing needs to be done: microscopy (positive is positive, negative is further testing). Further testing can be done with a rapid diagnostic test.

Question 42

What is very typical of schistosomiasis?

Answer 42

The focal distribution. The majority (at least 85%) of the 250 million cases around the world lives in sub-Saharan Africa. The majority is infected with Schistosoma haematobium or Schistosoma mansoni.

Question 43

What method plays a central role in diagnosis of schistosomiasis and soil-transmitted helminths?

Answer 43

Microscopy!

Question 44

How are schistosomiasis and soil-transmitted helminths diagnosed by microscope?

Answer 44

From a urine sample (10 mL) for Schistosoma haematobium or a Kato-Katz slide examination of the stool(Schistosoma mansoni or soil-transmitted helminths).

Question 45

What is often done for population-based surveys in regions endemic for schistosomiasis or soil-transmitted helminths?

Answer 45

The number of eggs is counted, making it quantified. The number of eggs is counted either per 10 mL of urine or a fixed amount of stool. The counting of the eggs gives an approximation of the worm burden. Because of this, it is recommended by the WHO.

Question 46

What are the advantages and disadvantages of diagnosing schistosomiasis and soil-transmitted helminths using a microscope?

Answer 46

Advantages: high specificity, quantifiable, low technology, fast, available, affordable, simple. Disadvantages: low sensitivity, operator dependent, training needed, fluctuation in egg secretion by the worms that can influence outcomes.

Question 47

What is the result of the low sensitivity of microscopy for schistosomiasis and soil-transmitted helminth diagnosis?

Answer 47

Light infections are missed, meaning that the effectiveness of the treatment is over-estimated if microscopy is used to measure treatment effectiveness (a lot of worms before treatment and a little after, but by microscopy you might see no eggs after).

Question 48

What can solve the problem of fluctuation in egg secretion?

Answer 48

Examining stool on three consecutive days

Question 48

What can solve the problem of fluctuation in egg secretion?

Answer 48

Examining stool on three consecutive days

Question 49

What are the advantages and disadvantages of diagnosing schistosomiasis and soil-transmitted helminths using serology?

Answer 49

Not useful for soil-transmitted helminths, as they hardly induce an antibody response within the body. It can be used for schistosomiasis. Advantages: sensitive, specific (good for travellers!). Disadvantages: no correlation with intensity of infection, not useful for evaluation of treatment (as soon as people are positive, they stay positive for many many months or even years).

Question 50

Why could antigen detection be useful for diagnosing schistosomiasis and soil-transmitted helminths?

Answer 50

It can detect an active infection and worm load, it is highly field applicable and user friendly, it is highly sensitive and specific and very useful for case detection and the monitoring of interventions

Question 51

How does the antigen detection test for diagnosing schistosomiasis work?

Answer 51

The target antigens are circulating Schistosoma antigens called CCA (Circulating Cathodic Antigen) and CAA (Circulating Anodic Antigen). These antigens are glycans derived from the gut of the young juvenile or adult worm. These antigens are regurgitated by the parasite and excreted into the blood circulation of the host and eventually from the blood circulation excreted into the urine. This makes it possible to do non-invasive sampling (urine! Just pee in a jar!). The antigen levels reflect parasite load and are only positive in active infection. As soon as you treat a patient with praziquantel, the parasites die and the antigens are cleared from the blood circulation and urine in a couple of weeks. This makes this method very suitable to monitor praziquantel treatment.

Question 52

How has the antigen detection test for diagnosing schistosomiasis developed?

Answer 52

When very specific antibodies for CCA were developed, first an ELISA was used for diagnosis. Later, it was used as a rapid diagnostic test (POC-CCA test) (think COVID self test).

Question 53

How good is the POC-CCA test?

Answer 53

It is more sensitive than one stool test, which is quite sensitive. It is less sensitive for Schistosoma species other than Schistosoma mansoni. The specificity is around 98% with some batch-to-batch variation. It can be used in endemic settings for e.g.: surveys, mapping, test-and-treat, post-mass drug administration (MDA) screening

Question 54

Is the POC-CCA test observer dependent?

Answer 54

Yes, because you have to determine whether you see a test line or not (and these lines can be very vague, but even a vague line is positive)

Question 55

What are two disadvantages of POC-CCA?

Answer 55

It is observer dependent and time-sensitive (the tests must be read exactly 20 minutes after adding the urine sample). The time-sensitivity also makes it hard to get a second opinion on the presence of a line from an expert.

Question 56

What is done to further standardize the POC-CCA?

Answer 56

A standardization model with pre-printed cards labelled with their correct interpretation.

Question 57

What can still be improved in the POC-CCA test?

Answer 57

Differentiation of all Schistosoma species

Question 58

What is the improved version of the POC-CCA test?

Answer 58

The UCP-LF CAA assay. It is based on ultra-sensitive Up Converting Phosphor (UCP) technology. It can differentiate all Schistosoma species from serum or urine. A disadvantage is that this technique has limited availability, as it is laboratory-based. It needs a special reader, as the lines cannot be seen with the naked eye. In addition, to make this test sensitive a sample pre-treatment is required, which requires a centrifuge.

Question 59

What needs to be controlled when the disease prevalence is high? What is the most important for a diagnostic tool in a situation like this? What is a test that is most suitable in this situation?

Answer 59

Morbidity needs to be controlled and the diagnostic tool needs to be simple and low cost. The POC-CCA test for S. mansoni is an example of a suitable test for this situation

Question 60

What needs to be controlled when the disease prevalence is less high and what is the most important for a diagnostic tool in a situation like this?

Answer 60

Transmission needs to be controlled and the diagnostic tool needs to be sensitive. A PCR test (Schistosoma and soil transmitted helminths) or a UCP-LF-CAA (only differentiate different Schistosoma species) is most suitable in this situation

Question 61

What is the measure of control when the disease prevalence is intermediate and what is the most important for a diagnostic tool in a situation like this?

Answer 61

The measure of control is surveillance and the diagnostic tool needs to be sensitive and specific. A PCR test (Schistosoma and soil transmitted helminths) or a UCP-LF-CAA (only differentiate different Schistosoma species) is most suitable in this situation

Question 62

What is the measure of control when the disease prevalence is low-zero and what is the most important for a diagnostic tool in a situation like this?

Answer 62

The measure of control is elimination and case detection and there is a heavy focus of the diagnostic tool to be sensitive and specific. A UCP-LF-CAA (only differentiate different Schistosoma species) is most suitable when there is very low transmission (elimination). Antibody detection is suitable for case detection if there is no transmission.

Question 63

How can a real-time PCR be used for diagnosis of soil-transmitted helminths?

Answer 63

As a multiplex PCR. Different parasites can be tested for at the same time. This is an advantage compared to testing circulating antigens, as there is only a test for circulating antigens for Schistosomiasis. Over the years it has been shown that if you mix stool with ethanol it is no longer necessary to store it in the freezer or fridge. This eliminates the cold chain and thus gives opportunity to do surveys in remote areas in the tropics (analysing happens in a central laboratory).

Question 64

What is the ANAS-PCR?

Answer 64

A multiplex PCR that analyses A. lumbricoides, N. americanus, A. duodenale, T. trichiura and S. stercoralis. It can also be used as a quality control for microscopy