The assessment of nematodes as they generate and die is not a simple thing to do due in part to the complexity of the organism, and the fact that still relatively little is known about their physiology and internal biology. Indeed, the pathological changes in the internal organs of the worms are still only recognized in general terms. Obviously dead worms are easily recognized (when fractured, or calcified, etc.) but the lesser obvious changes can be difficult to detect and interpret. The point at which a worm can be defined as dead is not a simple matter; cessation of motility is currently the most commonly used parameter for this but it is not always a robust indicator and better indicators are needed. Various methods can be used to assess the presence, viability, and functionality of nematodes but these must be used with an understanding of the situation at hand and the specific questions being addressed. Careful use of appropriate statistics is essential given the complex nature of the target organism and the variability in the changes that can be seen within even one anatomical component of these worms. Histological assessment of the parasites present in both parasitized host tissues and isolated worms used in in vitro experiments can provide information that gives a more detailed understanding of the changes in nematodes as they degenerate and die. Understanding of the pathways nematodes follows as they degenerate naturally or under various external pressures, such as chemotherapy, remains a fascinating and potentially productive goal for investigation. Likewise, a complete understanding and definition of specific indicators that reflect parasite load, parasite viability, and damage, or reduced fecundity, will greatly help the fight against those nematode infections that currently cause significant burdens of disease in humans and animals.
- filarial nematodes
Nematodes commonly infect humans, animals, and plants in all the ecosystems from the tropics to the polar regions; they can cause significant damage and consequently are responsible for some of the major chronic infections of these hosts. This being the case there is a great need to develop better treatment and prophylactic procedures to reduce the pathological effects of these infections, much of which are caused by events associated with the degeneration and death of the causative parasites in sensitive host tissues. Successful development of new effective and safe chemotherapeutic agents, a leading approach in controlling these detrimental effects, necessarily requires improved and more accurate assessment of the viability of these organisms. This is needed to both develop control mechanisms as well as to determine the epidemiological nature of these parasites.
In general terms, much is known about the effects of many parasitic nematodes on their human and animal hosts but comparatively little about the effects of the hosts, or chemotherapeutic agents, on the parasites themselves, i.e. the pathology of the parasites. There is, however increasing knowledge about model nematodes such as
Many examples exist in disease management suggest that it is important that we gain a clearer understanding of the biology of the infecting nematodes and the effects that the host and drugs induce in these parasites. Plant parasitic nematodes cause very significant problems to major crops throughout the world including vegetables, fruits and grain crops . Some of the most devastating of chronic tropical diseases in human medicine are caused by nematodes; indeed, a major global health effort has been underway for some years aimed to control and eliminating a few of these tropical diseases. Two of the most successful programs today involve filarial nematodes, one that causes “river blindness” (onchocerciasis) and a second that is responsible for “tropical elephantiasis” (lymphatic filariasis). Well over 100 million people are affected by these parasites, and many more people still are at risk of these infections. The veterinary world has long understood the importance of parasitic infections, especially the persistent intestinal nematode parasites, with their ability to compromise growth and development of domestic animals. In both human and animal infections, the primary approach to reduce and eliminate these parasites has been for almost a century using chemotherapeutic agents - agents that either primarily damage and destroy the infecting organism, an event that can often induce a reactive pathological response in the host, especially with tissue based nematodes.
Central to measuring, understanding and treating parasitic infections in animals and humans are two fundamental parameters–worm load (i.e. number) and the worm viability; for many years, these have been assessed by active counting the number of viable worms (or a more easily detectable parasitic stage such as eggs, etc.) present in the host. Nematode infections in humans and animals that cause significant disease are essentially found in three major locations: in organs (e.g. digestive and respiratory tract lumens and ducts), in connective tissues, or in the circulatory vessels (lymphatics and blood vessels). Those parasites that lie in the gut are perhaps the most well‐known and in many ways the most studied in human and animals, and are arguably more commonly seen because their cycles include detectable faecal stages. Thus, the time‐honored test for assessing loads of these parasites has been the measurement of their egg production by the parasites (i.e. fecal egg counts through a variety of well‐described methods: McMaster, Kato‐Katz, mini‐Flotac and other various egg concentrating methods) [2–5]. In recent years, there has been the gradual development and validation of molecular (PCR) approaches for estimating intestinal nematode presence and load, and it is likely that this type of technique will be used more commonly in the future.
In the case of
2. Filarial nematodes
Filariae are a very diverse group of nematodes that infect a very wide range of specific hosts. The three major filarial human nematode infections are river blindness or onchocerciasis (
Filarial worms are one of the best examples of a group of parasitic nematodes where a better understanding of the biological status of the parasite, or at least of certain parasitic stages, is essential to establishing optimal effective and safe therapies, and to obtain a clearer understanding the pathogenesis of the disease in the host. Such an increased understanding is particularly important since these parasites infect and effect internal tissues such as connective tissues and circulatory vessels; this makes them more difficult targets for control compared to those nematodes that reside in the intestine where often treatment results in complete rejection of the damaged organism from the body.
3. Assessment of parasitic nematodes
In assessing a nematode infection in a host, it is important to select the most appropriate stage of these five‐stage parasites to focus on and use as an indicator. This depends very much on the life‐cycle in the host, as well as the availability of suitable techniques for assessment. For example, if one is trying to break infection transmission to a vector then the crucial stage that provides the most important information in terms of epidemiological control is usually the stage entering the vector; in the filarial infections, this is almost exclusively the microfilarial stage. However, it is possible that permanent alterations in an earlier phase or stage may also be a strong and useful indicator that can predict termination of transmission. In our example here, evidence of a destroyed capability to reproduce and produce the first stage forms (the microfilariae) seen in significant uterine damage, and thus a lack of production of microfilariae, is an important indicator of the breaking of transmission. Observing such changes in adult female worms may indeed be more practically feasible than detecting the presence of transmissible microfilariae in the host or in the blood‐feeding vectors. The effective break in the parasite’s cycle, seen here in the permanent uterine damage and ceased reproductive ability, also shows that the actual death of the female worm is not the necessary target endpoint for defining a successful therapeutic agent or intervention: female worms may still be thought to be alive but have permanently lost the capability of reproducing. This also demonstrates the fact the simple counting of worm numbers often needs to be supported by an assessment of the internal anatomy of worms (e.g. the uteri or other vital structures) rather than just the number of whole worms present; a sterile worm is functionally as important as a dead worm. Thus, it is important to have methods of assessing the functional anatomy of worms. In both gut‐residing nematodes and the tissue/vessel filariae, it is arguably more important to understand the functional state and reproductive capability of adult worms than just their physical, or numerical, presence. Measuring such parameters can either be done through histopathology examination as described below, or by the identification of products from components of the worms such as the uterus that may be released and be able to be detected in body fluids.
Assessment of the reproductive organs of male worms may also be a useful target for assessment; male filarial parasites are in the minority, and are probably fertilizing more than one female. Given that the reproductive cycle of filariae is comparatively long (months), it is likely that the female–the producer of the transmissible stage–is overall a better indicator of the host’s infection status than is the male. The other stages that occur in hosts after infection, the third and fourth parasitic stages, are in general both hard to detect physically and exist for relatively short periods compared to the other stages; they have not been to date very useful targets for parasitological assessment. However, an indirect assessment of these stages through stage‐specific antibodies or specific circulating antigens is becoming more feasible as the reagents for this type of test are improving rapidly.
It is important to re‐emphasize, in the context of monitoring nematode infections, the distinction between using functional parameters and assessment that is through simple numerical quantification. In most cases a more detailed assessment that involves more functional parameters (biochemistry, fecundity etc.) is preferable. However, an exception where an estimation of the circulating load of the parasite is crucial and preferred is loiasis, caused by the filarial nematode
The most common approach to assessing the viability of worms
Deciding on the optimal means of assessing worm viability and number requires a consideration of the specific question being addressed, as well as the circumstances at hand. As mentioned above assessing nematodes
4. Technical aspects of assessing nematodes
The various techniques used to assess nematodes infections
4.1. Indirect (clinical) assessment of population endemicity
Adult worms are central to the assessment the longevity of nematode infections, and this is particularly so in filarial infections, e.g. over five years with most filariae. The female adult worm is often used as the indicator worm rather than males because of its potential to produce large numbers of offspring and its remarkable longevity–arguably over 12 years in the case of
Therefore, manual palpation and clinical examination by experienced observers can be used to detect certain specific presentations of the main two filarial infections under discussion, with at least a moderate degree of reliability. The most reliable test being the assessment of the typical subcutaneous “nodules” (containing coiled adult worms and host inflammatory cells and tissues) in onchocerciasis; their presence in people living in a known endemic area has been to estimate the level of endemicity in the population of a defined geographic area. There are other causes of dermal nodules (e.g. dermal cysts, cysticercosis), but the typical location of onchocercal nodules on the body (iliac crest, the base of the spine, the chest wall or the head) increases the likelihood that such a mass is due to
4.2. Imaging of parasites
There are certain specific occasions in filarial infections where the adult worms can be seen by careful direct observation or using a diagnostic instrument. Loiasis has often been identified in people through the observation by the patient themselves of the migrating adult worm passing across the conjunctiva of the eye (thereby giving this parasite the common name “eye worm”). Questionnaire‐based surveys using photographs of these worms active in the eye have been used with endemic populations to estimate the degree of endemicity of geographic area; such a frightening experience as watching a several centimeters long parasite pass across one’s external eye–for example with women using a mirror to put on makeup in the morning–is dramatically memorable. Another example of the use of direct observation of the presence of worms is the identification by ophthalmologists using Slit Lamps to detect and count
Another non‐invasive technique that has been used in both onchocerciasis and lymphatic filariasis to observe the movement of worms, and thus their presence and viability, is ultrasound imaging. Motile adult worms can be visualized in the lymphatic vessels of the inguinal canal of males infected with
4.3. Histopathological examination of infected host tissues
The histological assessment of parasite‐containing tissues removed from patients is a very commonly used approach for assessing tissue (i.e. internally located) parasites, the subcutaneous nodules in onchocerciasis being a prime example in this present discussion. Most well‐trained pathologists can identify the presence of a parasite in these tissues and do this by using some very simple characteristics that indicate the infecting organism (such as obvious outer walls containing non‐mammalian cells), or by typical signs of a specific host reaction to these nematodes i.e. eosinophil and macrophage dominant host inflammatory responses. The viability status of the infecting nematode is usually only classified through major changes (e.g. its overall anatomical integrity, the breaking of the parasite wall, calcification etc.). More subtle changes in the parasite as it degenerates are still much less understood, therefore much less described in regular histopathological reports of parasite‐induced pathology. Nevertheless, such histopathological material is a vital window that can be used to describe the biology of the worm and its own pathology as it degenerates and dies. A good example is seen in advances in the understanding of the biology of many nematodes that has come in recent years from histological studies of the filarial endosymbiont
One of the characteristics of filarial nematodes that has inhibited studies at the histological level is the fact that these organisms are comparatively long and very narrow; the name “filarial” comes from the Latin word for “thread”. There are many anatomical differences at different places along this almost 200–300 μm long adult worm. Indeed, it has been noted that the degree of change and degeneration in these worms can vary considerably from place to place within a single worm; this makes an assessment of developing degenerative changes in the worms hard to detect unless the worm has reached the stage of almost complete anatomical degeneration and the whole organism is changed. The more subtle changes that take place within this long organism as it progresses and degenerates towards obvious physical finality are poorly described to date. In fact, what change or changes that can be defined as the irreversible point(s) of degeneration of the worm remains unclear, and indeed may vary from species to species.
Many of the early morphological studies on the effects of drugs on filariae were focused on electron microscopic (EM) studies and although changes were seen using this technique, observations such as these (which are carried out at a very high power magnification) are notoriously poor for defining the overall changes in the observed worm or a group of worms. EM images usually only look at a very small proportion of the worm’s complex anatomy, and thus do not give a good overall assessment of the status of the whole organism. Such detailed level techniques are however useful for defining specific anatomical characteristics such as those of the endosymbiont bacteria
A characteristic that has often being sought in filarial worms, and particularly with worms in adult filarial worm nests, is the age of the worm or worms being observed; such information with these long living worms is useful to those studying epidemiological questions. As these parasites ingest blood, and the products of its breakdown accumulate in the parasite’s intestines over time, the presence of hemosiderin (often defined histochemically) in the gut has been used as an indicator of an older worm. Degeneration has been commonly defined by simple anatomical change although some histological markers, usually immunomarkers, have been used to reflect biochemical degeneration. One such example is the reduction in the molecule Nras, an important component in cell cycle maintenance, which occurs in adult female
There is a major challenge in making statistically relevant observations on nematodes, organisms that have a lengthy anatomy and that often are coiled
A caution must also be made here in that although nodules are usually removed by the surgeons as “single usually ovoid” structures, or collection of ovoid structures in heavy infections, and then these masses typically bisected through the longest axis for histopathological and other studies. These “halves” are not always equivalent in parasitic content; it has been noted that after long‐term use of ivermectin the remaining adult worms often clustered in nests to one side of the ovoid nodule rather than being in the center as seen in untreated nodules. Thus, simple bisection does not necessarily provide equal content in these cases, and if one is bisecting a nodule to use two different assays to determining worm status–for example one‐half for pathology and the other for molecular assay,–one cannot assume that the two halves are equal of similar in terms of parasitic content.
These basic principles in the preparation and assessment of onchocercal nodules (Figure 3) also apply to the examination of worm nests in lymphatic filariasis, although this is rarely done in human infections it is used in experimental models and in studies of other natural filarial infections where the worms are present in nests and nodules a such a
4.4. Isolation of worms from host tissues
In the case of onchocerciasis, the surgical removal of the fibrous subcutaneous nodules, and is in fact regarded as a therapeutic step, as it removes at least some of the fecund females; removal of nodules is also in some cases also a definitive diagnostic step. In many field situations, material collected this way also provides the opportunity for assessment of the viability of the worms in these nodules. This approach remains one of few readily available approaches to assessing the presence and status of worms
As these parasites are embedded in chronic host inflammatory responses, which in the case of filarial parasites usually contains a large amount of collagen, the general approach utilized for isolation is the use of enzymes to digest the worms free from the tissue. The most commonly used enzyme is collagenase which targets the fibrous material ; the addition of dispase, a protease which targets fibronectin, collagen IV, and to a lesser extent collagen, is beneficial. It usually takes some 10–12 h to free the parasites in the case of
4.5. Assessing the changes in parasites
Alterations in worms that are extensively changed can often be seen by directly looking at the whole worm either
It is the more subtle changes in nematodes that need better description and understanding of their significance. It is essential in this goal to understand the normal anatomy of the nematode before defining histologically changes that indicate degeneration and death. A summary of the major anatomical components that can be assessed is given in Figure 3 and representative images in Figure 5. As already mentioned it is important to recognize that pathological changes within a degenerating worm can present differently at different points along the length of the organism, with changes occurring in one location and not in another within a single worm.
Different ways of assessing the state of worms in histological sections have been used. A scoring system that has been developed and used successfully for the investigation of new chemotherapeutic agents [11, 12] and the type of degenerating forms seen in such studies shown in Figure 6. The use of a four‐level score (0–3) for subjective assessments has been generally accepted in the realm of anatomical pathologists as minimizing, as much as possible, differences between observers and is a suitable formula for assessing nematodes.
4.6. The assessment of
Much of what has been described for assessing parasites in
A useful approach to preparing
In situmarkers of viability
The use of
There are several studies that have described several enzymes that appear to be present in filarial worms , and it is likely that specific enzymes will be identified soon whose presence could act as reliable indicators of worm viability. Biochemical approaches have been used to assess the viability of
4.8. Indirect markers
Although it is not a major purview of this discussion here to go into the wider area of laboratory and rapid test systems, it is nevertheless important to note that there is a considerable amount of experience over many years with the use of immunological markers, such as circulating antigens and host antibody responses for the diagnosis and epidemiological assessment of filarial infections, both in humans, dogs, and other animals . In fact, with human lymphatic filariasis, the major diagnostic tool used in major public health control programs is a rapid diagnostic test for detecting circulating antigen in finger‐prick sampled blood. In human onchocerciasis, the current approach is to use the presence of parasite‐specific antibody (Ov16) to indicate the status of infection in an endemic community. The use of samples of urine and saliva for these assays has been attempted but with varying and unfortunately rather unuseful results to date.
There is an ever‐increasing number of studies considering whether or not circulating specific products of parasites (e.g. protein microRNAs, etc) can reflect both the presence infection (interms of the presence of different parasitic stages) or perhaps the load of infection (the intensity of infection). This is an area of research that is vital to the efforts to eliminate the major parasitic diseases across the world. It is likely that in the next few years specific circulating markers will be identified in blood, or hopefully (for ease of collection) in urine or in saliva. This would provide a more practical way to assesses populations in epidemiological studies and lead us more quickly to the global goal of eliminating nematodes for affected populations.
There are still many aspects of measuring the viability of nematodes that need improving. A major challenge is to determine when a population of worms that have been subjected to an intervention, e.g. chemotherapy, an immune response, etc., and are on an irreversible pathway to death, and thus the parasite no longer can contribute to the infection in question. To achieve this, it is necessary at the level of the worm itself to understand what are the actual changes, or pathological events, within the worm’s anatomy and biology that reflect permanent irreversible damage.
As described above it is relatively easy to detect alterations if they are physically obvious (e.g. calcified, broken and obviously damaged entities) but it is the interpretation of the less obvious changes in worms or the deceased levels of a marker indicator that is difficult; what level of damage is irreversible? Optimally it would be extremely useful to define a single change or a simple collection of changes that reflect permanent irreversible damage. In the case of filariae it is likely that the uterus (the biggest organ in the female) is a useful indicator site for detecting damage and defining permanent damage. The body wall is also an important target organ for this role but it is an organ that is easily artificially altered by many of the isolation techniques used in preparing the worm samples for studies, especially those for
The assessment approach used needs to be driven by the question being asked when deciding on the method and focus of any assessment. The complexity of the life cycle of nematodes necessitates carefully focusing investigations on stages or anatomical components that are likely to prove useful and provide practical information. The approaches used for assessment need in most cases to be closely associated with the programmatic question being addressed, and take into consideration the environmental situation at play; for example, the breaking of infection transmission or understanding the direct effect of a chemotherapeutic agent on the worm’s reproductive capability. It is also important to distinguish between estimating parasite loads in an infected host and the measuring of direct effects on a parasite stage; although these two questions may be intimately linked they are not necessarily the same nor necessarily use the same method for assessment.
An obvious area of basic research that would greatly enhance the needs for developing better techniques for assessing parasite viability is advancing the knowledge of the basic physiological, biochemical and functional characteristics of nematodes and any species differences. Using