Electron Microscopy and the Investigation of New Infectious Diseases

Review

Electron microscopy and the investigation of new infectious diseases

Alan Curry@)

Objectives: To review and assess the role of electron microscopy in the investigation of new infectious diseases. Design: To design a screening strategy to maximize the likelihood of detecting new or emerging pathogens in clinical samples. Results: Electron microscopy remains a useful method of investigating some viral infections (infantile gastroenteritis, virus-induced outbreaks of gastroenteritis and skin lesions) using the negative staining technique. In addition, it remains an essential technique for the investigation of new and emerging parasitic protozoan infections in the immunocompromised patients from resin-embedded tissue biopsies. Electron microscopy can also have a useful role in the investigation of certain bacterial infections. Conclusions: Electron microscopy still has much to contribute to the investigation of new and emerging pathogens, and should be perceived as capable of producing different, but equally relevant, information compared to other investigative techniques. It is the application of a combined investigative approach using several different techniques that will further our understanding of new infectious diseases. Int J Infect Dis 2003; 7: 251-258

INTRODUCTION at individually by a skilled microscopist have con- The electron microscope was developed just before tributed to the decline of electron microscopy. Against World War II in several countries, but particularly in this background, the inevitable question must be Germany.l The dramatic increase in resolution available asked-does electron microscopy still have a useful in comparison with light microscopy promised to role to play in the investigation of emerging or new revolutionize many aspects of cell biology, virology, infectious diseases? bacteriology, mycology and protozoan parasitology. New preparative and staining methods were developed and ADVANTAGES OF ELECTRON MICROSCOPY perfected, allowing greater insights into many aspects of the biology of microorganisms. Commercially available The major advantage of electron microscopy is the electron microscopes, from several manufacturers, ability to see fine detail in samples. The detail resolved became widely available during the 1960s and 1970s. can be many hundreds of times smaller than what can be Biological and medical journals of that era feature large seen under a light microscope, even when it is optimally numbers of ultrastructural investigations into many adjusted. Although of secondary importance in com- cells, organs, microorganisms and diseases. However, parison to the resolution of the microscope, the ability by the 1990s with the advent of other techniques such to magnify structures seen in samples is also of use. In as immunofluorescence, immunoperoxidase staining, addition to the performance of the microscope, good immunoassays and, particularly, molecular biology, the specimen preparation technique is of great importance use of electron microscopy had dramatically diminished. when trying to achieve the best results from electron Additional factors such as cost, availability, preparation microscopy. The two most commonly used preparation time and the fact that every specimen must be looked techniques are negative staining and resin embedding/ thin sectioning.2 Negative staining is often used in virology and microbiology laboratories for examination of viruses and bacteria. This method reveals surface (l)Electron Microscopy Unit, Manchester Royal Infirmary, Central structures, whereas thin sectioning shows internal Manchester Healthcare Trust, Manchester, UK. structures. Thin sectioning is often restricted to histology Address correspondence to: A. Curry PhD, Electron Microscopy laboratories, which examine tissue biopsies. A number Unit, Clinical Sciences Building, Manchester Royal Infirmary, Central of centralized electron microscopy units utilize both of Manchester Healthcare Trust, Oxford Road, Manchester, Ml3 9WL, these basic preparative techniques, and this could be UK. advantageous when investigating some infections. If E-mail: [email protected] both of these preparative methods are available within, Corresponding Editor: Michael Ellis, AI Ain, UAE for example, a microbiology laboratory, cultured bacteria 252 Znternational Journal of Znfectious Diseases I Volume 7, Number 4,2003 can be examined after negative staining to reveal diag- phosphate-buffered formalin can be substituted.3 This nostic features on the bacterial surface (such as number can give results that are almost indistinguishable from and arrangement of flagella), and with the use of the thin those obtained with glutaraldehyde, but the results sectioning technique on some clinical samples, diag- are more variable. A possible advantage of phosphate- nostic ultrastructural details of non-cultivable bacteria buffered formalin is that it is compatible with conven- can sometimes be revealed. tional histologic processing into wax, and therefore offers flexibility in the route of processing into either LIMITATIONS OF ELECTRON MICROSCOPY wax or resin, or both. After this primary fixation, a second fixative (normally osmium tetroxide) is normally Electron microscopes are expensive precision instru- used in the laboratory as part of the processing of the ments. Every specimen must be examined individually, biopsy into resin. and the usefulness of the results is critically dependent on the skill, experience and knowledge of the microscopist. These factors and others, such as preparation CURRENT USE OF ELECTRON MICROSCOPY time for some specimens, mean that electron microscopy IN THE INVESTIGATION OF VIRAL is an expensive technique to use. Specimen preparation GASTROENTERITIS time is variable and depends on the type of specimen Infantile gastroenteritis and the investigation required. For example, a skin lesion can be prepared for examination in about lo-15 min, Electron microscopy still has an important role in the whereas a biopsy may take several days to embed and investigation of viral gastroenteritis, particularly that section before examination. In addition, electron micro- associated with infants and community outbreaks. scopy is a relatively insensitive technique, particularly in Several viruses are known to cause symptoms in infants, detecting some viruses, such as the Norwalk-like viruses including rotaviruses, enteric adenoviruses, astroviruses, (NLVs), and this has also contributed to the reduction in Sapporo-like viruses (classical caliciviruses) (Figure 1) its use. and NLVs (sometimes referred to as small round structured viruses (SRSVS)).~ Electron microscopy remains a ‘catch-all’ method of detecting these viruses, but enzyme METHODS immunoassays (EIAs) have been developed for some, but not all, of these enteric viruses, such as rotaviruses, Negative staining adenoviruses, astroviruses and NLVs. Such EIAs are cheaper, are easier, require less skilled staff to use, and, Generally, viruses and bacteria are examined under the electron microscope after being stained with heavy in some cases, are more sensitive than electron micro- metal salts that produce images showing negative scopy. The role of electron microscopy in the investig- contrast (negative staining). The stain surrounds, for ation of infantile gastroenteritis is diminishing, but it example, virus particles and penetrates surface features. should be retained to monitor any changes in the preva- This not only allows the virus to stand out from the lence of enteric viruses from a public health standpoint background, but also gives morphologic information and to validate the results of EIAs (quality assurance). such as symmetry and capsomere arrangement, which Here, the main advantage of the electron microscopy can allow specific identification or placement into result is that it is based on morphology and not the groups of morphologically similar viruses. A commonly used negative stain is phosphotungstic acid (PTA), but several similar stains are available. These can have similar staining properties to PTA, but may be specifically required for certain labile structures or very fine structures, which can be obscured by PTA. In virology, negative staining of viruses may be preceded by centrifugation (or ultracentrifugation), which can both partially purify and concentrate particles from clinical samples or cell cultures.

Resin embedding/thin sectioning With biopsies or cell preparations, good quality fixation is essential if the full potential of electron microscopy is to be realized. Tissue biopsies must be fixed as quickly as possible after being taken. The fixative of choice Figure 1. A group of Sapporo-like viruses from the stool of an for excellent ultrastructural preservation is buffered infant with symptoms of gastroenteritis. Note characteristic glutaraldehyde. If glutaraldehyde is not available, then surface hollows which fill with stain. Scale bar=100 nm. Electron microscopy and the investigation of new infectious diseases I Curry 253 presence of a particular antigen or nucleic acid type. herpesviruses from poxviruses and papilloma viruses if Arguably, the intact virions seen with electron micro- present in a skin scraping or lesion smear. Such samples scopy may be indicative of infectious virus, whereas can be prepared for examination in about lo-15 min. detection of antigen or nucleic acid may not always Such investigation is of particular importance if the indicate the presence of viable infectious virus particles. patient is immunocompromised or on a ward with other immunocompromised patients. Outbreaks of viral gastroenteritis FUTURE USE OF ELECTRON MICROSCOPY In many industrialized countries, NLVs (Figure 2) may IN VIROLOGY be responsible for the majority of outbreaks of gastroenteritis.5 Electron microscopy remains an important Monitoring possible changes in the pattern of technique for the detection of NLVS,~ but it is not ideal virus excretion following the introduction of a for this purpose, as it is relatively insensitive. Polymerase safe rotavirus vaccine chain reaction (PCR) tests are available in some centers, but these only detect certain strains. EIAs are also being If and when an acceptable rotavirus vaccine’ is developed developed, but, again, only detect certain genotypes. and becomes widely used, electron microscopy should Electron microscopy detects whole virions, and is there- be used to monitor any possible changes in the pattern fore not dependent on detecting antigens or nucleic acid of infection by enteric viruses and particularly rota- present in a sample. Along with the introduction of viruses. Current rotavirus EIAs detect group A, but not immunoassays or molecular detection, electron micro- group B and C, viruses. Children, under the age of 5 scopy should be retained for the examination of samples years, with symptoms of gastroenteritis and who have found to be negative by these methods. It should also be been vaccinated against rotavirus infection, should have appreciated that not all outbreaks of viral gastroenteritis stool samples examined by electron microscopy. If such in adults involve NLVs. Outbreaks in homes for the an organized surveillance exercise were put in place, elderly often involve viruses found in infantile cases then it would monitor any change in the prevalence of of gastroenteritis, such as rotaviruses. In the elderly, enteric viruses. the immune system goes into decline, rendering such individuals susceptible to reinfection by infantile gastro- Monitoring the viruses associated with skin lesions enteritis viruses. Samples from outbreaks in homes for the elderly should be examined with electron micro- and deliberate release scopy to determine whether the infectious agent The release of anthrax in the USA during 20018 has involved is an NLV or a virus normally associated with alerted many countries to the possible consequences of infantile gastroenteritis. deliberate release of other infectious agents, including smallpox, and their potential for causing epidemics within the human population. Measures need to be in Skin lesions place to diagnose, rapidly, potential poxvirus infection Electron microscopy still has a useful role to play in the (and differentiate orthopox viruses from parapox investigation of skin lesions. Here, it can distinguish viruses) (Figure 3) and also to differentiate other causes

Figure 2. A group of Norwalk-like viruses from the stool of an Figure 3. Orthopoxvirus particles (Molluscum contagiosum) adult involved in an outbreak of gastroenteritis. Note that the from a skin lesion. Molluscum contagiosum is a large virus, the surfaces of virions appear to be covered in short spikes, giving surface of which appears to have thread-like structures running the particles a ‘fuzzy’ appearance. Scale bar=100 nm. over it. Scale bar=250 nm. 254 International Journal of Infectious Diseases I Volume 7, Number 4,2003 of skin lesions, such as herpesvirus infection, so that unnecessary public panic can be avoided. Electron microscopy can provide such a rapid diagnostic service, so that only orthopox samples are referred to centers specializing in the molecular determination of poxvirus types. For this to be effective, a network of regionally based electron microscopy units, with the necessary expertise, needs to be in place to facilitate early warning of deliberate release of smallpox.

New viruses The possibility remains that new viral agents may be detected and, as in the past, electron microscopy should play its part in the investigation of such agents, in parallel with other investigative techniques. In an era Figure 4. Meront of Cryptosporidium muris in the stomach of when tests are becoming very specific, the major the common house mouse (Mus muscuh-). This is an advantage of electron microscopy is that this technique intracellular parasite that prefers an extracytoplasmic location can visualize a new viral agent in a sample and that within the crypt lumen. The interface between parasite and host cell appears to be restricted (narrowed) compared to C. this process is not dependent on detection of antigen parvum-infected cells. Scale bar= 1 pm. present or nucleic acid product produced. It should be noted that, although, many viruses are visualized by the negative staining technique, some enveloped viruses THE MICROSPORIDIA (e.g. HIV) require thin sectioning of infected cells or tissues to be visualized. Such electron microscopic Microsporidia are small, obligate, intracellular parasitic (EM) investigations can provide both morphologic and protozoa with a unique method of infecting cells.17 developmental information. Within the highly resistant spore is a coiled polar tube, which, when activated, everts and penetrates susceptible cells. The contents of the spore travel through the CURRENT USE OF ELECTRON MICROSCOPY everted polar tube, and replication begins. This is a two- FOR THE DETECTION OF EMERGING AND stage process, which ultimately produces many new NEWLY RECOGNIZED PROTOZOAN PARASITES infectious spores. Pre-HIV, only a very few human The acquired immunodeficiency syndrome (AIDS) was infections with microsporidia had been documented, first recognized in the early 1980s. The cause of the despite the widespread occurrence of these organisms in underlying immunodeficiency in the cellular arm of the nature.17J8 In 1985, Desportes et all9 reported a new immune system was a newly recognized virus, which we species of microsporidian found in an AIDS patient now know as human immunodeficiency virus (HIV). A with symptoms of chronic diarrhea, and gave an ultra- whole range of pathogens are currently known to infect structural description of the development of this such HIV-positive individuals, but possibly the biggest organism, Enterocytozoon bieneusi, within the entero- surprise has been the rise in prominence of Microsporidia cytes of a small intestine biopsy from this AIDS patient. (see below), from being merely a medical curiosity to Enteric involvement is the commonest manifestation of frequently detected and important opportunistic patho- microsporidial infection in humans, and Enterocytozoon gens. Other parasitic protozoa, such as Isospora belli, bieneusi the commonest microsporidian to infect humans have become more commonly detected in patients with (Figure 5). A second enteric microsporidian species, AIDS.9-11 Cryptosporidium causes important infections Encephalitozoon (Septata) intestinalis, was described in humans, and is particularly problematic in immuno- from a small intestine biopsy in 1993.20 Other new suppressed individuals. Most human infections involve microsporidian species have been described from Cryptosporidium parvum, but other species have now muscle,21-23,conjunctiva24-26 and individuals with multiple been detected,12 possibly including C. baileyi13 and organ infection. 27We now recognize 14 species of micro- C. muris.14,15 Species such as C. muris (Figure 4) are sporidian that infect humans, and electron microscopy ultrastructurally distinguishable from C. parvum, and has had a fundamental role in providing descriptions those studying biopsies should be aware of the differences of most of these newly recognized human parasites. between these species. l6 EM investigation of biopsies Microsporidians have been identified in specific (particularly small intestine biopsies) from this group of organs, but some species can disseminate. Species of immunocompromised patients should be considered, as Encephalitozoon, in particular, can disseminate, and, if this technique can reveal the characteristic ultrastructure they are detected in one organ system, consideration of such parasites and thus influence the treatment of must be given to the possibility of systemic infection.28 such infections. Several cases of dual microsporidial infection have been Electron microscopy and the investigation of new infectious diseases I Curry 255

rence in humans have appeared recently.3942 Most of these reports are of the incidental finding of myxozoan spores in stool samples, but one of these reports describes the finding of myxozoan spores in stool samples from patients with gastrointestinal symptoms.41 Three cases are described, and all had consumed freshwater fish. Fish muscle was available for two of the described cases, and both muscle samples were found to be parasitized by the myxozoan Myxobolus sp. Although myxozoans are not currently regarded as pathogens of humans, it is possible that they could become established in immunocompromised humans exposed to infected fish muscle. Some evidence for this possibility comes from the recent finding of a myxozoan-like parasite in the brain of the European mole (Talpa europaea),43 and raises the possibility that these organisms could be found in other Figure 5. An early sporogonic stage of the microsporidian, Enterocytozoon bieneusi, in a small intestine enterocyte. Note endothermic animals, including humans, particularly if the disk-shaped organelles (arrowheads) that eventually severely immunocompromised.42 coalesce to form the characteristic polar tube found within the EM examination has revealed fundamental new spores of these parasites. Such developmental stages are difficult to identify by light microscopy. Scale bar=400 nm. details for some of these organisms, which allow insights into their evolution, biology and life cycles. For example, nematode worms are common organisms in nature and reported.29z30 Although most of the newly described are also significant parasites of humans and animals. species of microsporidian are from AIDS cases, these Buddenbrockia, a seemingly insignificant, small (about infections are now being recognized in immunocom- 1 mm) nematode-like parasite of certain bryozoan promised individuals not infected by HIV, such as animals, has recently been found by electron microscopy transplant patients,31,32 and in immunocompetent to contain polar capsules.44 The presence of these individuals.33 Indeed, Wttuforma comeae was originally structures in both Myxozoa (Figure 6) and the isolated from an immunocompetent patient and nematode-like worm Buddenbrockia has added to the described after becoming established in athymic mice.34 evidence that Myxozoa are parasites that have evolved Subsequently, it has been identified in an AIDS patient.30 by reduction in cellular complexity from ancestral Inevitably, molecular tests for the diagnosis of nematode worms.44,45 the commoner microsporidial infections have been One species of myxozoan, Tetracapsuloides developed, but in laboratories that have not yet (Tetracupsula) bryosalmonae (previously named PKX), established molecular-based techniques, electron micro- causes proliferative kidney disease in salmon.46$47 scopy remains the ‘gold standard’ for diagnosis and Recently, important new details of the life cycle of this species confirmation of microsporidians.35-37 Considera- organism have been revealed by electron microscopy in tion should be given to the EM examination of biopsies an alternative bryozoan host for this myxozoan parasite. from immunocompromised individuals with symptoms Characteristic synaptonemal complexes, indicative of thought to be caused by infection.

THE FUTURE-AREAS WHERE ELECTRON MICROSCOPY EXCELS IN THE INVESTIGATION OF PARASITES As about 10 new species of microsporidia have been detected in humans since the advent of AIDS, it is likely that other species will be detected in future, and, as before, electron microscopy will play a fundamental role in descriptions of these organisms. It is difficult to predict what new human parasites will emerge, but Myxozoa must be candidates. Myxozoan spores characteristically contain several polar capsules, within each of which is a coiled filament.38 Discharge of this filament is thought to facilitate attachment to new hosts. The Myxozoa comprises a group of parasites that mainly infect fish Figure 6. A myxozoan spore showing a characteristic polar and invertebrates,38 but a few reports of their occur- capsule (arrowhead) found in these parasites. Scale bar=1 km 256 International Journal of Infectious Diseases I Volume 7, Number 4,2003 pairing (synapse) of homologous chromosomes during and examine by light microscopy. This step aids selection meiotic division, have been found (Figure 7).47 Meiosis of appropriate areas for EM examination and can is a reduction division of the nuclear material and is confirm that likely target cells, which may be infected, indicative of a sexual cycle. Such information adds to our are present in the sample. Ultrathin sections, stained understanding of the biology and development of with uranyl acetate and lead citrate, can then be parasites. carefully examined under the electron microscope. Here, knowledge of the ultrastructure of the tissue being A STRATEGY FOR DETECTING NEW examined and of the organisms likely to be encountered PATHOGENS BY ELECTRON MICROSCOPY is important. The microscopist must remain ‘open- IN TISSUE BIOPSIES minded’ when examining the sample, and screen it at a suitable magnification. If protozoan parasites are The detection of new or emerging agents in biopsies by thought to be involved, then a screening magnification electron microscopy requires a procedure in order to of 10 000-16 000 times is recommended, but if viruses maximize the likehood of detection. The use of electron microscopy should be considered if: are involved, then a higher screening magnification is likely to be required, depending on the size of the virus. 1. the patient is immunocompromised If an organism is detected, then pathognomic features 2. an infection is suspected should be looked for (such as the coiled polar tube 3. a biopsy is to be taken for other investigations. found in the spores of Microsporidia). In AIDS patients, some infections can disseminate,20 and some organisms The site from which the biopsy is taken is an important may therefore be found in unusual sites. Dual infections consideration. For example, diarrhea and malabsorption have also been reported,2g,“0 and if one organism is are common clinical symptoms in AIDS patients. To detected, then examination should be continued until investigate such symptoms, a rectal biopsy may be there is confidence that a second different organism has considered, as it is relatively easy to obtain. However, not been overlooked. It is also good practice to examine some emerging pathogens, such as the microsporidian the sample more than once and to examine further Enterocytozoon bieneusi, do not normally colonize this sections, as sometimes the bars of the specimen support terminal segment of the gut. Although less easy to grid can obscure organisms that are focal or scanty in obtain, a small intestine biopsy would be more appro- distribution. priate for the detection of enteric microsporidians. Once a biopsy is obtained, pieces from it may be required for several other tests. The sample for electron UNEXPECTED RESULTS IN THE microscopy should be chemically fixed as quickly as EXAMINATION OF BIOPSIES possible. The fixative of choice for electron microscopy Electron microscopy can produce unexpected and is buffered glutaraldehyde, but if this is not readily significant results when other investigative methods, available, then phosphate-buffered formalin can be such as light microscopy, are inconclusive or reveal used, and this should be readily available in any unusual appearances, as the following example shows. histopathology laboratory. Once the biopsy is embedded An AIDS patient with chronic diarrhea had a stool in resin, it is a good practice to cut semithin (l+m) examination that failed to detect any pathogens. A small sections onto glass slides, stain with toluidine blue stain, intestine biopsy was taken, and light microscopy revealed no infectious agents. Fortunately, part of this biopsy was sent to be examined by electron microscopy for microsporidia. EM examination revealed no evidence of microsporidial infection, but another parasitic infection was detected. Zoites of a coccidian parasite were present within enterocytes (Figure S), and tissue cysts of the same organism were present within the lamina propria. Unusually, the vacuolar membrane (parasitophorous vacuolar membrane) surrounding the zoites was closely adherent to the parasite wall, and this was thought to be the reason why the parasites had been overlooked by light microscopic examination. Because of the EM findings, a further stool examination was requested, and staff were asked specifically to look for the presence of oocysts of Isospora. Typical large, oval- shaped oocysts of the rare parasite Isospora belli were detected.48 These had been overlooked on initial Figure 7. A synaptonemal complex (arrowhead) indicative of meiosis seen in the developmental stages of the myxozoan screening, as the staff involved in the screening were parasite Tetracapsuloides bryosalmonae. Scale bar=500 nm. looking for oocysts of Cryptosporidium. Subsequently, Electron microscopy and the investigation of new infectious diseases I Curry 257

must undertake succession planning to ensure the long- term survival of electron microscopy. Electron microscopy still has much to contribute to the investigation of emerging and any potential new pathogens of both animals and humans. The method should be seen as capable of producing different, but equally relevant, information about infectious agents. Electron microscopy is thus complementary to other investigative techniques, and it is the application of a combined approach using several different techniques that will elucidate many aspects of the biology of emerging and new infectious diseases.

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