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

Design: To design a screening strategy to maximize the likelihood samples.

of new infectious diseases.

of detecting new or emerging pathogens in clinical

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 The electron microscope was developed just before World War II in several countries, but particularly in Germany.l The dramatic increase in resolution available in comparison with light microscopy promised to revolutionize many aspects of cell biology, virology, bacteriology, mycology and protozoan parasitology. New preparative and staining methods were developed and perfected, allowing greater insights into many aspects of the biology of microorganisms. Commercially available electron microscopes, from several manufacturers, became widely available during the 1960s and 1970s. Biological and medical journals of that era feature large numbers of ultrastructural investigations into many cells, organs, microorganisms and diseases. However, by the 1990s with the advent of other techniques such as immunofluorescence, immunoperoxidase staining, immunoassays and, particularly, molecular biology, the use of electron microscopy had dramatically diminished. Additional factors such as cost, availability, preparation time and the fact that every specimen must be looked

(l)Electron Manchester

Microscopy Healthcare

Address correspondence Unit, Clinical Sciences Manchester Healthcare UK. E-mail:

Unit, Trust,

Manchester Manchester,

Royal UK.

Editor:

Central

to: A. Curry PhD, Electron Microscopy Building, Manchester Royal Infirmary, Central Trust, Oxford Road, Manchester, Ml3 9WL,

[email protected]

Corresponding

Infirmary,

Michael

Ellis,

AI Ain,

UAE

at individually by a skilled microscopist have contributed to the decline of electron microscopy. Against this background, the inevitable question must be asked-does electron microscopy still have a useful role to play in the investigation of emerging or new infectious diseases? ADVANTAGES

OF ELECTRON

MICROSCOPY

The major advantage of electron microscopy is the ability to see fine detail in samples. The detail resolved can be many hundreds of times smaller than what can be seen under a light microscope, even when it is optimally adjusted. Although of secondary importance in comparison to the resolution of the microscope, the ability to magnify structures seen in samples is also of use. In addition to the performance of the microscope, good specimen preparation technique is of great importance when trying to achieve the best results from electron microscopy. The two most commonly used preparation 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 structures, whereas thin sectioning shows internal structures. Thin sectioning is often restricted to histology laboratories, which examine tissue biopsies. A number of centralized electron microscopy units utilize both of these basic preparative techniques, and this could be advantageous when investigating some infections. If both of these preparative methods are available within, 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 diagnostic features on the bacterial surface (such as number and arrangement of flagella), and with the use of the thin sectioning technique on some clinical samples, diagnostic ultrastructural details of non-cultivable bacteria can sometimes be revealed. LIMITATIONS

OF ELECTRON

MICROSCOPY

Electron microscopes are expensive precision instruments. Every specimen must be examined individually, 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 time for some specimens, mean that electron microscopy is an expensive technique to use. Specimen preparation time is variable and depends on the type of specimen and the investigation required. For example, a skin lesion can be prepared for examination in about lo-15 min, whereas a biopsy may take several days to embed and section before examination. In addition, electron microscopy is a relatively insensitive technique, particularly in detecting some viruses, such as the Norwalk-like viruses (NLVs), and this has also contributed to the reduction in its use. METHODS Negative staining Generally, viruses and bacteria are examined under the electron microscope after being stained with heavy metal salts that produce images showing negative contrast (negative staining). The stain surrounds, for example, virus particles and penetrates surface features. This not only allows the virus to stand out from the background, but also gives morphologic information such as symmetry and capsomere arrangement, which can allow specific identification or placement into 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

phosphate-buffered formalin can be substituted.3 This can give results that are almost indistinguishable from those obtained with glutaraldehyde, but the results are more variable. A possible advantage of phosphatebuffered formalin is that it is compatible with conventional histologic processing into wax, and therefore offers flexibility in the route of processing into either wax or resin, or both. After this primary fixation, a second fixative (normally osmium tetroxide) is normally used in the laboratory as part of the processing of the biopsy into resin. CURRENT USE OF ELECTRON MICROSCOPY IN THE INVESTIGATION OF VIRAL GASTROENTERITIS Infantile

gastroenteritis

Electron microscopy still has an important role in the investigation of viral gastroenteritis, particularly that associated with infants and community outbreaks. Several viruses are known to cause symptoms in infants, including rotaviruses, enteric adenoviruses, astroviruses, Sapporo-like viruses (classical caliciviruses) (Figure 1) and NLVs (sometimes referred to as small round structured viruses (SRSVS)).~ Electron microscopy remains a ‘catch-all’ method of detecting these viruses, but enzyme immunoassays (EIAs) have been developed for some, but not all, of these enteric viruses, such as rotaviruses, adenoviruses, astroviruses and NLVs. Such EIAs are cheaper, are easier, require less skilled staff to use, and, in some cases, are more sensitive than electron microscopy. The role of electron microscopy in the investigation of infantile gastroenteritis is diminishing, but it should be retained to monitor any changes in the prevalence of enteric viruses from a public health standpoint and to validate the results of EIAs (quality assurance). Here, the main advantage of the electron microscopy result is that it is based on morphology and not the

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 for excellent ultrastructural preservation is buffered glutaraldehyde. If glutaraldehyde is not available, then

Figure infant surface

1. A group of Sapporo-like viruses from the stool with symptoms of gastroenteritis. Note characteristic hollows which fill with stain. Scale bar=100 nm.

of an

Electron microscopy and the investigation of new infectious diseases I Curry presence of a particular antigen or nucleic acid type. Arguably, the intact virions seen with electron microscopy may be indicative of infectious virus, whereas detection of antigen or nucleic acid may not always indicate the presence of viable infectious virus particles.

253

herpesviruses from poxviruses and papilloma viruses if present in a skin scraping or lesion smear. Such samples can be prepared for examination in about lo-15 min. Such investigation is of particular importance if the patient is immunocompromised or on a ward with other immunocompromised patients.

Outbreaks of viral gastroenteritis

In many industrialized countries, NLVs (Figure 2) may be responsible for the majority of outbreaks of gastroenteritis.5 Electron microscopy remains an important technique for the detection of NLVS,~ but it is not ideal for this purpose, as it is relatively insensitive. Polymerase chain reaction (PCR) tests are available in some centers, but these only detect certain strains. EIAs are also being developed, but, again, only detect certain genotypes. Electron microscopy detects whole virions, and is therefore not dependent on detecting antigens or nucleic acid present in a sample. Along with the introduction of immunoassays or molecular detection, electron microscopy should be retained for the examination of samples found to be negative by these methods. It should also be appreciated that not all outbreaks of viral gastroenteritis in adults involve NLVs. Outbreaks in homes for the elderly often involve viruses found in infantile cases of gastroenteritis, such as rotaviruses. In the elderly, the immune system goes into decline, rendering such individuals susceptible to reinfection by infantile gastroenteritis viruses. Samples from outbreaks in homes for the elderly should be examined with electron microscopy to determine whether the infectious agent involved is an NLV or a virus normally associated with infantile gastroenteritis.

FUTURE USE OF ELECTRON IN VIROLOGY

MICROSCOPY

Monitoring possible changes in the pattern of virus excretion following the introduction of a safe rotavirus vaccine

If and when an acceptable rotavirus vaccine’ is developed and becomes widely used, electron microscopy should be used to monitor any possible changes in the pattern of infection by enteric viruses and particularly rotaviruses. Current rotavirus EIAs detect group A, but not group B and C, viruses. Children, under the age of 5 years, with symptoms of gastroenteritis and who have been vaccinated against rotavirus infection, should have stool samples examined by electron microscopy. If such an organized surveillance exercise were put in place, then it would monitor any change in the prevalence of enteric viruses. Monitoring the viruses associated with skin lesions and deliberate release

Electron microscopy still has a useful role to play in the investigation of skin lesions. Here, it can distinguish

The release of anthrax in the USA during 20018 has alerted many countries to the possible consequences of deliberate release of other infectious agents, including smallpox, and their potential for causing epidemics within the human population. Measures need to be in place to diagnose, rapidly, potential poxvirus infection (and differentiate orthopox viruses from parapox viruses) (Figure 3) and also to differentiate other causes

Figure 2. A group of Norwalk-like viruses from adult involved in an outbreak of gastroenteritis. surfaces of virions appear to be covered in short the particles a ‘fuzzy’ appearance. Scale bar=100

Figure 3. Orthopoxvirus particles (Molluscum from a skin lesion. Molluscum contagiosum surface of which appears to have thread-like over it. Scale bar=250 nm.

Skin lesions

the stool of an Note that the spikes, giving nm.

contagiosum) is a large virus, the structures running

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 when tests are becoming very specific, the major advantage of electron microscopy is that this technique can visualize a new viral agent in a sample and that this process is not dependent on detection of antigen present or nucleic acid product produced. It should be noted that, although, many viruses are visualized by the negative staining technique, some enveloped viruses (e.g. HIV) require thin sectioning of infected cells or tissues to be visualized. Such electron microscopic (EM) investigations can provide both morphologic and developmental information. CURRENT USE OF ELECTRON MICROSCOPY FOR THE DETECTION OF EMERGING AND NEWLY RECOGNIZED PROTOZOAN PARASITES The acquired immunodeficiency syndrome (AIDS) was first recognized in the early 1980s. The cause of the underlying immunodeficiency in the cellular arm of the immune system was a newly recognized virus, which we now know as human immunodeficiency virus (HIV). A whole range of pathogens are currently known to infect such HIV-positive individuals, but possibly the biggest surprise has been the rise in prominence of Microsporidia (see below), from being merely a medical curiosity to frequently detected and important opportunistic pathogens. Other parasitic protozoa, such as Isospora belli, have become more commonly detected in patients with AIDS.9-11 Cryptosporidium causes important infections in humans, and is particularly problematic in immunosuppressed individuals. Most human infections involve Cryptosporidium parvum, but other species have now been detected,12 possibly including C. baileyi13 and C. muris.14,15 Species such as C. muris (Figure 4) are ultrastructurally distinguishable from C. parvum, and those studying biopsies should be aware of the differences between these species. l6 EM investigation of biopsies (particularly small intestine biopsies) from this group of immunocompromised patients should be considered, as this technique can reveal the characteristic ultrastructure of such parasites and thus influence the treatment of such infections.

Figure 4. Meront of Cryptosporidium muris in the stomach of the common house mouse (Mus muscuh-). This is an intracellular parasite that prefers an extracytoplasmic location within the crypt lumen. The interface between parasite and host cell appears to be restricted (narrowed) compared to C. parvum-infected cells. Scale bar= 1 pm.

THE MICROSPORIDIA Microsporidia are small, obligate, intracellular parasitic protozoa with a unique method of infecting cells.17 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 everted polar tube, and replication begins. This is a twostage process, which ultimately produces many new infectious spores. Pre-HIV, only a very few human infections with microsporidia had been documented, despite the widespread occurrence of these organisms in nature.17J8 In 1985, Desportes et all9 reported a new species of microsporidian found in an AIDS patient with symptoms of chronic diarrhea, and gave an ultrastructural description of the development of this organism, Enterocytozoon bieneusi, within the enterocytes of a small intestine biopsy from this AIDS patient. Enteric involvement is the commonest manifestation of microsporidial infection in humans, and Enterocytozoon bieneusi the commonest microsporidian to infect humans (Figure 5). A second enteric microsporidian species, Encephalitozoon (Septata) intestinalis, was described from a small intestine biopsy in 1993.20 Other new microsporidian species have been described from muscle,21-23,conjunctiva24-26 and individuals with multiple organ infection. 27We now recognize 14 species of microsporidian that infect humans, and electron microscopy has had a fundamental role in providing descriptions of most of these newly recognized human parasites. Microsporidians have been identified in specific organs, but some species can disseminate. Species of Encephalitozoon, in particular, can disseminate, and, if they are detected in one organ system, consideration must be given to the possibility of systemic infection.28 Several cases of dual microsporidial infection have been

Electron

microscopy

Figure 5. An early sporogonic stage of the microsporidian, Enterocytozoon bieneusi, in a small intestine enterocyte. Note the disk-shaped organelles (arrowheads) that eventually coalesce to form the characteristic polar tube found within the spores of these parasites. Such developmental stages are difficult to identify by light microscopy. Scale bar=400 nm.

reported.29z30 Although most of the newly described species of microsporidian are from AIDS cases, these infections are now being recognized in immunocompromised individuals not infected by HIV, such as transplant patients,31,32 and in immunocompetent individuals.33 Indeed, Wttuforma comeae was originally isolated from an immunocompetent patient and described after becoming established in athymic mice.34 Subsequently, it has been identified in an AIDS patient.30 Inevitably, molecular tests for the diagnosis of the commoner microsporidial infections have been developed, but in laboratories that have not yet established molecular-based techniques, electron microscopy remains the ‘gold standard’ for diagnosis and species confirmation of microsporidians.35-37 Consideration should be given to the EM examination of biopsies from immunocompromised individuals with symptoms thought to be caused by infection.

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 endothermic animals, including humans, particularly if severely immunocompromised.42 EM examination has revealed fundamental new 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 are also significant parasites of humans and animals. Buddenbrockia, a seemingly insignificant, small (about 1 mm) nematode-like parasite of certain bryozoan animals, has recently been found by electron microscopy to contain polar capsules.44 The presence of these structures in both Myxozoa (Figure 6) and the nematode-like worm Buddenbrockia has added to the evidence that Myxozoa are parasites that have evolved by reduction in cellular complexity from ancestral nematode worms.44,45 One species of myxozoan, Tetracapsuloides (Tetracupsula) bryosalmonae (previously named PKX), causes proliferative kidney disease in salmon.46$47 Recently, important new details of the life cycle of this organism have been revealed by electron microscopy in an alternative bryozoan host for this myxozoan parasite. Characteristic synaptonemal complexes, indicative of

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 and invertebrates,38 but a few reports of their occur-

Figure capsule

6. A myxozoan spore showing a characteristic (arrowhead) found in these parasites. Scale

polar bar=1 km

256

International Journal of Infectious Diseases I Volume 7, Number 4,2003

pairing (synapse) of homologous chromosomes during meiotic division, have been found (Figure 7).47 Meiosis is a reduction division of the nuclear material and is indicative of a sexual cycle. Such information adds to our understanding of the biology and development of parasites.

A STRATEGY FOR DETECTING NEW PATHOGENS BY ELECTRON MICROSCOPY IN TISSUE BIOPSIES The detection of new or emerging agents in biopsies by electron microscopy requires a procedure in order to maximize the likehood of detection. The use of electron microscopy should be considered if: 1. the patient is immunocompromised 2. an infection is suspected 3. a biopsy is to be taken for other investigations. The site from which the biopsy is taken is an important consideration. For example, diarrhea and malabsorption are common clinical symptoms in AIDS patients. To investigate such symptoms, a rectal biopsy may be considered, as it is relatively easy to obtain. However, some emerging pathogens, such as the microsporidian Enterocytozoon bieneusi, do not normally colonize this terminal segment of the gut. Although less easy to obtain, a small intestine biopsy would be more appropriate 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 microscopy should be chemically fixed as quickly as possible. The fixative of choice for electron microscopy is buffered glutaraldehyde, but if this is not readily available, then phosphate-buffered formalin can be used, and this should be readily available in any histopathology laboratory. Once the biopsy is embedded in resin, it is a good practice to cut semithin (l+m) sections onto glass slides, stain with toluidine blue stain,

Figure meiosis parasite

7. A synaptonemal complex (arrowhead) indicative of seen in the developmental stages of the myxozoan Tetracapsuloides bryosalmonae. Scale bar=500 nm.

and examine by light microscopy. This step aids selection of appropriate areas for EM examination and can confirm that likely target cells, which may be infected, are present in the sample. Ultrathin sections, stained with uranyl acetate and lead citrate, can then be carefully examined under the electron microscope. Here, knowledge of the ultrastructure of the tissue being examined and of the organisms likely to be encountered is important. The microscopist must remain ‘openminded’ when examining the sample, and screen it at a suitable magnification. If protozoan parasites are thought to be involved, then a screening magnification of 10 000-16 000 times is recommended, but if viruses are involved, then a higher screening magnification is likely to be required, depending on the size of the virus. If an organism is detected, then pathognomic features should be looked for (such as the coiled polar tube found in the spores of Microsporidia). In AIDS patients, some infections can disseminate,20 and some organisms may therefore be found in unusual sites. Dual infections have also been reported,2g,“0 and if one organism is detected, then examination should be continued until there is confidence that a second different organism has not been overlooked. It is also good practice to examine the sample more than once and to examine further sections, as sometimes the bars of the specimen support grid can obscure organisms that are focal or scanty in distribution.

UNEXPECTED EXAMINATION

RESULTS IN THE OF BIOPSIES

Electron microscopy can produce unexpected and significant results when other investigative methods, such as light microscopy, are inconclusive or reveal unusual appearances, as the following example shows. An AIDS patient with chronic diarrhea had a stool examination that failed to detect any pathogens. A small 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, ovalshaped oocysts of the rare parasite Isospora belli were detected.48 These had been overlooked on initial screening, as the staff involved in the screening were 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 longterm 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.

REFERENCES Figure 8. Zoites of lsospora be//i in an enterocyte of a small intestine biopsy. Note the characteristic apical structures (conoid and rhopteries) at the anterior end of the zoites (arrowhead). These organelles are characteristic features of these apicomplexan parasites and are involved in cell invasion. This parasite could not be seen in enterocytes by light microscopy, and this may be related to the close adherence of the parasitophorous vacuolar membrane surrounding these organisms. Scale bar=1.5 pm.

EM examination of an esophageal biopsy from this patient also revealed an Isospora infection. This unusual site of infection again demonstrated the value of electron microscopy in the examination of biopsies from immunocompromised individuals. EM examination may reveal little of significance, but, as the above example shows, such examination can be rewarding, if investigation by electron microscopy is encouraged and the microscopist has the ultrastructural expertise to identify any organisms found.

CONCLUSION Electron microscopy has little use in diagnostic bacteriology and mycology, but can still have a positive role in the investigation of diseases caused by these infections. Within the field of diagnostic virology, EM investigation is useful, but its role is diminishing. By contrast, electron microscopy remains an important technique in the investigation of parasitic protozoan diseases. Also, electron microscopy still has an important role in research into many established and emerging infectious agents. Electron microscopy can and should have a continuing role in microbiological investigations, but this requires that the use of electron microscopy is supported and encouraged. We may have to centralize electron microscopy facilities, so that a sufficient volume of work is undertaken to keep costs down, and this may mean that associations with users who still require electron microscopy, such as centers specializing in renal investigations, need to be encouraged. Skills and experience need to be passed on to a new generation of electron microscopists, and this means that institutions

1. Agar AW. The story of European electron microscopes. In: Mulvey T, ed. Advances in imaging and electron physics, 1st edn. London: Academic Press, 1996:415-584. 2. Doane FW, Anderson N. Electron microscopy in diagnostic virology-a practical guide and atlas, 1st edn. Cambridge: Cambridge University Press, 1987:1-178. 3. Carson FL, Martin JH, Lynn JA. Formalin fixation for electron microscopy: a re-evaluation. Am J Clin Path01 1973; 59:365-371. 4. Hart CA, Cunliffe NA. Viral gastroenteritis. Current Opin Infect Dis 1999; 12:447-457. 5. Fankhauser RL, Monroe SS, Noel JS, et al. Epidemiologic and molecular trends of ‘Norwalk-like viruses’ associated with outbreaks of gastroenteritis in the United States. J Infect Dis 2002; 186:1-7. 6. Chadwick PR, Beards G, Brown D, et al. Management of hospital outbreaks of gastro-enteritis due to small round structured viruses. J Hosp Infect 2000; 45:1-10. 7. Baoming J, Gentsch JR, Glass RI. The role of serum antibodies in the protection against rotavirus disease: an overview. Clin Infect Dis 2002; 34:1351-1361. 8. Jernigan JA, Stephens DS,Ashford DA, et al. Bioterrorismrelated inhalation anthrax: the first 10 cases reported in the United States. Emerg Infect Dis 2001; 7:933-944. 9. Restrepo C, Macher AM, Radany EH. Disseminated extraintestinal isosporiasis in a patient with acquired immune deficiency syndrome. Am J Clin Path01 1987; 87:536-542. 10. Michiels JF, Hofman P, Bernard E, et al. Intestinal and extraintestinal Zsosporu belli infection in an AIDS patient. A second case report. Path01 Res Pratt 1994; 190:10891093. 11. Sarathchandra P, Curry A, Kapembwa MS. Intestinal

isosporiasis in African HIV positive woman with chronic diarrhoea.

CME Bull SexTransm

Infect HIV 2000;4: 51-53.

12. Xiao L, Bern C, Limor J, et al. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. J Infect Dis 2001; 183:492-497. 13. Ditrich 0, Palkovic L, Sterba J, Prokopic J, Loudova J, Giboda M. The first finding of Cryptosporidium baileyi in man. Parasitol Res 1991; 77:44-47. 14. Katsumata T, Hosea D, Ranuh IG, Uga S, Yanagi T, Kohno S. Short report: possible Cryptosporidium muris infection in humans. Am J Trop Med Hyg 2000; 62:70-72. 15. Gatei W, Ashford RW, Beeching NJ, Kang’ethe Kamwati S, Greensill J, Hart CA. Cryptosporidium muris infection in an HIV-infected adult, Kenya. Emerg Infect Dis 2002; 8:204-206.

258

International

Journal

of Infectious

Diseases

I Volume

16. Chalmers RM, Sturdee AP, Casemore DP Cryptosporidium muris in wild house mice (Mus musculus): first report in the UK. Em J Protistol 1994; 30:151-155. 17. Canning EU. Microsporidia. In: Kreier JP, ed. Parasitic protozoa, Vol. 6, 1st edn. London: Academic Press, 1993: 299-370. 18. Canning EU, Lom J. The Microsporidia of vertebrates, 1st edn. London: Academic Press, 1986:1-289. 19. Desportes I, Le Charpentier Y, Galian A, et al. Occurrence of a new microsporidian: Enterocytozoon bieneusi n. g., n. sp., in the enterocytes of a human patient with AIDS. J Protozool 1985; 321250-254. 20. Cali A, Kotler DP, Orenstein JM. Septata intestinalis n.g. n.sp., an intestinal microsporidian associated with chronic diarrhea and dissemination in AIDS patients. J Eukaryot Microbial 1993; 40:101-112. 21. Field AS, Marriott DJ, Milliken ST, et al. Myositis associated with a newly described microsporidian, Trachipleistophora hominis, in a patient with AIDS. J Clin Microbial 1996; 34:2803-2811. 22. Hollister WS, Canning EU, Weidner E, Field AS, Kench J, Marriott DJ. Development and ultrastructure of Trachipleistophora hominis n.g., n.sp. after in vitro isolation from an AIDS patient and inoculation into athymic mice. Parasitol 1996; 112:143-154. 23. Cali A,Takvorian PM, Lewin S, et al. Brachiola vesicularum, N. G., N. Sp., a new microsporidian associated with AIDS and myositis. J Eukaryot Microbial 1998; 45:240-251. 24. Visvesvara GS, Belloso M, Moura H, et al. Isolation of Nosema algerae from the cornea of an immunocompetent patient. J Eukaryot Microbial 1999; 46:lOS. 25. Didier ES, Didier PJ, Friedberg DN, et al. Isolation and characterization of a new microsporidian, Encephalitozoon hellem (n. sp.) from three AIDS patients with keratoconjunctivitis. J Infect Dis 1991; 163:617-621. 26. Didier PJ, Didier ES, Orenstein JM, Shadduck JA. Fine structure of a new human microsporidian, Erzcephalitozoon hellem, in culture. J Protozooll991; 38:502-507. 27. Vavra J, Yachnis AT, Shadduck JA, Orenstein JM. Microsporidia of the genus Trachipleistophora-causative agents of human microsporidiosis: description of Trachipleistophora anthropophthera N. Sp. (Protozoa: Microsporidia). J Eukaryot Microbial 1998; 45:273-283. 28. Molina J-M, Oksenhendler E, Beauvais B, et al. Disseminated microsporidiosis due to Septata intestinalis in patients with AIDS: clinical features and response to albendazole therapy. J Infect Dis 1995; 171:245-249. 29. Blanshard C, Hollister WS, Peacock CS, et al. Simultaneous infection with two types of intestinal microsporidia in a patient with AIDS. Gut 1992; 33:418-420. 30. Deplazes P, Mathis A, Van Saanen M, et al. Dual microsporidian infection due to Vittaforma corneae and Encephalitozoon hellem in an AIDS patient. Clin Infect Dis 1998; 27:1521-1524. 31. Sax PE, Rich JD, Pieciak, WS, Trnka YM. Intestinal microsporidiosis occurring in a liver transplant recipient. Transplantation 1995; 60:617-618. 32. Rabodonirina M, Bertocchi M, Desportes-Livage I. Enterocytozoon bieneusi as a cause of chronic diarrhea in a heart-lung transplant recipient who was seronegative for human immunodeficiency virus. Clin Infect Dis 1996; 23: 114-117.

7, Number

4,2003

33. Wanke CA, De Girolami P Federman M. Enterocytozoon bieneusi infection and diarrhea1 disease in patients who were not infected with human immunodeficiency virus: case report and review. Clin Infect Dis 1996; 23:816-818. 34. Silveira H, Canning EU. Vittaforma corneae n. comb. for the human microsporidium Nosema corneum Shadduck, Meccoli, Davis & Font, 1990, based on its ultrastructure in the liver of experimentally infected athymic mice. J Eukaryot Microbial 1995; 42:158-165. 35. Franzen C, Muller A. Microsporidiosis: human diseases and diagnosis. Microbes Infect 2001; 3:389-400. 36. Field AS, Canning EU, Hing MC, Verre J, Marriott DJ. Microsporidia in HIV-infected patients in Sydney, Australia: a report of 37 cases, a new diagnostic technique and the light microscopy and ultrastructure of a disseminated species. AIDS 1993; 7(suppl3):S27-S33. 37. Rabeneck L, Gyorkey F, Genta RM, Gyorkey P, Foote LW, Risser JMH. The role of microsporidia in the pathogenesis of HIV-related chronic diarrhoea. Ann Intern Med 1993; 119:895-899. 38. Lom J. Phylum Myxozoa. In: Margulis L, Corliss JO, Melkonian M, Chapman DJ, eds. Handbook of protoctista. Boston: Jones & Bartlett, 1990:36-52. 39. McClelland RS, Murphy DM, Cone DK. Report of spores of Henneguya salminicola (Myxozoa) in human stool specimens: possible source of confusion with human spermatozoa. J Clin Microbial 1997; 35:2815-2818. 40. Lebbad M, Willcox M. Spores of Henneguya salminicola in human stool specimens. J Clin Microbial 1998; 36:1820. 41. Boreham RE, Hendrick S: O’Donoghue PJ, Stenzel DJ. Incidental finding of Myxobolus spores (Protozoa: Myxozoa) in stool samples from patients with gastrointestinal symptoms. J Clin Microbial 1998; 36:3728-3730. 42. Moncada LI, Lopez MC, Murcia Ml, et al. Myxobolus sp., another opportunistic parasite in immunosuppressed patients? J Clin Microbial 2001; 39:1938-1940. 43. Friedrich C, Ingolic E, Freitag B, et al. A myxozoan-like parasite causing xenomas in the brain of the mole, Talpa europaea L., 1758 (Vertebrata, Mammalia). Parasitology 2000; 121:483-492. 44. Canning EU, Curry A, Feist SW, Longshaw M, Okamura B. Tetracapsula bryosalmonae n.sp. for PKX organism the cause of PKD in salmonid fish. Bull Eur Assoc Fish Path01 1999; 19:1-4. 45. Canning EU, Curry A, Feist SW, Longshaw M, Okamura B. A new class and order of myxozoans to accommodate parasites of bryozoans with ultrastructural observations on Tetracapsula bryosalmonae (PKX organism). J Eukaryot Microbial 2000; 47:456-468. 46. Okamura B, Curry A, Wood TS, Canning EU. Ultrastructure of Buddenbrockia identifies it as a myxozoan and verifies the bilatarian origin of myxozoa. Parasitology 2002; 124:215-223. 47. Canning EU, Tops S, Curry A, Wood TS, Okamura B. Ecology, development and pathogenicity of Buddenbrockia plumatellae Schroder, 1910 (Myxozoa, Malacosporea) (syn. Tetracapsula bryozoides) and establishment of Tetracapsuloides n. gen. for Tetracapsula bryosalmonae. J Eukaryot Microbial 2002; 49:280-295. 48. Hamour A, Curry A, Ridge A, Baily G, Wilson G, Mandal B. Zsospora belli in a patient with AIDS. J Infect 1997; 35:94-95.