Download Scrape Procedure

Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
CHAPTER 7: CHARACTERIZATION OF CORNEAL MICROSPORIDIOSIS
BY ELECTRON MICROSCOPIC STUDIES, CYTOKINE RESPONSES AND
ASSESSMENT OF INFECTIVITY OF DONOR CORNEAS
7.1
ELECTRON MICROSCOPIC OBSERVATIONS OF MICROSPORIDIAL
KERATITIS
7.1.1
Introduction
Diagnostic electron microscopy (EM) can contribute decisively to the laboratory
diagnosis of infectious diseases and is of particular importance if emerging infectious
agents are to be characterized (Goldsmith et al. 2004, Morens et al. 2004). Definite genus
identification of microsporidial ocular infections requires examination by electron
microscope to demonstrate the pathognomonic coiled polar filament or tubule. Other
features, such as arrangement and complexity of polar tubule, number and size of nuclei
and relationship to the host cell cytoplasm allow more precise species diagnosis (Wittner
1999). Hence, transmission electron microscopy (TEM) is essential for viewing internal
structures of microsporidian cells and till date remains the gold standard for the
identification and speciation of these organisms (Franzen and Muller 1999). However, in
recent years, studies of microsporidia have focused on the molecular biology, and
detailed analysis of the cytology is often neglected. One reason for this might be that the
detailed cytological investigation requires expertise and is time-consuming (Larsson
2005).
Though EM has a great diagnostic potential, with the wide availability of
alternative diagnostic methods, a change of paradigms in using diagnostic EM occurred.
However, the “modern” techniques also show limitations: nucleic acid amplification
techniques detect exclusively genomic sequences that are already known, and the
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
128
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
reactions may be inhibited by ingredients of the sample. Therefore, whenever a safe
diagnosis appears urgent, the “open view” of diagnostic EM should be used in parallel to
other methods (Hazelton and Gelderblom 2003). The objectives of this study were:
-
To perform TEM on corneal tissues as well as corneal scrapings to understand the
pathobiology of microsporidia in the eye
-
To evaluate the TEM for the speciation of microsporidia associated with keratitis
7.1.2
Materials and Methods
7.1.2.1 Patients
Five patients with microbiologically proven microsporidial keratitis and five patients with
histopathologically proven microsporidial keratitis seen at the L.V. Prasad Eye Institute,
Hyderabad, India between January 2002 and December 2005 were included in the study.
The corneal scrapings from these patients were collected in 1 ml PBS and centrifuged at
1500g for 3 minutes. The pellet was washed twice using 5 ml of PBS buffer and sent to
the electron microscopy laboratory (CCMB, Hyderabad and RUSKA Lab, College of
Veterinary Sciences, ANGRAU, Hyderabad, India). In cases that underwent PK, the
corneal button was bisected and sent for electron microscopic evaluation.
7.1.2.2 Transmission Electron Microscopy
Tissue preparation of the clinical sample for identification, involves the fixation of the
tissue, dehydration and infiltration with a medium that can be hardened to give a material
suitable for thin sectioning.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
129
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
Fixation
The tissue was fixed in 2.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) for 24
hours at 40C and post fixed in 2% aqueous Osmium tetroxide in the same buffer for 2
hours. Double fixation was adopted with glutaraldehyde as the primary fixative followed
by OsO4. Glutaraldehyde stabilizes tissues by cross-linking proteins rendering them
insoluble and immobile. Osmium tetroxide reacts with lipids and certain proteins but also
provides electron density to the tissue. Therefore, OsO4 acts as both a second fixation
step and an electron stain.
Infiltration and Embedding
This final process in tissue preparation is to infiltrate the specimens with a liquid
embedding medium, which is then polymerized to produce a solid block. After
dehydration, specimens are infiltrated with Spurr’s resin (Spur 1969) by passing them
through a series of graded alcohol (50% - 99%) until the dehydrating agent has been
completely replaced by the final embedding medium. This is done in small vials on a
shaker at room temperature. Activator must be included in all dilutions, and after the
tissues are transferred to capsules, filled with pure resins, and polymerized in an oven.
Post-staining Procedure
Both semithin and ultrathin sections were cut with a glass knife on a Leica Ultra cut
UCT-GA_D/E –1/00 ultra microtome, semithin of 200-300 nm knife were stained with
toludine blue and ultra thin sections (50-70 nm) were mounted on grids. Conventional
double-staining of thin-sections of biological material involves staining first in uranyl
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
130
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
acetate followed by lead citrate. A drop of uranyl acetate stain is placed onto a clean
surface in a chamber (petri dish). The grid with sections is floated on the drop, section
side down (Fig. 7.1B). After the desired time (normally five minutes), the grid is washed
three times by immersion in a rinse made of the same solution as the stain (for example,
if 50% methanolic uranyl acetate is the stain, the rinse should be 50% methanol) and then
air dried. A drop of lead citrate stain is placed onto a clean surface in a petri dish and the
grid placed into the drop with the section side up and stained for 5 minutes. The grid is
washed three times by immersion in a rinse made of boiled, distilled water and then air
dried. Then the ultra thin sections were observed at various magnifications under
transmission electron microscope (Model: Hitachi, H-7500, Fig. 7.1A)
A
B
Figure 7.1 : (A) A picture of the transmission electron microscope (Model: Hitachi, H-7500) used
in the study. (B) Petri dish containing ultra thin sections mounted on grids after staining
with uranyl acetate.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
131
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
7.1.3
Results
Thick sections of corneal scrapings showed many extracellular spores with scattered
degenerated structures with a vacuolated appearance and were surrounded by a thick
capsule. The degenerated spores had a thick wall, but no internal structures were
identified. The electron microscopic observations from the keratoplasty specimens
revealed masses of round to oval sporoblasts, measuring 3.5 - 5.0 m in length by 2.5 to
3.0 m in width, dissecting amongst the corneal lamellae. Vegetative and spore stages of
the organisms can be found in the host epithelial cells as the parasite undergoes
merogony and sporogony, resulting in the production of the infective spore stage of the
parasite. These stages together gave an understanding into the development of
microsporidia in the eye.
7.1.3.1 Ultrastructure of development of microsporidia in the eye
After entry of microsporidia on the surface of the eye, the polar tubule of the spore is
extruded (Fig 7.2 A-B) penetrating the epithelial cell in patients with keratoconjunctivitis.
The empty spore coat can be visualized on the surface of the epithelial cells after
discharge of sporoplasm inside the host epithelial cells (Fig 7.2 C). In patients with
stromal keratitis, the spores due to trauma are directly lodged onto the stromal cells of the
cornea, where they begin their development by extruding their polar tubule, penetrating
the host stromal cells (Fig 7.2 D). Contact of the end of the tubule with a host cell
membrane allows the spore to transfer its contents (sporoplasm) to initiate infection,
where in some cells the parasites lay in direct contact with the host cell cytoplasm, in
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
132
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
others, the parasites are situated within a clearly defined parasitophorous vacuole (PV) or
lay in direct contact with the PV membrane within the new host cell. The earliest
developmental stage seen are the meronts which are irregular in outline and bounded by a
plasma membrane, and occur randomly in the host cell cytoplasm (Fig 7.2 E-F). The
meronts divide by binary fission and the resultant daughter cells appear to be thickened
with dense particles external to the limiting membrane. The daughter cells transform into
sporonts (Fig 7.3 A-B), which in more advanced stages are easily recognized by their
elongated appearance and their wrinkled surface. The boundary layer of sporonts are
thickened and electron-dense and appear to have been formed by the deposition of dense
material between the two membranes seen in the daughter cells. Sporonts contained a
nucleus and numerous free ribosomes. Chains of sporonts appear to form by repeated
fission, later separate to form sporoblasts. Sporoblasts are bounded by a distinct wall
composed of two membranes, the outermost of which is covered by a flocculent dense
layer (Fig 7.3 C-D). Each sporoblast has a nucleus, ribosomes, and accumulation of
dense, membranous Golgi like material, the latter of which appear to contribute to the
formation of the polar tubule (Fig 7.3 E-F). In mature spores the polar tubule is arranged
into six to twelve coils in the posterior region of the spore (Fig 7.4 A - D). Closely
packed ribosomes impart a dense appearance to the cytoplasm of the mature spores. The
wall of mature spores is thickened. External to the plasma membrane of the parasite’s
cytoplasm is a broad, electron-lucent layer, the endospore. This layer is overlain by the
outer layer of the spore wall, the exospore, which is covered with a dense flocculent
material. The surface of mature spores are smooth.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
133
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
A
B
C
D
E
F
Fig 7.2: Electron micrograph of ‘‘early stages’’ of microsporidial infection in human corneal
epithelial and stromal cells. (A) An activated spore whose polar filament has started the process
of translocation inside the epithelial cell from patient (L2096/05) diagnosed as microsporidial
keratoconjunctivitis (B) Polar tube arising from an anchoring disk, being extruded through the
disrupted spore wall found in patient (L1835/05) diagnosed as microsporidial keratoconjunctivitis
(C)The remnants of membranes, still remain in the empty spore found on the surface of the
epithelial cell of a patient (L1820/03) diagnosed as microsporidial keratoconjunctivitis (D) An
activated spore whose polar filament has started the process of translocation inside the stromal
cell of a patient (H490/02) diagnosed as microsporidial stromal keratitis (E) The parasite is in
direct contact with the host cell cytoplasm and several meronts are interspersed in the epithelial
cells in case (L1841/03) (F) Meronts are interspersed in the stromal cells in (H 490/02)
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
134
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
A
B
C
D
E
F
Figure 7.3: Electron micrograph of intracellular events and development of microsporidia (A)
Meronts transformed to sporonts in epithelial cells of patient (L1835/05) (B) Phagocytized
sporonts in stromal cells of a patient (L 2457/05) (C) Sporoblasts have a "thick" dense surface
coat and a more complex and dense cytoplasm found in patient (L1786/05) diagnosed as
microsporidial keratoconjunctivitis (D) Sporoblast containing portions of the developing polar
tube, nucleus and a "thick" surface coat within a phagosome extending into the remains of the
cytoplasm of the infected stromal cell of patient (L 2457/05) (E) Characteristic electron-dense
outer and electron-lucent inner layers of the wall of a mature spore, as well as various sections of
coils of the polar tube The spore (S) contains a polar tube, Note the irregular shape of the cell and
the more complex cytoplasm found in a patient (L 2631/05) diagnosed as microsporidial
keratoconjunctivitis (F)Maturation of spores in the stromal cells showing coils of the polar tubule
and two nucleus found in a patient (H 374/02) diagnosed as microsporidial stromal keratitis.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
135
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
A
B
C
D
Figure 7.4: Electron micrograph of (A) a mature spore enclosed by a wide electron-lucent
endospore, which is in turn covered by a dense, thick exospore and displaying approx 10 coils of
the polar tubule giving a possible identification of Vittaforma corneae from a patient (V 367/05)
with stromal keratitis. (B) Transverse section of coils of polar tubule of a spore present in the
stromal cells of the corneal tissue from a patient (H 374/02) with microsporidial stromal keratits
(C) A mature spore showing electron dense cytoplasm with polar tubule and ribosomes and
nucleus from a patient (L 2828/05) with keratoconjunctivitis (D) a mature spore enclosed by a
wide electron-lucent endospore, which is in turn covered by a dense, thick exospore and
displaying approx 4-7 coils of the polar tubule giving a possible identification of Encephalitozoon
spp. from a patient (L 2631/05) with keratoconjunctivitis.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
136
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
7.1.3.2 Speciation of Microsporidial keratitis
Electron microscopy confirmed microsporidial spores within the stromal cells for the
keratoplasty specimens and on the epithelial cells for the corneal scrapings specimens.
Only the spore stage of development was easily recognizable for species identification.
Among the five cases of keratoconjunctivitis, the electron micrograph of only one case
showed the polar tube having a single row of 4-7 coils, a feature that is consistent with
the genus Encephalitozoon spp. Speciation of this genus was not possible as the
distinctive parasitophorous vacuole, characteristic of E. intestinalis could not be
visualized with certainty in the sample. Further sectioning was not possible due to the
small amount of material. Similarly, among the five cases of stromal keratitis, the
electron micrograph of only one case showed the polar tube having a single row of 10-14
coils, a feature that is consistent with Vittaforma corneae.
7.1.4
Discussion
The most familiar stage in the microsporidial developmental cycle is the spore, as it is
more easily detected than the intracellular proliferative stages. Following infection, a two
stage developmental cycle follows – an initial proliferative phase (merogony) followed
by a spore-forming phase (sporogony). Despite cumulative number of reports on corneal
infections due to microsporidia, few studies have given conclusive evidence of the
development of these organisms in the eye. This study has addressed this issue using ultra
structural studies and demonstrated the difference in their biology during infection on
both epithelial or stromal cells. The results suggest that different species of microsporidia
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
137
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
could possess a range of pathogenicity, tissue predilection and virulence to human host.
Species known to cause keratitis include E. intestinalis, E. hellem, E. cuniculi, Vittaforma
corneae, Nosema oculorum, Nosema connori, and Trachipleistophora hominis (Franzen
and Muller 1999). There remains a possibility that more species are yet to be identified if
detailed studies using electron microscopy coupled with molecular characterization are
employed. In some circumstances, inadequate information obtained from limited material
may be delineated by in vitro cultivation using appropriate cell lines that provides more
detail description and identification of microsporidial species.
Undoubtedly, definite species identification of microsporidia remains an important issue
in order to know the range of pathogenic species, the extent of disease profiles and
appropriate management of human microsporidiosis. The differentiation of Vittaforma
corneae species from Encephalitozoon is based on several electron microscopic features.
First, Vittaforma are larger than Encephalitozoon (Vittaforma organisms measure
approximately 3.5–5.0 mm in length versus 2.0 –3.0 mm in length for Encephalatozoon
organisms). Second, the absence of a parasitophorous vacuole surrounding the organism
within the host cell is more consistent with Vittaforma. Third, the coils of the filament
range from 10 to 13 in Vittaforma corneae versus 4 to 7 in Encephalitozoon (Pinnolis et
al. 1981). The electron microscopy findings in our study are consistent with Vittaforma
corneae species in another case of stromal keratitis. However, in one case of
keratoconjunctivitis, though we could make a possible genus identification of
Encephalitozoon, speciation was not possible as E. hellem and E. cuniculi are
morphologically indistinguishable,
whereas
E.
intestinalis
has
the
distinctive
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
138
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
parasitophorous vacuole (Chu and West 1996) which could not be visualized with
certainty in this study. Comparison of speciation by EM with the PCR and sequencing
results, described in Chapter 6, showed that while V. corneae was identified by both
methods, E. cuniculi could be identified by PCR alone as EM only suggested presence of
Encephalitozoon spp. In addition, PCR could also determine the species for the remaining
eight cases included in this study. Moreover, evaluation by EM is time-consuming,
expensive and preparation and evaluation cannot be performed in an automated way, they
are dependent on experienced staff. Another limitation is the relatively low sensitivity
(105-6 particles/ml) of diagnostic EM compared to other diagnostic methods (Hazelton
and Gelderblom 2003).
Conclusions

Though it was possible to identify the species involved in two of the ten cases in this
study, the recent impression is that EM is not very useful for speciation of MK as
corneal scrapings / corneal biopsies reveal more often the developing stages and not
mature spores which is necessary for proper identification of the species causing
keratitis. This suggests that the accurate speciation of an infecting parasite is possible
only by means of a molecular biology technique and EM may have limited
application as a diagnostic tool in clinical studies.

Nevertheless, this method can be useful to study the development and pathogenicity
of these parasites, as well as for the characterization of new species of this rare
entity.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
139
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
7.2
INVESTIGATION OF THE CYTOKINES RESPONSE OF HUMAN
CORNEAL EPITHELIAL CELLS TO MICROSPORIDIAL INFECTION
7.2.1
Introduction
Microsporidial keratitis is a disease initiated by infection of the epithelial and stromal
layers of the cornea with spores of microsporidia, resulting in infiltration of neutrophils
and mononuclear lymphocytes (Wittner 1999). Studies by Didier (1995) have shown that
cytokines released by sensitized T cells activate macrophages to kill E. cuniculi in vitro.
These findings suggest that a protective immune response to E. cuniculi is likely
dependent on cytokine-producing immune T cells. Studies with E. intestinalis, have
shown that mice lacking the IFN-γ gene are unable to clear the infection (Achbarou et al.
1996).
In a previous study (Khan et al. 1999), it was observed that treatment of
E. cuniculi-infected mice with neutralizing antibody against IFN-γ or IL-12 resulted in
increased mortality for these animals. Also, IL-10 has been reported to be involved in the
regulation of Th1 immune response in other infectious disease models (Gazzinelli et al.
1994, Trinchieri 1997), it is possible that it plays a similar role in infection caused by
microsporidia too. Most of what is known about mammalian microsporidiosis is based on
studies using E. cuniculi (Didier 1995), because this was the first microsporidian that
could be grown continuously on tissue culture (Shadduck 1969) and most of these in vitro
studies used murine culture cell systems (Didier 1995, Khan and Moretto 1999).
However in addition to E. cuniculi, Vittaforma corneae was also found to be an important
pathogen, in our series, causing microsporidial keratitis. Although the inflammatory
response is necessary to clear the organism from the infected tissue, it can also be
destructive to the host cornea, leading to scar formation and vision loss.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
140
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
Corneal epithelial cells constitute the first line of defense against microbial pathogens and
therefore should possess the ability to respond to their presence. It is reasonable to
propose that, in response to microsporidial infection, the epithelial cells send initial
signals, such as the release of proinflammatory cytokines, to recruit neutrophils and
mononuclear lymphocytes into the cornea. Epithelial cells could also produce factors to
enhance the antimicrobial activity of residential cells of the cornea. Hence, we
hypothesize that human corneal epithelial cells, which constitute the outermost layer of
the cornea, can be infected with microsporidia, and that the infection leads to the
activation of proinflammatory macromolecules. In the present study, we examined the
cytokine responses of human corneal epithelial (HCE) cells to Vittaforma corneae,
isolated from a patient with microsporidial keratoconjunctivitis.
7.2.2
Materials and Methods
7.2.2.1 Parasites
Microsporidial spores isolated from corneal scrapings of a patient with microsporidial
keratoconjunctivitis was collected in MEM, thawed, vortexed vigorously for 30 seconds
and an equal volume (0.5 ml/ vial) of the sample was inoculated into monkey kidney cells
(Vero cells) maintained on Eagle's minimal essential medium (EMEM) fortified with 2
mM glutamine and 10% fetal bovine serum (FBS). The culture medium from each flask
(Nunc, 25 cm2) was removed daily for the first week and twice weekly thereafter and
replaced with fresh medium containing the antibiotics. The cultures were incubated at
37°C.
Spores that were extruded into the culture medium were harvested by
centrifugation at 1500g for 20 min at 4 °C. The supernatant was removed and the spore
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
141
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
sediment was washed once with 0.25% sodium dodecyl sulfate in PBS and then washed
once more in PBS. Spores were purified by centrifugation over 50% Percoll (Amersham
Pharmacia Biotech, Piscataway, N.J) at 500g for 30 minutes at 4 °C. The purified
sediment was washed once again in PBS, counted in a hemocytometer (Neubauer-ruled
Bright Line counting chambers; Hausser Scientific, Horsham, Pa.), suspended in PBS to
obtain 108 spores/mL, and stored at 4 °C until use.
7.2.2.2 HCE Infection
Human corneal epithelial (HCE) cell line (Kind gift from Dr. Araki-Sasaki K, Osaka
University Medical School, Osaka, Japan) was established by immortalizing primary
cultured human corneal epithelial cells (obtained from a donor cornea) with a
recombinant SV40-adenovirus vector and cloned three times to obtain a continuously
growing cell line. This cell line has been shown to have properties similar to normal
human corneal epithelial cells (Araki-Sasaki 1995). HCE cells were grown using the
supplemented hormonal epithelial medium consisting of equal volumes of MEM and
Ham's nutrient mixture F-12 supplemented with 5% (vol./vol.) heat inactivated fetal
bovine serum (FBS), 5 μg/ml insulin, 10 ng/ml human epidermal growth factor, 0.5%
dimethyl sulfoxide and 40 μg/ml gentamicin. All the reagents were obtained from Sigma,
St. Louis. To infect cells, confluent epithelial cultures grown in Nunc 25 cm2 flasks were
inoculated with 1x108 spores/mL of microsporidia. Simultaneously, an uninoculated HCE
culture was also stored for analysis. At 0 and 24 hrs the culture supernatants of infected
culture as well as the control, were collected in duplicates for cytokine determination and
stored at -70°C for cytokine determination.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
142
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
7.2.2.3 Cytokine assays
The cytokine analysis was measured by the evidence investigator™ using a biochip,
according to the manufacturer’s directions (Randox laboratories ltd, United Kingdom)
which included Interleukin-1 alpha (IL-1), Interleukin-1 beta (IL-1), Interleukin-2 (IL2), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10),
Vascular Endothelial Growth Factor (VEGF), Tumour Necrosis Factor-alpha (TNF-)
Interferon-gamma (IFN-), Epidermal Growth Factor (EGF), Monocyte Chemotactic
Protein-1 (MCP-1) . Conventional immunoassay techniques are utilized for the
measurement of analytes on the surface of a biochip, which results in the specific and
simultaneous profiling of markers. Rather than having to divide and separately test a
patient sample to obtain each test result, evidence investigator™ offers a means for
simultaneous testing of a sample and thus provides a more complete diagnostic profile for
each patient. The cytokine and growth factors array measures 12 analytes from a single
sample.
The cytokines and growth factors array of tests are presented on a single
biochip. All twelve tests are performed simultaneously using just one assay diluent, one
panel-specific conjugate solution and one signal reagent. All reagents required to run
assays are provided in a single kit – assay buffer, multi-analyte conjugate, nine levels of
multi-analyte calibrator, wash buffer and signal reagent. Multi-analyte controls are
available as a separate kit. The evidence investigator™ CVs are below 10% thus
providing the user with confidence in results obtained. There is reduced variation as all
results are determined at the exact same time point. One vial of multi-analyte calibrator is
all that is required for each of the nine levels.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
143
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
Figure 7.5 : Picture of the evidence investigator™ (Randox Laboratories) which offers a means
for simultaneous testing of a sample and thus provides a more complete diagnostic profile. Insert
showing the cytokines and growth factors array of tests presented on a single biochip.
7.2.3
Results
The microsporidial spores inoculated in human corneal epithelial cells were identified as
Vittaforma corneae after sequencing the PCR product obtained using pan-microsporidial
primers based polymerase chain reaction described in Chapter 6. The levels of cytokines
and growth factors at 0 hour and after 24 hours of inoculation of microsporidial spores in
HCE culture supernatants are shown in Table 7.1. The levels of cytokines in both test
sample and control sample at 0 hour were comparable. Among the 12 cytokines screened,
six (IL-6, IL-8 , VEGF, IFN-γ, EGF, MCP-1) were induced after 24 hours of infection
with microsporidia. The levels of TNF-α, IL-10, IL-1 and IL-1 remained unchanged in
the control during the course of the study. Levels of IL-2 and IL-4 were not elevated
above control levels 24 hours post infection. IL-6 and VEGF were significantly elevated
in test sample infected with microsporidial spores (approximately two fold higher)
compared to uninfected HCE cells. Similarly, IL-8 and MCP-1 were significantly
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
144
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
elevated (approximately 10-fold) in infected HCE cells compared to uninfected cells.
Infection of HCE cells by microsporidia did not increase levels of Th1 type cytokines like
TNF-α and IL-2 (Table 7.1) above baseline levels.
Table 7.1: Levels of cytokines in culture supernatants of HCE cell line at 0 hour and 24
hours after infection.
Cytokines
IL2 (pg/ml)
IL4 (pg/ml)
IL6 (pg/ml)
IL8 (pg/ml)
IL10 (pg/ml)
VEGF (pg/ml)
IFN (pg/ml)
TNF (pg/ml)
IL1 (pg/ml)
IL1 (pg/ml)
MCP1 (pg/ml)
EGF (pg/ml)
0 hr control
0 hr test
24 hr control
24 hr test
<0.000
2.792
1.705
0.868
3.493
2.689
2.55
1.828
<0.000
<0.000
122.412
>275.000
<0.000
<0.000
178.241
1530.388
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
428.45
925.587
<0.000
<0.000
<0.000
9.261
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
<0.000
>1200.000
<0.000
<0.000
<0.000
1.438
IL-2 : Interleukin 2, IL-4: Interleukin 4, IL-6: Interleukin 6, IL-8: Interleukin 8, IL-10: Interleukin
10, VEGF : Vascular Endothelial Growth Factor, IFN : Interferon – gamma , TNF - Tumour
Necrosis Factor-alpha, EGF: Epidermal Growth Factor, MCP-1 : Monocyte Chemotactic Protein1, IL-1 : Interleukin-1 alpha, IL-1 : Interleukin-1 beta
7.2.4
Discussion
In the immunobiology of microsporidial infections the role of both humoral and cellular
immunity has been demonstrated (Braunfuchsona et al. 1999). As microsporidia are
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
145
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
intracellular parasites, the role of cell-mediated immunity is probably more significant
(Schmidt and Shadduck 1983). To understand the role of epithelial cells in the innate
response to microsporidial infection of the cornea, an in vitro infection model of human
corneal epithelial cells (HCE) grown in the laboratory was used. This study reveal that
corneal epithelial cells respond to the presence of microsporidial parasites and initiate a
rapid innate immune response, leading to the production of pro-inflammatory cytokines.
The most striking result in this study was the abundant expression of MCP-1 and IFN-γ
which is a strong activator of monocytes and macrophages suggesting that these
mediators contribute to the cellular infiltration of the corneal cells. The significance of
IFN-γ in the control of microsporidial infections has been previously demonstrated
(Didier et al. 1994, Achbarou et al. 1996). Macrophages activated by IFN-γ kill
microsporidia by the nitric oxide-dependent mechanism (Didier 1995). This study also
demonstrated the production of IL-6 and IL-8 in our study which has been linked to the
recruitment of neutrophils and lymphocytes, suggesting that corneal epithelial cells play a
role in inducing neutrophil chemotaxis to the parasite infection site. Like IL-6, TNF-α is
a potent proinflammatory cytokine and is produced mainly by activated macrophages and
T cells and has been reported to be released by the epithelial cells of the mouse central
cornea in response to lipopolysaccharide (Sekine-Okano et al. 1996). However, this study
showed the absence of TNF-α expression, the reason for this could not be estabilished.
Previous studies have demonstrated the essential role of IL-6 as a potent stimulator of
VEGF production (Nakahara et al. 2003, Wei et al. 2003) from corneal epithelial cells
after HSV infection. In addition, the intrastromal injection of IL-6 resulted in corneal
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
146
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
neovascularisation in a VEGF-dependent process (Banerjee et al. 2004). This data
substantiates the close relationship between proinflammatory cytokines and VEGFinduced corneal neovascularisation. In addition, this study also showed minimal EGF
expression that has been reported to stimulate epithelial cell proliferation (Klenkler and
Sheardown 2004). Nevertheless, the ability of the epithelium to produce multiple
proinflammatory cytokines in response to microsporidial infection indicates that the
epithelium is an important part of the innate response system for the cornea and plays a
role in eliciting the infiltration of inflammatory cells into the tissue.
Studies of parasite infections have played a major role in establishing the veracity of the
Th subset paradigm. The balance between Th1 and Th2 helper cells and their associated
cytokine patterns can in an immune response influence the phenotype and progression of
several clinical diseases (Alexander and Bryson 2005). Th1 cells produce type 1
cytokines (IL-2, TNF-α, IFN-γ), while Th2 cells produce type 2 cytokines (IL-4, IL-6,
IL-10, IL13). In this study, IL-6 and IFN-γ are coexpressed, the expression of IL-2 and
IL-4 were down-regulated. In addition, TNF-α, IL-1 and IL-10 are suppressed, suggesting
a skewed cytokine production and a mixed pattern of Th response. This is the first study
in the literature to investigate the balance of Th1/Th2 in microsporidiosis in corneal cells.
Further investigations should be focused on the polarization of the immune response and
the role of various subpopulations of lymphocytes in the elimination of microsporidia
from the host cornea. Selective receptor blockage of highly expressed chemokines or
proinflammatory cytokines in inflamed eye tissue could then be explored as a reasonable
strategy to inhibit the recruitment of leukocytes.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
147
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
7.3 INVESTIGATION OF THE POTENTIAL INFECTIVITY OF DONOR
CORNEAS LEADING TO MICROSPORIDIAL KERATITIS FOLLOWING
KERATOPLASTY
7.3.1
Introduction
The infection of the corneal graft is one of the most serious complications following
keratoplasty (Morris and Bates 1989, Varley and Meisler 1991). In many instances, it can
be treated successfully with intensive topical and sub-conjunctival antibiotics. However,
when this therapy is ineffective, a surgical approach is considered. Two cases of
microsporidial epithelial keratoconjunctivitis occurring in the corneal graft of individuals
who were locally immunocompromised have been already described in Chapter 3. These
patients had no history of owning pets, river swimming, or wearing contact lenses. The
only possible associated risk in both these cases was the use of topical steroids, leading to
a localized immunosuppressed state, resulting in secondary infection by microsporidia.
The use of immunosuppression as a therapeutic goal following keratoplasty, therefore,
may increase the risk for acquiring microsporidia infections in these patients. It is
tempting to speculate the possible presence of these organisms in the donor corneas and
subsequent infection in these patients. To test this hypothesis, the presence of
microsporidia in donor corneas from the eye bank was evaluated by the most sensitive
nucleotide amplification method currently available in the laboratory. The study objective
was to determine the potential infectivity of donor corneas leading to microsporidial
keratitis following keratoplasty.
7.3.2
Materials and Methods
Postmortem donor corneas were harvested with the consent of families from 53 subjects
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
148
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
by in situ excision by the Ramayamma International Eye Bank at the L.V.Prasad Eye
Institute, Hyderabad, India. These tissues were considered unsuitable for use as donor
tissue by the eye bank as they were of poor quality or were from patients who had died of
causes contraindicated as suitable donors (septicemia, multiple myeloma) by eye bank
association of India guidelines. The corneal tissue was minced and washed in PBS buffer
for DNA isolation using the ‘UNSET procedure’ (described in Chapter 6). Polymerase
chain reaction (PCR) was performed using pan-microsporidia primers which is capable
of amplifying a conserved region of the small-subunit rRNA of V. corneae, E. cuniculi, E.
hellem, and E. intestinalis (Conners et al. 2004). The PCR conditions included 1 minute
denaturation at 94°C, 2 minute annealing at 55°C and 3 minute extension at 72°C for 35
cycles. The DNA from all corneal tissues were additionally spiked with 1µl of E. hellem
(ATCC 50504) to rule out the presence of PCR inhibitors in the samples.
7.3.3
Results
Thirty seven out of 53 donors were male and sixteen were female. The mean age of the
donors was 65.5 ± 18.9 (range 25-104) years. The lower limit of detection for the primers
was determined to be 10pg when using genomic DNA as a template. The primers when
tested for analytical specificity, amplified DNA from all microsporidia species tested (V.
corneae, E. cuniculi, E. hellem, and E. intestinalis ) but did not amplify any of the
selected mammalian, bacterial, or viral DNA. Microsporidial DNA was not detected in
any of the samples tested. On the other hand none of the samples in this study showed
presence of any inhibitors and an expected amplification corresponding to ~270 bp was
observed in all cases after spiking them.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
149
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
7.3.4
Discussion
Corneas have transmitted rabies, hepatitis B, cytomegalovirus, herpes simplex virus,
bacteria, and fungi (Eastlund 1995). Over the past several years, improvements in donor
screening criteria, such as excluding potential donors with infection for HIV-1 and
hepatitis B and syphilis have greatly reduced the risk of such infections. There have been
few reports of additional danger of transplanting active HCV and HSV if critical
assessment of the graft prior to surgery is not carried out (Sengler et al. 2001). Therefore
it was tempting to know if donor screening for microspordia would help in elimination of
graft associated microsporidial keratitis. Traditional methods for the diagnosis of
microsporidia in clinical laboratories are still reliant on histochemical staining and light
microscopy (Franzen and Muller 1999). PCR-based diagnostic methods have become
increasingly popular for pathogen identification and are particularly suitable for those
microbial organisms that are hard to culture in vitro, morphologically similar, and present
in low infectious doses. The detection limit and specificity of the pan-microsporidia
primers used in our PCR assay is satisfactory and we consider the assay advantageous,
because it makes it possible to rapidly screen samples collected from corneal donors for
potential microsporidial contamination.
Consistent with the low prevalence, there was no case of microsporidial infection in any
of the donor corneas that were screened in this study. In conjunction with the established
quality control for corneal storage in eye banks, there is potential for these PCR assays to
aid in rapid screening of clinical specimens for microsporidial contamination and in the
diagnosis of ocular infections. Possible causes of primary graft failure could include poor
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
150
Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies,
Cytokine Responses and Assessment of Infectivity of Donor Corneas
surgical technique, poor donor material and poor eye banking technique, inadequate
storage in organ culture, or infection of the donor material. However, the surgery in these
two cases of microsporidial infection after penetrating keratoplasty were straightforward
and routine. A qualitative and quantitative assessment of the donor endothelium in those
cases indicated good quality and microbiology of the medium during culture and at issue
was sterile. However, it is not sure if microsporidia can establish latent infection in the
cornea and therefore be transmissible by corneal transplantation as they are known to be
commonly associated with immunocompromised patients (Franzen and Muller 1999).
Given that immunosuppression probably is the major predisposing factor for prolonged
microsporidiosis in patients after organ transplantation, patients with systemic
immunosuppression and AIDS may be at higher risk of acquiring unusual infections,
especially in situations with hygiene deficiencies.
Conclusions

Considering the fact that the prevalence of microsporidial keratitis in L. V. Prasad
Eye Institute is around 0.4% (Joseph et al. 2006b), the number of donor corneas
examined in this study is too small for any conclusive results.

However, the absence of microsporidial DNA in all the donor corneal tissues tested
rules out the risk for transmission of microsporidia by corneal transplantation.

At this point of time it is concluded that screening of donor corneas for presence of
microsporidia is not warranted.
Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular
and Proteome Approach
151