IM~uNoDIAGNOSIS OF WEITHALMIC CYSTICERCOSIS AND EVALUATION

OF TEAR AS A SPECIMEN FOR DIAGNOSIS OF OPHTHALMIC

cYSTICERCOSIS

CHAPTER - III

6.1. BACKGROUND

~iqposis of ophthaimic cysticercosis is usually based on either one or more parameters such

rls histopathological observations, imaging features and less commonly serological tests,

along with clinical as well as epidemiological profile of the patients (Cano, 2001). The

picture of a living intravitreous or subretinal Cysticercus is practically

pathognomonic. Symptoms usually manifest in a later stage of the infection and the eye may

be destroyed by the chronic inflammatory reaction (Hutton, 1976). The inflammatory

reaction is possibly mediated by the toxins or degenerated products from the parasite and is

usually accentuated on its death. Intravitreous or subretinal cysts usually lead to blindness

within three years to five years unless the parasite is surgically removed from the eye (Cano,

2001). The clinical manifestations of orbital or adnexal cysticercosis are entirely different

and depend on the location, size, relation to adjacent structures and stage of evolution of the

cyst. Orbital cysticercosis may also frequently manifest as subconjunctival abscess (Grover

and Puri, 1996).

Helrninth infection of the retina usually elicits a delayed type hypersensitivity reaction. In

ophthalmic cysticercosis, the absence of reaction in the anterior chamber may be due to the

free mobile nature of the cyst, as well as the lack of immune response to the antigens of

Cysticercus at the early stage (Sachdeva et al., 1995). Serological assays have been

developed for detection of either specific antigens or antibodies in serum and tear for

diagnosis of parasitic diseases of eye such as onchocerciasis and acanthamoebiasis (Ngu et

al., 1998, Hassan et al., 2001; Ayong et al., 2005). The reports are very few on serological

study of ophthalmic cysticercosis (Sachdeva et al., 1995; Grover and Puri, 1996; Sekhar and

Lemke, 1997; Menon et al., 2000; Bajaj and Pushker, 2002).

Cysticercosis of eye is initially asymptomatic but in the later stage, symptoms develop due to

increase in cyst size or destruction of eyeball by the chronic inflammatory reaction.

Diagnosis of ophthalmic cysticercosis, either intraocular or extra ocular, depends on history

of tapeworm infection, ophthalmoscopy, ultrasonography, CT scan, biopsy and less

commonly on laboratory tests (Cano, 2001). Ultrasonography alone is not useful to confirm

the diagnosis. Although ultrasonography may suggest a probable cystic mass, it always does

CHAPTER - ZII

provide a confirmed diagnosis in case of orbital cysts unlike the floating intraocular

Ophthalmoscopy may not be helphl in diagnosis of peripherally located parasite

(~ger-Lei te et al., 1985) and also in cases of severe inflammation (Kapoor et al., 1977).

Moreover, in the cases of sub conjunctival cysts sometimes, parasitic cysts appear like

dermolipoma (Cano, 200 1).

There are conflicting reports on the usehlness of serology in diagnosis of ophthalmic

cysticercosi~. The poor sensitivity of serum antibody detection tests in ocular infections may

be due to the privileged situation of eye with regard to immunology. It may be due to

avascularity of the cornea and lens and the physiological selectivity of blood-aqueous barrier

as well as absence of lymphatic channels within the globe itself. Complement fixation

antibody titers are often negative (Junior, 1949). ELISA tests are also found to be negative,

as the Cysticercus antigens do not always give rise to an immune response (Sachdeva, 1995).

This may be due to the lack of an immune response to the Cysticercus antigens of particular

stage of the parasite or due to anatomic location of the parasite.

Seropositivity was found to correlate with clinical profile of ophthalmic cysticercosis in

studies conducted by Sekhar and Lernke (19971, Menon et al. (2000) and Bajaj and Pushker

(2002). However, Sachdeva et al. (1995) and Grover and Puri (1996) did not observe

serology to be useful for diagnosii of ophthalmic cysticercosis. Hence, in the absence of a

usell imaging method and a useful serological test, laboratory diagnosis of ophthalmic

cysticercosis remains far from satisfactory. Thereby, management of both intraocular and

extra ocular cysticercosis still continues to pose a serious challenge.

.4lthough antigen and antibody detection assays in serum are widely used for diagnosis of

many parasitic diseases such as Chagas' disease (Freilij et al., 1987; Corral et al., 1996),

leishmaniasis (Kohanteb et al., 1987; Saha et al., 2005), filariasis (Hoti et al., 2002) and

schistosomiasis (Wang et al., 1999), the collection of the blood for serum is an invasive

procedure. The method requires technical expertise and disposable synnges and when

stringent conditions are not carried out; these methods are associated with the risk of

acquiring blood-borne infections such as hepatitis B virus and human immunodeficiency

CHAPTER - 111

The specimens collected by non-invasive methods could therefore be of immense

in the diagnosis of parasitic diseases.

Recently, there has been much interest in the collection of body fluids other than serum for

diagnosis of parasitic infections. The other body fluids include urine, saliva and tear (Parija,

1998). Of these specimens, tear is being evaluated as a specimen alternate to blood for the

diagnosis of many microbial infections of the eye.

The indirect haemagglutination test (IHA) has been used with a sensitivity of 68% for the

demonstration of antibody to herpes simplex virus (HSV) in tear (Krichevskaya et al., 1980).

Detection of anti-Chlamydial IgG in tear is reported to be usehl for diagnosis of chlamydia1

conjunctivitis (Haller et al., 1997). The indirect fluorescent antibody test is used for detection

of Mol-axellu bovis antibodies in bovine lacrimal secretions (Killinger et al., 1978). HSV-

specific secretory IgA is also detected in the tear for diagnosis of herpes simplex keratitis

(HSK) with a sensitivity of 20.28% (Pramod et al., 1999). It is suggested that specific IgA

immunoglobulin may play an important role in the protection of the eye by limiting the levels

of Gram-positive normal microbiota and defending against the more pathogenic

Staphylococc~~s allreus (Lan et al., 1997). Antigens have been demonstrated in tear specimen

for diagnosis of Hepatitis B infection in humans (Gastaud et al., 1989). Tear is also used as a

specimen for detection of varicella zoster virus DNA in patients with Ramsay Hunt

syndrome (Hiroshige et al., 2002).

Tear specimens have also been used for diagnosis of few parasitic infections in eye such as

onchocerciasis, toxoplasmosis and Acanthamoebu infection (Ngu et al., 1998; Meek et al.,

2000; Ayong et al., 2005).

In onchocerciasis, both parasite specific antigen and antibodies have been detected in tear

specimen. Besides tear, Onchocerca specific antigens were also demonstrated in other body

fluids like dermal fluid and urine, employing antibodies to 0. volvulus specific recombinant

proteins, Oncho-C27 and OvD3B for monitoring the efficacy of control programs (Ngu et

al., 1998).

CHAPTER - 111

Secrerov IgA is the predominant immunoglobulin present in tears that protects the ocular

surface against various antigens. In order to investigate mucosal immune responses directed

against Toxoplasma gondii, non-stimulated tears are studied where the avidity of anti-l:

oOHdij. IgA antibodies in tears are found to be similar to that of IgG antibodies in serum

(Meek et al., 2000). However, it is not known whether these frequently observed antibody

responses are the result of common mucosal immune responses against T. gondii or represent

the natural antibody repertoire. A quantitative investigation of equine tear and aqueous

humor immunoglobulins is done using normal horses and ponies as well as horses and ponies

infected with 0. cewicalis. The preferential elevation in IgG antibodies in tears precedes the

development of corneal opacities observed in the same horses (Glaze et al., 1984). Tear

samples are also used in the diagnosis of Acanthamoeba keratitis by demonstration of

Acanthamoeba DNA by PCR (Lehmann et al., 1998).

Till date, however, no reports are available on the use of tear as a specimen for diagnosis of

ophthalmic cysticercosis. So an attempt is made in the present study to detect specific

Cysticercz~s antigens as well as antibodies in tear specimens for diagnosis of ophthalmic

cysticercosis.

6.2. OBJECTIVES

The main objectives of this phase of the study are:

1. To evaluate ELISA using C. cellulosae somatic and ES antigen for demonstration of

antibodies in serum for diagnosis of ophthalmic cysticercosis.

2 . To evaluate ELISA using affinity purified polyclonal C. cellulosae somatic and ES

antibodies for demonstration of antigens in serum for diagnosis of ophthalmic

cysticercosis.

3. To detect antigens in tear by ELISA using affinity purified polyclonal C. cellulosae

somatic and ES antibodies

4. To detect IgA antibodies in tear by ELISA using C. cellulosae complete somatic

homogenate antigen and ES antigen

5. To demonstrate antigens and antibodies in serum and tear for the post treatment

follow-up of ophthalmic cysticercosis.

CHAPTER - 111

6.3. MATERIALS AND METHODS

Patients

The present study is conducted in the Department of Microbiology of Jawaharlal Institute of

postgraduate Medical Education and Research (JIPMER), Pondicherry. Samples are

from the patients with ophthalmic cysticercosis and patients with other diseases

(disease controls), healthy students and blood donors (healthy controls). The informed

consent is obtained from all human adult participants.

A detail history is taken and a thorough clinical examination is done in each case and the

clinical presentation noted as per the patient information profonna (Appendix-IT).

Information about the complications in eye and other symptoms like seizure, were collected

from the patient as well from close relatives. General information, such as the illness, family

history, personal history, eating habits, sanitation etc, is collected on a questionnaire as

followed in case of NCC patients. All the clinical examinations were performed by

ophthalmologists in the hospital.

A presumptive diagnosis of cysticercosis of eye and adnexa is made based on observation of

a cystic lesion in the orbit or adnexa by ophthalmoscopy, further substantiated by

ultrasonography of eye in the Department of Ophthalmology. Imaging of brain and spinal

cord (either CT scan or MRI) is performed to check for CNS involvement. A total of 40

patients with a presumptive diagnosis of ophthalmic cysticercosis were included in this study

for evaluation of immunodiagnosis. There were 23 males and 17 females and the age

between 5 years to 65 years.

Study groups

Group4 Confirmed cases of cysticercosis in eye (n=6): This group included the cases of

ophthalmic cysticercosis that are confirmed by the characteristic features of Cysticercus cyst

by biopsy (cystic structure lined by a thick fibrous wall and containing the parasite; high

Power view of the scolex of the parasite shows suckers, multiple papillary infoldings of the

tegument; a tough host-tissue adventitious capsule outside the cyst wall; surrounding

CHAPTER - III

inflmatory cells) or ophthalmoscopy (large cyst with a denser white area of protoscolex in

the vitreous or subretinal area; the extended scolex area with hooklets is birefrigent to

po];trized iight) or direct visualization (subconjunctival protruding cysts).

~ ~ o u p - l l : Suspected cases of cysticercosis in eye (n=34): This group included

subconjunctival cysts, the orbital cystic mass with echogenic spot on ultrasonography

suggestive of fluid filled parasitic cyst. In these cases the scolex is not clearly visualized by

ultrasonography.

Croup-III: Disease controls (n=19): This group included the cases with other diseases of eye

(such as, conjunctivitis, 9; orbital hydatid cyst, 6; non-infectious disorders of eye-

dermolipoma, 2; conjunctival abscess, 2).

Group-Y: Healthy controls (n=20): This group included healthy volunteer students and blood

donors.

Specimens

Serum

Five milliliters of venous blood is collected from all the cases of ophthalmic cysticercosis

(n=40) and controls (n=39) under aseptic precautions and is allowed to clot. The serum is

separated and stored in pair at - 2 0 ' ~ till use.

Tear

Tear sample of 600pL to 700pL volume is collected from the inferior forrnix from the cases

of ophthalmic cysticercosis and controls by using sterile glass capillaries (Becton Dikinson),

causing minimal irritation to the eye as per the procedure described by Meek et al. (2000).

For further processing, the tear is then blown into the pre-coated micro titer well by applying

positive pressure at the opposite end of the tube with the help of a micropipette.

CHAPTER - III

The C. ceiluiosae antigen

C. cellulosae somatic antigen

The C. cellulosae somatic antigen is prepared from naturally infected porcine cysts following

the procedure described by Sreenivasamurthy et al. (1999) as mentioned earlier (Chapter-I)

C. cellitlosue ES antigen

The ES antigen is prepared by in vitro culture of C. cellulosae (obtained from naturally

infected pigs) in RPMI-1640 medium following the procedure described by D'Souza et al.

(1999) as mentioned earlier (Chapter-I).

C, celiulosae polyclonal antibodies

Polyclonal C. cellulosae somatic antibody and ES antibody are prepared from hyperimmune

sera raised against Cysticernls somatic and ES antigens by affinity chromatography using

protein A-Sepharose CL 4B column (Bangalore Genei, India) following the protocol as

descried earlier (Chapter-I).

Immunodiagnosis of ophthalmic cysticercosis

ELISA for Detection of Cysticercus IgG and IgA antibodies in serum

The ELISA using C. cellulosae somatic antigen and C. cellulosae ES antigen is carried out

separately for detection of IgG antibodies in serum samples from cases of ophthalmic

cysticercosis and control subjects. ELISA using C. cellulosue somatic antigen and C.

celltrlosae ES antigen are performed in parallel to detect the level of IgA in serum samples

from patients with ophthalmic cysticercosis and controls.

The optimum antigen concentration for coating plates and serum dilution is standardized by

checkerboard titration as mentioned before (Chapter-I). All the test sera are analyzed at the

dilution (1 :200) in PBS-T.

ELISA using the somatic and ES antigen for detection of IgG antibodies consisted of

following steps:

CHAPTER - III

r dnfigen coating: Polyvinyl high binding microtiter ELISA plates (NUVC, New . . Zealand) are coated with 100 pL1well of appropriate dilution of the somatic or ES

antigen (1.0 pGIwell) in carbonate bicarbonate buffer pH 9.6 and then incubated

at 4 ' ~ undisturbed.

2. washing: The un-adsorbed antigen is removed by washing the plates with washing

buffer (sterile PBS 7.2 containing 0.1% Tween-20 (PBS-T) is used as the washing

buffer).

3. Blocking: The uncoated reactive sites in the wells are blocked by PBS 7.2 containing

2% BSA by incubating for 3 hours at 3 7 ' ~ .

4. Washing: Plates are washed 3 times with PBS-T as before.

j. Sample serum dilution and incz~bation: Appropriate dilution of the patient sera

(1:200) is prepared in PBS-T. 100 pL of each diluted serum is added in duplicate and

incubated for 1.5 hours at 3 7 ' ~ .

6. Washing: The plates are washed 3 times with PBS-T as before to remove unbound

antibodies in sample serum.

7. Secondary antibody (conjugate) incubation: Rabbit anti-human-IgG-HRP conjugated

secondary antibody (Bangalore Genei, India) is used as per the manufacturer's

instruction (1:2000) with PBS 7.2 containing Tween-20 (0.05%) and 100pL volume

is dispensed to all the wells and incubated for 0.5 hours at 3 7 ' ~ in dark.

8. Washing: Plates are washed 3 times with PBS-T as before to remove unbound

conjugate.

9. Plate development: Substrate solution is prepared freshly by adding 6 mg of OPD

(S.D. Fine Chemicals, India) in 10 ml of PBS containing 0.05% Tween-20 and 10 pL

H202 is added just before adding to the wells. 100pL volume of the substrate solution

per well is dispensed to all the wells and incubated for 15-20 minutes at 3 7 ' ~ in dark

for the development of optimum color.

10. Stop reaction: The reaction is stopped by adding 50 pL of 2N H2S04 per well. The

absorbance is recorded at 492 nrn using ELISA reader (Biorad, USA).

For every run a positive control and another negative control serum are used. IgA antibodies

ln the same batch of sera &om cases of ophthalmic cysticercosis and control subjects is

detected by ELISA following the procedure as mentioned above excepting that anti-human

I& conjugate (DAKO) is used in place of anti-human IgG antibody conjugate (Step 7).

CYAPTER - IIl

.~,tigerr capture ELISA (Ag-ELISA) for detection of Cysticercus antigen in serum

The &-ELISA is evaluated by using polyclonal C. cellulosae somatic antibody and C.

cr!/irIosae ES antibody for detection of antigens in serum from the cases of ophthalmic

cysticercosis and control subjects. The optimum concentration of capturing antibody for

coating plates, detecting antibody dilution and sample serum dilution are standardized by

&eckerboard titration as described before (Chapter-I).

Biefly, the wells of polystyrene microtiter plates are coated with 100pL of purified rabbit C.

cellulosae somatic and C. cellulosae ES antibodies (concentration as standardized, mentioned

in Chapter-I) diluted in carbonate buffer, pH 9.6, and incubated overnight at 37 '~ . The wells

are then blocked with 2% BSA in PBS, pH 7.2 for 90 minutes. After washing for 4 times

with PBS-T, 100 ~Liwell of the test and control sera are added followed by incubation for 60

minutes at 37 '~ . The plates are washed as in the previous step and rabbit anti sera (C.

cellulosae Somatic antibody and C. cellz~losae ES antibody) is added at 1:200 dilution and

incubated for 1 hour. The plates are washed and 100 pL/well of 1: 1000 diluted goat-anti-

rabbit-IgG-HRP conjugate is added followed by incubation at 3 7 ' ~ for 30 minutes. Then the

plates are washed as before and developed with substrate solution as described before.

ELISA for detection of Cysticercus IgG and IgA antibodies in tear

ELISA using C. cell~dosae somatic antigen and C. cellulosae ES antigen is performed to

detect IgA antibody in tear specimens from patients with ophthalmic cysticercosis and

controls. In parallel ELISA is also done to detect IgG antibody in same batch of tear samples

from patients with ophthalmic cysticercosis and controls.

ELISAfor detection ofIgA antibody in tear

The initial standardization is done for antibody detection in tear using samples from

confumed cases of ophthalmic cysticercosis and healthy control subjects. The procedure

followed is the same as mentioned earlier for detection of IgA antibody in serum fiom

ophthalmic cysticercosis patients except that tear is used instead of serum. Tear sample is

Processed soon after collection for which micro titer wells pre-coated with C. cellulosae

CHAPTER - I11

,,tigens are used. Pre-coated wells are obtained in-house for which microtiter strip wells

i , l ~ ~ : ~ C ) are coated with the C. cellulosae somatic and ES antigens as described earlier. After

blocking with BSA, the microtiter well strips are washed and preserved at - 2 0 ' ~ till use

(chapter-I).

ELISA using C. cellulosae somatic antigen for detection of IgA antibodies consisted of

following steps:

1. Pre-coated ~nicrotiter well strips: The pre-coated microtiter well strips are brought to

normal temperature before to apply sample tear fluid.

1. Sample tear (lardiluted) application and incubation: Undiluted tear (100 yL/well) is

added to microtiter wells that are pre-coated with C. cellulosae somatic antigen and

then incubated for 90 minutes at 3 7 ' ~ .

3. Washing: The microtiter well strip is washed 3 times with PBS-T to remove unbound

antibodies in sample tear.

4. Secondary antibody (conjugate) inczibation: Rabbit anti-human-IgA-HRP conjugated

secondary antibody (DAKO) is used as per the manufacturer's instruction for dilution

(1:2000) in PBS pH 7.2 containing Tween-20 (0.05%) and lOOyL volume is

dispensed to all the wells and incubated for 30 minutes at 3 7 ' ~ in dark.

5. Washing: The microtiter well strip is then washed 3 times with PBS-T as mentioned

before, to remove unbound conjugate without exposure to bright light.

6. Plate development: Substrate solution is prepared freshly by adding 6 mg of OPD

(S.D. Fine Chemicals, India) in 10 ml of PBS pH 7.2 containing 0.05% Tween-20

and 10 pL H202. 100pL volume of the substrate solution per well is dispensed to all

the wells and incubated for 15-20 minutes at 3 7 ' ~ in dark for the development of

optimum color.

7. Stop reaction: The reaction is stopped by adding 50pL of 2N HzS04 per well. The

absorbance is recorded at 492 nm using ELISA reader (Biorad).

The same ELISA is repeated by using ES antigens of C. cellulosae to detect IgA antibodies

in the same batch of tear samples from cases of ophthalmic cysticercosis and control

subjects.

CHAPTER - III

FLJS,.I for detection of IgG antibody in teuv u .

The same ELISA is repeated by using somatic and ES antigens of C. cellulosae to detect IgG

in the same batch of tear samples from cases of ophthalmic cysticercosis and

subjects. IgG in tear is detected by using rabbit-anti-human-IgG-HRP conjugate

(~an~alore-Genei, India).

.@!?LISA for detection of antigen in tear

The Ag-ELISA is evaluated by using polyclonal C. cellulosae somatic antibody and C.

cell~~losae ES antibody for detection of antigens secreted in tear from patients with

ophthalmic cysticercosis and control subjects.

The optimum concentrations of capturing antibody for coating plates and of detecting

antibody are standardized as mentioned previously for detection of antigen in serum/CSF

(Chapter-I).

For antigen detection in tear, the sample is processed soon after its collection for which micro

titer wells pre-coated with the capturing antibody (affinity purified C. cellulosae somatic

antibody or C. celltllosae ES antibody) are used. Pre-coated wells are obtained in-house as

mentioned earlier (Chapter-I) for which micro titer well strips (NUVC) are coated with the

affinity purified polyclonal C. Cellulosae somatic antibody or C. cellulosae ES antibody.

After blocking with BSA, the micro titer well strips are washed and preserved at - 2 0 ' ~ till

use.

The Ag-capture ELISA procedure, for detection of parasite antigens secreted in tear, consists

of following steps:

1. Pre-coated microtiter well strips: The pre-coated microtiter well strips are brought to

normal temperature before to apply sample tear fluid.

2. Sample tear: 100 pL/well of undiluted tear from the cases of ophthalmic cysticercosis

and controls are applied to the wells pre-coated with C. cellulosae somatic antibody

or C. cellulosae ES antibody followed by incubation for 60 minutes at 37 '~ .

3. Washing: The plates are washed 3 times with PBS-T to remove unbound materials

6-om the tear sample.

CHAPTER - III

4. Detecting antibody: Polyclonal C. cellulosae Somatic antibody or C. celltilosae ES

antibody is then used at appropriate dilution (1 :200) in PBS pH 7.2 in order to bind to

the specific antigens captured by the capturing antibodies in the plate.

j. Washing: The plates are washed 3 times with PBS-T as mentioned before to remove

unbound detecting antibodies.

6. Secondaly antibody (conjugate): Goat anti-rabbit-IgG-HRP conjugated secondary

antibody (Bangalore Genei, India) diluted 1 in 1000 with PBS 7.2 containing Tween-

20 (0.05%) is used as per the manufacturer's instruction. 100 yL volume is dispensed

to all the wells and incubated for 0.5 hours at 3 7 ' ~ in dark.

7. Washing: Plates are washed 3 times with PBS-T as mentioned before to remove

unbound conjugate.

8 . Plate development: Substrate solution is prepared freshly by adding 6 mg of OPD

(S.D. Fine Chemicals, India) in 10 ml of PBS pH 7.2 containing 0.05% Tween-20.

10pL Hz02 is added just before adding substrate solution to the wells. 100pL volume

of the substrate solution per well is dispensed to all the wells. The plates are

incubated for 15-20 minutes at 3 7 ' ~ in dark for the development of optimum color.

9. Stop reaction: The reaction is stopped by adding 50pL of 2N H2SO4 per well. The

absorbance is recorded at 492 nm.

Statistical analysis

Sensitivity, specificity, positive predictive value, negative predictive value, and efficiency of

the diagnostic methods are calculated as per the standard method (Park and Park, 2001). The

difference in mean OD values between cases with a definitive diagnosis of ophthalmic

cysticercosis (Group-I) and control groups (Group-I11 and IV) are tested for statistical

significance using Mann-Whitney test (as the assumptions of normality and homogeneity of

variance are not satisfied). "P-value" <0.05 is considered as significant.

Post treatment follow-up of patients with ophthalmic cysticercosis

Albendmole is given to all the patients diagnosed ophthalmic cysticercosis. The albendazole

is given in a dosage of 15mgJkg body weightlday divided in three doses for a period of ten

days (Escobedo et al., 1987). A total of 18 cases are examined following treatment up to 3

CHAPTER - III

monhs clinically. Of 18 cases 6 cases are from Group-I and 12 cases from Group-11. The tear

serum samples collected at the intervals of 1 week, 1 month and 3 months are tested for

crsticer.ells antigen and antibodies. The patients are monitored for the presence of IgG

antibody in serum, IgA antibody in tear, and antigen in both serum and tear during the follow

up period. Prednisolone (1 mdkg per day) is given in addition to albendazole in the treatment

of orbital cysticercosis (Tandon et al., 1998).

6.4. RESULTS

Clinical information

Site of localization in the eye and adnexa In the present series of cases of ophthalmic cysticercosis, the major sites of localization of C.

cellulosae cyst in the eye are presented in Table-38. In the present series, extra ocular muscle

is found to be the predominant site (13 cases) of Cysticercus infection, followed by

conjunctiva (10 cases) including one cases each in lacrymal fossa, upper formix, and bellow

canthus. Other sites included three cases in vitreous chamber; two cases each in optic nerve,

macular (subretinal), eyelid, and uvea. Figure-32 depicts the picture of few cases of

ophthalmic cysticercosis in the present series of cases where the cyst is located in various

ocular and extra ocular sites. Figure-33a and 33b are ultrasonographic pictures of

Cysticereus cyst in two cases of orbital cysticercosis where the protoscolex is distinctly

visible within the fluid filled cavity. Figure-34a shows a surgically excised cyst of C.

cellzrlosae. The cyst wall of the larva shows an intense florescence by the immuno

fluorescent assay (Figure-34b).

Table-38 Summary of the sites of infection with respect to laboratory diagnosis for ophthalmic cysticercosis

Conjunctiva

Optic nerve

Vitreous chamber

[* EO muscle: Extraocular muscle infestation of @ticereus cellulosae is called as orbital cysticercosis]

10 (25)

2 (5)

3 (7.5)

Eye lid

Uvea

Not specified

2 (5)

2 (5)

6 (15)

I

Multiple Cysticercus cysts in the conjunctiva Conjunctival cyst

A cyst in the right conjunctiva in the outer canthus Multiple nodular cysts in the bulbar cunjunctiva region

Fundoscopic view of a free floating Fundoscopic view of an intra

Cysticercus cyst in vitreous chamber ocular cyst in the subretinal site I

Orbiil cyst causing proptoeis in right eye A lateral view of the right eye with prptosis

1 Figure-32 : Pictures showing Cysticercus infection in var ious ocular and extraocular s i tes in few representative cases i n the present series w i th ophthalmic cysticercosis.

Courtesy : Department of Ophthalmology, JIPMER, Pondicherry.

A3 1

I

Figure-33 : Ultrasonography of orbit showing Cysticercus cyst. A and B: Ultrasonographic picture of two cases of cysticercosis showing mass with echogenic spot, the typical appearance of fluid filled parasitic cyst.

Figure-34 : A: A Cysticercus cellulosae cyst surgically excised from the conjunctiva of one patient; PS- proto scolex, CW- cyst wall; B: The imunofluorescent assay using Polyclonal rabbit C. cellulosae antiserum showing intense fluorescence of the cyst wall of the excised Cysticercus cyst.

CHAPTER - IIZ

Clittical symptoms

The major clinical signs and symptoms of ophthalmic cysticercosis in the present series are

in Table-39. Ocular discomfort is the major complain in 52.5% cases. Other

s)imptoms included blurring of vision in 11 (27.5%) patients, proptosis in 12 (30%) patients,

restriction in extraocular movement in 8 (20%) patients. Other less frequent presentations

included binocular diplopia in 7.5% cases, ptosis in 5% cases, optic neuritis in 5% cases,

peretinal fibrosis in 2.5% cases, necrotizing chorioretinitis in 2.5% cases, swelling in 15%

cases, headache in 20% cases, and loss of vision in 1 case.

Table-39 Summary of the clinical signs and symptoms with respect to laboratory diagnosis for ophthalmic cysticercosis

Signs and symptoms

Ocular d~scomfort Blumng of vis~on Loss of vision Preretinal fibrosis Binocular diplopla Optic neuritis Restnction of extraocular

ELISA for demonstration of Cysticercus IgG and IgA antibodies in serum

movement

The cut-off OD492 value of the ELISA for detection of IgA antibody in serum samples is

estimated from the OD492 values obtained from 20 normal sera (Table-40). The cut-off OD492

value of the ELISA for detection of IgG antibody in serum was as estimated before for

detection of antibody in setum/CSF for diagnosis of NCC.

Frequency (%) of total cases (n-40)

21 (52.5) 11 (27.5) 01 (2.5) 01 2.5) 03 (7.5) 02 (5)

08 (20)

Table-40 Determination of cut-off ODdg2 value for detection of IgA antibody in serum

I conjunctiva

I C. cellulosae somatic antigen I C. cellulosae ESantigen I

Signs and symptoms

Necrotizing chorioretinit~s Proptosis Ptosis Swelling Headache Exotropia Extrusions from sub . .

Frequency (%) of total cases (n=40)

01 (2.5) 12 (30) 02 (5) 06 (15) 08 (20) 01 (2.5)

02 ( 5 )

CHAPTER - III

CsSticercus IgG antibody in serum

The results of ELISA with C. cellulosae somatic antigen and ES antigen, for detection of

Cystjcercus IgG antibody in serum from the cases of ophthalmic cysticercosis and controls,

summarized in Table-41 a.

ELISA using C. cellulosae somatic antigen demonstrated specific IgG antibodies in sera

from 2 of 6 (33.33%) cases with definitive diagnosis of ophthalmic cysticercosis (Group-I).

ne assay also demonstrated specific IgG antibodies in 11 of 34 (32.35%) sera from

suspected cases (Group-11). The test detected antibodies in 3 of 19 (15.78%) sera from the

disease controls (Group 111) but did not show specific IgG antibody in the sera from healthy

controls (Group IV). The ELISA using C. cellulosae ES antigen demonstrated diagnostic

level of IgG antibodies in 4 of 6 (66.66%) cases with definitive diagnosis of ophthalmic

cysticercosis (Group-I). The assay also demonstrated a diagnostic level of antibodies in 14 of

34 (41.17%) sera from suspected cases (Group-11). The test detected antibodies in 2 of 19

(10.52%) sera from the disease controls (Group 111) but did not show any diagnostic

antibodies in the sera from healthy controls (Group IV) (Table-4la).

The sensitivity, specificity, PPV, NPV and efficiency of the ELISA using C. cellulosae

somatic antigen and C. cellulosae ES antigen for detection of Cysticercus IgG antibody in

serum are mentioned in Table-41a. The scatter plot showing the levels of IgG antibodies

demonstrated in serum from various study groups are shown in Figure-35.

TabIe4la Demonstration of Cysticercus IgG antibodies in serum from the cases of ophthalmic cysticercosis and controls by ELISA

CHAPTER - III

~ ~ m n s t r a t i o n of IgG antibodies in serum by QISA using C. cellulosae somatic antigens for diagnosis of ophthalmic cysticercosis

I 0 9 5 . Group-l Group-ll Group-Ill Group-N

I 065

Mean OD 0.209 0.195 0.0.144 0.128 0 7 5 Range 0.112-0.334 0.093-0.51 2 0.1 11-0.242 0.097-0.1 99

g o s s ? SD + 0 091 + 0.105 + 0.045 - + 0.03

$ 0 5 5 1

0 4 6 n 0 0 3 5 1 I.

I

0 GROUP-I, n=6 GROUP-II, n=34 GROUP-Ill, n=l9 GROUP-IV, 4.5

Study groups

Figure-35a: Scatter plots of the relative distribution of OD492 values in the ELISA using C. cellulosae somatic antigen for detection of IgG antibodies in serum fiom cases with ophthalmic cysticercosis and controls. The difference in mean OD492 values of the ELISA between Group-I cases and disease controls is not significant (p-0.156). It is statistically significant beheen Group-I cases and healthy controls (p=0.013). The p values are derived by Mann-Whitney test. n: Number of subjects.

Demonstration of IgG antibodies in serum by ELISA using C. cellulosae excretory secretory antigens for diagnosis of ophthalmic cysticercosis

Mean OD 0.233 Range 0.118-0.361 SD 2 0.099

Group-ll Group-Ill Group-iV . 0.315 0.0.167 0,124 0.102-0.892 0.1 17-0.383 0.104-0.192 i 0.246 +0.081 + 0.027 . . . .

GROUP-I, n=6 GROUP-11, n=34 GROUP-Ill, 11-19 GROUP-IV, 4.5 . --,.

Study groups

Figure35b: Scatter plots of the relative distribution of OD492 values in the ELISA using C. cellulosae ES antigen for detection of IgG antibodies in serum from cases with ophthalmic cysticercosis and controls. The difference in mean OD492 values of the ELISA between Group-I cases and disease controls is not significant @=0.106). It is statistically significant between Group-I cases and healthy controls (p-0.003). The p values are derived by Mann-Whitney test. n: Number of subjects.

CHAPTER - III

Cjsticercus IgA antibody in serum

The results of ELISA using C. cellulosae somatic antigen and ES antigen canied out for

detection of Cysticercus IgA antibody in serum from the cases of ophthalmic cysticercosis

and controls is summarized in Table-41b.

ELISA using C. cellulosae somatic antigen did not demonstrate a diagnostic level of

Qsticercus IgA antibody in serum from any of the cases in Group-I. However, the assay

demonstrated Cysticercus IgA antibody in serum fi-om only 2 of 34 suspected cases (Group-

11). The Cysticercus IgA antibody is not detected in sera from any of the control groups.

ELISA using C. cellulosae ES antigen demonstrated a diagnostic level of Cysticercus IgA

antibody in serum from only 1 of 6 cases in Group-I. The assay also demonstrated

Cysticercus IgA antibody in 4 of 34 sera from suspected cases (Group-11), and in 1 serum

sample from the disease control group (Table-4lb).

The sensitivity, specificity, PPV, NPV and efficiency of the ELISA using C. cellulosae

somatic antigen and C. cellt~losae ES antigen for detection of Cysticercz~s IgA antibody in

serum are mentioned in Table-41b.

Table-41b Demonstration of Cysticercus IgA antibodies in serum from the cases of ophthalmic cysticercosis and controls by ELISA

, , ~fher fhan cysticercosis as control; &oupni: Healthy control: PPV: ~oiit ive *redictive Galue; ~ ~ ~ : h ' e g a t i v e predictive value]

Study groups

I I1 111

The scatter plot showing the levels of IgA antibodies demonstrated in serum from various

study groups are shown in Figure-36.

I IV 7n i nn

Number of subjects

6 34 19

Number (%) of serum samples positive for IgA antibody by ELISA using C. cellulosae somatic antigen

0 2 (5.88) 0

C. cellulosae excretory secretory antigen 1 (16.66) 4 (1 1.76) 1 (5.26)

CHAPTER - III

Demonstration of IgA antibodies in serum by ELISA using C. cellulosae somatic antigens for diagnosis of ophthalmic cysticercosis

05 Group-l Group-ll Group-Ill Group-IV

0 45

I Mean OD 0.085 0.087 0.086 0.085 0 4 ! Range 0.049-0.1 52 0.041-0.317 0.052-0.132 0.041-0.158

0 35 SD 2 0.036 + 0.054 - + 0.024 2 0.034

I

0 4 o GROUP-I, n=6 GROUP-II, n=34 GROUP-Ill, n=19 G R O ~ - I ~ = ~ < ~

Study groups

Figure36a: Scatter plots showing the relative distribution of values in the ELISA using C. cellulosae somatic antigen for detection of IgA antibodies in serum from cases with ophthalmic cysticercosis and coni~ols. n: Number of subjects. The differences between the mean OD492 value of the Ag-ELISA in Group-I cases is compared to either disease controls or healthy controls are found to be not significant statistically as derived by Mann-Whitney test.

Demonstration of IgA antibodies in serum by €LISA using C. ceNulosae excretory secretory antigens for diagnosis of ophthalmic cysticercosis

0 J

o GROUP-I, n=6 GROUP-II, n=34 GROUP-Ill, n=19 GROUP-IV, n=2IJ:,

O'I I Group-l Group-ll Group-Ill Group-IV 0.45

0 4 I MeanOD 0.156 0.103 0.101 0.081 Range 0.084-0.264 0.042-0.231 0.041-0.197 0.042-0.1 64

035! SD - + 0.062 - + 0.05 + 0.045 + 0.037 E 0 3 1

Study groups

0.25

0 0 0.2 -

0.15 -

Figure-36b: Scatter plots showing the relative distribution of OD492 values in the ELISA using C. cellulosae ES antigen for detection of IgA antibodies in serum from cases with ophthalmic cysticercosis and controls. n: Number of subjects. The mean OD4,, value of the Ag-ELISA in Group-I cases is significantly different compared to either disease controls or healthy mntrols by Mann-Whitney test.

. 8' . Cut-off : 0.174

• ! !

CHAPTER - III

,igELISA for detection of Cysticercus antigens in serum

The results of Ag-ELISA carried out with C. cellzllosae somatic antibody and C. cellulosae

ES antibody for detection of Cysticercus antigens in serum from the cases of ophthalmic

cysticercosis and controls is summarized in Table-42.

able-42 Demonstration of Cysticercus antigens in serum samples from the cases of ophthalmic cysticercosis and controls by ELISA

Ag-ELISA usingpolyclonal C. cellulosae somatic antibody

The Ag-ELISA using polyclonal C. cellulosae somatic antibody demonstrated Cysticerczls

antigens in 3 of 6 (50%) serum samples of the cases with definitive diagnosis of ophthalmic

cysticercosis (Group-I). The assay also demonstrated the antigens in 8 of 34 (23.52%) sera

from suspected cases (Group-11). The test detected antigen in 3 of 19 (15.78%) sera from the

disease control subjects (Group 111) but did not show any Cysticercus antigens in sera from

healthy controls (Group IV).

/ Subject I Group

I I1 I11 IV

Ag-ELISA usingpolyclonal C. cellulosae ES antibody

Cysticercus antigens, in sera from 4 of 6 (66.66%) of the cases with definitive diagnosis of

[Groupl Confmed cases of ophthalmic cystlcercosis; Group-11: Suspected cases of ophthalmic cyshcercosis, Group111 Disease of eye other than cysticercosls as control; Group-N Healthy control; C cellulosae somatic ant~body: anhbody rased aga~nst somatlc anhgen; Excretory secretory antibody antibody rased against Excretory secretory antigen, PPV Pos~tive predichve value, NPV Negatlve predtchve value]

Number of subjects

6 34 19 20

Number (%) of sera positive for Cysticercus antigen by Ag-ELISA using

ophthalmic cysticercosis (Group-I), are demonstrated by the Ag-ELISA using polyclonal C.

cellulosae ES antibody. The assay demonstrated the antigens in sera from 13 of 34 (38.23%)

Sensitivity (%) Specificity (%I

PPV (%) NPV (%)

Efficiency (%)

C cellulosae somatlc antibody 3(50) 8 (23.52) 3 (15.78)

0 (0) 50

92.3 50

suspected cases (Group-11) and in sera from 2 of 19 (10.52%) the disease control subjects

(Group 111) but did not show any Cysticercus antigens in the serum sample of healthy

controls (Group IV). The sensitivity, specificity, PPV, NPV and efficiency of the Ag-ELISA

using C. cellulosae somatic antibody and C. cellulosae ES antibody for detection of

C cellulosae excretory secretory antlbody 4 (66.66) 13 (38.23) 2 (10.52) 0 (0)

66.66 94.87 66.66

92.3 94.87 86.66 91.11

CHAPTER - III

Cyfticercus antigen in serum are presented in Table-42. The scatter plot showing the level

the parasite antigen demonstrated in serum from various groups are shown in Figure37

Demonstration of antigens in serum by Ag-ELISA using C. cellulosae somatic antibody for diagnosis of ophthalmic cysticercosis

Group-l Group-ll Group-Ill Group-IV 095

Mean OD 0.197 0.188 0.136 0.119 00s Range 0.143-0.305 0.093-0 685 0.094-0 343 0 088-0.153

SD + 0 076 - + 0.145 2 0 056 - + 0.018

$ 0 7 , . N 065

8 P 055

9 m 3 045

> I 8 . 8 """ .

Cut-off : 0.166 . . 0 15 025 I : I i I 005 C -

o GROUP-I, n=6 GROUP-II, n=34 GROUP-Ill, n=19 GROUP-iV, n=20 4 5

Study groups

Figure-37a: Scatter plot showing the relative distribution of ODdg2 values in the Ag-ELISA using C. cellulosae somatic antibody for detection of antigens in serum from cases with ophthalmic cysticercosis and controls. The mean OD492 value of the Ag-ELISA in Group-I cases is significantly different compared to that in either disease controls (p=0.017) or healthy controls (p=0.003) derived by Mann-Rlitney test. n: Number of subjects.

Demonstration of antigens in serum by Ag-ELISA using C. cellulosae excretory secretory antibody for diagnosis of ophthalmic cysticercosis

1 Group-l Group-ll Group-Ill Group-IV 0 5 1 Mean OD 0.235 0.191 0.142 0.123

04, . Range 0.126-0.41 9 0.098-0.412 0.1350.247 0.085-0.1 62 SD + 0.106 - + 0.09 + 0.039 + 0.021

04

o GROUP-I, n=6 GROUP-11, n=34 GROUP-Ill, n=19 GROUP-IV, n=20 4.5

Study groups

R~igure-37b: Scatter plot showing the relative distribution of OD492 values in the Ag-ELISA using C. cellulosae ES antibody for detection of antigens in serum from cases with ophthalmic cysticercosis and controls. The mean OD4g2 value of the Ag- ELISA in Group-I cases is significantly different compared to that in either disease controls (p=0.025) or healthy controls (P'0.003) derived by Mann-Whitney test. n: Number of subjects.

CHAPTER - III

ELISA for detection of Cysticereus IgG and IgA antibodies in tear

The optimal conditions for detection of anti-Cysticercus IgG and anti-Cysticercus IgA

rntibodies are standardized by checkerboard titration and the diagnostic OD (cut-off) value

for the ELISA using somatic antigen and ES antigen are shown in Table-43. The undiluted

sample tear volume of 100pL/well is found to be optimal for both the assays.

Table-43 Standardization of antibody detection in tear by ELISA

The cut-off OD value for detection of Cysticerczls IgG antibody is found to be 0.108 and

0.1 19 using C. cellulosae somatic antigen and C. cellulosae ES antigen respectively. The cut-

off OD value of the ELISA using C. cellt~losue somatic antigen and C. cellulosae ES antigen

for detection of specific I@ antibody is found to be 0.121 and 0.1 13 respectively (Table-

44).

Table-44 Determination of cut-off titer for detection of antibody in tear

ELISA for detection of C. cellulosae IgG antibody in tear

0Ddg2 of nonnal tear samples

(n=20) Absorbance range Mean average OD

SD *Cut-off titer

'be results of ELISA carried out with C. cellulosae somatic antigen and ES antigen for detection of

Cysticercus IgG antibody in tear from the cases of ophthaImic cysticercosis and controls is

Summarized in Table-45a. The ELISA demonstrated a diagnostic IgG antibody level in tear from 1

suspected case using C. cellulosae somatic antigen and 1 disease control using C. cellulosae ES

antigen.

Standard deviahon, OD optical density; *Cut-off titer = Mean average OD + 2SD]

Detection of IgG antibody in tear using

Detection of IgA antibody in tear using

Som antigen 0.042-0.1 0.07435 0 016962 0.108274

Som antigen 0.024-0 104

0.07645 0.022552 0.121554

ES antigen 0 046-0.108

0.0849 0.017399 0.119698

ES antigen 0.049-0.1 0.08655 0.013481 0.113512

CHAPTER - IIZ

~ ~ b l ~ - 4 j a Demonstration of Cysticercus IgG antibodies in tear from the cases of ophthalmic cysticercosis and controls by ELISA

[Group-I: Confmed cases of ophthalmic cysticercosis; Group-11: Suspected cases of ophthalmic cysticercosis; GroupIII: Disease of eye other than cysticercosis as control; GroupN: Healthy control; PPV: Positive predictive value; NPV: Negative predictive value]

The scatter plot of the OD values of the ELISA for detection of IgG antibodies in tear from

various study groups is shown in Figure-38.

Detection of IgG in tear by ELISA using C. cellulosae Somatic antigen

, ~ e a n OD 0,074 0.068 0.075 0.073

Range 0.04211.103 0.0346.138 0.042-0.102 0.M2-0.1 SO 10.026 + 0.022 20.019 + 0.017

b

0.08 i I

: I i 0.03 -

I I

- 7

0 Groupl, n=6 Group-11, n=34 Group-Ill, n=19 Group-IV, n=20 2.5

Study groups

agure-38a: Scatter plots showing the relative distribution of OD492 values in the ELISA using C. cellulosae somatic antigen for detection of IgG antibodies in tear from cases with ophthalmic cysticercosis and controls. n: Number of subjects. The difference in mean OD492 values of the ELISA between Group1 cases and both disease controls (~~0.798) and healthy controls k0.879) are not significant. The p values are derived by Mann-Whitney test.

CHAPTER - III

Detection of IgG antibody in tear by ELISA using ES antigen

0.38 Group-l Group-ll Group-Ill Group-IV

0.33 1 Mean OD 0.105 0.089 0.088 0.086 Range 0.095-0.116 0.0456.1 12 0.062-0.127 0.061-0.108

o,28 1 SO '0.007 +0.017 + 0.019 10.013

0.03 -- - -- 4 Group-I, n=6 Groupll, n=34 Group-Ill, n=19 Group-IV, n=20

Study group

Figure-38b: Scatter plots showing the relative distribution of ODdg2 values in the ELISA using C. cellulosae ES antigen for detection of IgG antibodies in tear from cases with ophthalmic cysticercosis and controls. n: Number of subjects. The difference in mean OD492 values of the ELISA between Group-I cases and controls are not estimated since none of the cases are positive for the test.

ELISA for detection of C. cellulosae IgA antibody in tear

The results of ELISA using C, cellulosae somatic antigen and ES antigen carried out for

detection of Cysticercus IgA antibody in tear from the cases of ophthalmic cysticercosis and

controls is summarized in Table-45b. The assay demonstrated a diagnostic level of IgA

antibodies in tear sample from 3 of 6 (50%) cases with definitive diagnosis of ophthalmic

cysticercosis (Group-I) and in tear from 11 of 34 (32.35%) the clinically suspected cases of

ophthalmic cysticercosis (Group-11). The assay demonstrated a diagnostic level of IgA

antibodies only in tear from 2 of 19 (10.52%) disease controls (Group-111). However, the test

did not show any diagnostic antibodies in the tear from healthy controls (Group IV).

ELISA using C. cellulosae ES antigen demonstrated a diagnostic level of IgA antibodies in

all (100%) tear samples from all the cases with definitive diagnosis of ophthalmic

cysticercosis (Group-I). The assay also demonstrated a diagnostic level of IgA antibodies in

tear from 25 of 34 (73.52%) clinically suspected cases of ophthalmic cysticercosis (Group-

11). The assay demonstrated IgA antibodies non-specifically in tear from 3 of 19 (15.78%)

disease controls (Group-111). The test did not show any diagnostic antibodies in the tear from

CHAPTER - III

healthy controls (Group IV). The sensitivity, specificity, PPV, NPV and efficiency of the

ELISA carried out with C. celllllosae somatic antigen and ES antigen for detection of

Cr~ticercLa IgA antibodies in tear are summarized in Table-45b. The scatter plots of the

level (OD192) of IgA antibodies in tear from various study groups is shown in Figure-39.

~~ble-45b Demonstration of Cysticercus IgA antibodies in tear from the cases of ophthalmic cysticercosis and controls by ELISA

Detection of IgA antibody in tear by ELISA using C. cellulosae somatic antigen

Group-I Group-ll Group-Ill Group-IV

Mean OD 0.132 0.114 0.09 0.087 Range 0.127-0.221 0.052-0.334 0.063-0.139 0.063-0.1 1 SD 20.057 + 0.057 2 0.024 +0.012 .

0.03 2 Group-), n=6 Group-ll, n=34 Group-Ill, n=19 Group-14, n=20

Study grwps

'eye

Figure-39a: Scatter plots showing the relative distribution of OD492 values in the ELISA using C. cellulosae somatic antigen for detection of IgA antibodies in tear from cases with ophthalmic cysticercosis and controls. n: Number of subjects. The difference in mean OD492 values of the ELISA between Group-I cases and both disease controls (p=0.121) and healthy conQol~ ( ~ 0 . 1 7 6 ) are not significant. Thep values are derived by Mann-Whitney test.

CHAPTER - III

Detection of IgA antibody in tear by ELISA using C. ceilulosae ES antigen

0.73 -, Group-l Group-ll Group-Ill Group-IV

I Mean OD 0.256 0.307 0.098 0.087

0.63 i Range 0.129-0.408 , 0.097-0.631 0.0560.159 0.067-0.1 + 0.059 SD - i0.184 2 0.039 + 0.011 .

0.03 - - - 6 Group-I, n=6 Group-ll, n=34 Group-Ill, n=19 Group-IV, n=20 8 5

Study groups

Figure-39b: Scatter plots showing the relative distribution of OD492 values in the ELISA using C. cellulosae ES antigen for detection of IgA antibodies in tear from cases with ophthalmic cysticercosis and controls. n: Number of subjects. The mean ODdY2 value of the ELISA in Group-I cases is significantly different compared to either controls with other parasitic diseases (p=0.001) or healthy controls (p=O.OOl) derived by Mann-Whitney test.

Ag-ELISA for detection of antigen in tear

The undiluted sample tear volume of 100pl/well is found to be optimal for the assays. The

absorbance value at both the concentrations is given in Table-46.

Table-46 Standardization and estimation of the optimal dilution of human tear for detection of antigens in tear by ELISA

- OD,,: !.slues in the .%g-capwe ELIS.4 for detection of antigen in tear samples using - ---- -

1 .-

1 Tear sample / C cellt~lasae somatic antibody / C. cellz~losae excretory secretory antibody / from

Infected eye

The cut-off OD value of the ELISA using C. cellulosae somatic antibody and C. cellztlosae

ES antibody for detection of antigen in tear is found to be 0.154 and 0.147 respectively

(Table-47).

I I I I Normal eye I 0.134 0.173 0.124

50 pL

0.483

0.173

100 pL

1.030

50 yL

0.530

100 pL

1.038

CHAPTER - III

~ ~ b ] ~ - 4 7 Estimation of the cut-off for the ELISA for detection of Cysticercus antigens in tear

,@-ELISA using poly clonal C. cellulosae somatic antibody

The results of Ag-ELISA carried out with C. cellulosae somatic antibody, and C. cellulosae

ES antibody for detection of Cysticercus antigens in tear from the cases of ophthalmic

cysticercosis and controls is summarized in Table-48. The Ag-ELISA using affinity purified

C. cellulosae Somatic antibody demonstrated Cysticercus antigen in tear samples from 3 of 6

(50%) definitive cases of ophthalmic cysticercosis (Group-I). The assay also demonstrated

Cysticerctis antigen in tear samples from 8 of 34 (23.52%) suspected cases (Group-11). A

non-specific demonstration of Cysticercus antigen is observed in tear samples from 2 of 19

(10.52%) disease control subjects but not in any of the healthy control tear.

Ag-ELISA usingpolyclonal C. cellulosae ES antibody

The results of Ag-ELISA canied out using C. cellulosae ES antibody, and C. cellulosae ES

antibody for detection of specific antigens in tear from the cases of ophthalmic cysticercosis

and controls are summarized in Table-48.

Table-48 Detection of Cysticereus antigens in tear from cases of ophthalmic cysticercosis

. - - "- --,.,, ". "Y"YLY"YL' * III,II.I ",,"( Y."YY... UY.,pIW., ". VyYYYLIYI- V,I I1I".-"IU, Y."Yp-I... Y.>"WI "I -,I

Other than Cysticercosis as control; Group-IV: Healthy control; C. cellulosae somatic anhbody: antibody raised agmst somatic antigen; E x c ~ b ~ secretory antibody: antibody raised against ES antigen; PPV: Positive predictive value; NPV: Negative predictive value]

245

CHAPTER - III

Demonstration of antigens in tear by Ag-ELK3 using C. celiulosae somatic antibody

Group-l Group-ll Group-Ill Group-N 0 . 4 , Mean OD 0.166 0.153 0.127 0.105

1 Range J

0.0850.266 0.093-0.31 7 0.094-0.209 0.053-0.141 0 3 5 , SD + 0.072 2 0.072 2 0.028 + 0.024

I

I

005 C--- a -- - -

o Group-l Group-ll Group-ill ~ r o u p - 1 v - 7 ~ n=6 n=34 n=19 n=20

Study groups

Figure-40a: Scatter plots showing the relative distribution of OD491 values in the Ag-ELISA using C. cellulosae somatic antibody for detection of antigens in tear from cases with ophthalmic cysticercosis and controls. n: Number of subjects. The difference in mean values of the Ag-ELISA between Group-I cases and both disease controls @-0.4) and healthy controls (p0.108) are not significant. The p values are derived by Mann-Whitney test.

Demonstration of antigens in tear by Ag-ELISA using C. cellulosae ES antibody

Gmup-l Group-II Group-Ill Gmup-IV Mean OD 0.223 0.198 0.13 0.115 ' 1 F 7 g e

0 45 0.106-0 312 0.068-0.402 0.098-0.196 0.075-0.145

2 0.071 2 0.089 2 0.028 - + 0.016 0 4 .

E ~ . 035 .

3 . .

, 03 1 .

. . 005 1

o G~O~-IGE~E~FJ~~-GII~W 4 5

n=6 n=34 n=19 n=20 Study groups

%ure-40b: Scatter plots showing the relative distribution of values in the Ag-ELISA using C. cel[ulosae ES antibody for detection of antigens in tear from cases with ophthalmic cysticercosis and controls. n: Number of subjects. The mean OD192 value of the Ag-ELISA in Group-I cases is significantly different compared to either disease controls ( ~ 0 . 0 0 7 ) Or healthy controls ( ~ 0 . 0 0 3 ) derived by Mann-Whitney test.

CHAPTER - III

The &-ELISA using affinity purified C. cellulosae ES antibody demonstrated Cysticercus

antigen in tear samples from 5 of 6 (83.33%) definitive cases of ophthalmic cysticercosis

(G~OUP-I). The assay also demonstrated Cysticevcus antigen in 19 of 34 (55.88%) tear

from suspected cases (Group-11) and in tear samples from 2 of 19 (10.52%) disease

control subjects but not in any of the healthy control tear (Table-51). The scatter plots

showing the OD492 values of the Ag-ELISA for detection of parasite antigens in tear from

various study groups are shown in Figure-40.

post treatment follow up of patients with ophthalmic cysticercosis

Collapse of the cyst cavity and obscuration of the scolex, are progressively seen as the cyst

reduced in size by USG, on treatment with albendazole orally in patients with orbital

cysticercosis. Complete resolution of the cyst is seen in all cases after 4 months to 5 months.

The results of ELISA for detection of Cysticercus antigens and antibodies in serum and tear

during the follow up period are summarized in Table-49 and Tabled0 respectively.

Serum

Demonstration of IgG antibodies in sertlm

ELISA using C. cellulosae somatic antigen demonstrated a diagnostic level of IgG antibodies

in serum in 6 of 18 cases included in the followed-up study. The IgG-ELISA using somatic

antigen demonstrated antibodies in sera from 6, 8 and 9 cases at 1 week, 1 month and 3

months respectively after treatment. The ELISA using C. cellulosae ES antigen demonstrated

a diagnostic level of IgG antibodies in serum from 8 of 18 cases. The IgG-ELISA using ES

antigen demonstrated antibodies in sera from 9, 8 and 8 cases at 1 week, 1 month and 3

months respectively following treatment (Table-52).

Table-49 ELISA for detection of Cysticercus antigens and IgG antibodies in serum from cases of ophthalmic cysticercosis following treatment

CHAPTER - III

~ ~ ~ ~ ~ s t r a t i o n of antigens in serum

The ~g-ELISA using polyclonal C. cellulosae ES antibody detected antigens in sera from 7

of 18 cases before start of chemotherapy however, none of the follow up sera is observed to

be positive for the antigen after the treatment. The Ag-ELISA using polyclonal C. cellulosae

somatic antibody is found to be positive in sera from 3 cases before start of chemotherapy.

n e Ag-ELISA is found to be positive in sera fiom 7 cases and I case at 1 week and 1 month

respectively, following treatment. The Ag-ELISA is found to be negative for all the 18 cases

in the subsequent follow up at 3 months post treatment (Table-49).

Tear

Demonstrution of IgA antibodies in tear

The ELISA using C. cellulosae somatic antigen demonstrated a diagnostic level of IgA

antibodies in tear from 7 of 18 cases included in the follow up study. The IgA-ELISA using

somatic antigen demonstrated IgA antibodies in tear from 8 and 3 cases at 1 week and 1

month, respectively, after treatment. The ELISA using C. cellulosae somatic antigen tested

negative in all the follow up cases at 3 months following treatment. The IgA-ELISA using

ES antigen demonstrated IgA antibodies in tear from 13 cases of the total 18 cases before the

initiation of treatment. The ELISA using ES antigen detected IgA antibodies in tear from 9

cases at 1 week after treatment, but none of the cases in the subsequent follow up (Table-50).

Tabled0 ELISA for detection of Cysticercus antigens and IgA antibodies in tear from cases of ophthalmic cysticercosis following treatment

Uemonstration of antigens in tear

Durat~on of , follow up 1 cases (n=18) Before treatment

After 1 week After 1 month After 3 months -

The results of the Ag-ELISA using polyclonal C. cellulosae somatic and ES antibodies for

detection of antigens in tear are presented in Table-50. Before treatment by chemotherapy,

antigen is detected in tear from 1 I of 18 cases by ELISA using polyclonal C. cellulosae ES

antibody. The ELISA detected antigens in tear from 2 cases after 1 week following

beatment, however, none of the tear sample is observed to be positive for the antigen at 1

Number (%) of tear from follow-up cases of ophthalmic cysticercosis positive for ELISA for detection of IgA antlbody uslng ELISA for detection of ant~gen using

. Somatic antigen 7 (3 8.88) 8 (44.44) 3 (16.66)

0 (0)

Somatic anhbody 4 (22.22) 3 (16.66) 0 (0) 0 (0)

ES antigen 13 (72.22)

9 (50) 0 (0) 0 (0)

ES antibody 11 (61 11) 2(11.11)

0 (0) 0 (0)

CHAPTER - ZII

after the treatment. The Ag-ELISA using polyclonal C. cellulosae somatic antibody

detected antigen in tear specimen from 4 cases before start of chemotherapy. The Ag-ELISA

is found to be positive in tear from 3 cases at 1 week following treatment. The same Ag-

ELISA is found to be negative for all the 18 cases in the subsequent follow up at 3 months

post treatment.

6.5. DISCUSSION

Ophthalmic cysticercosis has been reported from different parts of India with variable

frequency. A few recently reported series of cases from India include 11 cases from

Pondicheny (Kaliaperumal et al., In Press), 25 cases from Vellore (David and Mathai, 2000),

44 from Chennai (Sharma et al., 2003), 35 cases in a more recent report from Chennai

(Sundaram et al., 2004), 18 from New Delhi (Chowdhury et al., 2003) and 43 from

Chandigarh (Mohan et al., 2005). A case of AIDS patient with subretinal cysticercosis has

also been reported £?om Chennai (George et al., 1999). Some earlier studies also described

many more cases of ocular cysticercosis from South India (Reddy et al., 1980; Bhaskaran et

al., 1978).

Intraocular and extraocular cysticercosis has also been reported from North India during last

many decades including recent reports from New Delhi and Uttar Pradesh (Aganval and

h t a v a , 2000; Verrna et al., 2002; Nainiwal et al., 2002; Sodhi et al., 2004; Pushker et al.,

2004; Sharma et al., 2004). Cases of intraocular and orbital cysticercosis have also been

reported from Bombay (Ursekar et al., 1998; Natarajan et al., 1999).

All these studies show an increased reporting of ophthalmic cysticercosis from different parts

of India including South India, which indicates that ophthalmic cysticercosis, is emerging as

a far commoner disease than previously considered.

CHAPTER - III

clinical information

in the present study the cysticerci are found in the orbit in one-third of cases (13 out of 40

cases). The conjunctiva and sub conjunctiva are the other major sites (25% cases) (Table-

38). The site of infection differs in different geographical locations; most common site of

localization being the posterior segment of the eye in most Western reports (Cano, 1989)

\~here as the ocular adnexa is the common site of localization in most Indian studies (Malik,

1968). Ocular cysticercosis involving both the anterior and posterior segments of the eye is

more frequent but anterior chamber cysticercosis is considered rare in India (Das et al.,

2002). No case of anterior chamber cysticercosis is recorded in the present series.

Orbital cysticercosis

.A total of 13 cases of orbital cysticercosis, constituting nearly one third of the total number

of cases of ophthalmic cysticercosis, were documented in the present study. The first

suspicion of the Cysticercus cyst in the orbit on ultrasonography is typically a cystic mass

with echo suggestive of the protoscolex within the fluid filled cyst. Reports from other parts

of India also document a high incidence of orbital cysticercosis (Pushkar et al., 2002; Mohan

et al., 2005). However, within the orbit, hydatid cysts are probably more common than the

extraorbital cysts such as the orbital cysticercosis (Grover and Puri, 1996; Sekhar et al.,

1996; Ursekar et al., 1998; Sekhar and Honavar, 1999; Pandey et al., 2000; Pandey et al.,

2001). The cases of orbital and adnexal cysticercosis are being found more frequently in

children and in young adults with no definite sex predilection (Pushkar et al., 2002).

Optic nerve cysticercosis

Only a few cases of optic nerve infection had been reported in the world literature, of whlch

most of the cases are from India (Madan et al., 199 1 ; Gurha et al., 1999; Tandon et d., 1998;

Betharia et al., 1999; Chandra et al., 1999; Gulliani et al., 2001; Menon et al., 2000; Bajaj et

a]., 2002; Bajaj and Pushker, 2002; Verma et al. 2002; Das et d. 2005).

In the present study optic nuritis is observed to be presented in 2 cases. The patients

Presented with pain, diminution of vision and proptosis. One of the two cases is diagnosed as

Optic nerve cysticercosis based on ultrasonography and radioimaging. Both the cases were

CHAPTER - I11

positive for Cysticercz~s antibody in serum by the ELISA test, only one of the two cases is

psitive for C'sticernls antibody in tear by ELISA. This is the first report of optic nerve

infection of Cysticercus from Pondicherry.

Recently, two cases of optic nerve cysticercosis are reported from New Delhi (Bajaj and

hshker, 2002). Gulliani et al. (2001) have reported a case of bilateral cysticercosis of the

optic nerve from New Delhi. An earlier case of optic nerve cysticercosis in a 50-year-old

woman with atypical optic neuritis is also reported from New Delhi based on CT and a

positive ELISA test (Menon et al., 2000). MRI findings revealed a case of optic nerve

cysticercosis in a fifteen-year-old female patient in New Delhi where the cyst was located in

the optic canal, beneath the sheath of the optic nerve (Gurha et al., 1999). Optic neuritis

following albendazole therapy due to disorganization of the cyst with inflammation of the

adjacent optic nerve has also been observed in Indian studies (Tandon et al., 1998).

0p)ithal~nic cysticercosis and associated cysts in the brain parenchyma

Cysticerci can lodge themselves in any part of the ocular and extra ocular tissue, but

associated brain parenchyma involvement is known to be quite rare. In the present study 2

out of 40 cases showed associated brain parenchymal cyst on CT. A recent study has reported

the association of orbital cysticercosis with multiple C. cellulosae cysts in the brain and

subcutaneous tissue (Pushker et d., 2005). In one study, two out of twenty patients had

associated cysts in the brain parenchyma (Pushker et al., 2001). Gulliani et al. (2001) have

observed a case of subretinal cyst present in one eye, which on MRI of the brain and orbit

revealed multiple cysticerci in the brain, orbit, and eye. Shekhar and Lemke (1997) from

Hyderabad had reported 1 out of 11 subjects of orbital cysticercosis with associated NCC.

Albendazole treatment is advised for the sub-conjunctival cysts or other orbital cases of

cysticercosis, even after surgical interventions, because of associated NCC that normally

detected by CT scanning of brain (Santos et al., 1984; Chandrashekhar and Lemke, 1997).

The high cost of the CT scan however may be a major economic constraint for use in all

patients. The ELISA test may be of diagnostic use if the scolex is not visible on CT scan or

echography.

CHAPTER - III

~jthough subcutaneous cysticerci are inconsequential, their diagnosis is important in the

dia@osis of more severe cases of NCC. The subcutaneous cysticerci may be confused with

other painless swellings such as lymphadenopathies, ne~uofibromas, and epidermoid cysts.

Gupta et al. (2000) from Haryana, India reported two cases of subcutaneous cysticercosis

involving the eyelid based on sonographic diagnosis. Both of them had hundreds of scattered

subcutaneous cysticerci. Such clustering of cysticerci is highly suggestive of CNS

involvement. But in the present series no such clinically detectable subcutaneous cysticerci

were reported.

The overall ocular discomfort was the major complaint (52.5%) of the patients with

ophthalmic cysticercosis in the present study (Table-39). Other clinical symptoms included

blurring of vision (27.5%), proptosis (30%), and restriction in extraocular movement in 20%

patients. Other less frequent presentations included binocular diplopia (7.5%), ptosis (5%),

optic neuritis (5%), preretinal fibrosis (2.5%), necrotizing chorioretinitis (2.5%), swelling

(15%), headache (20%), and loss of vision (2.5%).

Ocular motility disorder is observed to be the major clinical presentation (20% cases) in the

present series. This is because of relatively more number of the cases of orbital cysticercosis

(32.5% cases) documented in the present study. Of 13 orbital cysticercosis cases, eight cases

presented with ocular motility disorder. Seven cases of these eight cases tested positive for

Cysticerctrs antibody in either serum or tear or both by ELISA. Reports from other parts of

the country also highlight different presentations of orbital cysticercosis, which may lead to

the acquired ocular motility disorders (Pandey et al., 2001). From New Delhi, ten cases of

acquired motility disorders examined in one-year period were diagnosed as extraocular

muscle cysticercosis by CT. The inferior rectus muscle is most commonly affected with

double elevator palsy being the most common clinical presentation (Pandey et al., 2000).

Twenty-six consecutive patients in Hyderabad, India have been diagnosed as

myocysticercosis, based on imaging methods (Sekhar and Honavar, 1999). Cystic lesions of

the extraocular muscles resolving with steroids have been reported for the fmt time from a

retrospective study on six patients with a cystic lesion in an extraocular muscle, as observed

by CT scanning, by the same author (Sekhar et al., 1996).

CHAPTER - 111

in summary, in the present study, ocular motility disorder is observed to be the major clinical

pentation (20% cases) in the present series. This is because of relatively more number of

the cases of orbital cysticercosis (32.5% cases) documented in the present study. The study

also documents for the first time two cases of optic nerve cysticercosis from Pondicherry.

Immunodiagnosis of ophthalmic cysticercosis

Several serological assays such as the EITB, ELISA, dot-ELISA, LAT, CoA, IHA, and CIEP

have been developed and evaluated for detection of C'vticercus antigen and antibody in

serum for diagnosis of NCC in human with varying sensitivity and specificity (Garcia et al.,

2000; Shiguekawa et al., 2000; Barcelos et al., 2001; Mittal et al., 2001; Das et al., 2002;

Odashima et al., 2002; Promo-Narvaez et al., 2002; Domy et al., 2003; Fleury et al., 2003;

Garcia et al., 2003b; Parija and Sahu, 2003; Biswas et al., 2004; Espindola et al., 2005).

However, reports on serodiagnosis of ophthalmic cysticercosis are only few and restricted to

detection of antibody in serum only (Sachdeva et al., 1995; Grover and Puri, 1996; Sekhar

and Lemke, 1997; Menon et al., 2000; Bajaj and Pushker, 2002).

In the present study, the ELISA is able to detect Cysticereus antibodies in serum with

sensitivity in the range of 76.4% to 88.2% and specificity in the range of 96.96% to 97.47%

for diagnosis of NCC (Table-lob in Chapter-I). The ELISA for detec.tion of Cysticereus

antigens in serum showed a sensitivity of 29.4% to 44.11% and specificity of 95.95% as

mentioned earlier (Table-1Sb in Chapter-I). Therefore, in the present study, the ELISA is

evaluated for detection of Cysticereus antigens and antibodies in serum and tear samples for

serodiagnosis of ophthalmic cysticercosis

ELISA for detection of C. cellulosae antibody in serum

The ELISA detected C. cellulosae IgG antibodies in serum with a higher sensitivity than that

of the ELISA detecting C. cellulosae IgA antibodies in serum for diagnosis of ophthalmic

c~sticercosis. The ELISA using C. cellulosae somatic and ES antigens detected serum IgG

antibodies in 32.35% and 41.17% cases respectively in the suspected group (Group-11). The

ELISA showed a sensitivity of 33.33% and 66.66% and specificity of 92.3% and 94.87%

using somatic and ES antigens respectively for demons.tration of antibody in the serum

CHAPTER - III

(Tab1e-4la). A low sensitivity for demonstration of IgA antibodies compared to IgG

rntibodies in serum is reported earlier for diagnosis of NCC (Carpio et al., 1998).

The result of the present study shows that, the ELISA could detect C. cellulosae IgG

antibodies in serum with a comparatively higher sensitivity using ES antigens than somatic

antigens for diagnosis of ophthalmic cysticercosis, although cross reactivity is observed with

sera froin 3 and 2 control subjects using C. cellz~losae somatic antigen and ES antigen

respectively (Table-4la).

The IgA-ELISA using C. cellulosae somatic antigen detected IgA antibody in serum from 2

of 34 suspected case only. The ELISA using C. cellulosae ES antigen detected IgA antibody

in serum from 5 cases (including 1 confirmed case and 4 suspected cases) and 1 disease

control. The ELISA using somatic and ES antigens for detection of IgA antibodies in serum

showed a very low sensitivity (0% and 16.66% respectively) though the specificity of the test

is observed to be 100% and 97.43% (Table-4lb). Hence, the IgA antibody ELISA in serum

may not be useful for diagnosis of ophthalmic cysticercosis.

In the present study, the ELISA tests for detection of IgG antibodies in serum, shows lower

sensitivities in diagnosis of ophthalmic cysticercosis than the ELISA tests for detection of

IgG antibodies in serum for diagnosis of NCC (Chapter-I). Earlier serological studies on

ocular cysticercosis also revealed a similar very poor sensitivity (Sachdeva et al., 1995;

Grover and Puri, 1996).

ELISA for detection of C. cellulosae antigens in serum

In the present study, the ELISA using C, cellulosae somatic antibody shows 50% sensitivity

and 92.3% specificity whereas, the ELISA using C. cellulosae ES antibody shows 66.66%

sensitivity and 94.87% specificity for detection of antigen in the serum for the diagnosis of

ophthalmic cysticercosis. Low sensitivity of serum antigen detection methods in ophthalmic

onchocerciasis has also been reported in earlier studies (Vincent et al., 2000). Sensitivity and

Specificity of earlier technique like CoA detecting Cysticercus antigen in serum is observed

to be 57.1% and 95% in this laboratory. In this study, serum samples from 3 out of 20 (15%)

non-cysticer~al parasitic infection controls and serum samples from 1 out of 20 (5%) healthy

controls showed a false positive reaction for the antigen by the Co-A test (Parija and Reddy,

ln Press). In the present study, demonstration of somatic antigen in 3 and 7 cases of

confirmed and suspected (Group-I and 11) cases may be due to partial shading of cyst wall in

he later stage of the infection or even homology of somatic antigens with the ES substances

ofthe parasite. In the present study also as discussed earlier in Chapter-I, sensitivity of serum

antigen detection was low when compared with the sensitivity of serum antibody detection in

the cases of NCC.

ELISA for detection of C. cellulosae antibodies in tear

Demonstration of antibody in tear sample from cases of ophthalmic cysticercosis is not

reported earlier, although the same has been demonstrated in other diseases such as, IgA

antibodies in toxoplasmosis (Meek et al., 2000), and in herpes simplex keratitis (Pramod et

al., 1999); and IgG antibodies in 0. cervicalis (Glaze et al., 1984), herpes simplex virus

(Krichevskaya et al., 1980) Chlamydia (Haller et al., 1997) and Moraxella infections

(Killinger et al., 1978).

Results of the present study, for the first time documents the presence of IgA antibodies in

tear specimen from human with ophthalmic cysticercosis. The IgA antibody is detected in

tears from 50% cases of ocular cysticercosis by ELISA using Cysticercus somatic antigen but

in 100% cases using Cysticercus ES antigen. The ELISA using C. cellulosae somatic and ES

antigens for detection of IgA antibodies in tear showed a specificity of 94.87% and 92.3%

using C. cellulosae respectively (Table-45). The ELISA using somatic antigen and ES

antigen demonstrated false positive reactions by detecting IgA antibodies in tear from 2 and 3

control subjects with hydatid cysts of the eye respectively.

The presence of HSV-specific tear IgA is reported to be diagnostic in 20.28% of cases of

HSV infections (Pramod et al., 1999). In case of ocular toxoplasmosis, nonstimulated tears

and blood analyses showed an anti-l: gondii IgA response in tears from 81% of the

individuals tested, whereas only 23% had evidence of systemic immunity against the

Parasite. The avidity of the anti-T gondii IgA antibodies in tears were similar to the avidity

of serum IgG antibodies (Meek et al., 2000).

CHAPTER - III

the overall efficiency of the ELISA for detection of IgA antibodies in tear, the

asay using C. cellulosae ES antigens showed a higher test efficiency (93.33%) compared to

bat of the ELISA using C. cellulosae somatic antigens (88.88%). The PPV and the NPV of

the ELISA using C. cellulosae ES antigens are found to be 66.66% and 92.3% respectively,

while the PPV and the NPV of the ELISA using C. cellulosae somatic antigens are 60% and

92.5% respectively (Table-45b).

Higher sensitivity of the ELISA using C. cellulosue ES antigen for detection of IgA

antibodies, as demonstrated in the present study, may be due to an elevated production of

antibodies against ES antigens of the living parasite in the eye (Table-45b and Figure-39).

A positive ELISA using C. celltllosae somatic antigen for detection of IgA antibodies may be

due to the partial shedding of the cyst wall components of the live parasite that elicit an

increased IgA antibody level locally specific to the somatic antigen. When the sensitivity of

the ELISA for detection of IgA antibodies is compared with the sensitivity of the ELISA for

IgG antibodies, it is observed that the local secretory antibody level to the parasite antigens to

be higher than the serum IgG level using C. cellt~losue somatic and ES antigens (Table-41).

It may be due the anatomically compactness of the eye.

Although it is known that tear contains antibodies, very little is known about the antibody

repertoires in tears (Grus et al., 2001). In few studies, IgA has been found as the major

immunoglobulin in response to parasitic infection of eye as demonstrated in the cases of

ocular toxoplasmosis (Meek et al., 2000). Also it is observed that most plasma cells are

positive for secretory IgA in lacrimal drainage-associated lymphoid tissue, which comprises

the common mucosal secretory immune system (Knop and Knop, 200 1).

The result of the present study also revealed the predominant secretory IgA antibody level in

tear by ELISA using both somatic as well as ES antigens of C. cellulosae. In other studies,

IgG has been found to be the major immunoglobulin in tear in the diagnosis of chlamydia1

conjunctivitis (Haller et al., 1997) and in Moraxella bovis infection (Killinger et al., 1978).

The highest and most persistent M bovis antibody titers were of the immunoglobulin IgG,

followed by IgM and secretory IgA in bovine lacrimal secretions (Killinger et al., 1978).

In conclusion, in the present study, the sensitivities of the ELISA for detection of IgA

CHAPTER - 111

antibodies in tear specimen is observed to be higher than the sensitivities of the ELISA for

detection of IgG antibodies in serum using either somatic or ES antigens of C. cellulosae for

diagnosis of ophthalmic cysticercosis

ELISA for detection of C. cellulosae antigens in tear

III the present study, an attempt is made to detect the C. cellulosae released antigens in tear as

well as to identify the nature of the antigens of C. cellt~losae whether somatic or ES by using

affinity purified polyclonal antibodies.

The present study is the first study of the kind where affinity purified polyclonal antibodies

are used in the Ag-ELISA for detection of Cysticercus antigens in serum as well tear

specimens for the diagnosis of ophthalmic cysticercosis. Using affinity purified polyclonal

antibodies, the Ag-ELISA using C. cellulosae somatic and ES antigens detected antigens in

tear with sensitivity of 50% and 83.33% respectively. The Ag-ELISA detected antigens in

tear with specificity of 94.87% using either C. celltllosae somatic antibody or ES antibody

(Table-48).

By comparing the overall efficiency of the Ag-ELISA for detection of antigen in tear, the

ELISA using C. cel2ulosae ES antibodies shows a higher efficiency (93.33%) compared to

that using C. cellulosae somatic antibodies (88.88%). The PPV and the NPV of the Ag-

ELISA using C. cellzllosae ES antibodies are 71.42% and 97.36% respectively. The PPV and

the NPV of the Ag-ELISA using C. cellulosae somatic antibodies are 60% and 92.5%

respectively (Table-48). None of the healthy control tears samples (Group-IV) showed any

false positive reaction. However, the Ag-ELISA using C. cellulosae somatic and ES

antibodies detected antigen in tear from two patients with hydatid cyst of the orbit. It may be

due to true antigenic homology between C. cellulosae and E. granulosus.

Antigens are demonstrated in tear specimens for diagnosis of various other parasitic and viral

infections such as onchocerciasis (Ngu et al., 1998), hepatitis B (Gastaud et al., 1989) and

herpes simplex virus infection (Dunkel et al., 1988). Techniques like dipstick

CHAPTER - 111

immunobinding assay and transblot irnmunobinding assay are reported to show sensitivities

of 100% and 82.5% respectively for detection of antigen in tear for diagnosis of

onchocerciasis (Ngu et a]., 1998). In a more recent study a MAb based dipstick assay

developed and evaluated which detected antigen in tear with sensitivity of 92% and

specificity of 100% for diagnosis of onchocerciasis (Ayong et al., 2005). Hepatitis B virus

surface antigen has been detected in tear samples in 80% acute, 80% chronic and 41%

asymptomatic individuals infected with Hepatitis B virus (Gastaud et al., 1989).

The Ag-ELISA using polyclonal Cysticercz~s ES antibody shows a relatively higher

sensitivity for demonstration of antigens in tear (83.33%) than in serum (66.66%) for

diagnosis of ophthalmic cysticercosis, however, the specificity of the Ag-ELISA is

observed to be same (94.87%) for both the specimens (Table42 and Table-48). This is

possibly due to the lower quantity of Cysticercus ES antigen excreted in a large volume of

systemic circulation compared to the proximity of the ES antigens in locally secreted tear

fluid. However, when Cysticercus somatic antibody is used, the sensitivity of the ELISA is

observed to be same (50%) in both the specimens. The specificity of the ELISA using

Cysticercz[s somatic antibody is observed to be comparatively lower in serum (92.3%) than

in tear (94.87%) (Table-42 and Table-48). Thus the differences in sensitivity between

serum and tear antigen assays could be explained by a possible difference in the clearance

dynamics of this serum antigen due to the very recent nature of infection (Hafid et al.,

1995). It is also suggested that the sensitivity of the assay is not dependent on the intensity

of infection as reported in case of hydatid disease but also on the prevalence in the area

where the patient comes from (Corral et al., 1996).

In conclusion, results of the present study shows that Ag-ELISA using affinity purified

polyclonal antibo&es, particularly, Cysticercus ES antibody, is a sensitive (83.33%) and

specific (94.87%) method to detect Cysticercus antigen in tear for the diagnosis of

ophthalmic cysticercosis. The Ag-ELISA using Cysticercus somatic antibody is also useful

for the diagnosis of cases since the specificity of the test is high (94.87%).

CHAPTER - III

post treatment follow-up of ophthalmic cysticercosis

Medical therapy is the recommended mode of treatment for the cases of extraocular and

retro-orbital cysticercosis (Pandey et al., 2000). In the present study, the albendazole therapy

is found to be effective in patients with orbital cysticercosis. Reports of few earlier Indian

studies have also shown the promising results in treatment of extraocular cysticercosis with

oral albendazole (Sihota and Honavar, 1994). The orbit with a better blood supply may prove

to be more amenable to medical therapy. But medical therapy in intraocular cysticercosis has

been observed to be a failure due to the drug producing a toxic but reversible effect on the

parasite, possibly due to insufficient concentration of the drug due to the blood ocular barrier

(Santos et al., 1984). Antihelminthics may destroy the eye in 80% of cases of intravitreous or

subretinal cysticerci because of endotoxins produced by dying cysticerci within the globe

(Santos et al., 1984). Hence, the most effective means recognized for preserving function in

an eye with intravitreous or subretinal cysticerci is found to be surgical removal of the

Cysticel-cus larva (Bartholomew, 1975). Surgical removal is also recommended for

subconjunctival and eyelid cysts (Sen, 1980; Gupta et al., 2002). However, surgery is noted

to be a fraught with inevitable postoperative fibrotic response, which may result in restrictive

ocular motility disorder (Hutton, 1976).

The post treatment follow up of ophthalmic cysticercosis is usually done by monitoring the

cases clinically based on physical examination, ophthalmoscopy or imaging (Sihota and

Honavar, 1994; Sekhar and Honavar, 1999; Puri and Grover, 2001; Mohan et al., 2005). No

information is available on serological monitoring of the cases of ophthalmic cysticercosis

following therapy. Nevertheless, serological monitoring has been found to be useful in

monitoring the cases of NCC following treatment, by detection of both antigen (Estrada and

Khun, 1985; Tellez-Giron et al., 1987; Corea et al., 1989; Estrada et al., 1989; Harrison et al.,

1989; Choromanski et al., 1990; Chen et al., 1991; Garcia et al., 2000) as well as antibodies

(Nguekam et al., 2003; Garcia et al., 2000). A monoclonal antibody-based parasite antigen

detection ELISA that specifically detects the products of living cysticerci in human serum

showed a sensitivity of 85%, and a specificity of 92% (Garcia et al., 2000). The utility of

serological monitoring of NCC is also discussed earlier in the present study (Chapter-I).

CHAPTER - 111

in the present study, serological follow up of the medically treated cases of ophthalmic

c4isticerc~~is patients is carried out by detection of C. cellz~losae IgG antibodies in serum and

antibodies in tear specimen by ELISA. Also Cysticercus antigens in both serum and tear - specimens are demonstrated in this follow-up study.

C. cellulosae IgG antibodies and C. cellulosae antigens in serum

Antibody detection assays, although sufficiently sensitive, are usually not used in follow up

studies of parasitic diseases after treatment since they are unable to distinguish between

active and past infections. In the other hand antigen detection assays are increasingly used in

follow up studies of parasitic diseases such as onchocerciasis (Ngu et al., 1998), cystic

echinococcosis (Devi and Parija, 2000; Ravinder et al., 1997) and schstosomiasis (Nibbeling

et al., 1998) following treatment, even though these may not be sufficiently sensitive and

specific or are laborious and time consuming (Vincent et al., 2000). The antigen detection

ELISA has been reported to be sensitive and useful in post treatment follow up of NCC

(Garcia et al., 2000; Nguekam et al., 2003)

In the present study, ELISA using C. cellulosae ES antibody did not detect antigens in serum

from any of the 18 patients in the follow up study up to 3 months. However, ELISA using C.

cellulosae somatic antibody detected antigens in serum from 7 cases at 1 week and then only

in 1 case at 1 month but none at 3 months of post treatment (Table-49 and Tabledo).

Disappearance of ES antigens in serum after a week time demonstrates the possible efficacy

of the therapy and thus it may be used as a marker following treatment. Nevertheless,

demonstration of antigens by ELISA-using C. cellulosae somatic antibody may also be

considered informative about the treatment efficacy. The detection of this antigen in serum

persists for a longer period compared to the ES antigens, which may be due to continued

release of the Cysticercus degenerated components (Chavaria et al., 2003).

C. cellulosae IgA antibodies and C. cellulosae antigens in tear

Reports are lacking on the use of serological monitoring of the cases of ophthalmic

cysticercosis by demonstration of antibodies and antigens in tear, following treatment by

CHAPTER - III

chemotherapy or surgery. In the present study, detection of IgA antibody in tear during post

treatment follow up of ophthalmic cysticercosis cases for the first time shows a correlation

with the efficacy of the albendazole treatment.

The ELISA using C. cellulosae somatic antigen detected IgA antibodies in tear with an

increased frequency at 1 week after the start of treatment compared to that of pre-treatment

(Table-50). In this study, 7 of 18 follow-up cases were positive for IgA antibody in tear by

ELISA using C. cellz~losae somatic antigen before the start of treatment. Three of these 7

cases appeared negative for the test at one week following treatment. At 1 week post

treatment, tear IgA antibody is demonstrated in 5 of 11 cases, which were negative for the

test before the start of treatment. This may be due to release of degenerated antigenic

components of C. cellulosae after the start of chemotherapy, which possibly stimulated for

the production of an elevated the level of secreted IgA antibodies specific to the released

somatic components in tear. During subsequent follow up, the frequency of serum samples

positive for IgA antibody by ELISA is observed to be only 3 at 1 month but none at 3 months

(Table-50). The IgA antibody is demonstrated by ELISA using C. cellulosae ES antigen in

tear from 9 cases only up to 1 week but none at 1 month and 3 months post treatment. This

observation may indicate the possible antigenic clearance over a 3 months period, which is

further authenticated by the observation of the disappearance of Cysticereus antigens in both

serum and tear.

In the present study, disappearance of ES antigens, both in serum and tear, at 1 week post

treatment is suggestive of efficacy of the therapy and thus it may be needed to follow up

patients as a marker of viability of the parasite in eye. The demonstration of antigens in tear

by ELISA using C. ceZluIosae somatic antibody is also useful in treatment monitoring where

these antigens are also detected up to one week following treatment.

CHAPTER - III

7.6. SUMMARY

The present study reports the largest series of cases of ophthalmic cysticercosis diagnosed

from Pondicheny, a southern state of India. Ocular motility disorder is observed to be the

major clinical presentation (20% cases) in the present series. This is because of relativeIy

more number of the cases of orbital cysticercosis (32.5% cases) documented in the present

study. The study also documents for the first time the case of optic nerve cysticercosis from

Pondicheny.

The ELISA using C. cellulosae somatic and ES antigens is evaluated for the first time for

diagnosis of ophthalmic cysticercosis.

The ELISA showed a sensitivity of 33.33% and 66.66% and specificity of 92.3% and 94.87%

using somatic and ES antigens respectively for demonstration of antibody in the serum. The

ELISA using C. cellzdosae somatic and ES antigens detected serum IgG antibodies in

32.35% and 41.17% cases respectively in the suspected group (Group-11).

The ELISA using C. cellulosae somatic and ES antigens for detection of IgA antibodies in

senun showed a very low sensitivity (0% and 16.66% respectively) though the specificity of

the test is observed to be 100% and 97.43%. In the present study, the ELISA tests for

detection of IgG antibodies in serum, shows lower sensitivities in diagnosis of ophthalmic

cysticercosis than the ELISA tests for detection of IgG antibodies in serum for diagnosis of

NCC (Chapter-I).

The Ag-ELISA using C. cellulosae somatic antibody shows 50% sensitivity and 92.3%

specificity whereas, the ELISA using C, cellulo$ae ES antibody shows 66.66% sensitivity

and 94.87% specificity for detection of antigen in the s e m for the diagnosis of ophthalmic

~ysticerwsis.

CHAPTER - III

In the present study, for the fist time, tear is used as a specimen alternate to serum for

diagnosis and post treatment monitoring of ophthalmic cysticercosis.

The present study, for the first time documents the presence of locally secreted IgA

antibodies in tear specimen from human with ophthalmic cysticercosis. The sensitivity of the

ELISA using both somatic and ES antigens for demonstration of local secretory IgA

antibodies in tear specimen is observed to be higher than the sensitivity of the ELISA for

demonstration of IgG antibodies in serum.

The sensitivities of the ELISA for detection of IgA antibodies in tear specimen is observed to

be higher than the sensitivities of the ELISA for detection of IgG antibodies in serum using

either somatic or ES antigens of C. cellz~losae for diagnosis of ophthalmic cysticercosis. The

ELISA using C. cellulosae ES antigens for detection of IgA antibodies in tear shows a higher

diagnostic efficiency (93.33%) compared to that of the ELISA using C. cellzrlosae somatic

antigens (88.88%). The PPV and the NPV of the ELISA using C. cellulosae ES antigens are

found to be 66.66% and 92.3%, while the PPV and the NPV of the ELISA using C.

cellulosae somatic antigens are 60% and 92.5% respectively for detection of IgA antibodies

in tear.

Results of the present study shows that Ag-ELISA using affinity purified polyclonal

antibodies, particularly, Cysticercus ES antibody, is a sensitive (83.33%) and specific

(94.87%) method to detect Cysticercus antigen in tear for the diagnosis of ophthalmic

cysticercosis. The Ag-ELISA using Cysticercus somatic antibody is also usehl for the

diagnosis of cases since the specificity of the test is high (94.87%).

In the present study, ELISA using C. cellulosae ES antibody did not detect antigens in serum

from any of the 18 patients with ophthalmic cysticercosis in the follow-up study upto 3

months. The ELISA using C. cellubsae somatic antibody detected antigens in serum from 7

cases at 1 week and then only in 1 case at 1 month but none at 3 months of post treatment.

The demonstration of somatic antigens in serum persists for a longer period compared to the

CHAPTER - III

ES antigen, which may be due to continued release of the Cysticercus degenerated somatic

components.

~n the present study, disappearance of ES antigens, both in serum and tear after a week time

is suggestive of efficacy of the therapy and thus it may be needed to follow up patients as a

marker of viability of the parasite in eye. The demonstration of antigens in tear by ELISA

using C. cellulosae somatic antibody is also useful in monitoring the treatment where these

antigens are also detected upto one week following treatment.