Molecular added in 400 µl of blood in

Molecular identification

DNA isolation from blood samples

DNA
isolation from whole blood samples were carried out as per protocol of Martins et al. (2008) with some modifications.
Briefly, 1 ml of RBC lysis buffer (0.22% NaCl, 0.015% Saponin, 1mM EDTA; pH
7.5) was added in 400 µl of blood in a 1.5 ml microcentrifuge tube. The
contents were properly mixed and centrifuged at 10000 rpm for 3 minutes (Remi,
India). The supernatant was discarded and the pellet was suspended in 750 µl of
RBC lysis buffer. The contents were properly mixed and centrifuged at 10000 rpm
for 3 minutes. The supernatant was discarded and the procedure was repeated
until the pellet was clear of haemoglobin. After obtaining a clear pellet, it
was resuspended in 100 µl of PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.0,
0.5% Tween-20 and 100 µg proteinase K/ ml). The tube was then incubated in a
water bath at 56oC for 2 hours and then stored at -20?C to be used
as DNA template.

Polymerase Chain Reaction

Oligonucleotide Primers

For
molecular identification of cysticercosis, conventional PCR was targeted
against the large subunit rRNA gene (LSU rRNA) of Taenia solium using the primers TBR-3
(5?-GGCTTGTTTGAATGGTTTGACG-3?; positions 34–55) as forward and TBR-6
(5?-GCTACTACACCTAAATTCTAACC-3?; positions 319–297) as reverse (Jardim et al., 2006; Sreedevi et al., 2012). The oligonucleotide
primers were synthesized at Eurofins Genomics India Pvt. Ltd. (Bengaluru,
India).

Cycling conditions

Amplification
reactions were carried out taking 3 ?l of DNA template, 10 pM of each TBR
primer (forward and reverse) and 2X Master Mix (Promega) in a volume of 25 ?l
in 0.2 ml PCR tubes in a thermal cycler (Applied Biosystems, USA) with the
cycling conditions as initial denaturation at 94?C for 2 minutes followed by 40
cycles of denaturation (30 s at 94°C), annealing (30 s at 59°C), and extension
(60 s at 72°C), plus one cycle of final extension of 5 min at 72°C.
Subsequently, PCR-amplified products were electrophoresed on 1.5 % agarose
gels.

Results

In
our study, maximum number of patients 11 (42.30%) belonged to 15-29 years age
group followed by 30-44 year age group 10 (38.46 %) with a mean age of 26.84
± 11.62 years.  Majority of participants 19
(73.07%) were males (table 1) and the ratio of male to female was 2.71:1. In accordance with the food habits, 38.46%
patients were vegetarian while 61.54 % had food habit of taking non-vegetarian
diet.

Majority
of the lesions were of calcified granular type (13 cases) followed by ring
enhancing lesions (6 cases), granular nodular lesions (4 cases) and calcified
spots (3 cases) (table 2). As far as the distribution of the lesions were
concerned, left parietal lobe was found to be the most common site of the
lesions (53.84%), followed by left frontal (15.38%),  right frontal (11.53%) and right parietal
lobe (7.69%) of the brain (table 3).

The diagnostic OD value was decided to be
0.0798 ± 0.0073, 0.0805 ± 0.0132 and 0.0788 ± 0.0101 by indirect IgG-ELISA for
scolex antigen (SA), excretory secretory antigen (ESA) and membrane body
antigen (MBA) respectively as calculated by taking 10 negative human sera
samples collected from volunteers having no neurological symptoms. A positive
ELISA result was defined as two standard deviations above the mean OD value of
confirmed known negative sera. After applying statistical
analysis, 10 patients were found to be seropositive for scolex antigen (SA) figure
1, 8 for excretory secretory antigen (ESA) figure 2 and 4 for membrane body
antigen (MBA) figure 3 revealing a seroprevalence of 38.46%, 30.76% and
15.38% respectively against their respective antigens. In addition, samples
from 12 epileptic patients turned seronegative against any of the
antigens.  Three patients were
seropositive for all the three antigens, five for both SA & ESA, three for
both SA & MBA while three patients were seropositive for ESA & MBA.
Seroprevalence of neurocysticercosis among epileptic patients (n=26) by
considering positivity against any one antigen was found out to be 53.85% (14
out of 26). No such correlations could be observed taking gender and age
distribution with the IgG-ELISA positivity against any of the antigens (table
4).

The
patients on CT scan/ MRI showed various types of lesions like granular nodular
lesion 4 cases (15.38%), calcified granular lesion 13 cases (50.0 %), ring
enhancing lesion 6 cases (23.07 %) and calcified spots 3 cases (11.53 %)
which is shown in table 2 along with the seropositivity against antigens. Amongst
the patients with calcified granular lesions, 46.15% had anti-cysticercus
antibodies against SA whereas 38.46% against ESA; and with ring enhancing
lesions, the seroreactivity against SA and ESA were found to be in equal
proportions (table 2). Based on CT and/or MRI findings, 19 (73.07%) epileptic
patients had solitary brain lesion while 7 (26.93%) patients had multiple
lesions out of a total 26 patients recruited in this study (Table 5). The
patients with solitary brain lesion were found to be more seroreactive against
SA (42.14%) while those with multiple brain lesions were found to be more seroreactive
against ESA (57.14%) (table 5). The association between seropositivity for NCC
by IgG- ELISA, and the number of lesions in brain was found to be statistically
non-significant in case of any of the antigens employed for serodiagnosis since
the P value was found out to be 0.7150, 0.2837 and 0.9432 in case of SA, ESA
and MBA respectively.

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