Biological Forum – An International Journal 7(1): 1742-1749(2015)

ISSN No. (Print): 0975-1130

ISSN No. (Online): 2249-3239

Seroprevalence of HIV among Hospital Based Patients around

Indore with Research Recommendations
Jaipal Singh Vishwakarma*, Shubham Bhawsar* and Kripal Singh Vishwakarma**
*
School of study in Zoology, Vikram University Ujjain, (MP), INDIA
**
Department of Environmental Sciences & Limnology,
Barkatullah University, Bhopal, (MP), INDIA
(Corresponding author: Kripal Singh Vishwakarma)
(Received 27 April, 2015, Accepted 07 June, 2015)
(Published by Research Trend, Website: www.researchtrend.net)
ABSTRACT: Several useful reviews on the epidemiology of AIDS have been published. Our studies are
focused on seroprevalence of HIV among hospital based patient in and around Indore. This study was
conducted in the Department of Microbiology, Central Lab Indore, located at Yashvant Plaza in front of
Railway station, Indore, with its associated CHL hospital (CHL). CHL is an 800 bedded multi especially
tertiary care hospital, located at Nehru Nagar Indore, providing health care services to both rural and urban
population of nearly 80 lakh, in and around Indore. A total number of 32534 patients of both gender, visiting
the out patients department of Microbiology Central Lab, Indore for sacking medical treatment during the
period of four year, that is, from April 2009 to March 2012, were included in the present study. These studies
will check out the sensitivity and specificity of ALISA compared to Rapid tests. Research also include various
data like retrospective analysis of HIV seropositivity data during April 2008-09 to March 2011-12, revealed
that seropositivity is increasing (doubled up), in this geographical area. Studies conclude some
recommendations’ like educating young person’s about HIV before they begin engaging in behaviours that
place them at risk for HIV infection at school and collage levels. Studies lighted graphically on distribution of
HIV according to age and gender wise, hence it will help to make policies, programmes etc. to government for
citizens.
Key words: Hospital, Seroprevalence, HIV, AIDS
INTRODUCTION patients with AIDS.
In the beginning AIDS was recognized in Africa MATERIAL AND METHODS
through the study of patients with the disease,
A total number of 32534 patients of both gender,
(Clumeck et al., (1983), Offenstadt et al., (1983),
visiting the out patients department of Microbiology
Clumeck (1984), Perre et al., (1984), Piot et al. (1984))
Central Lab, Indore for sicking medical treatment
and the subsequent documentation of infection with the
during the period of four year, that is , from April 2009
human immunodeficiency virus (HIV) (Bayley et al.,
to March 2012, were included in the present study.
(1985), Clumeck et al., (1985). Epidemiologic studies
have used case surveillance (Mann et al. (1986), A. Study Groups
Bigger, (1986) and serologic surveys (Mann, et al., Both OPD and admitted patients, who were screened
(1986), Melbye et al. (1986) to show the pattern of for HIV, constitute our study group.
heterosexual transmission, (Mann et al. (1986) risk
B. Study Centre
factors for acquisition, (Van de Perre et al. (1985),
Present study was conducted in the Department of
Kreiss et al. (1986), Van de Perre et al., (1987), Piot et
Microbiology, Central Lab Indore, located at Yashvant
al. (1987), Simonsen, et al., (1984), N’Galy et al.
Plaza in front of Railway station, Indore, with its
(1988), and the prevalence of infection. Several useful
reviews on the epidemiology of AIDS have been associated CHL hospital (CHL). CHL is an 800 bedded
published (Quinn et al., (1986), Mann et al., (1988), multi especially tertiary care hospital, located at Nehru
nagar Indore, providing health care services to both
Piot and Carael (1988), Piot et al., (1986). Although
rural and urban population of nearly 80 lakh, in and
intensive efforts have rightly focused on preventing
around Indore.
further transmission; the large number of cases of AIDS
will bring attention back to the clinical problems of
 Vishwakarma, Bhawsar and Vishwakarma 1743
C. Collection of Sample antibodies. Post final wash a positive reaction is
As per the policy laid by the hospital infection control visualized by the appearance of purple coloured bands
committee (HICC) of CRGH, all patients undergoing at the last region ‘1’ and ‘2’ is a negative test result.
any major / minor operative or invasive procedures are The band serves appearance of control to validate
routinely screened for HIV, BHsAG and HCV. Also sample addition, reagent and assay performance.
patients with high risk group, such as, those visiting Reagents and Materials Supplied:
STD clinic, and those suffering from TB, were also 1. Ready to use individually pouched, flow through test
screened or HIV/ HBsAg /HCV were included in the devices striped with HIV 1 specific purified synthetic
present study. Three CC blood was collected from peptides at test region ‘1’ and HIV ‘2’ specific purified
antecuboital vein in clean, plain vails, with proper synthetic peptides at test region ‘2’ and a blue dyed
antisepsis of the skin. All samples were allowed to clot protein a based control band a region ‘c’ along with a
and clear, non-haemolysed, non lipaemic serum specimen dropper and desiccant.
samples were collected in clean vials. All patient 2. Dropper bottle with ready buffer solution.
demographic data including name, age, sex, address, 3. Dropper bottle ready to use protein a conjugate gold
OPD / IPD no. and that of the samples collection etc. solution.
All serum samples were checked by ELISA and rapid 4. Package insert.
test for HIV, within 24 hours of collection. (a) Test Procedure (Microlisa - HIV Microwell
ELISA)
D. Separation of Serum Samples
1. Bring all reagent and specimen to room temperature
The important step after the collection of sample is the
(25-30) before use.
serum separation from the collection blood. Generally
2. Tear open the soil pouches and retrieve the required
in these cases the blood samples were collected a day
of retroquick HIV membrane teat device and label
before and were kept in the refrigerator and were
appropriately.
allowed to settle down, and a after a period of 24 hours.
3. Add two drop of wash buffer into the reaction port of
Mostly the separated serum was seen clearly and was
the device and allow soak through completely.
taken in the required amount with the help of the
4. Use the sample dropper provided add one drop of the
pipette, but in certain cases where serum separation was
serum plasma (25microlitre) specimen into the reaction
not successfully by this method another method was
port. Allow to soak through completely.
used that is as follows:
5. Add 3 drops of wash buffer to the reaction port and
Centrifugation. The centrifugation technique basically
allow soak through completely.
works on the principle of settlement of the heavier
6. Add 2 drops of wash buffer and allow the wash
particles down and the lighter particles or the lighter
buffer to soak through completely.
matter is above and from there it is easily separated.
7. Read and record the result immediately.
The similar process was followed in the test tubes, the
Interpretation of Results
test samples, which were to the centrifuged, were taken
Positive
and the balance was deliberately maintained by using
Two distinct red lines appear. One line should be in the
test tubes with filled water and the RPM (1900 RPM)
control region (C) and another line should be in the test
and the time was also set (2 min.).
region (T).
1. Rapid Test. Retro quick HIV is a membrane based
Negative
flow through immunoassay for the detection of
One red line appears in the control region (C) no
antibodies to HIV-1 and HIV-2 in human serum and
apparent red or pink line appears in the test region (T).
plasma. Highly purified synthetic peptides of gp 41 ad
Invalid
36, corresponding to the immunodominant regions of
Control line fails to appear. Insufficient specimen
HIV-1 and HIV-2 utilised in the test system assist in
volume or incorrect procedural techniques are the most
visual, qualitative, simultaneous detection of antibodies
likely reasons for control line failure.
to HIV-1 and HIV-2.
2. ELISA Test
Principle. Retro quick HIV test comprises of a test
ELISA is the most commonly performed screening test.
device striped with distinct band of purified gp120 and
Screening assay must detect all positive sera that should
gp41 synthetic peptide to HIV-1 and gp36 synthetic
be highly sensitive even if some false positives do
peptide specific to HIV-2. At test region ‘2’ the third
occurs. However result of a screening test could never
band striped at region ‘c’ corresponds to the assay
be used as the final interpretation of HIV status. And
performance control. First the membrane assembly is
individual identified on the basis of 1 screening assay
hydrated with wash buffer and then the specimen is
as technical errors can occur. The serum reactive in
added. Antibodies to HIV-1 and /or 2 if present are
screening assay is subjected to confirmatory test (as per
captured by the respective antigens, after washing with
policy and strategy) of testing to be classified as
buffer, with protein a conjugated gold sol reagent is
reactive only if is reactive in repeated assay.
added to reveal the presence and absence of bound
 Vishwakarma, Bhawsar and Vishwakarma 1744
Material Required (b) 50 ml of buffer was prepared (2 ml concentrate
ELISA kit and Serum etc. buffer with 48ml. water) for each micro Lisa strip used
Components in Each MICROLISA HIV Kit and was mixed well before use.
Store all components at 2-8℃ when not in use. Expiry (c) 20 ml. 25 x wash buffer concentrated was mixed
date on the kit indicates that beyond which the kit with 480 ml. of deionizer water. Wash buffer is stable
should not be used. for 2 months when stored at 2-8℃.
1. Micro Lisa – HIV Strip plates – 12 strips (12x8 4. Preparation of Working Conjugate
wells) Conjugate concentrated was diluted by 1:100 in
2. Sample diluent-1 bottle (20ml). conjugated diluent, working conjugate was not stored.
3. Enzyme conjugate concentrate (100x) 1vial (0.25ml) A fresh dilution for each assay in a clean glass vessel
4. Conjugate diluent _ 1 bottle (15ml) was prepared.
5. Wash buffer concentrate (25x) – 1 bottle (50ml) 5. Preparation of Working Substrate Solution
(PBS with surfactant, Dilute 1:254 with distilled water TMB concentrate was diluted by 1: 100 in substrate
before use). TMB diluent. The 100X solution was crystallized
6. TMB Concentrate (100x) – 1 vial (0.25ml) during storage. Crystals were checked before use, if
7. Substrate (TMB diluent) – 1 bottle (20ml) crystals were present, solubilize by warming at room
8. Control - !- 1 vial (2.0ml) temperature.
(Ready to use normal human serum; negative for HIV, Test Procedure
HCV and HBsAg) Once the assay has started, complete the procedure
9. Control - !- 1 vial (2.0ml) (Ready to use, inactivated without interruption. All the reagents should be
and diluted human serum; positive for HIV antibodies) dispensed in the centre of the well and the tip of the
10. Stop solution – 1 vial (6ml) (Ready to use, 2 N pipette should not touch the wall of the micro well.
sulphuric acid) Fit the strip holder with the required number of
11. Plate sealers– Adhesive baked sheets for sealing MICROLISA-HIV strips. The sequence of the
micro well plate/strips procedure must be carefully followed. Arrange the
Preparation of Reagents assay control wells so that well A-1 is the reagent
The following reagents were prepared before or during blank. From well A-1 arrange all controls in a
assay procedures. Reagents and samples were kept at horizontal or vertical configuration. Configuration is
room temperature (20-30℃) before beginning the assay dependent upon reader software.
and left at room temperature during testing. After use 1. Add 100 µl sample diluent to A-1 well as blank.
return reagents to 2-8℃ all containers used for 2. Add 100µl Negative Control in each well no. B-1 &
preparation of reagents were cleaned thoroughly and C-1 respectively. Negative Control is ready to use and
rinsed with distilled or deionized water. The incubator hence no dilution is required.
was pre-warmed at 37℃. 3. Add 100µl Positive Control in D-1, E-1 & F-1 wells.
Sample Preparation Positive Control is ready to use and hence no dilution is
1. Tube Dilution required.
The tubes were marked carefully for the proper 4. Add 100 µl sample diluent in each well starting from
identification of the samples. The serum samples were G-1 followed by addition of 10µl sample. (Refer
diluted to be tested, with sample diluent (1:11 dilution) MICROWELL DILUTION) Alternatively Transfer 100
in separate tubes (20µl diluent + 20µl sample). A µl of each sample diluted in sample diluent (1:11), in
separate tip was used for each sample and then each well starting from G1 well. (Refer TUBE
discarded as biohazards waste. DILUTION).
2. Micro Well Dilution 5. Apply cover seal.
(a) 100µl of sample diluent was pippete into the micro 6. Incubate at 370C + 20C for 30 min. + 2 min.
well. 7. While the samples are incubating, prepare Working
(b) 10 µl of serum sample to be tested was added. Wash Solution and Working Conjugate as specified in
(c) Ensure thorough mixing of the sample to be tested. Preparation of Reagents.
3. Preparation of Wash Buffer 8. Take out the plate form the incubator after the
(a) The buffer concentration was checked for the incubation time is over and, wash the wells 5 times with
presence of salt crystals. The crystals resobulized by Working Wash solution according to the wash
warming at 37® C until all crystals dissolved, if the procedure given in the previous section (wash
crystals were present in the solution. procedure).
 Vishwakarma, Bhawsar and Vishwakarma 1745
9. Add 100 µl of Working Conjugate Solution in each CUT OFF VALUE
well including A-1. Absorbance
10. Apply cover seal.
11. Incubate at 370C + 20C for 30 min. + 2 min.
12. Aspirate and wash as described in step no. 8.
13. Add 100 µl of working substrate solution in each
well including A-1.
14. Incubate at room temperature (20 - 300C) for 30
min. in dark.
15. Add 100 µl of stop solution.
The cut off value is calculated by adding Mean
16. Read absorbance at 450 nm within 30 minutes in
Negative Control (NCx) and Mean Positive Control
ELISA READER after blanking A-1 well. (Bichromatic
(PCx) as calculated above and the sum is divided by 6.
absorbance measurement with a reference wavelength
600 - 650 nm is recommended when available).
CALCULATION OF RESULTS
Abbreviations
NC - Absorbance of the Negative Control
NCx - Mean Negative Control
PC - Absorbance of the Positive Control
RESULTS AND DISSCUTION
PCx - Mean Positive Control
TEST VALIDITY: 1. Test specimens with absorbance value less than the
Blank acceptance Criteria cut off value are non-reactive and may be considered as
Blank must be <0.100 in case of differential filter being negative for anti-HIV.
used. In case differential filter is not available in the 2. Test specimens with absorbance value greater than or
reader the blank value may go higher. equal to the cut off value are reactive for anti-HIV by
Negative Control Acceptance Criteria: MICROLISA-HIV.
NC must be < 0.150. If it is not so, the run is invalid 3. The O. D. for Crystal clear negative samples can be
and must be repeated. in minus. However, the minus (-) O.D. does not in any
Positive Control Acceptance Criteria: way affect the result interpretation. It rather gives better
1. PC must be > 0.50 specificity.
2. Determine the mean (PCx) value if one of three Observations
positive control values is outside of these limits, Observations of present study are as followings:
recalculate PCx based upon the two acceptable positive The highest number of patients (8991) tested for HIV
control values. was in the year 2009-10. The HIV reactivity is found to
3. If two of the three positive control values are outside be gradually increasing from 0.83% to 1.70%, during
the limits, the assay is invalid and the test must be the four years period from 2008-09 to 2011-12.
repeated

Table 1: Showing year wise HIV Seropositivity among Hospital based patients.
No. Year(April No. of No. of HIV %
to March) Patients Reactive
1. 2008-09 6952 58 0.83
2. 2009-10 8991 88 0.97
3. 2010-11 8274 112 1.35
4. 2011-12 8317 142 1.70
 Vishwakarma, Bhawsar and Vishwakarma 1746

150 142

112

No. of Reactive
100 88

58
50

0
2008- 2009- 2010- 2011-
09 10 Year 11 12
Fig. 1. Number of reactive increasing with year.
Table 2: Showing Gender wise distribution of HIV seropositivity among patients (Year 2008-09 to 2011-
12).
Year(April to Total Total Total No. Total No. Male Female
March) No. of No. of of Female of Reactive Reactive Reactive
Tested Male Tested
Tested
2008-09 6952 4518 2434 58 34 24
2009-10 8991 5484 3507 88 60 28
2010-11 8274 3888 4383 112 80 32
2011-12 8317 4408 3909 142 88 54
Total 32534 18298 14239 400 262 138

Out of 32534 patients screened for HIV during four years, 18298 were males and remaining 14239 were females.
Out of 18298 males tested, 262 were found reactive and of 14239 females tested, 138 were found reactive for HIV.
88
90
80
80
No. of Reactive

70
60
60 54
50
No. of Male
40 34 32 Reactive
28
30 24
No. of Female
20 Reactive
10
0
2008-09 2009-10 2010-11 2011-12

Year
Fig. 2. Gender wise distribution of HIV Reactive Patients (From April 2008-09 to March 2011-12).
 Vishwakarma, Bhawsar and Vishwakarma 1747
Table 3: Age wise Distribution of HIV Seropositivity in Male and Female Patients.
AGE GROUPS
Month 1-10 year 11-20 year 21-30 year 31-40 year 41-50 year 51-60 60
year above
M F M F M F M F M F M F M F
April - - - - 2 4 - - - - - - - -
May - - - - - 6 2 - 2 - - - - -
June - - - - - - - - 2 - 2 - - -
July - 2 - - 4 2 2 2 - - - - - -
August - - - - 2 4 2 - - 2 - - 2 -
September 4 - 2 - 2 - 4 2 4 - 2 - 2 -
October - - 2 - 2 2 2 4 - 2 - - - -
November - - 2 - 4 2 - 2 2 - - - - -
December - - - 2 6 2 4 2 - 2 4 - - -
January - - - - - 4 4 - 2 - - - 2 -
February - - - - 2 - - - - - - - - -
March 2 - - - 2 2 2 - 2 2 - 2 - -
Total 6 2 6 2 26 28 22 12 14 8 8 2 6 0

30 28
26
25
22

20

14
15 No. of male reactive
12
No. of female reactive
10 8 8
6 6 6
5
2 2 2
0
0
0to10 11to20 21to30 31to40 41to50 51to60 61
above
Fig. 3. Number of male and female in different age group.
Table 4: Showing Comparative Evaluation of HIV testing by ELISA and RAPID Test.
Test Positive Negative Total
ELISA 400 32134 32534
RAPID TEST 410 32144 32534

Out of 32534 patients tested for HIV 400 were Reactive A total of 32534 patients of both genders and various
by ELISA as compared to 410 by Rapid Test, while age groups ranging from 1 year to 60 years above
32134 were found Non-Reactive by ELISA in visiting to Central Lab Indore were tested for HIV by
compared with by Rapid Test 32144. two different methods namely Rapid and ELISA test
during a period of four years i.e. from April 2008-09 to
CONCLUSION
March 2011-12, conclusions of the present study are as
follows:
Vishwakarma, Bhawsar and Vishwakarma 1748
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