in renal surgery, which may even necessitate removal of kidney. Hence the knowledge of vascular pattern of the kidney is indispensable for the Urosurgeon.
Although the renal vascular segments were first described by John Hunter long ago in 18th century, the present day concepts are largely due to work of Graves4 (1954). He gave the detailed account of primary pattern of renal vascular segments. He divided the renal parenchyma on the basis of arterial distribution into five segments _ Apical, Upper, Middle, Lower & Posterior. Each of them is supplied by its own segmental artery.
Ligation of segmental artery supplying the area of operation produces an almost bloodless operating field. Therefore it can be inferred that there is no collateral circulation between segments, & these segmental arteries are `end arteries'.
Since the knowledge of renal vasculature is of paramount importance during various renal surgeries, the present study was undertaken.
As most of the cadavers used in the present study are received from Maharashtra, this can be a regional study. It will be interesting to note whether incidence of these variations is different from other studies carried out in other parts of the world.
Material & Methods
The study was based upon 212 adult cadaveric kidneys. They were obtained from department of Anatomy and from department of Pathology (Autopsy section). It comprised of 124 male & 88 female kidney specimens. The specimens were collected as a `Kidney
block'. It consisted of both the kidneys with renal vessels, aorta and inferior vena cava.
Meticulous dissection of the kidneys and the renal pedicles was done. The origin and course of the renal artery was observed till it divided into segmental arteries. The variations in branching were noted.
The suspension of gelatin powder was prepared in lukewarm water. It was applied to the structure in the renal pedicle. It was allowed to dry for two hours and then each structure was painted with oil paint using the following color code. —
Arteries - Red
Veins - Blue
Pelvis and ureters - Green
The color photographs were taken which clearly demonstrated the various tructures in the renal pedicle.
Observations
Table No. 1 shows the sex wise distribution of the segmental branches from anterior division of the renal artery.
TABLE No. 1
Number of branches going to anterior aspect
NO. OF MALE FEMALE
BRANCHES RIGHT(%) LEFT(%) RIGHT LEFT(%)
1 00.00 00.00 00.00 00.00
2 01.61 03.22 0.4.54 04.54
3 12.90 12.90 09.09 06.81
4 69.35 72.58 72.72 77.27
5 14.51 11.29 11.36 06.81
6 01.61 00.00 02.27 04.54
Table No. 2 shows the sex wise distribution of the segmental branches from posterior division of the renal artery.
TABLE No. 2
Number of branches going to posterior aspect
NO. OF MALE FEMALE
BRANCHES RIGHT(%) LEFT(%) RIGHT LEFT(%)
1 58.04 62.90 65.90 68.18
2 24.19 22.58 20.45 18.18
3 17.74 14.51 13.63 13.63
The incidence of the standard pattern of segmental arteries is shown as a graph in an illustration below.
Illustration
Standard pattern of Segmental arteries
Discussion
Normally a renal artery gives rise to anterior and posterior divisions. These divisions then divide to form the segmental branches. These branches pass either to the anterior or to the posterior aspect of the kidney.
The segmental arteries going to the anterior aspect were commonly four in number (72.63%) in the present study, although variations from two to six branches were noted. The posterior aspect of kidney commonly showed a single segmental artery (63.19%), though variations up to three branches were observed. The number of these primary branches & their area of distribution suggests the presence of vascular segmentation in the kidney.
Standard pattern of segmental arteries : -
Commonly the renal artery gives rise to four anterior and one posterior segmental branch. F.T. Graves4 first described this in 1954. This is often referred as standard pattern of segmental arteries & is commonly followed.
Sykes6 in 1963 found that 83% of cases, the renal artery divided into anterior and posterior branches and finally giving rise to five segmental arteries. In the present study
the standard pattern segment arteries was found in 59.43% of specimens. The details are as follows:
Total 59.43%
Males (57.25%) Right 54.83%
Left 59.67%
Females (62.49%) Right 61.36%
Left 63.63%
It is evident from the figures that the presence of standard pattern of segmental arteries is more in Females & that too on the Left side. Therefore it can be concluded that the variations are more common in Males and on the Right side.
In 1966, Fine H. & Keen E.H.3 had introduced a concept of primary & secondary branching pattern. According to them, the renal artery was divided into three primary branches viz. Posterior, Lower & Upper. The secondary branches (Intermediate & Middle) were sufficiently constant, apart from suprahilar (Apical) artery, which could be primary or secondary in origin. But this pattern was not taken into account for the present study.
The study conclusively proved, the complex nature of renal vascular distribution and the necessity of the knowledge of renal vasculature to a surgeon operating on the kidney to have a bloodless field. The detailed information of the vasculature will reduce the chance of hemorrhage, due to accidental trauma. Thereby, he can avoid the unwanted postoperative morbidity.
The results of the present study are helpful for radiologists performing renal artery embolisation.
Also, for the upcoming renal transplants, the precise knowledge of the
vascular segments of the kidney and the 'end artery' nature of its blood supply is indispensable for the urosurgeon. It is very important for him to make an anastomosis with all the arteries of the donor kidney during transplantation. Otherwise, infarction of a segment can lead to serious postoperative complications.
References
1. Decker G.A.G., D.J.du Plessis: Lee Mc'Gregor's Synopsis of Surgical Anatomy. 12th edition, Indian Ed. - K.M. Varghese Co. Bombay. 1986: 289-300.
2. Goldstein A.E. Accidents in renal surgery. Surg. Gynec. And Obstet. 1937, 65:575.
3. Fine H., Keen E.H. The arteries of human kidney. J. Anat. 1966, 100 : 113-137.
4. Graves F.T. The anatomy of intrarenal arteries and its application to segmental resection of kidney. B.J. Surgery 1954, 42: 132-139.
5. Hollinshead W.H. Anatomy for Surgens, Vol. 2:2nd Edition, — Harper and Row, New York 1971: 527-530.
6. Sykes D. The arterial supply of the human kidney with special reference to accessory renal arteries. B.J. Surg. 1963, 50: 368-374.
7. Williams P.L., Bannister L.H., Berry M.M., Collins P., Dyson M., Dussek. J.E. Ferguson M.W.J. Gray's Anatomy, In: Urinary System.-38th edition, Churchill Livingstone, U.K. 1995: 1826.
Original Article
Immunogenicity and Bioefficacy Study of Purified Duck Embryo Vaccine (PDEV) manufactured by Cadila Healthcare Ltd. (VaxiRab), in Indian Cohort
Renu Singh * Sweta Kothari **
R. Mahajan @ R. Mittal#
S. Sapatnekar##
ABSTRACT
This study on Purified
Duck Embryo Vaccine (PDEV)/ VaxiRab of Cadila Healthcare Ltd., was a prerequisite
for approval of the vaccine from the office of the Drugs Controller General of India
(DCGI).
It was carried out with 21 volunteers and 32 post-exposure cases using VaxiRab Lot
No.: MA - 1425, in 2002. It involved vaccination as per the WHO schedules for pre- and
post-exposure cases. The volunteers/patients were bled on day 0, 14 and 35.
Anti-rabies antibodies were detected in sera by ELISA
and Mouse Neutralization Test (MNT). Mean antibody titre by MNT for VaxiRab were 5.33
IU/ ml on day 14 and 11.86 IU/ml on day 35 in volunteers; and 7.22 IU/ml and 12.23 IU/ml respectively in patients, indicating satisfactory immunogenicity of the vaccine, 10 to 23 times more than the minimum protective level of 0.5 IU/ml (WHO standard). No adverse reactions were observed in any of the recipients.
Key words: Rabies, PDEV, VaxiRab, immunogenicity, antibody
INTRODUCTION
According to the WHO estimates, 35,000 to 50,000 persons die of rabies encephalitis in the world and nearly 99% of them are from India (1, 2). Among the tools available to
* Research Fellow, Department of Virology, Haffkine Institute For Training, Research and Testing Parel, Mumbai -400 012 • Tel: +91-22-24160947/61/62 Fax: +91-22-24161787 • E-mail: renusingh283@rediffmail.com
** Research Fellow, Department of Virology, Haffkine Institute For Training, Research and Testing
Parel,
Mumbai -400 012 • Tel: +91-22-24160947/61/62 Fax: +91-22-24161787 • E-mail:
kotharisweta@yahoo.co.in
@ Research Fellow, Department of Virology, Haffkine Institute For Training, Research and Testing
Parel,
Mumbai -400 012 • Tel: +91-22-24160947/61/62 Fax: +91-22-24161787
# Medical Advisor & Head - Regulatory Affairs, Cadila Healthcare Limited, Sarkhej - Gandhinagar Highway Ahmedabad - 380052 Tel: +91-79-26868211 • Fax: +91-79-26862362
## Director, Haffkine Institute For Training, Research and Testing Parel, Mumbai -400 012
Tel: +91-22-24160947/61/62 Fax: +91-22-24161787
Corresponding author:
R. Deshmukh, Head, Department of Virology, Haffkine Institute For Training, Research and Testing
Parel,
Mumbai - 400 012 • Tel: +91-22-24160947/61/62 Fax: +91-22-24161787 •
control rabies, vaccines rank highest in effectiveness and economic feasibility (3). Some of the early 20th century antirabies vaccines worked very well, while other vaccines were either poorly antigenic or showed adverse reactions (4) Despite the availability of modern tissue culture vaccines viz. Human Diploid Cell Vaccine (HDCV), Purified Vero Cell Rabies Vaccine (PVRV), and Purified Chicken Embryo Cell Vaccine (PCECV), there still remains a great need for newer, effective and cheaper anti rabies vaccines in India (5).
The efficacy of any vaccine depends on the immunological response in humans involving primary and secondary lymphoid organs, the cells inhabiting them, various types of antibodies, cytokines, and the genes coding for B and T cell receptors. Hence the thorough understanding of immune induction, immunorecognition, immune effector mechanism, immune potency and immune evasion mechanism used by the infectious organism is needed Based on this basic immunological principal, Kenneth et al reported the decreased antibody response in persons from developing countries when they were given HDCV (6) Inspite of the vast data collected over the past decades on immunological processes, vaccine development remains to some extent a process of trial and error.
The Purified Duck Embryo antirabies Vaccine (PDEV) was indigenously produced by Cadila Healthcare Limited, Ahmedabad, India, under the brand name VaxiRab, by the
technology transfer from the Swiss Serum and Vaccine Institute. When VaxiRab was being planned to be introduced in the Indian market, the following study in the Indian cohort was mandatory for obtaining the marketing permission from the Office of the Drugs Controller General of India (DCGI).
MATERIALS
Due permission and clearance were obtained from the Animal Ethics Committee and the Institutional Ethical Committee were obtained before the commencement of the study. For the pre-exposure study, healthy adult volunteers were enrolled while patients with history of Grade I/II animal bites were included in the post-exposure study. The volunteers were explained the details of the study to their satisfaction and an informed written consent of the patients / relatives was obtained prior to their enrolment. Their inclusions and exclusions were based on the following criteria
Inclusion criteria:
Indian patients/volunteers above
18 years of age were included in the study. They had no history of animal bite(s) for
pre-exposure study, while only patients having grade I or II animal bites as per
WHO classification were included in the post-exposure study
Exclusion criteria:
Pregnant women or lactating mothers were excluded from the study. Any patient who had received any type of rabies vaccination
in the past, or had received any dose of rabies immunoglobulin (human/equine) within the previous three months was also excluded. Patients suffering from chronic illness, being treated with steroids, any other immuno-suppressants, concomitant anti-malarials, or known to be HIV positive were excluded. Patients planning for a surgery in the subsequent three months, or having a severe history of allergy were also excluded. Patients not likely to be available for follow up or having participated in another clinical trial in the previous 3 months were also excluded.
Volunteers:
For VaxiRab, 21 adult healthy volunteers of either sex were enrolled out of which 12 were males and 9 were females with a mean age of 19 years. Upon enrolment, previous medical history and vital signs were recorded. Inclusion and exclusion criteria mentioned in the protocol were meticulously followed.
Patients:
Thirty-two adult male patients with Grade II wounds were enrolled for study with VaxiRab, with a mean age of 26 years. Of these 32 post-exposure cases, 28 had been bitten by stray dogs, three by stray cats and the remaining one by a wild monkey. They were given wound care, anti-tetanus prophylaxis and antibiotics. Upon enrollment, previous medical history and vital signs were recorded. Inclusion and exclusion criteria mentioned in the protocol were meticulously followed. All the volunteers and patients were
monitored for a period of one year for their antirabies antibody titres.
Enzyme linked Immuno Sorbent Assay (ELISA):
The ELISA-KIT (Platelia-Rage, BioRad) was used for detection of rabies virus antiglycoprotein antibody in the serum of all the vaccinees. The test is based on the principle of solid phase ELISA, prepared with the glycoprotein extracted from the inactivated and purified virus membrane, and an enzyme conjugate (protein A from Staph. aureus coupled with peroxidase enzyme)
Mice:
For the present study, Swiss albino mice, weighing 14 gms. were used. All the mice needed were procured from Haffkine Biopharma Corporation Ltd. (HBPCL), Mumbai and Mahavira Enterprises (Hyderabad) Both the organizations are Governments recognized animal breeders.
Vaccine:
Vaxirab Lot no: MA-1425 (Cadila Healthcare Ltd.), India, was the vaccine under investigation for the said study. The vaccine is free from myelin and myelin-like proteins due to absence of mechanical shear in their manufacturing process as the vaccine is prepared by the following method:
The Pitman Moore strain of the rabies virus is inoculated into the yolk sac of 7-day old duck eggs. After incubation for 14 days, the embryo are aseptically removed and decapitated. The heads are stored under
sterile conditions in liquid nitrogen. A batch of 40-60 heads is used for further processes to extract the antigen and subsequent centrifugation, removal of non-viral lipids using n-heptane, and final concentration, purification and elution of the viral particles. The virus is inactivated using b-propiolactone, and formulated to yield a dose of 107.5 MLD50/ml, generating an antibody titre of >2.5 I.U.per ml.
METHODS
Vaccination schedule:
The volunteers were vaccinated on day 0, day 7, and day 28 for the pre-exposure study, while the patients enrolled in the post-exposure study were vaccinated on day 0, day 3, day 7, day 14 and day 30.
Bleeding schedule for patients and volunteers:
All the subjects were bled on days 0 (before vaccination), and day 14 and day 35 post vaccination. The sera were separated using centrifugation. The sera samples were stored at -20°C till further use.
ELISA:
ELISA was carried out as per the pack-insert in the kit.
Mouse Neutralization Test (MNT):
The test was carried out as per WHO laboratory techniques. Titers were reported as the highest dilution in which complete neutralization was observed. For more critical
comparison of the end-point titers serial
2-fold dilution, 1:20, 1:40, 1:80, 1:160 and 1:320 were done and end-point between2-fold dilution were estimated by Reed- Muench method. Antirabies antibody titer
was expressed in International Units per ml (IU/ml) (7).
Challenge virus:
For each test, the rabies challenge virus (RV-CVS) standard was diluted in the range of 0.1, 0.01, and 0.001. For the above test 50 LD50 rabies challenge virus standard was used.
Standard antibody-sera:
The International Standard Reference Serum was obtained from Central Research Institute, Kasauli. The serum of titre >300IU/ml was titrated with each batch of test sera.
RESULTS AND DISCUSSION
Any new vaccine whenever introduced to a new cohort of genetically and ethnically different population may result in a different immunogenic response (1). Keeping this in mind, the pre- and post-exposure study for VaxiRab was carried to assess the immunogenicity in Indian population. In previous studies (8, 9) PDEV (Lyssavac N Berna) manufactured by Swiss Serum & Vaccine Institute, Berne, Switzerland has been reported as the most successful anti-rabies vaccine with good immunogenicity and bioefficacy and is accepted worldwide. In Western population, the response has' been excellent but it was the need of the time to
check it in Indian cohort, as the vaccine has now been proposed for the Indian market. Similarly, since PDEV \ (Vaxirab) has been manufactured indigenously by Cadila Healthcare Limited, Ahmedabad, India under a technology transfer agreement with Swiss Serum & Vaccine Institute, this vaccine needs to be assessed for immunogenicity and bioefficacy before it is marketed in the country. The study with Lyssavac N Berna was undertaken in 1998 by the same Department (14). To study the antibody assessment at earliest in study population both, in-vitro (ELISA) and in-vivo (MNT) tests were carried out for all the subjects.
Pre-exposure Cases:
No baseline antirabies antibody titre was detected on day O. The antibody titers by - ELISA were between the range of 0.8 IU/ml to 24.24 IU/ml with a mean value of 6.42 IU/ml on day 14 and 1.17 IU/ml to 22.19 IU/ml with a mean value of 10.0 IU/ml on day 35. The titers by MNT were 0.6 IU/ml to >20 IU/ml with a mean value of 5.0 IU/ml on day 14 and 1.66 IU/ml to >20 IU/ml with a mean value of 11.38 IU/ml on day 35.
Post-exposure Cases:
No baseline antirabies antibody titre was detected on day 0 in post-exposure cases, thus confirming their history of absence of exposure to previous similar vaccination. The antibody titers demonstrated by ELISA were 0.86 IU/ml to 24.27 IU/ml with a mean value of 7.89 IU/ml on day 14 and 3.18 IU/ml to 28.08 IU/ml with a mean of 11.85
IU/ml on day 35. For MNT, the values observed were between 1.0 IU/ml to 22.4 IU/ml with a mean of 7.22 IU/ml on day 14 and 4.6 IU/ml to 28.9 IU/ml with a mean of 12.23 IU/ml on day 35.
Based on ELISA test findings, it is apparent that all the patients had sero-converted within 14 days of administration of the vaccine. The MNT results further confirmed the immunogenicity of the vaccine in our population.
Unpublished data with the administration of Lyssavac N Berna from the same department showed similarity in antibody levels in both pre-exposure and post- exposure cases, as was obtained with VaxiRab. This testifies a satisfactory technology transfer adopted by Cadila HealthCare Ltd. Shaul J F, et al., have observed that the currently obsolete Duck Embryo Vaccine was less immunogenic in nature, and usually lead to the allergy to the avian antigen (10). Compared to this, VaxiRab is free from any kind of avian antigens. As compared to other tissue culture vaccines viz. neural tissue vaccines (Semple type) and HDCV, which are reported to cause allergic reactions (11, 12, 13), PDEV appears to be safer. In our series, one of the patients complained of dislocation of left shoulder in our study and was treated by an orthopedic surgeon with analgesics. The dislocation was natural and was unrelated to the vaccination. Barring this, no adverse drug reactions were reported or observed.
Rabies Vaccine was not included in this study.
ACKNOWLEDGEMENT
The authors are grateful to Cadila Healthcare Limited, India, for providing the vaccines used in the study. We are also thankful to Mrs. A. Gandhi, Senior Laboratory Technician at the Department Of Virology, Haffkine Institute, for her valuable support during the study.
REFERENCES
1. Dutta A. K. and Kawal
S.N, Rabies
and its prevention, J ofAPCRI, 1999;
1:5-13
2. WHO estimate: http://www.who.int/emc/diseases/zoo/ rabies.htm
3. Requirement of rabies vaccine for human use (amendment 1992). WHO Expert Committee on Biological Standardization. Forty-third report. Geneva. World Health Organization, 1994 (WHO technical report series, No. 840) Annex. 4.
4. Datta J.K, Adverse reactions to purified chick embryo cell rabies vaccine. Vaccine 1994; vol. 12, No. 15:- 1484.
5. Datta J.K, Rabies prevention; Cost to an Indian Labour, JAMA, 1996; 226-32.
6. Bernard Kenneth W., Daniel B. Fishbein et al. Pre- exposure rabies immunization with human diploid cell vaccine: decreased antibody responses in persons immunized in developing countries, Am. J. Trop. Med. Hyg., 1985, 34 (3), 633-647.
7. Fitzgerald E.A, "Potency Test for
antirabies serum and immunoglobulin." In: Meslin F.X, Kaplan M.M and Koprowski H, Editors. Laboratory techniques in Rabies, 4th edition. WHO Geneva, 1996:417-422.
8. Brigs D. T, Dreesen D. W., Morgan
P., Chin J. E., Seadle C. D., Cryz
L., Glueck R., Cryz S. J., Safety and immunogenicity of Lyssavac
Berna human diploid cell rabies vaccine in healthy adults. Vaccine 1996, vol.
14: 1361-1365.
9. Khawplod P., Glueck R., et ai, Immunogenicity of purified duck embryo vaccine (Lyssa vac-N) with use of the WHO- approved intradermal post exposure regimen. Clin. Info Dis. 1995, 20, (3), 646-651.
10. Shaul J F, et al. Duck embryo rabies
antirabies serum and immunoglobulin." In: Meslin F.X, Kaplan M.M and Koprowski H, Editors. Laboratory techniques in Rabies, 4th edition. WHO Geneva, 1996:417-422.
8. Brigs D. T, Dreesen D. W., Morgan
P., Chin J. E., Seadle C. D., Cryz
L., Glueck R., Cryz S. J., Safety and immunogenicity of Lyssavac
Berna human diploid cell rabies vaccine in healthy adults. Vaccine 1996, vol.
14: 1361-1365.
9. Khawplod P., Glueck R., et ai, Immunogenicity of purified
duck embryo vaccine (Lyssa vac-N) with use of the WHO-
approved intradermal post exposure regimen. Clin. Info Dis. 1995, 20, (3), 646-651.
10. Shaul J F, et al. Duck embryo rabies
vaccines. Anaphylactic reaction following initial reaction. J S C Med Assoc 1969, 65(10): 359-361.
11. Ahasan H A, Chowdhary M A, Azhar M A, Rafiqueuddin A K, Neuroparalytic complications after anti-rabies vaccine (inactivated nervous Tissue vaccine).
Trop Doct 1995, 25(2): 94.
12. Hemachudha T, et al., Neurologic complications of semple type rabies vaccine: clinical and immunological studies, Neurology 1987; 37: 550-556.
13. Systemic allergic reactions following immunization with human diploid cell rabies vaccine. Morbidity and Mortality weekly report 1984; 33: 185-187.
APPLICATION OF PAGE IN THE EPIDEMIOLOGICAL STUDY OF ROTAVIRUS INFECTIONS IN PEDIATRIC POPULATION
Kirtikar A A*
Kulkarni M V $
Deshmukh R A #
Abstract
Objectives: To determine the prevalence of rotavirus infection in pediatric cases of diarrhea, three methods for detection of rotaviruses, namely latex agglutination, polyacrylamide gel electrophoresis, and virus isolation in cell cultures were compared in the present study.
The study was carried out in Lokmanya Tilak Memorial Hospital, Sion, K.E.M.Hospital, Parel, and Kasturba Hospital for Infectious Diseases, Mumbai Central. Subjects for the study were pediatric patients under five years of age suffering from diarrhea and were admitted to one of the above hospitals. 225 subjects were included in the study. Of the 225 patients, 36 tested positive for rotavirus, giving
a rotavirus positivity of 16%. No atypical rotaviruses were observed. PAGE was found to be the most effective diagnostic method and is recommended for accurate diagnosis of rotavirus infection.
Surveillance to study the epidemiology of rotavirus infections should be carried out on a regular basis to ascertain the strains of rotavirus predominant in the community, which will aid in the development of a vaccine against rotavirus.
KEY WORDS: Rotavirus, pediatrics, diagnostic methods, agglutination, diarrhea It has long been recognized that diarrheal disease is a leading cause of morbidity and mortality, especially in developing countries and acute infectious gastroenteritis is one of the
* Junior Research Fellow, Department Of Virology, Haffkine Institute.
$ Head, Dept. of Pediatrics, Lokmanya Tilak Hospital, Sion.
# Head, Dept of Virology, Haffkine Institute, Parel.
Corresponding author:
Dr.(Mrs.)R.A.Deshmukh, Dept. Of Virology, Haffkine Institute, Parel, 400 012 • Tel. No. (022) 4160961/62 ext. 229
• Fax: (022) 4161787 • E-mail: rad21350@yahoo.com
commonest causes of childhood illness throughout the world and especially in developing countries, malnutrition being a major contributing factor. Another factor contributing to the unchecked spread of diarrhea is poor hygiene and sanitation. Coupled with ignorance and poverty, it makes the developing countries a virtual breeding ground for diarrheal diseases. Diarrheal diseases thus assume phenomenal proportions in developing countries and demand immediate action for their control. Within five years of its discovery in 1973, rotavirus was recognized as the most common cause of severe vomiting and diarrhea in infants and young children worldwide, accounting for approximately one third of cases of severe diarrhea requiring hospitalization (1,2,3). Rotavirus infects virtually all children by five years of age. Some of these infections are severe and many children are infected more than once, although severity of disease decreases with each infection (4).
Rotaviruses affecting humans were once thought to be limited to one antigenic family termed Group A, whereas other antigenic groups (B-G) were thought to be strictly zoonotic. In 1982, however, an epidemic of Group B rotavirus affected millions of persons in China (including adults, children, and neonates) (5), and since then outbreaks have recurred, although affecting fewer persons.
Research to develop a safe and effective rotavirus vaccine began in the mid-1970s when investigators demonstrated that
previous infection with animal rotavirus strains protected laboratory animals from experimental infection with human rotaviruses. (6). However, the efficacy of these vaccines varied. Then exploiting the inherent property of rotaviruses to reassort their genome, multivalent vaccine candidates were developed. (7). On August 31st, 1998, a tetravalent rhesus-based rotavirus vaccine was licensed in the United States for vaccination of infants. (8).
Therefore, it becomes necessary to monitor the epidemiology of rotavirus infection in a population. This would not only enable us to foresee a possible epidemic of unusual rotavirus gastroenteritis but also to ascertain the prevalent strains with a view towards vaccine production.
Because the clinical features of rotavirus gastroenteritis are nonspecific, confirmation of rotavirus infection in children with gastroenteritis by laboratory testing of fecal specimens is necessary for reliable rotavirus surveillance (9). The most practical method taking into consideration accuracy and time required for the test is antigen detection by enzyme immunoassay. But this method is expensive. Another practical method that is also inexpensive is antigen detection by latex agglutination. This method, however, may compromise on accuracy. Other techniques including electron microscopy, reverse transcription-polymerase chain reaction, nucleic acid hybridization, polyacrylamide gel electrophoresis and culture are mainly used in research settings. Thus there is a lack of an appropriate diagnostic tool. A definitive viral
diagnosis can reduce indiscriminate use of antibiotics, eliminate necessity for other tests in search of causative agents and remove uncertainty, allowing the physician to make proper prognosis and remain alert to potential complications.
Taking all these points in consideration we undertook the present study.
Methodology
STUDY SUBJECTS
In this study, 225 patients and 25 control subjects were included.
The study subjects were admitted in the pediatric wards of Lokmanya Tilak Memorial Hospital, Sion, K.E.M.Hospital, Parel, and Kasturba Hospital for Infectious Diseases, Mumbai Central. All subjects were infants and children under 5 years of age.
SPECIMEN COLLECTION
PATIENTS
Stool samples were collected from children suffering from diarrhea and admitted in the pediatric wards of the hospitals. Children were excluded if they had an associated complicating illness. The samples were collected on the day of admittance.
CONTROLS
Control patients were children admitted to these hospitals with other illnesses and who showed no symptoms of gastroenteritis. Stools from these children were collected on any day after admittance to the hospitals.
The clinical features and personal history of all patients including controls was recorded. These include features like age, sex, family income, nature of current illness etc. The specimens were collected in sterile 15ml containers with wide mouths and screw caps. The specimens were labeled and stored at 4C until processed.
DETECTION OF ROTAVIRUS
Detection of rotavirus was done using three methods, namely, latex agglutination (LA), polyacrylamide gel electrophoresis (PAGE), and isolation of the virus in cell culture.
LATEX AGGLUTINATION
Commercially available latex agglutination kit for the detection of rotavirus manufactured by Sanofi Pasteur was used. The kit was stored at 2-8C as described. It is a rapid test based on latex agglutination, intended to detect rotavirus from feces in acute gastroenteritis.
The samples to be tested were kept at room temperature. The kit was also removed from the fridge to allow the reagents to reach room temperature. The stool specimens were mixed properly and the specimens were diluted 1:10 in rotalex buffer and mixed well with vortex mixer for 1min.
Two separate drops of about 25mml were taken on disposable slide (provided in the kit) with a micropipette. Contents of both latex reagent vials were well suspended before use by gently rolling the vials. One drop of rotalex reagent 1 was put on one of the sample drops with micropipette. Rotalex control suspension was added on to the other drop. Mixture was
carefully stirred by using fresh wooden stick for each drop. The drops were spread to cover the entire area of circle of test slide for interpretation of results. The slide was continuously tilted back and forth and observed against dark background for eventual development of precipitate. The detection of agglutination is done by the naked eye.
The result was considered to be negative if no agglutination was observed in the sample drop containing rotalex reagent within 2 mins. Result was considered to be positive if agglutination was detected in sample drop containing Rotalex reagent. Agglutination was either complete resulting in granules and a clear background, or partial when granules could be detected, but the background remained opaque. If the results were doubtful and the test could not be interpreted, the test was repeated. If agglutination was observed in the drop containing Rotalex control latex reagent, the particular specimen could not be studied by this method as instructed in the procedure with the kit. These samples were further tested by PAGE and cell culture.
POLYACRYLAMIDE GEL ELECTROPHO RESIS (PAGE)
The method followed for PAGE is a modification of the protocols standardized by AJ Herring et al (10)and SM Rodger et al (11). The modification was carried out by Krishnan T, Div. of Virology, National Institute for Cholera and Enteric Diseases, Calcutta.
The procedure for detection of rotaviruses by PAGE is separated into three
sections as follows:
A. Extraction of rotavirus dsRNA from fecal material.
B. Electrophoresis of dsRNA in polyacrylamide gels.
C. Silver-staining of dsRNA bands.
A. Extraction of rotavirus dsRNA from fecal samples
The stool samples were vortexed. In case of solid samples, a minimum amount of phosphate buffered saline (PBS) was added to the samples and the mixture was homogenized by vortexing. 0.5ml of the sample was added to 0.5ml of 0.1M sodium acetate buffer with 1% sodium dodecyl sulfate (SDS) in an eppendorf tube. To this mixture was added 0.5ml of phenol-chloroform mixture and vortexed. The tubes were then centrifuged at 10,000 rpm for 10 mins. The aqueous supernatant was collected in fresh eppendorf tubes and refrigerated until tested by PAGE.
B. Electrophoresis of rotavirus dsRNA in PAGE
Bio-Rad electrophoresis apparatus was used for PAGE and a discontinuous system of buffers was used. Separating gel mixture of 10% polyacrylamide was prepared and this solution was poured within glass plates with dimensions 14cm X 16cm. Spacers that were 0.75mm thick separated the glass plates. When the polyacrylamide was set, a 3% stacking gel solution was prepared. This solution was
poured over the separating gel till the glass sandwich was filled. A comb was placed onto the glass sandwich. When the gel was set, the comb was removed and the wells were washed with reservoir buffer. Approximately 30ml of the extracted supernatants were loaded in the wells. A current of 25mA was applied to each slab gel for 16hrs.
C. Silver staining
After the gel run was over, the spacers were removed from in between the glass plates. The stacking gel was removed and discarded. Also, the bottom left corner of each gel was cut off for orientation of the gel. The gel was gently lifted off the glass plate and placed in fixing solution in a glass tray. After rocking the tray for 30mins., the fixing solution was drained off and the silver stain was added. The tray was again rocked for 30mins., after which the stain was drained off. The gel was then rinsed with double distilled water to make sure that no traces of the silver stain were left behind. The developing solution was then added and the tray was rocked till the RNA bands were satisfactorily stained. To stop the reaction, the developing solution was drained off and the stopping solution was added. The bands were visualized against white light.
CELL CULTURE
The cell line used for the isolation of rotavirus was MA-104. This is an established cell line derived from the kidneys of embryonic rhesus monkeys. The cell line was supplied by The National Center for Cell Sciences, Pune.
Inoculation of flasks for cell culture:
The cells from the surface of the flask were dislodged by trypsin treatment. The medium from monolayer culture of cells in tissue culture flasks was aspirated. 1ml of trypsin solution was added and the flask was rocked 4-5 times quickly back and forth so that the trypsin coated the cells in the flask, and traces of medium and serum were diluted. The cells were not detached at this stage. The trypsin solution was aspirated. The flask was then kept at 37°C in an incubator for 2-5 mins. Cell detachment was checked by tapping the flask against the hand so that the small amount of fluid in the flask sheared the loosely attached cells off the surface. If no cells were detached, the flask was put back at 37°C for a few more minutes and the procedure was repeated until the cells were completely detached from the flask. The cells were then suspended in growth medium (MEM with 10% FCS) and triturated gently to disrupt cell clumps. The cell number was determined by counting the cells using a hemocytometer and viability staining was done with trypan blue. The cells were then diluted with growth medium to an approximate concentration of 2 X 104 to 4 X 105/ml. Aliquots of the cell suspension were then added to flasks containing growth medium. The cell suspension was rotated and evenly distributed on the surface of the culture flasks. The flasks were then incubated at 37°C until the formation of monolayers of cells. The growth medium was changed twice a week.
Isolation of the virus:
The isolation of rotavirus was done in tissue culture flasks and the protocol standardized by Kutsuzawa T et al was followed (12). The stool specimens that were positive for rotavirus by PAGE were used for virus inoculation.
The stool specimens were brought to room temperature. A 10% suspension in MEM of the specimens was then prepared. After centrifuging at 10,000rpm for 10mins., the clear supernatant fluids were collected. These supernatants were mixed with an equal amount of 20mg of trypsin per ml for 20 mins. at 37°C. This mixture was then diluted 1:20 with MEM before inoculation. Before these samples were inoculated, confluent MA104 cell monolayers were washed three times with MEM without serum. Excepting the controls, each well was inoculated with 0.1 ml of the diluted mixture. The plates were then incubated at 37°C for 60mins. The cultures were then washed three times with MEM without serum and fed with MEM without serum and containing 0.5mg of trypsin per ml. The plates were incubated at 37°C. Media were changed every other day. The plates were inspected everyday for the occurrence of cytopathic effects(CPE). When CPE was observed, the culture flasks were frozen, thawed, and then the culture lysates were centrifuged. The supernatants were collected and tested for the presence of rotavirus by PAGE. When no CPE was observed even after eleven days after inoculation, cultures were lysed by freeze-thawing and supernatents were obtained by centrifugation. These lysates were
trypsin treated as described above and inoculated into fresh MA104 cell cultures. This procedure was carried out till passage 6. All cell lysates were then tested by PAGE for rotavirus positivity regardless of the demonstration of CPE.
Results:
This study was carried out from March 1998 to January 2000. In this period, of the total 225 diarrhea samples collected, 36 samples were found to be positive for rotavirus by PAGE, giving rotavirus positivity of 16%.
Prevalence of rotavirus diarrhea cases:
Sex Total no. Percen- No. of Percen-
of diarrhea tage rotavirus tage
cases (n=225) diarrhea (n=36)
cases
225 36
Male 133 59.11 23 63.88
Female 92 40.88 13 36.11
It was found that all the detected rotaviruses belonged to group A. No atypical rotaviruses were observed.
Age-wise distribution of rotavirus diarrhea cases.
Age group Diarrhea Rotavirus Percentage
(months) cases diarrhea (n=36)
cases
<6 28 3 8.33
6-12 107 20 55.55
12-18 36 7 19.44
18-24 21 2 5.55
>24 33 4 11.11
Total no. of 225 36
patients
The age group of 6-12 months was found to be most susceptible to infection with
55.55% of the rotavirus cases occurring in this age group. Overall, the age group upto 24 months was more susceptible (88.88%) than the age group from 24 months to 5 years (11.11%).
Season-wise distribution of rotavirus diarrhea cases.
Seasons Total number Rotavirus Percentage
of cases cases (n=36)
Rainy (July-
October) 89 9 25
Winter
(November-
February) 74 17 47.22
Summer
(March-
June) 62 10 27.77
Total 225 36
Although rotavirus disease was recorded around the year, a slight seasonal variation was observed. 47.22% of the observed infections took place during the months of November to February.
Correlation of results by PAGE, latex agglutination, and in vitro isolation.
Samples Latex PAGE Cell culture
agglutination positive positive
positive
Patients 33 36 19
Controls Nil Nil nil
Rotavirus was detected in 36 samples
by PAGE, in 33 samples by latex agglutination, and in 19 samples by isolation in cell culture. Latex agglutination gave 4 false positive results and 7 false negative results as compared to the results given by PAGE. Considering PAGE as the standard test for detection of rotaviruses, latex agglutination showed a sensitivity of 87.87%. No rotavirus was detected by any of the methods in control subjects.
Discussion:
In the present study, the study subjects are pediatric patients admitted to three municipal hospitals in Mumbai. Though all these hospitals are in central Mumbai, they serve patients from all over the city. Our findings reflect incidence of rotavirus infections all over the city since municipal hospitals cater to lower and middle class society in Mumbai. The patients admitted to these hospitals all belong to the lower socio economic strata of society, with low literacy levels, no source of potable water, and poor hygiene and sanitary conditions. Hence it can be said that these factors aid in the spread of rotavirus disease. The factors that could be used to study the role of the virus in causing diarrhea in children under 5 years of age, and those that are taken into consideration in this study are the age and sex of the affected children and the seasonality of the virus.
In our study, the age group of 6 - 12 months was maximally represented with 55.55% of the rotavirus diarrhea cases occurring in this group. A maximum of cases might have occurred in this group as it is at this age that maternal antibody has just been lost and virus can cause disease. However, the rate
primarily in the American's. In the tropics, the seasonality of such infections is less distinct and within 10 degrees latitude of the equator, some locations exhibited no trend. A recent study carried out in Bangladesh has found no seasonal variation15.
In our study we have found the rate of proportion of rotavirus to be 16.0% by PAGE method. Since all the detected rotaviruses belonged to group A, we can safely assume that rotavirus infection in Mumbai as of yet is by the conventional strain. However, the absence of any atypical rotaviruses should not stop us from carrying out routine surveillance in order to keep a check on the strains of rotavirus circulating in the community.
We have considered PAGE to be the standard one because its sensitivity is more than 90% and its specificity is 100%16. Compared to this method, LA gave a sensitivity of 87.87% and specificity of 97.88%. LA can therefore be used as a bedside method for preliminary testing of stools for rotavirus. However, results from this test need to be confirmed by another method. Also, viruses belonging to groups B and C go undetected by this method of diagnosis.
Another method of diagnosis that we have tested is isolation of the virus in cell culture. The sensitivity of this method was calculated to be 52.77%. The specificity of this method could not be checked as only samples that were positive for rotavirus by PAGE were tested by this method. This method, besides being time consuming, has proved to be cumbersome as after every passage of the
virus in cells, PAGE had to be used to confirm the presence or absence of the virus as CPE is not always visible and hence is not very useful as a diagnostic tool. It is more suited to a research setting.
PAGE has been used to detect rotaviruses in many studies and PAGE of rotavirus can provide information on variation in rotavirus strain prevalent in the community. Also, in developing countries like India, where the cost of diagnostic kits like EIAs is prohibitive, PAGE becomes a handy tool in the hands of laboratory technicians. Besides being extremely sensitive and specific, it is also inexpensive with no requirement for fancy reagents.
If PAGE will be carried out as a matter of routine by representative hospitals in a city, the descriptive epidemiology of locally prevalent strains will be available to clinicians on a fortnightly or monthly basis. Such practice will yield laboratory surveillance of viral strains that will discriminate between bovine and human strains. Its potential is obvious when rotaviral vaccines will be needed to be manufactures on the basis of locally prevalent strains.
Key Message
A surveillance of this type if done as a matter of routine by representative hospitals in a city, can keep a check on the simultaneous co-circulation of multiple strains in the community, which may lead to extensive genomic variation in rotavirus strains. This information would be vital when rotaviral vaccines will be needed to be manufactured on the basis of locally prevalent strains.
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et al.
New complement-fixation test for the human reovirus-like agent of
infantile gastroenteritis. Nebraska calf diarrhea virus used as antigen.
Lancet 1975;1:1056-1061.
2. Kapikian AZ, Cline WL, Kim HW, et al.
Antigenic relationships among five reovirus-like (RVL) agents by complement fixation (CF) and development of new substitute CF antigens for the human RVL agent of infantile gastroenteritis.
Proc Soc Exp Biol Med 1976;152:535-539.
3. Yolken RH, Kim HW, Clem T, et al.
Enzyme-linked immunosorbent assay (ELISA) for detection of human reovirus-like agent of infantile gastroenteritis.
Lancet 1977;2:263-267.
4. Mata L, Simhon A, Urrutia JJ, Kronmal RA, Fernandez R, Garcia B. Epidemiology of rotaviruses in a cohort of 45 Guatemalan Mayan Indian children observed from birth to the age of three years.
J Infect Dis 1983;148:452-461.
5. Hung T, Chen G, Wang C, et al.
Waterborne outbreak of rotavirus diarrhea in adults in China caused by a novel rotavirus.
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heterologous resistance to human virus induced by bovine virus.
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7. Midthun K, Greenberg HB, Hoshino Y, Kapikian AZ, Wyatt RG, Chanock RM.
Reassortant rotaviruses as potential live rotavirus vaccine candidates.
J Virol 1985;53:949-954.
8. CDC.
Rotavirus vaccine for the prevention of rotavirus gastroenteritis among children - recommendations of the Advisory Committee on Immunization Practices.
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Rotaviruses.
In: Fields BN, Knipe DM, Howley PM, et al, eds. Fields Virology. 3rd ed. Philadelphia: Lippincott-Raven, 1996:1657-1708.
10. Herring AJ, Inglis NF, Ojeh OK, Snodgrass DR, Menzies JD.
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J Clin Microbiol 1982;16:473-477
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J Virol 1979;30:839-846
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Isolation of human rotavirus subgroups 1 and 2 in cell culture.
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Broor S.
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human rotavirus subgroups.
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Acknowledgements
We thank Dr.B.G.Khadse, ex-Director, Haffkine Institute, for making available excellent facilities to carry out the research.
We also thank Dr.(Mrs.)M.V.Kulkarni, Head, Dept. of Pediatrics, Lokmanya Tilak Memorial Hospital, Sion, Dr.(Mrs.)J.Kamat, Head, Dept. of K.E.M.Hospital, Parel, and Dr. Aigle, Medical Superintendent, Kasturba Hospital for Infectious Diseases, Mumbai Central.
EFFECTS OF RELAXATION TECHNIQUE ON SYMPATHETIC ACTIVITY IN PATIENTS OF MILD TO MODERATE HYPERTENSION
Dr. M.M. Jain, M.D.,*
Dr. V. Y. Deshpande, M.D.,**
Dr. Alka Deshpande, M.D.***
Dr. S. P. Gite, Ph.D.$
V.S. Keskar#
Abstract
Aim of the study
The aim of the study was to assess effects of Relaxation Technique (RT) on sympathetic activity in mild to moderate uncomplicated hypertensive patients.
Methodology
A prospective open label clinical study was undertaken. It was an ICMR sponsored short term undergraduate research projet. The project was approved by institutional Ethical Review Committee Subject for the study included mild to moderate, uncomplicated hypertensive patients who were attending medical OPD at JJ Hospital. After meeting inclusion criteria, each patient blood pressure pulse and respiratory rate was recorded. Patients were asked to practice mental and physical relaxation in the morning
as well as in the evening for about 20 minutes every day for two weeks. BP, pulse and respiratory rate was assessed every week for two weeks. The effect of relaxation was assessed by changes in BP, pulse and respiration before and after the intervention. Also, all the patients wre broadly divided in to sub-groups by direct questioning on their food habits and spirituality to evaluate effects of relaxation on such personal characteristics.
Results
There is significant decrease in diastolic BP and respiratory rate in all the patients. However, no significant change in systolic BP and pulse was observed. Among sub group of patients, spirituality and vegetarian food habits had significant reduction in diastolic BP, pulse and respiration while no significant difference was observed in non-vegetarian and no spirituality group.
* Associate profesor of Pharmacology.
** Professor of Pharmacology.
*** Professor of Medicine.
$ Statistician, Department of PSM
# Undergraduate student
At Grant Medical College & Sir J. J. Group of Hospitals, MUMBAI.
Reprint Request :
Dr. M. M. Jain, Associate Professor of Phermacology, Grant Medical College, Mumbai - 400 008.
e-mail : mangal_m_jain@hotmail.com
Conclusion
Physical mental relaxation might help in reducing sympathetic tone in some of the hypertensive patients within a short period of two weeks. This approach may be useful in management of some cases of hypertension.
INTRODUCTION
Indian Council of Medical Research encourage short term research project for undergraduate students to train them in research methodology under the guidance of their seniors1. It is desired that the research project so selected need to be simple, clinically relevant and ethically acceptable.
In recent years, hypertension has become one of the major cardiovascular disorders associated with increased morbidity and morality. According to one estimates as much as 5% of the urban population is suffering from various grades of hypertension with an estimated 10% below 35 years of age. Although, hypertension is due to multiple etiological factors ranging from neuropsychiatry and endocrine abnormalities to receptors and vascular defects2,3, sympathetic over activity seems to be a common denominator in most of the patients4. Sympathetic Nervous System is highly sensitive to acute stress known as Acute Phase Response characterized by rise in blood pressure, tachycardia, tachypnoea, hyperglycemia, sweating, restlessness, and anxiety5. Chronic stress may increase sympathetic tone and set it at higher level6. Thus it can be postulated that hypertension, in some patients, is the consequence of chronic physical or mental stress which is amenable to those measures that provide physical and mental relief7. Incidentally, ancient techniques such as transcendental meditation, yoga,asked to visit Medical OPD at weekly interval for 3 weeks. At the end of one week, baseline measurements for BP, Pulse & RR were made. Subsequently, patients were given a lecture cum demonstration on Relaxation Technique at Clinical Pharmacology Unit of the department. In addition, they also received a written hand out in their own mother tongue
pranayam and simple procedures to restore mental peace have shown beneficial effects in patients with hypertension8, Ischemic Heart Disease9,10, Hypercholesterolemia".
In the present study, we have tried to evaluate effects of Relaxation Technique (RT) on sympathetic activity in mild to moderate hypertensive patients using BP, Pulse and Respiratory Rate (RR) as parameters. We also evaluated effects of RT among subgroups of patients based on their food habits and spiritually Subgroups were formed by direct questioning and consisted of either Veg-Non veg or Spiritual-No spiritual groups.
MATERIAL AND METHODS
The study was carried out at Medical (OPD) of JJ Hospital. The protocol was approved by Institutional Ethics Review Committee.
A formal request was made to all the
doctors working in medical OPD to refer cases of mild
to moderate uncomplicated hypertension which is defined as Systolic BP between 130 to 180
mm of Hg and Diastolic BP between 90 to 110 mm of Hg. All patients were taking
antihypertensive drugs at least for one month and asked
to continue same treatment during entire period of the study. All patients participated in the
study have approval from their physician and also
gave informed written consent.
After screening and verification of eligibility criteria (Figure-1), patients were
asked to give details about their food habits and spiritual practices by asking direct
questions. Their BP, Pulse & RR were recorded after
they were made to sit quitely for 10 minutes. BP
was measured in supine position in right upper arm using same instrument by same
investigator taking muffling sound (Vth Korotkoffs
sound) as point for Diastolic BP. The patients were describing details of RT. The patients
were advised to practice relaxation technique for
20 minutes every day in the morning and evening. At the end of the second week the patients
were called for compliance reports about
relaxation practice. On third visit, i.e. at the end of
third week, the compliance report was reassessed. BP, pulse and respiration was recorded. (Figure
Fig. 1.: Selection criteria for the patients
Age - < 30 - 80> years
SBP - <130 - 180> mm of Hg
DBP - <090 - 110> mm of Hg
INCLUSION Informed written content
permission from the
treating physician 1 month on
SCREENING antihypertension treatment.
Hypertension related complications
EXCLUSION Associated Diseases.