Standardization of serum free media for cryopreservation of RK13 cells.

Foetal calf serum present in the media used for cryopreservation was replaced by various synthetic polymer such as gelatin, glycerol, carboxymethyl cellulose and dimethyl sulphoxide at various concentration. Growth pattern of cells, % survival and karyological studies have been done in the present study. It was found that optimum concentration of carboxymethyl cellulose was 0.1% in combination with 10% glycerol and 10% DMSO. At this concentration percentage survival of cells was found maximum and karyotype was found normal without any abnormality in the chromosomes. It was concluded from the study that serum free media can be employed for the cryopreservation of these cells which are further used for production of tissue culture vaccines without causing any adverse affects.

Epidemiological characteristics of rabies in Delhi and surrounding areas, 1998.

OBJECTIVE: To describe the epidemiological characteristics of rabies in Delhi in 1998. METHODS: Analysis of the records of hydrophobia cases admitted to the Infectious Diseases Hospital, Delhi (IDH) in 1998. RESULTS: About 46 percent (99/215) of the hydrophobia cases admitted to the IDH in 1998 belonged to Delhi. The remaining came from the adjoining states, both urban and rural areas. In Delhi residents, overall hospitalization rate was 0.81 per 100,000 population. It was significantly higher in 5-14 year old than in other age groups and in males than in females (p <0.0009). Cases occurred round the year. Almost 96 percent cases (206/215) gave history of animal exposure, 13 days to 10 years (median 60 days) before hospitalization. Majority (195/206) had class III exposure. Animals involved were stray dog (193/206 = 90 percent), pet dog, cat, jackal, mongoose, monkey and fox. Most of cases were never vaccinated (78 percent) or inadequately vaccinated (22 percent); only 1 percent each received appropriate wound treatment, or rabies immunoglobulin. CONCLUSIONS: Rabies is a major public health problem in Delhi. Its incidence is significantly higher in 5-14 year old children than in other age groups. The results indicate the need to educate the community and health care workers about the importance of immediate and adequate post-exposure treatment and to start an effective control program for dogs, the principal vector of rabies.

Community studies on prevalence of HBsAg in two urban populations of southern India.

OBJECTIVE: To find out prevalence of HBsAg in general population, especially in under-five children. SETTING: Bangalore and Rajahmundry towns in southern India. METHODS: Localities were chosen as the sampling units in each town. About 10-20 households were randomly selected from each locality. Only the youngest but apparently healthy person present in the household was interviewed for age, sex and history of jaundice any time in life. Mothers were interviewed to collect data for children below 15 years of age. Blood samples were collected from these persons on filter paper strips (18-mm diameter disc, Whatman filter paper No. 3) by finger prick method. The samples were tested for HBsAg by Micro ELISA (Ortho-Clinical Diagnostics). RESULTS: Overall, 3.3% (95% CI, 2.0-4.5) of 737 persons in Rajahmundry and 4.2% (95% CI, 2.8-5.5) of 816 persons in Bangalore were found carriers of HBsAg. Age-specific or sex specific carrier rates were similar in Rajahmundry as well as in Bangalore. Most of the carriers (96%) denied having jaundice ever in life. CONCLUSIONS: The results from this community based study are in agreement with the historical data from hospital based studies that about 3-5% of persons may be carriers of HBsAg and that the pool of chronic carriers of hepatitis B virus in India is built up in childhood and is then maintained in older children and adults. The results highlight the need of completing hepatitis B immunization during the infancy.

Epidemiological characteristics of poliomyelitis in Delhi, 1997.

OBJECTIVE: To describe the epidemiological characteristics of poliomyelitis in Delhi in 1997 after four consecutive statewide immunization campaigns with oral polio vaccine (OPV). METHODS: Stool samples were collected from 158 cases of acute flaccid paralysis (AFP) along with their age, sex, residential address, immunization history and dates of onset of paralysis, reporting and investigation. The samples were processed for isolation of polioviruses. In addition, historical data on vaccination coverage surveys and OPV testing were reviewed. These data were analyzed to understand the epidemiological patterns of poliomyelitis in Delhi. RESULTS: Of 158 cases of AFP, about 23% were investigated within 2 days of onset of paralysis. Two samples each were collected from 97 (61%) cases, and one each from the remaining cases. Detection of 158 cases of AFP gave an incidence of 1.34 per 100,000 population. About 36% (57/158) of AFP cases excreted poliovirus, mostly (53/158) wild poliovirus. Of the wild poliovirus isolates, 72% (38/53) and 25% (13/53) were serotypes P1 and P3 respectively; 2 isolates were P2. Almost 95% (146/154) of AFP cases and all the laboratory confirmed cases (excreting wild poliovirus) occurred in children below 5 years of age. Only one-third of AFP (55/158) or laboratory confirmed cases (18/53) had received 3 or more doses of OPV before onset of paralysis. About one-fourth of cases in both the categories were totally unvaccinated. AFP cases occurred round the year but peaked in November-December. Peaks were always observed during July-August in the past. The cases were widely scattered without any obvious clustering in any locality. CONCLUSIONS: Poliomyelitis has declined substantially in Delhi. The study underscores the need for further efforts to improve vaccine coverage levels, AFP surveillance, and cold chain maintenance to achieve the complete interruption of transmission.

Transplacental transfer of measles antibody in Delhi.

OBJECTIVE: To find out the patterns of and the factors, if any, affecting the transplacental transfer of measles antibody. DESIGN: Comparison of measles antibody titres in mothers with titres in cord blood samples. METHODS: Maternal and cord blood samples from 174 full-term pregnant women of middle socio-economic status were tested for hemagglutination inhibition (HI) antibody against measles in Delhi during October 1993 to January 1995. None of the mothers had been immunized against measles. RESULTS: Antibody were undetectable in both maternal and cord samples in only 4 (2.3%) pairs. Mean maternal titre was found to be 2.94 Log2. Transplacental concentration and dilution were respectively observed in 34% and 26% of the samples. Cord titres were more often higher than the maternal values only if the maternal values were low. Overall, cord/maternal ratio of mean titre (Log2) was found to be 1.06. Although the age of the mother and parity had had no significant bearing on the transplacental transfer of measles antibody, cord titres were significantly more often higher than the maternal values as the birth weight increased (Chi-square for linear trend = 5.4; p = 0.02). CONCLUSIONS: The study failed to show appreciable concentration of measles antibodies across the placenta.

Merit of mobilization technique in transfer of non-autotransferable R-plasmids.

A total of 200 urinary isolates of Esch.coli received at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli during the years 1995 to 1997 were studied for transferable drug resistance. Out of 188 strains, 134 strains showing resistance to either Nalidixic acid or Rifampicin were subjected to autotransferable resistance studies. Of these 134 strains 131 showed either partial or enbloc transfer of R-factor. Mobilization experiment successfully transferred resistance marker in 14 of the 68 isolates in which resistance to one or more drugs could not be transferred during conjugation experiment.

Prevalance and resistance pattern of Salmonella serotypes in India.

During the period 1990-91, 3222 Salmonella strains were identified at the National Salmonella and Escherichia Centre (NSEC) at Central Research Institute, Kasauli. Of these, 2894 were from humans, 226 from poultry, 84 from animals and remaining 18 from reptiles, birds and other sources. These strains belonged to 53 different serotypes. These include 4 serotypes reported for the first time in India, namely S. kedogou, S. VP. bornheim, S. kisarawe and S. madras. Drug resistance studies revealed that 573 strains were sensitive to all the antibiotics commonly used, 1351 single drug resistant, 594 resistant to two drugs and 704 were multidrug resistant. One strain from human stool was resistant to all the antibiotics used. Prevalence of various Salmonella serotypes and their response to various drugs is discussed.

Snake bites in the hills of north India.

BACKGROUND: Snake bites are a common cause of morbidity and mortality in the hills. The risk of snake bite is high due to the presence of a huge herpeto fauna flourishing in a favourable climate--low environmental temperature and heavy rainfall. In India, there are 236 species of snakes of which four are dangerously venomous. This study was undertaken to determine the risk factors exposing the population to snake bite and the common types of snakes causing them. METHODS: We studied 243 patients of snake bite over a period of 24 months. All patients were examined for evidence of snake bite and, where possible, the snakes were identified based on description, identification (if the snake was brought) and symptoms of envenomation. RESULTS: Seasonal variation in snake bite was seen, with a peak in the months of August and September. No bites were recorded in December, January and February. Eighty-four per cent of the bites were on the hands and feet (up to the ankle). Bites on the hand were more common in females with a left hand preponderance (3.5 times higher). The age group most affected was between 11 and 40 years (73.7%). Most bites occurred while the person was cutting grass, working in the fields or walking in the hills (75.3%). Snake bites while sleeping were at uncommon sites. Non-poisonous snakes were the most common (90.5%). Kraits caused 60% of bites with envenomation. CONCLUSION: Snake bites occur frequently in the hills of Himachal Pradesh. Although snake bites are a cause for concern, most of them are caused by non-poisonous snakes.

An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

Quantitation of tetanus and diphtheria antitoxins in mouse sera by indirect haemagglutination.

Serum samples obtained from 75 groups of mice immunized with various doses of adsorbed tetanus vaccine, adsorbed diphtheria-tetanus vaccine and adsorbed diphtheria-tetanus-pertussis vaccine were titrated for tetanus antitoxin content by an in-vitro indirect haemagglutination (IHA) and by toxin neutralization test (TN) in mice. From these serum samples of 49 groups of mice which were immunized with combined vaccine containing diphtheria toxoid were titrated for their diphtheria antitoxin content by IHA and by i.d. toxin neutralization test (TN) in guinea pigs. Good correlations were found between the estimates obtained by in-vitro IHA and in vivo TN tests in both tetanus and diphtheria antitoxin titrations. The minimum level of tetanus or diphtheria antitoxin detectable by IHA was 0.00039 IU/ml. It is concluded that IHA is a simple, sensitive and reproducible alternative test which can replace the animal TN tests for the estimation of tetanus and diphtheria antitoxins and could reliably be used in the potency assay of tetanus and diphtheria toxoids of combined vaccines based on antibody induction in mice.

Alternative approach and evaluation of three potency assay methods for testing adsorbed tetanus toxoid.

Thirteen batches of adsorbed Tetanus Toxoid (TT) from different manufacturers were tested for potency by three different methods viz: (i) An Antibody Induction Method (AIM) developed in mice: (ii) WHO lethal challenge in mice; and (iii) Conventional Antibody Induction (I.P). Method in guinea pigs. The potency results obtained in AIM, by serological evaluation of immunized mice were found identical and correlated significantly with those obtained by WHO recommended lethal challenge test in mice. The potency data obtained in the present study was found comparable with other studies. An AIM in mice thus offers an alternative to lethal challenge tests and can replace guinea pig model. Out of 107 serum samples obtained from immunized guinea pigs in the conventional antibody induction method, 90% samples contained more than 4 units of tetanus antitoxin per ml. End point titres of 42 serum samples belonging to 5 batches of TT also showed much higher tetanus antitoxin content when determined by TN test. The potency data obtained thus suggest revision of the minimum requirement in Indian Pharmacopoeia which is too low and which may be increased as indicated by the present study.

Isolation of poliovirus from cases of acute flaccid paralysis.

OBJECTIVE: To evaluate the frequency of isolation of polio and other viruses from fecal samples in subjects with acute flaccid paralysis in northern parts of India. SETTING: Rural and Urban Immunization Centers used for Expanded Programme on Immunization. METHODS: 219 fecal samples were collected by anal tube in 1992 from Punjab, Haryana, Himachal Pradesh and Chandigarh and processed for virus isolation. RESULTS: Of 219 fecal samples, 103 (47%) were positive for viruses in the age group of 3 mo to 5 yr. Amongst the positive samples, the predominant isolated was poliovirus type 1 (70.9%) from all the States. Non polio enteroviruses were isolated from 20.4% of fecal samples. CONCLUSION: Even though poliovirus is still the most common etiologic agent for acute flaccid paralysis in northern India, non polio enteroviruses are also emerging as important causal pathogens in this condition.

Adjuvant effect of DEAE-dextran and tetanus toxoid on whole cell heat inactivated phenol preserved typhoid vaccine.

Active mouse protection test (AMPT) and enzyme linked immunosorbent assay (ELISA) were used to determine the immunogenicity of whole cell typhoid vaccine when administered in conjunction with either tetanus toxoid (TT) or DEAE-Dextran (DD). Immunization of mice with whole cell typhoid vaccine showed enhanced potency either when administered in conjunction with TT or DD and values were statistically significant (p < 0.05) in comparison to conventional or standard typhoid vaccines. For ELISA, the mice were immunized with 2 different schedules, one in which a single dose of 0.25 ml subcutaneously (s/c) was administered and in another two doses of 0.25 ml each s/c, 14 days apart. In case of single dose schedule of immunization D vaccine (Whole cell typhoid + 5 mg/ml DD) showed significant increase of immune response (3.201 log10) as compared to plain vaccine (2.550 log10). Two dose schedule further increased the titres to 3.856 log10. DD adjuvanted vaccine showed higher potency by AMPT as compared to the TT adjuvanted vaccine or plain vaccine. The present study clearly demonstrates that a single dose of 0.25 ml which is equivalent to half of the conventionally used single human dose of typhoid vaccine adjuvanted with DD can significantly improve the immunogenicity of the vaccine.

Adverse reactions after measles vaccination in India.

BACKGROUND. One hundred and sixty million doses of measles vaccine have been used since the Expanded Programme of Immunization began in 1985. Mild-to-moderate vaccine reactions are not infrequent and are accepted by parents but occasionally severe reactions and even death occur. We studied these severe reactions. METHODS. From 1986 to 1994, 1762 batches of measles vaccine were tested and found to be satisfactory by the World Health Organization criteria. These were released for mass immunization. RESULTS. After 40 reported incidents of severe reactions or deaths in the field, 59 intact samples of vaccine produced by different manufacturers were tested and found to be safe, i.e. they were not toxic and were sterile. However, on testing reconstituted or used vials a few were found to be toxic and many were unsterile. Reactions occurred in 115 vaccinees resulting in death of 79 children. These reactions were characterized by high fever, vomiting and profuse watery diarrhoea resulting in death within 24 hours. CONCLUSIONS. Reactions to the vaccines were more likely to be related to the toxic shock syndrome due to the use of unsterile syringes and needles and perhaps the use of reconstituted vaccines beyond their specified time for administration resulting in contamination.