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.

A survey on community perceptions of jaundice in east Delhi: implications for the prevention and control of viral hepatitis.

Using senior health professionals as interviewers, a 30-cluster sampling survey was carried out to investigate community perceptions of pilia (the local word for jaundice) in east Delhi (India). Of 416 persons (mostly mothers of children aged < 2 years) interviewed, 339 (81%) were aware of pilia as an illness. Only 322 (77%), 164 (39%), 73 (18%) and 71 (17%) people knew about correct symptoms, dangers, causes and prevention of pilia. Most of the correct responses were related to the faeco-orally transmitted viral hepatitis. Literate respondents were significantly more aware of pilia (chi 2 52.81, P < 0.0001), its symptoms (chi 2 48.88, P < 0.0001), causes (chi 2 39.34, P < 0.0001), dangers (chi 2 19.3, P = 0.0007), and prevention (chi 2 60.8, P < 0.0001). However, age of the respondents had no significant bearing (P > or = 0.05) on the correctness of responses. About 293 (70%) subjects considered pilia as a treatable illness; of them, 193 (66%) and 77 (26%) respectively expressed their preference for the 'modern' and indigenous systems of medicine for its treatment. In contrast, 110 (38%) respondents said that they would prefer faith healers for the treatment of pilia. Although only 31 (7%) persons were aware of a vaccine against pilia (hepatitis B vaccine), virtually all agreed to have their children immunized if such a vaccine were made available. The study underscores the usefulness of pilia in lay-reporting of viral hepatitis and epidemiological studies on jaundice-associated illnesses and the need for educating the community about its causes and prevention to increase people's participation in controlling viral hepatitis and other diseases that mainly manifest as jaundice.

Evaluation of potency of inactivated cholera vaccine by mouse protection assay & antibody induction method.

Twenty one batches of whole cell inactivated cholera vaccine manufactured at Central Research Institute, Kasauli were evaluated for potency by mouse protection assay (MPA) and antibody induction method. In the antibody induction method the sera of immunized mice were screened for the presence of antibodies against Vibrio cholerae by microagglutination (MA) test and IgG ELISA. The number of organisms estimated by MPA were correlated with agglutinating and neutralizing antibodies against individual serotypes by MA and ELISA respectively. Correlation coefficient(r) of 0.692 and 0.815 were observed for the titres evaluated by MA and ELISA when compared with standard MPA method for the serotype Ogawa. Similarly r values of 0.925 and 0.849 were observed for titres evaluated by MA and ELISA when compared with standard MPA method for the serotype Inaba. Antibody induction method can be as an alternative method for determining the potency of inactivated cholera vaccine.

Insecticide susceptibility status of Culex tritaeniorhynchus giles, vector of Japanese encephalitis in Delhi.

Laboratory studies were carried out to ascertain the current susceptibility status of adult and larval stages of the Culex tritaeniorhynchus mosquito, vector of Japanese encephalitis, to various insecticides used under public health programs in India. The present study revealed that exposure of adult mosquitoes to diagnostic concentrations of DDT - 4.0%, malathion - 5.0%, fenitrothion - 1.0%, and propoxur - 0.1% could induce only 50.0, 10. 0, 15.0, and 5.0% mortality, respectively, indicating that the species was resistant to all of these insecticides. The LT50 and LT95 values calculated using diagnostic concentrations of DDT, malathion, fenitrothion, and propoxur were found to be 56.4 and 136, 138 and 272, 185 and 258, and 187 and 249 min, respectively. However, when adult mosquitoes were exposed to the diagnostic concentration of synthetic pyrethroids, viz., deltamethrin - 0.025%, permethrin - 0.25%, and lambdacyhalothrin - 0.1%, 100.0% mortality was observed, indicating that the species was highly susceptible to these adulticides. Larval susceptibility tests carried out using diagnostic dosages of DDT- 0.008, temephos- 0.02, fenthion- 0.008, fenitrothion- 0.125, and malathion- 0.005 mg/l failed to induce any mortality, indicating that larvae were resistant to these larvicides. The LC50 and LC90 values calculated for commonly used larvicides, viz., temephos and fenthion, were 0.1511 and 1.9098, and 0.6151 and 2.395 mg/l, respectively. Increase in tolerance level were estimated at 95.5- and 299.4-fold when these LC90 values were compared with diagnostic dosages of temephos and fenthion, respectively.

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.

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.

Induction of interferon in mice and cell cultures by Mycoplasma pneumoniae.

Inoculation of mice and L and human embryonic lung (HEL) cell cultures with Mycoplasma pneumoniae failed to induce the production of interferon. M. pneumoniae multiplied in these cell cultures without a marked cytopathic effect. M. pneumoniae induced interferon in human peripheral blood leukocytes with maximum titres of 32 units/ml at 48 hours after infection.

Factors affecting the sensitivity and reproducibility of passive haemagglutination test for the quantitation of measles-specific antibodies.

Various factors affecting the passive haemagglutination test (PHA) for the quantitation of measles-specific antibodies have been evaluated with the aim to obtain maximum sensitivity and reproducibility of the test. The antigen used for sensitization was prepared in Vero cells using Edmonston 245 strain of measles virus. Sheep red blood cells (SRBCs) were found most sensitive for use in PHA test. The optimum dilution of tannic acid was found to be 1:40,000 for tanning of fixed and 1:10,000 for unfixed SRBCs, when the tanning was carried out at 4 degrees C overnight. Fixed and tanned SRBCs sensitized with 32 HA units of measles HA antigen at 56 degrees C for 30 mins were found optimal. SRBCs from different sheep affected the sensitivity of the assay. Stability study of SRBCs showed that storage at -70 degrees C of glutaraldehyde-fixed and sensitized SRBCs gave better results as compared to those stored at -20 degrees C and +4 degrees C. Tanned SRBCs could be stored at -70 degrees C only up to 15 days. Sensitized SRBCs with-stood two cycles of freezing and thawing after removal from -20 degrees C and -70 degrees C. Sensitized SRBCs could be stored for 120 days without any significant loss of titer at -20 degrees C, +4 degrees C or 22 degrees C; when lyophilized with stabilizers, there was a slight decline in the titer after exposure at 37 degrees C for 30 days. The lyophilized sensitized SRBCs after reconstitution were found to be stable at +4 degrees C for 3 days without any loss in the titer.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplacental immunity and waning of maternal antibody in measles.

Since transplacental immunity and waning of maternally derived measles specific antibodies play an important role in determining the optimum age for vaccination of infants against measles, a study was carried out in which 150 paired samples and 581 infant serum samples were tested for measles specific antibodies. Out of these paired samples, 132 pairs showed measles antibodies in both mother and cord. HAI antibody was absent in 3 paired samples whereas, 5 mothers could not pass on the antibodies in the cord samples. In the remaining 10 serum samples only cord blood showed the presence of antibodies without the detectable level of antibodies in mother. Statistically no significant difference between the mother and cord blood titers was observed by applying the student 't' test for comparison of the mean (t = 0.01). Analysis of 581 infant serum samples for prevalence of maternal antibodies indicated that 83% of the samples at the age of 3 months or below had measles antibodies but with the increase in age there was tremendous loss with only 19-20% at the age of 6-7 months. After 7 months the percentage of infants which had antibody varied from 11-13%. There was negative correlation between age and seropositivity (r = -0.72) which was highly significant (p < 0.05).

Adverse events following immunization: 1990.

The vaccines used under the Immunization Programme are safe and effective. However, as no vaccine is 100% effective, none is entirely without risk. The benefits of immunization greatly exceed the risks because of the large number of complications and deaths prevented. Although, the risks of vaccine-associated adverse events are extremely low, the occurrence of such events in areas with high immunization coverage levels and low incidence of vaccine preventable diseases can influence public acceptance of immunization services. Moreover, since infections and neurological syndromes are common in the age-groups in which immunizations are given and there are a number of contacts with an infant under the programme, there is a risk of temporally-related severe adverse medical events being attributed to immunization. Monitoring of adverse events is essential to document low risks and to identify programmatic errors, if any, for corrective action. An important, aim of the monitoring system is to disseminate such information to the medical professionals and others associated with the immunization programme. The paper summarizes reports received in 1990, including reports of temporal events where the cause of death was other than immunization.

Control of neonatal tetanus in India.

PIP: Neonatal tetanus (NNT), a disease preventable by immunization, is a major problem and a leading cause of neonatal mortality. Sample surveys were organized in 1981 and 1982 by the Directorate General of Health Services in India, in collaboration with the state health authorities, to collect reliable baseline data on NNT. The surveys were carried out by the 30 cluster sampling technique. Rural and urban populations were surveyed separately. The survey was conducted by restrospective house to house visits. In each unit, a sample of 2000 live births was studied; 67 live births being recorded in each cluster. Based on the survey results, it is estimated that 2.3-2.5 lakh infants died within the 1st month of life due to NNT during 1981; nearly 2/3 of these were in Uttar Pradesh. Although the NNT mortality rates varied from state to state, they were consistently higher in the rural areas. Tetanus as a cause of neonatal deaths ranged from 0-68.7% in the urban areas and from 16.4-72.5% in the rural areas. The control of tetanus in general and neonatal tetanus, in particular, is receiving a high priority in the government programs. 2 major programs are in operation for the prevention of NNT in the country -- the immunization of pregnant women with tetanus toxoid vaccine (TT) under the expanded program on immunization (EPI) and the training of dais under the rural health program. NNT will be prevented if the women and the dais (who are still associated with almost 70-75% of the deliveries in many areas with high NNT mortality rates) are convinced of the need for TT vaccination during the antenatal period and practice the basic principles of cutting cord and keeping the umbilical stump free of unclean dressings. The vaccination of pregnant women was started as a national program in 1975-76 with a modest coverage of 14.5 lakhs. The number of pregnant women given either 2 or a booster dose in 1982-83 had risen to 74.0 lakhs. The plan is to cover 110 lakhs pregnant women in 1983-84 and 130 lakhs in 1984-85. Besides the vaccination of pregnant women, TT immunization services are provided to infants and children. Since the protection received from the mother will last only for a few months, infants are given TT as a combined vaccine with diphtheria toxoid and pertussis vaccine (DPT) at 3 months of age. The immunization program is a part of primary health care and the services are provided through the existing health delivery systems. The vaccination services are available at the hospitals, maternal and child health clinics, and dispensaries in the urban areas and in the primary health centers and the subcenters in the rural areas. The effectiveness of the control measures will be evaluated by determining the vaccination coverage of the eligible population and by the documentation of the reduction of NNT mortality rates in the area.^ieng

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.

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.

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 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.

Elimination of neonatal tetanus: 1995.

India aims at the elimination of neonatal tetanus by 1995 by providing TT immunization services to all pregnant women in the country. Strengthening of the surveillance system is part of the strategy for the elimination of neonatal tetanus. There is a wide range of performance at state and district level. Many states have the potential of achieving the difficult and challenging goal of neonatal tetanus elimination within a few years.

PIP: The situation of neonatal tetanus in India is discussed in terms of the status of the immunization program, surveillance of neonatal tetanus and its contribution to the infant mortality rate, and goals of the neonatal tetanus prevention program for the early 1990s. Neonatal tetanus is atypical because it is often underreported, its risks and rates are highly non-uniform, and a high coverage of immunization does not confer "herd" immunity. In India before 1980 tetanus immunization coverage was negligible. Tetanus immunization was integrated with the Immunization Programme in 1981-1982, emphasizing school children, especially girls in their last classes and pregnant women. People in rural areas who live near animals, deliver at home, and are not attended by trained dais are at highest risk. Neonatal tetanus has been reported separately by age since 1989, when 7096 cases were reported, probably 5% of the total. Special tetanus vaccination and neonatal tetanus surveys estimate that the neonatal tetanus mortality rate is 1/1000 live births, or 10% of the infant mortality rate in areas surveyed. The NNT mortality rate ranges from 1.9 per 1000 live births in Kerala, a state where most deliveries are institutional, to 66.7 per 1000, or 71.7% of all neonatal mortality, in rural areas of Uttar Pradesh. As a result of the immunization program, tetanus coverage has surpassed 70% in half the states. Since 95% of the NNT mortality occurs in 3 states, the near-term goals are to maintain the surveillance system of neonatal tetanus and poliomyelitis, to identify districts with low coverage and intensify efforts there, and to investigate suspect cases and target unimmunized women with vaccine and better perinatal care.

Immunogenicity in monkeys of two polio type 3 seed viruses (SO + 2 and SOR + 1) presently used for production of oral polio vaccine.

Rhesus monkeys were immunized orally and intramuscularly with polio type 3 seed viruses (SO + 2 and SOR + 1). No immune response was detected after oral feeding. Geometric mean titres of 891.4 and 237.02 were obtained after intramuscular administration of six and eight doses of SO + 2 and SOR + 1 respectively. All the monkeys immunized with SO + 2 and SOR + 1 were positive for antibodies after third and fifth dose respectively. High antibody titre (1:1024) was reached in 80 per cent monkeys after sixth dose with SO + 2 whereas this antibody titre was obtained in 20 per cent monkeys after eight doses of SOR + 1. SOR + 1 was found to be a poor immunogen as compared to SO + 2 as it had produced low titre antibody response in monkeys even after administration of eight doses.

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.

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.