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Crump J. A. et al. The global burden of typhoid fever. Bulletin of the World Health Organisation (2004),82: 346-353.

Felix A. and Pitt R.M. Virulence and immunogenic activities of B.Typhosus in relation to its antigenic constituents. J. Hyg. (1935), 35: 428-436.

Gupta, A .Swarnkar ,N.K. et al. Changing antibiotic sensitivity in enteric fever J. Tropical Pediadtr.(2001), 147:369-398.

Hoffman, S. et  al. Bone marrow aspirate culture superior to streptokinase clot culture and 8 ml 1:10 blood- to- broth ratio blood culture for diagnosis of Typhoid fever .Am J.Trop. Med. Hyg(1986), 35: 836-839

Kossaczka, Z. et al. Synthesis and immunological properties of Vi and Di-O-Acetyl Pectin protein conjugates with adipic acid dihydrazide as the linker. Infect immunity (1997),65:2088-2093.

Kossaczka, Z. et al. Safety and immunogenicity  of Vi conjugate vaccine for Typhoid fever in adults, teenagers and 2 to 4 years old children in Vietnam. Infect Immunity (1999), 67:5806-5810.

Lin F. Y. et al. The efficacy of a Salmonella typhi Vi conjugate vaccine in 2 to 5 year old children.  Engl J.Med,(2001) 344: 1263-1269.

Lanh, M. N., et al. Persistent efficacy of Vi conjugate vaccine against typhoid fever in young children. N .Engl. J.Med. (2003) 349 :1390-1391.

Robbins J.D.,Robbins J.B. Reexamination of the protective role of the capsular polysaccharide (Vi antigen)of Salmonella typhi. The journal of infectious Diseases(1984),150 :436-449.

Saha ,M. R. et al. A note on incidence of Typhoid fever in diverse age groups in Kolkata, India. Jpn. J . infect. Dis.(2003),56: 121-122.

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Introduction 
The studies were undertaken to assess the immunogenicity and safety in infants and older children of a new vaccine-Vi polysaccharide conjugated with Tetanus toxoid protein, developed by Dr. Puneet Garg & his associates at BIO MED(P) ltd, Ghaziabad, INDIA. This new technology has avoided the use of recombinant proteins for conjugation. The vaccine has passed the required safety & immunological parameters in animals. The permission to conduct clinical trial Phase III in human was cleared by the Drugs Controller General (India) after necessary evaluation.

Methods & Materials
Open , multicentric, controlled & comparative study was undertaken. The three consecutive batches of Vi conjugate typhoid vaccine (Peda Typh^TM), used for conducting the clinical trial, were found to be of standard quality by Central Drugs Laboratory, Central Research Institute, Kasauli, H.P. of Government of India. The Vi polysaccharide typhoid vaccine manufactured by BIO-MED (P) LTD., was also tested for comparative assessment studies. The study protocols were approved by the ethics committee of eminent medical colleges located in three widely separated zones of India.

Details of three medical colleges where the clinical trials were conducted.

Selection of volunteers
The subjects were selected randomly from different economic strata of the society. All children taken in the study were normal, in good health & clinical state.

Group A-Vi conjugate typhoid vaccine (Peda Typh^TM)
Peda TyphTM was administered to infants > 12 weeks of age by intramuscular route. 169 volunteers were vaccinated and observed for local and systematic side effects, paired serum samples from 145 volunteers were collected.

Group B-Vi polysaccharide typhoid vaccine
Vaccine was administered to children = 2 years of age by intramuscular route. Thirty seven volunteers were vaccinated and observed for local and systemic side effects, paired serum samples from 29 volunteers were collected.

Evaluation of safety
Local side effects (at the site of injection) : Pain, erythema and inflammation and
Systemic side effects : Fever. diarrhoea, vomiting and any other were recorded 30 minutes, 4 to 6 hours and 48 hours post vaccination. Scaling of adverse reactions was done as following :

Scaling of adverse reactions was done as following :

Of the total 169 subjects vaccinated with Peda Typh^TM-147 subjects were in category “excellent”, 22 subjects in category “good”. There were no case in “fair” and “bad” categories on the scale of adverse reactions.

There were no serious adverse event leading to premature withdrawal from the study. None of the subjects had inflammation, one subject had erythema at site of injection. Local reactions were confined to mild transient pain which resolved without any sequella.

Three subjects had fever >38.30 C. The temperature rise became normal within 24 hours post vaccination without any medication.

Based on the above data, the principle investigators concluded that Peda Typh^TM (Vi Conjugated Typhoid vaccine) was safe & well tolerated in all age groups including infants.

Immunological Response
Evaluation of Immunogenicity :
Blood samples(1-2ml) were collected by venipuncture before vaccination on day 0(pre-immune) and 4 weeks after vaccination (post immune). lgG anti Vi antibodies were detemined by ELISA.

ELISA test : (Assay for assessment of immune response in paired serum samples)
ELISA test kit was validated and calibrated as per the guidelines of good laboratory practice.

Serum lgG Vi antibodies were assayed by ELISA and expressed in ELISA units relative to a standard reference. The unitage of the standard reference was assigned by NICHD, NIH, USA.

Preparation of standard reference curve and calculation of lgG antibodies was done by program of ELISA version 2.0, Centers for Disease control , Atlanta(USA).

Biostatistical Analysis : Biostatistical analysis of the clinical trial of Peda Typh^TM was done by Dr.R.M. Pandey Ph.D.,FRSS(U.K.),Professor and Head, Department of Bio-Statistics, All India Institute of Medical Sciences ,N.Delhi. Biostatistical analysis proved that Peda Typh^TM vaccine is safe and immunogenic. The antibody titers of the sera from 100% of the subjects (from all three centers) showed a four fold or greater rise in antibody titer of each group after immunization .No statistically significant differences were found in male and female children.

Results are graphically presented in figure 2. The lgG anti-Vi antibodies of Peda Typh^TM were 97.425 ELISA units in infants and children 3 months to 24 months of age. The geometric mean (95% confidence interval) of all volunteers was 70.32(62.86-78.66).

The Vi conjugate typhoid vaccine (Peda Typh^TM) under clinical trial have been found to be highly immunogenic in infants and children less than 2 years of age in which unconjugated Vi polysaccharide typhoid vaccine is known to induce very low or nil immunogenic response.

The efficacy of Vi conjugate typhoid vaccine in clinical trials conducted in Vietnam have been found to be 89% over the 46 months period.

On comparing the data of clinical trial of Vi conjugate typhoid vaccine developed by N.I.H (Published in The New England Journal vol.344 no. 17, 2001 pg.No.1263-1269) with Peda Typh^TM manufactured by BIO-MED(P)LTD., it was found that the geometric mean post immune lgG(25-75 percentile) are statistically equivalent.

Immunizations not only prevent mortality and morbidity. They also reduce the expenditure of public and private resources. The latest generation Vi conjugate typhoid vaccine is an effective tool to control the emerging pattern of typhoid fever in children and infants <2 years of age.

The above study has been published in :
Garg P., Garg S., Sharma M.K (2014), Clinical trial of Tetanus Toxoid Conjugated Vi Polysaccharide Typhoid Vaccine in infants and young children, Sharma et al (2014) Biotechnology International 7(4) : 90-100.

Further post licensure studies on Peda Typh^TM has been done in SRM Medical college Chennai by Dr. Balaji Chinnasami et al. Dr. Monjori Mitra et.al conducted a large scale Safety Immunogenicity & Efficacy study in Municipal school children in Kolkata (highly endemic area).

The studies have been published in :

1. Chinnasami B., Mangayarkarasi V., Prema A., Sadasivam K. & Davis M.J. (2013). Safety and immunogenicity of Salmonella typhi Vi conjugate vaccine (Peda Typh™) is children upto five years. International Journal of Scientific and Research Publication, Volume 3, issue 2, February 2013.
2. Chinnasami B., Sadasivam K., Vivekanandhan A., Arunachalam P. & Pasupathy S. (2015). A study on Longevity of Immune Response after vaccination with Salmonella typhi Vi Conjugate Vaccine (Peda Typh™) in children. Journal of Clinical & Diagnostic Research. 2015 May, Vol-9(5).
3. Mitra M., Shah N., Ghosh A., et al (2015). Efficacy and Safety of Vi-Tetanus Toxoid Conjugated Typhoid Vaccine (Peda Typh^TM) in Indian Children: School Based Cluster Randomized study. Human Vaccine & immunotherapeutics 2016, VoI. 0,No. 0, 1-7

The highlights of the result of the studies are as under :

• Efficacy of Peda Typh has been found to be 100% versus 33 cases of typhoid in control group over a follow up period of 1 year.
• One dose of the vaccine was found to give protective immunity in infants & children . No significant advantage of two doses regimen over one dose was found.”as per Dr. Chinnasami in his study “ A study on Longevity of Immune Response after vaccination with Salmonella typhi Vi Conjugate Vaccine (Peda Typh™) in children.
• Serum analysis of post licensure follow up study at SRM Medical College using Peda Typh™ showed adequate immune response 30 months post vaccination with Single dose – 14 (4.8 – 29.8) µg/ml (which is much greater than earlier seroprotective level 3.52 Elisa unit equivalent to 4.36mcg/ml or current seroprotective level 1.4 µg/ml-2µg/ml).

Typhoid vaccines are known to the medical science for more than a century. Wright in 1896 introduced typhoid vaccine for the first time. Vi polysaccharide of S. typhi was recognized as the only important antigen involved in inducing protective antibodies Felix & Pitt(1935).The new type of typhoid vaccines were developed by reexamination of the protective role of the capsular polysaccharide (Vi antigen) Robbins J.D., Robbins J.B.(1984).

Vi Polysaccharide typhoid vaccine

Vi antigen also called as the virulence antigen of Salmonella typhi  has been described as an important antigen capable of inducing immune response in children >2 years of age. Extensive clinical trial conducted in Nepal showed that the Vi polysaccharide is immunogenic and efficacious.

This vaccine was introduced in India in 1995.It contains a very small component of the outer layer of the bacteria (Vi polysaccharide), which provides high degree of protection , with almost minimal adverse effects profile. Additionally instead of two injections with whole cell type vaccine ,Vi polysaccharide typhoid need only one injection. This vaccine provides protection which lasts for at least  2 years. Hence booster injection is recommended every 3 years.

Since Vi Polysaccharide typhoid vaccine does not induce immune response in infants and children below 2 years of age, hence they had remained unprotected against typhoid fever so far.

Research by Szu, S.C. et al. (1987),Kossaczka Z.et al.(1997,1999) led to the development & availability of Vi conjugate typhoid vaccine in India (Peda Typh^TM) for the first time in the world by Bio-Med (P) Ltd.

Vi conjugate typhoid Vaccine -Peda Typh^TM

The need to find an alternative has been on for past several years. The scientists of the N.I.H.,U.S.A. & the A.I.I.M.S., New Delhi, India had been successful in developing Vi polysaccharide conjugated with recombinant protein antigens e.g.

1. Recombinant exotoxin A of P.aeruginosa.
2. ‘OMP’ of S.typhi produced in E.Coli.

Research by Szu, S.C. et al .(1987),Kossaczka Z.et al.(1997,1999) led to the development and availability of Vi conjugate typhoid vaccine in India (Peda Typh^TM) and for the first time in the world.

BIO-MED scientists have developed a unique process of conjugation using a time tested natural protein Tetanus toxoid. This method has proved very effective and safe as tested in animal studies and in the multicentric clinical trials on infants, children and adults.

The Vi conjugated typhoid vaccines have been demonstrated to induce ‘T’ cell dependent response with much higher antibody levels providing protection in >90% Lin F.Y.et al .(2001), Lanh M.N. et al.(2003).

The Peda Typh ^TM availability promises control of typhoid in infants, children and adults. This vaccine is amenable to booster vaccination and is expected to confer much better grade immunity in much higher percentage of vaccinees.

Until recently , typhoid was considered a disease of children above 3-5 years since it presented itself in an atypical clinical picture in infants and young children. This was further hampered due to difficulty in getting enough blood samples for appropriate culture isolation. The laboratories were not well equipped with good quality media, reagents & trained personnel. Introduction of bone marrow sampling using very sharp needles for culture identification has made detection of typhoid cases more amenable Hoffman S.et al.( 1986).

Reports on a typical clinical picture of typhoid in infants :

• Sinha, A.et al.(2004) defined typhoid fever in infants as fever which lasted more than 5 days, with no response to antimalarial therapy, coupled with bradycardia and splenomegaly. Almost 30% of such cases suspected for typhoid fever were found to be due to para-typhoid.

• Sen, S.K. and Mahakur, A.C.(1972) reported that continuous temperature hepato- splenomegaly and bradycardia were less frequent in children,however pulmonary signs, lymphadenopathy, hyperpyrexia and toxic tongue were more frequent. Children suffered more from complications like constipation followed by diarrhoea and tympanitis. Pulmonary complications e.g. bronchitis, bronchopneumonia, pneumonia and neurological complications like meningitis, encephalopathy, convulsions, typhoid state were more frequent in children.

Due to these atypical clinical presentation , clinicians usually resort to arrive at a diagnosis by a process of elimination of possible disease like malaria etc. to call the case as of typhoid fever.

Recent reports confirm that infants and children below 2 years of age are highly susceptible to typhoid fever and constitute a high percentage of cases of typhoid fever.

Reports of typhoid fever in infants and children

A large number of studies have revealed high incidence of typhoid in infants and children below 2 years of age :

• Sinha,A. et al.(1999) reported in their studies in Delhi area, the highest number of typhoid cases in children below 5 years (27.3%) compared to 11.7% in 5-19 years and only 1.1% in 14-40 years age group.
• Saha ,M.R. et al.(2003) reported in their studies in Kolkata area that children between 2-3 years age group are the most susceptible(35.6%).
• Gupta, A. et al.(2001) reported significant number of patients were below 3 years age, the youngest being an infant of 7 months in Jaipur area.
• Saha, S.K., Baqui, A.H. et al.(2001) reported in Bangladesh that majority (54.5%) of culture positve typhoid cases were from children younger than 5 years. Out of these 27% were in first two years of life (0-5 months=0.8%, 6-12 months=8.7%, 13-24 months=17.5%)

All the above referred studies are strong pointers to the fact that typhoid causes serious disease in infants for which there was no specific vaccine available for active immunization till now.

The hygiene is very difficult to maintain for infants & toddlers, thus exposing them to more chances of getting infected. The extra cautious approach in the use of antibiotics in children makes the job of pediatrician very complicated.

Typhoid fever exhibits in various forms. Typically, it starts with malaise, anorexia, muscular  aches and fevers,that increases everyday by 0.25-0.5⁰C to reach to 39 to 41⁰ C coupled with abdominal pain & headaches. Fever rises to 41⁰ C in 5-7 days where it stays for 10-15 days if untreated, then fever decreases slowly over several days.

During the period of rise in fever, some patients (20%) with fair skin show “rose spots” of 2-4 mm in the chest, abdomen & back areas. Constipation is typically observed.

In blood – leukocytes are below 4500/mm³ & platelets less 80,000/mm³ . Liver dysfunction may be present and can be detected by elevated serum transaminase values.

Two of most important complications are perforations & hemorrhage as a result of intestinal lesions (See figure).

Other uncommon complications are hepatitis, empyema, osteomyelitis & psychosis. Particularly severe form of typhoid can cause cerebral dysfunction, delirium or coma and shock, case fatality in such cases can exceed 20%.

About 2% to 5% patients become chronic gallbladder carriers of the organism where it has been implicated as a cause of cancer.

The epidemiology of the typhoid fever primarily involves person to person spread because this organism lacks an animal reservoir. Faecal contamination is the major mode of spread and the usual vehicle is contaminated water. An asymptomatic human carrier state exists.

The carrier state may last for many weeks to years providing human reservoir. The incidence of chronic carrier state is around 3% Christie, A.B. et al.(1974). Occasionally food handled by an individual who is carrier of S.typhi may be the vehicle.

The famous case study of “Typhoid Mary” who gave rise to hundreds of typhoid cases during her life time is a classic example of chronic carrier.

Typhoid fever has been reported from all parts of the world. With the improvement in sanitation and standard of living, the disease is now uncommon in developed countries. However, in developing and under-developed countries, it is still one of the major infectious disease having significant mortality and morbidity. It has been estimated that globally about 16 million cases of typhoid fever are reported annually causing about 6,00,000 deaths Crump J.A. et al.(2004).In addition to this, the socio-economic implications of the disease are huge because of following reasons :-

1. Complete recovery takes several weeks.

2. Emergence of multi-drug resistant strains contributing to escalating cost of treatment. It may also contribute to higher morbidity and increased mortality.

3. Recent research reveal that the INFANTS and children up to 5 years of age suffer most from typhoid fever than in any other age group.

A large population particularly infants remained unprotected against typhoid fever Since there was no vaccine available for this age group and children less than 2 years of age.

Spread of the disease can be controlled by improving sanitation, water supply and standard of living, however, this is almost impossible to implement in many of the countries including India because of financial , social and technical reasons. Under these circumstances, the ideal way is to provide through active immunization.

Introduction of Vi polysaccharide typhoid vaccine with much lower reactogenicity. has led to marked increase in acceptance of vaccination as a preferred method to prevent typhoid fever. Still a large population particularly infants remained uncovered since there was no vaccine available for children and infants less than 2 years of age.

Further the Vi antigen includes immunity for about 2-3 years only, thus revaccination is required.

Conjugated Vi polysaccharide typhoid vaccine can be used to vaccinate 3 month or older infants with very high levels of protection. With Vi conjugated vaccine, it is expected that immunity will last for much longer period due to establishment of memory, and immunity may be boosted by natural exposure to S.typhi.