This was a community based, cross-sectional study which was conducted in the Department of Microbiology and Immunology, Veer Chandra Singh Garhwali Government Medical Sciences and Research Institute and the attached H.N.B. Base Hospital, Srinagar, Garhwal, Uttarakhand, India from February 2011 to January 2012. The Garhwal region is located at the foothills of the Himalayas.

Our aim was to determine the average baseline antibody titre against the Salmonella enterica serotypes among the healthy people of various age groups in the Garhwal region. The study protocol and objectives were duly explained and after obtaining a written consent from the apparently healthy volunteers of both the sexes and of the age groups which ranged from 18 to 50 years, non-repetitive blood samples were collected (n= 2164).

The health screening was done by using a semi structured survey questionnaire. Those who were vaccinated for enteric fever in the preceding 3 years and those with a recent history of fever were excluded from the study.

Commercially available antigens which contained the Salmonella enterica subspecies enterica serovar Typhi O and H antigens, the Salmonella enterica subspecies enterica serovar Paratyphi AH antigen and the Paratyphi BH antigen were used (Span Diagnostics Ltd). Briefly, 0.5 ml of the 2 fold serially diluted sera (dilutions from 1:20 to 1:320) in 0.9% normal saline were tested by adding an equal amount of antigen and the tubes were than incubated overnight at 37°C in a water bath. The results were interpreted and analyzed as per the standard guidelines. A negative control was included in each batch of the tests. The Widal anti-O agglutinin (TO) and the anti-H agglutinin (TH) titres were taken as the highest dilutions of serum with a visible agglutination.

A total of 2164 healthy volunteers were screened for the agglutinins against the Salmonella enterica subspecies enterica serotypes, Typhi, Paratyphi A and Paratyphi B by the Widal tube agglutination test. 922 (42.60%) samples were positive for the agglutinins (≥1 in 20) whereas 1242 (57.40%) samples did not show agglutinins (≤ 1 in 20) [Table/Fig-1].

[Table/Fig-2] shows the distribution of the Salmonella agglutinin titre in the 922 sera of the healthy volunteers. The agglutinins to S. typhi were the most prevalent among the sera of various dilutions (42.6% for the H antigen and 32.25% for the O antigen) which were tested. The levels of the agglutinins for Salmonella paratyphi AH and paratyphi BH were low (only 3.04% and 1.3% for the AH and the BH antigens respectively).

[Table/Fig-3] depicts that the distribution of 698 samples with the anti-O titre of ≥1:20 to the Salmonella enterica serotype, Typhi showed that 182 samples (8.4%) had a titre of 1:20, 465 samples (21.5%) had a titre of 1:40 and 46 samples (2.1%) had a titre of 1:80, while only 5 samples (0.2%) had the highest titre of 1:160. Similarly, among the 922 samples which showed the anti-H titre of ≥1:20 to the Salmonella enterica serotype, Typhi, 213 samples (9.8%) had a titre of 1:20, 78 samples had a titre of 1:40 and 590 samples (27.3%) had a titre of 1:80. The highest titre of 1:160 was found in 41 samples (1.9%).

Altogether, 66 samples (3.04%) showed an agglutination titre of ≥1:20 against the H antigen of the Salmonella enterica serotype, Paratyphi A, among which 12 samples (0.6%) had a titre of 1:40 and the rest of the 54 samples (2.5%) had a titre of 1:20. Only 29 samples (1.3%) had an anti-H titre of 1:20 for the Salmonella enterica serotype, Paratyphi B.

The isolation of the various strains of Salmonella enterica subspecies enterica from blood remains the gold standard for the diagnosis of enteric/typhoid fever. However, in the modern era, there is an alarming upsurge in the empirical use of broad spectrum antibiotics, the practice of self-medication and the lack of proper timing for the specimen collection, that attributes to the reduced productivity of the blood culture technique. Also, in the developing countries, such as the Indian subcontinent, many clinics and hospitals do not have a ready access to the blood culture method, thus making the Widal tube agglutination test the most common alternative laboratory procedure for the diagnosis of enteric fever. The serological diagnosis relies classically on the demonstration of the rising titre of the antibodies in paired samples, 10 to 14 days apart. In typhoid fever, however, such a rise is not always demonstrable, even in the blood culture confirmed cases. This situation may occur because of the acute phase sample which is obtained late in the natural history of the disease, because of the high levels of the background antibody in a region of endemicity or because in some individuals, the antibody response is blunted by the early administration of an antibiotic [11]. Furthermore, the patient treatment cannot wait for long. For practical purposes, the treatment decision must be made on the basis of the results which are obtained with a single acute phase sample [12]. The cut off titre in a particular population depends on the background level of the typhoid antibodies and the level of the typhoid vaccination, which may vary with time [13].

The variation depends on the degree to which typhoid is endemic in each area, a fact which may change over time [7]. So, each country or region should have a baseline titre of their healthy population, which should be updated with time [4–10]. This was the first study which was done in the Garhwal region of Uttarakhand, India, to estimate the baseline antibody titre in the healthy population against various serotypes of S. enterica by using the Widal tube agglutination test. The results of this study showed that the sera of a significant proportion of healthy individuals in this area contained antibodies which were capable of reacting to the variable titres in the Widal test. Among the 2164 samples of the healthy volunteers among the local population, 42.6 % were positive for the agglutinins against various serotypes of Salmonella enterica. The agglutinins to S. typhi were the most prevalent among the sera which were tested at various dilutions. The most frequently recorded titre of the reactive samples was found to be 1:40 and this was considered as the cut off titre, as 21.5% of the study population had provided samples which were reacting at this level. For the H agglutinins, we observed that a majority of the study population (27.3%) had a titre of 1:80 and this was taken as the cut off titre. This study concludes that the current baseline titre for the diagnosis of typhoid fever in the Garhwal region is 1:40 for the anti-O agglutinins and that it is 1:80 for the anti-H agglutinins. Based on this finding, we have set our own laboratory guidelines of the H and O agglutinin Widal titres of 1:160 and 1:80 as being of diagnostic significance. The baseline anti-H agglutinin titre of the paratyphoid A and B groups was found to be 1:20 in 2.5 % and 1.3 % of the healthy population respectively, which suggested that the paratyphoid groups were less prevalent in this area as compared to S. typhi.

Our results were in concordance with those of the studies which were reported by some researchers in other endemic states of India [8,14,7]. For the anti-TH antibodies, an agglutinin titre of up to 1:80 was discovered in the apparently healthy study population, whereas for the anti-TO antibodies, our result was lower (a titre of 1:40), which was in contrast to the reports other workers [8,14, 7,15] but it was in agreement with the reports of some previous studies [9,4]. Several factors may have contributed to this discrepancy, because the differences in the antibody response may be due to the poorly standardized antigen preparation and the sharing of the antigen determinants with other Salmonellae [7]. A widespread antibiotic abuse can dampen the antibody response, giving a low titre in the Widal test and a previous immunization with the TAB vaccine and technical differences may be the other contributory factors. With respect to the last point, the Widal test which was performed on the same serum specimen in four laboratories gave widely different results [4]. The countries which have 439enough resources to support highly developed national typhoid fever surveillance systems generally experience a low incidence of typhoid fever as compared to the developing countries and they contribute little to the global burden of typhoid fever [16,17]. Proper hygiene and sanitation are the keys to a low prevalence of enteric fever in the developed countries, which can result in a low antibody titre [3].

It has been evident from the various studies which have been conducted across our country [Table/Fig-4] that the baseline titre is subject to variations, depending on the geographical area and the sanitary conditions of the region. Hence, the baseline titre of a particular area should be known. The probable reason for the low titre in our study could be the better health and hygiene conditions.

Several studies have highlighted the limitations of using the Widal serological test in the laboratory diagnosis of Salmonella, the worst being its nonspecificity. Despite this fact, considering the low cost and the absence of comparatively cheap tests, the Widal tube agglutination test is likely to remain the test of choice in many developing countries, as of ours, provided a baseline antibody titre of healthy individual in the population, is known.