This cross sectional study was conducted in the Department of Microbiology, Dr. Vikhe Patil Medical College and Hospital, which is a tertiary care hospital with 700 beds, which is located at Ahmednagar, Maharashtra, India. Approval of the institutional ethical committee was obtained for carrying out this study.

Informed written consent were taken from the donors for the study.

Participants who were apparently healthy were only included as study subjects. All participants were given questionnaires to fill. All the donors who did not have any obvious signs and symptoms of infectious diseases were included in the study.

Those with obvious signs and symptoms were excluded.

Participants who were already vaccinated for Salmonella were excluded from study.

The following study was undertaken to determine the baseline Widal titre (titre of the antibodies to the ‘O’ and ‘H’ antigens of Salmonella typhi and the ‘H’ antigens of Salmonella paratyphi A and B) amongst apparently healthy individuals in and around Ahmednagar. It also aimed to define the significant titre for the Widal agglutination test for the diagnosis of enteric fever in an endemic area, in a single serum test.

Health screening of the volunteer donors, was done by using a questionnaire. Randomly selected, non-repetitive 103(n=103) blood samples were collected from healthy blood donors who were in the age group of 18 to 60 years, of both the genders, who attended our blood bank from January 2012 to February, 2013.

About 2ml of blood was taken from the tube of each bag, that was not diluted with CPDA-1 which was present inside the blood bag. Serum was separated immediately, labeled and stored at 4°C for further processing. The serum samples were processed according to standard tube dilution method of the Widal test. Commercially available antigens which contained the “O” and “H” antigens of S. typhi and “H” antigens of S. paratyphi A and B were used, which were procured from Span Diagnostics Ltd. The test used two fold serially diluted sera of patients, dilution being from 1:20 to 1:320. All serum samples were first diluted in a 1:20 ratio with isotonic normal saline (NaCl 8.5gm./liter), in such a way that final volume contained a total of 1ml. For each sample, further 4 dilutions of 1:40, 1:80, 1:160 and 1:320 were made. Then, one drop (0.03ml) each of the appropriate antigen suspensions was added to the corresponding tubes. A known positive control, negative control and antigen control were also set up with each batch of the test. All the tubes were mixed well and they were incubated at 37°C overnight (16-20 hours.). After completion of incubation period, the tubes were examined for agglutination. When antigen suspensions were mixed and incubated, anti-Salmonella antibodies which were present in the serum reacted with the corresponding antigens to give agglutination. The ‘O’ antigen being a somatic antigen, brought about a coarse, compact, granular agglutination, whereas ‘H’ antigen being a flagellar antigen, brought about a large, loose, fluffy, agglutination. The antibody titre was reported as the highest dilution of serum which showed distinct agglutination.

The purpose of this study was to find out the baseline antibody titre in the population and to develop a local recommendation for interpretation of the Widal test results. This was the first study of its type which was done at Ahmednagar, to the best of our knowledge. A total of 103 sera were tested. Among these, 84 (81.55%) showed agglutinations at a titre of ≥1:20 for the “O” and “H” antibodies against Salmonella typhi, Salmonella paratyphi A or Salmonella paratyphi B. The rest of the 19 did not show agglutination. The number and percentage of sera with corresponding end titres for agglutinins against Salmonella typhi, Salmonella paratyphi A and Salmonella paratyphi B, has been presented in [Table/Fig-1].

Eighty four (81.55%) of the sera were found to be positive for agglutinins for the Salmonella serotypes. Our findings were quite higher than those which were reported by other investigators. A comparative analysis of samples which were positive for agglutinins for Salmonella serotypes has been presented in [Table/Fig-2].

The highest level of titre for TO, TH, AH and BH was found to be 1:320. Our findings were in accordance with those of Madhusudhan NS [12], who observed that the highest level of titres for O, H and AH was 1:320. High titre of antibodies in our study may be caused by infections with cross reacting organisms other than Salmonella. For example, cross reaction between a normally harmless gut bacterium, Citrobacter freundii, can confuse the diagnosis of typhoid bacilli which share antigenic properties [13].

Sera of 31.64% and 33.87% samples showed titres of ≥ 1:40 for TO and TH respectively, whereas highest number of samples i.e. 38.46% and 36.84% respectively, showed titres of ≥ 1:80 for AH and BH. Therefore, as is evident from the above findings, the baseline titre for “O” and “H” antibodies of Salmonella typhi was found to be 1:40. Shekhar Pal et al., [10] and Madhusudhan NS [12] also reported a baseline titre of 1:40 for TO, but of 1:80 for TH. A comparative analysis of baseline titres of O and H agglutinins has been presented in [Table/Fig-3].

The cutoff level for a single antibody titre in the present study was ≥1:80 for TO and TH, which was in accordance with the finding of Prashant Peshattiwar [11] but which varied from that of Bahadur et al., [14], who reported that the cutoff level for TO and TH was ≥1:320. The cutoff level for AH and BH was ≥1: 160. The variations in the titres could have been caused by differences in endemicity of typhoid in each area.

The baseline titre for antibodies to “O” and “H” antigens of Salmonella enterica serotype typhi was 1:40 and hence, based on the above results, it could be recommended to use a cutoff level of ≥1:80 for a single antibody test titre. Similarly, baseline titre for antibody to H antigen of Salmonella enterica serotype paratyphi A and paratyphi B was 1:80 and the cutoff level was ≥1:160 for a single antibody test titre.

It is clear that Salmonella agglutinins are common among apparently healthy people and as endemicity of typhoid in an area may change over time, more studies should be carried out to determine Salmonella agglutinin titre in apparently healthy populations, so that a better judgment which is based on the prevailing agglutinin titres can be made.