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Introduction: Typhoid fever is a prevalent endemic disease in India, with the Widal test being a frequently utilized diagnostic tool. Given the widespread nature of typhoid in the region, healthy individuals may exhibit varying levels of antibodies that respond to the Widal test. This variation may result from previous exposure to the infection, TAB vaccination, or cross-reacting antigens from other diseases, which can differ across locations {1}. This study intends to assess the prevalence of the disease in the peripheral region of Maharashtra. Materials and Methods: Blood samples were obtained from blood donors between the period of February 2024 and March 2024. The samples were collected in plain tubes and tested for the presence of Salmonella antibodies using the Widal Tube agglutination test. Results: Out of 1150 serum specimens analysed, 461 (40.08%) tested positive for the Widal tube agglutination test, while 689 were negative. The predominant reactions observed were at 1:40 for anti-O antibodies, 1:80 for anti-H antibodies, and 1:40 for both anti-AH and anti-BH antibodies. The findings indicated that the diagnostic titres in the peripheral region of Maharashtra were 1:80 for anti-O antibodies and 1:160 for anti-H antibodies for enteric fever, along with a titre of 1:80 for paratyphoid fever. Conclusion: The study revealed that the diagnostic titres for enteric fever in the peripheral region of Maharashtra were identified as 1:80 for anti-O antibodies and 1:160 for anti-H antibodies, while for paratyphoid fever, the diagnostic titre was noted as 1:80.
Typhoid fever is a serious infection caused by the bacterium Salmonella enterica subsp. enterica serotype Typhi. contaminated food or water is the usual reason for transmission. After ingestion, the Salmonella Typhi bacteria reproduce and enter the bloodstream. This infection is a significant health concern in India and other developing nations, particularly in tropical and subtropical regions. It is estimated that over 100,000 deaths occur annually from paratyphoid fever, with around 9 million cases of typhoid reported each year, over 90% of which are in Asia.{2} Factors such as poor sanitation, contaminated food, and unsafe water sources heighten the risk of infection in these locations. Furthermore, atypical presentations of the disease in developing countries can complicate diagnosis, as patients often seek medical care later in their illness or self-medicate with over-the-counter drugs before consulting a healthcare professional. The definitive method for diagnosing enteric fever involves culturing S. Typhi or S. Paratyphi from a sterile site, such as blood or bone marrow.{3} However, the extensive use of cultures is constrained by cost and technical limitations. In many developing countries, the Widal test is frequently employed for diagnosis due to its simplicity, speed, and affordability. The interpretation of this test relies on the baseline titres of agglutinins to the O and H antigens of S. Typhi and the H antigen of S. Paratyphi, which are found in healthy individuals within specific geographic regions.{4} These baseline titres vary by location, influenced by the endemicity of enteric fever in those areas. Therefore, it is essential to regularly update the baseline Widal titres in a population for accurate test interpretation.
Objectives:
Type of Study: Cross-sectional
Place of Study: The research was conducted in the serology section of the microbiology department at a tertiary healthcare centre.
Subjects: A total of 1150 samples were collected from healthy blood donors who participated in various blood donation camps organized by the institution between February 2024 and March 2024. Blood samples were collected following the acquisition of written informed consent from the donors.
Inclusion Criteria: Samples were collected from both male and female donors. Blood donors were screened using a survey questionnaire and consented in writing. Blood samples were obtained from healthy donors who had not received the TAB (Typhoid and Paratyphoid A and B) vaccine or the oral typhoid (Vi) vaccine. Individuals with any active or recent infections, including hepatitis B, hepatitis C, malaria, dengue fever, brucellosis, and HIV/AIDS, were not considered for participation.
Exclusion criteria: Individuals who experienced any type of fever within the last 6 months, as well as those who did not meet the aforementioned criteria, were excluded from the study.
Method: A 5 ml venous blood sample was collected in a plain tube and allowed to clot at room temperature for approximately 30 minutes. The samples were subsequently centrifuged at 3000 rpm for 10 minutes to separate the serum from the blood. The serum was transferred to clean, dry, sterile storage vials and stored in a refrigerator at −20º C. A stained Salmonella antigen test kit for tube testing was supplied by Beacon Diagnostics Pvt. Ltd., India. Serial dilutions of the serum were carried out, ranging from 1/20 to 1/640, followed by the addition of one drop of the appropriate antigen suspension. 'H' agglutinations were incubated for 4 hours at 37ºC in a water bath and were evaluated after standing on the bench for 30 minutes, while 'O' agglutinations were incubated for 4 hours at 37ºC and assessed after overnight refrigeration at 4ºC. The highest dilution of serum exhibiting visible agglutination was noted as the endpoint, with the titre represented as the reciprocal of the dilution. For quality control purposes, known positive and negative control sera were incorporated in each run.{5}
The baseline titre was determined using the Widal tube agglutination test. The quantity of antigen suspension used matched that of the diluted sera, resulting in a 2-fold dilution, initiating our readings at a titre of 1:40, by the manufacturer’s instructions and the standard lab protocol adhered to by the Microbiology department at the institution where the study was conducted.
Blood culture is considered the most reliable method for identifying different strains of salmonella enterica subspecies and diagnosing enteric/typhoid fever. Unfortunately, the growing reliance on antibiotics is detrimental and a crucial time for collecting samples has diminished the accuracy of microbial isolates traditionally obtained from blood culture procedures. In developing countries like India, a significant number of hospitals lack convenient access to blood culture.However, the diagnosis relies on monitoring the increase in titres in samples collected at 10-14 day intervals. Consequently, the treatment of patients cannot be delayed for an extended period. Consequently, the choice of treatment should be based on the serological response results, making the Widal tube agglutination test the most commonly used and widely accepted test for diagnosing enteric fever. The occurrence of typhoid fever is influenced by various factors that can fluctuate over time, including the availability and usage of typhoid vaccines. This modification in typhoid prevalence in each region can vary and may evolve. {6}
Based on our research, this is the first study conducted in the peripheral region of Maharashtra to determine the initial antibody levels in humans for salmonella enterica serotypes typhi, paratyphi A, and paratyphi B. Our research indicates that there are varying levels of antibodies against salmonella enterica in healthy individuals, and a substantial number of healthy individuals in this region possess antibodies that can recognize the different titres in the Widal test.
Among the 1150 samples of healthy blood donors in the local population, 40.08% of the sera tested positive for the agglutinins against different serotypes of salmonella enterica. The antigens to S.typhi were the most common among the sera that were tested at different dilutions. Out of 461 (40.08%) healthy individuals with positive titres for at least one antigen. we found that 197 (42.73%) had anti-O antibody titres of ≥ 1: 40 and 179(38.82%) had antibody titres of ≥ 1: 40 against anti-H antibody Salmonella Typhi antigen. The most frequently observed baseline antibody titres against the O antigen and H antigen of salmonella enterica serotype were 1: 40 and 1: 80, respectively. Our findings align with the research conducted by scientists in other states of India, such as Karnataka and Uttarakhand {7}{8} where they measured the baseline titres of S and H antigens in S. Typhi were discovered to be 1: 40 and 1: 80, respectively, in both states. In the research carried out by Sreenath et al. at Kollam Kerala, the majority of samples showing seropositivity were positive in dilutions 1:40 for TO as well as TH antigen{9}. We were able to observe that 5. 85% and 12. 58% of individuals were showing antibodies against the paratyphi A and paratyphi B antigen respectively. In the peripheral region of Maharashtra, the anti-H antigen was lower than it was in the central region. A total of 58 people showing antibodies against Paratyphi B. A total of 27 individuals showed high levels of anti-H against Paratyphi A, which indicates that it is not a usual type in the peripheral region of Maharashtra. Nineteen per cent of the study population was reacting at this level. The majority of agglutination for O antigen was 1:40 in the study population and this is considered a cut-off for O antigen. For H antigen majority of baseline titre was 1:80. We have set our laboratory guidelines of the O and H agglutinin Widal titres of 1:80 and 1:160 as being of diagnostic significance based on this finding. An agglutinin ratio as high as 1:80 was observed in the seemingly healthy study population, while the anti-TO antibody levels in our findings were lower, differing from the reports of other workers.{10} The reports of previous studies were in correlation. Various factors may have led to this inconsistency, primarily due to differences in the antibody response, which could stem from inadequately standardized antigen preparations and the overlap of antigen determinants with other Salmonella species. (11) Additionally, widespread misuse of antibiotics can diminish the antibody response, significantly contributing to the rise of antibiotic resistance in cases of enteric fever. Many food handlers who don’t follow all the personal hygiene measures can also be carriers of Salmonella. (12) Proper preventive measures shall be taken by all of them. Lack of preventive measures and hospital admissions can increase the healthcare expenditure in cases, many comorbidities like diabetes, immunodeficiency etc can make patients susceptible to these infections. (13) Many public health measures should be taken for increasing awareness and utilization of preventive measures by community. (14-16)
This disease is predominantly transmitted via the faecal-oral route, and maintaining proper hygiene and sanitation is crucial for minimizing its prevalence in developed nations. Although the effectiveness of the Widal agglutination test can vary, it remains a valuable diagnostic tool when used in suitable contexts. (17, 18) Therefore, it is essential to establish and periodically update the baseline Widal titre for specific regions to ensure accurate interpretation of Widal test results. Consequently, regular assessment of the Widal baseline titre among healthy individuals in a defined geographical area should be conducted. (19, 20)
The study revealed that a significant proportion (40.08%) of healthy individuals in the peripheral region of Maharashtra exhibited a positive Widal test, indicating widespread baseline antibody titres likely due to past exposure, vaccination, or cross-reactivity. Based on the observed titre distribution, the diagnostic cut-off values for enteric fever in this region were identified as 1:80 for anti-O antibodies and 1:160 for anti-H antibodies. For paratyphoid fever, the diagnostic titre was determined to be 1:80 for both anti-AH and anti-BH antibodies. These findings underscore the importance of establishing region-specific baseline titres to enhance the diagnostic accuracy of the Widal test. In endemic settings like Maharashtra, using uniform national or international cut-off values without local validation may lead to misinterpretation of results, overdiagnosis, or unnecessary treatment. Therefore, this study contributes valuable epidemiological data that can help refine diagnostic criteria for typhoid and paratyphoid fevers in similar endemic zones.
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