Culture positivity and antibiotic sensitivity pattern of typhoid fever in children aged 1 to 10 years: A 10-year retrospective study from a tertiary care centre of Eastern India

Background: Over the last few decades there has been a change in the trend of antibiotic sensitivity of Salmonella typhi from being sensitive to first line drugs like chloramphenicol, ampicillin and cotrimoxazole to developing multi-drug resistance. Most strains are presently sensitive to third generation cephalosporins and azithromycin but are developing resistance against fluoroquinolones. Objectives: To determine the culture positivity and antibiotic sensitivity pattern of Salmonella typhi Method: This retrospective s tudy was conducted at the Institute of Child Health, Kolkata from July 2014 to December 2020. Nine hundred and twenty children aged 1-10 years were included in the study, all with fever for more than 5 days. These children were suspected to have typhoid fever clinically i.e., with history, suggestive physical examination and after excluding other common febrile illnesses with routine blood test as per hospital protocol. Blood culture was done to confirm the diagnosis of typhoid fever, determine the proportion of blood culture positive cases and to find out the antibiotic susceptibility of Salmonella typhi in our hospital set _________________________________________ 1


Introduction
Typhoid fever is a multisystem infection caused by Salmonella enterica serovar typhi. Each year around 27 million cases get affected worldwide with around 1% mortality 1 . Majority of these cases are from the developing countries of Asia 2 . Over the last few decades there has been a change in the trend of antibiotic sensitivity of Salmonella typhi from being sensitive to first line drugs like chloramphenicol, ampicillin and cotrimoxazole to developing multidrug resistance (MDR). Most strains are presently sensitive to third generation cephalosporins and azithromycin but are developing resistance against fluoroquinolones 3.4 .
Diagnosis of typhoid fever may be initially challenging due to its varied presentation 4 . Blood culture is the gold standard for diagnosis and the highest yield is in the 1st week of illness (90%) and is 75%, 60% and 25% in 2nd, 3rd and 4th week respectively 5,6 . However, due to inadequate laboratory facilities, cost and technical difficulties to obtain a good culture sample from smaller children, culture is not frequently done in developing countries. Positivity of blood culture may be further compromised by prior antibiotic usage. Sensitivity and specificity of the Widal test being low, in most cases, treatment is empirical 6 . This leads to improper and inadequate use of antibiotics and the development of MDR typhoid.
In the last two decades MDR Salmonella typhi has become a serious threat 7 . These strains are resistant to ampicillin, chloramphenicol and cotrimoxazole. Thus, the antibiotics of choice are 3rd generation cephalosporins and fluoroquinolones 8 . Unfortunately, due to indiscriminate use of these drugs in the last few years, resistance has emerged against fluoroquinolones. There are no recent studies regarding the antibiotic sensitivity pattern of salmonella typhi from this part of India.

Objectives
To analyse the antibiotic sensitivity pattern of Salmonella typhi and also to document the changing sensitivity pattern over a period of 10 years.

Method
This retrospective study was conducted at the Institute of Child Health, Kolkata from July 2014 to December 2020. Nine hundred and twenty children aged 1-10 years were included in the study, all with a history of fever for more than 5 days. These children were suspected to have typhoid fever clinically i.e., with history, suggestive physical examination and after excluding other common febrile illnesses with routine blood test as per hospital protocol. Widal test was done routinely. Blood culture was done to confirm the diagnosis of typhoid fever, determine the proportion of blood culture positive cases and to find out the antibiotic sensitivity pattern of Salmonella typhi in our hospital set-up. Children were included in the study irrespective of prior antibiotic treatment.
Ethical issues: Approval for the study was obtained from the Institutional Ethics Committee for Biomedical and Health Research, Institute of Child Health, Kolkata, India (No. ICH/ IECBMHR/ 20/ 2022 dated 29.06.2022). As this was a retrospective study informed consent was not a possibility.

Statistical analysis: Data entry was done in
Microsoft Excel and was statistically analysed using SPSS software. Descriptive statistical data like means, medians and standard deviations were calculated for continuous variables. Table 1 shows the gender distribution of cases. There was no statistically significant difference of gender between the study population and culture positive cases and no gender predilection of typhoid fever in our study. Table 2 shows the age distribution of cases. Table 3 shows the distribution of culture positive cases among the total study population.  To compare the number of culture positive cases in children less than 2 years of age and 2-10 years of age we did Chi square test. The result was not significant (p>0.05). Whilst 65% of cases <2 years of age were culture positive compared to 51% of cases 2-10 years of age, this was not statistically significant. Table 4 is a comparison of the duration of fever at presentation in culture positive and negative cases. There was no significant difference between mean duration of fever between study population and culture positive cases (p= 0.24). Table 5 and Figure 1 show the pattern of antibiotic sensitivity of Salmonella typhi among culture positive cases. In this study Salmonella typhi was fully sensitive to ampicillin, chloramphenicol, cefixime, ceftriaxone and azithromycin. It was found to be highly sensitive to cotrimoxazole. It was highly resistant to ciprofloxacin and nalidixic acid.

Discussion
In our study, out of total 920 children 504 (54.8%) were male and 416 (45.2%) were female. Among total 488 blood culture positive cases, 280 (57.4%) were male and 208 (42.6%) were female. However, we did not find any significant gender difference like most of the previous studies.
We included children aged 1 to 10 years with a mean age of 4.87 years and grouped them as less than 2 years and 2-10 years. We excluded infants from our study. In present study we found that 136 (14.8%) cases were 1-2 years old and 784 (85.2%) were 2 to 10 years old. Among total 488 culture positive cases, 88 (18%) were 1-2 years old and 400 (82%) were 2 to 10 years old. We observed that 64.7% had culture positivity in children less than 2 years of age compared to 50% in the older group. However, this was not statistically significant. We found that for culture positive cases the mean duration of fever was 8.44 ± 2.72 days and for culture negative cases 9.59 ± 2.97 days. In contrast to the general belief that blood culture is usually positive in 1 st week of illness, we found it to be also positive in the 2 nd week. Thus, blood culture should not be discouraged if the patient presents late or with prior antibiotic treatment.
We found that Salmonella typhi in our set-up was fully sensitive to ampicillin (100%) and chloramphenicol (100%) and highly sensitive to cotrimoxazole (95% Thus, there is increased resistance to fluoroquinolones due to their over-the-counter use in the last few years. In the present study we found complete sensitivity to third generation cephalosporins and azithromycin like most of the recent studies. With these findings, use of empirical antibiotic use may have to be revised and use of first line drugs should be re-considered to prevent rise of drug resistance and complications.
Typhoid in children remains a significant health issue. Though limited with small number of sample size our study attempted to address few emerging issues of typhoid fever. A good number of children came to be culture positive even after late presentation and prior antibiotics especially in the younger age group. Re-emergence of susceptibility of Salmonella typhi was documented with reversal of MDR. However, rise of fluoroquinolone resistance was threatening. Larger population-based studies are essential to assess the current situation in a deeper and more extensive manner.

Conclusions
There was complete sensitivity of Salmonella typhi to third generation cephalosporins and azithromycin. It was also sensitive to older and less commonly used antibiotics like chloramphenicol, cotrimoxazole and ampicillin. There was increased resistance to fluroquinolones.