Introduction

Typhoid is a life-threatening systemic infection caused by Salmonella enterica serovar Typhi (S. Typhi), which causes an estimated 11–21 million illnesses and 135,000–200,000 deaths annually in low- and middle-income countries (LMICs) [1–3]. The majority of cases and deaths occur in LMIC settings with limited access to clean water, poor sanitation, and overcrowded living conditions. In 2018, the World Health Organization recommended that countries with a high burden of typhoid introduce typhoid conjugate vaccines (TCVs) [4], which have demonstrated high efficacy in randomized trials [5–7]. Several countries have now introduced typhoid conjugate vaccines (TCVs) into national immunization programs. However, the population level impact of such introductions is not yet clear.

Nepal is a high typhoid burden country, with estimated incidence between 250 and 1,000 cases annually per 100,000 persons [8,9]. One of the pivotal Phase 3 trials assessing the effectiveness of TCVs was completed in Lalitpur, in the Kathmandu Valley [5] and on April 13, 2022, Nepal became the fourth country in the world to introduce TCVs nationally. The TCV roll-out began with a campaign targeting children aged 15 months to 15 years and was followed by introduction into the routine immunization program alongside the second dose measles and rubella (MR) vaccine at 15 months [10]. The catch-up campaign was estimated to have achieved greater than 90% coverage nationally [11].

In the two years prior to Nepal’s introduction of TCV, the COVID-19 pandemic impacted the country’s health services and disease surveillance systems. Mitigation measures including lockdowns, control of public gatherings, and closures of educational institutions and public spaces (restaurants, malls, etc.) may have altered transmission patterns of many pathogens beyond SARS-CoV-2, including typhoidal Salmonella. Reductions in enteric disease notifications during the COVID-19 pandemic have been reported in several countries [12–19]. Distinguishing reductions in infections from declines in notifications due to altered health care-seeking behaviors or weakening of surveillance systems can be difficult. In this context, understanding the impact of TCV introduction on typhoid incidence requires careful analysis.

In Nepal, the Surveillance for Enteric Fever in Asia Project (SEAP) performed systematic surveillance for typhoid at multiple hospitals in and around the Kathmandu Valley from 2016 – 2024 [8]. We leveraged this system to investigate the impact of TCV introduction amid changing typhoid burden during the COVID-19 pandemic. This study provides a unique opportunity to assess typhoid trends over a seven-year period encompassing the pre-pandemic, pandemic, and post-vaccine introduction phases, using comprehensive blood culture surveillance data from multiple healthcare facilities in and nearby the Kathmandu Valley.

Methods

Results

A total of 62,236 blood cultures were performed across four sites during the study period of January 2018 to April 2024. Of these, 31,307 (50.30%) were performed pre-pandemic (January 2018 to March 2020), 10,051 (16.15%) were performed during the pandemic (April 2020 to March 2022), and 20,878 (33.55%) were performed post-TCV introduction (April 2022 to April 2024). Among the participants, 46.30% (28,817/62,236) were female. The majority of participants (87%) were ages 16 or older (Table 1). Overall, 3.56% (2,216/62,236) cultures were positive for a pathogen. S. Typhi was the most common pathogen identified with 865 cases (39.03% of blood cultures with a pathogen), followed by Acinetobacter spp (n = 346), Escherichia coli (n = 326) and S. Paratyphi (n = 155).

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Table 1. Demographic characteristics and blood culture outcomes by study period among individuals with blood cultures in Nepal (N = 62,236).

https://doi.org/10.1371/journal.pntd.0013242.t001

Blood culture testing exhibited a seasonal pattern, with higher numbers of testing during the summer (rainy season) and lower numbers of testing during the winter. A sharp decline in the number of blood cultures performed was observed during the COVID-19 pandemic (from a mean of 1,663 per month to 670 per month across sites), followed by gradual recovery toward pre-pandemic levels (Fig 1). The trend of other bacteremia cases followed a very similar temporal pattern to blood culture testing, including a decline during the COVID-19 pandemic (from a mean of 23 per month to 10 per month across sites) and a subsequent increase. Before the pandemic, S. Typhi cases peaked during the summer season and declined from October to January. However, after the onset of COVID-19 pandemic, the expected peak in the summer months was absent, and case numbers remained low throughout the pandemic and continued to remain low after the introduction of the TCV vaccine, despite the gradual increase in blood culture testing (Fig 1). After TCV introduction, cases were further lower in children under 15 while they increased in those over 15 years (S1 Fig).

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Fig 1. Trends of monthly COVID-19 cases, blood culture testing, Salmonella Typhi cases, and other bacteremia cases in Nepal (2016-2024).

https://doi.org/10.1371/journal.pntd.0013242.g001

The gray shaded areas reflect the time periods of the COVID-19 related lockdowns in Nepal. Purple: total COVID-19 cases, blue: total blood cultures collected, green: total other bacteremia cases, red: total other typhoid cases.

Before the pandemic, 2.11% (662/31,307) of blood cultures were positive for S. Typhi, which decreased to 0.59% (59/10,051) during the pandemic and continued to be low at 0.69% (144/20,878) following TCV introduction. In contrast to the decline in S. Typhi positivity, positivity for other pathogenic bacteria increased slightly during the pandemic (2.49%) compared to pre-pandemic (2.01%) and remained slightly higher (2.26%) during the period post-TCV introduction compared to the pre-pandemic period. S. Paratyphi A cases followed a similar trend to S. Typhi, decreasing from 0.41% (128/31,307) pre-pandemic to 0.08% (8/10,051) during the pandemic, and remained low at 0.09% (19/20,878) post-TCV introduction (Table 1).

Among children ages 15 months to 15 years, the odds of blood cultures testing positive for S. Typhi during the pandemic were 47% lower (adjusted odds ratio [aOR] 0.53, 95% confidence interval [CI] 0.29-0.90) than the pre-pandemic period. After TCV introduction, compared the pandemic period, the odds of culture positivity for S. Typhi further decreased by 75% (aOR 0.25, 95% CI 0.11-0.55). For participants 16 years and older, the odds of blood cultures testing positive for S. Typhi during the pandemic were 77% lower (aOR 0.22, 95% CI 0.16-0.31) than the pre-pandemic period. However, the odds of S. Typhi culture positivity were 59% higher (aOR 1.59, 95% CI 1.14-2.27) following TCV introduction for this age group compared to the pandemic period (Table 2).

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Table 2. Multivariate logistic regression analysis of Salmonella Typhi blood culture positivity by study period and age group (Nepal).

https://doi.org/10.1371/journal.pntd.0013242.t002

In sensitivity analysis, when restricting the analysis to blood cultures containing any pathogen, the results were similar. The odds of blood culture positivity for S. Typhi were 26% lower but not statistically significant (aOR 0.74, 95% CI 0.30-1.83) in those ages 15 months to 15 years and 82% lower (aOR 0.18, 95% CI 0.12-0.26) in those 16 and older during the pandemic compared to the pre-pandemic period. After TCV introduction, compared to the pandemic period, the odds of culture positivity were 57% lower but not statistically significant (aOR 0.43, 95% CI 0.11-1.58) in those ages 15 months to 15 years and 100% higher (aOR 1.88, 95% CI 1.26-2.85) in those 16 years and older (Table 2).

Discussion

While clinical trials demonstrated that TCVs are highly efficacious in preventing typhoid fever in those who receive them, understanding their population-level impact following introduction into national immunization programs is critical. The COVID-19 pandemic disrupted healthcare-seeking behavior, disease surveillance and possibly transmission patterns of Salmonella Typhi, complicating efforts to evaluate the impact of TCV introductions. Through systematic surveillance spanning the pre-pandemic, pandemic, and post-TCV introduction periods in Nepal, we found a sharp decline in the number of blood cultures and blood culture–confirmed typhoid cases before TCV rollout during the pandemic, likely reflecting effects of pandemic control measures on typhoid epidemiology and healthcare utilization. After introduction of vaccination, blood culture positivity for S. Typhi continued to significantly decrease in the younger age group, whereas in those ages 16 years and above, positivity increased after TCV rollout. Taken together, these findings indicate that TCV introduction reduced typhoid burden in the age-eligible population, but that disease burden is rebounded in older age groups following relaxation of COVID-19 measures.

The decline in the total number of blood cultures performed during the COVID-19 pandemic is likely a result of several factors, including reduced hospital visits due to travel restrictions, fear of nosocomial COVID-19 transmission, redirection of laboratory resources towards COVID-19 testing and modifications in clinical practice, such as empiric management to reduce movement of febrile patients in the hospital [26]. As the pandemic waned, the total number of blood cultures performed gradually recovered but remained below pre-pandemic levels. A similar pattern of decline during COVID-19 restrictions, followed by a return to pre-pandemic levels, were observed in Bangladesh and Pakistan [27].

The trend of blood cultures positive for bacterial pathogens other than S. Typhi closely mirrored the trend of blood culture testing. The identification of other bacteremia was similar in pre-COVID, during COVID and post-vaccination periods, suggesting that the reductions in blood culture utilization were not driven primarily by alterations in typhoid epidemiology, but rather in overall reductions in healthcare utilization.

S. Typhi positivity among blood cultures declined after the onset of the pandemic and before the introduction of vaccine in both the younger age group eligible for the TCV vaccine as well as the older age group who was not eligible. Moreover, there were larger decreases in the older age group in comparison to the younger age group. During the COVID-pandemic, improved hygiene behavior and practices may have contributed to reduced transmission of other pathogens including S. Typhi [28,29]. Additionally, pandemic-era social restrictions limited eating or drinking outside of the home, thereby decreasing exposure to S. Typhi. It is possible that older individuals were more likely to be exposed to S. Typhi outside the household in the pre-pandemic period, as they are more likely to eat at restaurants or consume street food, and that the reduction in this exposure caused greater reductions in typhoid risk. Similar declines in S. Typhi blood culture positivity during the COVID-19 pandemic were observed in Pakistan [30] and Bangladesh [24]. Reductions in reported cases of other enteric infections during COVID-19 have been observed in the United States [15], Israel [12], England [31], China [19], Spain [16], Taiwan [17], and Japan [18]. However, in Madagascar, delays in seeking care led to a surge of typhoid intestinal perforation cases [32].

In Nepal, after the introduction of TCV, typhoid positivity further decreased in the vaccine-eligible age group while it increased in the ineligible age group. While three randomized controlled trials and several observational studies have demonstrated TCVs to be highly effective at preventing typhoid fever among recipients [33], one cluster-randomized trial did not show clear evidence of indirect effects [6]. In the present study, although S. Typhi culture positivity declined in the vaccine eligible population following TCV introduction, it increased in older, vaccine-ineligible age groups during the post-COVID-19 period, when non-pharmaceutical pandemic interventions were scaled back and social activities started to return to pre-COVID patterns. While this pattern does not exclude the possibility that indirect effects blunted the increase in the unvaccinated individuals, it does not provide evidence for effects in this age group and does highlight the ongoing risk that this group faces in typhoid-endemic settings. Additionally, our study period ends approximately two years following TCV introduction, limiting our ability to assess longer-term vaccine effectiveness and durability of protection.

This study has several limitations. First, TCV vaccination status of the participants was not available for the vast majority of participants, precluding analysis of the direct effects of TCV on typhoid risk. This is particularly important as, to date, there are no published data on the clinical effectiveness of the TYPHIBEV (Vi-CRM197) vaccine, which was the TCV introduced in Nepal. A separate, smaller sub-study is being performed to evaluate the effectiveness of this vaccine in Nepal. Second, antibiotic use prior to presentation to the study facilities might have varied over the study periods and could have influenced blood culture sensitivity; however, we did not find differences in self-reported antibiotic utilization over the study period [27,34,35]. Third, S. Typhi cases were unusually high in 2018 compared with prior years, such that the decline during and after 2020 was accentuated; nevertheless, compared with data from earlier years (Fig 1), S. Typhi cases were still markedly lower during the COVID-19 period. Fourth, although our study included both urban and peri-urban hospitals, the findings may not be fully generalizable to all regions of Nepal, particularly rural areas with different healthcare access and transmission dynamics. However, these sites reflect risk in and around the Kathmandu Valley and provide early evidence on typhoid trends following TCV introduction. Our analysis included sites that had both prospective enrollment of febrile patients and retrospective enrollment based on blood cultures routinely performed by the laboratory. Prospective enrollment increased the number of blood cultures performed at a site. We used fixed effects for each site to account for differences between sites and analyzed culture positivity rather than case counts, but differences over time might have still impacted the results.

Lastly, the low number of S. Typhi cases during the pandemic and post-TCV introduction period limit statistical power to definitively attribute the lower burden to vaccination. S. Paratyphi A would make for an excellent negative control outcome due to shared transmission pathways, but when stratifying by age, we had too few cases to evaluate trends by study period. Although the reduction in S. Typhi positivity among vaccine-eligible individuals following TCV introduction is consistent with vaccine impact, we also observed a concurrent decline in positivity for other bacteria in this age group (S1 Table). This pattern indicates that some of the observed reduction may reflect broader changes beyond TCV rollout, and we are unable to fully explain these findings. We therefore interpret the post-TCV decline with caution and acknowledge this as a major limitation of our analysis.

In conclusion, blood culture utilization and positivity for S. Typhi declined substantially during the COVID-19 pandemic, but S. Typhi positivity rebounded after 2022 in vaccine-ineligible older age groups. In contrast, blood culture positivity for S. Typhi further declined in vaccine-eligible age groups after the national introduction of TCV. This study reinforces the critical role of TCV in typhoid control in Nepal and provides valuable evidence to support the integration of the WHO pre-qualified typhoid vaccine in immunization programs. The persistent burden in older age groups, however, indicates need for further intervention. Comprehensive control requires sustainable investments in water and sanitation infrastructure [36], alongside consideration of expanded age eligibility for typhoid vaccination.

Supporting information

Acknowledgments

The authors thank Sudan Maharjan, Melina Thapa, Suraj Jakibanjar, Barsha Koirala, Neeru Suwal, Sarita Gosain, Mamata Maharjan, Nisha Shrestha, Natasha Shrestha, Nisha Ale, and Suvash Tamang (Dhulikhel Hospital); Himadri Neupane, Mitra Rai, and Renu Rey (Helping Hands Hospital); Archana Lamichhane and Bipin Gopali (Bir Hospital); Rishma Jal Pun and Bijay Dangol (Kathmandu Model Hospital) for their valuable support in the research. We also gratefully acknowledge all participants for their time and involvement.