10.1 Tricycle
Tricycle, as illustrated in Figure 12, is a standardised global surveillance approach designed by WHO to monitor the prevalence and spread of ESBL-producing E. coli across the One Health sectors: humans, animals (livestock) and the environment. The protocol focuses on ESBL-producing E. coli because of its high clinical relevance, its role as an indicator organism and its ability to spread between domains.
The aim of the Tricycle protocol is to provide countries with a common, simplified and integrated multisectoral surveillance system to detect, and then estimate the prevalence of ESBL-producing E. coli in humans, animals (livestock) and the environment. For these specific goals the protocol gives detailed advice about sampling locations and procedures. Remember that:
- occurrence of ESBL-producing E. coli in wastewater can give information about its prevalence in the general human population
- occurrence of ESBL-producing E. coli in surface water can be relevant for exposure.
Sampling of surface water reflects the sources of ESBL-producing E. coli and its prevalence. Therefore, sampling locations should be near:
- human waste emissions, e.g. city waterways (canals, rivers, etc.) or wastewater treatment plants
- animal waste emissions, e.g. wastewater from wet markets/slaughterhouses.
Sampling of surface water also reflects the environment and possible exposure for humans. Therefore, sampling locations should be selected carefully and include controls, such as:
- rivers, before (upstream) and after (downstream) human and animal contamination
- drinking water (a recent extension – protocol development is underway).
Besides environmental sampling of surface water, the Tricycle protocol includes human sampling, including a hospital patient population (such as those positive for a blood stream infection) and a community population (such as healthy pregnant women). It also addresses food animal sampling by including poultry (chicken) caecal samples from fresh carcasses at wet markets and/or slaughterhouses.
In the following activity you will look at an example of how the Tricycle protocol has been used in practice.
Activity 9: An example application of the Tricycle protocol
Read the following summary of a Tricycle study in Madagascar, in which the ESBL-producing E. coli isolates were subsequently subjected to WGS to allow comparison with each other (Milenkov et al., 2024 ). (WGS and its use in AMR surveillance is explained further in the course Whole genome sequencing in AMR surveillance.)
When you have read the summary, answer the following questions:
- Are nearly identical ESBL-producing E. coli found in the three different sectors?
- Does this finding prove that exposure through (a) animals and (b) the environment contributes to human ESBL carriage?
The researchers implemented the Tricycle protocol in Madagascar to assess ESBL-producing E. coli prevalence and describe intrasector and intersector circulation of ESBL-producing E. coli and plasmids. In their prospective study, the researchers collected blood culture data from hospitalised patients with a suspected bloodstream infection and rectal swabs from healthy pregnant women from three hospitals in Antananarivo, Madagascar. They also collected caeca from farm chickens, water from the Ikopa river, wastewater and slaughterhouse effluents in the same area.
In total 1056 blood cultures were collected, and 289 pregnant women, 246 chickens and 28 surface waters were sampled. All the samples were tested for ESBL-producing E. coli and the genomes of all isolates were sequenced, characterised and compared.
Of the blood cultures, 18 contained E. coli, of which seven (39%) were ESBL. ESBL-producing E. coli was present in samples from 86 (30%) of 289 pregnant women, 140 (57%) of 246 chickens and 28 (100%) of 28 surface water samples. When the researchers looked at the DNA from 277 bacterial samples they found a great deal of variety: 90 different types. However, some bacteria were shared between two or even three of the compartments, with similar plasmids carrying the ARGs being found in all three compartments.
The researchers found that:
- ESBL-producing E. coli strains and plasmids were circulating among humans, chickens and the environment in Antananarivo, Madagascar
- the Tricycle protocol can be implemented in a low-income country, where it represents a powerful tool for investigating dissemination of AMR from a One Health perspective.
Answer
- Yes: genetically similar ESBL-producing E. coli were found in all three sectors.
- No, you cannot conclude anything about directionality. The epidemiological design of the sampling protocols was neither intended nor sufficient for attribution of dissemination from one sector to another, such as from animals to humans and vice versa. Repeated over time, however, trends in ESBL-producing E. coli resistance in each sector would provide strong evidence for the magnitude of selection pressure applied in each sector and, in combination with molecular characterisation, may provide evidence for directionality of flows of resistance elements among sectors.
You can read the full Tricycle report with detailed protocols for the full study (WHO, 2021). Optionally, you may like to see if you can complete the first four columns of the table from Activity 8 for the three compartments in the Tricycle project (humans/patients, animals [chicken carcass] and surface water). You could use another copy of the table template from Activity 8 if you’d like to complete this activity, but note that this is not a formal part of your study on this course.
Other initiatives
As well as Tricycle, other collaborative initiatives are ongoing at the time of writing this course in 2025. You can find out more about these by following the links in the further resources section at the end of this course.
10 Integrated surveillance
