Target Product Profile for a Diagnostic Assay to Differentiate between Bacterial and Non-Bacterial Infections and Reduce Antimicrobial Overuse in Resource-Limited Settings: An Expert Consensus

Acute fever is one of the most common presenting symptoms globally. In order to reduce the empiric use of antimicrobial drugs and improve outcomes, it is essential to improve diagnostic capabilities. In the absence of microbiology facilities in low-income settings, an assay to distinguish bacterial from non-bacterial causes would be a critical first step. To ensure that patient and market needs are met, the requirements of such a test should be specified in a target product profile (TPP). To identify minimal/optimal characteristics for a bacterial vs. non-bacterial fever test, experts from academia and international organizations with expertise in infectious diseases, diagnostic test development, laboratory medicine, global health, and health economics were convened. Proposed TPPs were reviewed by this working group, and consensus characteristics were defined. The working group defined non-severely ill, non-malaria infected children as the target population for the desired assay. To provide access to the most patients, the test should be deployable to community health centers and informal health settings, and staff should require <2 days of training to perform the assay. Further, given that the aim is to reduce inappropriate antimicrobial use as well as to deliver appropriate treatment for patients with bacterial infections, the group agreed on minimal diagnostic performance requirements of >90% and >80% for sensitivity and specificity, respectively. Other key characteristics, to account for the challenging environment at which the test is targeted, included: i) time-to-result <10 min (but maximally <2 hrs); ii) storage conditions at 0–40°C, ≤90% non-condensing humidity with a minimal shelf life of 12 months; iii) operational conditions of 5–40°C, ≤90% non-condensing humidity; and iv) minimal sample collection needs (50–100μL, capillary blood). This expert approach to define assay requirements for a bacterial vs. non-bacterial assay should guide product development, and enable targeted and timely efforts by industry partners and academic institutions.

The targeted population will present to outpatient clinics, health centers or lower tier health facilities (e.g. district level). The ease of use and characteristics of the biomarker test will determine how broad a test is applicable. Optimal requirements: Total febrile population presenting at community level with suspected non-malarial fever.

Target user of test Minimal requirements: Health workers with laboratory and medical training
Under the ideal circumstances a biomarker-test can be used at village outpatient level and at this level the health providers have likely had very limited education without specific medical training in biomarker diagnostic. Optimal requirements: Trained lay person without medical training Price of individual testing Minimal requirements: <10USD No good evidence based data are available to estimate a minimal price requirement. However, given that it would replace repeated individual pathogen testing a minimum price of ~2x the average dengue RDT (23) is assumed as the minimum price requirement.
Under ideal circumstances a simple biomarker-test is deployable in a variety of geographical settings. The resulting large market would contribute to reduced production costs.

Analytical sensitivity / Limit of detection (LoD)
Minimal requirements: Depending on biomarker tested.
The analytical sensitivity describes the ability of the test to detect small quantities of the biomarker/molecule in question. This parameter will largely depend on the chosen biomarker and has to be determined for each biomarker of interest in order to determine correct cut-off points.

Optimal requirements:
Depending on biomarker tested.

Diagnostic sensitivity to differentiate bacterial and viral infections Minimal requirements:
Equal or better than 90% The diagnostic sensitivity will very much depend on the target population, background levels of disease/biomarker and the day of presentation at the health facility. What is an acceptable sensitivity to detect a bacterial infections is very much depended on what is considered an acceptable 'false negativity', which would result in under treatment or severe disease outcomes and death. With a test that would be 90% sensitivity to correctly identify a bacterial infection 100 false negatives would be considered acceptable in a population of 1000 patients.

Optimal requirements:
Equal or better than 98% Diagnostic specificity Minimal requirements: Equal or better than 90% Similar to the sensitivity, the specificity will likely be depended on the population, background illness and the day of illness the patient presents to the health facility. Optimal requirements: Equal or better than 99% It the case of a biomarker test that aims to differentiate between non-bacterial and bacterial infections, limited specificity would refer to the misclassification of a nonbacterial as a bacterial infection, or vice versa.

Quantitation Minimal requirements: Qualitative
The read-out of a simple biomarker test should be simple without the need for further analysis of quantitative data. Optimal requirements: Qualitative Sample type/collection Minimal requirements: Whole blood from finger prick collected with a lancet Lancet selection should consider trade-offs between cost, safety, user preference, and blood volume requirements.
Biomarkers might be also found in saliva or buccal swabs (24), which represent less invasive samples, and the possibility of such should be explored, particularly for community use.

Optimal requirements:
Less invasive samples like saliva or buccal not requiring finger pricking

Sample volume/sample transfer device
Minimal requirements: 10-75µL of finger prick blood.

~0.2-1mL for saliva. Transfer device included in kit
The sample volume needed for the test should be as small as possible to accommodate possible small blood or sputum volumes obtained from individual subjects. A number of different transfer devices for finger prick blood have been developed and evaluated (25) for malaria RDTs. Optimal requirements: 5-50μL of finger prick blood. ~0.2-1mL for saliva. Transfer device included in kit Additional sample preparation Minimal requirements: 1-2 sample-processing steps Sample preparation prior to applying the sample to the biomarker test should be kept to a minimum, as this will reduce the likelihood of error and will ensure higher reproducibility of results.
Further given that limited laboratory facilities as well as human capacity will be available at community level any additionally required equipment will make the test more expensive and less likely to be performed at small health centers.

Minimal requirements:
Alone or in conjunction with a simple battery powered reader Simple visual read-outs, which don't require additional interpretation, will be most suited for deployment at the community level. Reading of the tests should ideally be possible without extra equipment, however an additional reader could be included with the device.

Optimal requirements:
No additional reader or equipment necessary

Additional reagents needed Minimal requirements:
No additional supplies or reagents are needed. All supplies are provided in self-contained kit All reagents and tools needed to perform the biomarker test should be included in the provided in the kit. Example from malaria RDTs which include the test cassette in a sealed sachet and buffer.

Optimal requirements: Same
Time to result Minimal requirements: Less than 1h with less hands-on time At community level the turn-around time needs to be quick to allow the result and patient management within the same visit.

Optimal requirements:
Less than 20 minutes Sample capacity

Minimal requirements:
One sample at a time Ideally it should be possible to perform multiple tests at the same time to allow for patient influx due to seasonality.

Optimal requirements:
Biomarker testing in combination with specific detection of pathogens of local importance and/or pathogens that would require specialized treatment.

Ease of test performance
Minimal requirements: Not more than 2 timed steps during assay performance; Instructions should include a diagram of the method and result interpretation.
As tests are aimed at staff or lay personal with limited training the number of steps to perform the test need to be kept to a minimum. This will reduce the error rate and increase reproducibility of results. Optimal requirements: One or no timed step during the assay. Instructions should include a diagram of the method and result interpretation.

Storage conditions
Minimal requirements: 18 months at temperatures between 5°C and 35°C; no cold chain required Stock control and the expiration of reagents is a major problem in resource-poor laboratories therefor a biomarker assay needs to have a reasonable long shelf life to allow stock piling on site and centrally. Optimal requirements: 36 months at temperatures between 5°C and 45°C; stable for 2 weeks at 50°C; timetemperature monitors included on each kit; no cold chain required Operating temperature Minimal requirements: Between 15°C and 40°C; up to 70% humidity No specialized facilities with air-conditioning are available at community level. Tests need to withstand the temperature fluctuations in the field without quality loss.

Optimal requirements:
Between 15°C and 45°C ; up to 90% humidity

Shipping conditions Minimal requirements:
All included in the kit (including water, alcohol, lancet, swab, test); shipping without cold chain It is important that the biomarker test can be shipped without the need for cold transport (dry ice). Optimal requirements: All included in the kit (including water, alcohol, lancet, swab, test); shipping without cold chain Training & education needs Minimal requirements: <3 days, healthcare worker Low training and education needs are necessary given high staff turn-around. Optimal requirements: <1 day, healthcare worker Instrumentation requirements Minimal requirements: Single tool used alone or in conjunction with a reader Only minimal additional instruments should be required at the community level. Any further device might reduce the usability of a test in the field. Optimal requirements: Preferably instrument free. If instrument: Small, portable or hand-held instrument (<1kg) that can operate on battery or solar in places with interrupted power supply Power requirement Minimal requirements: Optional battery or solar operation As stable power supply cannot be expected at community level the device needs to be independent of the grid. The less infrastructural requirements are needed the more widely applicable will the biomarker test be.

Optimal requirements:
None Water requirement Minimal requirements: No water required All buffers and solutions need to be included in the device as continuing water supply cannot be expected at community level.

Optimal requirements:
No water required Maintenance (external) Minimal requirements: Preventative maintenance at 1 year or >1000 samples; simple with only minimal expertise; Maintenance alert should be included Maintenance of instruments is important and necessary, however in resource poor settings regular technical is rarely available. A timely alert for the user will help to keep within maintenance schedules. Such an alert is particularly important if the device is not always used by the same person and accurate record keeping is unlikely.

Optimal requirements:
Disposal, no maintenance required

Instrumentation requirements
Minimal requirements: Single tool used alone or in conjunction with a reader Only minimal additional instruments should be required at the community level. Any further device might reduce the usability of a test in the field. Optimal requirements: Preferably instrument free. If instrument: Small, portable or hand-held instrument (<1kg) that can operate on battery or solar in places with interrupted power supply Power requirement Minimal requirements: Optional battery or solar operation As stable power supply cannot be expected at community level the device needs to be independent of the grid. The less infrastructural requirements are needed the more widely applicable will the biomarker test be.

Optimal requirements:
None Water requirement Minimal requirements: No water required All buffers and solutions need to be included in the device as continuing water supply cannot be expected at community level.

Optimal requirements:
No water required Maintenance (external) Minimal requirements: Preventative maintenance at 1 year or >1000 samples; simple with only minimal expertise; Maintenance alert should be included Maintenance of instruments is important and necessary, however in resource poor settings regular technical is rarely available. A timely alert for the user will help to keep within maintenance schedules. Such an alert is particularly important if the device is not always used by the same person and accurate record keeping is unlikely.

Optimal requirements:
Disposal, no maintenance required

Minimal requirements:
Remote calibration or auto-calibration Onsite calibration should not be required as it leaves additional room for error which might subsequently result in misinterpretation of biomarker results. Optimal requirements: None required Quality control/Internal controls Minimal requirements: Easily visible process control Internal quality control to rule out false negative testing needs to be included for quality assurance reasons. After every valid test a control line appears to ensure the test has been correctly performed. Only if the control line is visible the test can be reported as positive or negative. In addition to internal controls, EQA is important.

Optimal requirements:
Easily visible process and performance control

Data display/Result capturing Minimal requirements:
In case instrumentation is needed to read the results, the instrument should have a simple LCD screen, key pad or touch screen with a display that visually guides the user (accommodating limited literacy)