Ethics statement

For work in Peru, the study protocol was approved by the U.S. Naval Medical Research Unit No. 6 (Protocol #NAMRU6.2014.0003) Institutional Review Board (IRB), which includes Peruvian representation and complies with U.S. Federal and Peruvian regulations governing the protection of human subjects. IRB Reliance Agreements were established between U.S. Naval Medical Research Unit No. 6 (NAMRU-6), and the University of California, Davis, as well as Tulane University School of Public Health and Tropical Medicine for the NAMRU-6 IRB to be the reviewing IRB. Additionally, the protocol was reviewed and approved by the Loreto Regional Health Department, which oversees health research in Iquitos. For work in Cambodia, the study protocol was approved by the National Ethics Committee in Cambodia and the University of California, Los Angeles IRB. For the first research question involving participant observation while applying the RDT to assess competency with tests of different complexity, written consent was obtained from all adult participants (≥18 years of age) that were either self-applying the RDT, or having another participant administer the RDT on them. Children who participated in the role of “patient” provided written assent (8–17 years of age) and a parent or guardian provided written parental consent for children age 2–17. For the second research question, RDTs were provided to higher-risk households to assess whether people would choose to use them (“RDT adoption” from now on) on a febrile household member. All eligible family members (≥2 years of age) provided written consent to self-administer the RDT or have a family member administer the RDT if they displayed febrile symptoms.

Study setting

This study took place in Iquitos, Peru, and Phnom Penh, Cambodia, described in detail previously [9,10]. Iquitos is a large urban center (population ~ 400,000) where residents have easy access to a network of government health facilities and hospitals, but they do not have a network of health workers within the city. Although malaria is endemic in villages outside Iquitos, it does not circulate inside the city [11]. At the time of this competency observation study, the Asian-American genotype of dengue virus serotype 2 was circulating and Zika virus had not been detected in the city [12]. Study subjects were recruited between May 2015 and March 2016 from ten city blocks selected at random from four neighborhoods, two with ongoing prospective cohort studies [13–15] and two with no exposure to human research studies to control for the possibility that prior exposure as a research participant might improve competency. Within these randomly selected blocks, our research team recruited participants door-to-door, with one adult per household, until we reached a target of 50 participants per neighborhood. When a participant agreed to participate, our staff would call an administrator who provided the assignment of the two- or five- line device from a randomized list. Related to the question of RDT use adoption, Iquitos has ongoing cohort studies for dengue virus that monitor residents from houses in study neighborhoods three times per week for febrile illness, with subsequent laboratory confirmation if that illness is due to dengue or Zika virus [16,17]. The latter triggers contact cluster investigations where laboratory confirmed cases are interviewed to identify residential locations where the individual spent time in the 15-days prior to infection [14,18,19]. All residents in those locations are then monitored for secondary cases. We leveraged our ongoing cluster investigations to assess RDT use adoption (research question #2) in Iquitos between September 2016-September 2018.

The Cambodian sites were peri-urban areas on the outskirts of Phnom Penh with an active community health worker (CHW) program. CHWs were integrated in both dengue and malaria surveillance programs and provided basic health care to neighborhood residents. During our previous study examining acceptability of RDT use at home, participants revealed a preference for having CHWs apply RDTs in their communities [9]. As a result, both community members and CHWs were selected by convenience sampling, then invited to a local health center to apply the RDTs on others, and their competency was assessed by our Cambodian team.

Rapid diagnostic devices and application

Instructional materials

In both locations, detailed systematic instructions with pictures or drawings were developed for low literacy users (S1 Text), piloted in the community prior to use during FGDs, and finalized in both sites–one in real time during the first phase (Iquitos) and one right after the first phase (Phnom Penh)—prior to their application in the second phase. Based on overly complicated instructions that came with the devices, the Peruvian field team developed a one-page document consisting of images and minimal text descriptions of each step. These initial instruction drafts were piloted among the local research assistants, staff, and community members, followed by feedback from FGD participants, who were able try using RDT [9]. The lead scientists from the Peruvian research team (VPS, ACM) led the FGDs, and based on participant feedback and their previous experience producing video instructions [10,22], the potential value of video instructions was recognized; a video was immediately developed and validated in the subsequent FGDs. A DVD containing the final version of the video was included with the test kits to observation study participants (S2 Text. https://youtu.be/5RQtc9KNA5A, https://youtu.be/adv-yQaI_iA, https://youtu.be/arOSOaeulIw). In Cambodia, draft instructions and graphic images were developed from the manufacturer’s instructions, translated to Khmer, piloted with local community members and health center staff prior to their use in FGDs. In contrast to Peru, RDT use by FGD participants was not approved by local IRBs, so adaptation materials in real-time and video development was not possible; instead FGDs used the original piloted written materials.

Study procedures

Objective 2: Would community members use RDTs to test their own or family member’s febrile illness? (Peru only)

Participants responding to a hypothetical question about the option to use an RDT for febrile illness are likely to respond with what they perceive to be socially desirable. Hence, to determine whether community members would use an RDT in case of febrile illness, we decided to distribute commercially available dengue RDTs to families perceived to be at high risk for dengue or Zika based on proximity to other confirmed dengue or Zika cases in their communities. Leveraging the research infrastructure in Iquitos, where our research team has been conducting contact cluster investigations since 2008 [14], we identified laboratory-confirmed dengue or Zika cases (“index cases”), then carried out an interview to find out what locations the infected person had visited during the 15 days prior to becoming ill [23]. We then visited the index case’s home and contact locations, and provided a kit with two RDTs (SD Bioline Dengue Duo), written and video instructions (S1 and S2 Texts), and all additional materials needed to carry out the test to each consenting household in the cluster. The kits were removed from each house after a 30-day follow up period if not used. During the follow up period, each household was visited every 1–2 days to identify new febrile cases which were tested by PCR. Our team carried out confirmatory testing if the person used the RDT. We recorded how many individuals showed symptoms of fever, whether they used the RDT, and if not, explored their rationale for not using the RDT.

Analysis

Research assistants provided a score, ranging from one (unable to complete the procedure without asking for help) to three (able to complete the step correctly without assistance) for each individual step needed to complete the test (12 steps were scored). An intermediate score of two was given when the participant demonstrated some degree of competency but needed some assistance or reassurance. To assess factors contributing to correct use of the RDT, we developed an overall “competency score” for each participant’s use of one of the two RDTs. The scores for each of the 12 steps that both tests had in common were summed for an overall competency score (range 12–36) and divided by the 12 common steps evaluated (range 1–3). A binary response variable of > 2.9 defined as “highly competent” was used to construct separate logistic regression models for Peru and Cambodia. All analyses were conducted in SAS/STAT software, version 9.4 of the SAS system for Windows (SAS Institute, Cary, NC).

Participant characteristics

The demographic characteristics of the study participants who applied an RDT are summarized in Table 1. In Peru, only community members were enrolled in the study; 203 participants were originally observed using an RDT, and one observation was excluded because the participant was a NAMRU-6 nurse technician with extensive experience using RDTs. In Cambodia, due to a stated preference for relying on CHWs, we observed RDTs being applied by both CHWs (n = 45) and community members (n = 50). In Iquitos, females comprised a significantly higher percentage of the participants and those recruited were significantly younger than in Phnom Penh (gender: χ2 = 4.5, df = 1; p = 0.0349; age: χ2 = 26.5, df = 1 p<0.0001). Education levels, however, were higher overall in Peru than Cambodia (χ2 = 96.2, df = 2; p<0.0001). Forty-one percent of the participants were housewives in Peru compared to 17% in Cambodia (χ2 = 16.5, df = 1; p<0.0001), where most of the participants were low-skilled laborers. The majority of RDTs were self-administered in both countries, but when administered on others, they were done on family members and friends, including young children (as low as 5 years of age).

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Table 1. Summary of study population of device application by community members (excluding the 8 Community Health Workers in Cambodia that participated in a separate experiment described below).

https://doi.org/10.1371/journal.pntd.0009307.t001

In Iquitos, >80% (range 80–95%) of residents testing the two-line test were able to perform 11 out of 12 necessary steps competently without requesting help (scored a ‘3’ [high in Table 2] by a trained observer). The most challenging step was using the retractable lancet (69% used the lancet correctly without help) (see Table 2). The steps associated with starting the procedure (recognizing test materials) and blood collection (locate puncture site, capillary tube use, blood collection) required more help than the remaining steps. The five-line test, with additional steps, was more difficult for residents than the two-line test. High competency was observed for >80% (range 80–91%) for only 7 of 15 steps evaluated.

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Table 2. Observations of community members’ competency applying two rapid diagnostic tests (two-line, five-line) in Iquitos, Peru and Phnom Penh, Cambodia.

The number of participants scored in each competency category, high = 3, medium (med) = 2, and poor = 1, are shown for each testing step.

https://doi.org/10.1371/journal.pntd.0009307.t002

In Phnom Penh, users universally struggled to perform the required tasks (every category, both devices). The percentage of participants demonstrating high competency on their ability to execute the steps appropriately without asking for help, ranged from 26–76% and 23–72%, for the two-line and five-line tests, respectively. As in Iquitos, Phnom Penh participants had more problems with the initial steps of the procedures, specifically, recognition of the test kit materials and blood collection steps (Table 2). Although CHWs tended to do slightly better than community members, the overall difference was not statistically significant, so the aggregated results are presented in Table 2.

To assess factors contributing to correct RDT use, we developed an overall “competency” score for each participant’s use of one of the two RDTs. For each of the steps observed, the scores were summed and divided by the number of steps assessed, providing a final competency score between one and three. The competency scores were higher overall in Peru, ranging from 2.00 to 3.00 (IQR 2.75–3.00), than Cambodia where scores ranged from 1.08 to 3.00 (IQR 1.92–2.75). To examine factors associated with competency, we created a dichotomous variable for competency: high (competency score >2.9) and low to medium competency score ≤ 2.9).

In, Iquitos household members performed the two-line test significantly better than the five-line test; the odds of a high competency score were 4.00 (95% CI 2.13–7.54) for the two-line test compared to the five-line test. Younger household residents (18–34 years of age) were 2.28 (95% CI 1.21–4.28) times more likely to perform the test at a high level than older household residents (34–78 years of age). Education level was also an important factor; people reporting any post-secondary education (university or technical school) were 2.69 times (95% CI 1.45–5.00) more likely to perform the test at a higher competency level than participants reporting primary or secondary education only.

In Cambodia, no significant difference in ability to perform the test was observed between the two devices, between CHWs and householders, or by age. Only education level had a statistically significant effect: participants with post-secondary education were 12.49 times (95% CI 1.17–133) more likely to perform the test at a high competency level compared to participants with less education (none, primary, or secondary).

To assess how many repetitions of the test would be necessary to perform the test competently, eight CHWs (different from those who participated in the earlier study) carried out the two-line (n = 4) and five-line (n = 4) tests up to six times on different individuals. The only feedback provided was during the testing process, when the observer intervened if requested or a significant mistake was imminent. As expected, the learning curve was fast; by the sixth trial, all four CHWs using the two-line RDT had perfect scores in all steps, without needing any help. The steps that required the most practice were waiting for results (one took five attempts before doing this correctly), reading results, capillary tube use, and blood collection (three CHWs took four attempts before doing these steps correctly). Scores also dramatically improved for the five-line device, although one CHW still struggled with reading and interpreting the results even at the sixth attempt. Additional steps that required various attempts prior to achieving perfect scores were blood collection, blood transfer, adding buffer, and waiting for results.

Use of commercially available RDTs by community members with febrile illness (Peru only)

In Iquitos, we provided FDA approved and commercially available SD Bioline Dengue Duo test kits (NS1 test), including all the items needed for their use and written and video instructions, to 74 households participating in 56 cluster investigations [14]. Seventy-four (72%) of the 103 houses consented to participate. Of these 74 households, 17 of the clusters were initiated by a dengue case, and 39 clusters were initiated by a febrile case that was negative for dengue but with a presumptive or laboratory confirmed Zika case. When Zika virus was circulating (June 2016 to April 2017), we explained to the participants that there was still a possibility that any febrile illness could be dengue, but that the test might also be positive for Zika because of cross reactivity [24]. Within participating households, 293 of 445 (66%) residents provided consent. Out of these 74 households and 293 individuals consented, there were 14 reports of individuals who developed dengue-like symptoms, from 10 households, that could have used the NS1 test. Of these, seven of the NS1 tests were applied to six individuals: one on an adult, five on children between the ages of three and 13, and in one participant, a second test was repeated by the family member. Eight individuals who could have used the available test in their home decided not to use it.

The primary reasons for not using the NS1 test were fear of performing the test incorrectly or “wasting it”, or they reported a lack of fever with their other symptoms so they assumed it was Zika and should not use the test. They preferred to wait for the phlebotomist who was part of our research team to test them rather than self-administer the test. One individual forgot that they had the test altogether.