Accuracy of commercially available c-reactive protein rapid tests in the context of undifferentiated fevers in rural Laos (2024)

Abstract

Keywords: C Reactive Protein, Diagnostic accuracy, Southeast Asia; Laos

Background

In most febrile patients presenting with non-specific symptoms an accurate clinical diagnosis is not possible. This is particularly challenging in the presence of diverse aetiologies and inadequate diagnostic facilities, characteristic of low income, malaria-endemic settings. While malaria can be diagnosed with the aid of rapid diagnostic tests (RDTs), the majority of fevers across much of the malaria-endemic world have other causes [1–5]. These patients commonly receive antibiotics when they are not indicated, often for mild, self-limiting viral infections. Conversely, antibiotics are often withheld from patients they could benefit. This irrational and poorly targeted use of antibiotics is a known contributor to the ever increasing threat of antimicrobial resistance that plagues global health [6].

Improving the management of non-malarial fevers is also crucial for malaria elimination campaigns that necessitate diagnosis and treatment of all remaining malaria cases. In many areas in Asia community health worker networks are being extended to support this aim. The utilization of their services, however, is likely to suffer if they are unable to influence the care of the majority of patients with non-malarial causes of fever.

C-reactive protein (CRP) is a non-specific marker of inflammation. Use of CRP point-of-care tests to guide the use of antibiotics in fevers and respiratory infections in primary care is standard practice in a number of high-income countries [7–10]. A recent analysis of over 1400 samples from fever studies in the Mekong region confirmed the ability of CRP to distinguish between viral and bacterial infections in these settings [11]. The quantitative readers used in high-income facilities, however, might not be practical or affordable in rural tropical settings. We evaluated three commercially available CRP immunochromatographic rapid tests of a format that could be used by community health workers in remote settings to improve the management of non-malarial fevers. This evaluation aims to provide a general assessment of the reliability of the existing technology underlying these tests to correctly classify patients as having CRP values above or below the rapid test threshold(s). Whether or not CRP is an effective guide for antibiotic treatment and the identification of appropriate threshold(s) to inform patient management is the subject of further clinical outcome studies.

Methods

Samples for this investigation were obtained from a fever study in rural Laos aiming to provide a prospective description of the clinical features and aetiology of fever among consecutive outpatients attending three provincial hospitals in the provinces of Salavan, Luang Namtha, and Xieng Khouang over one year. Adults giving informed consent or children (<18years) whose parent or guardian gave informed consent who presented with a history of fever for ≤8days and/or admission body temperature ≥38°c were recruited. CRP readings were not used to inform patient recruitment or management as the role of CRP in guiding antibiotics in these settings is still under investigation.

Two individuals carried out independent searches for commercially available CRP rapid tests with analytical ranges and thresholds relevant to the detection of bacterial infections (>10mg/L). Three of five available tests were evaluated in this study, with two further tests under current evaluation in another study. In each site one of the three CRP RDTs and the reference test was performed at point-of-care immediately after patient presentation. Two of the sites used serum for both the RDT and the reference test (DTS233, Creative Diagnostics, USA; WD-23, Assure Tech, China). In a third site whole blood was used for the RDT (bioNexia CRPplus, bioMérieux S.A., France) and EDTA plasma was used for the reference test (see Table1 for test characteristics). All tests were performed and interpreted by a laboratory technician according to the manufacturers’ instructions. In all sites a NycoCard Reader II (Alere/Axis-Shield, Oslo, Norway) for quantitative CRP readouts was used as a reference test. The NycoCard Reader II has an analytical range of 5-120mg/L using serum/plasma or 8-120mg/L in whole blood and its accuracy has previously been confirmed to be high [12], including in the context of resource-poor settings [13]. Due to limited study personnel at the sites, the RDTs and the reference tests were carried out in an unblinded manner by the same individual, with the RDT being interpreted prior to the reference test to minimise the risk of bias.

Table 1

Rapid test characteristics

	Manufacturer	Recommended reading time	Format and thresholds	Visual interpretation
DTS233	Creative Diagnostics, USA	10 minutes, no more than 15 minutes.	Qualitative, single threshold of 10mg/L
WD-23	Assure Tech, China	5 minutes; no more than 7 minutes.	Semi-quantitative; <10mg/L; 10-30mg/L; 30-80mg/L; >80mg/L
				A positive result and the classification determined by the appearance and relative strength of the test band as compared with the control and reference bands.
bioNexia CRPplus	bioMerieux, France	Exactly 5 minutes	Semi-quantitative; <10mg/L; 10-40mg/L; 40-80mg/L; >80mg/L

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Results

From the introduction of the RDTs in March 2015 up until the end of July 2015, a total of 837 patients were recruited into the fever study. Demographic and clinical characteristics of these patients are described in Table2. In all sites fever was only present in one third of patients at enrolment (35, 25.6, and 39.2 % in Salavan, Luang Namtha and Xieng Khouang respectively). Respiratory syndrome was the most frequent presentation, occurring in 81.7 % of cases (82.1 % in Salavan, 85.2 % in Luang Namtha, and 75.6 % in Xieng Khouang).

The overall prescription rate of antibiotics was 58.8 % in all sites (44 % in Salavan, 72 % in Luang Namtha, and 51.5 % in Xieng Khouang). Overall, penicillin A was the most prescribed antimicrobial in 55.9 % of cases, followed by cephalosporins (11.8 %). The Xieng Khouang site also had the highest rate of pre-attendance antibiotics consumption (31.1 %), compared with Salavan and Luang Namtha (15.3 and 23.8 % respectively).

Of these 837 patients, 82 % had fresh-blood samples taken for CRP assays for immediate testing, at point-of-care. The distributions of CRP levels in the three sites according to the reference test are shown in Fig.1. In two of the sites (Salavan and Luang Namtha) the majority of patients had CRP levels <10mg/L, while in Xieng Khouang 86 % of patients had CRP levels above this threshold. A total of 167, 363 and 157 tests were carried out for the DTS233, WD-23 and bioNexia CRPplus tests, respectively. These data are plotted in Fig2, along with the manufacturer specified ranges with which each test result is supposed to correspond.

Using the shared threshold of 10mg/L and a binary interpretation of the RDT results (either negative or positive regardless of positivity category in the semi-quantitative tests), all three RDTs had high sensitivity and specificity, as shown in Table3. The two semi-quantitative tests also showed high agreement with the reference test result, with weighted kappa values of 0.7 and 0.8 for the WD-23 and the bioNexia CRPplus tests, respectively.

Discussion

Use of CRP rapid tests in remote settings could help ensure that patients in need of antibacterial treatment are identified as such, while reducing unnecessary use of antimicrobials in viral infections. All three tests included in this analysis exhibited high accuracy in line with their manufacturers’ specified thresholds. Other tests are available on the market; the selection of the three tests was arbitrary, based on their accessibility to our research units in Thailand and Laos.

All three tests had high accuracy when interpreted in a binary manner. The semi-quantitative tests also performed well in classifying patients into the specified range. In doing so they provide richer information that could feed into more nuanced algorithms to guide the use of antibiotics and decisions on referral. On the other hand, the interpretation of semi-quantitative tests can be more challenging and in the absence of a dedicated reader a binary test might be preferred.

Whether a single threshold is sufficient and the optimal value(s) to indicate the need for antibiotics and/or referral is not well established. The threshold of 10mg/L is lower than most recommendations in high income settings [7, 15], although for remote settings with limited ability to follow patients up and obtain emergency care it might be appropriate to accept a higher rate of false positives. Further studies of CRP-guided treatment algorithms are required to identify optimal threshold(s).

There are limitations to this study. First, the RDTs used fresh serum and EDTA whole blood whereas if implemented in routine care CRP RDTs are likely to be performed on fresh whole blood. Second, in one site the reference test used a different matrix than the RDT. Third, due to restricted study personnel, the RDTs and the reference test were performed by the same individual. Further work is being carried out on a broader range of tests to address these limitations. Fourth, while patients were recruited prospectively into the study, in one site many children did not have a blood sample taken. This might partly explain the elevated CRP levels in this site as compared with the other two sites; consultations of children in primary settings often occur at an earlier stage than adults because of parental behaviour [16]. The limited number of individuals with low levels of CRP in this site resulted in a wide confidence interval of 71–99 % for the specificity of the RDT. Lastly, the NycoCard Reader II was used as a reference test although ELISA testing in reference laboratories might have provided more accurate measurements. The NycoCard Reader has, however, been used in similar remote settings, with excellent correlation with laboratory-based CRP [13].

Several questions are highlighted for further research. The use of rapid CRP tests needs to be prospectively evaluated in routine settings, including estimating their accuracy using fresh whole blood; inter-observer agreement; and the importance of timing. In a previous evaluation of CRP tests it was shown that even small delays in reading the test results can be detrimental to their accuracy [17]. Most important, however, are investigations into the clinical implications of CRP-guided treatment algorithms, to gauge whether they can safely reduce antibiotic prescriptions without impacting adversely on outcomes. The thresholds used in currently available qualitative or semi-quantitative tests might not be optimal for these purposes; further clinical and economic evaluations are required to identify optimal thresholds. Whether health workers and patients adhere to CRP test results will also need to be determined. Notwithstanding these questions, this analysis provides further support to the possible implementation of CRP rapid tests at point-of-care, in order to inform the management of febrile patients with a negative malaria test.

Conclusions

Over the last few years, widespread use of malaria rapid test transformed the management of fevers in tropical settings. Improved targeting of antimalarials, however, has been accompanied by increasing misuse of antibiotics, often prescribed to patients they will not benefit, while treatable infections go undetected. There is increasing evidence from aetiological and clinical studies indicating that CRP testing could contribute to better identification of patients that require treatment, while reducing overall drug pressure and mitigate the spread of antimicrobial resistance.

This study evaluated simple lateral flow CRP tests that could potentially be used in the most remote settings to guide referral and treatment of non-malarial fevers. The three tests performed well as compared with a quantitative validated reader in detecting elevated levels of CRP. While further research is required into the performance of such tests in field conditions and to determine the optimal cut-off threshold(s) for CRP guided treatment, the timeframe to introduction of such tests could be relatively short. New pathogen specific multiplex testing and/or other biomarkers could again revolutionize the management of fever in the long run, but until these become available current practice can and should evolve using available tests that are already routinely used in other settings.

Acknowledgements

We thank staff and patients at the research sites for their involvement in this study, especially Aphaphone Atsamoud, Thongsavanh Lasajuk, and Valanouth Luanglath from LOMWRU who performed the tests. The study was supported by a Grand Challenges Canada Stars in Global Health Award (Round 5, Grant number S5 0443–01) and by our colleagues Lisa White, Wirichada Pan-Ngum and Susie Dunachie from their independent research grants, for which we are very grateful. The Mahidol-Oxford Tropical Medicine Research Unit and the Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU) are supported by the Wellcome Trust of Great Britain (grant number 077166/Z/05/Z). We thank the U.S. Navy and the Naval Medical Research Center-Asia for their continued financial and scientific support for fever surveillance in Laos.

Abbreviations

Footnotes

Contributor Information

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References