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Point of care rapid test for diagnosis of syphilis infection in pregnant women

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Abstract

This is a protocol for a Cochrane Review (Diagnostic test accuracy). The objectives are as follows:

To determine the diagnostic accuracy of rapid tests at point of care (POC) for detecting syphilis infection in pregnant women, as verified with the combination of both reactive non‐treponemal and treponemal tests as the reference standard.

Background

Target condition being diagnosed

Syphilis is a complex, curable sexually‐transmitted infection caused by the Treponema pallidum bacterium that has a variable clinical course (CDC 2014). Mother‐to‐child‐transmission may occur if the mother has syphilis. If the disease remains untreated, perinatal adverse outcomes are 52% higher in women with syphilis than women without syphilis (Gomez 2013).

In 2012, approximately 350,000 adverse pregnancy outcomes worldwide were attributed to syphilis, of these 40% were early fetal deaths/stillbirths, 18% neonatal deaths, 13% preterm/low‐birthweight babies and 29% infected infants (Wijesooriya 2016).
The majority of adverse perinatal outcomes (87%) were seen in Africa and Asia. Approximately 66% of all adverse outcomes occurred in women who had attended antenatal care and who were not tested or were not treated for syphilis (Newman 2013). Syphilis infects 1% or more of antenatal care attendees in over 55 countries (WHO 2016). The severity of adverse outcomes associated with congenital syphilis is usually determined by the length of the maternal infection as well as the stage of pregnancy; in the early stages, the spirochetaemia (presence of spirochetes in the blood) is higher, therefore fetus infection is more frequent. Mothers with primary syphilis can transmit the infection to the fetus in 70% to 100% of cases, with secondary infection in 90%, and with latent syphilis in 30% (Berman 2004). Women screened and treated in the first two trimesters of their pregnancy are more likely to have a healthy infant, compared to women screened and treated in the third trimester (odds ratio (OR) 2.24; 95% confidence interval (CI) 1.28 to 3.93) (Hawkes 2013). Thus, diagnosis and treatment of syphilis must be carried out early in the pregnancy.

Syphilis testing and treatment can effectively prevent adverse pregnancy outcomes related to syphilis; neonates who survive with congenital syphilis are at risk of congenital anomalies, active syphilis, and longer‐term sequelae, including deafness and neurologic impairment (Gloyd 2001).

In 2007, the World Health Organization (WHO) released a strategy for the elimination of congenital syphilis as a public health problem through reduction of the prevalence of syphilis in pregnant women and the prevention of mother‐to‐child transmission of syphilis. This strategy rests on four distinct pillars, namely: (1) ensuring sustained political commitment and advocacy; (2) increasing access to, and quality of, maternal and newborn health services; (3) screening of all pregnant women and treatment of all positive cases and their partner; and (4) establishing surveillance, monitoring and evaluation systems. To eliminate mother‐to‐child transmission of syphilis, countries should test at least 95% of pregnant women for syphilis at the first antenatal control visit (WHO 2007).

Current diagnostic strategies

The diagnosis of syphilis is suspected by clinical presentation, or by the risk factors of infection. Most patients do not have symptoms or signs at presentation, and the diagnosis has to be made by diagnostic test. The treponemal infection produces an inflammatory response in which the damaged host cells release lipoproteins such as cardiolipins and lecithin, the non‐treponemal test detects these substances, but can produce a false‐positive reaction with some clinical conditions such as collagen diseases, antiphospholipid syndrome, other treponemical infections (for example, pian, leprosy), as well as pregnancy (Cohen 2013). The sensitivity is higher in secondary (100%) and latent syphilis (96% to 98%), but the sensitivity is lower in primary (78% to 86%, dark‐field confirmed cases as this was mentioned in (Creegan 2007), and in late syphilis (71% to 73%). The specificity of the test is 98% in all stages, compared with the Treponema pallidum hemagglutination (TPHA) test (Naidu 2012). The reports of the non‐treponemal test are in dilutions or in titres, the higher the production of antibodies, the higher the titres; a result of 1:8 titres is very suggestive of recent or active infection.

People with syphilis develop specific antibodies against treponema. The detection of these antibodies can be made by different techniques such as fluorescent agglutination, hemagglutination or chemiluminescence, which comprise the treponemal test, and the more often used are: the fluorescent treponemal antibody absorbed (FTA‐ABS) test, Treponema pallidum particle agglutination (TP‐PA) assay, enzyme immunoassay (EIA), and chemiluminescence immunoassay (CIA). These test have a moderate sensitivity in primary syphilis (84% to 96%), and a high sensitivity in the other stages; and they have high specificity (98%) in all stages. The result is either positive or negative (Naidu 2012).

Due to the importance of infection in pregnancy, given the risk of infection to the fetus, all pregnant women are screened for syphilis infection. The traditional algorithm of diagnosis begins with a non‐treponemal test, and if this test is positive, the infection is confirmed with a treponemal test (CDC 2014; PAHO 2015; WHO 2007). As the non‐treponemal tests have false‐positive results, and low titles have to be confirmed, with a delay in the diagnosis, some authors recommend the reverse algorithm (PAHO 2015). This strategy consists in first performing the treponemal test, if it is positive, given its high specificity, antibiotic treatment is started and the non‐treponemal test is requested to confirm the infection activity (CDC 2008).

The diagnosis of syphilis is made when the pregnant women has clinical signs of infection, such as chancre, skin ulcer, or rash, and the non‐treponemal and treponemal syphilis tests are reactive. The most common clinical scenario is the absence of symptoms, in this case the diagnosis of active or recent infection is made when the two tests are positive. In the non‐active syphilis or immunological memory, only the treponemal tests are positive. When only the non‐treponemal test is positive, it is considered to be a false positive for infection (PAHO 2015).

Treponemal tests require more technology and are more expensive, therefore, they are not available in all healthcare settings (Peeling 2004). In some countries or healthcare settings, when the diagnosis begins with the non‐treponemal test, if the test is positive, the prenatal control personnel will request the treponemal test. However, due to the difficulties mentioned with this test, many patients do not return for the result and the patient is left without the appropriate treatment for the infection (Jafari 2013). This results in cases of congenital syphilis, which could have been avoided. In low‐ and middle‐income countries in Asia, Africa and Latin America, roughly one out of three women is not tested for syphilis during pregnancy, with wide variations from country to country (WHO 2014).

Index test(s)

In pregnancy, it is necessary to make a timely diagnosis of all pathologies that affect the mother or the fetus. Perinatal infections cause severe lesions in the fetus that produce sequelae in neonates. When gestational syphilis is diagnosed and treated early, the fetal morbidity and mortality decrease significantly (Hawkes 2013).

In order to improve the opportunity for diagnosis, rapid diagnosis tests (RDTs) have been developed. These tests consist of a kit, with all the necessary components included, and they can be applied anywhere, by a trained person. The RDT applied at the point of care (POC) has made syphilis testing possible even in remote settings (WHO 2016 B), because they do not need refrigeration or laboratory equipment. The syphilis RDTs use for diagnosis, a drop of blood, that can be obtained by puncture of one finger, and they provide results in 10 to 15 minutes (Jafari 2013) expressed in a qualitative way (positive or negative).

The RDTs developed for syphilis are treponemal and non‐treponemal. The technology base of the test is inmunochromatographic or agglutinant. A positive treponemic test indicates an old or recent infection. The non‐treponemal test indicates an active infection. Studies with RDTs (treponemal and non‐treponemal) show a specificity from 94% to more than 99% and a sensitivity between 60% to 100%, depending on the test (Gaydos 2014; Jafari 2013). In the prenatal control clinic, it is advisable not to miss the opportunity of treatment of gestational syphilis, therefore in a patient with a positive treponemal test, it is important to start antibiotic treatment as soon as possible (Binnicker 2012). This strategy could result in over‐treatment as a result of false positives due to old infections, but is less dangerous than the secondary risk of not treating the infection in pregnant women (Jafari 2013). In high‐prevalence gestational syphilis communities, the combined tests may significantly reduce over‐treatment (Greer 2008). Another test, that is very useful in pregnancy is the HIV/syphilis Duo test, that is a method of qualitative detection of both infections, by inmunochromatography to detect immunoglobulin (Ig) IgG, IgM and IgA antibodies for specific‐HIV antigens and the Treponema pallidum recombinant antigen in serum (Bristow 2016; Gaitán‐Duarte 2016).

In some settings these RDTs could be more cost‐effective in screening and treating syphilis than laboratory‐based testing methods such as the rapid plasma reagin (RPR) or venereal disease research laboratory (VDRL) test (Terris‐Prestholt 2015). The implementation of rapid testing must be accompanied by quality assurance systems and technical competence (Benzaken 2014). Currently there are numerous commercial trades for POC tests, being the most commonly used the Abbott Determine, Omega Visitect, Qualpro Syphicheck, Standard Bioline and Trinity Syphilis Health Check (Mabey 2006)

In recent meta‐analysis the sensitivity of treponemal POC tests varied form 74.26% to 90.04% and specificity from 94.15% to 99.58% (Jafari 2013). The combined test has a sensitivity of 98.4% when the standard is a non‐treponemal test (RPR) greater than 1:8 dilutions, but it has been seen that sensitivity falls to 88% when RPR is negative (Singh 2015). Nevertheless, these recent analysis are made with studies that use frozen samples with the serologic status already known (STD clinic attendees, female sex workers and antenatal clinic attendees) and a majority used a TP specific reference standard, resulting in high sensitivities and specificities, inducing biases during the process. Our review will include only studies in pregnant women made on the field, with fresh samples (Jafari 2013)

Clinical pathway

The diagnosis of gestational syphilis, early in pregnancy is very important, in order to prevent congenital syphilis. The majority of pregnant women with syphilis are in the latent stage of infection, so they do not present symptoms or signs. The diagnosis is based on laboratory tests (Cohen 2013). It is known that prevention of mother‐to‐child syphilis transmission is feasible, even in low‐resource settings. In pregnant women, early screening and detection of syphilis with a rapid treatment for positive patients will prevent transmission to the child (CDC 2008; Taylor 2017). Based on this, in 2011 the WHO launched strategies for prevention of congenital syphilis (WHO 2007). In some places, it is not possible to perform or to wait for the results of a laboratory test, therefore diagnosis depends on the availability of RDTs, and their use at POC helps to improve early detection and they contribute to achieving this goal (Peeling 2010).The RDT for syphilis can be implemented by clinicians under different scenarios regardless of healthcare levels, covering a broad patient spectrum.

In prenatal control clinics, the diagnosis of syphilis can be made, beginning with non‐treponemal test, and then confirming with the treponemal test. Syphilis is diagnosed, when the two tests are positive. In recent years, some countries have used the inverse algorithm, beginning with the treponemal test, followed by the non‐treponemal test to determine the activity of the disease. The RDT is a treponemal test that could be done at the POC, and the results obtained in the same prenatal control visit. A positive treponemal RDT confirms the diagnosis of old or active syphilis and the necessary treatment must be applied. In addition, in the same consultation, the non‐treponemal test should be requested, to know the activity of the infection, and then to continue the treatment according to the stage of syphilis (Binnicker 2012). A negative treponemal RDT, rejects the diagnosis of infection, and control with rapid treponemal test should be continued, each trimester of gestation. When the patient has had one positive treponemal test, the control has to be carried out with a non‐treponemal test.

The introduction of a rapid test for syphilis in pregnant women, in low‐ and middle‐income countries, has resulted in substantial increases in antenatal syphilis testing (Swartzendruber 2015). Studies in six countries found significant increases in the proportion of pregnant women screened for syphilis from pre‐ to post‐introduction of screening: Perú from 61% to 100% (Flores 2014); Kenya from 18% to 70% (Fleming 2013); Uganda from 1.7% to 90.3%; Zambia from 79.9% to 95.6% (Strasser 2012); Tanzania 17.8% to 100% (Mabey 2012); and Cambodia from 0% to 77% (Delvaux 2011). In India, a prospective cross‐sectional study, showed an increased proportion of pregnant women screened for HIV, syphilis and Hepatitis B from 9% to 96% (Pai 2012).

Rationale

The WHO sexually transmitted infections (STI) guidelines recommend treatment for early and late congenital syphilis, considering the associated burden and economic costs. Consequently, it is highly desirable to undertake a critical appraisal of the available evidence of the diagnostic accuracy of the different RDTs. There is therefore, a need for high‐quality systematic reviews to improve the diagnosis of syphilis. This systematic review will facilitate the synthesis of the current evidence, and recognise the strengths and weaknesses, address the uncertainty of the current knowledge and make it possible to assess the effectiveness and safety of this intervention.

The most recent systematic review that evaluates the diagnostic accuracy of the rapid POC tests concluded that immunochromatographic tests such as Determine and Sd Bioline Syphilis 3.0 are acceptable options in antenatal testing for syphilis (Rogozinska 2017). Nevertheless, there is a need for a systematic review that strengthens these findings. One of the main limitations of Rogozinska 2017 systematic review was the broad limits for the search, wide enough to be less practical. Another limitation was the bivariate analysis applied only in the RPR, weakened by a marked heterogeneity of sensitivity between studies.

Knowing the performance, advantages and limitations of the RDTs, could facilitate the generation of a public policy based on testing and treatment in the first two trimesters of all pregnant women and also contribute to goal three of sustainable development goals of the United Nations Organization. This goal is to ensure healthy lives and promote well‐being all ages, and includes one point that is to reduce neonatal mortality to at least as low as 12 per 1000 live births, by 2030 (United Nations 2016).

Objectives

To determine the diagnostic accuracy of rapid tests at point of care (POC) for detecting syphilis infection in pregnant women, as verified with the combination of both reactive non‐treponemal and treponemal tests as the reference standard.

Secondary objectives

To assess the accuracy of rapid POC testing according to type (non‐treponemal and treponemal), infection stage (active versus inactive) and setting (low/middle‐income vs high‐income countries). In addition, we will to determine the diagnostic accuracy of POC test by brand without realize a formal comparison.

Methods

Criteria for considering studies for this review

Types of studies

We will include accuracy studies. Participants in included studies should have been enrolled under field conditions, prospectively and consecutively or through random sampling. Only studies reporting that all participants received the index test and the reference standard and presenting 2 × 2 data will be eligible for inclusion. We will exclude diagnostic case‐control studies, because the diagnosis of infection should not be known, before performing the diagnostic tests.

Participants

We will include pregnant women, recruited at primary‐ or secondary‐care facilities without previous diagnostic testing, who are attending an outpatient facility, or at the point of labour.

Index tests

Rapid tests at POC from whole blood, serum or plasma, regardless of the type of POC test (non‐treponemal or treponemal) or technique (immunochromatographic, agglutination or any other technology).

Target conditions

Syphilis at any infection stage (active versus inactive).

Reference standards

A combination of both reactive non‐treponemal (positive at any titre) and treponemal test (positive result).

Search methods for identification of studies

We will develop a highly‐sensitive, systematic search strategy to identify as many relevant accuracy or validity studies, irrespective of their language, and publication status (published, unpublished, in press, and in progress). We will use both electronic searching in bibliographic databases and handsearching, as described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011).

The results of all searches will be downloaded and managed using Endnote bibliographic software. Duplicate records of the same study will be deleted.

Electronic searches

We contacted the Information Specialist of the Cochrane Sexually Transmitted Infections (STI) Review Group in order to implement a comprehensive search strategy to capture as many relevant studies as possible in electronic databases. For this purpose, we will use a combination of exploded controlled vocabulary (MeSH, Emtree, DeCS) and free‐text terms (considering spelling variants, plurals, synonyms, acronyms and abbreviations) for the index tests (point‐of‐care test, point of care, point of care testing, point of care devices, point of care diagnostic, point of care laboratory, POC, POCT, rapid test, rapid test device, self testing, self test, patient self testing) and the target condition (syphilis, Treponema pallidum), with field labels, truncation, proximity operators and boolean operators. The search strategies and their results can be found in Appendix 1 (Electronic search strategies).

Specifically, we will search the following electronic databases.

  • MEDLINE, Ovid platform: inception to present.

  • MEDLINE In‐Process & Other Non‐Indexed Citations, Ovid platform: inception to present.

  • MEDLINE Daily Update, Ovid platform: inception to present.

  • Embase.com: inception to present.

  • Cochrane Central Register of Controlled Trials, Ovid platform: inception to present.

  • LILACS, iAHx interface: inception to present.

Searching other resources

We will attempt to identify additional relevant studies by using of the following methods.

  • Searching in the Cochrane STI Review Group’s Specialized Register, which includes randomised controlled trials (RCTs) and controlled clinical trials (CCTs), from 1944 to 2014, located through the following.

    • Electronic searching in MEDLINE, Embase and CENTRAL.

    • Online handsearching in those journals not indexed in MEDLINE or Embase, according to the journals’ master list of the Cochrane STI Review Group.

  • Searching in trials registers.

  • Searching in Web of Science®: inception to present.

  • Searching for grey literature in System for Information on Grey Literature in Europe “OpenGrey” (http://www.opengrey.eu/): inception to present.

  • Searching in Health Services Research Projects in Progress (HSRProj), and the Database of Abstracts of Reviews of Effect (DARE) for additional articles.

  • Handsearching of conference proceeding abstracts in the following events.

    • The International Society for Sexually Transmitted Diseases Research ‐ ISSTDR (http://www.isstdr.org/): 2007, 2009, 2011, 2013 and 2015.

    • The British Association for Sexual Health and HIV ‐ BASHH (http://www.bashh.org/): 2014 and 2015

    • International Congress on Infectious Diseases ‐ ICID (http://www.isid.org/): 2010, 2012 and 2014.

    • The International Union against Sexually Transmitted Infections ‐ IUSTI (http://www.iusti.org/): 2011, 2012, 2013, 2014 and 2015.

    • International Society for Infectious Diseases ‐ ISID (http://www.isid.org/): 2011, 2012, 2013, 2014 and 2015.

    • International Meeting on Emerging Diseases and Surveillance ‐ IMED (http://www.isid.org/): 2007, 2009, 2011, 2013 and 2014.

    • Interscience Conference on Antimicrobial Agents and Chemotherapy ‐ ICAAC (http://www.icaac.org/): 2011, 2012, 2013, 2014 and 2015.

    • The International Federation of Gynecology and Obstetrics ‐ FIGO (http://www.figo2012.org/home/): 2009, 2012 and 2015.

  • Handsearching within previous systematic reviews and other relevant publications on the same topic.

  • Handsearching within reference lists of all relevant studies identified by others methods. Finally, we will search the citation lists from reviewed articles.

Data collection and analysis

Selection of studies

Two review authors (NT‐M, LV‐V) will independently selecting the titles and abstracts of studies retrieved as a result of the search. Disagreements will be resolved through consensus or, if required, by consultation with a third review author (CFG‐A). We will retrieve the full text of a study if we have doubts about whether the study should be included or excluded. Two authors (EA‐M, JA‐G) will review independently the full text of selected articles in order to defined their inclusion. Disagreements will be resolved through consensus or, if required, by consultation with a third review author (CFG‐A).

Data extraction and management

We will design a data extraction form. For eligible studies, four review authors (JA‐G, NT‐M, EA‐M, LV‐V) will extract data independently using the form. Discrepancies will be resolved through consensus or, if required, by consultation with a fifth review author (CFG‐A). The data extraction form will include the following information.

  • Methods

    • Country of the study. Setting.

    • Basic study design.

    • Power calculation.

    • Number of participants and sampling of patients.

    • Ethical issues.

  • Participants

    • Inclusion and exclusion criteria. Participants in included studies should have been enrolled under field conditions.

    • Baseline information on participants: presentation at recruitment and characteristics (e.g. gestational age, symptoms, presence of risk factors, sociodemographic characteristics and infection stage).

    • Proportion of participants included in the analysis.

  • Index test

    • POC specimen: whole blood, serum or plasma.

    • POC technology: Immunochromatographic, agglutination or any other technology.

    • POC type: single non‐treponemal, single treponemal or combined tests.

    • POC staff: trained technicians, nurses, social workers or others.

    • POC timing: before, after or simultaneously with the Reference standard.

    • POC application: laboratory or field.

    • POC origin: fresh or frozen sample.

  • Reference standard

    • Reference standard specimen: whole blood, serum or plasma.

    • Reference standard technology: Immunochromatographic, agglutination or any other technology.

    • Reference standard type: Treponemal test (TPPA, TPHA, FTA‐Abs, other), non treponemal test (VDRL, RPR).

    • Reference standard timing: before, after or simultaneously with the POC.

    • Reference standard staff: trained technicians, nurses, social workers or others.

  • Outcomes

    • True positives, false positives, false negatives, true negatives.

    • Sensitivity and specificity.

    • Acceptability of the test.

    • Cost‐effectiveness.

We will collate and present this information in 'Characteristics of included studies' tables. We will add the data to Review Manager 5.3 (Revman 2014), and two review authors (CFG‐A, JA‐G) will independently assess the accuracy of the data. Differences will be resolved through consensus or by evaluation by a third review author (EA‐M). When information regarding any of the above is unclear, we will contact the authors of the original reports to request further details.

Assessment of methodological quality

We will assess the quality of included articles by using a modified version of the Quality Assessment of Diagnostic Accuracy Studies‐2 (QUADAS‐2) tool (Whiting 2011). Two review authors (JA‐G,EA‐M ) will independently perform the quality assessment using the four key domains to assess risk of bias and concerns regarding the applicability to the research question (patient selection, index test, reference standard, and flow‐timing domains). We will score the papers as having a ’low’, ’high’ or ’unclear’ risk of bias for each of four domains, and for the patient selection, index test and reference standard applicability. Studies classified at high or unclear risk of bias and/or high concern regarding applicability in at least one domain will be regarded as having low methodological design. The questions for these domains can be found in Appendix 2. In the case of disagreements, differences will be resolved through consensus or by consultation with a third review author (CFG‐A). The results will be presented in graphics according to each study an as a summary of all the studies.

Statistical analysis and data synthesis

We will to assess the accuracy of all POC available test for detecting syphilis infection regardless their type: treponemal or non‐treponemal. In this sense we plan to look at their accuracy grouped. On the other hand, because currently there are two common approaches to the diagnosis of syphilis using serological tests: the traditional algorithm (begins the screening with a non‐treponemal test and confirms a positive result with a treponemal test) and the reverse algorithm (utilises a treponemal primary screening assay followed by a non‐treponemal test if the primary treponemal assay is positive) we plan to report POC test accuracy using as gold standard both approaches grouped by type and sequence.

We will summarise diagnostic test accuracy by creating a 2 × 2 table for each study based on information retrieved directly from the papers. Each table will contain false positive, false negative, true positive, and true negative rates. Two review authors (CFG‐A, JA‐G) will independently enter the data into (Revman 2014). Discrepancies will be resolved by consensus or, if required, by consultation with a third review author (EA‐M).

In the first instance, we will analyse in a descriptive way all data retrieved from the included studies. For this purpose and given that results of the POC are reported qualitatively (positive or negative), we will present the results by plotting their sensitivity and specificity (and their 95% confidence intervals) both in forest plots and in a scatter plot in receiver operating characteristic (ROC) space. For the meta‐analysis of diagnostic accuracy measures, we will use the bivariate model (Reitsma 2005). For studies with a common threshold, this model takes into account within‐study variation and between‐study variation and focuses on estimating a summary operating point (i.e. a summary value for sensitivity and specificity). In addition, we will estimate the 95% confidence region and the 95% prediction region around the summary operating point. We will perform these analyses using the command xtmelogit in STATA, according to the licenses available.

We will include a 'Summary of findings' table using the GRADE (Grades of Recommendation, Assessment, Development and Evaluation) approach to diagnostic test accuracy (Hsu 2011), using the template provided in the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy (DTA) (Bossuyt 2013). We will present this summary table to provide a more accessible perspective of diagnostic information to healthcare providers and other end users.

Investigations of heterogeneity

We will explore heterogeneity initially by performing visual inspection of forest plots of sensitivities and specificities and visual examination of the prediction region. We will formally assess the source of heterogeneity by examining differences in diagnostic accuracy between subgroups of studies. Again, we will use the bivariate method to analyse how the summary estimate of sensitivity and specificity varies according to study level covariate. For this purpose, we will create a factor variable with N categories and will generate an N‐1 dummy that will be entered into the bivariate model to test the effects of covariate on both sensitivity and specificity (Macaskill 2010).

We will define the sources of heterogeneity a priori and will include the following factors: POC type (non‐treponemal and treponemal), infection stage (active versus inactive) and setting (low/middle‐income vs high‐income countries). In addition, we will to realize a subgroup analyses in order to determine the diagnostic accuracy of POC test by brand, without incurring a formal comparison.

Sensitivity analyses

We will perform sensitivity analysis for aspects of the review that might affect the results, such as risk of bias associated with the quality of included trials based on an overall 'Risk of bias' assessment (low versus unclear and high risk of bias) according to QUADAS‐2 patient selection, index test, reference standard, and flow‐timing domains.

Assessment of reporting bias

We will investigate publication bias if we find 10 or more studies for inclusion in this systematic review. We will investigate reporting bias by using the natural logarithm of the DOR (lnDOR) and plot it against its variance or SE. We will assess asymmetry visually, and if asymmetry is suggested by a visual assessment, we will perform exploratory analyses using Deeks’ test to investigate the asymmetry and using diagnostic odds ratio (DOR) as a measure of test accuracy (van Enst 2014).