Rapid diagnostic tests for typhoid and paratyphoid (enteric) ...

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In 37 studies that evaluated the diagnostic accuracy of RDTs for enteric fever, few studies were at a low risk of bias. The three main RDT tests and variants had moderate diagnostic accuracy. There was no evidence of a difference between the average sensitivity and specificity of the three main RDT tests. More robust evaluations of alternative RDTs for enteric fever are needed.

The quality of the data for each study was evaluated using a standardized checklist called QUADAS&#;2. Overall, the certainty of the evidence in the studies that evaluated enteric fever RDTs was low.

In a hypothetical cohort of patients presenting with fever where 30% (300 patients) have enteric fever, on average Typhidot tests reporting indeterminate results or where tests do not produce indeterminate results will miss the diagnosis in 66 patients with enteric fever, TUBEX will miss 66, and Test&#;It Typhoid and prototype (KIT) tests will miss 93. In the 700 people without enteric fever, the number of people incorrectly diagnosed with enteric fever would be 161 with Typhidot tests, 91 with TUBEX, and 70 with Test&#;It Typhoid and prototype (KIT) tests. The CIs around these estimates were wide, with no difference in false positive results shown between tests.

Most studies evaluated three RDTs and their variants: TUBEX in 14 studies; Typhidot (Typhidot, Typhidot&#;M, and TyphiRapid&#;Tr02) in 22 studies; and the Test&#;It Typhoid immunochromatographic lateral flow assay, and its earlier prototypes (dipstick, latex agglutination) developed by the Royal Tropical Institute, Amsterdam (KIT) in nine studies. Meta&#;analyses showed an average sensitivity of 78% (95% confidence interval (CI) 71% to 85%) and specificity of 87% (95% CI 82% to 91%) for TUBEX; and an average sensitivity of 69% (95% CI 59% to 78%) and specificity of 90% (95% CI 78% to 93%) for all Test&#;It Typhoid and prototype tests (KIT). Across all forms of the Typhidot test, the average sensitivity was 84% (95% CI 73% to 91%) and specificity was 79% (95% CI 70% to 87%). When we based the analysis on the 13 studies of the Typhidot test that either reported indeterminate test results or where the test format means there are no indeterminate results, the average sensitivity was 78% (95% CI 65% to 87%) and specificity was 77% (95% CI 66% to 86%). We did not identify any difference in either sensitivity or specificity between TUBEX, Typhidot, and Test&#;it Typhoid tests when based on comparison to the 13 Typhidot studies where indeterminate results are either reported or not applicable. If TUBEX and Test&#;it Typhoid are compared to all Typhidot studies, the sensitivity of Typhidot was higher than Test&#;it Typhoid (15% (95% CI 2% to 28%), but other comparisons did not show a difference at the 95% level of CIs.

Thirty&#;seven studies met the inclusion criteria and included a total of participants (range 50 to ). Enteric fever prevalence rates in the study populations ranged from 1% to 75% (median prevalence 24%, interquartile range (IQR) 11% to 46%). The included studies evaluated 16 different RDTs, and 16 studies compared two or more different RDTs. Only three studies used the Grade 1 reference standard, and only 11 studies recruited unselected febrile patients. Most included studies were from Asia, with five studies from sub&#;Saharan Africa. All of the RDTs were designed to detect S.Typhi infection only.

Two review authors independently extracted the test result data. We used a modified QUADAS&#;2 extraction form to assess methodological quality. We performed a meta&#;analysis when there were sufficient studies for the test and heterogeneity was reasonable.

We included diagnostic accuracy studies of enteric fever RDTs in patients with fever or with symptoms suggestive of enteric fever living in endemic areas. We classified the reference standard used as either Grade 1 (result from a blood culture and a bone marrow culture) or Grade 2 (result from blood culture and blood polymerase chain reaction, or from blood culture alone).

We searched the Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, IndMED, African Index Medicus, LILACS, ClinicalTrials.gov, and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) up to 4 March . We manually searched WHO reports, and papers from international conferences on Salmonella infections. We also contacted test manufacturers to identify studies.

Differentiating both typhoid (Salmonella Typhi) and paratyphoid (Salmonella Paratyphi A) infection from other causes of fever in endemic areas is a diagnostic challenge. Although commercial point&#;of&#;care rapid diagnostic tests (RDTs) for enteric fever are available as alternatives to the current reference standard test of blood or bone marrow culture, or to the widely used Widal Test, their diagnostic accuracy is unclear. If accurate, they could potentially replace blood culture as the World Health Organization (WHO)&#;recommended main diagnostic test for enteric fever.

Based on these results, in patients with fever where 30% (300 patients) have enteric fever, we would expect Typhidot tests reporting indeterminate results or where tests do not produce indeterminate results to, on average, miss the diagnosis (give a false negative result) in 66 patients with enteric fever, TUBEX to miss 66, and Test&#;It Typhoid and prototypes (KIT) to miss 93. In the 700 people without enteric fever, the number of people incorrectly given a diagnosis of enteric fever (a false positive result) would be on average 161 with these Typhidot tests, 91 with TUBEX, and 70 with the Test&#;It Typhoid and prototypes (KIT). These differences in the number of false negative and false positive results in patients from the different tests are not statistically important. The RDTs evaluated are not sufficiently accurate to replace blood culture as a diagnostic test for enteric fever.

Sensitivity indicates the percentage of patients with a positive test result who are correctly diagnosed with disease. Specificity indicates the percentage of patients who are correctly identified as not having disease. TUBEX showed an average sensitivity of 78% and specificity of 87%. Typhidot studies, grouped together to include Typhidot, Typhidot&#;M, and TyphiRapid&#;Tr02, showed an average sensitivity of 84% and specificity of 79%. When Typhidot studies with clear reporting of indeterminate results are considered, the average sensitivity and specificity of Typhidot was 78% and 77% respectively. Test&#;It Typhoid and prototypes (KIT) showed an average sensitivity of 69% and specificity of 90%.

The Cochrane researchers evaluated the quality of the data for each study using a standardized checklist called QUADAS&#;2. High quality studies that compared different types of RDT in the same patients were few in number. Two&#;thirds of the included studies did not evaluate the RDTs in the context of patients who are typically tested for the disease. Many studies utilized a particular study design (a case control study) which risks overestimating RDT accuracy. In the studies evaluating the Typhidot RDT, it was often unclear how many test results were indeterminate, when the test cannot distinguish a current episode of infection from a previous disease episode. Overall, the certainty of the evidence in the studies that evaluated enteric fever RDTs was low.

Cochrane researchers searched the available literature up to 4 March and included 37 studies. Most studies recruited participants from South Asia. Most participants were adults, with 22 studies including children. All of the RDTs evaluated detected Salmonella Typhi (typhoid fever) only.

Rapid diagnostic tests (RDTs) are designed to be easy to use, and to deliver a quick result without the need for a blood culture laboratory. The cost of an enteric fever RDT would be significantly less than a blood culture, and requires less training to perform.

The recommended test to confirm if a person has enteric fever is to grow the Salmonella from their blood. It takes at least 48 hours to give a result, so cannot help healthcare workers make a diagnosis the same day the blood culture is taken. Blood cultures may give a negative result even though a person has enteric fever. The test also requires a laboratory and trained staff, which are often unavailable in communities where enteric fever is common.

Typhoid fever and paratyphoid fever are infections caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A respectively. The term &#;enteric fever&#; is used to describe both infections. Enteric fever can be difficult to diagnose as the signs and symptoms are similar to those of other infectious diseases that cause fever such as malaria.

Cochrane researchers assessed the accuracy of commercially&#;available rapid diagnostic tests and their prototypes (including TUBEX, Typhidot, Typhidot&#;M, Test&#;it Typhoid, and other tests) for detecting typhoid and paratyphoid (enteric) fever in people living in countries where the estimated number of individuals with the disease at any one time is greater than 10 per 100,000 population. If accurate, they could replace the current World Health Organization (WHO)&#;recommended diagnostic test: culture (growing the bacteria that causes the infection from a patient&#;s blood or bone marrow).

Summary of findings

It is quite possible that RDTs are more sensitive than the current reference standards for enteric fever. If laboratory isolation of the causative organisms is neither cost&#;effective nor reliable, then there is a potential role for RDTs to replace microbiological culture as the main diagnostic test ( Parry ). If no single reference standard test exists, use of a composite reference standard (CRS) could improve estimation of diagnostic test accuracy ( Storey ).

The reference standard for diagnosing enteric fever has been culture of S. Typhi or Paratyphi A from bone marrow, peripheral blood, or other sterile sites. The mainstay of diagnosis in clinical practice is a positive blood culture, although the test is only positive in 40% to 80% of cases, usually in the first two weeks of the disease ( Parry ; WHO ). This lack of sensitivity is due to the low number of bacteria circulating in the blood, and may also be affected by: prior antimicrobial therapy ( Wain ); the type of culture medium used; the ratio of blood to broth; stage of illness at the time of presentation; and the duration of incubation ( Mogasale ). Bone marrow culture gives a higher culture&#;positive rate, probably because the concentration of organisms is higher than in the blood, and may remain positive even after antibiotic therapy has been started ( Wain ). Bone marrow culture is positive in 80% to 95% of patients with enteric fever, including in patients who have been taking antibiotics for several days regardless of the duration of the illness ( Parry ). Although bone marrow culture is more sensitive, it is difficult to obtain, relatively invasive, and is of little use in public health settings ( Wain ). Even with sophisticated laboratories, confirming the diagnosis of enteric fever can be difficult with negative blood or bone marrow cultures despite a patient actually having enteric fever ( Baker ).

RDTs have the potential to be useful to clinicians working in resource&#;limited settings in LMICs. Differentiating the common causes of the febrile patient by clinical criteria is challenging without the laboratory support for blood films, serology, or blood cultures ( Bhutta ). A diagnostic test in such a setting must be cheap, simple to perform, and able to quickly deliver a result. Such a test should correctly identify true enteric fever cases among febrile patients, ensuring prompt and specific treatment, allowing the avoidance of broad&#;spectrum medication that cover all common causes of fever. In many endemic areas, treatment for enteric fever may be given to all patients with fever ( Larsson ). The diagnosis of enteric fever by an RDT could reduce unnecessary prescription of antimicrobials, reduce drug expenditure, and limit the development of antimicrobial resistance ( Andrews ). The role of an enteric fever RDT in practice is to identify those febrile patients who warrant anti&#;Salmonella antibiotic treatment as opposed to conservative management, antimalarial treatment, or treatment for other bacterial infections ( Parry ).

A new group of diagnostic tests rely on the metabolites produced by the host in response to infection. Metabolites induced by specific infections could be measured in the blood and urine of affected patients ( Baker ). By comparing the metabolite profiles from healthy patients to profiles of patients with typhoid and paratyphoid infections, thresholds could be determined to identify those with acute enteric fever ( McKinnon ). Similar studies have used metabolomics to identify diagnostic markers of malaria and dengue fever ( Andrews ). The use of metabolomic tests currently requires specialized laboratory infrastructure, so use of these tests in both developed and developing countries is likely to have very restricted applicability.

Nucleic acid amplification tests (NAATs) for enteric fever diagnosis, such as polymerase chain reaction (PCR), and real&#;time PCR are being explored. Theoretically, NAATs could amplify DNA from dead or unculturable bacteria, thus addressing the concern of poor culture positivity because of pre&#;treatment with antimicrobials ( Wain ). One study found that a novel three&#;colour real&#;time PCR technique had the same limitations in test sensitivity as culture and deemed it an unsuitable methodology for the routine diagnosis of enteric fever ( Nga ). Methods that combine culture and PCR methods have been also been tested ( Zhou ). The use of NAATs in developing countries will most likely be limited in the medium&#;term because of high cost and the lack of laboratory infrastructure ( Olsen ).

The role of the WT is controversial because the sensitivity, specificity, and predictive values vary considerably between geographical areas ( Parry ). Test results need to be interpreted carefully in the light of previous history of enteric fever and vaccination. Interpretation of the result is also greatly helped by knowledge of the background levels of antibodies in the local healthy population ( House ). The increasing use of enteric fever vaccines and the occurrence of infection with other Salmonella enterica serovars lower the specificity of the WT ( Waddington ). Infection with non&#;Salmonella organisms (for example, malaria, dengue, brucellosis) also leads to cross&#;reactivity in the WT in enteric fever&#;endemic regions ( Olopoenia ). There is considerable variation in agglutinin levels among non&#;infected populations. These levels are susceptible to change over time and depend on the degree of endemicity ( Parry ). Despite these shortcomings of both sensitivity and specificity, because the WT is simple and inexpensive, it is still widely used as a diagnostic test ( Fadeel ).

The Widal test (WT) is a serological test that detects agglutinating antibodies to LPS (O antigen) and flagella (H antigen). The WT is the principal alternative test and is widely used but is neither sensitive nor specific ( Olopoenia ). In its original format the WT required both acute and convalescent&#;phase serum samples taken approximately 10 days apart. The test has also been evaluated as a single, acute&#;phase serum sample ( Saha ). In people with enteric fever, titres often rise before the clinical onset, making it very difficult to demonstrate the diagnostic four&#;fold rise between initial and subsequent samples ( Gill ).

It is anticipated that in low&#;resource settings endemic for enteric fever, a robust RDT could be utilized instead of blood or bone marrow cultures in a febrile patient, that is to replace the expensive reference standard test in daily clinical practice. A positive RDT result at the point&#;of&#;care would prompt treatment with appropriate antimicrobials. A negative result would prompt consideration of other illnesses as the cause of the patient&#;s fever ( Parry ). Simple, accurate, and robust RDTs would be of considerable help to clinicians managing patients in areas where enteric fever is common ( Baker ). In addition, an enteric fever RDT could be used as a triage tool to trigger further testing, such as blood culture, in settings where microbiological culture is less accessible. In secondary or tertiary care settings a positive RDT could warrant the collection of a peripheral blood culture prior to starting antimicrobial therapy ( Parry ).

The definitive diagnosis of enteric fever requires confirmation with a laboratory test to distinguish it from other infections (such as dengue, malaria, rickettsial infections, leptospirosis, and melioidosis) that present with similar symptoms ( Waddington ). The current recommendation is to use blood culture to diagnose enteric fever ( WHO ). This test is specific, but lacks sensitivity and so will miss patients who actually have the disease ( Mogasale ). A bone marrow culture, although more sensitive, is impractical for routine use ( Wain ). Furthermore, bacterial culture requires a relatively sophisticated laboratory usually unavailable in areas where enteric fever is common ( Parry ).

A RDT for enteric fever should be used in a patient who presents with fever who currently lives in, or has recently visited, an area of medium to high endemicity. It is likely that patients would not have received any prior testing. However, it is more likely that a patient may have been given a clinical diagnosis, or indeed empirical antimicrobial treatment, based on history and examination ( Darton ). The setting could be primary, secondary, or even tertiary care, but more commonly in a setting that has limited diagnostic laboratory facilities. Unfortunately the clinical diagnosis of the disease is imprecise, so any patient with a fever from endemic regions should be subject to an enteric fever RDT, not just those with classical signs and symptoms of the target conditions ( Parry ). In areas endemic for HIV, dengue, and malaria as well as enteric fever, patients may have had other point&#;of&#;care testing performed ( Abba ).

With the Mega Salmonella test, patient antibodies bind to unspecified S. Typhi antigens insolubilized on microplates, and are quantitatively detected by ELISA with both an IgM and IgG&#;specific peroxidase&#;labelled reagent ( Kawano ).

The PanBio test utilizes a direct ELISA format. Unspecified S. Typhi antigen&#;coated microwell strips are incubated with a patient&#;s serum for 20 minutes. The absorbance readings at a wavelength of 450 nm are converted into 'PanBio units' with greater than 10 PanBio units considered positive, and less than 10 PanBio units as negative ( Gopalakrishnan ).

The Multi&#;Test Dip&#;S&#;Tick is also a qualitative test, but in a dipstick format that detects IgG antibodies against S. Typhi O, H, and Vi antigens. It is part of a fever stick which tests for five other pathogens in addition to S. Typhi ( Olsen ).

SD Bioline similarly utilizes an ICT method to visually and qualitatively detect IgG and IgM antibodies to unspecified S. Typhi antigens which are indirectly labelled with colloidal gold (via an antibody). The immune complexes are captured by anti&#;IgM or anti&#;IgG antibodies immobilized on the test strip to give a qualitatively positive or negative result ( Kawano ).

Enterocheck WB ® detects S. Typhi&#;specific antibodies to LPS antigen in an ICT lateral flow format. As the patient sample flows through the cassette, the antibody&#;antigen complexes are immobilized by a coated membrane leading to the formation of a pink to pink&#;purple coloured band. The absence of this coloured band in the test region indicates a negative test result ( Anusha ; Anagha ).

The tests developed by KIT detect IgM antibodies against the S. Typhi LPS O9 antigen. The test has been applied in different formats as a prototype RDT using a dipstick and latex agglutination format, and an ICT lateral flow assay. The ICT lateral flow format is now commercially available as the Test&#;it Typhoid test.

The TUBEX TF tests for antibodies against S. Typhi lipopolysaccharide (LPS) antigen by quantifying inhibition of binding between O9 monoclonal antibodies and LPS&#;coupled magnetic particles. A visible decolourization of patient serum in the test reagent solution through magnetic particle separation indicates a positive result. Samples are graded as 0 to 10 according to the colour of the reaction mixture at the end of the procedure. Those with a grade greater than 2 are considered positive. Unlike the Typhidot test there has been a single version of the TUBEX test, although there may have been minor test modifications not made public by the manufacturer ( Thriemer ).

The three commercially available index tests that have most commonly been evaluated in published studies are: Typhidot (including Typhidot&#;M, and TyphiRapid Tr&#;02); TUBEX; and Test&#;It Typhoid and its earlier prototypes developed by the Royal Tropical Institute (KIT), Amsterdam. The Typhidot test measures both IgM and IgG antibodies against a 50 kDa outer membrane protein (OMP) antigen in a miniaturized dot&#;blot enzyme&#;linked immunosorbent assay (ELISA) format. The test is considered positive if the IgM is positive, and indeterminate if the IgG is positive but IgM negative. The Typhidot&#;M test measures IgM against the same 50 kDa antigen in the same dot&#;blot format after removal of the total IgG. The TyphiRapid Tr&#;02 test measures IgM antibodies against the 50 kD antigen in an immunochromatographic (ICT) format.

Current enteric fever rapid diagnostic tests (RDTs) include a variety of different methods and formats. RDTs can be applied to blood or urine samples, with blood RDTs (using either venous or capillary samples, or both) most common. Test formats are based on lateral flow, flow&#;through, agglutination, or solid phase methods ( Pastoor ). RDTs may detect antigens (components of the causative Salmonella organism) or antibodies (markers of the person's immune response to the antigen). The type of antibody class or immunoglobulin detected could be either immunoglobulin&#;M (IgM), which may be indicative of recent exposure, or immunoglobulin&#;G (IgG), which can indicate recent or previous exposure. Examples of commercial RDTs for enteric fever that have been undergoing evaluation in recent years include Typhidot ® , Typhidot&#;M ® , and TUBEX TM ( Baker ; Thriemer ). Future RDTs are also likely to take a serological approach, although the identification of novel antigens that are free of cross&#;reacting epitopes is a major challenge ( Baker ).

There is antimicrobial resistance to S. enterica serovar Typhi and Paratyphi A worldwide ( Kariuki ). Health professionals in the tropics overprescribe antimicrobials for many reasons, including cultural factors and patient expectation ( Okeke ). The purchase of drugs such as antimicrobials from untrained vendors and unlicensed pharmacists is commonplace in the developing world ( Larsson ). A major challenge is the inability to confirm diagnoses in resource&#;limited settings where traditional laboratory methods of diagnosing enteric fever are unavailable. Healthcare workers are therefore reliant on their clinical skills to make an educated guess of the cause of illness or to prescribe an antimicrobial that targets several bacteria, or both ( Shetty ). This over treatment has contributed to increasing resistance to fluoroquinolones (for example, ciprofloxacin) and multiple drug resistance (resistance to chloramphenicol, ampicillin, and co&#;trimoxazole) in S. enterica serovar Typhi and Paratyphi A in endemic Asian countries ( Chuang ).

The clinical presentation of enteric fever varies from a mild illness with a low&#;grade fever, malaise, and slight dry cough to a severe clinical illness with multiple complications including intestinal perforation ( Ismail ). Toxic apathy, blanching 'rose spots' on the trunk, abdominal organomegaly, and diarrhoea are also associated with enteric fever, but the clinical picture is highly variable between geographical location and age groups. Enteric fever can present in many different and non&#;specific ways, thus posing a diagnostic challenge for the health professional. Enteric fever is usually diagnosed on clinical grounds and treated presumptively. The diagnosis may be delayed or missed, while other febrile illnesses are being considered ( Parry ).

Typhoid and paratyphoid (enteric) fever are diseases caused by Salmonella enterica serovar Typhi and Paratyphi A respectively. Typhoid, the more common infection, is an important infectious disease in low&#; and middle&#;income countries (LMICs) with over 22 million new cases worldwide and an estimated 200,000 deaths annually ( WHO ). South and South&#;East Asia are the most affected areas of the world, with an estimated annual incidence in some areas of greater than 100 people per 100,000 population ( Crump ). Enteric fever is common in areas with inadequate sanitation and hygiene, particularly regarding food, water, and disposal of human excrement, and only to this extent are these diseases tropical ( Gill ). Despite advances in technology and public health strategies, enteric fever remains a major cause of morbidity in the developing world ( Bhutta ). Urbanization, global warming, and traditional methods of waterside living have created even greater demands for clean water in developing countries ( UNICEF ). We will use the term 'enteric fever' throughout this Cochrane Review to include both typhoid and paratyphoid fever, unless specified. The causative organisms are Gram&#;negative bacilli that are transmitted by the faecal&#;oral route when a person ingests food or water that is contaminated with infected human faeces. The most important reservoirs of infection are short&#;term convalescents or chronic human carriers. Food handlers who are carriers are a particularly important source of transmission ( Gill ; Andrews ).

The rationale for distinguishing sub&#;Saharan Africa from the rest of the world was that non&#;typhoidal Salmonellae (NTS) are an important cause of bacteraemia in sub&#;Saharan Africa ( Parry ), and may affect the performance of enteric fever RDTs in these settings.

For PanBio Multi&#;test Dip&#;S&#;Tick, Mega Salmonella, and SD Bioline tests, where the only included data is from comparisons of tests with fewer than four studies, we compared individual tests with results from Typhidot and TUBEX on the same participants as available. We based comparisons on conservative estimates from unpaired comparisons of proportions, as paired data were not available from articles. Where 95% CIs did not overlap between test estimates, we established statistical significance without formal testing. Where 95% CI overlapped, we reported the differences in unpaired proportions with 95% CIs for the differences.

For meta&#;analysis we used the bivariate random&#;effects models of sensitivity and specificity ( Reitsma ; Chu ). We exported the data from RevMan 5 ( Review Manager ) into STATA models fitted using xtmelogit with all three main test types included in a single model allowing for unequal variances between tests and allowing correlation of sensitivity and specificity for each test in the random effects. Within xtmelogit we calculated pairwise comparisons of the difference between sensitivity and difference in specificity with 95% CIs of the three tests. We also used xtmelogit for heterogeneity analyses to compare sensitivity and specificity for the subgroup of studies where the Typhidot test reported indeterminate test results or not. We entered meta&#;analysis parameter estimates (bivariate model parameter estimates and confidence and prediction region parameters) into RevMan 5 ( Review Manager ).

For Test&#;It Typhoid and prototypes (KIT) studies, we performed a meta&#;analysis for the threshold of > 1+ only as this was the manufacturer's recommendation. Data from the same study may contribute to different comparisons (for example, RDT versus blood culture; RDT versus bone marrow and blood culture), but we only combined one set of data from each study in an individual meta&#;analysis.

The statistical analysis focused on sensitivity and specificity at average operating points for the three main commercially&#;available RDTs and their prototypes: TUBEX; Typhidot (including Typhidot&#;M); and Test&#;it Typhoid (and KIT prototypes). We included each test in a separate meta&#;analysis. For other tests we identified fewer than four studies, so we did not complete any meta&#;analysis summary. Where sufficient data were available, we performed meta&#;analyses to estimate and compare the performance of the tests.

We entered all 2 x 2 table data from all RDTs in included articles into Review Manager 5 (RevMan 5) ( Review Manager ), which calculates sensitivity and specificity with 95% confidence intervals (CIs). We used forest plots and summary receiver operating characteristic (SROC) plots to present the variation in sensitivities and specificities between studies. In the description of studies we recorded the number of uninterpretable or invalid test results.

Two review authors (LW and CMP) independently assessed the quality of each individual study using a modified QUADAS&#;2 tool ( Whiting ; see Appendix 3 ). We answered each quality indicator on the checklist with a 'yes', 'no', or 'unclear' response for each study, and we provided the reason for our judgment.

For Moore and Maude , two review authors (LW and CMP) were the study authors, so one review author (SM) independently extracted data using individual participant data (from CMP) as we could not extract ideal data for review from the published articles. In Fadeel , the article did not report results summarized across the cohort. For both Typhidot and TUBEX tests, for nested case control results within a cohort of patients, we back calculated 2 x 2 tables to reflect cohort composition (see the ' Strengths and weaknesses of the review ' section).

Where a study applied multiple index tests or reference standards, we extracted data for each test. Since blood culture, bone marrow culture, and blood PCR are imperfect reference standards, where possible we extracted the results of a composite reference standard (blood culture and bone marrow culture, or blood culture and blood PCR), such that we documented a negative result if bone marrow culture, blood culture, PCR, or all three, were negative ( Reitsma ). We extracted the number of uninterpretable or invalid test results.

We extracted the number of true positives, true negatives, false positives, and false negatives based only on the Salmonella enterica serovars the test was designed to detect (Typhi or Paratyphi A) as a 2 x 2 table for each study along with the corresponding threshold value. If data for multiple 2 x 2 tables were presented based on more than one threshold for a single study, we extracted each table and the threshold values. If this data (2 x 2 table) was also available for a subgroup of patients in the study, we extracted this data if the subgroup of patients was of interest (that is, grouped by patient age). For studies that we only included a subgroup of participants in the review, we only extracted this data and presented it for that particular subgroup. In case control design studies, we restricted negative controls to febrile participants, and we excluded healthy control participants from the 2 x 2 table data.

Two review authors (LW and CMP) independently extracted a standard set of data from each study article (see Appendix 2 ), using a pre&#;piloted specifically designed data extraction form. A third review author (SM) cross checked the data extraction and resolved any discrepancies by discussion with the two review authors (LW and CMP). If information was missing or not clear, we contacted the study investigators.

One review author (LW) screened the titles and abstracts of articles identified by the search strategy. We coded articles that did not fulfil the inclusion criteria as 'do not retrieve'. In the case of potentially eligible articles or if we were unclear whether the articles met the inclusion criteria or not, we coded these articles as 'retrieve'. We retrieved the full&#;text texts of articles in the 'retrieve' category. Two review authors (LW and CMP) independently assessed the full&#;text articles for inclusion and consulted a third review author (SM) in case of disagreement. We listed all studies excluded after full&#;text assessments and their reasons for exclusion in the ' Characteristics of excluded studies ' section. We presented the study selection process in a study flow diagram.

We checked the reference lists of all studies identified by the above methods, and we manually searched World Health Organization (WHO) reports. In addition we manually searched papers from the 3rd () to the 7th () International Conferences on Typhoid Fever and other Salmonellosis. We contacted test manufacturers to identify ongoing or unpublished studies.

We searched the following databases using the search terms and strategy described in Appendix 1 : the Cochrane Infectious Diseases Group Specialized Register (4 March ); MEDLINE (OVID, to 1 March ); Embase (OVID, to 4 March ); Science Citation Index&#;expanded (Web of Science, to 4 March ), IndMED; African Index Medicus, and LILACS ( to 4 March ). We also searched ClinicalTrials.gov and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/trialsearch). for trials in progress, using "typhoid", "paratyphoid", "enteric fever", "rapid diagnostic test", "RDT", and "diagnostics" as search terms.

As overall estimates of accuracy ignoring the use of different reference standards are difficult to interpret, we reported the results separately for each grade of reference standard ( Reitsma ).

We defined a Grade 2 study as one that used either peripheral blood culture only as the reference standard, or peripheral blood culture and peripheral blood PCR as the composite reference standard. In Grade 2 studies, we considered either blood culture or blood PCR positivity a positive composite reference standard.

We defined a Grade 1 study as one that used both bone marrow culture and peripheral blood culture as the reference standard. In Grade 1 studies, we considered either bone marrow or peripheral blood culture positivity a positive reference standard.

When only a subgroup of participants in a study was eligible for inclusion in the review, we included the study provided that we were able to extract relevant data specific to that subgroup. Subgroups included participants enrolled as separate groups, for example a clinical cohort subgroup without healthy control patient subgroup ( Fadeel ).

Patients living in enteric fever&#;endemic areas attending a healthcare facility with fever were eligible. This may or may not have included patients with a clinical suspicion of enteric fever.

Retrospective case control studies that compare a group of patients with laboratory&#;confirmed enteric fever cases (positive reference standard) and a group of patients without enteric fever (negative reference standard). In case control design studies, we only extracted data relating to the index test(s) from control groups participants with fever, and not from healthy control participants without fever.

Prospective cohort studies in which a series of patients from a given population are recruited and receive one or more index test and a reference standard.

There were insufficient studies for formal heterogeneity analysis using meta&#;analysis of test subgroups, except for a comparison of Typhidot test studies at lower risk of bias due to clear reporting of indeterminate results. For other potential sources of heterogeneity (' Investigations of heterogeneity ' and ' Secondary objectives ' sections) where individual study characteristics could be investigated, such as study design, prevalence, and study reference standard, we presented results for visual examination of heterogeneity in summary ROC (SROC) plots and forest plots.

Two included studies assessed Onsite Typhoid (CTK Biotech). In Maude , it was compared with both Test&#;It Typhoid (Life Assay) and the SD Bioline test. Onesite Typhoid had similar results to the Test&#;It Typhoid test, which were superior in sensitivity to SD Bioline. However, SD Bioline had significantly higher specificity (97%, 95% CI 95% to 99%) than both Test&#;It Typhoid test (61%, 95% CI 55% to 67%) and Onsite Typhoid (74%, 95% CI 68% to 79%). Tarupiwa evaluated Onsite Typhoid alongside TUBEX, where the performances of both tests were closely comparable. We note that these results are based on two studies and further research is needed.

Three included studies evaluated SD Bioline ( Kawano ; Limpitikul ; Maude ), and all three studies reported the preferred IgM test format. In Kawano , SD Bioline IgM had an inferior performance to TUBEX when tested on the same participants. SD Bioline had significantly lower sensitivity to TUBEX (51% (95% CI 58% to 72%) versus 95% (95% CI 87% to 99%) respectively) and similar specificity (76% versus 80% respectively). In Maude , SD Bioline IgM had significantly lower sensitivity at 21% (95% CI 9% to 38%) compared to both Test&#;It Typhoid (Life Assay) and Onsite Typhoid (CTK Biotech), both with a reported sensitivity of 59% (95% CI 41% to 75%), indicated as the 95% CIs did not overlap.

A single study compared Mega Salmonella to Typhidot, TUBEX, and SD Bioline using the same participants ( Kawano ). Mega Salmonella had superior sensitivity to Typhidot and SD Bioline but significantly lower specificity (the 95% CI for specificity did not overlap with those from TUBEX or SD Bioline). In this study TUBEX has similar sensitivity to Mega Salmonella (95% and 91% respectively) and significantly higher specificity (80%, 95% CI 71 to 88) versus 49% (95% CI 39 to 59) respectively). Mega Salmonella had an inferior performance to TUBEX, SD Bioline, and Typhidot, although this was only based on evidence from one included study.

Multi&#;test Dip&#;S&#;Tick was tested in the same study participants as TUBEX and Typhidot ( Olsen ). There was no significant difference in sensitivity between the tests, but a clinically and statistically inferior specificity in Multi&#;test&#;Dip&#;S&#;Tick (specificity: 50%, 95% CI 26% to 74%) compared in the same participants with both TUBEX (TUBEX higher specificity; difference in specificity of 44%, 95% CI 19% to 69%) and Typhidot (Typhidot higher specificity; difference in specificity of 39% (95% CI 12% to 66%).

Gopalakrishnan tested both PanBio and Typhidot in the same study. While the sensitivity of the tests was similar (78% and 82% respectively), the specificity of PanBio was superior in this study (81% versus 68%; 13% difference in conservative unpaired proportions with 95% CI 0.6% to 25%. We noted that there was insufficient data for more appropriate paired comparison).

Enteroscreen was only tested in one case control study ( Prasad ), where it was compared to Typhdot in overlapping participants. In this single case control study, Enteroscreen had a significantly lower sensitivity (Typhidot higher sensitivity based on conservative estimate of unpaired proportions; difference in sensitivity 9%, 95% CI 3% to 16%) but a significantly higher specificity (Typhidot lower specificity; difference 17%, 95% CI 14% to 20%).

Enterocheck WB was not compared with any other index tests in the two included studies ( Anusha ; Anagha ), so only lower quality indirect evidence is available to compare test performance to other tests ( ). For both studies, both sensitivity and specificity were reasonably high ( Anagha : sensitivity 89%, 95% CI 67% to 99%; specificity 97%, 95% CI 89% to 100%; Anusha : sensitivity 85%, 95% CI 73 to 94%; specificity 89%, 95% CI 85% to 92%).

There were seven other commercial RDTs that were evaluated by only 1, 2, or 3 studies, and therefore we did include them in the meta&#;analyses (' Methodological quality of included studies ' section). We have presented the results of these individual studies and tests in the ' Data and analyses ' section and . Further research is needed before there is sufficient data to recommend these tests. From the current studies, the most promising tests are Enterocheck WB, Enteroscreen, and PanBio.

Eleven studies compared different RDTs within the same study. There were 10 paired comparisons of Typhidot/Typhidot&#;M and TUBEX ( ), and one study compared TUBEX and Test&#;It Typhoid (and KIT prototypes) ( House ), although it is unclear whether or not these were on the same patients ( ). There were no paired comparisons of Test&#;It Typhoid (and KIT prototypes) and Typhidot tests. There was no statistically significant difference in either average sensitivity nor average specificity between Typhidot and TUBEX tests, with a lower sensitivity in Typhidot (&#;7.6%, 95% CI &#;19.8% to 4.6%) and a lower specificity in Typhidot (&#;3.7%, 95% CI &#;13.9% to 6.5%). This is supported by , where no consistent direction is evident for differences between these tests.

Direct comparison of diagnostic tests in the same patients in the same study provides the highest level of evidence to compare tests ( Rutter ; Takwoingi ).

Comparing Typhidot to TUBEX, Typhidot had a slightly higher average sensitivity when all studies were compared to TUBEX but this was not statistically significant (5.0%, 95% CI &#;6.1% to 16.1%). When TUBEX was compared to Typhidot tests with a lower risk of bias due to clear reporting of indeterminates, Typhidot had a slightly lower, but not significant, average sensitivity (&#;0.7%, 95% CI &#;13.6% to 12.0%). The average specificity was lower for Typhidot compared with TUBEX based on all studies (&#;8.2%, 95% CI &#;17.7% to 1.4%) and based on Typhidot studies with lower risk of bias due to clear reporting of indeterminates (&#;10.1%, 95% CI &#;20.6% to 0.5%). In neither case was the difference in specificity statistically significant.

Comparing Typhidot to Test&#;It Typhoid (KIT), there was a statistically significant difference in average sensitivity when compared to all Typhidot tests (Typhidot higher sensitivity 15.0%, 95% CI 2.0% to 28.1%) but the difference in sensitivity was not statistically significant when Test&#;It Typhoid was compared to Typhidot tests with a lower risk of bias, due to clear reporting of indeterminates (9.3%, 95% CI &#;5.2% to 23.7%). The differences in average specificity were not statistically significant for either comparison (22 Typhidot studies: lower Typhidot specificity of &#;7.6%, 95% CI &#;18.6% to 3.4%; 13 Typhidot studies: lower Typhidot specificity of &#;9.5%, 95% CI &#;21.5% to 2.4%).

Using all 37 studies including all 22 studies with Typhidot results to compare Typhidot, TUBEX, and Test&#;It Typhoid (KIT) tests, TUBEX had a 10% higher average sensitivity than Test&#;It Typhoid (KIT) (95% CI &#;1.6% to 21.7%) although this was not a statistically significant difference. The specificity was similar between tests with TUBEX having a slightly lower average specificity of 0.5% (95% CI &#;7.7% to 8.9%). This also was not a statistically significant difference.

When comparing the three main tests (Typhidot, TUBEX, and Test&#;it Typhoid (KIT ICT)) we used two different groups of comparator Typhidot test because of the risk of bias introduced when studies at risk of indeterminates do not report whether indeterminates were present or how they were treated in study results. Our primary analysis related to all Typhidot tests (based on 22 studies) with a sensitivity analysis based on restricting to the 13 Typhidot studies with lower risk of bias due to clear reporting of indeterminates.

Combining all different formats, the median sample size was 300 (range 85 to 502). Studies were published from to . Sensitivities ranged from 42% to 92%, and specificities ranged from 61% to 97% ( ). The meta&#;analytical average sensitivity and specificity across all nine studies of KIT RDTs based on a threshold of > +1 was 69% (95% CI 59% to 78%) and 90% (95% CI 78% to 93%) respectively ( ).

Included studies evaluated these assays against different reference standards: Grade 2 (peripheral blood culture only); and Grade 2 (peripheral blood culture and blood PCR) ( Moore ; Maude ). One study was a Grade 1 study (peripheral blood culture, or bone marrow culture, or both) although less than half (61/127) had a bone marrow culture performed, with the remainder using blood culture only as the reference standard ( Gasem ). illustrates the performance of the ICT lateral flow assay by these different reference standards. present study results according to case control or non&#;case control design.

The results for both thresholds (1+ versus 2+ when we could extract these results from the same study) are illustrated in . Increasing the threshold to greater or equal to 2 (&#; 2+) decreases the sensitivity of the index test but increases the specificity. One study suggested the diagnostic accuracy was improved by using a threshold of 2+ or more ( Moore ).

All studies evaluating this test plotted in ROC space by different test types (1+ result classified as positive) are presented in . Although the dipstick and ICT RDTs appear to perform better with higher average sensitivities, most studies adopted a case control design ( ).

Nine studies evaluated the performance of the Test&#;it Typhoid index test and its earlier KIT prototype formats: five as a dipstick assay; one as a latex agglutination test; and three as the ICT lateral flow assay. The KIT ICT lateral flow assay is now commercially available as Test&#;It Typhoid (LifeAssay) and two studies evaluated this ( Moore ; Maude ). In the dipstick and lateral flow assay formats, the test gives a semi&#;quantitative result scored as 1+, 2+, 3+, or 4+ dependent on the intensity of the band on the test strip. The manufacturer's recommended threshold that is considered positive is 1+ or more. A few studies have additionally evaluated a threshold of 2+ or more.

The median sample size was 158 (range 73 to ). The earliest study was published in , and the most recent study published in . Sensitivities ranged from 56% to 100%, and specificities ranged from 69% to 96% ( ). The meta&#;analytical average sensitivity and specificity (95% CI) were 78% (71% to 85%) and 87% (82% to 91%) respectively ( ).

Fourteen studies evaluated TUBEX. We have presented the study results plotted in ROC space and as a forest plot in and , which illustrate heterogeneity in test performance between studies. All included studies were Grade 2 (peripheral blood culture only as reference standard), with one study using both blood culture and blood PCR ( Siba ). This heterogeneity is mirrored when the TUBEX test results are presented by those with and without a case control study design ( ). One study used two different reference tests ( ). As with the Typhidot studies, the composite reference standard of blood culture and PCR lowered sensitivity by around 25%.

The median sample size of all studies of Typhidot and its variants was 127 (range 50 to ). The earliest study was published in , with the remainder being published in the s. The latest study was published in . Sensitivities ranged from 27% to 100%, and specificities ranged from 38% to 99% ( ). The meta&#;analytical average sensitivity and specificity for all three Typhidot test types were 84% (95% confidence interval (CI) 73% to 91%) and 79% (70% to 87%) respectively based on 22 studies ( ). However, based on the 13 Typhidot studies where indeterminates were reported or were not produced by the test (Typhidot&#;M and TyphiRapid Tr&#;02) which have a lower risk of bias, the average sensitivity was 78% (95% CI 65% to 87%) and specificity was 77% (95% CI 66% to 86%). Comparing the 13 studies at lower risk of bias with the nine studies that did not report indeterminates, the difference in sensitivity was &#;9.8% (95% CI &#;26.1% to 6.4%) and specificity of &#;8.0% (95% CI &#;24.2% to 8.3%). Studies where indeterminates were not reported are at a higher risk of bias and have both higher average sensitivity and specificity, although neither difference is statistically significant.

The included studies used three different grades of reference test: Grade 1 (peripheral blood culture or bone marrow culture, or both); Grade 2 (peripheral blood culture only); and Grade 2 (peripheral blood culture, nucleic acid amplification (blood PCR), or both). To determine the impact of the reference test on accuracy, we plotted the study results in ROC space according to the reference test used in . In the study that used both blood culture alone, and blood culture combined with blood PCR on the same patients ( Siba ), use of the composite reference standard of PCR and blood culture lowered test sensitivity results by about 25%.

The study results plotted in receiver operating characteristic (ROC) space are shown in . The Typhidot variant studies did not perform consistently across studies. shows the forest plots of studies evaluating Typhidot RDTs by various test type, and by whether indeterminate results were reported or not. There is no obvious visually distinguishable trend in test performance with prevalence across non&#;case control studies.

For the Typhidot test, indeterminate results can be produced which are classified as both IgM test negative but IgG test positive ( Olsen ; Naheed ). Some studies explicitly classified indeterminate results, where others did not clearly report indeterminate results ( Siba ), or only presented the IgM data without the IgG data ( Khan ). We attempted to separately extract the IgM and IgG positive data from each study and, where possible, used the IgM data only to allow comparison of results between all three types of Typhidot test by classifying the indeterminate results as negative (see the ' Differences between protocol and review ' section).

Only 11 studies recruited unselected febrile patients. Most included studies selected patients on the basis of a clinical suspicion of enteric fever, although the criteria for suspecting enteric fever were usually not stated. Only three studies employed the Grade 1 reference standard, with blood and bone marrow culture ( Bhutta ; Gasem ; Khan ). All studied used peripheral blood culture. Three studies also used blood PCR ( Siba ; Moore ; Maude ). One study used stool culture, and another used the Widal Test in a composite reference standard ( Gopalakrishnan ; Pastoor ). Only half of the included studies reported that the index test results were interpreted without knowledge of the reference standard results. Patients were recruited prospectively in 26 of the 37 included studies. Index tests were performed retrospectively on stored samples in 18 studies. Twenty&#;three studies reported enrolling a consecutive or random group of patients (see the ' Characteristics of included studies ' section). Sixteen studies used a case control design where diagnostic accuracy results can be overestimated, although all these studies reported results separately for control groups from febrile patients. Nineteen studies used cohort (not case control) designs, and in two studies the reporting was unclear.

We have summarized the methodological quality of the 37 included studies in . We extracted this data using a modified QUADAS&#;2 criteria proforma ( Appendix 3 ) that focused on four domains of methodological quality: patient selection; index test; reference standard; and flow and timing. The domain with the highest level of risk for bias across all studies was that of patient selection (> 50%). We have summarized the risk of bias and the review authors' judgements about the applicability concerns of these domains for each included study in .

The included studies evaluated 13 index tests in total ( ). The most commonly evaluated RDTs were Typhidot and its variants (Typhidot; Typhidot&#;M; TyphiRapid Tr&#;02; Malaysian Biodiagnostic Research SDN BHD, Malaysia) in 22 studies, and TUBEX TF (IDL Biotech, Sollentuna, Sweden) in 14 studies. An index test created by the Royal Tropical Institute, Amsterdam (KIT), and now commercially available as the Test&#;it&#;Typhoid test (LifeAssay Diagnostics, South Africa) was evaluated in three different test formats in nine studies (dipstick assay; latex agglutination assay; lateral flow immunochromatographic test (ICT)). Other index tests evaluated included: Enterocheck WB (Zephyr Biomedicals, Tulip Group, Goa, India) in two studies; Enteroscreen (Zephyr Biomedicals, Tulip Group, Goa, India); SD Bioline (Standard Diagnostics, Kyonggi&#;do, Korea); Mega Salmonella (Mega Diagnostics, Los Angeles,USA); Multi&#;Test Dip&#;S&#;Tick (PANBIO INDX Inc., Baltimore, USA); and Onsite Typhoid IgG/IgM combo (CTK Biotech Inc., San Diego, California, USA) in one study each.

All of the RDTs evaluated were antibody tests on blood designed to detect S. Typhi infection. None of the included studies evaluated a RDT that detected S. Paratyphi A infection. All the RDTs evaluated used venous blood as the biological sample with one study additionally using capillary blood samples ( Anusha ). There were no suitable studies that evaluated RDTs using other biological samples such as saliva or urine.

Eighteen of the studies included both adults and children, and seven studies included children only. The age distribution of recruited patients was not clear in 14 of the included studies. Thirty&#;three studies included participants attending a tertiary healthcare facility, 15 studies included secondary (district) healthcare attendees, and seven studies included primary healthcare attendees. Twenty studies recruited inpatients, 12 studies recruited outpatients, while 10 studies did not state the point of recruitment.

Most included studies recruited participants from the Asia&#;Pacific. The South Asian study locations included: India (10 studies); Bangladesh (five studies); and Pakistan (four studies). In South&#;East Asia, the study locations included: Indonesia (five studies); Vietnam (two studies); Malaysia (one study); Cambodia (one study); Thailand (one study), and Papua New Guinea (one study). East Asian countries included China (one study) and the Philippines (one study). From Africa, two studies were from the north (Egypt), and five studies were from sub&#;Saharan countries (Kenya, Tanzania, Zimbabwe, and South Africa) where non&#;typhoidal Salmonellae (NTS) are also an important cause of bacteraemia. Six studies recruited patients from areas of medium enteric fever endemicity ( Crump ). Most study participants were from areas considered highly endemic for enteric fever ( Crump ).

We have summarized the study selection process in a PRISMA flow&#;chart ( ). We performed a literature search up to 4 March and identified a total of titles and abstracts. There were articles after we removed duplicates. We retrieved 95 full&#;text articles for assessment. From the total number of 95 full&#;text articles retrieved and assessed, we included a total of 37 studies for qualitative analysis in the Cochrane Review. We did not include two of the studies ( Anagha and Anusha ) in the quantitative analysis as together they were not powered sufficiently for a meta&#;analysis of the single index test (Enterocheck WB) they evaluated ( ; ). The number of included studies in the quantitative analysis after full&#;text assessment was 35.

Discussion

The principal findings of this systematic review were that the diagnostic accuracy of the three main groups of commercially available rapid diagnostic tests (RDTs) for enteric fever (Typhidot and its variants, TUBEX, Test&#;It Typhoid and prototype (KIT) tests) was moderate. There was no statistically significant difference in the average sensitivity between Typhidot, TUBEX, or Test&#;It Typhoid tests, except when we compared all Typhidot tests to Test&#;It Typhoid (84% all Typhidot studies, 78% Typhidot studies with low risk of bias due to clear reporting of indeterminates, 78% TUBEX, 69% Test&#;It Typhoid). There was no statistically significant difference for average specificity between these tests (79% all Typhidot studies, 77% Typhidot with low risk of bias due to clear reporting of indeterminates, 87% TUBEX, 90% Test&#;It Typhoid); see 'Summary of findings' table 1 ( ).

A clinically useful test requires high values for both sensitivity and specificity. There was no statistical evidence to demonstrate that one group of tests was significantly better than the other ( ; ; ; ; ). The quality of studies that evaluated the diagnostic accuracy of RDTs for enteric fever was generally low. Only three of the 37 included studies used the Grade 1 reference standard requiring a bone marrow and blood culture result, and less than one&#;third of studies recruited unselected febrile patients.

In a hypothetical cohort of patients presenting with fever where 30% (300 patients) have enteric fever: on average, and based on all the test results, Typhidot will miss the diagnosis in 48 of the 300 patients with enteric fever (66 missed based on Typhidot studies with low risk of bias due to clear reporting of indeterminates); TUBEX will miss 66; and Test&#;It Typhoid and prototype (KIT) tests will miss 93. In the 700 people without enteric fever the average number of patients with a false positive diagnosis of enteric fever would be 147 with Typhidot tests, (161 in Typhidot tests with low risk of bias due to clear reporting of indeterminates), 91 with TUBEX, and 70 with Test&#;It Typhoid and prototype (KIT) tests. The target product profile of an enteric fever RDT has not been defined. A sensitivity of > 90% and specificity of > 95% are probably minimum targets. In our hypothetical cohort of patients a test with our minimum target product profile would miss on average 30 of 300 enteric fever patients and give a false positive diagnosis in 35 of 700 without enteric fever.

RDTs for other febrile illnesses, such as malaria and dengue, already have been tested extensively in standardized evaluations that have provided an evidence base for World Health Organization (WHO) guidance and for the diagnostic algorithms used in endemic regions (WHO ; Abba ). The diagnostic tests for acute enteric fever have not been evaluated with the same rigorous methods. A diagnostic test to detect chronic (asymptomatic) carriers and individuals who have had prior exposure to the causative pathogens may also be of considerable epidemiological value. Such tests could potentially strengthen surveillance programmes aimed at identifying populations with a high&#;burden of enteric fever that might benefit from vaccination initiatives (Andrews ). The lack of such diagnostics obscures the true burden and impact of the disease; crucial information needed for policymakers, Ministries of Health, and others (Baker ; Crump ).

It is important to highlight the heterogeneity among the included studies. Patient selection (unselected febrile patients versus those suspected to have enteric fever) is a major source of heterogeneity. The variation in how indeterminate results in evaluations of Typhidot (IgG positivity, IgM positivity, or both) were treated and reported was also considerable (see the 'Strengths and weaknesses of the review' section). Most included studies took place in tertiary centres in South&#;Asian settings highly endemic for enteric fever. There were also studies set in medium&#;endemic regions but relatively few in sub&#;Saharan Africa (Crump ).

Thriemer review

Thriemer and colleagues published a systematic review of TUBEX and Typhidot for the diagnosis of acute enteric fever (Thriemer ). They reported a meta&#;analysis average sensitivity and specificity of TUBEX of 69% (95% CI 45% to 85%) and 88% (95% CI 83% to 91%) respectively. The Thriemer review authors also reported Typhidot sensitivity and specificity estimates of between 56% and 84% and 31 and 97% respectively (Thriemer ). They did not perform a meta&#;analysis for Typhidot due to the limited data available. These results are comparable to the findings of this Cochrane Review: TUBEX sensitivity of between 71% to 85% and specificity 82% to 91%; Typhidot sensitivity 73% to 91% and specificity 70% to 87% ( ). There are however a number of methodological differences between the two reviews.

Thriemer only included studies that used a commercial blood culture system with automated detection of positive cultures, and excluded studies using an 'in&#;house' blood culture system with manual detection of positive cultures. The number of studies of these tests using commercial blood culture systems was limited, which meant a meta&#;analysis was not possible. Commercial blood culture systems ensure that the reference test has been performed in a consistent and quality assured manner. If the 'in&#;house' blood culture system employs accepted media formulations and is subjected to appropriate quality control testing, it should be as sensitive as commercial systems (Wilson ). The major difference between the commercial automated and 'in&#;house' manual blood culture systems relates to the speed of result, with the automated systems detecting bacterial growth earlier.

Thriemer did not include test accuracy data for the Typhidot&#;M test. The Thriemer review authors explored various classifications of how to treat the indeterminate results when describing the statistical approach to analysing the Typhidot test data. In our Cochrane Review we have included studies that looked at Typhidot&#;M and classified indeterminate results as negative. To allow a clearer comparison between the Typhidot and Typhidot&#;M test results, we extracted the IgM antibody data from the Typhidot studies when given in the report.

The Thriemer review only included commercially available RDTs at the time of the literature search. We included the Test&#;it Typhoid ICT lateral flow assay (LifeAssay Diagnostics), which is now commercially available. This test was developed from several prototype RDTs by the Royal Tropical Institute (KIT) in Amsterdam. The Test&#;it Typhoid test and the KIT protypes all measure IgM antibodies against an lipopolysaccharide (LPS) antigen in various formats. In this review we have evaluated both the KIT prototypes and the commercial RDT.

Reference standard

The evaluation of RDTs in enteric fever is complicated by the lack of a suitable reference standard (Baker ). The quality of the reference standard used in these studies affects the diagnostic accuracy results of each RDT. Combinations of peripheral blood culture, bone marrow culture, and blood PCR positivity have been used to indicate a true positive result (enteric fever case). If these reference tests are negative then we have described these as a non&#;enteric fever case. Blood culture lacks sensitivity (WHO ; Mogasale ), so it is likely some of the culture&#;negative patients will actually have enteric fever. It must be acknowledged that culture&#;negative patients with a positive RDT result may actually be true positives rather than false positives. Most Grade 2 studies used blood culture only as the reference standard ( ; ). The stronger studies were those where index tests were evaluated against more than one different reference test (Siba ; Moore ). Studies with more robust reference standards demonstrated reduced RDT sensitivity. The Grade 1 studies using bone marrow culture were conducted in higher prevalence populations (Khan : 54%; Bhutta : 47%), and perhaps in those with more severe disease. This correlates with the reduced index test performance in other high prevalence studies (Olsen : 75%). In the TUBEX ( ) and Typhidot ( ) studies, there seem to be a common 20% to 25% reduction in sensitivity when the blood polymerase chain reaction (PCR) result was combined with blood culture as a composite reference standard. PCR has the potential ability to increase the number of typhoid cases identified by detecting dead bacteria or bacteria that cannot be cultured (Massi ; Nga ). It appears that these patients are less likely to be antibody positive in the RDTs, which explains the decrease in sensitivity when a PCR reference test is used.

Study design

The identification of studies that use or avoid a case control design formed part of the assessment of methodological quality (Whiting ). Case control designs can introduce bias and increase apparent accuracy as more severe disease is often compared to healthy patients. Studies that avoid a case control design by recruiting a cohort of unselected febrile patients have a lower risk of bias relating to patient selection. Over a third (16) of the 37 studies used a case control study design. , , and are receiver operating characteristic (ROC) plots for Typhidot, TUBEX, and Test&#;It Typhoid and KIT prototypes respectively. Each study is plotted indicating whether they adopted or avoided a case control design. Across all three index test groups, case control studies had higher apparent accuracy, with results having a higher combination of sensitivity and specificity. This highlights the importance of robust study designs in the evaluation of diagnostic test accuracy.

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Only 11 of the 37 included studies recruited unselected febrile patients. Most of the other studies used a clinical suspicion of enteric fever as the major entry criteria, but rarely specified the precise clinical criteria used to suspect the disease. The choice of the optimum non&#;disease control group is also difficult. Unselected febrile patients with another confirmed diagnosis are the optimum control group, but difficult to recruit. Thriemer also discussed this control group issue (Type 1 control). Patients with suspected enteric fever or non&#;specific fever but who are blood culture negative are less satisfactory as a non&#;disease control group (Type 2 control) and will decrease the apparent specificity of the test (Thriemer ). Cases in this group may actually have enteric fever despite testing negative on both index and reference tests. In addition to this, studies that analysed index tests in healthy afebrile controls are likely to have overestimated specificity.

Comparisons between tests

Comparisons of diagnostic tests are typically based on a combination of both direct comparisons where the tests are compared in the same patients, and indirect comparisons, where the tests being compared are conducted on different patients. Direct comparisons are at lower risk of bias as when the same patients at the same time point are tested as patients are tested with the same disease severity and comorbidities, and other features of study design that may give rise to potential for bias are also the same.

We compared Typhidot, TUBEX, and Test&#;It Typhoid based on a combination of direct and indirect test comparisons. We did not detect any statistically significant difference between these tests when the comparisons were based on Typhidot tests at lower risk of bias due to clear reporting of indeterminates.

There were 11 studies with direct comparisons of different RDTs within the same study ( ; ). TUBEX and Typhidot/Typhidot&#;M were the most common comparisons. There was no statistical difference detected and no consistent direction of difference found between these two groups of index tests (Typhidot and variants versus TUBEX).

Summary of main results

We have summarized the main quantitative diagnostic test accuracy results in 'Summary of findings' table 1 ( ).

• The number of high quality studies that evaluated the diagnostic accuracy of RDTs for enteric fever was low, as many studies adopted a case control study design.

• Only 3/37 included studies used the Grade 1 reference standard of bone marrow culture.

• Less than one&#;third of the included studies (11/37) recruited unselected febrile patients. Most used a clinical suspicion of enteric fever as the major inclusion criterion.

• Most included studies (86%) recruited patients from the Asia&#;Pacific region, and 50% of studies recruited from South Asia.

• The three main groups of RDTs for enteric fever evaluated were: Typhidot and its variants; TUBEX; and the Test&#;it Typhoid test with its earlier dipstick/latex agglutination/lateral flow assays prototypes developed by the Royal Tropical Institute (KIT), Amsterdam.

• The diagnostic accuracy for enteric fever of the three main RDT groups was moderate. TUBEX performed the most consistently with moderate average sensitivity (78%) and better specificity (87%), but when compared to Typhidot there was no evidence to suggest that one was better than the other.

• The Test&#;it Typhoid tests and KIT protypes demonstrated moderate sensitivity, but higher levels of specificity (average 90%).

• For Enterocheck WB, Enteroscreen, PanBio Multi&#;test Dip&#;S&#;Tick, Mega Salmonella, SD Bioline,and Onsite Typhoid, there is insufficient evidence to recommend these tests, as there are only results from 1, 2, or 3 included studies. Several of these RDTs had inferior performance to either Typhidot or TUBEX, based on comparison of sensitivity in the same participants in single studies.

• We did not find any statistically significant differences in sensitivity or specificity between Typhidot tests evaluated with low risk of bias due to clear reporting of indeterminates and the TUBEX and Test&#;It Typhoid tests, based on combined data from both direct and indirect test comparisons (comparisons of test on either the same patients or different patients).

• Analysis of direct paired (comparative) data was possible across 10 studies comparing Typhidot and TUBEX, but we did not find any statistically significant difference between the two tests. It is not possible to state that one group of index tests has higher accuracy than another. Within individual studies data was available to compare other commercial tests, and further studies are needed to substantiate findings from single studies.

• There was insufficient data to formally investigate sources of heterogeneity as listed in the ' Secondary objectives ' and ' Investigations of heterogeneity ' sections.

• There were no eligible studies that evaluated RDTs exclusively for detecting paratyphoid disease.

Strengths and weaknesses of the review

A major problem with most included studies was the use of a relatively weak reference standard. Blood culture has an estimated sensitivity of between 40% to 80% (WHO ), with a more recent systematic review estimating sensitivity to be around 60% (Mogasale ). Only three studies used the best reference standard currently available (blood culture and bone marrow culture). Bone marrow culture is estimated to increase the number of true positives by an additional 10% over blood culture alone (WHO ; Mogasale ). The additional benefit of a blood PCR result is undefined, and the testing methodology has not yet been standardized (Smits ). A weak reference standard means that a number of true positive results were classified as false negatives (Reitsma ). There was a great variation in the reporting of the accreditation and quality of microbiology laboratories where the cultures were processed.

Statistical analysis of Typhidot and its variants was complicated, given the evolution of the product target from measuring both IgM and IgG antibodies to just IgM alone. This was compounded by the inadequate clarity of the reported results. Many of the included studies were not well reported, and did not perform well under the scrutiny of the modified QUADAS&#;2 tool. The data for a number of studies was incomplete, and could not be clarified despite contacting corresponding authors. Only a few studies reported blinding of the index and reference tests.

A weakness of the review related to the classification and subsequent analysis of indeterminate results for Typhidot tests. When we could extract both IgG and IgM data for Typhidot, we classified a case that was IgG positive and IgM negative as indeterminate. This differed from the treatment of indeterminate results of some included studies (Fadeel ; Olsen ).

Thriemer described the differences in sensitivity and specificity from one study (Kawano ) in three different ways: when indeterminate results were excluded; when indeterminate results were considered negative; and when indeterminate results were included in the denominator. In our Cochrane Review, this is illustrated in and . These demonstrate a roughly 20% decrease in sensitivity when we included indeterminate results in the analysis. It is important to acknowledge variation in the classification of indeterminate results as a limitation in the analysis of results for Typhidot.

Data extraction from certain case control studies, Fadeel , required careful recalculation where different categories of negative patients were described, for example, blood culture negative and Widal Test positive, versus known negatives. Index tests were then tested against different sub&#;groups within the cohort. This change in sampling meant that the prevalence of disease changed depending on which subgroup the index test was used in.

This review covers both typhoid and paratyphoid fever, but there were no suitable studies related to paratyphoid alone. Another weakness of this review was the variability in the treatment of paratyphoid cases as part of the diagnostic test accuracy data between studies. In one study, authors excluded cases of blood culture positive Salmonella Paratyphi A (Jesudason ). A number of studies classified blood culture positive cases of paratyphoid as true negatives (Gasem ; Dutta ; Hosamani ; Sanjeev ). In contrast, paratyphoid fever was classified as a target condition along with typhoid fever in two studies (Dong ; Prasad ).

Applicability of findings to the review question

A low number of studies have evaluated the diagnostic test accuracy of enteric fever RDTs. Furthermore, the number of good quality studies was low. The main issues relating to quality include: utility of a second&#;class reference standard; recruitment of clinically suspected enteric fever patients as opposed to unselected febrile patients; poor reporting of whether investigators were blinded to reference test results when interpreting the index tests; and frequent use of a case control design. The sensitivity and specificity of TUBEX, Typhidot and its variants, and Test&#;it Typhoid test and its KIT protypes are not robust enough to replace existing diagnostic tools in enteric fever.

Abstract

Typhoid is an enteric disease caused by Salmonella Typhi. Like many febrile illnesses, typhoid presents with nonspecific symptoms. In routine healthcare settings in low- and middle-income countries, typhoid fever is suspected and treated empirically. Though many diagnostic tests are available for typhoid diagnosis, there are currently no diagnostic tests that meet ideal requirements for sensitivity, specificity, speed, and cost-effectiveness. With introduction of typhoid conjugate vaccine, it is essential to explore the current and future typhoid approach in the context of use case and access to ensure their utilization for disease control.

Regional estimates of the burden of typhoid fever cannot be measured accurately without improved disease diagnostics; this lack of diagnostics and data impacts the ability of governments to plan and appropriately intervene. Given the need for disease control, funding typhoid diagnostic capacity, including availability and use of improved typhoid test kits, should be increased, especially where the incidence of typhoid is unknown [1]. Challenges regarding typhoid diagnostics may also impact the implementation of new-generation typhoid vaccines in endemic regions due to lack of surveillance tools [2]. Multidrug resistance (MDR) has been spread intercontinentally due to an increase in travel connectivity, affecting those living in endemic regions and travelers alike [3, 4]. Notably, multidrug-resistant and fluoroquinolone-resistant variants of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) may be associated with more severe disease with potentially adverse outcomes, therefore creating clinical management challenges [3]. The spread of drug-resistant organisms as well as an expected reemergence of typhoid in currently nonendemic settings due to climate change make improved diagnostics key for tracking incidence to inform public health policy in additional to ensuring individual patients get appropriate treatment. Here, we aim to review gaps in the diagnostic landscape for typhoid and explore new technological and access developments that could improve the diagnostic landscape in the future and in the context of existing target product profile drafts. We explore different areas of typhoid diagnostic challenges, including current shortcomings in an example setting, expanding understanding of existing rapid diagnostic tests (RDTs), the future of diagnostics as part of surveillance, and access-related considerations that aim to improve the availability of quality diagnostic tests in the future.

CHALLENGES IN TYPHOID DIAGNOSIS: EXAMPLE FROM LAOS

The Lao People&#;s Democratic Republic (Laos) is a landlocked country in Southeast Asia with a population of approximately 7 million people [5]. Typhoid fever is endemic in Laos, but there are limited epidemiological data. In a hospital-based study examining blood cultures at Mahosot hospital in the capital, Vientiane, between and , there were a total of 913 culture-confirmed typhoid cases from just over 60 000 blood cultures (&#;1.5% positivity). Most cases originated in rural areas with the majority of patients recruited into research studies; there were limited specimen requests outside of these studies, particularly outside of the capital city [6].

These data suggest that the amount of typhoid in Laos is underestimated; detection relies on blood culture, with the laboratory capacity to process blood cultures being limited outside of Vientiane (capacity is increasing as a consequence of a UK Fleming Fund grant). Provincial hospital laboratories in the network perform manual blood cultures in-house and then send positive cultures, including suspected S. Typhi, to a central government or reference laboratory in Vientiane for identification or confirmation. The shipping process can take several days mostly due to infrastructure challenges (eg, lack of transportation/staff, unpassable roads) with blood culture bottles sitting at room temperature without any additional temperature regulation. This leads to isolates not being recovered in the reference laboratory in Vientiane due to high temperatures and transportation conditions. A previous study recorded temperatures as high as 41°C in a transportation box used for sample shipment [7]. The cost and availability of confirmation tests also impacts the diagnostic result, as high costs of identification reagents, antisera, and shipment may be prohibitive for laboratories in low- and middle- income countries (LMICs); for example, API 20E identification strips cost approximately US$6 per test and antisera cost approximately $3 per test and can take months to be shipped to the country. Blood culture is recognized to be the gold standard for typhoid diagnosis; however, it is a complex and expensive process as highlighted by the example from Laos. In this example use case, financial and logistic support can be provided due to research affiliation of the laboratories and even here things are not working in a way that provides reliable, high-quality data. The result of such imperfect data is the underestimation of typhoid cases nationally but also in the areas with access to research network.

This example from Laos shows that without local capacity and appropriate (cheap, long shelf life, simple to use) diagnostic tests, provincial hospitals have to continue to rely on sample transport over large distances. This type of centralized testing leads to lost time and quality and as a result clinicians are inclined to skip the laboratory sample and rather go ahead with treatments without laboratory confirmation. However, realistically, blood or bone marrow cultures, which are highly specific and considered the gold standard, are not suitable for use outside of well-established centers and not truly close to the point of care. This lack of accurate diagnostic testing has a negative impact on patient care and reliable incidence data.

THE LACK OF SUITABLE RAPID DIAGNOSTIC TESTS

Ultimately to most of the challenges described in the case study, well-performing, high-quality tests are needed to be performed in a decentralized manner at the point of contact and not at a central facility that requires sample shipment. While many tests exist and are used at point of care (POC) by minimally trained staffs, unfortunately few meet the &#;well performing&#; or &#;high quality&#; bar that is equally essential. Various RDTs and different forms of the Widal test are commonly used in health facilities around the world to diagnose typhoid. These tests are cheap and simple, do not require sophisticated laboratories, and deliver results in a shorter time frame than blood culture, making them very popular. However, such tests lack sensitivity and specificity and thus are not of sufficient accuracy to replace blood culture as the main diagnostic approach for typhoid fever. The Widal test is the most used test to diagnose typhoid despite a low performance (sensitivity range, 57%&#;74%; specificity range, 43%&#;83% [8]) reported in several studies. A Cochrane review summarized the evidence on diagnostic accuracy of available RDTs for enteric (typhoid) fever (mostly TUBEX, Typhidot, and KIT Test-It) [9]. The result of the meta-analysis found TUBEX to have an average sensitivity of 78% and a specificity of 87%; Typhidot had an average sensitivity of 84% and a specificity of 79%, and Test-It (KIT) was found to have an average sensitivity of 69% and a specificity of 90% [8]. Numerous studies had been conducted to assess commercial diagnostic tests for typhoid [8]; however, key opinion leaders highlighted that these studies are difficult to compare due to a lack of comparable case definitions and a lack of geographical diversity. To address these data shortcomings, FIND established a head-to-head comparison of commercial typhoid tests and simultaneously generated a sample set that could be used in the evaluation of emerging technologies [10]. Typhoid positives and negatives were analyzed in both regions with 205 positives and 205 negatives from Asia and 59 positives and 59 negatives from Africa. Nine different RDTs were evaluated against blood culture as the reference standard. The tests used were SD Bioline Salmonella Typhi immunoglobulin G (IgG)/immunoglobulin M (IgM), Typhidot Rapid IgG/IgM, Enterocheck WB, Test-It Typhoid IgM, CTK Typhoid IgG/IgM Combo Rapid Test CE, Spectrum Typhoid IgG/IgM Rapid cassette, TUBEX-TF, Diaquick S.Typhi/Paratyphi antigen cassette, and the Widal test. The sensitivity values varied widely between the different tests, from 0% with the Diaquick antigen cassette to 78.8% with the IgG component of the CTK Typhoid IgG/IgM Combo Rapid Test CE. Overall, the study confirmed that no test currently meets the desired accuracy criteria [11] and diagnostic innovation is critical.

THE FUTURE OF TYPHOID DIAGNOSTICS

While a variety of techniques are currently in use for the diagnosis of typhoid, no single technique satisfies the requirement for sensitivity and specificity while being rapid and cost-effective. This was again confirmed in the most recent data generated by FIND and partners [9], and the need for innovations was once again made obvious. However, future innovation for typhoid diagnosis should not only focus on disease diagnosis for immediate treatment purposes but also disease surveillance and the detection of carriers, to support public health interventions. Ultimately both aspects are different sides of the same coin and need to be advanced simultaneously to accelerate disease elimination as a whole.

RDTs using selected antigens such as the protein HlyE and sugars in the lipopolysaccharide are under investigation and exhibit some potential [12, 13]. Furthermore, studies using metabolomic platforms have sought to identify biomarkers specific to typhoid. Identifying a single or a combination of metabolites during the course of typhoid illness could provide several promising biomarkers [14&#;16]. Polymerase chain reaction (PCR)&#;based detection of typhoid in the blood generally shows poor sensitivity. Conventional to real-time PCR and nested and multiplex PCR using different targets have been used to diagnose typhoid with sensitivity ranges of 40%&#;100% [17]. However a more recent study using machine-learning algorithms to identify expression signatures of host-associated genes showed some promise. This study identified the transcripts of 5 key genes (STAT1, SLAMF8, PSME2, WARS, and ALDH1A1) that can differentiate enteric fever from other febrile illness; this approach may have some traction for a multipathogen diagnostic approach [18]. The latter 2 approaches might provide better value and may aid in identifying the cause of undifferentiated febrile disease (including typhoid) in resource-limited LMICs for better patient management. At this point, however, this is not yet the case as none of the existing tools meet the needs of resource-poor settings, both in terms of cost and performance [17]. A tool would have to be cheap and simple to use (akin to the GeneXpert) to really make it suitable for hospitals in lower-resource settings. While simple molecular tools to be used at the POC level were scarce pre-, after the scientific advancements and investments made linked to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic it might be more feasible to think about a workable POC device that can be used to identify a magnitude of possible fever causes [18]. Given the complexities of typhoid diagnosis in patients or carriers using simply accessible samples, public health agencies might have to resort to identifying the pathogens in the environment as a proxy for patients.

Molecular approaches look promising to detect S. Typhi in environmental samples. These methods are not meant as tools for healthcare workers to inform patient management so will not advance this area, yet they might open up a more promising area for public health surveillance, similar to cholera.

When thinking about and envisioning the next generation of diagnostic tools, it is critical that we do not confuse the different use cases and that we make sure the future Target Product Profiles account for all. As of now, the ideal approach unfortunately remains elusive as it needs to be low-cost and simple to use even when deployed for environmental surveillance. Looking toward a future with increased focus on typhoid, the most likely scenario is a combined approach where more high-tech approaches are developed by research and public health authorities and individual patient management remains to be guided by culture as well as improved RDTs.

IMPROVED DIAGNOSTICS ACCESS TO IMPROVE VACCINE DELIVERY

Arguably, one of the biggest consequences of the current limitations in typhoid diagnostics and the resulting data gap on true prevalence is the ability of governments to determine whether and where to use typhoid conjugate vaccine (TCV). Two TCVs have been prequalified by the World Health Organization (WHO) [19]. The WHO Strategic Advisory Group of Experts recommends prioritizing TCV introduction in areas with high typhoid fever burden and areas with high prevalence of antimicrobial resistance (AMR) [20]. Although the introduction is good news for many countries, due to the lack of quality diagnostics both at the central (eg, blood culture or environmental surveillance data) or decentralized level (eg, reliable RDT data, as part of the surveillance data set), the same countries often struggle to justify the use of TCVs due to missing data. The lack of data links both to typhoid as well as AMR data, the latter also requiring microbiology facilities.

Since , a TCV vaccine has been approved for funding support by Gavi, the Vaccine Alliance, and will be made available to eligible countries [21]. Liberia, Malawi, Nepal, Pakistan, and Zimbabwe have been approved for funding support and related TCV introduction support. Sixteen countries theoretically eligible for Gavi support do not have reliable typhoid surveillance data in the public domain (ie, blood culture confirmation, since at least ) [22]. In light of the outlined limitations of all currently available typhoid RDTs, the WHO recommends that blood culture be used as the preferred diagnostic test for guiding immunization program decisions [23]. Gavi is working to help make improved typhoid diagnostic tests available to countries eligible for Gavi funding support. Such improved tests should facilitate country efforts to scale up reliable typhoid diagnostic testing as part of multidisease surveillance systems, which in turn should lead to improved availability and use of information to ensure that immunization programs are more effective, efficient, and equitable [24].

CONCLUSIONS

Reviewing the past, present, and future of typhoid diagnostic tests highlights that we really have not progressed rapidly since the introduction of the Widal test. There are many advances still to be made to enable the timely and reliable diagnosis of typhoid infections. Our overview of use cases ranging from patient management to environmental surveillance and vaccine allocation highlight the critical need for research and product development work. We argue that we are in the right period to solve these problems, with the ongoing SARS-CoV-2 pandemic showing the importance of diagnostics for disease mitigation. Additionally, the fact that Gavi is a committed ally for diagnostics is encouraging and may help to steer additional resources toward the development of pragmatic tools.

Notes

Financial support. The study mentioned on &#;The lack of suitable rapid diagnostic tests&#; was conducted by FIND through DFID grant.

Supplement sponsorship. This article appears as part of the supplement &#;Charting the Course to Meet the Challenges Ahead: Research and Developments on Typhoid and Other Invasive Salmonelloses&#; sponsored by the Coalition against Typhoid Secretariat, housed at the Sabin Vaccine Institute in Washington, DC and made possible by a grant from the Bill & Melinda Gates Foundation.

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© The Author(s) . Published by Oxford University Press on behalf of Infectious Diseases Society of America.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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