How accurate are nucleic and antigen detection tests in diagnosing leptospirosis?

What was studied in this review?

Leptospirosis is an infectious disease, caused by bacteria called Leptospira that can be found in soil, freshwater, or in the infected urine of certain animals. It is mainly a problem in humid, tropical countries in Southeast Asia, and Central and South America, but it can also occur in temperate regions.

Leptospirosis causes fever and headache, and in some cases kidney, lung, or heart problems. Often, the symptoms are not unique for the disease, which makes it difficult to diagnose, and is therefore frequently missed.

Laboratory tests confirm diagnosis. These tests are based on demonstration of the presence of Leptospira, its DNA, or antibodies against Leptospira. Nucleic acid and antigen detection tests, such as conventional polymerase chain reaction (PCR) and real-time PCR, identify the bacterium or its DNA directly in blood or urine. Nucleic acid and antigen detection tests may detect Leptospira better in the early days of an infection, so that people can be treated earlier with antibiotics – resulting in better outcomes – and can provide useful information in outbreak situations. In outbreak situations, nucleic acid and antigen detection tests could serve as early warning systems.

What was the aim of this review?

The aim was to assess how well nucleic acid and antigen tests perform in detecting leptospirosis. In other words, to assess how many mistakes these tests make by either missing people with leptospirosis or misidentifying people without leptospirosis (healthy people or people with another disease).

What were the main results in this review?

The review included information from 41 studies with 5981 participants. We identified nine nucleic acid and antigen detection tests, of which PCR and real-time PCR were most often investigated.

An important finding was that the accuracy of both PCR and real-time PCR varied strongly between studies. We presented average accuracies for both tests, but there was great uncertainty around these averages. PCR often correctly identified people without leptospirosis (averaging 95 in 100 people), but frequently missed people with leptospirosis (averaging 30 in 100 people). The accuracy of the real-time PCR depended on the cut-off value for a positive test result. At a cut-off value where real-time PCR often correctly identified people without leptospirosis (averaging 95 in 100 people), it also frequently missed people with leptospirosis (averaging 71 in 100 people). If a person tests positive or negative for PCR or real-time PCR, the chance of the person actually having the disease depends on whether the suspicion of leptospirosis in that person was already high before taking the test. So, when interpreting the results of any of these tests, one must consider the strength of suspicion of leptospirosis in an individual, and how often leptospirosis occurs in the setting in which the test will be used.

It was uncertain whether PCR or real-time PCR performed better in detecting leptospirosis, since studies directly comparing these two tests were lacking. The results of other nucleic and antigen detection tests are described in the main text of the review.

How reliable were the results of the studies in this review?

Not all studies were conducted according to the highest scientific standards. This means that the results of some studies may have been overestimated or underestimated. Furthermore, the tests used to verify whether a person truly had leptospirosis or not (called the reference standard) may not accurately distinguish people with or without leptospirosis. For these reasons, more high-quality studies are needed to confirm the reliability of these results.

Who do the results of this review apply to?

The results may apply to people who may have leptospirosis. However, the performance of the PCR and real-time PCR vary considerably among studies and it is yet unclear what causes this difference in performances. It is probable that the test performs better or worse depending on how prevalent leptospirosis is in the region, and depending on the time between the onset of symptoms and time of testing. Therefore, it is difficult to generalise the results of this review to all settings.

How up-to-date is this review?

The review authors searched for and used studies published up to 6 July 2018.

Authors' conclusions: 

The validity of review findings are limited and should be interpreted with caution. There is a substantial between-study variability in the accuracy of PCR and real-time PCR, as well as a substantial variability in the prevalence of leptospirosis. Consequently, the position of PCR and real-time PCR in the clinical pathway depends on regional considerations such as disease prevalence, factors that are likely to influence accuracy, and downstream consequences of test results. There is insufficient evidence to conclude which of the nucleic acid and antigen detection tests is the most accurate. There is preliminary evidence that PCR and real-time PCR are more sensitive on blood samples collected early in the disease stage, but this needs to be confirmed in future studies.

Read the full abstract...
Background: 

Early diagnosis of leptospirosis may contribute to the effectiveness of antimicrobial therapy and early outbreak recognition. Nucleic acid and antigen detection tests have the potential for early diagnosis of leptospirosis. With this systematic review, we assessed the sensitivity and specificity of nucleic acid and antigen detection tests.

Objectives: 

To determine the diagnostic test accuracy of nucleic acid and antigen detection tests for the diagnosis of human symptomatic leptospirosis.

Search strategy: 

We searched electronic databases including MEDLINE, Embase, the Cochrane Library, and regional databases from inception to 6 July 2018. We did not apply restrictions to language or time of publication.

Selection criteria: 

We included diagnostic cross-sectional studies and case-control studies of tests that made use of nucleic acid and antigen detection methods in people suspected of systemic leptospirosis. As reference standards, we considered the microscopic agglutination test alone (which detects antibodies against leptospirosis) or in a composite reference standard with culturing or other serological tests. Studies were excluded when the controls were healthy individuals or when there were insufficient data to calculate sensitivity and specificity.

Data collection and analysis: 

At least two review authors independently extracted data from each study. We used the revised Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2) to assess risk of bias. We calculated study-specific values for sensitivity and specificity with 95% confidence intervals (CI) and pooled the results in a meta-analysis when appropriate. We used the bivariate model for index tests with one positivity threshold, and we used the hierarchical summary receiver operating characteristic model for index tests with multiple positivity thresholds. As possible sources of heterogeneity, we explored: timing of index test, disease prevalence, blood sample type, primers or target genes, and the real-time polymerase chain reaction (PCR) visualisation method. These were added as covariates to the meta-regression models.

Main results: 

We included 41 studies evaluating nine index tests (conventional PCR (in short: PCR), real-time PCR, nested PCR, PCR performed twice, loop-mediated isothermal amplification, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, immunochromatography-based lateral flow assay, and dipstick assay) with 5981 participants (1834 with and 4147 without leptospirosis). Methodological quality criteria were often not reported, and the risk of bias of the reference standard was generally considered high. The applicability of findings was limited by the frequent use of frozen samples. We conducted meta-analyses for the PCR and the real-time PCR on blood products.

The pooled sensitivity of the PCR was 70% (95% CI 37% to 90%) and the pooled specificity was 95% (95% CI 75% to 99%). When studies with a high risk of bias in the reference standard domain were excluded, the pooled sensitivity was 87% (95% CI 44% to 98%) and the pooled specificity was 97% (95% CI 60% to 100%). For the real-time PCR, we estimated a summary receiver operating characteristic curve. To illustrate, a point on the curve with 85% specificity had a sensitivity of 49% (95% CI 30% to 68%). Likewise, at 90% specificity, sensitivity was 40% (95% CI 24% to 59%) and at 95% specificity, sensitivity was 29% (95% CI 15% to 49%). The median specificity of real-time PCR on blood products was 92%. We did not formally compare the diagnostic test accuracy of PCR and real-time PCR, as direct comparison studies were lacking. Three of 15 studies analysing PCR on blood products reported the timing of sample collection in the studies included in the meta-analyses (range 1 to 7 days postonset of symptoms), and nine out of 16 studies analysing real-time PCR on blood products (range 1 to 19 days postonset of symptoms). In PCR studies, specificity was lower in settings with high leptospirosis prevalence. Other investigations of heterogeneity did not identify statistically significant associations. Two studies suggested that PCR and real-time PCR may be more sensitive on blood samples collected early in the disease stage. Results of other index tests were described narratively.

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