Sunday, July 22, 2012

Not so golden? Microscopic agglutination test for diagnosis of leptospirosis

The microscopic agglutination test (MAT) is designated the "gold standard" for the laboratory diagnosis of leptospirosis, a spirochete disease that can cause severe illness if not promptly treated.  Although imperfect, MAT is used as the benchmark when the performance of another diagnostic test for leptospirosis is being assessed.  It is also used to determine the prevalence of leptospirosis in a population.  How imperfect is MAT?  A recent study by Limmathurotsakul and colleagues, published in the journal Clinical Infectious Diseases, claims that its performance is much worse than scientists previously thought.

MAT involves mixing serial dilutions of patient sera with live suspensions of Leptospira.  If agglutinating antibodies against Leptospira are present, the spirochetes will clump.  The clumps can be seen by darkfield microscopy.  Although the idea behind MAT is simple to understand, the technique itself is cumbersome.  Since agglutinating antibodies react best with the specific Leptospira serovar infecting the patient, cultures of at least one serovar from each of the ~20 major Leptospira serogroups must be maintained.  To perform the assay, each serum dilution is mixed individually with a suspension from each culture and examined by microscopy one at a time.  The assay is time consuming, laborious, and potentially hazardous to laboratory personnel.  For these reasons MAT is not routinely employed for diagnostic testing outside of the research setting.

The performance of a diagnostic test is judged by its sensitivity and specificity.  The problem with leptospirosis is figuring out how many actually have the disease so that the sensitivity can be calculated accurately.  Since the sensitivity of culture is poor, researchers rely on antibody tests such as MAT to identify leptospirosis cases.  This approach assumes that the sensitivity and specificity of MAT are 100%.

In general there are two problems with using antibody tests for diagnostics.  The first is that it takes time for the immune system to generate enough antibody that can be detected.  The second problem is that those with previous exposure to the pathogen will test positive even if they are not currently infected.  To minimize these problems, patients with the signs and symptoms of leptospirosis are deemed to have a positive MAT if they fulfill one of the following criteria.
  • At least a four-fold increase in MAT titer between paired sera. 
  • At least a 1:400 MAT titer when only a single specimen is available.  This cutoff is sometimes adjusted based on the prevalence of leptospirosis in the population being examined.

Since some false negative MAT cases can be identified by culture, one way to calculate the sensitivity of MAT is to add the number of MAT-positive and culture-positive (but MAT-negative) cases together to estimate the number of patients with leptospirosis and then calculate the percentage of MAT-positive cases among these patients.  Limmathurotsakul and colleagues performed these calculations with data from their four earlier studies conducted in Thailand.  A total of 413 patients tested positive by MAT or culture (or both).  They found that the sensitivity of MAT was 86%-96% across the four sets of data.  The remaining 4-14% were false negatives, having tested positive by culture but not by MAT.

The authors next calculated the true sensitivity and specificity of MAT with a statistical tool called latent class analysis, which does not assume any perfect gold standard.  Since there is no perfect test, the true disease status of each patient is the unknown or "latent" variable.  Results from multiple diagnostic tests are related to the latent variable using statistical models.  The calculations go beyond the scope of this blog post, but the bottom line is that the true sensitivity and specificity of each diagnostic test can be estimated with these models.  In addition to MAT and culture, the authors tested some of their patients with an immunofluorescence assay (IFA), lateral flow test (LF), and/or PCR. Latent class analysis is more powerful when the diagnostic tests being evaluated detect different features of the infection.  MAT, IFA, and LF are antibody tests, and culture and PCR detect the pathogen itself.

The sensitivity of MAT calculated by this method turned out to be only 49.8%, much lower than the 86%-96% calculated using the standard method that assumes a perfect gold standard.  The sensitivity of culture alone was 10.5%.  Combining culture with MAT did not help much; the sensitivity of the combined approach was only 55.5%.  The low sensitivity of "MAT plus culture" suggests that the specificities calculated for the alternative tests may be underestimated by the standard method.  This is because some of the many false-negative cases may be correctly identified as having leptospirosis by the alternative tests.  This turned out to be the case for two of the tests.   Specificities for all tests were over 95% by latent class analysis.  However, the specificities for PCR (82.5%) and the lateral flow test (70.5%) were lower when "MAT plus culture" was assumed to be the perfect gold standard.

You can see that the accuracy of alternative leptospirosis tests is underestimated when MAT (or MAT plus culture) is assumed to be the perfect reference test.  Another implication of the study is that the prevalence of leptospirosis has been underestimated, at least in Thailand.  The only other study to evaluate the performance of MAT by latent class analysis was conducted by the CDC here in the U.S almost a decade ago.  In contrast to the Limmathurostsakul study, the CDC study determined that the sensitivity of MAT was high, at 98.2%.  There were many differences between the two studies, including the patient population, the alternative tests evaluated, the time interval between collection of paired sera, and the number of serovars included for MAT.  The poor performance of MAT in the Thailand study may therefore not be a universal finding.


Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ, Cooper B, Peacock SJ (August 2012).  Fool's gold: why imperfect reference tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis.  Clinical Infectious Diseases 55(3):322-331.  DOI: 10.1093/cid/cis403

Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA, Plikaytis BD, Perkins BA, Phelan M, Levett PN, and Weyant RS (February 2003).  Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis.  Journal of Clinical Microbiology 41(2):803-809.  DOI:  10.1128/JCM.41.2.803-809.2003

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