Saturday, March 31, 2012

qPCR testing for Leptospira infection reveals sex differences in bacterial load

The pitfall of serological testing is that patients test negative during the early days of infection because time is needed for the immune response to ramp up antibody production against the pathogen.  Unfortunately clinical laboratories are stuck with serological methods for detecting Leptospira since direct tests for the spirochete are not available.

Since Leptospira has to enter the bloodstream to spread throughout the body, PCR testing of blood is one tool that may aid prompt diagnosis of leptospirosis.  In a study published in Clinical Infectious Diseases, Agampodi and colleagues conducted quantitative real-time PCR (qPCR) on patient sera collected during a 2008 leptospirosis outbreak in Sri Lanka, where leptospirosis is endemic.  With a reported annual incidence of 5.4 cases per 100,000, Sri Lanka has the sixth highest incidence of leptospirosis in the world.

The acute-phase sera tested for the study came from patients with confirmed leptospirosis.  The patients had clinical evidence of leptospirosis, and a second blood sample drawn at least a week later indicated that they had rising antibody titers or had seroconverted.

The PCR primers targeted the DNA encoding the 16S rRNA of Leptospira.  The sensitivity of qPCR turned out to be 51% (25/49) with acute-phase sera collected during the first ten days of illness.  This doesn't sound impressive at all, but the sensitivity of the microscopic agglutination test (MAT) was much worse.  MAT testing is done by mixing dilutions of serum with suspensions of Leptospira.  If anti-Leptospira antibodies are present, they will cause the bacteria to clump.  The sensitivity of MAT with acute-phase sera collected up to 15 days after symptoms began was a miserable 18% (13/73).  Past studies conducted in Sri Lanka and elsewhere around the world have also demonstrated poor performance of serological testing for leptospirosis.  Although the specificity of qPCR wasn't determined, it's clear that qPCR is an improvement over serological testing for the prompt diagnosis of leptospirosis.

One interesting observation came out of the qPCR data.  Men who were qPCR positive had a higher bacterial load than women who tested positive (median of 15,640 bacteria/ml in men vs. 5,611 bacteria/ml in women, P = 0.022, Mann-Whitney U test).  Based on this observation, the authors raised the possibility that men are biologically more susceptible to leptospirosis than women.  On the other hand, the difference in bacterial load may have nothing to do with biology.  It may simply reflect differences in when men and women sought medical care, or it may mean men were exposed to environmental sources that were more heavily contaminated with Leptospira.

A 2007 study conducted in Germany revealed sex differences in leptospirosis severity.  Men diagnosed with leptospirosis were more likely than women to have hemorrhage, jaundice, and renal impairment, all signs of severe disease (see table below).   The difference in disease severity could not be accounted for by differences in exposure risk, the infecting serogroup, or interaction with the health care system.  Could the difference be due to biological variation between the sexes?

Table 1 from Jansen et al., 2007.  OR adjusted for age.

Note: Here's a recent post in the blog Camp Other that examined sex differences in another disease of spirochetes, Lyme disease.

Main references

Agampodi SB, Matthias MA, Moreno AC, and Vinetz JM (March 12, 2012).  Utility of quantitative polymerase chain reaction in leptospirosis diagnosis: association of level of leptospiremia and clinical manifestations in Sri Lanka.  Clinical Infectious Diseases (published online ahead of print February 21, 2012).  DOI: 10.1093/cid/cis035

Jansen A, Stark K, Schneider T, and Schöneberg I (May 1, 2007).  Sex differences in clinical leptospirosis in Germany: 1997-2005.  Clinical Infectious Diseases 44(9):e69-e72.  DOI: 10.1016/j.ijid.2007.09.011

Other references

Pappas G, Papadimitriou P, Siozopoulou V, Christou L, and Akritidis N (July 2008).  The globalization of leptospirosis: worldwide incidence trend.  International Journal of Infectious Diseases 12(4):351-357.  DOI: 10.1016/j.ijid.2007.09.011

Reller ME, Bodinayake C, Nagahawatte A, Devasiri V, Kodikara-Arachichi W, Strouse JJ, Flom JE, Dumier JS, and Woods CW (September 9, 2011).  Leptospirosis as frequent cause of acute febrile illness in southern Sri Lanka.  Emerging Infectious Diseases 17(9):1678-1684.  DOI: 10.3201/eid1709.100915