Sunday, July 22, 2012

100 (micro)meter dash

A fun read in the current issue of Nature Reviews Microbiology is an essay entitled "The Microbial Olympics, " just in time for the Summer Olympics.  You will find stories about microbes competing in boxing, javelin, pathogen relay, diving, and other Olympic events.  Flagellated bacteria compete in the 100 micrometer dash, which you can watch below.  (Note that some of the contestants were genetically modified.)

Lane assignments:
  1. E. coli chimera (has sodium-driven flagellar motors instead of its normal proton-driven motors)
  2. E. coli (proton-driven flagellar motors)
  3. Vibrio alginolyticus, puller (clockwise-locked flagellum "pulls" cell body from front)
  4. Vibrio alginolyticus, pusher (counterclockwise-locked flagellum "pushes" cell body from back)
  5. Pseudomonas aeruginosa
  6. Rhodobacter sphaeroides
  7. Rhodospirillum rubrum
  8. Yersinia enterocolitica


Youle M, Rohwer F, Stacy A, Whiteley M, Steel BC, Delalez NJ, Nord AL, Berry RM, Armitage JP, Kamoun S, Hogenhout S, Diggle SP, Gurney J, Pollitt EJG, Boetius A, and Cary SC (August 2012).  Nature Reviews Microbiology 10(8):583-588.  DOI: 10.1038/nrmicro2837

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

Thursday, July 12, 2012

Borrelia burgdorferi needs the alternative sigma factor RpoS to flee from the tick's midgut

The alternative sigma factor RpoS is a key player in the life cycle of Borrelia burgdorferi, the Lyme disease spirochete.  RpoS directs RNA polymerase to transcribe genes with promoters recognized by the alternative sigma factor.  B. burgdorferi deploys RpoS to directly or indirectly boost transcription of 103 out of its ~1400 genes while inside a mammalian host.  The most famous RpoS-dependent gene is ospC, which encodes a surface protein that enables B. burgdorferi to survive the early stages of infection.  Not surprisingly, RpoS is essential for B. burgdorferi to establish infections in mammals.  On the other hand, B. burgdorferi does not bother to make RpoS while living in the midgut of Ixodes ticks since RpoS-dependent gene products are not needed in this stage of its life cycle.  The rpoS gene is turned on only after the tick attaches to an animal and begins sipping its blood.  B.burgdorferi is transmitted to the victim as the tick feeds.

A study published by Justin Radolf's group in the February issue of PLoS Pathogens showed that RpoS is needed by B. burgdorferi to be transmitted from the tick to a mammal.  Transmission is a multistep process for B. burgdorferi.  Although the spirochetes proliferate to large numbers in the midgut while the tick feeds, only a few of them escape through the wall of the midgut into the hemocoel, the tick's body cavity.  From there the spirochetes invade the salivary glands, which produces the saliva that carries the spirochetes into the victim's skin.  The authors found that B. burgdorferi mutants missing their rpoS gene failed to even make it out of the midgut.  None of the hemolymph samples extracted from the hemocoel of 39 feeding ticks carrying the rpoS mutant were culture positive, whereas the hemolyph from 21 of 25 feeding ticks harboring the wild-type strain were culture positive.

The researchers also viewed the activity of the rpoS mutant in the midgut by fluorescence microscopy.  From an earlier study (described in this blog post), they already knew how wild-type B. burgdorferi behaved within the midgut of feeding ticks.  In brief, the multiplying spirochetes remained firmly attached to the epithelial cells.  A mesh of spirochetes eventually surrounded the cells.  Since an unknown substance in the midgut was inhibiting motility, only the few spirochetes at the base of the epithelial cells detached and managed to wiggle their way into the surrounding hemocoel.

The rpoS mutant behaved quite differently from the wild-type strain in feeding ticks.  Instead of remaining stuck to the surface of the gut epithelial cells, the mutant spirochetes detached and accumulated in the lumen of the midgut.  Since the spirochetes were immotile, they were too far away from the base of the epithelium lining to escape into the hemocoel.

To get a better look of the spirochetes, the researchers examined silver-stained sections of the midgut contents by microscopy.  Here they saw something fascinating.  With the wild-type B. burgdorferi, they saw tufts of spirochetes attached to the epithelial cells, as expected from their earlier studies (panels D and G below).  With the rpoS mutant, they found midguts packed with round bodies (rpoS mutant, panels E and H).  The round bodies were not dead.  When the investigators removed the midguts and released the contents into Borrelia culture medium, the round bodies reverted back to the spiral shape within minutes.

Extracted from Figure 4 of Dunham-Ems et al., 2012.  Midguts of Ixodes ticks after feeding on mice for 72 hour.  Panels D and G, wild-type B. burgdorferi.  Panels E and H, rpoS mutant.  Bars, D and E, 25 µm; G and H, 10 µm.  Source

Spirochetes in culture change shape into round bodies when their nutritional demands fail to be met.  The authors suspected that the rpoS mutant had a metabolic defect that caused the spirochete to round up while rapidly proliferating in the feeding tick's midgut.  They suspected that limited expression of the enzyme coenzyme A disulfide reductase (CoADR) was the source of the metabolic defect since they knew from earlier work that transcription of cdr was partially dependent on RpoS.  (The "housekeeping" sigma factor σ70 also transcribes cdr.)  CoADR couples the oxidation of NADH to NAD+ with the reduction of the disulfide bond linking two molecules of coenzyme A together.  A major role of this reaction is to replenish the NAD+ that is reduced during glycolysis, the primary means for energy generation in B. burgdorferi.

To test their prediction, the researchers knocked out the cdr gene.  Next, they inoculated the mutant into culture medium lacking nutrients needed by B. burgdorferi to grow.  As predicted, they found that starved cdr mutants formed round bodies at an even higher frequency than wild-type B. burgdorferi.  This result supported the notion that the failure of the rpoS mutant to produce enough CoADR is what triggered round bodiy formation in feeding ticks.

ResearchBlogging.orgDo the round bodies serve any biological role in the life cycle of Borrelia burgdorferi, or are they a laboratory artifact generated by knocking the rpoS gene out?  The investigators even saw a few round bodies among the many spiral-shaped spirochetes in feeding ticks harboring wild-type B. burgdorferi. This observation may suggest that round bodies indeed do have a role.  In the final sentence of their paper, the authors leave us to ponder the following: "We propose that round body formation has evolved to support the tick phase of the cycle and predict that there are circumstances, as yet undefined, when spirochetes within the tick resosrt to this survival program on a large scale in order to maintain a population of transmissible organisms."

Main reference

Dunham-Ems SM, Caimano MJ, Eggers CH, & Radolf JD (2012). Borrelia burgdorferi requires the alternative sigma factor RpoS for dissemination within the vector during tick-to-mammal transmission. PLoS pathogens, 8 (2) PMID: 22359504, DOI: 10.1371/journal.ppat.1002532

Other helpful references

Caimano MJ, Iyer R, Eggers CH, Gonzalez C, Morton EA, Gilbert MA, Schwartz I, and Radolf JD (September 2007).  Analysis of the RpoS regulon in Borrelia burgdorferi in response to mammalian host signals provides insight into RpoS function during the enzootic cycle.  Molecular Microbiology 65(5):1193-1217.   DOI: 10.1111/j.1365-2958.2007.05860.x

Eggers CH, Caimano MJ, Malizia RA, Kariu T, Cusack B, Desrosiers DC, Hazlett KRO, Claiborne A, Pal U, and Radolf JD (November 2011).  The coenzyme A disulphide reductase of Borrelia burgdorferi is important for rapid growth throughout the enzootic cycle and essential for infection of the mammalian host.  Molecular Microbiology 82(3):679-697.  DOI: 10.1111/j.1365-2958.2011.07845.x

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