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

Life after leptospirosis, a pilot study

The signs and symptoms associated with acute human leptospirosis are well known.  Less is known about how well patients manage after they begin recovering from the illness.  Most who survive the illness, even those who had severe illness, appear to regain full health eventually.  However careful examination may reveal subtle deficiencies in organ function.²  Moreover the spectrum of health problems that could crop up later is not known.  The only late complication of leptospirosis that has been well documented is uveitis, which can lead to eye pain and vision problems.³  Old case studies have also reported recurring headaches, general malaise, and even dementia, aggression, depression, and psychosis in those surviving leptospirosis,^4,5 although these observations would need to be confirmed in well-designed clinical studies.

To gain a better understanding of the long-term outcomes of leptospirosis, a large systematic study is needed to monitor the health of leptospirosis patients as they heal from the illness.  Such a study was attempted by Spichler and colleagues.¹  Their pilot study targeted patients hospitalized with leptospirosis at any time from December 2008 to May 2009 in São Paulo, Brazil, where leptospirosis is endemic.  There were 180 patients hospitalized with leptospirosis during this period, but for a variety of reasons only 47 of the 180 could be enrolled in the study.  All 47 had been treated for at least seven days with antibiotics (penicillin or ceftriaxone) during their hospitalization, and all had their diagnosis confirmed by serologic testing.  17 of the 47 had severe leptospirosis, defined as those who had experienced jaundice, kidney failure, bleeding (hemorrhage), pulmonary (lung) involvement, or shock while hospitalized.  Fortunately no patient died.

The first outpatient visit was conducted an average of 22 ± 12 days after the patients were discharged from the hospital.  23 of the 47 (49%) continued to experience one or more of the following health problems:  general malaise, headache, muscle pain, dizziness, bronchitis, and abdominal pain.  Three patients still had jaundice.

Only 22 of the 47 patients came back for a second visit, which took place a mean of 40 ± 21 days after they were discharged from the hospital.  Two of the 22 patients continued to suffer from medical problems.

One individual was experiencing general malaise, which wasn't a problem for him before being stricken with leptospirosis.  He did always have high blood pressure, which may or may not have been a contributing factor to his malaise. An ECG showed some abnormalities with his heartbeat.  Leptospira is known to cause myocarditis, an inflammation of the heart muscle.  This patient continued to suffer from profound general malaise when examined one year later.

The second patient started to experience panic attacks between the two clinic visits.  Were the panic attacks related to his earlier bout with leptospirosis?  It's difficult to conclude anything from this single patient.  Although the patient had severe leptospirosis, did not suffer from any of the known neurologic features of leptospirosis during his acute illness⁶ and had not been diagnosed with any neurologic or psychiatric condition before contracting leptospirosis.  Panic disorder has never been described in leptospirosis patients in the scientific literature.

Since this was a pilot study, the investigators probably lacked the resources to address the problems that cropped up during the study.  As pointed out by the authors, the major limitations of the study were that few of the eligible patients were enrolled in the study and that many dropped out between visits, resulting in a potentially biased sampling of leptospirosis patients. Additionally the follow-up visits did not include any laboratory testing to detect lingering functional deficiencies in the kidney, liver, and other organs.  For these reason it's impossible to make any definitive conclusions about the recovery of these subjects.  Despite the preliminary nature of this pilot study, the possible outcomes of acute leptospirosis identified in this study and earlier case studies beg for a future prospective study with a larger number of individuals living in an area where leptospirosis is endemic.  A longer time frame for follow up is also necessary since uveitis can first appear up to four years after recovery from leptospirosis.³

Any future study should also follow those with mild or asymptomatic Leptospira infections.  The reason is that the long-term outcome of mild disease is unknown.  Since those with mild or asymptomatic infections are unlikely to seek medical attention, identifying such individuals will require investigators to monitor healthy high-risk individuals by serologic testing so that the newly infected could be identified as those with increasing anti-Leptospira antibody titers.

In light of the recent discovery of chronically infected individuals in the Peruvian Amazon,⁷ it would also be prudent to test the urine of healthy individuals for Leptospira being shed from the kidney tubules so that the findings of the Peruvian study could be confirmed.  The long-term effects of chronic infection, if any, could also be identified.

Where could a future study examining the long-term outcomes of Leptospira infection be conducted?  Brazil and India may be best suited for this type of study.  Leptospirosis is highly endemic in those countries, and multiple research teams are already investigating the epidemiology of leptospirosis in those countries.

10-30-2011, edits for clarity.

Featured paper

1.  Spichler A., Athanazio D, Seguro AC, and Vinetz JM (July 2011).  Outpatient follow-up of patients hospitalized for acute leptospirosis.  International Journal of Infectious Diseases 15(7):e486-e490.  DOI:  10.1016/j.ijid.2011.03.020

References

2.  de Francesco Daher E, Zanetta DMT, and Abdulkader RCRM (September 2004).  Pattern of renal function recovery after leptospirosis acute renal failure.  Nephron, Clinical Practice 98(1):c8-c14.  DOI: 10.1159/000079922

3.  Shukla D, Rathinam SR, and Cunningham ET (Spring 2010).  Leptospiral uveitis in the developing world.  International Ophthalmology Clinics 50(2):113-124.  DOI:

4.  Shpilberg O, Shaked Y, Maier MK, Samra D, and Samra Y (April 1990).  Long-term follow-up after leptospirosis.  Southern Medical Journal 83(4):405-407.  Link

5.  Avery TL (July 27, 1983).  Leptospirosis and mental illness.  New Zealand Medical Journal 96(736):589 (Letter).

6.  Panicker JN, Mammachan R, and Jayakumar RV (September 2001).  Primary neuroleptospirosis.  Postgraduate Medical Journal 77(911):589-590. DOI: 10.1136/pmj.77.911.589

7. Ganoza CA, Matthias MA, Collins-Richards D, Brouwer KC, Cunningham CB, Segura ER, Gilman RH, Gotuzzo E, and Vinetz JM (2006). Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira. PLoS Medicine 3(8):e308.  DOI: 10.1371/journal.pmed.0030308

Related post

• Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon

Congenital syphilis, upward trend (again) in the United States

Syphilis can be deadly if passed from an infected mother to her unborn child. The most recent CDC data show that 6.5% of U.S. infants with congenital syphilis (CS) in 2008 were stillborn or died within 30 days of birth.¹

Newborns with CS who are destined to live begin to show signs of disease within the first few weeks of life.  The main features of CS in early infancy include fever, skin lesions, enlarged liver and spleen, and a chronic runny nose ("snuffles"), which may be tinged with blood.  Bone lesions may lead to Parrot's pseudoparalysis, a condition so painful that the infant will refuse to move the affected extremities.  Ongoing damage to bony tissue may later lead to childhood deformities including saddle nose, sabre shins, and Hutchinson incisors (notched central incisors).  Other late signs of CS include inflammation of the cornea and sudden hearing loss.

The lesions and deformities associated with congenital syphilis are sparked by Treponema pallidum, a spirochete that can cross the placenta from the mother's bloodstream.  The probability of transmission to the fetus depends on how long the mother has been infected with T. pallidum.  The risk of transmission is lower in mothers at later stages of syphilis.  After crossing the placenta, the spirochete invades the fetal organs.  The continuing immune response to persistent T. pallidum infection causes the damage seen in CS.  Early treatment of the mother with penicillin, at least 30 days before delivery, is essential to stop the disease.

The rate of congenital syphilis in the United States has started to creep back up after plummeting over two decades.¹  The incidence of congenital syphilis has gone up from 8.2 cases per 100,000 live births in 2005 to 10.1 in 2008 with most of the increase having occurred in the South.  CS rates in infants born to black mothers have gone up from 26.6 in 2005 to 34.6 per 100,000 live births in 2008 and now account for half of all CS cases.  Since CS is transmitted from mothers with syphilis, CS rates have historically tracked the combined primary and secondary syphilis rate seen in women, which has also started to climb (see figure below).  What factors account for the increased incidence of syphilis?  In one Alabama county, increased syphilis rates in black women were linked to crack cocaine use and the exchange of sex for money or drugs.²  Although more studies are needed to determine whether the same factors are linked to syphilis throughout the South, it should be pointed out that the same factors were associated with the previous syphilis epidemic that peaked in the early 1990s, when there were several thousand yearly cases of CS as opposed to the several hundred seen today.³

Figure from CDC¹

Now that the upward trend in the CS rate has been recognized, public health authorities in partnership with community-based groups must allocate some of their scarce resources to reverse the trend.  With prenatal care and prompt treatment, congenital syphilis can be prevented.

1. Centers for Disease Control and Prevention (CDC) (April 16, 2010). Congenital syphilis - United States, 2003-2008. MMWR Morbidity and Mortality Weekly Report 59(14):413-417.  link

2. Centers for Disease Control and Prevention (CDC) (May 8, 2009).  Primary and secondary syphilis - Jefferson County, Alabama, 2002-2007.  MMWR Morbidity and Mortality Weekly Report 58(17):463-467.  link

3. Nakashima, A.K., Rolfs, R.T., Flock, M.L., Kilmarx, P., and Greenspan, J.R. (Jan-Feb 1996). Epidemiology of syphilis in the United States, 1941-1993.  Sexually Transmitted Diseases 23(1):16-23.  PMID: 8801638

Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon

The spirochete Leptospira is the agent of leptospirosis, a zoonosis that primarily burdens tropical regions of the world.  Moist conditions promote the survival of Leptospira in soil and fresh water. Although Leptospira could survive out in wet environments if they had to, they thrive in the kidneys of rats and other maintenance hosts, where they form dense masses lining the inner surface of the kidney tubules.  The spirochetes spill into the urine that forms in the tubules, which drain into the bladder.  Animals colonized by their "preferred" serovar (immune type) shed Leptospira throughout their lives without ever showing signs of illness.  The tainted urine ends up contaminating soil and water with infectious Leptospira.

Humans aren't regarded as long-term carriers of Leptospira.  Rather, they are deemed "accidental" (incidental) hosts who may suffer serious complications of acute disease, including kidney failure and lung hemorrhage.  Humans get infected when they come into contact with contaminated water or soil or following direct exposure to infectious animal urine or tissue.  Leptospira enters through cuts in the skin or mucous membranes.  From there the motile spirochete spreads via the bloodstream and invades internal organs, including the kidneys, where they remain for the duration of the disease.  Patients typically stop releasing Leptospira into their urine after they recover from the illness, presumably because the spirochetes have been eliminated from their kidneys.  However, there have been a few reports of Leptospira excreted in urine months or even years following recovery from leptospirosis.  The truth is that no one has ever done a systematic study to determine how common the chronic carrier state is in humans.

A team of investigators from the United States and Peru set out to find long-term carriers of Leptospira.  Their study is described in the February issue of PLoS Neglected Tropical Diseases.  The authors examined the inhabitants of a rural Amazon village of Padrecocha near the city of Iquitos, Peru, where leptospirosis is endemic. The tropical climate is ideal for the survival of Leptospira in the moist environment favored by the spirochete.  Indeed, in an earlier study the authors detected infectious strains of Leptospira in the streams and wells serving the village.  Cattle, pigs, dogs, and rats, all potential carriers, freely roam the area.

Ganoza and colleagues wanted to determine what percentage of the villagers were chronic carriers of Leptospira.  They first identified villagers who were not recently infected with Leptospira.  Out of the 314 healthy villagers enrolled in the study, 102 (32.5%) had no clinical or serological evidence of recent infection; they did not recall experiencing a fever during the previous year (fever is a typical symptom of leptospirosis), and they tested negative for newly-acquired Leptospira infection by IgM ELISA.

The investigators next identified those whose kidney were colonized by Leptospira among the 102 who were not newly infected.  Since Leptospira living in the kidney tubules are shed into urine, they screened urine samples by nested PCR using primers targeting the 16S rRNA gene of Leptospira.  To exclude false-positive signals, the investigators screened the PCR-generated DNA (amplicon) by dot blot analysis with a Leptospira 16S rRNA probe.  Many false positive signals occurred because their Leptospira PCR primers also hybridized to the 16S rRNA gene from Atopobium vaginae, a bacterium recently found to be associated with vaginosis. 

When urine from the 102 "long-term" healthy individuals was screened, Leptospira DNA was found in 6 (5.9%).  Sequencing of the 16S rRNA gene revealed that the carriers were colonized with L. interrogans, L. fainei, and L. licerasiae.  So it turns out that the asymptomatic carrier state is not as rare as initially believed.  More than 1 in 20 individuals who had been healthy for at least a year were colonized with Leptospira in their kidneys.

The investigators found seven additional individuals colonized with Leptospira by screening urine from the other 212 individuals in the study.  Overall the percentage of shedders of Leptospira among all healthy individuals, irrespective of when they were infected, was 4.1% (13/314).  The concentration of Leptospira in the urine of shedders, as measured by quantitative PCR, was low, in the 10²-10⁴/ml range.  In contrast, rats may shed up to 10⁸ spirochetes/ml!

The study unearthed another surprise.  All 13 individuals who were shedding Leptospira at the time of the study (including the 6 chronic carriers) were women.  The proportion of women with Leptospira DNA in their urine (13/13, 100%) was significantly higher than the proportion of women in the group lacking detectable DNA in their urine (199/301, 66%, p = 0.011).  This result raises the possibility that women are more likely to become persistent carriers than men.  However, the authors pointed out that men were underrepresented in the study sample.  Less than one third of the villagers enrolled in the study were men.  Most of the other men were away at work when the authors were recruiting people for the study.  Agricultural occupations, which bring workers into contact with environmental sources of Leptospira, are well-known risk factors for infection by the spirochete in endemic areas.  Hence, male shedders of Leptospira may have been inadvertently excluded from the study.

Another surprising result was that sera from all six chronic carriers failed to agglutinate Leptospira by MAT (microscopic agglutination test), a standard serological test used to check for Leptospira infection whether it occurred recently or years ago.  The authors mentioned that this was entirely consistent with old studies failing to detect agglutinating antibodies in the sera of some maintenance host animals excreting Leptospira. However, another possibility is that the "chronic human carriers" may have actually acquired asymptomatic infections very recently.  They could have been enrolled in the study before the anti-Leptospira IgM and agglutinating antibodies had enough time the accumulate to the cut-off values selected for the IgM ELISA and MAT, respectively.  Although the authors discounted the possibility of newly acquired asymptomatic infections accounting for the seronegativity of the shedders, they recommended a longitudinal study to clarify the issue.

The authors posed several questions raised by their study:

• Does persistent Leptospira infection of human kidneys have any subtle effect on their function? If so, is antibiotic treatment warranted?
• Are some strains of Leptospira more likely than others to persistently infect the kidneys of humans?  .
• Can persistent human shedders be a source of transmission of Leptospira to other humans (and animals)?

Future studies will need to include urine cultures to demonstrate that Leptospira shed by human carriers are alive.

In conclusion, this is an important study that challenges the simplistic notion that humans are incidental hosts of Leptospira.  The reality appears to be more complicated.

Featured paper

Ganoza, C.A., Matthias, M.A., Saito, M., Cespedes, M., Gotuzzo, E., & Vinetz, J.M. (2010). Asymptomatic renal colonization of humans in the Peruvian Amazon by Leptospira. PLoS Neglected Tropical Diseases, 4 (2) DOI: 10.1371/journal.pntd.0000612

Related paper

Ganoza, C.A., Matthias, M.A., Collins-Richards, D., Brouwer, K.C., Cunningham, C.B., Segura, E.R., Gilman, R.H., Gotuzzo, E., & Vinetz, J.M. (2006). Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira. PLoS Medicine, 3 (8) DOI: 10.1371/journal.pmed.0030308

Does male circumcision protect against syphilis?

Circumcision has been shown to reduce the risk of men contracting several sexually transmitted infections (STIs). Three randomized controlled trials (RCTs) published over the last few years have demonstrated that removing the foreskin of adult men diminished the risk of HIV infection by at least 50%. An article by Tobian and colleagues in last week's issue of the New England Journal of Medicine revealed a weak protective effect of circumcision against two other infections, herpes simplex virus 2 (HSV-2) and human papilloma virus (HPV), which cause genital herpes and penile warts, respectively. The same study showed no effect of circumcision on acquisition of Treponema pallidum, the agent of syphilis. You can find a nice critical analysis of the study here. Because I am interested in diseases caused by spirochetes, I will focus on the syphilis data.

Tobian et al. conducted two RCTs with similar designs. When the data were combined, they found that 50 of 2083 (2.4%) male adolescents and adults in Uganda who underwent circumcision became infected with T. pallidum over the following 24 month period. Similarly, 45 of 2143 (2.1%) control subjects became infected within the same time period, suggesting that circumcision had no effect on contracting T. pallidum. An editorial in the same journal issue points out that the study may have been underpowered to detect a protective effect (i.e., not enough subjects in the study).

Since the Tobian et al. study failed to give a simple answer to the question, I thought it would be illuminating to look at the older observational studies that examined the effects of male circumcision on syphilis transmission. Fortunately, I found a meta-analysis that compiled data from 14 research papers, most of which described cross-sectional studies.

The meta-analysis presented by Weiss and colleagues revealed a slight protective effect of male circumcision. The relative risks (RRs) along with the 95% confidence intervals (CI) are plotted in the graph. The RR in 11 of the 14 studies were adjusted for potential confounding factors such as age. The summary statistics listed at the bottom of the graph indicate a small protective effect (summary RR, 0.67; 95% CI, 0.54-0.83).

Ideally, all studies included in a meta-analysis would have similar RRs. However, if you look carefully at the graph, you will notice a wide variation in the RRs with some of the 95% confidence intervals failing to overlap. Using standard statistical calculations, the investigators determined that it was unlikely that the variation of the RR among the studies was due to chance (P = 0.01). In other words, differences in how the studies were designed and conducted led to significant variation in the outcomes. Consequently, the authors declared that there was significant heterogeneity among the studies and warned that the summary RR "should be interpreted cautiously."

The authors described one potential source of the heterogeneity. Looking at the plots again, you will note that the Cook and Parker studies demonstrated the largest statistically significant protective effect of male circumcision. Those two studies were conducted in the United States and Australia, respectively, where males are circumcised as infants. In contrast, the two largest studies, authored by Gray and Urassa, showed no effect of circumcision on the risk of becoming infected with T. pallidum. Those studies were conducted in Uganda and Tanzania, respectively, where males are not circumcised until they are adolescents or young adults. Many of the circumcised males examined in the two African studies, which were cross-sectional and case-control studies, could have contracted syphilis before being circumcised. Weiss et al. excluded subjects who were circumcised after their first sexual intercourse or after age eleven, but this information was not available for all studies. This would lead to an underestimate of the protective effects of circumcision.

You may look at the large protective effects of infant circumcision observed in the U.S. and Australian studies (RR = 0.25 and 0.19, respectively) and conclude that mass infant circumcision would be beneficial (at least for protection against syphilis). However, both studies involved men visiting STD clinics, and the results may not apply to the general population in those countries.

References

Tobian, A.A.R., Serwadda, D., Quinn, T.C., Kigozi, G., Gravitt, P.E., Laeyendecker, O., Charvat, B., Ssempijja, V., Riedesel, M., Oliver, A.E., Nowak, R.G., Moulton, L.H., Chen M.Z., Reynolds, S.J., Wawer, M.J., Gray, R.H. (2009). Male circumcision for the prevention of HSV-2 and HPV infections and syphilis. The New England Journal of Medicine 360(13):1298-1309.

Golden M.R. and Wasserheit, J.N. (2009) Prevention of viral sexually transmitted infections--foreskin at the forefront (Editorial). The New England Journal of Medicine 360(13):1349-1350.

Weiss, H.A., Thomas, S.L., Munabi, S.K., and Hayes, R.J. (2006). Male circumcision and risk of syphilis, chancroid, and genital herpes: a systematic review and meta-analysis. Sexually Transmitted Infections 82(2):101-109.

Leptospirosis outbreak among strawberry harvesters in Germany

Humans can become infected with the spirochete Leptospira if they have wounds that contact contaminated soil or water. Leptospira live in the kidneys of reservoir animals and are released in urine into the environment. The majority of leptospirosis cases occurs in tropical locales, where the warm, moist conditions promote the survival of Leptospira in the environment. Flooding following heavy rainfall can cause large epidemics of leptospirosis in these regions of the world. A research article published in the March 15th issue of Clinical Infectious Diseases by Desai and colleagues describes an unexpected outbreak of leptospirosis among strawberry harvesters in Germany, a country not known for tropical weather. The July 2007 outbreak, while small by the standards of those that occur in tropical countries, was Germany's largest since the 1960s. The authors searched for factors that may have caused the epidemic.

The outbreak occurred among laborers who worked on a strawberry farm near Düren. The strawberry harvesters were seasonal workers from Romania, Slovakia, and Poland. Among the 153 who were employed on the farm during the outbreak, 24 were stricken with leptospirosis, and 13 were hospitalized. Fortunately, no one died. Most who became sick had antibodies against the Leptospira serogroup Grippotyphosa in their blood.

In September 2007, the authors conducted a retrospective cohort study to identify risk factors for acquiring leptospirosis. The strawberry harvesters were questioned about possible sources of exposure to Leptospira before the outbreak, including the presence of wounds, contact with rodents, and consumption of unwashed strawberries. Logistic regression of the data revealed two statistically significant risk factors. First, the odds of the harvesters acquiring leptospirosis increased with the number of days worked with unprotected hand lesions (OR, 1.1; 95% CI, 1.04-1.1). Leptospira probably entered the lesions as the workers reached into the water-logged soil. "Accidental contact with rodents" was listed as the other significant risk factor (OR, 4.8; 95% CI, 1.5-15.9), although the nature of the contact was not described by the authors. Of course recall bias may have skewed the results of the retrospective study. However, there is nothing unusual about these risk factors; contact of skin wounds with moist soil laden with Leptospira and exposure to infected rodents are typical means of acquiring leptospirosis.

The common vole was the main suspect in contaminating the strawberry field with Leptospira. More than 10 mouse holes per square meter were found in the field and surrounding areas when the site was examined in September 2007. Kidneys of voles captured in and near the field were infested with the spirochete. Leptospira was successfully cultured from the kidneys and were identified as members of the serogroup Grippotyphosa, the same serogroup that the antibodies in the patients' blood were targeting.

The strawberry field outbreak is reminiscent of the large epidemics of leptospirosis that sickened thousands of German agriculture workers from the 1920s through the 1960s, with the Leptospira serogroup Grippotyphosa carried by voles and hamsters responsible for many cases. In contrast, most German cases of leptospirosis of late are related to activities outside of work. Recreational activities involving water and exposure at home to infected pets and contaminated soil while gardening are common risk factors. Icterohaemorrhagiae, with rats as the reservoir host, is now the most prevalent serogroup in Germany. So why did a 1960s-style leptospirosis outbreak occur in 2007? The authors propose that the unusual weather in the months preceding the outbreak was a critical factor.

Europe experienced its warmest autumn in 2006 since recordings began, and the warm weather continued through the winter into 2007. In Germany, heavy rainfall accompanied the high temperatures in the months preceding the outbreak. May and June 2007 were Germany's wettest months since 1901. The tropical conditions may have augmented the amount of food available to the voles. In addition, the vole population, which naturally fluctuates every 3-5 years, may have already been at the peak of its cycle in 2007. A population explosion of infected voles may have increased the density of Leptospira residing in the strawberry field. Because the wet, warm conditions were favorable to survival of Leptospira, it is easy to imagine how an outbreak would have occurred among workers who spent days reaching into the contaminated soil with unprotected hands.

In conclusion, the authors present leptospirosis as a potential model for an infectious disease affected by global warming. Only time will tell whether the leptospirosis outbreak caused by the confluence of warm weather and heavy rainfall in a normally temperate region was a statistical anomaly or a sign of things to come.

Featured paper

Desai, S., van Treeck, U., Lierz, M., Espelage, W., Zota, L., Sarbu, A., Czerwinski, M., Sadkowska‐Todys, M., Avdicová, M., Reetz, J., Luge, E., Guerra, B., Nöckler, K., & Jansen, A. (2009). Resurgence of Field Fever in a Temperate Country: An Epidemic of Leptospirosis among Seasonal Strawberry Harvesters in Germany in 2007 Clinical Infectious Diseases, 48 (6), 691-697 DOI: 10.1086/597036

Other references

Jansen, A., Schöneberg, I., Frank, C., Alpers, K., Schneider, T., and Stark, K. (2005). Leptospirosis in Germany, 1962-2003. Emerging Infectious Diseases 11(7):1048-1054.

Pappas, G., Papadimitriou, P., Siozopoulou, V., Christou, L., Akritidis, N. (2008). The globalization of leptospirosis: worldwide incidence trends. International Journal of Infectious Diseases 12(4):351-357.