GENERAL CHARACTERISTICS
The leptospires include both free-living and parasitic forms. The organisms are spiral-shaped, right-handed helices with hooked ends. The organisms contain two axial filaments and exhibit either a spinning motility or a rapid back-and-forth movement. Leptospira species are typically classified into two major groups, with Leptospira interrogans sensu stricto being the main species associated with human leptospirosis; in France, this organism is responsible for about 60% of human cases. Leptospira biflexa contains the saprophytic environmental strains. Molecular classification using 16srRNA sequencing currently separates the genus into three distinct groups of pathogens, environmental saprophytes, and other species of uncertain pathogenicity.
The pathogens include more than 260 serologically defined types that were formerly designated as species and are now referred to as serovars, or serotypes, of L. interrogans sensu stricto. Each serovar is usually associated with a particular animal host and therefore serovar identification is important for epidemiology studies and prevention strategies. The genotypic classification scheme now includes approximately 20 species, which incorporates all current serovars. Serovars cross species lines as a result of the horizontal transfer of genetic elements, making it difficult to fully classify species phenotypically.
EPIDEMIOLOGY AND PATHOGENESIS
Leptospirosis, a zoonosis, has a worldwide distribution but is most common in developing countries and warm climates where contact with infected animals or water contaminated with urine is likely to occur. L. interrogans can infect most mammals throughout the world, as well as reptiles, amphibians, fish, birds, and invertebrates. The organism is maintained in nature by virtue of persistent colonization of renal tubules of carrier animals. Humans become infected through direct or indirect contact with the urine or blood of infected animals. Leptospires enter the human host through breaks in the skin, mucous membranes, or conjunctivae. Infection can be acquired in home and recreational settings (e.g., swimming, hunting, canoeing) or in people who work in certain occupational settings (e.g., farmers, ranchers, abattoir workers, trappers, veterinarians).
Pathogenic leptospires rapidly invade the bloodstream after entry and spread throughout all sites in the body such as the central nervous system and kidneys. Virulent strains show chemotaxis toward hemoglobin as well as the ability to migrate through host tissues. A number of potential virulence factors that might facilitate this process are shown in Box 1. Precisely how L. interrogans causes disease is not completely understood, but it appears that the presence of endotoxin and other toxins may play a role in which hemostasis pathways are activated as is an autoimmune response in the human host.
Box1. Potential Virulence Factors of Leptospira
SPECTRUM OF DISEASE
Symptoms begin abruptly 2 to 20 days after infection and include fever, headache, and myalgia. The most common clinical syndrome is anicteric leptospirosis, which is a self-limiting illness consisting of a septicemic stage, with high fever and severe headache that lasts 3 to 7 days, followed by the immune stage. Symptoms associated with the immune stage (onset coincides with the appearance of IgM) are varied but in general are milder than those associated with the septicemic stage. The hallmark of the immune stage is aseptic meningitis. Weil’s disease, or icteric leptospirosis, is generally the most severe illness, with symptoms caused by liver, kidney, or vascular dysfunction with lethal pulmonary hemorrhage; death can occur in up to 10% of cases. Unfortunately, the clinical presentations of leptospirosis mimic those of many other diseases.
LABORATORY DIAGNOSIS
Specimen Collection, Transport, and Processing During the first 10 days of illness, leptospires are present in the blood, CSF, and peritoneal dialysate. Urine specimens can be obtained beginning in the second week of illness and up to 30 days after the onset of symptoms. Specimens may be collected in citrate, heparin, or oxalate anticoagulants. There are no other special requirements for specimen collection, transport, or processing. Citrate or ethylenedaminetetraacetic acid (EDTA) is the preferred anticoagulant for molecular testing. Urine specimens should not be placed in preservatives and should be processed within 1 hour for optimal results. Specimens should be transported at room temperature and inoculated for culture within 24 hours.
Direct Detection
Blood, CSF, and urine may be examined directly by dark field microscopy examination. Detection of motile leptospires in these specimens is optimized following centrifuging at 1500× g for 30 minutes; sodium oxalate or heparin-treated blood is initially spun at 500× g for 15 minutes to remove blood cells. Other techniques, such as fluorescent antibody staining and hybridization techniques using leptospira-specific DNA probes, have also detected leptospires in clinical specimens. Conventional and real-time PCR assays have been used to detect leptospires in clinical and environmental samples.
Molecular Diagnostics
Several nucleic acid–based amplification methods have been developed. Studies indicate that the sensitivity is comparable to paired sera testing using serologic methods. These methods are not readily available in routine clinical laboratories. However, PCR methodologies are not useful for the differentiation of serovars and therefore are of limited utility in epidemiologic studies. Highly complex and labor-intensive techniques such as pulsed field electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) are more useful for the identification of serovars.
Cultivation
Albeit insensitive, the definitive method for laboratory diagnosis of leptospirosis is to culture the organisms from blood, CSF, or urine. A few drops of heparinized or sodium oxalate–anticoagulated blood are inoculated into tubes of semisolid media enriched with rabbit serum (Fletcher’s or Stuart’s) or bovine serum albumin. Urine should be inoculated soon after collection, because acidity (diluted out in the broth medium) may harm the spirochetes. One or 2 drops of undiluted urine and a 1 : 10 dilution of urine are added to 5 mL of medium. The addition of 200 µg/mL of 5-fluorouracil (an anticancer drug) may prevent contamination by other bacteria without harming the leptospires. Commercial media such as Ellinghausen-McCullough-Johnson-Harris (EMJH) or Fletcher’s Medicum (Difco EMJH or Difco Fletcher’s medium; BD Diagnostic Systems, Sparks, Maryland) are available that contain 5-fluorouracil for use at the patient’s bedside. Tissue specimens, especially from the liver and kidney, may be aseptically macerated and inoculated in dilutions of 1 : 1, 1 : 10, and 1 : 100 as for urine cultures.
All cultures are incubated at room temperature or 30° C in the dark for up to 6 to 8 weeks. Because organisms grow below the surface, material collected from a few centimeters below the surface of broth cultures should be examined weekly for the presence of growth, using a direct wet preparation under dark-field illumination. Leptospires exhibit corkscrew-like motility.
Approach to Identification
Based on the number of coils and hooked ends, leptospires can be distinguished from other spirochetes. Physiologically, the saprophytes can be differentiated from pathogens by their ability to grow to 10° C and lower, or at least 5° C lower than the growth temperature of pathogenic leptospires. Leptospires may also be visualized using dark-field or immunofluorescence.
Serodiagnosis
Serodiagnosis of leptospirosis requires a fourfold or greater rise in titer of agglutinating antibodies. The microscopic agglutination (MA) test using live cells is the standard serologic procedure. Serologic diagnosis of leptospirosis is performed using pools of bacterial antigens containing many serotypes in each pool. Positive results are indicated by the presence of agglutination using dark-field microscopy. However, a macroscopic agglutination procedure is more readily accessible to routine clinical laboratories. Reagents are available commercially. Indirect hemagglutination and an ELISA test for IgM antibody are also available; IgM-detection assays are primarily used because IgM antibodies become detect able during the first week of illness.
Molecular Testing
Recently several nucleic acid-based testing methods have been developed for detection of Leptospires. These techniques include traditional PCR, real-time PCR, and loop-mediated isothermal amplification. To date, no commercial molecular assays are available for diagnostic use.
ANTIBIOTIC SUSCEPTIBILITY AND THERAPY
Treatment of leptospirosis is supportive management and the use of appropriate antibiotics. Ceftriaxone, penicillin, amoxicillin, doxycycline, and tetracycline are recommended for treatment of leptospirosis. Standardized procedures for antibiotic susceptibility are limited by the slow growth of the organisms and the need for serum during cultivation.
PREVENTION
General preventive measures include the vaccination of domestic livestock and pet dogs. In addition, protective clothing, rodent control measures, and preventing recreational exposures, such as avoiding freshwater ponds, are indicated in preventing leptospirosis.
