Approach Considerations
Leptospires grow slowly in culture, and recovery rates are low. Serologic tests are available only in specialized laboratories, and the sensitivity of acute serologic tests is low. Consequently, those tests should not be the basis on which treatment is initiated. In a patient with compatible symptoms and a plausible exposure history, empiric therapy should be started.
Laboratory studies are used for two purposes: to confirm the diagnosis and to determine the extent of organ involvement and severity of complications. Laboratory confirmation of leptospirosis can be accomplished through isolation of the pathogen or by serologic testing.
Isolation of the leptospires from human tissue or body fluids is the criterion standard, but culture is not routinely available; thus, molecular assays such as DNA PCR are more commonly used, if available. Consultation with the local microbiology laboratory is essential, because processing requires specialized techniques. Urine is the most reliable body fluid to study because the urine contains leptospires from the onset of clinical symptoms until at least the third week of infection.
Other body fluids contain the organism, but the window of opportunity to isolate them is shorter. Blood and CSF may produce positive PCR or cultures during the first 7-10 days of symptoms.
Tissues (ie, liver, muscle, kidney, skin, eyes) are also sources of identification of the leptospires but are obviously more complicated to acquire.
Most often, paired acute and convalescent serum specimens are used to confirm the diagnosis. Again, this is a delayed means of confirmation because the acute sera are collected 1-2 weeks after onset of symptoms, and the convalescent sera are collected 2 weeks afterward.
Antileptospire antibodies in these samples are detected using the microscopic agglutination test (MAT). The Centers for Disease Control and Prevention (CDC) laboratory in Atlanta, Georgia, performs the MAT using 23 leptospire antigens. A 4-fold rise in MAT titer between acute and convalescent sera with any of these antigens confirms the diagnosis of leptospirosis.
Faster laboratory methods may strongly suggest the diagnosis of leptospirosis, but they may be no more readily available than the CDC laboratory in Atlanta. A single MAT titer of 1:200 on any sera or identification of spirochetes on dark-field microscopy, when accompanied by the appropriate clinical scenario, is strongly suggestive.
In suspected leptospirosis, further laboratory studies should be routinely performed to determine the extent and severity of organ involvement after the acute phase of illness. A complete blood cell count (CBC) is necessary. Findings on general laboratory studies are as follows:
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In patients with mild disease, elevated erythrocyte sedimentation rates and peripheral leukocytosis (3,000-26,000 x 109/L) with a left shift are noted
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Significant anemia due to pulmonary and gastrointestinal hemorrhage can occur
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The platelet count may be diminished as a component of disseminated intravascular coagulation (DIC)
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Levels of blood urea nitrogen and serum creatinine may be profoundly elevated in the anuric or oliguric phase
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Serum creatine kinase levels (MM fraction) are often elevated in patients with muscular involvement.
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Coagulation times may be prolonged in patients with hepatic dysfunction and/or DIC On liver function testing, serum bilirubin levels elevate as part of the obstructive disease due to capillaritis in the liver. Levels of hepatocellular transaminases are elevated less often and less significantly (usually < 200 U/L). Jaundice and bilirubinemia disproportional to hepatocellular damage is common in leptospirosis; alkaline phosphatase levels may be elevated 10-fold.
On urinalysis, proteinuria may be present. Leukocytes, erythrocytes, hyaline casts, and granular casts may be present in the urinary sediment. Rapid urine antigen test using lateral flow assay is under development.
Analysis of the CSF is useful only in excluding other causes of bacterial meningitis. When the CNS becomes involved in leptospirosis, polymorphonuclear leukocytes initially predominate and are later replaced by monocytes. CSF protein may be normal or elevated, whereas glucose levels remain normal. CSF pressure is normal, but a lumbar puncture can relieve the headache. Leptospires are routinely isolated from the CSF, but this finding does not change management of the disease.
Imaging studies are useful in determining the extent and severity of organ involvement. This may include chest radiography or computed tomography to evaluate lung disease and biliary tract ultrasonography in suspected acalculous cholecystitis.
Electrocardiographic (ECG) abnormalities are common during the leptospiremic phase of Weil syndrome. In severe cases, congestive heart failure and cardiogenic shock may occur.
Culture
Isolating the organism by culture allows definitive diagnosis. Leptospires remain viable in anticoagulated blood for as long as 11 days; hence, specimens can be mailed to a reference laboratory for culture. The infecting serovar can be isolated only by culture. [41]
Blood cultures may be negative if drawn too early or too late. Leptospires may not be detected in the blood until 4 days after the onset of symptoms (7-14 d after exposure). Once the immune system is activated, blood cultures may again become negative. Leptospires may be isolated from the cerebrospinal fluid (CSF) within the first 10 days.
Leptospires may be isolated from the urine for several weeks after the initial infection. In some patients, urine cultures may remain positive for months or years after the onset of illness. Positive urine cultures may take as long as 8 weeks to grow.
Microscopic Agglutination Testing
Microscopic agglutination testing (MAT) uses a battery of antigens taken from common (frequently locally endemic) leptospire serovars. MAT is available only at reference laboratories, such as the Centers for Disease Control and Prevention (CDC).
In a patient with clinical findings consistent with the disease, a single titer exceeding 1:200 or serial titers exceeding 1:100 suggest leptospirosis; however, neither is diagnostic. A 4-fold rise in titer between acute and convalescent specimens is considered a positive result. The antibody response does not reach detectable levels until the second week of illness, and it can be affected by treatment.
False-negative MAT findings may result from testing a single specimen obtained before the immune phase of disease. Test accuracy is also affected by appropriate selection of antigens for the battery, necessitating discussion with the laboratory about which serovars are suspected or predominate in the region where the case originated. False-positive MAT results may occur with cases of Legionella infection, Lyme disease, and syphilis.
Other Tests
Screening tests for leptospirosis, which are easy to perform and provide results relatively rapidly, include the macroscopic slide agglutination test, the Patoc-slide agglutination test, the microcapsule agglutination test, latex agglutination tests, dipstick tests, and the indirect hemagglutination test. Confirmation of screening test results (positive or negative) is advisable, however, preferably with MAT. [42]
An immunoglobulin M (IgM) enzyme-linked immunoabsorbent assay (ELISA) has been developed. The ELISA uses a broadly reactive antigen and is a standard serologic procedure, as is the MAT. [43] Because it detects IgM, it may be useful for diagnosis of new infections within 3-5 days. Positive results should be referred for confirmatory testing.
Nucleic acid amplification (polymerase chain reaction [PCR])–based techniques have been developed to diagnose leptospirosis. PCR can confirm the diagnosis rapidly during the early phase of the disease, when leptospires may be present and before antibody titers are detectable, but it requires adequate infrastructure such as appropriate equipment, laboratory space, and skilled personnel. In addition, PCR-based techniques are unable to identify the infecting serovar, which reduce their epidemiologic and public health value.
Dark-field examination of blood or urine has been used to identify leptospires. However, this technique is insensitive and nonspecific on its own.
Chest Radiography
The most common abnormality on chest radiography is bilateral diffuse airspace disease. Chest radiography may also reveal cardiomegaly and pulmonary edema due to myocarditis. In patients with alveolar hemorrhage due to pulmonary capillaritis, the lung parenchyma may contain multiple patchy infiltrates on computed tomography.
Histologic Findings
Shortly after inoculation and during the incubation period, leptospires actively replicate in the liver. The leptospires then disseminate throughout the body and infect multiple tissues.
Silver staining and immunofluorescence can identify leptospires in the liver, spleen, kidney, CNS, muscles, and heart. During the acute phase of leptospirosis, histology reveals these organisms without much inflammatory infiltrate. In addition to the finding of leptospires during histologic examination, the pathologic effects of leptospiral toxins are also apparent.
Silver stain, liver, fatal human leptospirosis. Courtesy of the Centers for Disease Control and Prevention (CDC), Dr Martin Hicklin.
Leptospirosis may be seen as an infective systemic vasculitis. [19] Leptospiral toxins break down endothelial cell membranes of capillaries. This toxin-mediated process allows for extravasation of blood and leptospires from blood vessels into the supported parenchyma. Secondarily, because the capillaries are no longer functional, ischemia and cell death can occur. Later in infection, mononuclear cells predominate in the areas of this focal cell necrosis.
Leptospires can be identified in immunologically privileged sites, such as renal tubules, CNS, and the anterior chamber of the eyes, for weeks to months after the initial infection. In nonhuman animals, the intended hosts of infection, the leptospires establish residence in these immunologically privileged sites. Provided that the animal survives the initial infection, a chronic carrier state is then established, and histology reveals leptospires at these sites for years after initial infection.
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Darkfield microscopy of leptospiral microscopic agglutination test. (This image is in the public domain and thus free of any copyright restrictions. Courtesy of the Centers for Disease Control and Prevention (CDC), M Gatton.
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A scanning electron micrograph depicting Leptospira atop a 0.1-µm polycarbonate filter. (This image is in the public domain and thus free of any copyright restrictions. Courtesy of the Centers for Disease Control and Prevention (CDC), Rob Weyant.
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Silver stain, liver, fatal human leptospirosis. Courtesy of the Centers for Disease Control and Prevention (CDC), Dr Martin Hicklin.
