Leptospirosis Page : 1

Leptospira & Leptospirosis

Leptospirosis is a zoonosis of worldwide distribution. It is caused by spirochetes of the genus Leptospira. The traditional classification system is based on biochemical and serologic specificity to differentiate between the pathogenic species, Leptospira interrogans, and the free-living nonpathogenic species, Leptospira biflexa. The species are further broken down to more than 200 serovars of L interrogans and more than 60 serovars of L biflexa. The serovars are further organized into serogroups of L interrogans and serogroups of L biflexa. The serogroups are based on shared antigenicity and are primarily for laboratory use. A second classification system is based on DNA-DNA hybridization studies, which have demonstrated a high degree of heterogeneity within the two species of the traditional classification. The molecular classification of leptospires is done by 16S rRNA sequencing. Phylogenetic analysis of these sequences indicates that there are three clades of leptospires, pathogens, saprophytes, and some of uncertain pathogenicity. The species do not correspond to the species in the traditional serologic classification. Indeed some serovars in the traditional classification occur in multiple species in the molecular classification and the serologic classification cannot be used to predict the molecular classification. The discussion below uses the traditional serologic classification.

Epidemiology Leptospirosis is a zoonotic disease. Human-to-human transmission does not occur. Although more than 100 different mammals can be infected, the most important sources of transmission to humans are rats, dogs, cattle, and pigs. Rats do not become ill from leptospiral infection, but dogs often develop severe disease similar to that in humans; infection can cause reproductive failure in cattle and pigs. Even when vaccinated, asymptomatic dogs, cattle, and pigs can be leptospiruric and thus transmit infection to humans. Classic (but not exclusive) serovar-animal associations include Icterohaemorrhagiae and Copenhageni in domestic rats (Rattus norvegicus, R. rattus), Grippotyphosa in opossums and raccoons (emerging in the United States and Canada in the absence of a vaccine that covers this serovar), Canicola in dogs, Hardjo in cattle and buffalo, and Pomona in pigs. Patterns of leptospirosis transmission are characterized as epidemic, endemic, and sporadic. Factors that facilitate human infection are those that bring susceptible persons into indirect contact with contaminated animal urine through surface waters, moist soil, or other wet environments or into direct contact with urine and other excreta (e.g., products of parturition, placenta) of infected animals. In recent years, fewer occupation-related cases and more cases related to environmental exposure have been seen. Seasonal rains and seasonal flooding are the most important factors in the occurrence of epidemic leptospirosis. Tropical humid environments, poor sanitation leading to rodent infestation, and uncontrolled dog populations are important for endemic transmission. Sporadic leptospirosis is associated with human contact with contaminated environments in various settings: on the job (veterinary, sewer, and slaughterhouse workers), in poorly hygienic inner-city alleys and slums, during adventure travel and other non-work-related outdoor activities, and during military training exercises in endemic regions.

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Reliable data on the incidence of leptospirosis and on rates of associated morbidity and mortality remain scant and are generally drawn from biased hospital-based series or from governmental registries including passively reported serologic results. In the United States, leptospirosis was removed from the list of notifiable diseases in the 1990s. The ~50100 cases passively reported annually to the Centers for Disease Control and Prevention (CDC) clearly represent an underestimate; the majority of these cases are from Hawaii, and others are sporadically acquired in inner-city settings or in association with environments such as farms, lakes, and adventure-sport locales. In large urban centers in Brazil that are subject to seasonal flooding (e.g., So Paulo, Rio de Janeiro, and Salvador), tens of thousands of cases are estimated to occur annually. Prospective, population-based cohort studies in Salvador indicate that 5% of slum dwellers are infected annually and that some people are reinfected. Case-fatality rates among hospitalized patients in So Paulo can be as high as ~20% despite state-of-the-art intensive care unit and supportive care. In the Peruvian Amazon, ~3050% of patients with acute undifferentiated fever have been identified as having leptospirosis; the disease is severe in a small minority of such cases (<2%), and these severe cases are often associated with urban acquisition of infection. Men are affected more often by clinical disease than are women. Infection by Leptospira does not occur via inhalation, and leptospirosis is a rare cause of laboratory-acquired infection. Laboratory strains usually used for serologic diagnosis have been serially passaged for long periods and usually have lost their virulence. Morphology & Identification
Typical Organisms Leptospirae are tightly coiled, thin, flexible spirochetes 515 m long, with very fine spirals 0.10.2 m wide; one end is often bent, forming a hook. They are actively motile, which is best seen using a dark-field microscope. Electron micrographs show a thin axial filament and a delicate membrane. The spirochete is so delicate that in the dark-field view it may appear only as a chain of minute cocci. It does not stain readily but can be impregnated with silver. Culture Leptospires grow best under aerobic conditions at 2830C in semisolid medium (EMJH, others) in 10 mL test tubes with 0.1% agar and 5-fluorouracil. See also Diagnostic Laboratory Tests (below). After 12 weeks, the leptospires produce a diffuse zone of growth near the top of the tube and later a ring of growth at a level in the tube corresponding to the level of the optimal oxygen tension for the organisms. Growth Requirements Leptospirae derive energy from oxidation of long-chain fatty acids and cannot use amino acids or carbohydrates as major energy sources. Ammonium salts are a main source of nitrogen. Leptospirae can survive for weeks in water, particularly at alkaline pH. Antigenic Structure The main strains ("serovars") of L interrogans isolated from humans or animals in different parts of the world (Table 24 1) are all serologically related and exhibit cross-reactivity in serologic tests. This indicates considerable overlapping in

Leptospirosis Page : 3 antigenic structure, and quantitative tests and antibody absorption studies are necessary for a specific serologic diagnosis. The outer envelope contains large amounts of lipopolysaccharide of antigenic structure that is variable from one strain to another. This variation forms the basis for the serologic classification of the Leptospira species. It also determines the specificity of the human immune response to leptospirae.

Pathogenesis & Clinical Findings

Human infection usually results from leptospires, often in bodies of water, entering the body through breaks in the skin (cuts and abrasions) and mucus membranes (mouth, nose, conjunctivae). Ingestion is considered to be less important. After an incubation period of 12 weeks, there is a variable febrile onset during which spirochetes are present in the bloodstream. They then establish themselves in the parenchymatous organs (particularly liver and kidneys), producing hemorrhage and necrosis of tissue and resulting in dysfunction of those organs (jaundice, hemorrhage, nitrogen retention). The illness is often biphasic. After initial improvement, the second phase develops when the IgM antibody titer rises. It manifests itself often as "aseptic meningitis" with intense headache, stiff neck, and pleocytosis of the CSF. Nephritis and hepatitis may also recur, and there may be skin, muscle, and eye lesions. The degree and distribution of organ involvement vary in the different diseases produced by different leptospirae in various parts of the world (Table 241). Many infections are mild or subclinical. Hepatitis is frequent in patients with leptospirosis. Kidney involvement in many animal species is chronic and results in the shedding of large numbers of leptospirae in the urine; this is probably the main source of environmental contamination resulting in infection of humans. Human urine also may contain spirochetes in the second and third weeks of disease. Agglutinating, complement-fixing, and lytic antibodies develop during the infection. Serum from convalescent patients protects experimental animals against an otherwise fatal infection. The immunity resulting from infection in humans and animals appears to be serovar-specific.

Clinical Manifestations
The clinical expression of infection by Leptospira, which is related to diverse focal organ dysfunction, includes subclinical infection, an undifferentiated febrile illness, and Weil's diseasethe most severe form. Leptospirosis is classically described as biphasic (Fig. 171-3). Acute fever in the initial leptospiremic phase lasts for 310 days, during which period the organism may be cultured from blood. In a later immune phase, fever is not responsive to antibiotic therapy but leptospires can be isolated from urine. Unlike milder forms, Weil's disease may also be monophasic and fulminant.

Leptospirosis Page : 4 Physical examination may include any of the following findings, none of which is pathognomonic for leptospirosis: conjunctival suffusion (dilated conjunctival blood vessels in the absence of discharge); pharyngeal erythema without exudate; muscle tenderness; rales on lung auscultation or dullness on chest percussion over areas of pleural hemorrhage; rash (which may be macular, maculopapular, erythematous, petechial, or ecchymotic); jaundice; meningismus; and hypo- or areflexia, particularly in the legs. The natural course of mild uncomplicated leptospirosis usually ends in spontaneous resolution within 710 days without sequelae, but the difficulties of rapid diagnosis do not permit the initiation of specific antimicrobial therapy. Biomarkers to predict progression to severe disease are not available. Some patients experience a return of fever, headache, and other systemic symptoms after 310 days (the immune phase) in association with the clearance of leptospires from the blood and the appearance of antibodies; this phase of illness does not respond to antibiotic therapy.

Weil's disease is characterized by variable combinations of jaundice, acute kidney injury, hypotension, and hemorrhagemostcommonly involving the lungs (Fig. 171-4) but also potentially affecting the gastrointestinal tract, retroperitoneum, pericardium, and brain. Other syndromes include aseptic meningitis, uveitis, cholecystitis, acute abdomen, and pancreatitis (with hypo- or hyperglycemia). Jaundice is not associated with fulminant hepatic necrosis or hepatocellular damage, but rather with abnormal laboratory values (see "Diagnosis," below). The liver can be enlarged and tender; splenomegaly is reported in a minority of cases. Acute kidney injury manifests after several days of illness and can be nonoliguric or oliguric, with serum electrolyte abnormalities reflecting proximal renal tubular dysfunction. Hypokalemia and hypomagnesemia are common in nonoliguric renal failure; hypomagnesemia can cause severe muscle weakness. Hypotension is associated with acute tubular necrosis and oliguria, requiring fluid resuscitation, sometimes pressors, and hemodialysis. Renal function typically returns to normal in survivors of severe disease. Severe pulmonary hemorrhage in leptospirosis is a clinical problem wherever the disease is endemic, manifesting with cough, chest pain, and hemoptysis but without purulent sputum. Cardiac involvement is commonly reflected on the electrocardiogram as nonspecific ST and T wave changes but also asright-bundle-branch block and right- and/or left-sided ventricular dilation indicating myocarditis. Skeletal muscle involvement manifests as severe myalgia, typically of the legs (especially the calves) but also of the abdominal muscles (mimicking acute abdomen); these symptoms are associated with a moderately elevated serum concentration of creatine kinase that, by itself, is not sufficient to result in acute kidney injury. Skin abnormalities include petechiae and ecchymosis as well as macular and maculopapular rash. Neurologic findings include aseptic meningitis (in which CSF pleocytosis can range from

Diagnostic Laboratory Tests

Specimens Specimens consist of aseptically collected blood in a heparin tube, CSF, or tissues for microscopic examination and culture. Urine should be collected using great care to avoid contamination. Serum is collected for agglutination tests. Laboratory Findings The leukocyte count may be normal or as high as 50,000/mcL (0.05/L), with neutrophils predominating. The urine may contain bile, protein, casts, and red cells. Oliguria is common, and in severe cases uremia may occur. Elevated bilirubin

Leptospirosis Page : 5 and aminotransferases are seen in 75%, and elevated creatinine (> 1.5 mg/dL) (> 132.6 mcmol/L) is seen in 50% of cases. In cases with meningeal involvement, organisms may be found in the CSF during the first 10 days of illness. Early in the disease, the organism may be identified by darkfield examination of the patient's blood (a test requiring expertise since false-positives are frequent in inexperienced hands) or by culture on a semisolid medium (eg, Fletcher EMJH). Cultures take 16 weeks to become positive. The organism may also be grown from the urine from the tenth day to the sixth week. Diagnosis is usually made by means of serologic tests. Agglutination tests (microscopic, using live organisms, and macroscopic, using killed antigen) become positive after 710 days of illness, peak at 34 weeks, and may persist at high levels for many years. Thus, to make a diagnosis, a fourfold or greater rise in titer must be documented. The agglutination tests are cumbersome to perform and require trained personnel. Indirect hemagglutination and enzymelinked immunosorbent assay (ELISA) tests are also available. The IgM EIA is particularly useful in making an early diagnosis, since it is positive as early as 2 days into illness, a time when the clinical manifestations may be nonspecific, and it is extremely sensitive and specific (93%). PCR methods (presently investigational) appear to be sensitive, specific, positive early in disease, and able to detect leptospiral DNA in blood, urine, CSF, and aqueous humor. Serum creatine kinase (CK) is usually elevated in persons with leptospirosis and normal in persons with hepatitis. Microscopic Examination Dark-field examination or thick smears stained by the Giemsa technique occasionally show leptospirae in fresh blood from early infections. Dark-field examination of centrifuged urine may also be positive. Fluorescein-conjugated antibodies or other immunohistochemical techniques can be used also. Culture Whole fresh blood or urine can be cultured in a semisolid medium. Because of inhibitory substances in blood, only 1 or 2 drops should be placed in each of five tubes containing 5 or 10 mL of medium. Up to 0.5 mL of CSF can be used. One drop of undiluted urine can be used followed by one drop each of 10-fold serially diluted urinefor a total of four tubes. Tissue approximately 5 mm in diameter should be crushed and used as the inoculum. Growth is slow, and cultures should be kept for at least 8 weeks. Serology The diagnosis of leptospirosis in most cases is confirmed serologically. Agglutinating antibodies first appear 57 days after infection and develop slowly reaching a peak at 58 weeks. Very high titers may be attained (>1:10,000). The reference laboratory standard for detection of leptospiral antibody uses microscopic agglutination of live organisms, which can be hazardous. The test is highly sensitive, but it is difficult to standardize; the end point is 50% agglutination, which is difficult to determine. Agglutination of the live suspensions is most specific for the serovar of the infecting leptospires. Agglutination tests are generally performed only in reference laboratories. Paired sera that show a significant change in titer or a single serum with high titer agglutinins plus a compatible clinical illness can be diagnostic. Because of the difficulty in performing the definitive agglutination tests a variety of other tests have been developed for use primarily as screening tests. Immunity Serovar-specific immunity follows infection, but reinfection with different serovars may occur. Treatment

Leptospirosis Page : 6 Treatment of mild leptospirosis should be with oral doxycycline, ampicillin, or amoxicillin. Treatment of moderate or severe disease should be with intravenous penicillin or ampicillin. Epidemiology, Prevention, & Control The leptospiroses are essentially animal infections; human infection is only accidental, following contact with water or other materials contaminated with the excreta of animal hosts. Rats, mice, wild rodents, dogs, swine, and cattle are the principal sources of human infection. They excrete leptospirae in urine both during the active illness and during the asymptomatic carrier state. Leptospirae remain viable in stagnant water for several weeks; drinking, swimming, bathing, or food contamination may lead to human infection. Persons most likely to come in contact with water contaminated by rats (eg, miners, sewer workers, farmers, and fishermen) run the greatest risk of infection. Children acquire the infection from dogs more frequently than do adults. Control consists of preventing exposure to potentially contaminated water and reducing contamination by rodent control. Doxycycline, 200 mg orally once weekly during heavy exposure, is effective prophylaxis. Dogs can receive distemper-hepatitis-leptospirosis vaccinations. Prognosis No vaccine is available for human leptospirosis. Preventive strategies, including prophylaxis with doxycycline, have been variably effective in different settings. Antibiotic prophylaxis can be considered for anticipated short-term, well-defined exposures, such as those incurred during military training or specific adventure travel (with, for example, fresh-water swimming). Long-term antibiotic prophylaxis has not been shown to be effective in preventing infection in hightransmission endemic settings. General sanitation approaches (e.g., rodent control) and avoidance of swimming in potentially contaminated places (e.g., for recreational use) are recommended, but these measures are difficult to apply consistently. The severity of illness in terms of pulmonary and renal dysfunction is the most important determinant of prognosis. Advanced age, clinically evident pulmonary involvement, elevated serum creatinine level, oliguria, and thrombocytopenia are associated with a poor prognosis; liver dysfunction in acute leptospirosis has not been confirmed to be an independent risk factor for death. Chronic alcoholism seems to be associated with severe disease. Reported mortality rates among hospitalized patients have varied from <5% to >20%. Leptospirosis is generally considered to leave no permanent sequelae, although renal dysfunction, as manifested by electrolyte imbalances, may persist for days or weeks after acute illness resolves. Severe pulmonary hemorrhage and liver disease are not known to lead to persistent or progressive organ dysfunction. Some authorities have suggested that neuropsychiatric disturbance may follow severe disease; such conclusions must be assessed in a prospective clinical investigation.