Diagnostic approach in leptospirosis patients

Leptospirosis is as a worldwide zoonotic disease, spread by pathogenic species of the bacterial genus Leptospira that occurs most commonly in tropical and subtropical regions which are one of endemic diseases in some places in Indonesia. The leptospira serovars are naturally carried in the renal tubules of rodents, wild and domestic animals. Human can be infected either through direct contact with urine of infected animals or indirect contact through with contaminated water and soil. Clinical manifestation is highly variable. The most cases are with a mild flu-like illness which may mimic many other diseases. Weil’s disease is the name given to severe illness and is characterized by a severe febrile illness with bleeding, jaundice and renal failure with high mortality rate. Leptospirosis has been frequently underdiagnosed and underreported. The diagnosis of leptospirosis is difficult to confirm and laboratory test is rarely available even in endemic areas.


Introduction
Leptospirosis is a neglected zoonotic disease caused by pathogenic spirochetes of the genus leptospira. The pathogenesis particularly that of severe disease, remains poorly understood even though Weil published a clinical description of leptospirosis in 1866.It is considered the most common zoonosis in the world and is associated with rodent in setting of poor sanitation, especially after heavy rain or flooding due to storm, agricultural occupations, and increasingly adventure sport involving fresh water, mud, or soil exposure. The organism can remain in renal tubules of rodent and pass through the urine for long period of time, even for the lifetime of the animal. Human infection follows exposure to infected animals, either directly or indirectly through contaminated water and soil. Characteristic of disease is a broad spectrum of clinical manifestation, varying from asymptomatic infection to fulminant, fatal disease. Most of cases are mild form, leptospirosis may present as an acute febrile illness with a biphasic course. Nonspecific symptoms such as fever, headache, myalgia, nausea and vomiting are often confused with viral illness. while a minority develop a severe illness, characterized by jaundice, renal dysfunction, and hemorrhagic diathesis is known as Weil's syndrome. It is often rapidly progressive and is associated with high mortality rate. [1][2][3][4]

Etiology
The causative agents belong to the genus leptospira, highly-motile, fine spiral bacteria of 0.1 μm in diameter and x 6-25 μm in length. Under dark ground microscopy, the organism appears straight with oneor both ends hooked. [1] The genus of leptospira comprises two species: L interrogans (pathogenic) and L. biflexa (saprophytic). Pathogenic Leptospira species are divided into serovars according to their antigenic composition. L. interrogans included more than 250 serovars. [4] Inada isolated and identified leptospira as the etiologic agent also linked rats to disease transmission. Many

Pathogenesis
Leptospirosis is caused by spirochetes of the genus called Leptospira, particularly the pathogenic Leptospira interrogans species. This pathogenic species is further subdivided into different serogroups, serovars and strains based on the embedded antigens. Transmission of the pathogen typically includes contact of infected rodent urine through mucous membranes (ie. eye, nose, mouth) and skin lesions. Following the entry, pathogen disseminate through systemic circulation to distant vessels and organs. Virulence factors exist that favors invasion of Leptospira, such as hyaluronidase as well as a structural hooked ends and axial flagella, enabling 'burrowing motility' for deep penetration. Bacterial endotoxin, hemolysin and lipase also triggers inflammatory cascade leading to various disease manifestations. Leptospira is found in large, or medium vessels or capillaries. On a cellular level, endothelial damage is commonly observed and serves as an underlying cause of endothelial edema, necrosis, or lymphocytic infiltration which constitute systemic vasculitis. Capillary vasculitis is also prominent in all affected organs. Consequently, erythrocyte and fluid loss occurs through enlarged junctions and fenestrae leading to secondary injury. Organ involvement is frequent, mainly kidneys, lungs and liver. In kidneys, Leptospira migrate to interstitium, renal tubules and tubular lumen causing diffuse tubule interstitial inflammation as well as tubular necrosis. Within the lungs, Leptospira causes alveolar and interstitial vascular damage with subsequent hemorrhage. This lung involvement is typically associated with leptospirosis-related death. Liver involvement is also common, characterized by centrilobular necrosis and kupffer cell proliferation which may cause jaundice due to hepatocellular dysfunction. Other than aforementioned organs, in severe disease, other organs may also be affected causing myocarditis, meningoencephalitis or uveitis. [1][2][3][4]

Clinical Features
Manifestations of Leptospirosis is categorized into two distinct types: anicteric and icteric. In anicteric manifestation, infection is typically self-limited, with mild flu-like syndromes that do not require specific therapy. Whilst in the icteric type, known as Weil disease, infection is severe and typically involves multi organ dysfunction or even failure. Clinical course of leptospirosis is described as biphasic, involving acute bacteremia phase and secondary immune phase. Incubation period generally lasts from 5 -12 days (ranging from 2 -30 days).

5.1.The acute bacteremia phase
Following the bacterial migration into the blood stream, leptospiremia occurs. Clinically this phase is characterized by a sudden onset of fever (could up to 39 0 -40 0 ), with other accompanied flu-like symptoms such as chillness, headache, sore throat, myalgia, nausea, and vomiting. More burdensome symptoms such as cough, hemoptysis, dyspnea, abdominal pain with persistent vomiting may also occurs. In some severe form, aseptic meningitis can develop. During this leptospiremia, pathogen can be isolated through blood, cerebrospinal fluid and other tissues, but not from urine. Serology test frequently remains negative of up to at least 5 days prior onset of symptoms. This bacteremia phase lasts approximately for 4 -7 days. Subsequent dissemination to organs may follow in this phase, which might include meninges. Myalgia is severe particularly in calves, back and abdominal muscles. Liver is moderately enlarged in this stage commonly without spleen enlargement. Hematologically, platelet count typically falls potentially causing thrombocytopenic purpura or frank bleeding. Serum creatinine increases, with normal creatinine clearance unless tubular necrosis or glomerulonephritis already develop; with accompanied proteinuria in urinalysis.

5.2.The secondary (immune) phase
In this later immune phase, which occurs immediately or within 2-3 days after, the patient generates antibodies towards Leptospira. The antibody response mainly involveIgM class that displays strong agglutination properties and could last for up to several months. During mild infection, signs and symptoms may not be as apparent. However in more severe cases, meningeal or hepatorenal manifestation is predominant. In such severe form, septicemia and immune phase typically merge and is difficult to distinguish clinically, showing persistently high fever, jaundice, frank hemorrhage into skin, mucous membranes or lungs. Hepatomegaly and icteric sclera are clinically more noticeable in this phase. The suffused vessels become orange. Purpura and ecchymosis are visible. Renal failure develop, causing oliguria, with accompanied shock, and myocarditis. Pulmonary edema and pulmonary hemorrhage with hemoptysis may also occur which highly associated with mortality.
In oliguric or later anuric patients, plasma creatinin is commonly elevated that typically requires renal dialysis. Potassium level is contradictory low. Bilirubin level is high but liver enzyme usually remains normal or mildly elevated. Thus the presence of high bilirubin and creatinine level should raise possibility of leptospirosis infection. Renal failure commonly cause further deterioration that leads to death, however other underlying causes include myocarditis (showing abnormal electrocardiography), hemorrhage, adrenal failure and cerebral artery thrombosis. In surviving patients without dialysis support, creatinin level begins to decline at the end of the second week prior onset of disease. This process occurs as rapid resolution of tubular necrosis begins. Additionally, renal function parameter is expected to return to normal in 6 months, except the urine-concentrating ability [1][2][3][4][5].
The clinical manifestations are highly variable. In general, the disease presents in four broad clinical categories [6]:  A mild, influenza-like illness  Weil's syndrome characterized by jaundice, renal failure, hemorrhage and myocarditis with arrhythmias  Meningitis/meningoencephalitis  Pulmonary hemorrhage with respiratory failure The typical course of leptospirosis with an acute septicemic phase followed by the immune phase as shown in Figure 1. antileptospiral antibodies lead to clearance of the organism from most tissues except kidney tubules; leptospiresmaycontinueto shed in the urine for long periods Figure 1. Typical course of leptospirosis [6].

Diagnosis
A good clinical history is often the key to accurate diagnosis in leptospirosis. The problem in the diagnosis of leptospirosis are most cases underdiagnosed because symptoms and sign are often nonspecific and serologic confirmation is rarely available. Laboratory studies are used for the purposes [4,5]:  To confirm the diagnosis and to determine the extent of organ involvement and severity of complication  For epidemiological and public health reasons, namely to determine which serovar caused the infection, the likely source of infection and the potential reservoir and its location. This help control strategy Laboratory studies used to diagnosis of leptospirosis include the following:  Leptospira immunoglobulin M (IgM) ELISA or IgM/immunoglobulin G (IgG) enzyme linked immune absorbant assay (ELISA), including rapid diagnostic kits usable in the field  Real-time DNA polymerase chain reaction (PCR) of blood, urine, and cerebrospinal fluid (CSF)  Microscopic agglutination testing (MAT); criteria standard for serologic identification of leptospira, available at reference laboratories, Single titer ≥1:200 or 4-fold rise in serum drawn between the first and fourth week of illness is considered diagnostic  DNA PCR of blood, urine, CSF, tissue  Culture of leptospira from body fluids or tissue (criterion standard, but requires specificmedia and several weeks' incubation, thus usually limited to reference laboratory) Studies to determine the extent of organ involvement and severity of complications may include the following, depending on the clinical presentation: complete blood cell (CBC) count, renal function test, coagulation test, liver function test, CSF analysis, chest radiography, biliary tract ultrasonography, Electrocardiography (ECG). [1,2,4,5]

Treatment
Antimicrobial therapy is indicated for the severe leptospirosis, but its use is controversial for the mild form of leptospirosis. [4][5][6][7]  Peritoneal dialysis or hemodialysis should be provided to patients with oliguric renal failure that has been shown to reduce mortality risk and typically and necessary only for short periods.