Leptospira interrogans: Characteristics, Pathogenesis, Lab Diagnosis

Leptospira interrogans is a member of the class Spirochaetes order Spirochaetales and the family Leptospiraceae. There are two species of Leptospira; Leptospira interrogans are the pathogenic species and are the causative agents of leptospirosis or Weil’s disease, whereas L. biflexa is saprophytic.

Morphological Characteristics

  • Tightly coiled fine spirochete:
    • L. interrogans have a size of 6-12 μm in length x 0.1 μm in width, allowing them to pass through filters used to sterilize the culture medium.
    • They are tightly and regularly coiled, with characteristic hooked ends (hence the species name interrogans resembling interrogation or question mark).
    • Spirals have a wavelength (interval between spirals) of 0.5μm and amplitude of 0.1μm.
    • They possess a single endoflagellum attached to the pole and are highly motile, exhibiting spinning and translational movements.
  • Not Stained with dyes
    • They cannot be seen under the light microscope due to their thinness (leptos, meaning fine or thin).
    • They do not take up ordinary stains but may be observed by dark ground or phase-contrast microscope or stained by silver impregnation method and by immunofluorescence.
  • Antigenically complex: Divided into serogroups which were further divided into serovars. On the basis of agglutination testing with specific antisera against the surface LPS antigens, Leptospira is divided into:
    • Leptospira interrrogans: Comprises 250 serogroups, consisting of over 250 serovars.
    • L. biflexa: 65 serovars arranged in 38 serogroups.

Pathogenesis

  • Source: Although more than 100 animals can be infected, important sources of infection are rats, dogs, cattle, and pigs. Even asymptomatic animals can transmit the infection via urine (persistent colonization of renal tubules of carrier animals.)
  • Transmission:
    • Zoonotic: A person may acquire leptospirosis if he/she comes in contact with the urine of infected animals (or other body fluids, except saliva) such as dogs, pigs, and cattle directly or through contaminated food water, or soils.  Abrasions or cuts in the skin increase the chances of infection.
    • It is an occupational hazard for many people who work outdoors or with animals, such as farmers, slaughterhouse workers, veterinarians, and animal caretakers. Leptospirosis has also been associated with swimming, kayaking, and rafting in contaminated lakes and rivers.
    • Person-to-person transmission is rare.

3R’s: The three important epidemiological determinants for leptospirosis include exposure to rodents, rainfall, and rice field.

  • Risk factors:
    • Lower socioeconomic status
    • Urban and rural slum areas
    • Rainfall and floods
    • Occupational exposure to animal urine, e.g., rice field workers and farmers.
  • Global distribution: Worldwide, the highest burden of the disease has a reported in urban slums of Brazil, India, and Thailand.

Signs and Symptoms

Leptospirosis can use many symptoms, some of which may be mistaken for other diseases such as dengue fever and other viral hemorrhagic diseases. Leptospirosis can lead to kidney damage, meningitis, liver failure, respiratory distress, and even death if left untreated.

Leptospirosis is often misdiagnosed as aseptic meningitis, influenza, dengue, hepatic disease, or pyrexia of unknown origin. Therefore, diagnosis is based on laboratory tests rather than clinical symptoms alone, but these tests are not always available, especially in developing countries.

There are two phases of Infections:

  1. The acute or bacteremic/septicemic phase
  2. Immune phase: Aseptic meningitis

First Phase: Acute or bacteremic/septicemic phase

  • Entry via mucous membranes of the eyes, nose, or mouth or through skin abrasion
  • Vascular damage: Spirochetes can be found in the walls of capillaries, medium, and large-sized vessels. The exact mechanism of vascular damage is not clear.
  • Penetration and invasion of tissues are due to active motility and release of hyaluronidase.
  • Invade bloodstream and hematogenous spread multiple sites of the body:
    • producing fever,
    • dysfunction of the liver (jaundice),
    • kidneys (uremia),
    • lungs (hemorrhage)
    • central nervous system (aseptic meningitis)

Second Phase (Immune Phase)

After seroconversion, Spirochetes disappear from the blood, but they may colonize in the kidney: Leptospira becomes adherent to the proximal tubular brush border and is excreted in the urine.

Clinical Manifestations

Incubation period: 5-14 days

Two distinct clinical syndromes:

  • Mild anicteric febrile illness: 90% of patients. Biphasic in nature; septicemic phase followed by immune phase.
  • Weil’s disease (hepato-renal-hemorrhagic syndrome)
Weil’s Syndrome:
  • a severe form of the leptospirosis infection (icteric) which occurs in approximately 10% of patients
  • jaundice and significant liver damage
  • kidney and/or vascular dysfunction
  • lethal pulmonary hemorrhages
  • Typical biphasic course may not be present.
  • death is up to 10% of cases

Laboratory Diagnosis

Leptospirosis is usually diagnosed in the laboratory by detecting antibodies, by culturing the bacteria from blood, CSF, urine, or tissues, or by demonstrating the presence of leptospires in tissues using antibodies labeled with fluorescent markers.

Detecting L. interrogans in a clinical specimen (urine, blood, or CSF) by immunofluorescence, impregnation stains such as Fontana stain, modified Steiner technique, or by using dark-ground microscopy or phase-contrast microscopy is a commonly used microscopic method. Other methods include polymerase chain reaction (PCR) and immunostaining.

(a) Growth from blood in EMJH semisolid media Leptospira is forming subsurface colonies in the tube on the left and no growth in the tube on the right; (b) dark field microscopy image of the culture showing spirochetes with morphology compatible with Leptospira; (c) Conventional PCR targeting lipL32 gene  (Image source: Christopher Ryan Larson)

Microscopy and Staining

1998 Rob Weyant This scanning electron micrograph (SEM) depicts a number of Leptospira sp. bacteria atop a 0.1. µm polycarbonate filter.

Demonstrating leptospires in the fluids using Dark-Field Microscopy is often described as a proper diagnostic method, but its significance is doubtful.  Serum protein and fibrin strands, and other cell debris in blood resemble leptospires, while the concentration of organisms in the urine of humans and animals is frequently too low to be detectable by this method. Care and great experience are therefore necessary to avoid mistaking artifacts for leptospires.

Leptospires are corkscrew-shaped bacteria that differ from other spirochaetes by the presence of end hooks. They are too thin to be visible under an ordinary microscope. All leptospires look-alike with minor differences, so morphology does not help differentiate between pathogenic and saprophytic leptospires or the various pathogenic leptospires.

Leptospiral microscopic agglutination test with live antigen using dark field microscopy

Culture

The culture of Leptospira is a problematic and relatively insensitive process; it is laborious and can take up to three months. In the acute phase, which lasts for about ten days, the leptospires can often be cultured from blood or cerebrospinal fluid (CSF). When a specific antibody response is detected (at approx. ten days), leptospires disappear from the blood. During the second phase, which may last up to several months, bacteriuria is often intermittent.

Leptospires grow in a variety of cultural media. Media with 14% (v/v) rabbit serum, such as Fletcher’s or EMJH (Ellinghausen, McCullough, Johnson, Harris) medium, is used for the isolation. Their growth is relatively slow, with a doubling time of about 6–8 hours at best. Incubation is done at 28-30°C for about 13 weeks, with culture examined every third day by darkfield microscopy.  In the dark field microscopy, leptospires are observed as thin, coiled, rapidly moving microorganisms.

Antibody detection

Antibodies may be detected in the blood within 5–7 days of symptom onset. Evidence of seroconversion between acute and convalescent-phase serum specimens confirms the diagnosis of leptospirosis, which can be demonstrated by a microscopic agglutination test (MAT).

Microscopic Agglutination Test (MAT)

MAT is a gold standard method for diagnosing leptospirosis, but it may only be available in reference laboratories. MAT has high sensitivity and detects serovar-specific antibodies.  Seroconversion with a 4-fold or larger rise in titer between acute- and convalescent-phase serum specimens obtained two weeks, or more apart confirms the diagnosis. A single elevated MAT titer in a patient with a compatible febrile illness and suspected exposure suggests acute leptospirosis.

Major disadvantages of Microscopic Agglutination Test (MAT)

  1. In the endemic regions, there may be a substantial proportion of the population with elevated titers of MAT.
  2. The performance of MAT is restricted to laboratories capable of maintaining strains for the preparation of live antigens.

Other serological methods

  1. Microplate IgM ELISA and
  2. IgM dot-ELISA dipstick test

Molecular Methods

Polymerase Chain Reaction (PCR) assay can be used on clinical samples such as CSF, urine, or blood to detect the presence of leptospiral DNA. It is based upon the amplification of 16S rRNA gene sequences of Leptospira.

Advantages and disadvantages of diagnostic tests for the detection of Leptospirosis

Tests Advantages Disadvantages
Dark Field Microscopy Visualize leptospira Lack of sensitivity and specificity. 10^4 Leptospires/ml is necessary for one organism/field to be visible under DFM.
IgM ELISA Most widely used IgM cannot be detected in the early stages of infection and can persist in the blood for years.
Microscopic Agglutination Test (MAT) Gold Standard Less sensitive in the early phase of the disease. Labor-intensive and complicated procedure.
Polymerase Chain Reaction (PCR) Successful in detecting Leptospira DNA in serum and urine samples of patients Reagents are expensive. It requires a large quantity of DNA but cannot identify the infecting serovar.

References and further reading

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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