Laboratory Diagnosis of Leptospirosis

Leptospirosis (also known as weil’s disease) is one of the widespread zoonoses caused by pathogenic spirochetes of the genus Leptospira and is found in virtually all tropical and temperate areas of the world.

Adolf Weil described leptospirosis as a disease in 1886. In 20th century, Leptospires were recognized by Inada and Ido in Japan and soon after, independently, in Germany by Uhlenhuth and Fromme as the cause of the disease.

A person may acquire leptospirosis if he/she comes in contact with urine of infected animals such as dogs, pigs, and cattle directly or through contaminated food, water or soils.  Abrasions or cuts in the skin increases the chances of infection. Human-to-human transmission is rare.

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.

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

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 on clinical symptoms alone but these tests are not always available especially in developing countries.

Laboratory Diagnosis of Leptospirosis

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 labelled with fluorescent markers. Other methods include polymerase chain reaction (PCR) and (immuno)staining.

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.


Demonstration of leptospires in the fluids using Dark-Field Microscopy is often described as useful 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 artefacts for leptospires

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

Leptospiral microscopic agglutination test with live antigen using dark field microscopy

Culture

Culture of Leptospira is difficult and relatively insensitive process; it is laborious and can take up to three months. Isolation and culture are primarily used for retrospective diagnosis.

In the acute phase, which lasts for about 10 days, the leptospires can often be cultured from blood or cerebrospinal fluid (CSF). When specific antibody response is detected (at approx. 10 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 culture 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 28-30oC for about 13 weeks with culture examined every third day by dark field 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.  Evidences of seroconversion between acute and convalescent phase serum specimens confirms diagnosis of leptospirosis which can be demonstrated by Microscopic Agglutination Test (MAT).

Microscopic Agglutination Test (MAT)

MAT is regarded as a gold standard method for the diagnosis of leptospirosis but it may be only 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 2 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 titres of MAT.
  2. The performance of MAT is restricted to laboratories that are 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 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 early stages of infection and can persist in blood for years.
Microscopic Agglutination Test (MAT) Gold Standard Less sensitive in early phase of disease. Labor intensive and complicated procedure.
Polymerase Chain Reaction (PCR) Successful in detecting Leptospira DNA in serum and urine samples of patients Reagents are expenseive. It requires large quantity of DNA but still cannot identify the infecting serovar.

References and further reading

About tankeshwar 386 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion, I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

1 Comment

  1. Very useful. I’m a lab tech. I need more information regarding Dipthroid isolation in CLED in urine culture

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