Leptospira – Serology and PCR

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.

Background
This page provides serology and PCR testing information for Leptospirosis (Weil’s Disease) through Public Health Ontario (PHO). Testing is referred out to the National Microbiology Laboratory (NML) - Leptospira - Guide to Services - CNPHI (canada.ca).

The causative agent(s) of Leptospirosis include bacteria of the genus Leptospira. The following testing options are available for Leptospirosis: Serology (ELISA, MAT) and PCR. This page is for information specific to Serology and PCR.

Updates

As of December 15, 2023, additional acceptance criteria (listed in Acceptance/Rejection criteria section below) are required to improve leptospirosis diagnosis.

*Uncontrolled print copy. Valid only on day of print: 01 May 2024.

Testing Indications

Testing is only advised for individuals with compatible exposure history and clinical presentation.

Symptoms of Leptospira spp. infection may include fever, headache, chills, severe malaise, skin rash, vomiting, diarrhoea, myalgia, conjunctival suffusion, jaundice, kidney and/or liver failure, meningitis and chest pains¹.
Contact with an infected animal (dogs, cattle, raccoons, rodents, etc.) or recent travel to locations known to be endemic for Leptospira spp¹.

See the Centres for Disease Prevention and Control (CDC) website for further information.

Acceptance/Rejection Criteria

Leptospira test requests will only by accepted in the following instances:

For PCR testing; specimen should be collected as soon as possible after symptom onset, but no later than 14 days following onset of illness.
Collection date (Mandatory) is included.

Specimens may be subject to rejection if they are not the appropriate sample type, have insufficient volume or are not accompanied by relevant patient information or clinical history. Detection of Leptospira species by PCR and sequencing - Guide to Services - CNPHI (canada.ca)

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Leptospirosis^1,2	General Test Requisition	Blood or serum	5.0 ml blood 1.0 ml serum	Blood, clotted – Serum Separator Tubes (SST)
Leptospira PCR^1,2,3,4	General Test Requisition	CSF	0.5 mL	sterile container
Leptospira PCR^1,2,3,4	General Test Requisition	EDTA/ Whole Blood	2.0-5.0 mL	EDTA tubes AVOID HEPARIN.
Leptospira PCR^1,2,3,4	General Test Requisition	Urine	5.0-10.0 mL (as fresh as possible to prevent degradation)	sterile container

Submission and Collection Notes

Complete all fields of the requisition form, including:

Test(s) requests and indications for testing
Patient setting/population/source
Clinical information including symptom onset date
Date of collection is Mandatory

For clinical specimens, label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Buffy coat, synovial fluid, wound fluid/pus, swabs, serum/plasma, tissue and paraffin blocks are NOT acceptable sample types for Leptospira PCR.

Medical microbiologist approval for Leptospira PCR testing is no longer required if the appropriate specimen is submitted with the appropriate clinical context and the rationale for the request is provided on the requisition.

Timing of Specimen Collection

Antibodies for leptospirosis develop between 3-10 days after symptom onset
Negative serologic test results from samples collected in the first week of illness do not rule out disease, and serologic testing should be repeated on a convalescent sample collected 7-14 days after the first
In the acute phase of illness, leptospires are present in the blood for approximately the first 4–6 days of illness. For PCR testing, whole blood should be collected in the first week of illness (ideally in the first 4 days)

Limitations

Grossly haemolysed, icteric, lipemic or contaminated sera or plasma are not recommended for testing.

Initiation of antibiotic treatment prior to testing may result in decreased antibody production which will affect the outcome of serological testing.¹

As with any laboratory test, results of the test should be interpreted with consideration of all available laboratory and clinical findings.¹

Storage and Transport

Centrifuge tube if using SST; Do not centrifuge EDTA tubes.

Place specimen in biohazard bag and seal. Specimens should be stored at 2-8°C following collection and shipped on ice packs to PHO’s laboratory as soon as possible. For PCR specimens stored > 5 days, freeze at -20°C and ship on dry ice.

All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Leptospira serology and PCR testing is forwarded to the National Microbiology Laboratory (NML) in Winnipeg. Turnaround time is typically up to 21 calendar days (PCR or ELISA result) but can be up to 30 calendar days (if specimen requires further confirmatory testing by MAT) upon receipt at PHO’s laboratory.

Test Methods

If Leptospira serology or PCR is requested with all acceptance criteria above met, samples will be forwarded to NML testing and results will be returned to PHO’s laboratory to be reported to the physician.

Leptospira serology testing is initially performed at NML using a Commercial IgM Enzyme-Linked ImmunoSorbent Assay (ELISA) kit. Samples, which are positive or equivocal by ELISA, are further tested by a Microagglutination test (MAT), for confirmation.

Leptospira molecular testing is done by PCR.

EIA¹:
Commercial IgM ELISA kit; this is a qualitative test for the detection of IgM antibodies to Leptospira spp. in serum.

MAT¹ (using up to 18 serovars):
Initial screening titre is 1:100 and if necessary, endpoint titres will be determined.

Supportive criteria for a probable case of Leptospirosis include a single MAT titre of ≥ 1:200, accompanied by a clinically compatible illness.

Criteria required for a confirmed diagnosis include a MAT titre of ≥ 1:800 in one or more serum samples or a four-fold or greater increase in a MAT titre between acute and convalescent serum samples.

PCR¹:
Extracted DNA is screened by real-time PCR using an assay that targets the lipL32 gene of pathogenic Leptospira spp. Samples that are positive by the screening assay are then tested by a confirmatory real-time PCR assay targeting Leptospira 16S rRNA and conventional PCR and sequencing of the citrate synthase gene.

Interpretation

The following table provides possible test results with associated interpretations:

Test	Result	Comments
EIA	Negative Positive, Equivocal	Note that a negative result does not exclude a diagnosis of leptospirosis since some individuals may be immunologically unresponsive, or the serum specimen may have been drawn too early in the course of the illness to detect antibodies. MAT testing done to confirm
Microagglutination test (Mat) up to 18 serovars	MAT titres of <100 are considered negative for anti-Leptospira antibodies* MAT titres of ≥100 are considered positive	Note that a negative result does not exclude a diagnosis of leptospirosis since some individuals may be immunologically unresponsive, or the serum specimen may have been drawn too early in the course of the illness to detect antibodies. A four-fold rise in titre between paired sera or a titre of ≥800 in a single specimen is considered diagnostic for leptospirosis. It may not be possible to determine the infecting serovar by serology due to cross reactivity.
Leptospira spp. PCR:	Negative or Positive

Test

Result

Comments

EIA

Negative

Positive, Equivocal

Note that a negative result does not exclude a diagnosis of leptospirosis since some individuals may be immunologically unresponsive, or the serum specimen may have been drawn too early in the course of the illness to detect antibodies.

MAT testing done to confirm

Microagglutination test (Mat)

up to 18 serovars

MAT titres of <100 are considered negative for anti-Leptospira antibodies*

MAT titres of ≥100 are considered positive

A four-fold rise in titre between paired sera or a titre of ≥800 in a single specimen is considered diagnostic for leptospirosis. It may not be possible to determine the infecting serovar by serology due to cross reactivity.

Leptospira spp. PCR:

Negative or Positive

Reporting

Results are received back at PHO’s laboratory, and reports are forwarded to the ordering physician or authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

References

Canada's National Public Health Institute. Pathogens - Start with "L" [internet]. Ottawa, ON: Canada's National Public Health Institute; [cited 2024 Jan 10]. Available from: https://cnphi.canada.ca/gts/pathogens-start-with/L