Laboratory diagnosis, treatment and prevention of Leptospira interrogans

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Laboratory diagnosis of Leptospira interrogans

Specimens

Specimens consist of aseptically collected blood in a heparin tube, CSF, urine, or tissues for microscopic examination and culture.
Serum is collected for agglutination tests.

Microscopy

As leptospires are thin, they are at the limit of the resolving power of a light microscope and thus cannot be seen by conventional light microscopy.
It cannot be detected by Gram stain or silver stain technique.
Darkfield microscopy is also relatively insensitive, capable of yielding nonspecific findings.
Fluorescein-conjugated antibodies or other immunohistochemical techniques can be used.

Laboratory diagnosis, treatment and prevention of Leptospira interrogans

Culture

Leptospira interrogans can be cultured on specially formulated media (e.g., Fletcher, EMJH [Ellinghausen-McCullough- Johnson-Harris], Stuart’s, or Tween 80-albumin media supplemented with neomycin and 5-fluorouracil.
They grow slowly (generation time, 6 to 16 hours), requiring incubation at 28° C to 30° C for as long as 4 months; however, most cultures are positive within 2 weeks.
Growth of the bacteria in culture is detected by darkfield microscopy.

Serology

The diagnosis of leptospirosis in most cases is confirmed serologically.
Agglutinating antibodies first appear 5–7 days after infection and develop slowly, reaching a peak at 5–8 weeks.
IgM antibodies appear early within one week of illness, reach peak levels in third or fourth week and then decline slowly and become undetectable within six month.
IgG antibodies appear later than IgM, reach peak level after few weeks of illness and may persist at low level for years.
Various tests available are: Macroscopic slide agglutination test(MAT), Microcapsule agglutination test (MCAT), Latex agglutination rest, ELISA- it detects IgM and IgG separately, Lepto dipstick assay – it detects IgM antibodies, Immunochromarographic test (ICT) – it detects IgM antibodies.
The microscopic agglutination test (MAT), which can indicate the likely infecting serogroup or serovar, is generally accepted as the ‘gold standard’.
Several enzyme-linked immunosorbent assay (ELISA) kits offer ease of use together with relatively good sensitivity and specificity.
Most detect IgM antibodies, which, are detectable in the acute phase of the illness and may remain for several months after infection.

Molecular methods

PCR has been found particularly useful in severe disease, before seroconversion occurs.
Various genes such as 16S or 23S rRNA or IS1533 insertion sequence are targeted.
However, PCR is not serovar-specific and hence, PCR-RFLP (Restriction fragment length polymorphism) or PFGE (Pulsed-field gel electrophoresis) are the methods followed to determine the genome of species of Leptospira.

Antimicrobial therapy is the mainstay of treatment of leptospirosis.
Oral doxycycline is the drug of choice for treatment of uncomplicated Leptospira infection.
Intravenous penicillin or ampicillin is recommended for hospitalized patients.
Streptomycin, tetracycline, or erythromycin are alternative choice for patients allergic to penicillin.

Prevention of exposure to potentially contaminated water and food helps control the transmission of L. interrogans and reducing contamination by rodent control.
Measures to reduce rodent populations in the vicinity of human activity, such as removing rubbish, especially waste food, and prevention of the access of rats into buildings is most important.
Simple measures to reduce the risks of acquiring infection also include covering cuts and abrasions with waterproof plasters and wearing protective footwear before exposure to surface waters.
Mass immunization of domestic livestock will prevent clinical disease in the animals and reduce the risk of human acquisition of infection.
Awareness of leptospirosis through the education of doctors, employers and the general public help to develop safer practices or procedures in the workplace and during recreational pursuits.