Laboratory diagnosis, treatment and prevention of Leptospira interrogans

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.

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.

Treatment of Leptospira interrogans (Leptospirosis)

• 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 and control of Leptospira interrogans (Leptospirosis)

• 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.