Molecular Approaches in the Detection and Characterization of Leptospira

Abstract

Conventional diagnosis of leptospirosis and characterization of Leptospira spp. relies mainly on serology. A major drawback of serology in diagnosis is that it needs sufficient levels of anti-Leptospira antibodies, thus jeopardizing confirmation in the early acute phase of disease. The cross agglutinin absorption test (CAAT) that determines Leptospira serovars is a technically demanding and laborious method and therefore is only performed at a few laboratories. Novel molecular diagnostic tests mainly rely on the polymerase chain reaction (PCR). PCR perfectly complements serological testing, since it is especially sensitive in the first 5 days after onset of symptoms. Real time PCR is rapid and has been validated for their high clinical accuracy. The introduction of molecular techniques for defining Leptospira species revolutionized the categorization of strains in this genus, as species and serogroups appeared to show little correlation. The reference test in molecular speciation is based on determining DNA homology. This approach is tedious and user-unfriendly and therefore is increasingly replaced by other techniques. To date, a wealth of molecular typing methods is available. Most attractive are those techniques that provide direct digital and electronically portable data. Such techniques comprise fluorescent amplified fragment length polymorphism (FAFLP), array typing, multilocus variable number of tandem repeats analysis (MLVA) and sequence-based phylogeny, and to some extent pulsed field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) is the most robust method for determining Leptospira strain diversity and in the future will probably only be surpassed by phylogeny on whole genome sequences.

Leptospirosis in Humans

“…An initial attempt to gather global data on the incidence of leptospirosis was published over 15 years ago (WHO 1999). Based on global data collected by International Leptospirosis Society surveys, the incidence was estimated to be 350,000–500,000 severe leptospirosis cases annually (Ahmed et al 2012). Despite these efforts, the global burden of leptospirosis was felt to be largely underestimated for a number of reasons, including the fact that the vast majority of countries either lack a notification system or notification is not mandatory (Ahmed et al 2012).…”

“…Based on global data collected by International Leptospirosis Society surveys, the incidence was estimated to be 350,000–500,000 severe leptospirosis cases annually (Ahmed et al 2012). Despite these efforts, the global burden of leptospirosis was felt to be largely underestimated for a number of reasons, including the fact that the vast majority of countries either lack a notification system or notification is not mandatory (Ahmed et al 2012). To address these shortcomings, the WHO established the Leptospirosis Burden Epidemiology Reference Group (LERG) (Abela-Ridder et al 2010).…”

“…Conventional PCR and other assays such as LAMP and NASBA were reviewed recently (Ahmed et al 2012) and will not be discussed further. Many quantitative PCR assays have been described, which target a number of different genes (Ahmed et al 2009; Merien et al 2005; Palaniappan et al 2005; Smythe et al 2002; Stoddard et al 2009).…”

“…Many quantitative PCR assays have been described, which target a number of different genes (Ahmed et al 2009; Merien et al 2005; Palaniappan et al 2005; Smythe et al 2002; Stoddard et al 2009). Assays developed for diagnostic use can be considered in two broad categories, targeting either housekeeping genes, such as rrs , gyrB , or secY, or pathogen-specific genes such as lipL32, lig, or lfb1 (Ahmed et al 2012). Examples of these two types of quantitative assay were evaluated in a large case-control study in a high-prevalence population in Thailand (Thaipadunpanit et al 2011), that confirmed earlier reports that PCR detection in blood samples collected at admission to hospital was more sensitive than culture, but serology using the microscopic agglutination test (MAT) ultimately detected more cases (Brown et al 1995).…”

Detection of infection with Leptospira spp. in manatees (Trichechus inunguis) of the Peruvian Amazon

“…The only way to assess strain is through culture and isolation of the organism (Levett, 2003). PCR of leptospires shed in the urine or colonizing the kidneys can be used to identify the infecting organism to the serovar level (Ahmed et al, 2012).…”