2.3 Discovery of antibiotic/antiviral medicine for the disease

 2.3 Discovery of antibiotic/antiviral medicine for the disease

Piscina, an L. interrogans isolate, was found in a southern Brazilian swimming pool that had been abandoned. With an LD50 of only two leptospires, it is extremely virulent in the hamster model and causes lesions that are frequently related to acute leptospirosis. This suggested that the local public health might be at risk from L. interrogans Piscina. This isolated strain has been employed as a challenging strain in studies to assess the level of security provided by new recombinant vaccine candidates against leptospirosis in vaccine lab facilities because of its high virulence.

2.3.1 Derivation of the technique

Since leptospirosis is mistakenly thought to be a disease that only affects rural areas, clinicians may fail to notice its transmission in urban settings. Therefore, rather than relying just on clinical symptoms, diagnosis is based on laboratory studies. Considering the disease's significant incidence in underdeveloped nations, the lab infrastructure is insufficient for diagnosis. Since MAT has great sensitivity and enables the identification of group-specific antibodies, it is the cornerstone of the serodiagnosis for leptospirosis. The efficacy of MAT is limited to laboratories that are capable of maintaining strains for the manufacture of live antigens. This test has two primary drawbacks: first, in areas where leptospirosis is endemic, a significant small subset may have increased titres of MAT. 

      Serological assays are still insufficient for detecting leptospirosis in clinical settings. The diagnostic techniques that show the existence of the pathogens are by far the most reliable. Leptospira cultivation is challenging, time-consuming, and can take up to three months. So the main methods for retrospective diagnostics are isolation and culture. Acknowledging the infection stage is essential for the cultivation of the organism from tissues or bodily fluids. Leptospires can frequently be cultured from blood or cerebrospinal fluid during the acute phase (10 days) (CSF). 

       Leptospires typically vanish from the bloodstream when a particular antibody reaction is found. Bacteriuria is frequently inconsistent throughout the second phase, which could extend for a few months. Sensitive assays for the screening of Leptospira DNA that are based upon amplification of the Leptospiras (16S) gene have been established. Molecular approaches to identify the presence of Leptospiral DNA in Blood, Urine, or Spinal Fluid have been demonstrated to be specific and sensitive. As a screening tool for situations of suspected leptospirosis, PCR assay can be applied to biological samples like CSF, urine, or blood.

2.3.2 Using DNA to design primer

Leptospira mPCR amplification was carried out using the oligonucleotide sequences of Lep F, Lep R, Lau01, and Lau02. A 330 bp region from the 16S rRNA gene was amplified by Lep F and Lep R primer. The LipL32 gene (accession number: NC 005823) was aligned using the ClustalW tool, and primers were chosen using the Primer3 programme, to create the Lau01 and Lau02 primer combination. The LipL32 gene's 660 bp sequence is amplified by the Lau01 and Lau02 primer combination. The Primer-Blast programme was used to bioinformatically confirm the specificity of the primers.

Figure 10. Primers for the mPCR amplification of Leptospira spp.

2.3.3 Genome correlated to leptospira

Leptospira interrogans is a bacterium that causes leptospirosis. The bacteria reside in the kidneys of numerous animals and are transported by them. Via their urine, it enters the land and groundwater. The Leptospira genome is made up of two circular chromosomes, chromosome I and chromosome II, with a combined length of 4,627,366 base pairs (bp). Similar to other bacterial genomes, the location of the big replicon's origin of replication was found to be between the dnaA and dnaN genes. The location of replication for the large replicon was validated by GC nucleotide skew (G C/G + C) analysis, which also identified two potential locations for the tiny replicon. 

        Classification based on serology; before 1989, the genus Leptospira was divided into 2 species: L. biflexa, which contained saprophytic strains obtained from the ecosystem, and L. interrogans, which included all pathogenic strains. By growing at 13°C and in the presence of 8-azaguanine (225 g/ml), L. biflexa was distinguished from L. interrogans, as well as by its difficulty forming spherical cells in 1 M NaCl (Levett P. N., 2001). 

Figure 11: Genome features of L. interrogans serovar by SciELO