Strategies

2.1 Identification of the disease

    After the formation of leptospira-specific antibodies, initially primarily of the IgM class, leptospira are present in the blood until they are eliminated after 4–7 days. IgM antibodies can persist for many months, which raises the issue of whether a positive IgM result truly detects a present infection. This is a drawback of IgM detection assays. There are five techniques used to identify Leptospira disease which are rapid leptocheck test, IgM ELISA test, microscopic agglutination test (MAT), real-time PCR assay, and blood culture. 

2.1.1 Rapid leptocheck tests 

    Following the idea of immunochromatography, a special membrane-based two-site immunoassay. The anti-human IgM colloidal gold conjugate combines with the IgM antibodies in the sample set as it passes through to the membrane of the testing machine. This compound is bound by the leptospira genus particular antigen covered on the membrane when it advances in the cassette to the test window "T," which causes the creation of a red to a deep purple coloured band at the test region. A positive test result is confirmed if the band is present in the "T" region. Negative results are indicated if there is no band at the test location. The anti-rabbit antibodies are coated at the "C" region. If there is any insoluble unbound compound in the sample, it will travel further along the membranes and become blocked at the "C" window, where it will form a red to a deep purple band. If no control band forms at this location, the test may not be legitimate.

Figure 3: Leptocheck WB Leptospirosis LFT by Selinion

2.1.2 IgM ELISA test

    According to the manufacturer's instructions, IgM ELISA was used to analyse all samples. This study's objective was to conduct a thorough literature review and meta-analysis in order to confirm the precision of the IgM ELISA for leptospirosis diagnosis (Silva et. al.) The lab provided duplicate sets of positive control, a negative control, and a cut-off calibrator on hand for each test run. Serion units of <40 were viewed as a negative result and ≥40 was regarded as a positive result in the calculation for Serion ELISA classic leptospira IgM.

Figure 4: IgM ELISA Test kit by Athenese Dx

2.1.3  Microscopic Agglutination Test (MAT)

    Due to its unrivalled diagnostic sensitivity, the microscopic agglutination test (MAT) is the gold standard for leptospirosis sero-diagnosis. In order to reflect the prevailing serovars from the region in which the patient contracted the infection, panels of live leptospires, ideally recent isolates, are used (Goris et. al., 2014) . Each test run of the MAT test uses an antigen from a different serogroup. These strains were acquired from the facility centre. A situation of leptospirosis was regarded to be made official if any serum demonstrated seroconversion, a fourfold increase in antibody titre in the MAT test between acute and convalescent-phase serum, or was positive by IgM ELISA.

Figure 5: Leptospirosis serodiagnosis by Current Protocols in Microbiology 

2.1.4 Real-time PCR assay

    The Taqman Universal PCR Master Mix was supplemented with a DNA sample. To check for the presence of contaminating DNA, a negative control was utilised in the reaction medium that didn't have any additional template. An ABI Prism 7300 sequence detector was used for the 50-cycle programme of proliferation and fluorescence imaging, with every cycle consisting of primer annealing, initial denaturation, and extension. The reporter 1 sequence was TCCGGCGCTTGTCCTG with reporter 1 dye FAM and reporter 1 quencher NFQ. The oligonucleotide primers being used are 5'-GGATCTGTGATCAACTATTAC-3' and reverse primer 3'-CGAACTCCCATTTCAGCGATTAC-5'.

Figure 6: Culture identification and PCR of pathogenic Leptospira spp. by American Society for Microbiology Journal

2.1.5 Blood culture

    Upon patient admission, whole blood samples were drawn and grown in an aerobic environment using EMJH liquid medium supplemented with enrichment medium and 5-fluorouracil. From the second week on, a drop out of each culture media was examined weekly by dark field microscopy and kept for 4 months before being discarded as negative.

Figure 7: Leptospira isolation on EMJH agar plates by ResearchGate

Figure 8: Advantages and disadvantages of diagnostic tests for Leptospirosis detection

 2.2 Possible ways to cure

    Antibiotics, such as doxycycline or penicillin, are used to cure leptospirosis and therefore should be administered early on in the course of the illness. antibiotics intravenously for those with serious conditions. Anyone who exhibits leptospirosis-like symptoms needs to see a doctor. Situations when medical advice is needed, are when there is direct or indirect contact with one's pet's urine, blood, or tissues while it is ill, helping to deliver newborns from an infectious agent and common symptoms, such as fever, muscle aches, and headaches, appear within three weeks of a high level of exposure. The interval between initial infection and the progression of the disease is typically 5 to 14 days but can be as short as a day. It is simple to contract leptospirosis from an infected animal and its surroundings. By vaccinating your pets, managing rodent populations, maintaining well hygienic practices, wearing protective gear, and getting care as soon as you feel sick to break the cycle of illness, a strong animal immunisation programme is essential, and this includes recognising modern farming's potential risks.

2.2.1 Test kit design

    The OnSite Leptospira IgG/IgM Combo Rapid Test is a lateral flow chromatographic immunoassay that may be carried out by workers with minimal training in 15-20 minutes without any need for laboratory apparatus. IgG and IgM antibodies to Leptospira interrogans (L. interrogans) in human serum, plasma, or whole blood are simultaneously detected and distinguished with this test. It is intended for use as a screening test by healthcare professionals and offers a preliminary test result to help with the diagnosis of L. interrogans infection. Any assessment or application of this test result must also take into account further clinical findings and the experts' clinical decision-making.

2.2.2 Leptospirosis test kit features: 

Figure 9. Leptospira IgG/IgM Combo Rapid Test by CTK Biotech

1. Accuracy: 100% specificity and 100% sensitivity in comparison with a reference rapid test on the market

2. Cross Reactivity: No false-positive results were observed on specimens from TP, HIV, Dengue, TB, Typhoid Ab, HBsAg, HCV Ab, HEV Ab, H.P Ab, HAV, HCG, RF, ANA, HAMA

3. Interference: No interference were seen with substances

4. Salicylic acid: 4.34 mmol/L; Glucose: 55 mmol/L

5. Sodium citrate: 3.8 %; Heparin: 3,000 U/L

6. Creatinine:  442 μmol/L 

7. Bilirubin:  20 mg/dL

8. EDTA: 3.4 μmol/L

9. Haemoglobin: 2g/L

10. Human IgG: 1000 mg/dL

11. AlbuminL 60 g/L

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