2.0 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