Acute febrile illness (undiffenerentiated fever, AFI) is common disease and problem in worldwide including Thailand. Symptoms in early phase of AFI are non-specific resulting in mistakenly diagnosing it in acute febrile illness group. Laboratory diagnosis is important to confirm the definite clinical diagnosis. A rapid and accurate diagnostic test is essential tests for Point-of-care diagnosis. The objective was to generate the template products for diagnosis of scrub typhus and leptospirosis based on the principle of immunochromatrogaphy with gold nanoparticles (strip test). In preparation of recombinant proteins, the specific primers of entire genes were designed. PCR product from extracted DNA was cloned into plasmid and then selected positive clones were performed by PCR and restriction fragment analysis. Recombinant plasmids were transformed into expression cells. The characterized and purified recombinant proteins for scrub typhus and leptospirosis were 56 kDa proteins from Orientia tsutsugamushi prototype strains of Karp, Kato and Gilliam and 25 kDa protein of (Electron transfer flavoprotein subunit beta, ETF_beta), respectively. In the development of scrub typhus test kit, the nucleotide sequences of the recombinant proteins of all three strains showed the identity percentage of 97.8, 97.7 and 97.4 respectively. IgM/IgG immunoreactivity indicated that the recombinant proteins could be used as excellent antigens for antibody detection against all three strains and others two strains of TA716 and TA763.
Evaluation of scrub typhus test kit IgM (Scrub_M) and IgG (Scrub_G) by using immunofluorescence assay (IFA) as a reference test. The titer >1:400 of IFA_M/G was considered to be positive. Scrub_M/G were tested in 318 sera from 100 scrub typhus patients and 218 non-scrub typhus patients (53 leptospirosis, 96 dengue, 30 murine typhus and 39 unknown). Scrub_M/G yielded the values of sensitivity, specificity; positive predictive value and negaitive predictive value were 97.0%, 100%, 100% and 98.6%, respectively. Scrub_M showed the values of sensitivity, specificity, positive predictive value (PPV) and negaitive predictive value (PNV) were 81%, 100%, 100 and 92.0, respectively. Scrub_G showed the values of sensitivity, specificity, positive predictive value (PPV) and negaitive predictive value (PNV) were 93.0%, 100%, 100 and 96.9, respectively. Evaluation of leptospirosis test kit IgM (LepProt_M) and IgG (LepProt_G) with recombinant ETF protein as antigen (version 1), was tested in 60 sera by using immunofluorescence assay (IFA) as a reference test. Correlation of (LepProt_M) and LepLPS_M with LPS as antigen was observed. The titer >1:400 of IFA_M/G was considered to be positive. LepProt_M and LepLPS_M gave the same negative results of 43.5 in sera with IFA_M (titer <1:50). In the group of positive results for IFA_M, LepProt_M gave the negative results of 44.4 (4/9 samples) while LepLPS_M yielded 100% of positive results. LepProt_M and LepProt_G which were further developed to be version 2 as compared with commercial kit SD BIOLINE Leptospira IgM/IgG (SD Lep_M/G) which demonstrating sensitivity of 96.2% and specificity of 95.7%, was evaluated The results showed that leptospiral isolation as reference standard test could not be used as a tool of evaluation. However, LepProt_M gave the higher rate than SD Lep_M in antibody detection of early phase of illness. Using microscopic agglutination test (MAT) as reference test, the sera in the positive group (titer > 1:400) showed that LepProt_M/G yieled the the values of sensitivity, PPV and PNV were higher than SD_M/G, except specificity of SD_M was higher than LepProt_M (100% vs 91.7%). In conclusion, two template products of rapid diagnostic tests were Scrub_M and Scrub_G (for Scrub typhus) and LepProt_M and IgG LepProt_G (for leptospirosis) with high sensitivity, specificity; positive predictive value and negaitive predictive value.