Initial Study On Laboratory Diagnostic Methods For Histoplasmosis

ObjectiveHistoplasmosis—a kind of strongly infective,transmissible and systemic mycoses, used to be rarely discovered in China, has increased in recent years, and there are no systematic methods for its laboratory diagnosis and standard. In this research, purified protein derivative of histoplasmin (P-HTPM) was successfully extracted from HC and studied the efection on skin test in mice immunized with Histoplasma(HC).In addition,we plan to develop systematic methods of immunologic and molecular biologic laboratory diagnosis for HP and provide evidence for clinical research in the future.Methods1. Biologic characteristics of HC were observed by culture and morphologies, and were identified by biochemical and antigen property.P-HTPM was abstracted and purified from culture medium through dilute acid and salting out method and prepared for experiment use. Skin reaction of P-HTPM were primary detected through guinea pig.2. To explore efficacy and sensitivity of skin reaction for guinea pig we use P-HTPM as delayed cutaneous reaction antigen and HIMN as a control. To explore specific of P-HTPM as a in vivo diagnostic product, TB-PPD and PPD-B were used as control to detect cross reaction.3. HC antibody were detected via serology test by P-HTPM antigen. The sensibility and efficacy of ELISA and Gold immunochromatography assay(GICA) to detect antibody were studied by exploring property test condition. A lot of kinds of immune serum with Monilia, Aspergillus,dimorphic fungi and Mycobacterium were used as control to test the specific of P-HTPM as antigen.4. Primer was designed based on the coding gene of M protein—a specific antigen of HC, and cDNA was obtained from the culture of HC,then detected the specificity of amplification, establish Real-Time Fluorescent Quantitive PCR. Grope and optimize the condition for PCR—concentration of Mg+ and primer, anneal temperature and additives of balance solution. Determine the sensitivity of amplification through gradient dilution of template. Templates of common fungi, other dimorphic fungi and Mycobacterium were also amplified, and the specificity were detected by electrophoretic analysis.Results1. protein extraction and preparationWe extracted the protein successfully through this method and the yield of protein was 18.30±3.27mg/L.The purity of protein reached 85%.P-HTPM could elicit specific skin reactions in guinea pigs immunized with HC.2. skin reactions①The ratio at 1μg/ml,2μg/ml,4μg/ml,8μg/ml P-HTPM and international standard substance elicited skin reactions was 0.9,1.0,1.1,and 1.2, so 2μg/ml P-HTPM induced the same skin reactions as international standard substance.②P-HTPM could elicit positive skin reactions in guinea pigs immunized with HC, and the average induration diameter was 11.2±1.0 mm.But P-HTPM failed to elicit postive skin reactions in guinea pigs immunized with BCG,M bovis,M avium,M xenopi,M terrae,M gastri,M nonchromogenicum,M shimoidei,M triviale,M farcinogenes,M kansasii,M marinum,M asiaticum,M scrofulaceum,M gordonae,M szulgai,M chclonae,M fortuitum,M smegmatis,M hubuense,M porcinum and M vacce,and the average induretion diameter was 0 mm.③TB-PPD could elicit positive skin reactions in guinea pigs immunized with Macobacterium, and the average induretion diameter was 12.9±1.9 mm.But TB-PPD failed to elicit postive skin reactions in guinea pigs immunized with HC.PPD-B could also elicit specific skin reactions in guinea pigs immunized with BCG,M bovis,M avium,M xenopi,M terrae,M gastri,M nonchromogenicum,M shimoidei,M triviale,M farcinogenes,M kansasii,M marinum,M asiaticum,M scrofulaceum,M gordonae,M szulgai,M chclonae,M fortuitum,M smegmatis,M hubuense,M porcinum and M vacce,but failed to elicit postive skin reactions in guinea pigs immunized with HC.3. ELISA①Anti-HC serum of low titer and high titer could be detected using the plate coated with P-HTPM in concentration of 0.5μg/well,1μg/well,2μg/well,4μg/well, and 1μg/well was set optimum working concentration for its maximum A value. Serum of high titer could be detected when diluted at 1:100,1:1000,1:10000, but the A value of low titer serum was low(minimum 0.187) when diluted at 1:10000, dilution of 1:1000 witnessed an A value of 0.396, and the optimum working concentration of Anti-HC serum was 1:1000.②Positive and negative results were separately seen after 20 samples of Anti-HC sera and anti-MTB sera were detected using ELISA in which P-HTPM was coating antigen, and the A value was 1.036±0.24 and 0.052±0.004; and there was a statistical significance between them with P<0.01. Negative results were seen when sera samples of other Mycobacterium, Candida, Aspergillus and Cryptococcus were detected(P<0.01) .8 samples of 10 were positive when sera of Paracoccidioides and Penicillium marneffi were detected; and all 10 samples were positive with A value of 1.103±0.478 when sera of Blastomyces dermatitidis dermatitidis were detected, no statistical significance(P<0.05)was seen when compared with positive control.③Positive results with A value of 1.614±0.272 were seen when ELISA was performed to detect anti-MTB sera using TB-PPD and PPD-B as coating antigen; sera of other Mycobacterium were positive (100%)and sera of HC were negative (positive ratio 0%).Positive ratio were compared , P<0.01.④ELISA using Coccidioides immitis and Blastomyces dermatitidis dermatitidis as coating antigen was performed to detect HC sera respectively, and A values were 0.135±0.053 and 0.229±0.035 with positive ratio of 40%(4/10) and 70%(7/10); when detect sera of their own, A values were 1.382±0.151 and 1.604±0.087 with positive ratio of 100%. Positive ratio were compared , P>0.05.4. Gold immunochromatographic assay(GICA)①At the optimum concentration of antigen of 2mg/ml, coloration is good and the strongly positive and weakly positive results could be discriminated.②Of the different BSA concentration for blocking antigen—0.5mg/ml,1mg/ml,2mg/ml,5mg/ml,10mg/ml, 5mg/ml functioned the best when all 10 negative samples displayed negative results, and 9 positive samples were not influenced except that the 2# sample was detected positive at antigen concentration of 0.5mg/ml,1mg/ml,2mg/ml.③The detection efficiency could be improved when serum was diluted with treating solution before detection, and the best dilution was 4:5, which witnessed fast affluxion , obvious coloration of C-line and T-line, clear borderline, clean background and exact determination of results.④All of 9 samples of rabbit anti-HC sera and 14 samples of mouse anti-HC sera were positive using this method and positive rate was 100%. T-line displayed colorations in different depth, and samples of higher titer produced deeper color, which was consistent with ELISA, and the coincidence was 100%. Strongly positive samples and weakly positive samples were still able to be detected when diluted at 1:4096 and 1:512 separately, which indicated high sensitivity of this GICA.⑤Using GICA to detect 16 mice samples of anti-MTB serum, 1 positive result; detect sera of Monilia and Aspergillus, no positive result; detect sera of dimorphic fungus of which Blastomyces dermatitidis dermatitidis and Coccidioides immitis displayed high positive rates—86%(6/7) and 40%(4/10) individually, and that was consistent with ELISA; detect 7 samples of anti-Penicillium marneffi sera, and the negative result was not consistent with ELISA.5.PCRThe PCR conditions that were optimized were listed: When low level of the templates were detected, the sensitivity of the detection was improved after adding suitable Formamide to the mixtures,Ct was 30.43±1.2.When the concentration of Mg2+ was higher than 5mmol/L, we obtained a better result. The templates of high concentration were amplified, Ct was 21.72±0.33,in a contrary, Ct was 29.96±2.4. Increasing the volume of Taq DNA polymerase favored the PCR amplification. The Taq DNA polymerase from Cytech was prior to two other corporations.When the primer concentration was low, the effect of the PCR amplification was limited and the intensity of fluorescence were weak. Annealing at 68℃,70℃,72℃, no significant effect were found. All production of PCR were negtive which templates of Mycobacterium and other fungi were detected with primer D1D2 by common PCR Technique .Conclusions1.The technology is effective and feasible which was used to extact P-HTPM from the culture of Histoplasma in our study.2.The skin test with P-HTPM is a useful adjunct dignostic method for Histpolasmasis and will be applied in epidemiology investigation on HC infection.There is a value of differential diagnosis between Histoplasmasis and tuberculosis combined with P-HTPM skin test and TB-PPD skin test.3.ELISA and GICA using P-HTPM to detect serum antibody are usful serological methods for diagnosis of Histoplasmasis.4.FQ-PCR using HC specific primer D1D2 is a good,rapid and precise method of diagnosis of Histoplasmasis.