Significance Of (1, 3)-β-D-glucan Detection And Nest PCR Method In The Auxiliary Diagnosis For Disseminated Trichosporonosis

Background and ObjectivesSince 2000 the first case of disseminated trichosporosis has been reported,the numerous disseminated mycoses,including disseminated trichosporosis,are attracting more and more attention,the disseminated trichosporosis is a disseminated mycoses which involve the skin,liver,spleen,lung and kidney and so on.The main pathogenic bacterium is T.asahii.The patient mainly die on the complicated condition,multi-organ infection,diagnosis unknown and imprompt treatment.Owning to have indefinite clinical situation,its diagnose mainly depend on the uxiliary examination.Traditional uxiliary examination including the fungus culture,identity and patho-examination need long time to complete.That condition not only impact the early diagnosis and treatment but also result in the misdiagnosis and mistreatmentRecently,many diagnostic technique such as detection of fungus composition and determination of metabolic product and specific DNA fragment amplification are drawing people's eyes.Fungus consist of cell wall,barrier diaphragm,cytolemma and cell organ. Their composition and metabolism production also changes in both content and activation along with the creation and propagating of fungus.(1,3)-β-D-glucan is an important composition of fungus cell wall.It is released into blood only when fungus being deeply infected and phagocytize by phagocytic cell.So it is vital to the identification of disseminated mycoses,the molecular biologcal method owned higher sensitivity and specificity are are attracting more attention.The common method consist of the hybridization of nucleic acida and nest PCR.This study is based on a disseminated trichosporosis(Trichosporon asahii,T.asahii) infection model.The contents of(1,3)-β-D-glucan in bronchoalveolar lavage fluid(BALF), urine and plasma are comparatively observed,then apply the nest PCR method to detect theT.asahii DNA fragment in three species humor samples,with the aim of finding new diagnose approach from body fluids other than blood.At the same time,further evaluation of the impaction of(1,3)-β-D-glucan and nest PCR in the early diagnosis and prognosis of disseminated T.asahii trichosporosis is also attempted in order to find more evidences for the research of the pathology and therapy prospect of T.asahii trichosporosis.Methods and results1.Seting up the T.asahii animal infection model.45 male rats will be randomly grouped into sample A,sample B,and comparison sample C,each sample consists of 15 rats. On the 0day,sample A and sample B was mainlined with 0.8ml fungus suspension,while sample C with same dose of physiological saline.Sample A was executed on day 10,while sample B and C on day 20.the BALF,urine and plasma of those sample rats was collected respectively and was cultured,meanwhile the(1,3)-β-D-glucan was tested under G method, the specific T.asahii DNA fragment amplification was tested by nest PCR.Eatch animal model's hepatic tissue and lung and nephridial tissue was collected.Some of them were disposed by 10%formalin fixation to imbed and chip and stained preparation,Others were cultured.2.Consequence of fungus eultuer.The positive reat of issue culture is 2%,the plasma culture is 3.33%,the culture of BALF and urine all are negative.3.Consequence of(1,3)-β-D-gluean test.The G test positive reat in the samples of BALF,urine and plasma are 76.67%and 80.00%and 10%.Compareing the G test consepuence of plasma and BALF with the control group result,they have significant deviation(P＜0.001).But the urine test result has not statistically significant.The rat model which had more postive rate on tissue culture own more(1,3)-β-D-glucan density than other samples in plasma.On the other words the(1,3)-β-D-glucan density and the infected number of animal viscera are positively correlated.There exists linear relationship between.The correlation is high(|r|＞0.7,P≤0.05).On the 10days,the(1,3)-β-D-glucan density of three species samples reached the peak value,plasma 54.565pg/ml,BALF 24.508 pg/ml and the urine 13.117pg/ml.The density and the infected time are direct ratio.But after 20days,(1,3)-β-D-glucan density descend to 27.077 pg/ml on plasma samples,21.339 pg/ml on BALF samples and 11.748pg/ml on urine samples.At this time,the(1,3)-β-D-glucan density and the infected time are inverse ratio.4.Consequence of nest PCR test.The average sensitivity of those 30 sample rats are: 66.67%on plasma,43.33%on BALF,and 20.00%on urine.Compareing the nest PCR test consepuence of plasma and BALF with the control group result,they have significant deviation(P＜0.001),and urine result have unobviously difference(0.001＜P＜0.05).The postive rate of nest PCR test on plasma and BALF are significantly different between hemoculture and BALF culture under X~2 test of paired comparison of enumeration data (P＜0.005).5.compareing the consequence of G test and nest PCT and fungus culture.G test result,nest PCR and fungus culture was compared with the infected animal model plasma. The positive percent of those 30 sample rats are:76.67%under G test,66.67%under nest PCR,and 3.33%under fungus culture.Among of them,the G test and hemoculture results are significantly different under chi-square test(X=21.04,P=0.0000and b＞c).At the same time the nest PCR and hemoculture results also are different under chi-square test(X~1=17.05, P~1=0.0005 and b＞c).There is no statistical difference between G test positive percentage and that of nest PCR(P＞0.05),although the first is higher than the latter.The BALF culture all are negative.The nest PCR average postive rate on BALF is 43.33%and the G test is 80.00%. Both of nest PCR and G test results on BALF have significant deviation with BALF cultrue (P＜0.005).Meanwhile there are statistical difference between tow test(X~2=9.60, P2=0.0019).Conclusions1.The observation of the sensitivity and specificness of early G test shows high sensitivity for three group samples.All comparison sample shows negative G test result, which means this method has low fake-positive rate and high specificness.Compared with plasma culture,(1,3)-β-D-glucan test has higher positive percentage and fewer volume of blood(0.5-1 ml),so it is more acceptable to patients.This test observes the(1,3)-β-D-glucan in BALF and urine for the first time.Results show that BALF G test has sensitivity of 80.00%, higher than other sample.The grounds maybe,first,lung is susceptible to T.asahii.second,the large amount of phagocytic cell in lung may release more(1,3)-β-D-glucan after swallow the pathogenic bacterium.The G test of urine has a low sensitivity of 10%,which also exists in those rats with more infected viscera.So the appearance of(1,3)-β-D-glucan in urine may suggest more serious and wide infection.The nest PCR also have good performance in detection of plasma and BALF.Those nest PCR consequence in A and B groups display that the nest PCR test have higher sensitivity and specificity under detecting the disseminated trichosporosis.Especialy the plasma detection with nest PCR test have more clinical application value.2.Although there is no statistical significant difference between the sensitivity of G test and nest PCR test with plasma sample,but we still think the further has its advantage:the rats has bacteremia soon after injecting the fungus suspension,when most sample show positive result under nest PCR test.In a period,most fungus was phagocytized by leukocyte in blood,and the remaining fungus was taken to each viscera and reproduced there.Then it may be phagocytized by phagocytic cell in organs and release large amount of(1,3)-β-D-glucan into blood.This process produces a higher positive percentage for G test than nest PCR test,which is the very advantage of G test.3.The density of(1,3)-β-D-glucan has positive correlation of the number of infected viscera.The higher the density,the more viscera was infected.Histotomy shows that the higher the density of(1,3)-β-D-glucan in a rat,the more microabscess and histoclasia will be seen on histopathologic slide.One notable thing is that,although the tissue culture has low positive percentage,the positive proportion for kidney culture is high.This is consistent to overseas reports about the lowest eliminate rate of T.asahii trichosporosis in kidney.So the search of kidney should be valued in the future.4.(1,3)-β-D-glucan density is changing in different stage of infection.On the early stage of inoculation,the fungus propagate vastly under immune suppression.The release of(1, 3)-β-D-glucan will rise greatly along with it.Ten days later,the immunologic mechanism of body recovers gradually,the phagocytosis ability of phagocytic cell to pathogenic bacterium will restore.The number of fungus in blood will fall noticeably.But the release of glucan from infected viscera compensates the reduction of(1,3)-β-D-glucan release from blood itself to some extent,the destiny of(1,3)-β-D-glucan will show a moderate down slope. Observation shows remarkable recover on meat and drink,activation and reaction ability of rats 2 weeks after the inoculation.This is coincident with the(1,3)-β-D-glucan density variation.So dynamic observation of(1,3)-β-D-glucan helps to evaluate the infection degree.