Clinical Study On Detection Of Fungi In Paraffin - Embedded Lung Tissues By Molecular Biology

In the worldwide, invasive fungal infections (IFIs), such as aspergillus infection, has been one of the main causes of death and hospitalization in patients with immunodeficiency. Although the development and application of new anti-fungal drugs, and the understanding of antifungal drugs pharmacokinetics/pharmacokinetics, but in patients with IFIs, the morbidity and mortality rates have been always high, bringing heavy economic burden to individuals and society.In clinical, rapidly and accurate diagnosis of fungal infection and identification the fungal species can give timely appropriate anti-fungal treatment, reducing the length of hospital stay, the cost of treatment and mortality.At present, the main method of diagnosis IFIs include pathological examination, imaging examination, fungal culture and fungal-specific antigen detection. Histopathological examination need the doctor who has rich clinical experience, but may not differentiate some uncommon fungus. The positive culture result from sterile sites (blood, lung tissue and cerebrospinal fluid) is the gold standard, but the positive rate is not high, at the same time, culture costs more time. The imaging examination does not have high specificity. Detection of fungal antigens have high sensitivity and specificity, but existing a little rate of false positive and false negative. The method of new molecular biology was born at 1980s, which used in the clinical diagnosis rapidly.Polymerase chain reaction (PCR) after it born at 1985 has always being used in clinical diagnosis, including diagnosis of bacteria, viruses, fungi, tuberculosis and flus. There are a large number of literatures showing that the PCR method in the diagnosis of IFIs is better than the traditional methods. The PCR method can amplify the conserved region of ribosomal genes to identify the fungi, and amplify the easily-mutation region to differentiate fungal species.The dideoxy chain termination methodsequencing (Sanger sequencing), which can analysis DNA sequence’homology and mutation, can identify fungi or bacterium. Sequencing the ribosome of fungi, comparing the sequence in Genbank, can identify the fungi species and find new mutation sites.Aspergillus, Candida, Cryptococcus and Mucor are the main clinical pathogenic fungus, at the same time there are some rare fungus, such as Alternaria alternate. In pulmonary fungal infections, Aspergillus and Cryptococcous infections are the main cause of lung resection.This research mainly arms at diagnosis and identification of fungus in paraffin-embedded lung tissues by PCR and Sanger sequencing.Part I Establishment and evaluation of molecular biology methods for detection of fungiObjective:Though detecting 12 spaces of fungus by PCR and Sanger secquencing, verifying the specificity of the primers and sensitivity and specificity of PCR and Sanger sequencing. Methods:The experimental group which include 12 standard strains and the control group which include 6 non-invasive fungal infection lung tissue which embedded in paraffin are detected by PCR and Sanger sequencing.Results:All of the 12 standard strains were amplified positive by fungi-specific primers, the results of amplified by Aspergillus-specific primers and Cryptococcus-specific primers shows that 4 spaces of Aspergillus fungus are amplifying positive by Aspergillus-specific primers, the rest are negative; 3 space of Cryptococcus are amplifying positive by Cryptococcus specific primers, the rest are negative. PCR method is 100% sensitive and 100% specific.6 cases of non-fungal infection are all negative. Blasting the 12 species fungus’secquence after secquencing shows homology with standard strains. Conclusion:This study confirmed that the primers are highly specific and the PCR method has high sensitivity and specificity, Sanger sequencing can also distinguish the fungus accurately.Part II The molecular biology methods for detect clinical fungus in paraffin-embedded lung tissueObjective:Using the PCR method and Sanger sequencing detecting fungus in the clinical samples. Methods:From 2000 July to 2014 October,40 samples are confirmed fungal infection and 6 samples are confirmed non-fungal infections which collected in Nanjing General Hospital, the method of PCR and Sanger sequencing are used to detect the samples. Results:In the detection of lung tissues, all the 40 samples are amplifying positive by pan-fungus primers,22 samples were amplifying positive by Aspergillus-specific primers,18 specimens are amplifying positive by Cryptococcus-specific primers, there is no cross positive results existing in the 40 samples; 6 non-fungal infection specimens were all amplified negative; The sensitivity and specificity of PCR is both 100%, PCR (100% vs 67.5%, P<0.05) can better distinguish fungi’spaces than microscopy, Sanger sequencing and microscopy have no obvious difference (87.5% vs 67.5%, P>0.05), but Sanger sequencing can distinguish fungus between spaces. The consistency of PCR detection and Sanger sequencing is an excellent response, show that the result of PCR detection and Sanger sequencing are reliable. Conclusion:For the diagnosis of fungal (Aspergillus and Cryptococcus) in lung tissue, PCR and Sanger sequencing are highly sensitive and specific methods.