The Detection And Molecular Identification Of Fungi In Respiratory Tract From Patients With Pulmonary Diseases

With the development of the medical technique, organ transplantation, invasive examination, unceasingly extended new treatment technique and the wide use of the broad-spectrum antibiotics, glucocorticoid plus immunodepressant in clinical treatment in recent years, the disease incidence of the opportunistic infection by fungi has tended to have a dramatic ascent. As the major deep pathomycetes, aspergilli and monilia have been concerned increasingly by more researchers in the world.The spores of aspergilli are so tiny that they can float in the air as well as implant into the nasal cavity of the normal individual. Aspergilli, as a kind of opportunistic pathogens, often invade those who have suffered from some basic diseases especially in pulmonary diseases and cause illness mainly through respiratory tract. Unfortunately, the exact source of pulmonary aspergillosis is unclear till now. The conclusion related to the aspergilli spores which invaded into lung whether from surroundings or from the spores imbedded in nasal cavity has not been seen yet as far as the domestic and foreign research is concerned. It may be prone to the surrounding environments. Through detecting the respiratory fungi of the pulmonary disease patients, analyzing the genotype and making the molecular identification by random amplified polymorphic DNA (RAPD), we anticipate exploring the source of aspergilli spores infection in the internal lung tissue, which may present some rational experimental basis on the clinical diagnosis, prevention and epidemiologic survey of the pulmonary aspergillosis.Through detecting 925 cases of pulmonary diseases'patients and 100 healthy individuals, the implantation and distribution of respiratory aspergilli were observed in our research. It has been found that A. flavus, A. niger and A. fumigatus were the chief aspergilli in the respiratory tract, besides a small quantity of A. clavatus, A. versicolor and some unidentified aspergilli were also found. The total detection rates of the former three aspergilli were respectively 53.8%, 41.5%, 23.1% in the pulmonary diseases'patients and 23.0%, 14.0%, 1.0% in the healthy individuals. There was a high rate of aspergilli implanting in the nasal cavity, in which the rate was about 68.8% in pulmonary diseases'patients and 38.0% in healthy individuals. The aspergilli existed in both normal individual and pulmonary disease patients. However, the detection rate of aspergilli in the nasal cavity of the latter is much higher than the former (P<0.01 ). It can be seen that the resistence to aspergilli implanting of respiratory system may be obviously weakened after the influences of many etiological factors.In the respiratory tract of 925 cases of pulmonary diseases'patients, 399 strains of yeasts were detected. The total detection rate of C.albicans was 18.9%, and the others'was 24.2%. In the respiratory tract of 100 healthy individuals, 9 strains of yeasts were detected. The total detection rate of C.albicans was 4.0%, and the others'was 5.0%. The detection rate of C. albicans was high in the pharyngeal portion within which the rate was respectively 30.4% and 9.0%. Moreover, in 54 strains of yeasts which were identified by API 20C AUX system, the proportion of C.albicans was the highest, about 32.0%. The detection rate of C. albicans in the pharyngeal portion was so high that it summed up to 70.6% of all the C.albicans. It demonstrates that the distribution of monilia should probably be different from aspergilli in the respiratory tract. The former mainly exist in the pharyngeal portion and may descend to the internal pulmonary tissue to cause endogenous monilia infection.The detection rate of aspergilli in the nasal cavity and BALF was respectively 77.0%, 14.5% in the old patients of pulmonary diseases and 64.8%, 9.6% in the young. The results showed that the detection rate of aspergilli in the nasal cavity and BALF of the old pulmonics is significantly higher than that in the young(P<0.05). Comparing with the young, the respiratory tract of the old pulmonic patients is more suitable for aspergilli to exist and grow. It suggests that various kinds of factors correlated with aging, such as the aging of the pulmonary function, the physiological lack of resistance, the obviously decline of the organs'function, and so on, offer a more advantageous condition for aspergilli to implant in the respiratory tract.Furthermore, the detection rate of aspergilli in BALF was 15.0% in the 386 cases of lung cancer patients and 8.3% in the 409 cases of pulmonary tuberculosis (TB), bacterial infection. The results showed that the detection rate of aspergilli in BALF of the pulmonary malignant tumor patients was remarkably higher than the patients suffered from TB, bacterial infection or other diseases of respiratory system(P<0.01). It suggests that the pulmonary malignant tumor may destroy the physiological barrier of the respiratory tract, weaken the resistence and lead to immune deficiency to some extent. All of the above make the aspergilli implant in the respiratory tract more easily and augment the danger of the opportunistic infection.In 925 cases of pulmoary diseases'patients, the relativity that the detection rate of aspergilli in BALF was associated with some factors including the age, gender, course of disease, leucocyte count, the use of antibiotics, immunodepressants, chemotherapy over 2 weeks and complicated with some systemic diseases etc, was studied by means of the logistic regression analysis. The results manifested that in all of the observed targets, the Exp(B) was respectively 11.906, 14.860 (P<0.01) in the factors of using antibiotics, immunodepressants, chemotherapy more than 2 weeks and complicated with hematological diseases, and the Exp(B) was 4.093 (P<0.05) in the diseases of endocrine system. It indicates that the predisposing factors which bring a significant impact on aspergilli implanting in the respiratory tract mainly include three aspects. First of all is the inappropriately long-term (over 2 weeks) application of some certain drugs, such as broad-spectrum antibiotics, glucocorticoid, immunodepressants and chemotherapy drugs, and so on. In addition, complicated with some systemic diseases which came from hematological system and endocrine system are the other two chief factors.The random primer R108 was selected to analyze the genotype of the same kind of aspergilli which were isolated from the patients'different parts of the respiratory tract by using the RAPD technic in our experiment. The results showed that in the RAPD electrophoresis maps of A. fumigatus, A. flavus and A. niger, the number of amplification bands ranged from 1 to 11, in which the smallest fragment is about 150bp and the largest more than 2kb. Among the different strains, the quantity and the size of the amplification bands were all in differences. Even though the strains exactly had the same bands, the difference of brightness also existed in some fragments. Subsequently, the similarity of intraspecies that isolated from the aspergilli implanting in the different parts of the patients'respiratory tract was compared. The results indicated that although the same kind of aspergilli existing in the different parts of the patients'respiratory tract had the same morphological features, they were differences in characteristics of the genotype. It suggested that the aspergilli implanting in the internal pulmonary tissue through respiration differed from the same kind of aspergilli existing in the nasal cavity of the patients. The above demonstrated that the infection source of pulmonary aspergilli should probably originate from the patients'surrounding environments. So effectively controlling the aspergilli pollution of the surroundings is the best means to prevent pulmonary aspergillosis.During the period of collecting samples, we discovered a rare case of disseminated Penicillium marneffei disease by oropharynx swab in a patient without human immunodeficiency virus. Based on clinical manifestations and laboratory examinations, comprehensive analyses were carried out. Finally, the patient was treated with amphotericin B and itraconazole, and both of them had a definitive therapeutic effect. The reports had not been seen that the cases of disseminated Penicillium marneffei infection were discovered by oropharynx swab. It could be presumed that the spores of PM could flow into the pulmonary tissue through oropharynx from the environment, and then disseminated throughout the body. Meanwhile, it further confirmed that the infection of PM could exist in the normal individual, and the infection might be relevant with some factors, just like the patients'professions and staying in the epidemic areas of PM. So, as far as those patients who suffered from an unknown origin fever, diffused pulmonary inflammatory infiltration and systemic lymphadenectasis are concerned, the relative examinations of mycology should be emphasized especially under the circumstance of the failure in broad-spectrum antibiotics and regular anti-tuberculosis treatments. Consequently, the aim to PM of early diagnosis, early treatment, reducing case fatality rate and improving the patients'qualities of life may be achieved.