Pathogenic Microorganism Detection in Invasive Pulmonary Aspergillosis(IPA) Patients Using Bronchoalveolar Lavage Fluid

Background: To evaluate the diagnostic values of different forms of pathogenic microorganism detection from the bronchoalveolar lavage fluid (BALF) for Invasive Pulmonary Aspergillosis (IPA) diagnosis. Methods: Bronchoalveolar lavage fluid (BALF) was collected from 100 patients with suspected clinical pulmonary Aspergillus infections by means of bronchoscopy. The smear microscopy, fungal culture and PCR were used to detect the Aspergillus from the BALF. Results: 15 cases were diagnosed by pathological data(the proven group), while 12 confirmed cases were supported by clinical examinations including radiology and etiology (the probable group). The 73 cases left were not IPA, among which 21 cases of common pneumonia were taken as the control group. For the practise of smear microscopy method, the sensitivity, specificity, and accuracy of the diagnoses were 40.7%, 100.0%, and 66.7% respectively. As for the fungal culture, the sensitivity, specificity, and accuracy were 37.0%,100.0%, and 64.6% respectively. For PCR, the sensitivity, specificity, and accuracy were 55.6%, 100.0%, and 77.1% respectively. With the combination of the three methods mentioned, the final results were 92.6%, 100.0% and 95.8%. Conclusion: The pathogenic detection of bronchoalveolar lavage fluid (BALF) is an ideal diagnostic method for Aspergillus infections. The smear microscopy method, the fungal culture and PCR has their own limitations in diagnosis. The detection of combining the three methods is proved to improve the efficiency of IPA diagnosis significantly. genic Microorganism Detection in Invasive Pulmonary Aspergillosis(IPA) Patients Using Bronchoalveolar Lavage Fluid. Bi omed the evaluation of its pathogenic detection for the diagnosis of fungal infection. In this study, BALF was collected from 100 suspected patients suffering from invasive pulmonary Aspergillus infection. Three pathogenic detection methods, smear and were used to quantify the


Introduction
The insufficiency of detective methods during early stage of Invasive Pulmonary Aspergillosis(IPA) has lead to severe misdiagnosis [1]. Surveys and researches had been conducted to proved that the uncertainty and improper treatment could result in relatively high mortality rate [2,3]. Bronchoalveolar lavage (BAL) is the method which can collect secretory fluid in the respiratory tract and alveoli. The fluid can be separated for further analysis so that the content of the components can be quantified. It is an common method in locating the lesion site in clinical practice.
Compared with other methods, for example, BALF has fewer chance of contamination rather than collecting sputum specimen.
Therefore, it can improve the specificity of etiological diagnosis. At present, this technology is mainly applied in the clinical diagnosis of the evaluation of its pathogenic detection for the diagnosis of fungal infection. In this study, BALF was collected from 100 suspected patients suffering from invasive pulmonary Aspergillus infection.
Three pathogenic detection methods, smear microscopy, fungal culture and PCR were used to quantify the Aspergillus examples.
Then the results of these three methods would be valued under the clinical criterion. To provide assistance for the clinical diagnosis of pulmonary aspergillosis.

Collection and Storage of the BALF Specimens
The ethical approval for this study was obtained by First Affiliated Hospital of Sun Yat-sen University. Before bronchoalveolar lavage fluid were collected, the patients involved in this research received detailed explanation and understood the benefits and potential danger, and had signed the consent letters for bronchoscopy procedures. The subject inclusion and exclusion criteria used in this study refer to the consensus of Chinese experts on the detections of pathogens in the bronchoalveolar lavage of pulmonary infectious diseases, which was proposed by respiratory disease branch of the Chinese Medical Association in 2017 [4]. The procedure of collecting bronchoalveolar lavage was as follows: a) 1-2mL of 2% lidocaine was injected through the biopsy hole for local anesthesia, after the bronchoscope reached the lung segment of the lavage site.

b)
The top of the bronchoscope was incarcerated in the opening of the target bronchial segment or sub segment. A dose of 20-50mL sterile normal saline at 37 ℃ or room temperature was injected through the biopsy hole of the bronchoscope. After each injection, the appropriate negative pressure (less than 100cmH 2 O) was used to recover the liquid, and the total recovery rate was secured to be more than 40%.

Detection of the Specimens
After     Patients' data were collected (Table 2) Table 3.  results showed that in the clinical diagnosis group, the BALF culture and PCR results were significantly different (p<0.05). Furthermore, in the proven group, there were no significant differences observed between the three detection methods (p＞0.05).

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A Kappa Consistency Test Method was adopted in this study for the pairwise comparison of the results of the three detection methods as a whole, and also within the different groups.
The results are shown in Table 4. As shown in the table, there was a weak consistency observed between the microscopy and culture methods. However, the results of both were found to be inconsistent with the PCR method.

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In accordance with the above-mentioned results of the Internal Kappa Consistency Test, the consistency between the smear microscopy and culture methods among the three test methods was weak. However, the results of the PCR were observed to be very inconsistent with those of the other two methods. However, it was speculated that if the microscopy, culture, and PCR methods were combined for diagnosis procedures, the diagnosis efficiency could be significantly improved. the calculation results are shown in the last row of Table 5.

Discussion
The molecular biological diagnosis method (represented by PCR) is found to be more sensitive and rapid than the traditional culture method. It could directly detect the nucleic acid molecules of the pathogens without the steps of isolation and culturing, which proved to be helpful for the early diagnoses of the disease [8]. It is known that PCR can be theoretically detected regardless of whether the pathogen are active or inactive. The positive results of the PCR were significantly greater in number than those of the culture method. However, there were no significant differences observed when compared with the smear microscopy (p> 0.05). The reason was that the IPA patients had received antifungal therapy, therefore, the positive rates of the culture method were lower than PCR. This showed fungal culture method is less the diagnosis efficiency than PCR, when antifungal therapy were conducted. However, we found that some patients were positive with BALF smear microscopy or fungal culture, but negative with PCR method. A possible cause of this is the fungal composition. The outer sides of Aspergillus (which belong to eukaryote) are wrapped by a layer of compact and hard cell wall components. The complete cell wall will inevitably reduce the extraction effects of nucleic acid substances [9].
Therefore, the interpretations of PCR results should be made in combination with other factors, such as clinical manifestations, other examination results. In our study, it was found that when smear microscopy techniques were performed for the lower sediment of BALF, the concentrations of pathogen components in those specimens were relatively high. The positive rates of smear microscopy were 40.0%, while that of common sputum was only 4.5% [10,11]. This showed the lower sediment of BALF was very high efficiency for microscopic examinations . In summary, the three examined diagnostic methods were found to have high specificity, but insufficient sensitivity. In the present study, considering the low degree of overlap in the results of the three detection methods, it was speculated that if a combination of microscopy, culture, and PCR methods was applied in diagnosis processes.

Conclusion
The pathogenic detections of bronchoalveolar lavage fluid (BALF) have been determined to be an ideal diagnostic method for Aspergillus infections. Because of the limitations of the different detection methods, The combination of BALF smear microscopy, culture, and PCR could significantly improve IPA diagnosis efficiency.