Due to non-specific clinical features, the diagnosis of PC depends mainly on pathological examination (4). The gold standard is lung tissue pathological biopsy, H&E staining, PAS and hexamine silver staining. The type of lesion is related to the immune status of the patients. Non-caseating granulomas are often formed in patients with normal immune function and consist of devoured cryptococci cells and capsules that are visible in the cytoplasm of macrophages. Granulomas are not likely to appear in patients with impaired immune function, although the alveolar cavity is filled with cryptococci spores. Specialized bacterial staining indicated PAS (+), AB (-), hexamine silver (+) and acid resistance (-). The majority of the etiological examinations aim to detect Cryptococcus neoformans by blood culture or punctured materials. The aforementioned examinations exhibit a long reporting time and a low positive rate, and the majority of them are risky and invasive, which renders them unsuitable to be implemented in critically ill patients and patients with blood coagulation dysfunction or lesions. Moreover, these examinations exhibit limited value for early diagnosis of the disease. Clinicians expect from non-invasive examinations ideal specificity and sensitivity metrics in addition to the rapid analysis time. In recent years, the determination of cryptococcal polysaccharide capsular antigen in serum samples has provided etiological evidence for the diagnosis of PC. Currently, the methods for detecting the cryptococcal capsular antigen in serum include latex agglutination (LA), enzyme-linked immunosorbent assay (EIA) and lateral chromatography (CrAg-LFA) (5). A previous study has shown that CrAg-LFA exhibited higher accuracy than LA and EIA, resulting in a significant clinical value. This conclusion was derived due to its optimal stability at room temperature, the rapid detection procedure (10 min), the ease of operation and its limited high demand on laboratory equipment (6).
Since the sample was from the target lung lesion area, a large number of clinical studies have proven that the sensitivity, specificity and clinical application value of galactomannan (GM) detection in BALF samples are significantly higher than those noted in serum for the diagnosis of invasive pulmonary aspergillosis (7,8). Based on these results, we extracted bronchoalveolar lavage (BAL) samples at the lesion of PC with high-resolution CT positioning by lung biopsy of patients diagnosed with this disease in the present study. Furthermore, we compared the CrAg-LFA data derived from BALF with those derived from serum samples and examined the diagnostic criteria for PC. The results demonstrated that the sensitivity and specificity of CrAg-LFA on BALF samples were 93.1% and 100.00%, respectively, while those of serum samples were 75.0% and 99.6%, respectively. Therefore, the diagnostic value of CrAg-LFA with regard to PC infection in BALF samples was significantly higher than that noted in serum samples. The conclusion was consistent with previous report (9).
In the present study, the colloidal gold method was used to test the cryptococcal capsular polysaccharide antigen. The colloidal gold test strip comprises a simple and rapid detection reagent that can detect all four (type A-D) serotypes of Cryptococcus neoformans. During the test, the sample is subjected to capillary action and is chromatographed by binding to a monoclonal antibody against the Cryptococcus antigen, which is considered a gold standard detection method. In the presence of cryptococcal antigen in the sample, binding to the gold standard anti-Cryptococcus antibody will occur. This gold standard antibody-antigen complex continues to be chromatographed on the membrane by capillary action and reacts with the test strip containing immobilized anti-Cryptococcus monoclonal antibody. The cryptococcal capsular polysaccharide antigen in the test sample can react with the colloidal gold-labeled antibody, and the antigen-antibody complex migrates on the surface of the test paper under capillary action, and ultimately binds to the monoclonal antibody immobilized on the nitrocellulose membrane. This binding produces a red line that is considered the test line (10). The test strip itself carries a quality control line. A positive result denotes simultaneous appearance of the quality control line and the test line, and a negative result denotes appearance of the quality control line alone. No negative control is required, and this method is considered more reliable.
In the present study, 13 of the 18 pathologically confirmed PC patients who had negative serum CrAg-LFA had positive BALF CrAg-LFA. In certain patients, no clinical symptoms appeared, although small lesions were found at the edge of the lung and even near to the pleura of the lung as determined by CT (Figures 1a-d). In one rare case with bronchial Cryptococcus, the lesion was found in the lateral bronchus of the right middle lobe. The serum CrAg-LFA test for this case was negative, while the bronchial flushing fluid CrAg-LFA test was positive. It has been shown that the false negative results of the capsular polysaccharide antigen detection occur mostly during the early stages of infection (11). The higher positive rate and sensitivity noted in the BALF samples as opposed to the serum samples may be due to the infiltration of cryptococci colonies to the lung, resulting in a slow release of the capsular polysaccharide antigen to the blood. These events occur in the early stage of infection, notably in patients without systemic symptoms. In addition, the infection is associated with a high local cryptococcal load noted in the lung lesions. These speculations need further confirmation. In the present study, both the serum and BALF results of 5 confirmed cases were negative and corresponded to patients with small lesions and no symptoms. No case was found to have positive serum CrAg-LFA results and negative BALF CrAg-LFA results.
Bronchoalveolar lavage (BAL) is known as liquid lung biopsy. It is a simple, safe and convenient compared to the histopathological biopsy that can be implemented in primary hospitals. It can be used for cryptococcal antigen detection in case of biopsy unavailability. However, the process of collecting BALF and the requirements of the operators need to be further standardized. These procedures require a high demand of operational skills and accuracy to reach the sub-segment where the lesion is located for lavage, notably for small lesions. In the present study, the location of BALF collection was accurately positioned by lung CT, and only 60 ml of liquid was perfused to avoid the dilution of specimens caused by excessive perfusion. This resulted in avoiding result bias, which was a significant addition from the requirements stated by previous studies. Since less than 1 ml of fluid is required for CrAg-LFA, this method can increase the positive rate by collection of the centrifuged precipitation of the lavage fluid. The CrAg-LFA test exhibits significantly higher sensitivity, specificity and application value in BALF than in serum samples in a similar manner with the GM. However, GM indicates false positive results in testing both serum and BALF samples, and can be influenced by several factors (12,13). It has been reported that both latex agglutination and colloidal gold methods can occasionally lead to false positive results with regard to the detection of serum cryptococcal capsular polysaccharide antigen. In the present study, one out of 236 cases of non-pulmonary cryptococcosis also appeared weakly positive with regard to the serum CrAg-LFA test, which suggested that potential cross-reactivity was caused by specific factors in case of a low serum cryptococcal antigen titer. Therefore, semi-quantitative determination is advised in such cases. Finally, this case was pathologically diagnosed as rheumatoid lung disease. Whether such rare diseases cross-react with serum cryptococcal antigen to a certain extent requires confirmation with large sample clinical trials. In the present study, no false positive results were found in BALF samples from the four centers tested by CrAg-LFA, and the test exhibited a specificity of 100.00%. Therefore, we suggest that the diagnostic value of the CrAg-LFA test in BALF samples exhibits equivalent positive performance in detecting cryptococcal antigen to that of the microbiological culture, and that it is only inferior to the clinical value of the histopathological biopsy. Furthermore, lung puncture and other traumatic examinations are required only in cases with negative results.
Several limitations should be mentioned in the present study. First, we did not compare the diagnostic efficiency between LFA and conventional methods, such as LA or EIA. Second, the BALF CrAg-LFA value after treatment was not detected, therefore, we failed to evaluate the effectiveness of anti-cryptococcal therapy.
In summary, the CrAg-LFA test in BALF samples is of great significance for the early diagnosis of PC with small lesions, in which histopathological biopsy is not appropriate, due to its rapid, simple and safe method of analysis.