Due to non-specific clinical features, the diagnosis of PC mainly depends on pathological examination, i.e, 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 do not commonly appear in patients with impaired immune function, although the alveolar cavity is filled with cryptococci spores. Specialized bacterial staining can be used to indicate PAS (+), AB (-), hexamine silver (+), and acid resistance (-). C. neoformans is mainly detected using blood culture or punctured materials. The aforementioned examinations have a long reporting time and a low positive rate. In addition, these methods are invasive, thus unsuitable for critically ill patients and patients with blood coagulation dysfunction. Moreover, these examinations have limited value for early diagnosis of the disease.
Over 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) . 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 for laboratory equipment .
A large number of clinical studies have proven that the sensitivity, specificity, and clinical application value of GM detection in BALF samples (target lung lesion area) are significantly higher than those noted in serum for the diagnosis of invasive pulmonary aspergillosis [8, 9]. In this study, we extracted BALF samples at the target lesion of PC patients. Thus we compared the CrAg-LFA sensitivity and specificity between BALF and serum samples. 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, which suggested that the diagnostic value of CrAg-LFA in detecting PC infection is higher when using BALF samples; this was consistent with a previous report .
In the present study, the colloidal gold method was used to test the cryptococcal capsular polysaccharide antigen. The colloidal gold test strip comprises a rapid and straightforward detection reagent that can detect all four (type A-D) serotypes of C. 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, binding to the gold standard anti-Cryptococcus antibody occurs. 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 . The test strip itself carries a quality control line. A positive result denotes the simultaneous appearance of the quality control line and the test line, and a negative result denotes the 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. In one 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 . 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. Besides, the infection is associated with a high local cryptococcal load noted in the lung lesions. Yet, 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 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. The procedure is complex and requires high operational skills. In addition, it may not be appropriate for small lesions. In the present study, the location of BALF collection was accurately positioned using lung CT, and only 60 ml of liquid was perfused to avoid the dilution of specimens caused by excessive perfusion. Since less than 1 ml of fluid is required for CrAg-LFA, this method can increase the positive rate by a 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 may generate false-positive results when testing both serum and BALF samples and can be influenced by several factors [13, 14]. It has been reported that both LA 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-PC resulted as weakly positive with regard to the serum CrAg-LFA test, which suggested that specific factors cause potential cross-reactivity in case of a low serum cryptococcal antigen titer. Therefore, a 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 needs to be confirmed by 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, our data suggest that the diagnostic value of the CrAg-LFA test in BALF samples has equivalent positive performance in detecting cryptococcal antigen as the microbiological culture, and 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.
This study has a few limitations. 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, CrAg-LFA is a rapid, simple, and safe experimental method, which can be used for early diagnosis of PC with small lesions. CrAg-LFA has a higher diagnostic value for PC when analyzing BALF samples compared to serum samples. Furthermore, the BALF positive results are equivalent to the microbiological culture-positive results in terms of diagnostic value.