Cryptococcal antigen testing of lung tissue homogenate improves pulmonary cryptococcosis diagnosis:Two case reports

lNTRODUCTlON

the aetiological cause of pulmonary cryptococcosis (PC), is globally prevalent and can cause a fatal, disseminated disease. As a potentially serious fungal infection, a timely and reliable diagnosis is very important for improving the prognosis of PC. The diagnosis of PC is based on a combination of clinical symptoms, radiological suspicion, and laboratory confirmation[1]. Culture,histopathology, and serology are the main laboratory methods used for confirmation[2]. However, PC may easily be misdiagnosed or underdiagnosed owing to the absence of clinical symptoms and falsenegative laboratory results[2-6]. Cryptococcal antigen (CrAg) testing is convenient, inexpensive, and effective in diagnosing PC[5,7]. Serum, bronchoalveolar lavage fluid (BAFL), pleural effusion,percutaneous pulmonary aspirates, urine and cerebrospinal fluid have been used for CrAg testing[7-10], while lung tissue homogenate has not been reported thus far. The following cases highlight the usefulness and sensitivity of CrAg testing of lung tissue homogenate in the confirmation of PC.

書包和袋子早已褪色，青草般的翠綠變成比黯淡再淡一點的綠。跟學校其他同學的書包比起來，我好像背著一個外校的書包。原本綠底白字的書包和袋子，由于綠色部分太淡，校名便模糊不清。如果第一次遇見她時背著現在的書包，她應該很難看出我就讀的學校。那么我當時的問句便不再是“鳥問句”，而是有意義的。

CASE PRESENTATlON

Chief complaints

A 59-year-old female patient was admitted to the hospital for haemoptysis on August 21, 2017.

Physical examination showed an RR of 18 breaths/min, HR of 89 beats/min, temperature of 37.2°C and BP of 139/79 mmHg. Normal sounds were heard on auscultation of the lungs.

我們可以把8千克作為孕期體重增重的最低值，那么最高值應該是多少呢？這個表顯示應該是18千克。有研究證明，不管孕前體重有多輕，也就是孕媽媽懷孕前不管有多瘦，整個孕期體重增加如果超過18千克，寶寶就存在過大的風險，所以孕期體重增重最多不應超過18千克。那么我們現在就知道了孕期體重增重范圍是8～18千克。

History of present illness

The patient complained of haemoptysis with no sputum. Chest computed tomography (CT)before admission revealed nodules in both lungs, and 7 d of antibacterial treatment was ineffective.

The patient had a repeating cough with no fever or sputum.

History of past illness

The patient was HIV-negative. She underwent CT-guided TNLB of a nodule in the lower lobe of the right lung 2 d after admission (Figure 2A). CrAg testing using lung biopsy tissue homogenate was positive (Figure 2B). Ink staining of her lung biopsy specimen was positive for(Figure 2C). Lumbar puncture ruled out Cryptococcus infection of the nervous system.

The patient had no other medical history.

Personal and family history

Both patients had no remarkable personal or family history.

Physical examination

陪同親屬從柳絮、楊絮紛飛的城市一路開車到達腫瘤醫院，路上甚至得知親人生病后很長時間都沉浸在擔心與焦慮里，偶然抬頭看到流蘇花的那一刻，心里覺得松了一口氣，之后留意到每個角落里都熱烈生長的植物，心里也愉快了些，尋味到一種“人間四月芳菲盡，山寺桃花始盛開”的意境。再觀看每一位穿梭在我身邊的人，雖然行色匆匆不曾駐足，卻也是面目如素，沒有預設中的“悲戚”。在CT室外看到大家都在討論顯影水多么難喝時，我覺得自己此前“談癌色變”的緊張都顯得多余，緊張兮兮、一直面有難色的我在人群里才是最不正常的存在。

2A 57-year-old female patient complained of worsening cough for 4 mo and was admitted to the hospital on July 20, 2020.

Laboratory examinations

Due to the potential risk of further dissemination of Cryptococcus infection and the long duration of antifungal therapy, it is necessary to combine multiple methods to improve the diagnostic certainty.According to our cases, lung tissue homogenate CrAg testing may help enhance the accuracy of the diagnosis, especially for serologically negative patients.

The patient had a 20-year history of immune thrombocytopenic purpura, 2-year history of secondary diabetes, and 1-month history of splenectomy. She had long-term steroid therapy indicated for systemic blood disease.

Imaging examinations

The final diagnosis of both cases was PC.

CT scan of the chest showed a nodule in the right lung (Figure 2A).

1）斷層。左岸斷層以NWW向為主，其中斷層F14、F17、F18規模較大，斜切水墊塘。其他斷層規模較小。

FlNAL DlAGNOSlS

Chest CT revealed multiple nodules in both lungs (Figure 1A).

TREATMENT

Antifungal therapy (fluconazole: 400 mg once daily for 3 mo and then voriconazole 200 mg twice daily for 9 mo) was initiated.

Antifungal therapy (fluconazole: 400 mg once daily for 3 mo) was initiated.

從測算的結果來看，結果負數，而且負值越來越大，也就是說從2002年開始到2011年，國內沒有社會游資，這顯然與現實不符合。

OUTCOME AND FOLLOW-UP

Antifungal therapy had to be discontinued because of hepatic impairment. The lung lesions were stable without apparent respiratory symptoms for one year after antifungal therapy was discontinued (Figure 1A).

Initial medical examination showed a respiratory rate (RR) of 17 breaths/min, heart rate (HR) of 85 beats/min, temperature of 37.2 °C, and blood pressure (BP) of 102/65 mmHg. No rales were detected in either lung.

Imaging follow-up after 3 mo of antifungal therapy revealed very good resolution of the nodule in the right lung seen previously (Figure 2A).

DlSCUSSlON

Early diagnosis of PC is crucial for timely and effective treatment. The diagnosis is based on a combination of clinical symptoms (, cough, expectoration, chest tightness, chest pain, fever, and dyspnoea), suspicious radiological findings, and laboratory confirmation[1]. PC may easily be misdiagnosed or underdiagnosed in the absence of clinical symptoms, especially for immunocompetent patients[6]. Clustered or solitary pulmonary nodules are the most commonly seen lung abnormalities in PC[11]. The radiographic similarities to pneumonia or neoplasms, however, can often confuse or delay the clinical diagnosis. Further diagnostic evaluation is often needed to rule out or confirm the diagnosis.The laboratory confirmation of PC is often the next step in evaluation and usually involves serology,histopathology, and/or mycological culture[2].

Lung tissue biopsies are an important method for a definitive diagnosis., an encapsulated organism, is a narrow-based budding yeast, as seen on histological staining with India ink,haematoxylin and eosin, Grocott-Gomori’s methenamine silver, or periodic acid-Schiff[1,12]. However,in several previous studies, Cryptococcus in some samples did not have a typical polysaccharide capsule[13]. In a previous study, the detection rate ofobserved by electron microscopy was 89.5%[3]. Samples obtained by CT-guided percutaneous lung biopsy may therefore not be adequate for staining and present a further diagnostic dilemma in confirming cryptococcosis.

(2)當電路發生d2點故障短路時，電源負極接地故障。按照電路基礎知識，我們知道：負極接地，則該電路負極電壓U- =0V，由于正極未接地，U+=220-0=220V。根據電路基礎知識，在負荷1至負荷4的兩端，仍然為220V電壓，各負荷供電電壓正常，由于電路接通，所以，電路不受影響。

GPS定位信息與電子地圖的匹配就是通過這種方法獲取GPS經緯度信息并存入相應數組結構JD[]和WD[]，并與電子地圖中經緯度位置對比后，顯示目前所處位置。圖3為電子地圖顯示界面截圖，黑點為農機手通過GPS定位后所在位置，左上角為其所在位置的經緯度坐標。

The culture of respiratory samples has a complementary role in confirming PC. However, Cryptococcus cultures often take several days to grow, and many factors may influence the culture results of lung tissue, such as the number of pathogens, previous antifungal agents administered and duration of culture. A study reported a diagnostic rate of 70.8% for lung tissue culture[5].

Serum CrAg testing is a convenient, sensitive and rapid method for diagnosing PC[5]. The overall sensitivity and specificity of CrAg testing in the diagnosis of cryptococcal infection were approximately 97.6% and 98.1%, respectively[7]. However, in the two presented cases, the serum CrAg tests were all falsely negative. A false-negative CrAg test result may be due to a prozone reaction due to high antigen titres, low fungal load, samples transported in inappropriate vials, the presence of immunocomplexes preventing the release of glucuronoxylomannan antigen, or hypocapsular or acapsular strains of Cryptococcus spp[14]. Patients with a single pulmonary nodule were less likely to have positive antigen testing than those with other radiographic presentations or concomitant extrapulmonary disease[4]. For cases with a single pulmonary nodule, cryptococcal capsular antigen possibly only exists in the nodule and is not released into the blood. This might be the reason why lung tissue homogenate CrAg testing is more sensitive than serum testing in these cases.

In a typical procedure, Cu2–xS powders were prepared by the sol-gel method. The operating mode is similar to that followed by Riyaz et al. who synthesized only the covellite phase CuS[14]. However, three different phases were obtained by annealing in this study. Fig. 1 displays the operating mode.

However, there is a limitation of CrAg testing invasion in lung tissue homogenates. Since there are only two cases, this detection methodology deserves further study in a large sample.

CONCLUSlON

The patient was HIV-negative. A CT-guided transthoracic needle lung biopsy (TNLB) of a nodule in the right lung was sent for evaluation 3 d after admission (Figure 1A). Her serum CrAg test was negative, while her lung biopsy revealedinfection. Lumbar puncture ruled out Cryptococcus infection of the nervous system. Therefore, she was investigated for PC, and antifungal therapy (fluconazole: 400 mg once daily for 3 mo and then voriconazole 200 mg twice daily for 9 mo) was initiated; however, her lung lesions did not resolve. A follow-up CT-guided TNLB on September 21, 2018 was positive foron ink staining (Figure 1B). At that time,CrAg testing of lung biopsy tissue homogenate was performed, which yielded a positive result(Figure 1C). Her serum CrAg test and lung tissue culture remained negative.

ACKNOWLEDGEMENTS

We acknowledge the contributions of Mr Jun-Min Cao and Mr Jian-Feng Wang for the research assistance.

FOOTNOTES

Jiang LB performed the postoperative evaluation and diagnosis; Wang WY and Zheng YL reviewed the literature and contributed to manuscript drafting; Wang WY collected the medical data; all authors issued final approval for the submitted version.

Informed written consent was obtained from the patient for publication of this report and any accompanying images.

The authors declare that they have no conflicts of interest.

The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016).

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Wei-Yi Wang 0000-0003-2563-6294; Yu-Lu Zheng 0000-0003-1218-9480; Li-Bin Jiang 0000-0002-4527-7770.

Liu JH

A

Liu JH