Comparisons of cryptococcal meningitis caused by Cryptococcus neoformans between HIV-negative patients with and without lung infection in two Chinese university hospitals

Background Lung infection may cause many symptoms, such as fever and headache, that may be confused with cryptococcal meningitis (CM) symptoms. This study aimed to investigate the discrepancy in clinical features and outcomes of CM between HIV-negative patients with and without lung infection. Methods We retrospectively reviewed the medical records of patients with CM admitted to two hospitals in Southwest China from 1 January 2014 to 31 December 2018. Results A total of 71 patients was included during the 5 years, among which 35 (49.3%) patients had lung disease. CM occurred more frequently in male (62.9% vs. 44.4%, P=0.12) and young ( ≤ 30 years, 31.4% vs. 16.7%, P=0.30) patients with lung infection than in the patients without lung infection, with more fever (77.1% vs. 30.6%, P=0.001) and less central nervous system symptoms (5.7% vs. 16.7%, P=0.28) and vomiting (25.0% vs. 14.3%, P=0.26). In addition, patients with lung infection presented higher percentages of white blood cell (WBC) counts (cid:0)20×106/L (45.7% vs. 22.2%, P=0.036) and lower percentages of ethmoid sinusitis, maxillary sinusitis, paranasal sinusitis, and otitis media than patients without lung infection (8.6% vs. 30.6%, P=0.02). The Cryptococcus neoformans isolates were sensitive to itraconazole, voriconazole, uconazole, and amphotericin B but resistant to ucytosine. Patients with lung infection had higher mortality at discharge compared with patients without lung infection (8.6% vs. 0, P=0.12). Multivariable analyses showed that WBC counts (cid:0)20×106/L was signicantly associated with treatment outcome (OR=0.01, 95% CI=0-0.833, P=0.041).


Background
Cryptococcal meningitis (CM) caused by Cryptococcus neoformans is a common opportunistic infection, the main cause of mortality in human immunode ciency virus (HIV)-infected patients, and increasingly observed among patients with non-HIV immunosuppression [1,2]. Mortality outcomes for CM in HIVnegative individuals seem to be no better than those in HIV-positive patients [3][4][5]. Lung infection is a common disease that can be caused by many pathogens, such as Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus in uenza, Pseudomonas aeruginosa, Mycoplasma pneumonia, and fungi; lung infection may cause many symptoms, such as fever and headache, that may be confused with central nervous system (CNS) infection. In immunosuppressed individuals, infection begins in the lung after inhalation of fungal spores and often spreads to other organs, particularly the brain [6]. Many CM patients also have concomitant lung involvement, which is often overlooked or misdiagnosed as tuberculosis [7].
The clinical characteristics and outcomes in CM have been shown to vary depending on the underlying condition [8][9][10][11][12][13][14]. The HBV-positive CM patients presented with lower initial complaints of visual symptoms, lower cerebrospinal uid (CSF) white blood cell (WBC) counts, lower percentages of the total protein in the CSF exceeding 0.45 g/L, higher glucose levels in the CSF, higher percentage of positive results for Cryptococcus culture in the CSF, more extraneural involvement sites, and a higher proportion of normal brain images compared with the HBV-negative CM patients [14]. Compared with the immunocompromised patients, CM was present in a younger population, with higher initial complaints of visual and auditory symptoms, higher CSF WBC counts, higher proportion of normal brain images in the immunocompetent patients [11]. In addition, the elderly patients (≥65 years) were more vulnerable to CM than adults aged <65 years, and had female predominance, higher rates of altered consciousness and recent cerebral infarction [15]. Therefore, HIV-negative patients with lung infection but with CM tend to present with atypical features, which can lead to signi cant delays in their diagnosis and poorer outcomes.
There are many HIV-negative CM patients with and without lung cryptococcosis in clinics, but few epidemiological study has been explored in this region.Here, we retrospectively reviewed the medical records of patients with CM who were admitted to two Chinese university hospitals in Southwest China in the past 5 years to investigate the discrepancies. Patients with pneumonia and tuberculosis were classi ed as having lung infection. Sex, age, length of hospital stay, CSF pro les, brain images, underlying diseases, initial presentation, drug resistance of Cryptococcus neoformans, antifungal therapy and outcomes were recorded. The outcomes were classi ed as "cured", "improved", "other", "untreated", "invalid", and "death" at discharge and further classi ed into satisfactory (cured or improved) and unsatisfactory (the others) outcomes in accordance with previous studies [11,16]. (1) "Cured," no reappearance of symptoms, and 2 sequential negative CSF cultures for C. neoformans with at least a 2-week interval; (2) "Improved," no reappearance of symptoms, but without sequential negative CSF cultures for C. neoformans with at least a 2-week interval; (3) "Invalid", the symptoms were not improved.

Study population and de nition
Statistical analysis SPSS version 17.0 (SPSS Inc., Chicago, IL) was used to analyze the data. Data are presented as the mean ± SD or median and range. Independent Student's t-test and Mann-Whitney U test were used to compare parametric and nonparametric continuous variables, respectively. Categorical variables were compared using the Chi-squared test or Fisher's exact test. Then the regression equations for predicting the probability of poor prognosis of CM were established. A P-value of <0.05 was considered statistically signi cant. All analyses were conducted as 2-sided tests.  16.7%), progressive disturbance of consciousness (4/36, 11.1%), vague speech, memory deterioration, and walking lability (4/36, 11.1%). Among CM patients with lung infection, the common initial symptoms were fever (77.1%), headache (77.1%), dizziness (14.3%), vomiting (14.3%), progressive disturbance of consciousness (17.1%), edema of lower extremities (28.6%), and cough and expectoration (28.6%). Compared with CM patients without lung infection, CM patients with lung infection presented more fever (P 0.01), more edema of lower extremities and cough and expectoration (P 0.01), less vomiting (14.3% vs. 25.0%), less central nervous system symptoms and less ventosity (both 5.7% vs. 16.7%).

Laboratory data
The positive rates of the Pandy test of CSF in CM patients with and without lung infection were both high (77.1% vs. 72.2%), and the chloride ion and glucose levels were both decreased, while the total protein was increased in both. However, the percentage of CM patients with lung infection with WBC counts 20×10 6 /L was higher than that of patients without lung infection (45.7% vs. 22.2%, P 0.05). The brain images detected by CT or MRI showed that the percentages of ethmoid sinusitis, maxillary sinusitis, paranasal sinusitis, and otitis media in CM patients without lung function were higher than those in patients with lung infection (30.6% vs. 8.6%, P 0.05) (

Risk factors for poor treatment outcome of HIV negative CM
We included eight factors lung infection, sex, age, fever, WBC counts 20×10 6 /L, chloride ion, protein, and glucose that may impacting the prognosis of CM patients in the regression. Multiple regression analysis showed that WBC counts 20×10 6 /L was signi cantly associated with treatment outcome (OR = 0.01, 95% CI = 0-0.833, P = 0.041), and lung infection showed a tendency of association with treatment outcome (OR = 0.026, 95% CI = 0.001-1.173, P = 0.06) (table 5).

Discussion
It is generally known that CM is an opportunistic infection in HIV-positive patients, but it also occurs in HIV-negative patients. In a population-based study in the United States, the incidence of cryptococcosis among HIV-negative patients was close to half of the overall cases reported [8]. In the current study, 72.4% (71/98) of the patients were HIV negative, which may be because the patients were transferred to a professional hospital once HIV was discovered.
Lung infection affects many people in China, and our study showed a high proportion (49.3%) of CM patients with lung infection. The clinical characteristics of CM varied depending on the underlying conditions, such as virus infection, immune state, fungal species or lineage differences, and age [15,17]. In the current studies, the overall sex and age in CM patients with and without lung infection were not signi cantly different; however, CM occurred more frequently in male and younger patients (aged ≤30 years) with lung infection than in patients without lung infection. In a United States series of over 300 HIV-negative patients with cryptococcal infection, half had CNS involvement, and of these, 24% had chronic liver, kidney or lung disease; 16% had a malignancy; and 15% had received a solid organ transplant [18]. However, in our data, among 71 HIV-negative patients with CM, half had lung infection, followed by intracranial infection (16.9%), systemic lupus erythematosus (12.7%), sepsis (12.7%), nephrotic syndrome (9.9%), type II diabetes mellitus (9.9%), hypertension (9.9%), epilepsy (8.5%), and decompensated hepatic cirrhosis of hepatitis B (8.5%), which were different from the previous studies. Patients with CM presented with neurological symptoms, most typically headache and altered mental status, as well as with fever, nausea and vomiting. In the current studies, the main symptoms of patients were neurological symptoms, such as headache, followed by fever, vomiting, dizziness, ventosity, progressive disturbance of consciousness, vague speech, memory deterioration, and walking lability, and the symptoms in the two groups had no signi cant differences.
A large proportion of HIV-negative patients may have a marked systemic in ammatory response and hydrocephalus [17]. In our data, patients with lung infection presented with a higher proportion of fever, cough and expectoration, a lower proportion of noncentral nervous system symptoms, and a higher percentage of WBC counts in CSF 20×10 6 /L than patients without lung infection, which may suggest a higher in ammatory response of the brain in patients with lung infection than in patients without lung infection. However, in the current studies, only 9.9% of patients had hydrocephalus. In addition, patients with lung infection had less vomiting, ventosity, ethmoid sinusitis, maxillary sinusitis, paranasal sinusitis, and otitis media than patients without lung infection, and 28.6% of patients had edema of lower extremities; clinicians should pay attention to this symptom.
In contrast to the rare resistance of Cryptococcus neoformans to ucytosine that showed a baseline uconazole resistance rate of 12% in previous studies [19,20], our results showed that Cryptococcus neoformans isolates from CSF were sensitive to uconazole but resistant to ucytosine, with a resistance rate of 22.5%. Most of the recommendations for the management of non-HIV CM patients are extrapolated from HIV studies, among which the combination therapy of amphotericin B and ucytosine for the treatment of CM was the most commonly used in clinical trials [21,22]. However, only 11.3% of patients were treated with amphotericin B + ucytosine in our data; most (62.0%) CM patients used uconazole + amphotericin B due to the drug resistance rate of Cryptococcus neoformans to ucytosine. Outcomes for patients with HIV-associated cryptococcal meningitis in Africa suggested a 3-month mortality of 70% [17]. In prospective research studies, for patients treated with uconazole, mortality at 10 weeks was 50-60% [23,24]. In the current studies, 67.6% of patients had satisfactory results at discharge, which was consistent with previous studies. In HIV-negative individuals, altered mental status, markers of a poor in ammatory response, and low CSF white cell count have been linked with poor prognosis [25]. According to our results, the mortality of patients in the lung infection group was increased, which was consistent with the results showing a reduced CSF white cell count, furthermore, multivariable analyses showed that WBC counts 20×10 6 /L was the risk factor of treatment outcome.
This study has some limitations. First, the study was performed in only two hospitals in Southwest China. Second, we only investigated the recent 5-year clinical records of CM patients, and the number of patients was relatively small. Third, this investigation is a retrospective study, some Cryptococcus neoformans isolates with unique genotype have higher virulence or azole-resistance, we could not provide genotype data of the related Cryptococcus neoformans isolates in the study, and we could not obtain the long-term outcome of patients. Further multicenter studies are needed to con rm our results and investigate more signi cant factors to improve the diagnosis and treatment of CM patients with lung infection.

Conclusions
In conclusion, we found that compared with the patients without lung infection, male and younger patients with lung infection presented with CM more frequently, with more fever, edema of lower extremities and cough and expectoration; less central nervous system symptoms, vomiting and ventosity; higher percentage of WBC counts 20×10 6