Abstract
Background
The clinical features of India ink-negative and culture-negative cryptococcal meningitis (CM) in HIV-negative patients remain unclear.
Methods
A total of 395 antifungal-naïve HIV-negative CM patients were enrolled in this retrospective study. The 90-day poor outcome and associated risk factors were compared between patients with positive and negative India ink, and those with positive and negative culture in cerebrospinal fluid (CSF).
Results
India ink-negative patients had a high level of glucose [mmol/L; 2.6 ± 1.5 vs. 2.1 ± 1.4, P = 0.005], total protein level [g/L; 1.1 (0.7–1.8) vs. 0.9 (0.6–1.2), P = 0.002] and WBC count [×106/L; 121.9 (36.5–210.0) vs. 67.5 (18.0-140.0), P = 0.001] in CSF but lower intracranial pressure (ICP) (mmH2O; 185.0 (130.0-250.0) vs. 270.0 (191.0-397.5), P < 0.001) at the time of CM diagnosis. Culture-negative patients showed a high CSF glucose level (mmol/L; 2.9 ± 1.6 vs. 2.1 ± 1.3, P < 0.001) and low ICP [mmH2O; 215.0 (150.0-277.3) vs. 250.0 (180.0-360.0), P = 0.006]. The 90-day poor outcome was 9.3% for India ink-negative patients, 24.5% for India ink-positive patients (log-rank P = 0.003), 21.7% for culture-negative patients and 19.8% for culture-positive patients (log-rank P = 0.579). ICP ≥ 300.0 mmH2O [adjusted odds ratio (AOR) (95% confidence interval): 4.6 (1.0-20.7), P = 0.045] and not going CrAg Assay [AOR:4.4(1.0-18.4), P = 0.045] were independent risk factors for India ink-negative patients. Low CSF count (< 20.0 × 106/L) [AOR: 2.7 (1.0-7.2), P = 0.047] and and positive India ink [AOR:5.3(1.2–23.5), P = 0.028] were independent risk factors for 90-day poor outcome in culture negative patients.
Conclusions
India ink-/culture-negative CM patients had mild CSF profile changes. The CrAg assay was associated with an increased survival rate of India ink- negative CM patients and positive India ink was associated with poor outcome of culture-negative CM patients. The mortality of patients with a positive India ink was significantly increased.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12879-026-12572-8.
Keywords: Cryptococcal meningitis, CrAg assay, India ink stain, Culture, Mortality
Background
An early diagnosis of Cryptococcus infection can be lifesaving in clinical practice. The current methods for diagnosing cryptococcal meningitis (CM) are mainly based on India ink staining, Cryptococcus culture and cryptococcal antigen (CrAg) testing of cerebrospinal fluid (CSF), alone or in combination [1]. Of those, CSF India ink staining and Cryptococcus culture are the traditional methods widely used in the laboratory, although the CrAg assay is widely available in some countries [2]. Among CM patients, approximately 15% exhibit negative India ink results, and 10%-20% exhibit negative CSF culture results [3–5]. Although negative India ink and culture results for Cryptococcus are probably related to missed microscopy findings and insufficient culture time, negative India ink and culture results can reflect a negligible amount of Cryptococcus in the CSF [6]. Furthermore, high rate of sterile India ink and/or culture of Cryptococcus is more likely to occur in patients without predisposing diseases, especially among those with high white blood cell count in CSF [7].The inability to identify Cryptococcus in CSF results in two challenges in practice: (i) The impacts of negative India ink and/or Cryptococcus culture results on the outcome of patients remain unclear. Negative results increase the risk of a missed diagnosis of CM, which might increase the risk of mortality in patients [8]. In contrast, a low CSF fungal burden is associated with a high survival rate in patients [7, 9]. (ii) The utility of India ink staining and culture should be reevaluated in the CrAg era. The CrAg assay is a rapid and highly sensitive method that significantly shortens the diagnosis time and increases the accuracy of CM diagnosis. The CrAg- Lateral Flow Assays (LFA) display a sensitivity of 96% (68%-100%) and specificity of 96% (84%-100%) on serum, and sensitivity of 99% (95%-100%) and specificity of 99% (95%-100%) on CSF [10]. The CrAg-LFA reagent is available since 2015 in Chinese market. Thus, the necessity and utility of India ink staining and CSF fungal culture in the CrAg era need further discussion.
To date, the characterization of CM patients with negative India ink and/or Cryptococcus culture has been poorly described [11]. Many studies have shown that high titers of CrAg in serum and CSF are positively associated with poor outcomes in CM patients [12, 13]. However, most studies have not disclosed the association between India ink staining or culture results (positive vs. negative) and the outcome in patients who was diagnosed via CrAg assay. We hypothesized that a heterogeneous CSF status was inevitably associated with different clinical features, the prognosis of CM and even the potential selection of antifungal regimens. For example, amphotericin B (AmB) plus 5-flucytosine (5FC) (AmB + 5FC) is the preferred regimen in HIV-positive patients with a high CSF fungal burden [9, 14]. However, whether AmB + 5FC is the optimal treatment for CM patients with negative CSF India ink and/or culture results is debatable. Thus, the characterization of CM populations with negative India ink and/or culture results is crucial for improving the understanding and treatment of CM.
In total, 395 antifungal-naïve HIV-negative CM patients were enrolled in the present retrospective study, with the following three aims: (1) observe the differences in outcomes among patients with different CSF cryptococcal statuses, (2) disclose the utility of the CrAg assay, India ink staining, culture and their associations, and (3) identify the optimal antifungal strategy for CM patients with negative CSF India ink and culture results.
Materials and methods
Study cohort
Between January 2010 and December 2021, 639 adult CM patients were hospitalized in two tertiary hospitals. There were 2 patients who were diagnosed by brain biopsy, 4 patients whose diagnosis information were unavailable, and 3 patients whose antifungal information were missing. Those 9 patients, 91 patients who received antifungal treatment at local hospitals and 144 HIV-infected CM patients were excluded from our study. Finally, 395 antifungal-naïve patients were included in our cohort. The selection chart of the patients is shown in Fig. 1.
Fig. 1.
Flowchart of patient selection
Diagnostic methods and criteria
We described the diagnostic criteria for CM in previous studies [7, 15]. Briefly, the criteria were as follows: (1) positive culture of Cryptococcus neoformans from cerebrospinal fluid (CSF); (2) positive CSF India ink smear of centrifuged sediment for Cryptococcus; and (3) probable meningitis and positive cryptococcal antigen titer in CSF, albeit without microbiological or pathological documentation.
India ink-negative patients were defined as those for whom no cryptococcal capsules were observed in CSF obtained from consecutive lumbar puncture during hospitalization by the India ink staining method.
Culture-negative patients were defined as those for whom no Cryptococcus was grown in a flask after 14-day culture.
Therapeutic strategies
According to clinical recommendations [16], AmB was administered to patients at a dose of 0.5–0.7 mg/kg/d via peripheral intravenous (IV) injection in 5% dextrose in a total volume of 500 ml. One liter of saline was administered intravenously immediately prior to AmB administration to minimize renal toxicity. The oral 5FC dosage was 80–100 mg/kg/d, and the voriconazole dosage was 6 mg/kg/d (divided into two doses) via IV on the first day and was maintained at 4 mg/kg/d (divided into two doses) via IV for at least 14 days. A ventricular-peritoneal shunt (VP shunt) was placed in patients with an intracranial pressure (ICP) ≥ 300 mmH2O and mental changes. Mannitol (100 g ivgtt q8h- q6h) was used as an assistant medicine to control the ICP of patients whose ICP ranged from 200 to 300 mmH2O [17].
Data collection, follow-up and primary endpoint
Demographic and clinical data were extracted from the electronic medical record systems (EMRSs) of the hospitals. Initial CM diagnostic data (such as CSF India ink test, CSF culture and CrAg assay results), CSF biochemical profiles, routine blood tests and biochemical profiles were collected from both the local hospital and the 1st Hospital of ZJU. Patient survival was confirmed by reviewing medical record, monitoring in the EMRS and/or conducting telephone interviews. The patients were followed from the first day of admission. The median length of follow-up for the patients was 93.0 (26.0-256.8) days.
The primary endpoint was defined as poor outcome, which referred as CM-related death or VP shunt for ICP ≥ 300 mmH2O in 90-day follow-up.
Statistical analysis
Normally distributed continuous variables are expressed as the means ± standard deviations, and nonnormally distributed continuous variables are expressed as the medians (interquartile ranges; IQRs). Categorical variables are expressed as numbers (percentages). Continuous variables were compared by Student’s t test or the Mann–Whitney U test, and categorical variables were compared by the chi-square test or Fisher’s exact test. A CM-related death was defined as an ‘event.’ Patient survival was analyzed by the Kaplan–Meier method and Cox proportional hazards models. Covariates were first analyzed using univariate Cox models. Thereafter, the covariates with P values < 0.150 in the univariate models were selected for inclusion in the multivariate Cox proportional hazards model using the forward (likelihood ratio) method. P ≤ 0.05 was considered statistically significant. Data analysis was performed using SPSS version 26.0 (IBM, Armonk, NY, USA) and GraphPad Prism version 7.0 (GraphPad Software, La Jolla, CA, USA).
Results
Patient characteristics and demographic details
Of the 395 patients in our cohort, 58.5% (231) were male, and 41.5% (164) were female. The mean age was 53.8 ± 15.5 years old, and the mean body mass index (BMI) was 21.6 ± 6.9 kg/m2. The positive India ink rate was 70.4% (278/395), and the CSF culture positivity rate was 75.4% (298/395). In all, 16.7% of patients had HBV infection, 14.9% had diabetes, 13.4% had chronic kidney disease (CKD), 7.8% had solid organ transplantation (SOT), 13.2% had connective tissue disease (CTD), 6.8% had chronic hematological disease (CHD), and 3.5% had malignancy. The negative India ink rate was 26.5% (45/170) in patients without predisposing diseases (PDs) and 32.0% (72/225) in patients with PDs (P = 0.233). Likewise, the negative culture rate was 27.1% (46/170) in patients without PDs and 22.7% (51/225) in patients with PDs (P = 0.315). A total of 8.9% (35/395) of patients died, and 8.9% (35/395) of patients accepted a VP shunt at the 90-day follow-up. Altogether, there were 15.9% (63/395) patients progressed into poor outcome during the follow-up. The patient characteristics and demographic details are shown in Table 1.
Table 1.
The demographic characteristics of CM patients (N = 395)
| Parameters | Results |
|---|---|
| Sex [(n(%)] | |
| Male | 231 (58.5) |
| Female | 164 (41.5) |
| Age (years old) | 53.8 ± 15.5 |
| BMI (kg/m 2 ) | 21.6 ± 6.9 |
| Predisposing diseases[n(%)] | 225(57.0) |
| HBV infection | 66 (16.7) |
| Liver cirrhosis | 36 (9.1) |
| Diabetes | 59 (14.9) |
| Chronic kidney disease | 53 (13.4) |
| Solid organ transplantation | 31 (7.8) |
| Connective tissue disease | 52 (13.2) |
| Chronic hematologic diseases | 27(6.8) |
| Malignancies | 14 (3.5) |
| Antifungal treatment [n(%)] | |
| AmB + 5-FC ± Flu | 205 (51.9) |
| Vori + 5-FC | 40 (10.1) |
| Flu + 5-FC | 119 (30.1) |
| Other regimen | 31 (7.9) |
| India ink stain [n(%)] | |
| Positive | 278 (70.4) |
| Negative | 117 (29.6) |
| CSF culture[n(%)] | |
| Positive | 298 (75.4) |
| Negative | 97 (24.6) |
| CrAg assay[n(%)] | |
| Yes | 180 (45.6) |
| No | 215 (54.5) |
| VP shunt[n(%)] | |
| Yes | 35 (8.9) |
| No | 360 (90.1) |
| 90-day poor outcome[n(%)] | 63(15.9) |
BMI: body mass index; HBV: hepatitis B virus; AmB: amphoteric B; Vori: voriconazole; Flu: fluconazole; 5-FC: 5-flucytosine; CSF: cerebrospinal fluid; CrAg: cryptococcal antigen
The frequency of fever was 55.3%(94/170) in patients without PDs, 69.7%(46/66) in those with HBV, 75.0%(27/36) in those with liver cirrhosis, 65.5%(38/58) in those with diabetes, 69.8%(37/53) in those with CKD, 67.7%(21/31) in those with SOT, 64.7%(33/51) in those with CTD, 74.1%(20/27) in those with CHD, and 78.6%(11/14) in those malignancy, respectively; the frequency of headache was 85.3%(145/170)in patients without PDs, 77.3%(51/66) in those with HBV, 69.4%(25/36) in those with liver cirrhosis, 60.3%(35/58) in those with diabetes, 64.2%(34/53) in those with CKD, 61.3%(19/31) in those with SOT, 76.5%(39/51) in those with CTD, 85.2%(23/27) in those with CHD, and 64.3%(9/14) in those with malignancy, respectively; and the frequency of nausea was 28.2%(48/168) in patients without PDs, 24.2%(16/66) in those with HBV, 19.4%(7/36) in those with liver cirrhosis, 21.1%(12/57) in those with diabetes, 23.1%(17/52) in those with CKD, 16.7%(5/30) in those with SOT, 31.4%(16/51) in those with CTD, 33.3%(9/27) in those with CHD, and 21.4%(3/14) in those malignancy, respectively. 1). The 90-day mortality was 7.1%(12/170) in patients without PDs, 12.1%(8/66) in those with HBV, 19.4%(7/36) in those with liver cirrhosis, 10.2%(6/59) in those with diabetes, 7.7%(4/52) in those with CKD, 9.7%(3/31) in those with SOT, 11.5%(6/52) in those with CTD, 11.1%(3/27) in those with CHD, and 0(0/10) in those malignancy, respectively (Fig. 2).
Fig. 2.
Different symptoms and mortality in patients with different predisposing diseases. NPD: patients with no-predisposing disease; HBV: hepatitis B virus; LC: liver cirrhosis; DM: diabetes mellitus; CKD: chronic kidney disease; SOT: solid organ transplantation; CTD: connective tissue disease; CHD: chronic hematological disease; Malig: malignancy
India ink-negative patients had divergent clinical features from India ink-positive patients
The major symptoms of fever, headache, and nausea were compared between each subgroup. The frequencies of headache, fever and nausea were 66.7% (78/117), 61.5% (72/117) and 18.8% (22/117) in India ink-negative patients and 82.0% (228/278), 60.4% (168/278) and 29.9% (83/278) in India ink-positive patients (P = 0.001, P = 0.837 and P = 0.022, respectively).
We compared the initial CSF profiles between 117 India ink-negative patients and 278 India ink-positive patients among the antifungal-naïve patients. The India ink-negative patients had a significantly higher CSF glucose level [mmol/L; 2.6 ± 1.5 vs. 2.1 ± 1.4, P = 0.005] (Fig. 3a), total protein level [g/L; 1.1 (0.7–1.8) vs. 0.9 (0.6–1.2), P = 0.002] (Fig. 3b) and CSF WBC count [×106/L; 121.9 (36.5–210.0) vs. 67.5 (18.0-140.0), P = 0.001] (Fig. 3c) but an obviously lower initial ICP (mmH2O; 185.0 (130.0-250.0) vs. 270.0 (191.0-397.5), P < 0.001) (Fig. 3d). No difference was found in CSF chlorine levels between the two groups (P = 0.760).
Fig. 3.
Cerebrospinal fluid profiles in patients with negative India ink and positive India ink. CSF: cerebrospinal fluid; WBC: white blood cell; ICP: intracranial pressure; -:negative India ink; +: positive India ink
The rate of patients with ICP ≥ 300.0 mmH2O was 14.9% (17/114) and with VP shunt was 4.3% (5/117) in India ink-negative patients, which were lower than 41.0% (114/278) and 10.8% (30/278) in India ink-positive patients (P < 0.001 and P = 0.037, respectively).
The 90-day poor outcome of patients was 9.3% in India ink-negative patients and 24.5% in India ink-positive patients (Log-rank P = 0.003) (Fig. 4a). Our data indicated that a high ICR ≥ 300.0 mmH2O [OR: 4.8(1.1–21.5), P = 0.040] was associated with 90-day poor outcome in the unadjusted model among India ink-negative patients. Hemoglobin < 110.0 g/L [OR: 3.2(0.8–13.6), P = 0.107], CRP ≥ 10.0 mg/L [OR: 3.3 (0.8–13.8), P = 0.103], CSF WBC (< 20.0 × 106/L) [OR: 3.1(0.8–12.4), P = 0.110] and not undergoing a CrAg assay [OR: 3.5(0.9–15.0), P = 0.081] were marginally associated with 90-day poor outcome. However, ICP ≥ 300.0 mmH2O [adjusted odds ratio (AOR): 4.6 (1.0-20.7), P = 0.045] and not going CrAg Assay [AOR:4.4(1.0-18.4), P = 0.045] were independent risk factors for 90-day poor outcome in India ink-negative patients in the multivariate regression model (Table 2).
Fig. 4.
90-day poor outcome in patients with different India ink or culture status.+: positive; -:negative
Table 2.
Risk factors for 90-day poor outcome in antifungal Naive patients: negative India ink vs. positive India ink
| Factor | India Ink negative patients (N = 117) | India Ink positive patients (N = 278) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Poor outcome (N = 8) | Univariate | Multivariate | Poor outcome (N = 55) | Univariate | Multivariate | |||||
| OR (95% CI) | P | AOR (95% CI) | P | OR (95% CI) | P | AOR (95% CI) | P | |||
| Sex | 0.997 | |||||||||
| Male | 5/72 | 1 | ‒ | 30/159 | 1 | 0.647 | ‒ | |||
| Female | 3/45 | 1.0(0.2–4.2) | 25/119 | 0.8(0.5–1.5) | ||||||
| Age (years) | 0.679 | |||||||||
| < 50.0 | 2/34 | 1 | ‒ | 24/116 | 1 | 0.786 | ‒ | |||
| ≥ 50.0 | 6/83 | 1.4(0.3-7.0) | 31/162 | 1.1(0.6–1.8) | ||||||
| BMI | ||||||||||
| Missing data | - | 0.234 | 3/11 | |||||||
| < 20.0 | 4/35 | 2.3(0.6–9.3) | 18/100 | 1.0(0.6–1.8) | 0.965 | ‒ | ||||
| ≥ 20.0 | 4/82 | 1 | 34/167 | 1 | ||||||
|
Serum albumin (g/L) |
||||||||||
| missing data | 0/2 | 3/4 | ||||||||
| <35.0 | 4/46 | 1.6(0.4–6.7) | 0.472 | 17/69 | 1.6(0.9–2.9) | 0.081 | 0.141 | |||
| ≥35.0 | 4/69 | 1 | 35/205 | 1 | ||||||
| CRP (mg/L) | 0.103 | |||||||||
| Missing data | - | - | 0.068 | 0/1 | - | |||||
| < 10.0 | 3/79 | 1 | 1 | 28/153 | 1 | 0.293 | ||||
| ≥ 10.0 | 5/38 | 3.3(0.8–13.8) | 3.8(0.9–15.9) | 27/123 | 1.5(0.9–2.7) | |||||
| WBC (×10 9 ) | 0.649 | |||||||||
| Missing data | - | - | 4/4 | - | 0.112 | 0.264 | ||||
| < 10.0 | 7/95 | 1 | 32/197 | 1 | ‒ | |||||
| ≥ 10.0 | 1/21 | 0.6(0.1-5.0) | 19/77 | 1.6(0.9–2.7) | ||||||
| Hemoglobin (g/L) | ||||||||||
| Missing data | 0/1 | 4/4 | ||||||||
| < 110.0 | 5/39 | 3.2(0.8–13.6) | 0.107 | 0.290 | 13/70 | 0.9(0.5–1.6) | 0.688 | |||
| ≥ 110.0 | 3/77 | 1 | 38/204 | 1 | ||||||
| ICP(mmH2O) | ||||||||||
| Missing data | - | |||||||||
| < 300.0 | 5/100 | 1 | 1 | 0.045 | 30/164 | 1 | ||||
| ≥ 300.0 | 3/17 | 4.8(1.1–21.5) | 0.040 | 4.6(1.0-20.7) | 25/114 | 1.2(0.7–2.1) | 0.412 | |||
| CSF glucose (mmol/L) | ||||||||||
| Missing data | - | 0/1 | ||||||||
| < 2.0 | 4/45 | 1.9(0.5–7.6) | 0.364 | 20/141 | 2.0(1.1–3.4) | 0.017 | 1.9(1.1–3.3) | 0.022 | ||
| ≥ 2.0 | 4/72 | 1 | 35/136 | 1 | 1 | |||||
| CSF WBC (×106/L) | ||||||||||
| Missing data | - | 0/2 | ||||||||
| < 20.0 | 4/26 | 3.1(0.8–12.4) | 0.110 | 20/79 | 1.4(0.8–2.4) | 0.249 | ||||
| ≥ 20.0 | 4/91 | 1 | 35/197 | 1 | ||||||
| CrAg assay | ||||||||||
| Yes | 3/82 | 1 | 1 | 22/98 | 1 | |||||
| No | 5/35 | 3.5(0.9–15.0) | 0.081 | 4.4(1.0-18.4) | 0.045 | 33/180 | 0.7(0.4–1.3) | 0.286 | ||
| Culture result | ||||||||||
| positive | 6/77 | 1.5(0.3–7.5) | 0.611 | 41/221 | 0.6(0.4–1.2) | 0.161 | 0.156 | |||
| negative | 2/40 | 1 | 14/57 | 1 | ||||||
| Mental change | 0.549 | |||||||||
| No | 7/106 | 1 | 41/243 | 1 | 1 | < 0.001 | ||||
| Yes | 1/11 | 1.9(0.3–16.0) | 14/35 | 3.5(1.9–6.4) | < 0.001 | 3.4(1.8–6.2) | ||||
| Treatment | 0.688 | |||||||||
| AmB + 5FC ± Flu | 3/46 | 1 | 30/159 | 1 | 0.282 | |||||
| Other regimen | 5/71 | 1.3(0.3–5.6) | 25/119 | 1.3(0.8–2.3) | ||||||
5FC: flucytosine; AmB: amphotericin B; BMI: body mass index; CrAg: cryptococcal antigen; CRP: C reactive protein; CSF: cerebrospinal fluid; Flu: fluconazole; ICP: intracranial pressure; WBC: white blood cell
Regarding India ink-positive patients, we found that mental change [OR: 3.5(1.9–6.4), P < 0.001], CSF glucose level (< 2.0 mmol/L) [OR: 2.0 (1.1–3.4), P = 0.017] were associated with 90-day poor outcome. Serum albumin < 35.0 g/L [OR: 1.6 (0.9–2.9), P = 0.081], WBC count (×109) [OR: 1.6 (0.9–2.7), P = 0.112] were weakly associated with 90-day poor outcome. In the multivariate model, a lower CSF glucose level (< 2.0 mmol/L) [AOR: 1.9(1.1–3.3), P = 0.022] and mental change [AOR: 3.4 (1.8–6.2), P < 0.001] were risk factors for 90-day poor outcome in India ink-positive patients (Table 2).
Different clinical features between culture-negative and culture-positive patients
The frequencies of headache, fever and nausea were 72.2% (70/97), 54.6% (53/97) and 26.8% (26/97) in culture-negative patients and 79.2% (236/298), 62.8% (187/298) and 26.5% (79/298) in culture-positive patients, respectively (P = 0.150, P = 0.155 and P = 0.955).
We compared the CSF profiles of the patients with negative and positive cultures. The CSF glucose level was 2.9 ± 1.6 mmol/L in the culture-negative patients and 2.1 ± 1.3 mmol/L in the culture-positive patients (P < 0.001) (Fig. 5a). No significant difference was found for the total protein level [g/L; 0.8 (0.5–1.4) vs. 1.0 (0.5–1.4), P = 0.095] (Fig. 5b) or CSF WBC count [×106/L; 70.0 (12.8-187.5) vs. 80.0 (20.0-180.0), P = 0.236] (Fig. 5c) between the culture-negative and culture-positive patients. The ICP of 215.0 (150.0-277.3) mmH2O in the culture-negative patients was markedly lower than that of 250.0 (180.0-360.0) mmH2O in the culture-positive patients (P = 0.006) (Fig. 5d). Likewise, the rate of patients with ICP ≥ 300.0 mmH2O was 22.9% (22/96) among culture-negative patients and 36.8% (109/296) among the 278 culture-positive patients (P = 0.012), but no difference was found in VP shunts (9.3% vs. 8.7%, P = 0.868).
Fig. 5.
Cerebrospinal fluid profiles in patients with negative culture and positive culture CSF: cerebrospinal fluid; WBC: white blood cell; ICP: intracranial pressure; -:negative culture; +: positive culture
The 90-day poor outcome rate was 21.7% in culture-negative patients and 19.8% in culture-positive patients (log-rank P = 0. 579) (Fig. 4b).
In the univariate model, positive India ink [OR: 5.2 (1.2–23.1), P = 0.029] was associated with 90-day poor outcome in culture negative patients. Not undergoing the CrAg assay [OR: 2.5 (0.9–6.6), P = 0.072], a low CSF WBC count (< 20 × 106/L) [OR: 2.2 (0.8–5.8), P = 0.119] and mental change [OR: 2.3(0.7–7.2), P = 0.121] were nearly associated with 90-day mortality in culture-negative patients. In the multivariate Cox proportional hazards model, a low CSF WBC count (< 20 × 106/L) [AOR: 2.7 (1.0-7.2), P = 0.047] and postitive India Ink [ AOR: 5.3(1.2–23.5), P = 0.028] were an independent risk factors for a poor 90-daymortality in culture-negative patients.
Additionally, CRP (≥ 10.0 mg/L) [OR:2.2(1.2–3.9), P = 0.009], ICP ≥ 300.0 mmH2O [OR: 2.0 (1.1–3.5), P = 0.021], CSF glucose level (< 2.0 mmol/L) [OR: 2.5 (1.4–4.7), P = 0.004], mental change [OR: 4.0(2.0-9.9), P = 0.001] were associated with 90-day poor outcome. Furthermore, serum albumin < 35.0 g/L [OR: 1.7 (1.0-3.3), P = 0.068], WBC count (×109) [OR: 1.6 (0.9-3.0), P = 0.102] and positive India Ink [OR:2.3(1.0-5.4), P = 0.057] were weakly associated with 90-day mortality. In the multivariate model, we found that CRP (≥ 10.0 mg/L) [OR:2.1(1.2–4.6), P = 0.012], ICP ≥ 300.0 mmH2O [OR: 1.9 (1.1–3.5), P = 0.022], CSF glucose level (< 2.0 mmol/L) [OR: 2.4 (1.3–4.5), P = 0.008] and mental change [OR: 3.1(1.6–6.2), P = 0.001] were independent risk factors for 90-day mortality among culture-positive patients (Table 3).
Table 3.
Risk factors for 90-day poor outcome in antifungal naïve CM patients: negative culture vs. positive culture
| Factor | Culture negative patients (N = 97) | Positive culture patients (N = 298) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Poor outcome (N = 16) | Univariate | Multivariate | Poor outcome (N = 47) | Univariate | Multivariate | |||||
| OR (95% CI) | P | AOR (95% CI) | P | OR (95% CI) | P | AOR (95% CI) | P | |||
| Sex | 0.771 | |||||||||
| Male | 9/57 | 0.9(0.3–2.3) | ‒ | 26/174 | 0.9(0.5–1.5) | 0.607 | ‒ | |||
| Female | 7/40 | 1 | 21/124 | 1 | ||||||
| Age (years) | 0.612 | |||||||||
| < 50.0 | 6/29 | 1 | ‒ | 20/121 | 1 | 0.817 | ||||
| ≥ 50.0 | 10/68 | 0.8(0.3–2.1) | 27/177 | 1.1(0.6-2.0) | ||||||
| Serum albumin(g/L) | ||||||||||
| Missing Data | 1/3 | 2/2 | ||||||||
| < 35.0 | 5/31 | 1.2(0.5–3.6) | 0.622 | 16/84 | 1.7(1.0-3.3) | 0.068 | 0.451 | |||
| ≥ 35.0 | 10/63 | 1 | 29/211 | 1 | ||||||
| BMI | ||||||||||
| Missing data | 0/5 | 0.611 | 3/7 | |||||||
| < 20.0 | 4/30 | 0.7(0.2–2.3) | ‒ | 18/105 | 1.4(0.7–2.5) | 0.328 | ||||
| ≥ 20.0 | 12/62 | 1 | 26/185 | 1 | ||||||
| CRP (mg/L) | 0.346 | |||||||||
| Missing data | - | - | - | 0/4 | - | |||||
| < 10.0 | 11/55 | 1 | 20/174 | 1 | 0.009 | 1 | 0.012 | |||
| ≥ 10.0 | 5/42 | 0.6(0.2–1.7) | 27/119 | 2.2(1.2–3.9) | 2.1(1.2–4.6) | |||||
| WBC (×10 9 ) | 0.646 | - | ||||||||
| Missing data | 1/1 | - | 3/3 | - | 0.102 | |||||
| < 10.0 | 11/75 | 1 | 28/217 | 1 | 0.275 | |||||
| ≥ 10.0 | 4/21 | 1.3(0.4–4.1) | 16/77 | 1.6(0.9-3.0) | ||||||
| Hemoglobin (g/L) | ||||||||||
| Missing data | 1/1 | 3/3 | ||||||||
| < 110.0 | 3/25 | 0.6(0.2–2.2) | 0.462 | 15/84 | 1.2(0.6–2.2) | 0.602 | ||||
| ≥ 110.0 | 12/71 | 1 | 1 | 29/210 | 1 | |||||
| India ink | 0.029 | 0.028 | 0.238 | |||||||
| Missing data | - | - | - | |||||||
| postive | 17/57 | 5.2(1.2–23.1) | 5.3(1.2–23.5) | 41/221 | 2.3(1.0-5.4) | 0.057 | ||||
| Negative | 2/40 | 1 | 1 | 6/77 | 1 | |||||
| CrAg assay | ||||||||||
| No | 8/29 | 2.5(0.9–6.6) | 0.072 | 0.622 | 30/186 | 1.0(0.5–1.8) | 0.980 | |||
| Yes | 8/68 | 1 | 17/112 | 1 | ||||||
| ICP (mmH2O) | ||||||||||
| 0/1 | 1/1 | |||||||||
| ≥ 300.0 | 4/22 | 1.2(0.4–3.9) | 0.701 | 22/187 | 2.0(1.1–3.5) | 0.021 | 1.9(1.1–3.5) | 0.022 | ||
| < 300.0 | 12/74 | 1 | 24/109 | 1 | 1 | |||||
| CSF glucose (mmol/L) | ||||||||||
| Missing data | 0/1 | |||||||||
| < 2.0 | 6/27 | 1.5(0.6–4.3) | 0.381 | 33/154 | 2.5(1.4–4.7) | 0.004 | 2.4(1.3–4.5) | 0.008 | ||
| ≥ 2.0 | 10/69 | 1 | 14/144 | 1 | 1 | |||||
| CSF WBC (×106/L) | ||||||||||
| Missing data | 0/2 | |||||||||
| < 20.0 | 8/29 | 2.2(0.8–5.8) | 0.119 | 2.7(1.0-7.2) | 0.047 | 16/76 | 1.4(0.8–2.6) | 0.245 | 0.424 | |
| ≥ 20.0 | 8/68 | 1 | 1 | 31/220 | 1 | |||||
| Mental change | 0.145 | |||||||||
| No | 12/82 | 1 | 0.121 | 36/267 | 1 | 1 | 0.001 | |||
| Yes | 4/15 | 2.3(0.7–7.2) | 11/31 | 4.0(2.0-9.9) | 0.001 | 3.1(1.6–6.2) | ||||
| Treatment | 0.661 | |||||||||
| AmB + 5FC ± Flu | 9/44 | 1 | 24/161 | 1 | 0.319 | |||||
| Other regimen | 7/53 | 0.8(0.3–2.2) | 23/137 | 1.3(0.8–2.4) | ||||||
BMI: body mass index; CRP: C reactive protein; WBC: white blood cell; AmB: amphotericin B; 5FC: flucytosine; Flu: fluconazole
Discussion
In the present study, we evaluated CM patients with negative India ink results and negative CSF cultures and found the following: (1) compared with India ink-positive patients, India ink-negative patients had a lower frequency of headache, nearly normal CSF chemical profiles, lower initial ICP, lower likelihood of VP shunts and a high 90-day better outcome, and (2) in contrast to culture-positive patients, CSF culture-negative patients merely displayed relatively lower ICP and higher CSF glucose levels. It seemed that culture-negative patients had a similar 90-day clinical outcome to culture-positive patients.
We found that India ink/culture status had drastic effects on the CSF profile of patients. Glucose levels were higher in India ink-/culture-negative patients than in India ink-/culture-positive patients. Moreover, the ICP in India ink- and culture-negative patients was obviously lower than that in India ink- and culture-positive patients. These data supported that CSF fungal burden was positively related to high ICP [20]. Our data indicated high fungal burden was negatively associated with low CSF glucose level [7], indicating that a high cryptococcal burden might accelerate glycolysis and cause abnormalities in glucose transport [21]. Our data were also consistent with those of previous studies that showed that a high CSF fungal burden was an important contributor to high ICP and mortality [7, 15, 20].
CSF India ink staining results were profoundly associated with the outcome of patients. The 90-day poor outcome rate was 9.3% in India ink-negative patients and 24.5% in India ink-positive patients (Log-rank P = 0.003). In contrast, it seemed that culture results had less association with the outcome of patients. Previous studies also indicated that 2-week CSF culture sterile was not associated with 12-week survival rate [18, 19].These data suggested that India ink-negative patients were at low risk for mortality and VP shunt. Notably, our data emphasized that not undergoing the CrAg assay was associated with increased mortality in India Ink negative patients, suggesting that early diagnosis of CM is critical for saving lives of those population. Usually, a low fungal burden is associated with low ICP and low mortality in CM patients [22].
Our study also indicated that the optimal regimen for HIV-negative CM patients was debatable. The AmB + 5FC regimen was not associated with an increased survival rate in HIV-negative patients in our study, which was consistent with previous studies showing that the AmB + 5FC regimen did not display superior Cryptococcus clearance and survival rates compared with the fluconazole + 5FC regimen in HIV-negative patients [7, 26]. A recent study including 523 HIV-negative CM patients also did not observe that the AmB + 5FC regimen was a determinant of patient mortality [27]. We speculated that a highly active antifungal regimen, VP shunt and even mannitol administration were key management strategies for high ICP and the treatment of CM [17, 22, 23]. However, the most studies didn’t disclose the effects of different predisposing diseases and heterogeneous immune state on mortality. Thus, an even large cohort including enough patients with predisposing diseases are needed to elucidate the relationships between predisposing diseases, antifungal regimen and mortality.
Of note, the non-AmB + 5FC regimen was not related to high mortality in India ink- and culture-negative patients, even in the unadjusted model. Our research prompts an interesting question: should the AmB + 5FC regimen be recommended for HIV-negative patients (especially for patients with negative India ink results) as a preferred induction treatment? Some studies have argued that a highly potential antifungal regimen of AmB + 5FC should be administered to CM patients with a high fungal burden [7, 9]. The present study clearly showed that the rate of 90-day mortality and VP shunt was relatively low in patients with a negative India ink test, revealing that patients with a hint of Cryptococcus in the CSF were at low risk for mortality. Although the AmB + 5FC regimen is associated with the fast clearance of Cryptococcus in the CSF [23], it is also associated with severe side effects, such as electrolyte abnormalities, nephrotoxicity, anemia and thrombosis [24, 25]. Due to fewer side effects and better tolerance, azoles (such as fluconazole) can be considered as alternative induction treatments in CM patients with negative India ink results.
In addition, we found that a low CSF glucose level, high serum CRP, high ICP and mental change were independent risk factors for poor outcome in culture-positive patients. These results supported the notion that dysfunction of glucose metabolism in the CSF compartment had a great impact on the mortality of patients [7, 28]. Similar to HIV-positive patients [9, 20], our data also illustrated that high ICP and mental change predicted the outcome of HIV-negative CM patients.
There were some limitations to our study. First, the relationship between CSF CrAg titers and patient outcomes was not determined in our study. Previous studies indicated that high CSF CrAg titers were associated with high fungal burden and high mortality in both HIV-infected and HIV-uninfected CM patients [12, 29]. Second, the Cryptococcus species (such as Cryptococcus neoformans or Cryptococcus gattii) were not determined. However, studies from China have indicated that Cryptococcus gattii is rare and occurs sparsely [30, 31], and there were only 1.7% Cryptococcus gattii isolation in Zhejiang Province [32]. Third, the impacts of predisposing diseases on the mortality of patients were not fully discussed. However, many studies, including our present study, have indicated that CM patients with different predisposing diseases have similar mortality [7, 15, 33, 34].
In conclusion, our data suggested that CM patients with different cryptococcal assay results had different clinical features. Patients with negative India ink results were at low risk for 90-day mortality. Individualized therapeutic strategies should be considered in patients with different cryptococcal statuses.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
We thank all the staffs contributing to this work at each Hospital participating in this study.
Author contributions
LJX designed the study and wrote the manuscript. LDC and GC collected the data and performed the study. SSY and JYQ rechecked the data. LDC and GC analyzed and interpreted the data. JYQ performed the follow-ups. GCH supervised the study. All authors reviewed and approved the final manuscript.
Funding
The work was supported by the National Key R&D Program of China, 2021YFC2301900-2021YFC2301901. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Data availability
The original data are available from Supplementary material.
Declarations
Ethical approval and consent to participate
The study protocol was in accordance with the 1975 Declaration of Helsinki guidelines and was approved by the Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China (No. 2021 − 599). All data were analyzed anonymously. The committee waived the requirement for written informed consent from the participants.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Longda Chen and Gong Chen joint the first author.
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Data Availability Statement
The original data are available from Supplementary material.