The CD4 cell count at which to initiate HIV-associated cryptococcal antigen (CrAg) screening and pre-emptive antifungal treatment among CrAg positive persons may need to be raised to 200 cells/μL. Evidence based on a meta-analysis

Background: Current WHO guidelines (2018) recommend screening for cryptococcal antigen (CrAg) in HIV-infected persons with CD4<100 cells/μL, followed by pre-emptive antifungal therapy among CrAg positive (CrAg+) persons, to prevent Cryptococcal meningitis related deaths. The strategy may also be considered for those persons with a CD4 count of<200 cells/uL according the WHO guidelines. However, there remains little evidence for doing so in those HIV-infected persons with this CD4 cell count. Objective: We aimed to assess the necessity of CrAg screening and the efficacy of pre-emptive antifungal therapy in CrAg+ persons with CD4<200 cells/µL. Methods: We conducted a meta-analysis using data obtained from randomized controlled studies (RCTs) and cohort studies found in Pubmed, Web of Science, Cochrane Library and EMBASE/MEDLINE. Results: The pooled prevalence of CrAg positivity in HIV-infected persons with CD4<200 cells/µL was 5% (95%CI: 3-6). The incidence of CM in CrAg+ persons was 7- fold (7%, 95%CI: 4-10) that of CrAg negative (CrAg-) persons (1%, 95%CI: 0-1). Among CrAg+ persons who did not receive any treatment or only received placebo, the incidence of CM was 9% (95%CI: 5-13), whereas the incidence of CM among those who received antifungal therapy was 2% (95%CI: 0-3), a highly statistically significant reduction of 78% (RR: 6.03, 95%CI: 2.74-13.24, p<0.00001). Conclusions: In our meta-analysis, the incidence of CM in CrAg+ persons were significantly higher than in CrAg- persons with CD4<200 cells/µL. Furthermore, the incidence of CM was significantly reduced by pre-emptive antifungal therapy in CrAg+ persons with CD4<200 cells/µL.


Introduction
Cryptococcal meningitis (CM) continues to cause significant mortality in HIV-infected individuals (1,2) resulting in 181100 deaths globally each year (3). In resource-limited regions such as sub-Saharan Africa, 75% of HIV-related deaths are due to CM (3). However, cryptococcal antigen (CrAg) can be detected in blood several weeks to months (22 days on average) before the onset of signs and symptoms of meningitis (4,5) and therefore, can be used as a trigger for pre-emptive antifungal therapy in HIV-infected individuals with low CD4 cell counts. According to previous studies, pre-emptive antifungal therapy in CrAg+ persons is imperative to preventing death (6)(7)(8). Firstly, the prevalence of CrAg positivity among HIV-infected individuals can be considerable, ranging between 1% to 16% in several African and Southeast Asian countries (9), and among persons with CD4<100 cells/µL, the prevalence of CrAg positivity averages 7% with regional variation (3). Secondly, CrAg positivity resulted in a 20% increase in mortality at the time of antiretroviral therapy (ART) initiation (10), and the risk of CM in CrAg+ persons was as high as 25% during the first year of ART if fluconazole pre-emptive therapy was not prescribed (11,12).
According to the 2018 version of the WHO guidelines, routine CrAg screening and pre-emptive antifungal therapy are recommended in treatment-naive HIV persons with CD4<100 cells/µL (13). The guidelines also state that these strategies may also be considered for HIV-infected persons with CD4<200 cells/µL (13), suggesting that the evidence for the necessity of CrAg screening and the efficacy of implementing pre-emptive antifungal therapy in HIV persons with a higher CD4 cell count level is not sufficient enough to include them deterministically in these recommendations. Therefore, we conducted a meta-analysis to investigate whether routine CrAg screening and pre-emptive antifungal treatment in HIV-infected persons with <200 cells/µL would be beneficial.

Search strategy and article screening
We searched relevant English articles in Pubmed, Cochrane Library, MEDLINE/EMBASE and Web of Science from inception until the end of September 15 th 2018. The search terms we used were as follows: 'acquired immunodeficiency syndrome', "HIV", "AIDS", "cryptococcosis", and "prophylaxis".
We combined these terms by using "and" or "or". To avoid missing significant articles, we also screened references of previous meta-analyses and their included studies for eligibility.
Two reviewers (Yao Li, Yuanyuan Qin) independently screened all obtained articles by titles and abstracts. After removing ineligible articles by inclusion and exclusion criteria, the remaining articles were further selected by full-text reviewing.

2.
CrAg was tested for study subjects.

3.
Fluconazole or other azole medications were used as the intervention.

Exclusion criteria
We excluded articles if: (1) all of the study subjects were diagnosed with CM or asymptomatic CM; (2) sample size was less than 50; or (3) the incidence of CM and all-cause mortality was unreported.

Data extraction and quality assessment
The data we extracted included first author, publication year, type of study, study duration, location,  (14). The potential bias risk of the RCT was assessed using the Cochrane "risk of bias" tool (15).

Data analysis
The proportion of CrAg positivity, the incidence of CM, and all-cause mortality were performed by STATA 14 (Statacorp, Texas, USA) with a 95% confidence interval (95%CI). We used random-effects or fixed-effects models in Review manager 5.3 (The Nordic Cochrane Center, Copenhagen) to compare the incidence of CM and all-cause mortality in CrAg+ persons.
We evaluated statistical heterogeneity through visual inspection of forest plots. Statistical heterogeneity was also assessed by I² statistics (16), which was considered non-negligible if I 2 >50%.
Herein, random-model was applied if I 2 >50% and fixed-model was used when I 2 <50% (17). Reporting bias was assessed by examining asymmetry of funnel plots (16).
The protocol was registered at the International Prospective Register of Systematic Reviews (PROSPERO), and the registration number is CRD42018110980.

Results
In total, 490 articles were obtained from 4 databases, among which 276 were from Pubmed, 106 were from Web of Science, 12 were from Cochrane Library, and 96 were from MEDLINE/EMBASE. 82 of the 490 articles were RCTs or cohort studies. Additional 12 articles (RCTs or cohort studies) were extracted from references of previous meta-analyses and their included studies.
All the 94 RCTs or cohort studies were included for screening. Firstly, 9 articles (6 from Web of Science and 3 from MEDLINE/EMBASE) were reduplicative and were therefore excluded from the 94 articles. Next, after screening titles and abstracts, 43 of the remaining 85 articles were excluded.
Then, 25 articles were excluded from the remaining 42 articles after screening full-text, among which 1 article was news report, 3 articles reported patients with cryptococcal disease, 2 articles reported HIV-negative patients with cryptococcal antigenemia, 4 articles reported patients with CM or asymptomatic CM, 6 articles reported HIV-infected patients with negative CrAg, 1 article reported the epidemiology of cryptococcosis and 8 articles did not report CD4 cell counts or primary outcomes.
Finally, 17 articles were included in our meta-analysis.
The characteristics of the included 17 studies were shown in Table 1. The assessment of quality and potential risk bias showed that the following may contribute to clinical and methodological  .0%), which is shown in Figure 4A and Table 2.
Nine studies reported all-cause mortality in persons who received antifungal therapy ( ) between 1144 CrAg+ persons who received an azole drug and 1138 CrAg+ persons who received placebo or no intervention ( Figure 5).
In addition, we estimated and compared the prevalence of CrAg positivity, the incidence of CM and    (16). From the above results, it is clear that regardless of whether the CD4 count is less than 200 or 100 cells/µL, the incidence of CM is higher in CrAg+ persons than in CrAg-persons. Therefore, antifungal prophylaxis seems necessary for HIV-infected persons with cryptococcal antigenemia and CD4<200 cells/µL.

Discussion
Our results showed that the risk ratio of CM events among persons who received placebo or no intervention was significant higher than those who received antifungal therapy, suggesting that antifungal prophylaxis still significantly reduced the risk of CM events in CrAg + persons with a higher In conclusion, our meta-analysis found that in HIV-infected persons with CD4<200 cells/μL, the incidence of CM was higher in those CrAg+ than in those CrAg-, and that azole pre-emptive treatment significantly reduced the incidence of CM in CrAg+ persons with CD4<200 cells/μL. Our results support that the CD4 cell count at which to initiate HIV-associated CrAg screening and preemptive antifungal treatment among CrAg+ persons may need to be raised to 200 cells/μL.

-Ethics approval and consent to participate
Not applicable.

-Consent to publish
Not applicable.

-Availability of data and materials
All the data and materials were available from Pubmed, Cochrane Library, MEDLINE/EMBASE and Web of Science.

-Competing interests
All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

-Funding
This work was supported by the national science and technology major project of China during the

-Authors' Contributions
YL, XJH, HW, XFY and YKC conceived and designed the protocol and study. YL, YYQ, JHH and AXL identified studies to be screened. XJH and HC identified studies for eligibility, extracted data and assessed the methodology quality of included studies. YL performed the analysis with assistance from XJH, and YKC. All authors read and approved the final manuscript.

-Acknowledgements
Thanks to all authors for their contributions to this manuscript.   Figure 1 Flow chart of the study selection process.   All-cause mortality among CrAg+ and CrAg-persons with CD4<200 cells/μL. All-cause mortality among CrAg+ and CrAg-persons (A) and all-cause mortality among CrAg+ persons with antifungal therapy and without antifungal therapy (B).

Figure 5
Forest plots of incidence of CM and all-cause mortality. Forest plots of incidence of CM and all-cause mortality among CrAg + persons receiving azole vs. no intervention or placebo.

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