CD4 Cell Count Threshold for Cryptococcal Antigen Screening of HIV-Infected Individuals: A Systematic Review and Meta-analysis
CD4 Cell Count Threshold for Cryptococcal Antigen Screening of HIV-Infected Individuals: A Systematic Review and Meta-analysis
Nathan Ford 2 3
Zara Shubber 1 2
Joseph N. Jarvis 0 2 6 7
Tom Chiller 2 5
Greg Greene 2 5
Chantal Migone 2 3
Marco Vitoria 2 3
Meg Doherty 2 3
Graeme Meintjes 2 4 8
0 Botswana-UPenn Partnership
1 Department of Infectious Disease Epidemiology, Imperial College London , United Kingdom
2 Geneva , Switzerland
3 HIV Department, World Health Organization , Geneva , Switzerland
4 Wellcome Trust Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine
5 Mycotic Diseases Branch, Centers for Disease Control and Prevention , Atlanta , Georgia
6 Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine , United Kingdom
7 Botswana Harvard AIDS Institute Partnership , Gaborone , Botswana
8 Department of Medicine, Faculty of Health Sciences, University of Cape Town , South Africa
Background. Current guidelines recommend screening all people living with human immunodeficiency virus (PLHIV) who have a CD4 count ≤100 cells/µL for cryptococcal antigen (CrAg) to identify those patients who could benefit from preemptive fluconazole treatment prior to the onset of meningitis. We conducted a systematic review to assess the prevalence of CrAg positivity at different CD4 cell counts. Methods. We searched 4 databases and abstracts from 3 conferences up to 1 September 2017 for studies reporting prevalence of CrAg positivity according to CD4 cell count strata. Prevalence estimates were pooled using random effects models. Results. Sixty studies met our inclusion criteria. The pooled prevalence of cryptococcal antigenemia was 6.5% (95% confidence interval [CI], 5.7%-7.3%; 54 studies) among patients with CD4 count ≤100 cells/µL and 2.0% (95% CI, 1.2%-2.7%; 21 studies) among patients with CD4 count 101-200 cells/µL. Twenty-one studies provided sufficient information to compare CrAg prevalence per strata; overall, 18.6% (95% CI, 15.4%-22.2%) of the CrAg-positive cases identified at ≤200 cells/µL (n = 11 823) were identified among individuals with a CD4 count 101-200 cells/µL. CrAg prevalence was higher among inpatients (9.8% [95% CI, 4.0%-15.5%]) compared with outpatients (6.3% [95% CI, 5.3%-7.4%]). Conclusions. The findings of this review support current recommendations to screen all PLHIV who have a CD4 count ≤100 cells/µL for CrAg and suggest that screening may be considered at CD4 cell count ≤200 cells/µL.
The burden of cryptococcal meningitis among people living
with human immunodeficiency virus (PLHIV) remains
substantial despite scale-up of antiretroviral therapy (ART) [
A recent review estimated that globally there were 223 100
incident cryptococcal meningitis cases (with 73% of the cases
occurring in sub-Saharan Africa), resulting in almost 200 000 deaths
in 2014 [
Current World Health Organization (WHO) guidelines
recommend screening all PLHIV who have a CD4 count ≤100 cells/
µL for cryptococcal antigen (CrAg) to identify those patients
with cryptococcal disease who could benefit from preemptive
fluconazole treatment prior to the onset of meningitis. CrAg may
be detected several weeks before clinical features of cryptococcal
meningitis become apparent [
]. Providing preemptive
fluconazole treatment during this period of antigenemia prior to onset
of meningitis symptoms has been found to be life saving and cost
effective across a range of settings [
]. Some countries have
chosen higher CD4 cell count thresholds for their cryptococcal
screening guidelines: Ethiopia has adopted a cutoff of 150 cells/
µL, whereas in Rwanda CrAg screening is done at a CD4 count of
Recent WHO guidelines advise that a CD4 threshold of
≤200 cells/µL be used to define patients who have advanced
HIV disease [
], and studies have suggested there may be
benefit to CrAg screening among PLHIV using a higher
CD4 count threshold of ≤200 cells/µL to identify additional
numbers of PLHIV at risk of developing cryptococcal
]. The current recommended threshold for CrAg
screening at CD4 count ≤100 cells/µL is based on evidence
from a limited number of studies, and there may be benefits
to simplifying screening strategies to target all patients with
advanced human immunodeficiency virus (HIV) disease. We
conducted a systematic review to assess prevalence of CrAg
positivity at CD4 count ≤100 cells/µL compared to 101–200
cells/µL across a range of settings.
Search Strategy and Selection Criteria
This systematic review was conducted according to the Preferred
Reporting Items for Systematic Reviews and Meta-Analyses
(PRISMA) guidelines . Using a study protocol
(available from the corresponding author), we sought randomized
and quasi-randomized controlled trials, and comparative and
noncomparative observational studies reporting prevalence of
CrAg positivity according to CD4 cell count strata.
Using a broad search strategy combining terms for HIV
infection and CrAg screening, 3 investigators (N. F., Z. S., C. M.),
working independently and in duplicate, screened titles and
abstracts from Medline via PubMed, Embase, and the Cochrane
library, from inception to 1 September 2017. Abstracts from
the International AIDS Society conferences, the Conferences
on Retroviruses and Opportunistic Infections, and the
International Conference on Cryptococcus and Cryptococcosis
were also screened from 2012 to 2017 to identify studies that
have been recently completed but not yet published in full.
Database searches were supplemented by screening
bibliographies of review articles and all included full-text articles. The
same investigators scanned all abstracts and full-text articles
and achieved consensus on final study inclusions.
Reasons for exclusions included studies using samples other
than serum, plasma, or whole blood. If studies included patients
with a history of cryptococcal disease or overt clinical
meningitis, then these patients were excluded from the study
denominators and numerators included in this review; where it was not
possible to remove these patients from the study population, the
studies were only included if <10% of patients met these
criteria. No language or geographical restrictions were applied.
The same 3 investigators extracted data following a predefined
protocol and using a standardized and piloted extraction form.
Study characteristics included design, year, population,
location, ART status, CrAg positivity by CD4 cell count stratum, and
active tuberculosis (TB) infection. Where reported, outcomes for
CrAg-positive patients who received or did not receive
fluconazole were also extracted. Additional information was extracted
to inform an assessment of risk of bias and the certainty of the
evidence using the Grading of Recommendations Assessment,
Development, and Evaluation (GRADE) approach [
To estimate CrAg prevalence by CD4 cell count stratum, point
estimates and corresponding 95% confidence intervals (CIs) were
calculated and data were pooled using random-effects meta-analysis
], following data transformation [
]. The same approach was
used to summarize clinical outcomes among CrAg-positive patients
started and not started on fluconazole. Prevalence odds ratios were
calculated to compare diagnostic yield by CD4 cell count strata (≤100
vs 101–200 cells/µL) using random effects models. Heterogeneity
was assessed though visual inspection of forest plots and subgroup
analyses to examine potential differences by geographical region,
clinical setting, type of CrAg screening test used, and sample type.
We analyzed all data with Stata version 13.0 software.
Characteristics of Included Studies
From an initial screen of 540 titles, 60 studies were included in
this review (Figure 1) [
6, 9–12, 18–70
]. Among these, 40 were
prospective studies (including one randomized trial) and 20
were retrospective studies; 42 studies were published in full,
16 were abstracts, and additional unpublished data were
provided from Médecins Sans Frontières (MSF)–supported HIV
programs in Kenya and the Democratic Republic of Congo.
Data came from 28 countries, with the majority of studies (41)
carried out in Africa. Median age of patients ranged from 30
to 47 years, and the proportion who were female ranged from
20% to 74%. Date of study end ranged from 2013–2017
(median 2014). Most studies (41 studies [66%]) used sera as the
sample type and a lateral flow assay (34 studies [57%]).
Thirtytwo studies reported that all patients screened (n = 18 657) were
ART naive, while 16 studies (n = 6950) reported that a
proportion of patients were ART experienced (median, 41.7%
[interquartile range], 18.4%–72.4%) (Supplementary Appendix).
Risk of bias overall was assessed as being moderate
(Supplementary Appendix). The majority of studies used a
prospective study design (40 studies), were published in full (42
studies), and had <10% missing data (52 studies). Only 2 studies
reported blinding of investigators and only 3 studies reported
random sampling of patients. In subgroup analysis, none of
these risk of bias indicators influenced CrAg prevalence
estimates. Overall, the certainty of evidence was rated as moderate.
Prevalence of Cryptococcal Antigenemia
The pooled prevalence of cryptococcal antigenemia as
determined by CrAg positivity was 6.5% (95% CI, 5.7%–7.3%; 54
studies) among patients with CD4 count ≤100 cells/µL and
2.0% (95% CI, 1.2%–2.7%; 21 studies) among patients with
CD4 count 101–200 cells/µL (Figures 2 and 3).
Twenty-one studies provided sufficient information to
compare CrAg prevalence at CD4 count ≤100 cells/µL vs 101–200
cells/µL within each study. The prevalence odds ratio
comparing CD4 count ≤100 cells/µL and CD4 count 101–200 cells/µL
was 2.5 (95% CI, 1.9–3.3) (Figure 4). Overall, 18.6% (95% CI,
15.4%–22.2%) of the total CrAg-positive cases identified in this
sample of patients with CD4 ≤200 cells/µL (n = 11 823) were
among individuals with a CD4 count 101–200 cells/µL.
Among patients with CD4 count ≤100 cells/µL, CrAg
positivity ranged from 0.3% (95% CI, 0.3%–2.5%) in Iran to 22.8%
(95% CI, 17%–29.2%) in DRC (MSF, unpublished data). In
subgroup analysis, there was substantial variability by geographical
Cryptococcal Antigen Screening • CID 2018:66 (Suppl 2) • S153
region, with CrAg prevalence highest in the Africa Region
(6.7% [95% CI, 5.7%–7.6%]), the South-East Asia Region (6.9%
[95% CI, 4.4%–9.5%]), and the Western Pacific Region (13.3%
[95% CI, 7.4%–19.1%]). CrAg prevalence was also higher
among inpatients (9.8% [95% CI, 4.0%–15.5%]) than
outpatients (6.3% [95% CI, 5.3%–7.4%]). A higher prevalence was
also seen in studies that used nonfrozen samples (7.8% [95%
CI, 6.5%–9.0%]) rather than stored samples (4.7% [95% CI,
3.8%–5.5%]). CrAg prevalence was similar comparing studies
that only enrolled ART-naive patients and those that included
both ART-naive and ART-experienced patients (Figure 5).
Nineteen studies reported outcomes among 353 CrAg-positive,
asymptomatic PLHIV who were started on fluconazole
9, 11, 19, 21, 24–27, 31, 38, 42, 52, 54, 55, 60–62, 64, 70
with clinical outcomes from one study [
] reported in a
separate report [
]. Median follow-up time was 9 months
(interquartile range, 6–12 months). Of these, 34 (9.6%) of patients
had died, among whom none were documented to have died
of cryptococcal meningitis. Nineteen (5.4%) developed
incident cryptococcal disease. Fourteen studies reported outcomes
among 118 CrAg-positive, asymptomatic PLHIV who were not
started on fluconazole prophylaxis [9, 11, 12, 20, 21, 25–27, 31,
S154 • CID 2018:66 (Suppl 2) • Ford et al
38, 41, 42, 70, 71]. Of these, 22 (18.6%) had died, among whom
2 were documented to have died of cryptococcal meningitis; 3
others developed incident cryptococcal disease.
Thirteen studies reported the prevalence of TB disease among
patients who were CrAg positive, using different TB screening
9, 11, 19–22, 26, 28, 33, 41, 46, 57, 72
]. Among 234
patients screened CrAg positive, 45 also were diagnosed with
TB, giving an overall prevalence of coexistent disease of 19.2%
(95% CI, 14.4%–24.9%).
Cryptococcal meningitis remains an important cause of morbidity
and mortality among people with HIV, despite major improvements
in access to HIV testing and treatment services [
]. This is largely
explained by an enduring burden of advanced HIV disease, either
because people present late for diagnosis and care or, increasingly,
because PLHIV interrupt ART for a period during which time their
CD4 cell count drops, placing them at risk of major opportunistic
infections including cryptococcal meningitis [
This review estimated prevalence from available studies, by
country and region, and found a high prevalence of CrAg
positivity among people with advanced HIV disease that, consistent with
expectations, was higher among those with a lower CD4 cell count.
These findings support current guidance to screen all individuals
presenting for care with a CD4 count ≤100 cells/µL. Prevalence
at CD4 count ≤100 cells/µL was highest in the Africa, South-East
Asia, and Western Pacific regions. The finding that one fifth of
CrAg-positive patients were also found to have TB supports the
inclusion of CrAg and TB testing as part of a package to manage
advanced HIV disease.
This review further suggests that there may be additional
benefit to screening individuals at CD4 cell count up to 200 cells/µL,
depending on availability of resources and considering the
practical advantage of providing the same package of care to all patients
with advanced HIV disease within a public health approach.
Almost one-fifth of CrAg-positive cases identified at CD4 count
≤200 cells/µL are identified at CD4 between 101 and 200 cells/
µL. Cost-effectiveness analyses have so far focused on the benefit
of CrAg screening at CD4 count ≤100 cells/µL, and there is some
evidence of benefit down to a prevalence of 0.6% of cryptococcal
]. Further cost-effectiveness research is needed to
assess the value of screening at a higher CD4 cell count threshold
of 200 cells/µL, which has already been suggested to be cost
saving if carried out in inpatient settings [
There is a growing evidence base supporting the clinical
benefit and cost effectiveness of CrAg screening in
combination with enhanced ART adherence and
delivery interventions. A trial conducted in the United Republic of Tanzania
and Zambia randomized 1999 ART-naive adults living with
HIV with a CD4 count <200 cells/µL to receive enhanced
clinic-based care with CrAg screening and preemptive
antifungal treatment for those who were CrAg positive;
importantly, additional community support including ART
delivery and adherence counseling was provided to the
intervention group. The trial reported a 28% reduction in
mortality (13% vs 18%) among people receiving the
intervention compared to standard care [
]. In an unpublished
post hoc analysis, a statistically significant mortality
reduction was found in both people with a CD4 count <100 cells/
Cryptococcal Antigen Screening • CID 2018:66 (Suppl 2) • S155
Figure 3. Prevalence of CrAg positivity among patients with CD4 count 100–200 cells/μL. Abbreviations: CI, confidence interval; CrAg, cryptococcal antigen; DRC,
Democratic Republic of the Congo; MSF, Médecins Sans Frontières.
S156 • CID 2018:66 (Suppl 2) • Ford et al
Figure 5. Factors associated with CrAg positivity at CD4 count ≤100 cells/μL. Abbreviations: ART, antiretroviral therapy; AFRO, Africa Region; AMRO N, North America
Region; AMRO S, South America Region; CI, confidence interval; CrAg, cryptococcal antigen; EMRO, Mediterranean Region; EURO, Europe Region; SEARO, South-East Asia
Region; WPRO, Western Pacific Region.
µL (mortality rate ratio, 0.75 [95% CI, .58–.95]) and those
with a CD4 cell count of 101–200 cells/µL (mortality rate
ratio, 0.56 [95% CI, .32–.97]).
A recent study from South Africa reported the numbers
of patients starting ART in 2016 at different CD4 cell count
thresholds using data from the national laboratory database
]. According to this analysis, 128 888 patients (16.8% of the
total) started ART at a CD4 count ≤100 cells/µL and 123 164
(16.1%) started at a CD4 count of 101–200 cells/µL. Applying
the pooled prevalence estimates from this review, 8249 patients
(95% CI, 7347–9280) would theoretically be identified as being
CrAg positive at a CD4 screening threshold of 100 cells/µL,
and an additional 2463 patients (95% CI, 1478–3325) would be
identified if a threshold of CD4 200 cells/µL were applied.
The majority of studies included in this review were
carried out in Africa, and the findings of this review are of
greatest relevance to settings with a high burden of HIV and
cryptococcal meningitis. Nevertheless, cryptococcal disease
remains an important cause of illness and death among
people with HIV in high-income settings, and the role of CrAg
screening and preemptive therapy should be considered in
these settings [
Strengths of this review include a broad and inclusive search
strategy that allowed for the identification of a large number
of studies for analysis. Heterogeneity was anticipated, and
explored using standard methods that increased confidence
in the overall findings. The main limitations to note are the
limited reporting of important information which may
influence CrAg prevalence, notably ART experience, which was
missing for one-fifth of studies included in this review, and
the limited reporting of clinical outcomes. Another limitation
relates to methodological quality, with a number of studies
being carried out retrospectively and only 3 studies
reporting random patient sampling. While methodological quality
did not appear to importantly influence the prevalence
estimates, future studies are encouraged to take steps to improve
This review highlights several directions for research,
including the cost effectiveness of screening at higher CD4 cell counts
and the appropriateness and cost effectiveness of screening
ART-experienced adults and adolescents with low CD4 counts.
In conclusion, the findings of this review support current
recommendations to screen all adults and adolescents who have a
CD4 count ≤100 cells/µL for CrAg, whether they are ART naive
or experienced, and provide preemptive fluconazole treatment
to those testing positive. Consideration should also be given to
screening at a higher CD4 count of ≤200 cells/µL in settings
where there are sufficient resources to implement such an
approach, or where a simplified package of care for advanced
disease is required based on a unified CD4 threshold.
Cryptococcal Antigen Screening • CID 2018:66 (Suppl 2) • S157
Supplementary materials are available at Clinical Infectious Diseases online.
Consisting of data provided by the authors to benefit the reader, the posted
materials are not copyedited and are the sole responsibility of the authors,
so questions or comments should be addressed to the corresponding author.
Acknowledgments. We thank Sayoki Mfinanga and Shabbar Jaffar
for providing mortality reduction data by CD4 count threshold from the
REMSTART trial, and David Maman for providing unpublished data from
Disclaimer. The findings and conclusions in this paper are those of
the author(s) and do not necessarily represent the views of the Centers for
Disease Control and Prevention (CDC) or the WHO.
Financial support. This work was supported by several sources of
funding to the HIV Department, mainly funding from the US President’s
Emergency Plan for AIDS Relief through the US CDC cooperative
agreement and the Bill & Melinda Gates Foundation. G. M. is supported by the
Wellcome Trust (grant number 098316) and the South African Research
Chairs Initiative of the Department of Science and Technology and
National Research Foundation of South Africa (grant number 64787).
J. N. J. is supported by the Penn Center for AIDS Research, a National
Institutes of Health–funded program (grant number P30 AI 045008). This
Supplement was supported by funds from the Bill & Melinda Gates
Supplement sponsorship. This article appears as part of the supplement
“Advanced HIV Disease,” sponsored by the World Health Organization.
Potential conflicts of interest. All authors: No reported conflicts of
interest. All authors have submitted the ICMJE Form for Disclosure of
Potential Conflicts of Interest. Conflicts that the editors consider relevant to
the content of the manuscript have been disclosed.
S158 • CID 2018:66 (Suppl 2) • Ford et al
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