An observational prospective cohort study was conducted. Individuals with HIV-‐ associated CM that met study entry criteria were enrolled shortly after presentation
to hospital [section 3.2.6, Table 3.2]. A clinical assessment was made and markers of
clinical severity recorded [section 3.2.8; Table 3.3]. Host immune response was
examined in CSF and blood using flow cytometry and cytokine analysis [section 3.4].
Subjects were followed for 24 weeks to monitor for study endpoints [section 3.2.10;
Table 3.5]. Data were analyzed as detailed in section 3.2.14.
3.2.6Entry criteria and enrolment
Study staff identified hospitalized patients with cryptococcal meningitis through daily review of laboratory CSF results. Cryptococcal meningitis was defined as a positive
CSF culture identified as C. neoformans or a positive CSF cryptococcal antigen (CrAg)
detection assay with a titre ≥1:4. Individuals with CM were approached by a member of the study staff and assessed for eligibility based on the criteria detailed below
[Table 3.2]; previous episodes of cryptococcal meningitis were identified by
questioning the patient and checking previous laboratory results. Patients were recruited irrespective of ART status.
Inclusion Criteria Exclusion Criteria
HIV-‐1 infection Age ≥18 years
Cryptococcal meningitis* ≤2 doses of amphotericin B
Previous cryptococcal meningitis Taking immune modulating medication Contraindication to LP
Pregnancy
Table 3.2 Entry criteria for IICD study. *Diagnostic criteria for cryptococcal meningitis is detailed above [section 3.2.6]. Immune modulating medication: steroids, chemotherapy, etc.
3.2.7Consent
Potential participants were approached by a member of the study team and provided with verbal and written information in English or Xhosa (the most commonly spoken first language amongst patients admitted to these hospitals). Written informed consent was obtained in Xhosa or English (see Appendix B) and the study discussed with the patient’s clinical team to ensure they had no objections. Informed consent included permission to store samples for the current and future
projects and the sharing of information and samples with collaborators. Any patients who were illiterate or unable to sign their name were asked to make a mark witnessed by an individual not related to the study. If patients could not provide consent due to their medical condition (e.g. impaired consciousness), their next of kin was approached to provide surrogate consent and patients re-‐consented if their condition improved. This policy was adopted to ensure the clinical cohort was representative of hospitalized patients (i.e. Including patients with severe cryptococcal meningitis) and was specifically approved by the ethics committees at both University of Cape Town and Liverpool School of Tropical Medicine.
3.2.8Clinical assessment and data collection (cohort study)
All subjects had clinical data recorded anonymously on dedicated case record forms (CRFs), included baseline demographics, past medical and medication history (particularly ART or TB treatment), details of the presenting history, conscious level and presence of any abnormal neurology. This allowed an assessment of CM severity [Table 3.3].
Markers of disease severity
Altered consciousness (Glasgow Coma Scale<15) CSF opening pressure >30 cmH2O during admission CSF Fungal burden (CFU/ml CSF) [section 3.4.11].
Table 3.3 Markers of disease severity in cryptococcal meningitis
3.2.9CSF and blood sampling
Samples of cerebrospinal fluid (CSF) were obtained from lumbar punctures performed for clinical reasons (e.g. to measure opening pressure and drain CSF). Depending on the opening pressure, between 5-‐20mls of CSF was drained and
analyzed as detailed in Table 3.4. Cell count and other diagnostic tests were
performed in the hospital laboratory and the remaining CSF was placed on ice and transported to the research laboratory within 2 hours. Venepuncture was also
performed at study enrolment as detailed below [Table 3.4]. Twice weekly blood
tests were performed during hospital admission to monitor for amphotericin B toxicity [Table 3.4; section 3.5.2].
Day CSF Blood 0 Cell count, protein, glucose (0.5mls)*
Quantitative fungal culture (0.5mls) TB culture (5mls)*
Flow cytometry (10mls)
FBC, U&E, Mg, CD4, VL, CRP, blood culture* Whole blood antigen stimulation
Flow cytometry Serum for storage Plasma for storage RNA (PAXGENE)
1-‐14 Quantitative culture (0.5mls) FBC, U&E, Mg (twice weekly)
Table 3.4 Inpatient research laboratory investigations. Detailed methodology is provided in section
3.4. Tests marked * were performed in a hospital diagnostic laboratory: FBC (full blood count), U&E (Urea & Electrolytes), Mg (magnesium), VL (HIV viral load).
3.2.10 Follow up
Subjects were followed during their hospital admission; repeat clinical assessment was performed and documented at days 1, 3, 7 and 14 (+/-‐ 1) post recruitment; additional assessments were made if a participant’s clinical state deteriorated. All LPs were recorded along with opening/closing pressures, and any new medications deemed significant. Following discharge from hospital, subjects were followed up in clinic at study weeks 4, 8, 12, 16, and 24 to monitor for any clinical deterioration; additional attendances were arranged depending on clinical indications. Subjects not taking ART at enrolment were initiated on ART at week 4 as described in section
3.5.4. Study end points are detailed below [Table 3.5]. Repeat blood sampling was
performed depending on ART status at enrolment [Tables 3.6, 3.7].
Primary Outcome Secondary Outcomes
14-‐day mortality
12-‐week mortality Time-‐to-‐death
Rate of fungal clearance from CSF Paradoxical IRIS
Cryptococcal relapse
Table 3.5 Outcomes for the IICD study. The method used to calculate rate of fungal clearance is described in section 3.4.11.
Week CSF Bloods
4†
No routine CSF samples taken
As day 0 (but no VL or blood culture)
8 As day 0 (but no VL or blood culture)
12 As day 0 (but no VL or blood culture)
16 As day 0 (but no blood culture)
26 CD4 + clinical bloods
Table 3.6 Outpatient investigations for subjects not taking ART. †ART initiated at week 4
Week CSF Bloods
4
No routine CSF samples taken
Clinical bloods
8 Clinical bloods
12 As day 0 (but no VL or blood culture)
16 Clinical bloods 26 CD4 + clinical bloods
Table 3.7 Outpatient investigations for subjects taking ART at enrolment
3.2.11 Clinical deterioration
Participants experiencing clinical deterioration were fully assessed to determine the
cause and classified as detailed below [Table 3.8]. Participants with neurological
deterioration on ART (and therefore possible CM-‐IRIS) were assessed using the INSHI
criteria for paradoxical cryptococcal IRIS (Haddow et al. 2010) [Table 1.3]. Repeat
lumbar puncture and blood sampling were performed as detailed below [Table 3.9].
Additional persons presenting to one of the study hospitals who met INSHI criteria
for neurological CM-‐IRIS [Table 1.3] were also enrolled an additional IRIS case even if
they had not been enrolled during their initial episode of CM.
Category Criteria
CM-‐disease Worsening CM despite treatment
CM-‐relapse Culture proven relapse
CM-‐IRIS Meets INSHI criteria for paradoxical CM-‐IRIS (Haddow et al. 2010) AmB toxicity Creatinine doubling, thrombophlebitis, anaemia + transfusion
Bacteraemia Positive blood culture
TB Microbiologically confirmed TB
Other Cause of deterioration known, not listed above
Unknown Cause of deterioration not known
Day CSF Blood
ND
Cell count, protein, glucose (0.5mls)* Gram Stain, bacterial culture* Quantitative fungal culture (0.5mls) TB culture (5mls)*
Flow cytometry (10mls)
FBC, U&E, Mg, CD4, VL, CRP, bloodculture* Whole blood antigen stimulation
Flow cytometry Serum for storage Plasma for storage RNA (PAXGENE)
Table 3.9 Investigations for subjects with neurological deterioration (ND). * performed in hospital
diagnostic laboratory
3.2.12 Study deaths
An attempt was made to ascribe cause of death for any participant who died during
the course of the study using the same categories detailed above [Table 3.8]. To help
determine this, consent was sought from families of deceased study participants to conduct a medical post-‐mortem. If willing, the process and practicalities of a post-‐ mortem were discussed with the next of kin and close family members in English or Xhosa. It was emphasized that there was no legal requirement for the examination, and the possible fates of organs were specifically discussed: retained for teaching or research, replaced after examination prior to burial, or returned to the family for burial at a later date. Only if a family were in full agreement would a post-‐mortem examination be performed. Written informed consent was obtained using a form from Groote Schuur Hospital, Department of Anatomical Pathology (Appendix B).
3.2.13 Enrolment of control subjects
To better understand the abnormalities in blood immune response among subjects with CM, two groups of controls were enrolled: HIV-‐uninfected (healthy) and HIV-‐
infected controls [see Table 3.10 for entry criteria]. Potential control subjects were
identified through liaison with clinical staff at a large local community health centre (CHC) located close to Khayelitsha hospital [see section 3.2.2]. HIV-‐infected controls were identified from patients attending the ART clinic (“Ubuntu clinic”), while healthy controls were identified from patients attending the general clinic (“Day Hospital”) for HIV testing or other reasons. HIV-‐infected subjects with low CD4 count were specifically recruited to allow closer comparison with individuals with CM.
Particular efforts were made to enroll only HIV-‐infected persons with no evidence of opportunistic infection.
HIV uninfected healthy controls HIV-‐1-‐infected controls
Negative HIV test within last month No intercurrent illness, meningitis or TB* No chronic health problems
(e.g. diabetes, hypertension, asthma)
Positive HIV test Not taking ART
Clinic CD4<200 cells/µL
Negative serum cryptococcal antigen No evidence of TB†
Table 3.10 Entry criteria for healthy and HIV-‐infected controls. *determined clinically, †Subjects were screened at enrollment with TB symptom screen and samples sent if appropriate; subjects were excluded at a later point if any sputum samples sent from clinic were positive for M.tuberculosis.
Clinical assessment and data collection
A short symptom questionnaire was completed on all control subjects, including a TB symptom screen, and a sputum sample was sent for TB testing if clinically indicated. Limited demographic and clinical information was recorded. Control subjects were not followed up but their laboratory record was checked 3 months post recruitment
and any subjects with a positive sputum culture for M. tuberculosis excluded.
Peripheral blood sampling
Control patients had a single blood draw. Investigations and assays were identical to CM patients at enrolment except blood cultures were not performed; a detailed
breakdown is presented below [Table 3.11]. In addition, HIV-‐1 infected controls had
blood sent for viral load and a serum cryptococcal antigen test was performed to ensure they did not have asymptomatic cryptococcal antigenaemia. No CSF samples were obtained for either control group.
Day CSF Blood
0
No CSF sent for control subjects
FBC, U&E, Mg, CD4, CRP,
HIV viral load + CrAg test (HIV-‐infected only) Whole blood antigen stimulation
Flow cytometry Serum for storage Plasma for storage RNA (PAXGENE)
Table 3.11 Investigations and assays for control subjects. Only HIV-‐infected controls had HIV-‐1 viral load and serum cryptococcal antigen (CrAg) sent