Darlisha A. Williams

Timing of antiretroviral therapy after diagnosis of cryptococcal meningitis

BACKGROUND Cryptococcal meningitis accounts for 20 to 25% of acquired immunodeficiency syndrome-related deaths in Africa. Antiretroviral therapy (ART) is essential for survival; however, the question of when ART should be initiated after diagnosis of cryptococcal meningitis remains unanswered. METHODS We assessed survival at 26 weeks among 177 human immunodeficiency virus-infected adults in Uganda and South Africa who had cryptococcal meningitis and had not previously received ART. We randomly assigned study participants to undergo either earlier ART initiation (1 to 2 weeks after diagnosis) or deferred ART initiation (5 weeks after diagnosis). Participants received amphotericin B (0.7 to 1.0 mg per kilogram of body weight per day) and fluconazole (800 mg per day) for 14 days, followed by consolidation therapy with fluconazole. RESULTS The 26-week mortality with earlier ART initiation was significantly higher than with deferred ART initiation (45% [40 of 88 patients] vs. 30% [27 of 89 patients]; hazard ratio for death, 1.73; 95% confidence interval [CI], 1.06 to 2.82; P=0.03). The excess deaths associated with earlier ART initiation occurred 2 to 5 weeks after diagnosis (P=0.007 for the comparison between groups); mortality was similar in the two groups thereafter. Among patients with few white cells in their cerebrospinal fluid (<5 per cubic millimeter) at randomization, mortality was particularly elevated with earlier ART as compared with deferred ART (hazard ratio, 3.87; 95% CI, 1.41 to 10.58; P=0.008). The incidence of recognized cryptococcal immune reconstitution inflammatory syndrome did not differ significantly between the earlier-ART group and the deferred-ART group (20% and 13%, respectively; P=0.32). All other clinical, immunologic, virologic, and microbiologic outcomes, as well as adverse events, were similar between the groups. CONCLUSIONS Deferring ART for 5 weeks after the diagnosis of cryptococcal meningitis was associated with significantly improved survival, as compared with initiating ART at 1 to 2 weeks, especially among patients with a paucity of white cells in cerebrospinal fluid. (Funded by the National Institute of Allergy and Infectious Diseases and others; COAT ClinicalTrials.gov number, NCT01075152.).

HuA and Tristetraprolin Are Induced following T Cell Activation and Display Distinct but Overlapping RNA Binding Specificities

AU-rich elements found in the 3′-untranslated regions of cytokine and proto-oncogene transcripts regulate mRNA degradation and function as binding sites for the mRNA-stabilizing protein HuA and the mRNA-destabilizing protein tristetraprolin. Experiments were performed to evaluate the expression of HuA and tristetraprolin in purified human T lymphocytes and to evaluate the ability of these proteins to recognize specific AU-rich sequences. HuA is a predominantly nuclear protein that can also be found in the cytoplasm of resting T lymphocytes. Within 1 h after stimulation of T lymphocytes with anti-T cell receptor antibodies or a combination of a phorbol myristate acetate and ionomycin, an increase in cytoplasmic HuA RNA-binding activity was observed. Although absent in resting cells, cytoplasmic tristetraprolin protein was detected 3–6 h following activation. HuA recognized specific AU-rich sequences found in c-jun or c-myc mRNA that were poorly recognized by tristetraprolin. In contrast, tristetraprolin recognized an AU-rich sequence in interleukin-2 mRNA that was poorly recognized by HuA. Both HuA and tristetraprolin, however, recognized AU-rich sequences from c-fos, interleukin-3, tumor necrosis factor-α, and granulocyte/macrophage colony-stimulating factor mRNA. HuA may transiently stabilize a subset of AU-rich element-containing transcripts following T lymphocyte activation, and tristetraprolin may subsequently mediate their degradation.

Tristetraprolin recruits functional mRNA decay complexes to ARE sequences

AU‐rich elements (AREs) in the 3′ untranslated region (UTR) of numerous mammalian transcripts function as instability elements that promote rapid mRNA degradation. Tristetraprolin (TTP) is an ARE‐binding protein that promotes rapid mRNA decay through mechanisms that are poorly understood. A 31 nucleotide ARE sequences from the TNF‐alpha 3′ UTR promoted TTP‐dependent mRNA decay when it was inserted into the 3′ UTR of a beta‐globin reporter transcript, indicating that this short sequence was sufficient for TTP function. We used a gel shift assay to identify a TTP‐containing complex in cytoplasmic extracts from TTP‐transfected HeLa cells that bound specifically to short ARE sequences. This TTP‐containing complex also contained the 5′–3′ exonuclease Xrn1 and the exosome component PM‐scl75 because it was super‐shifted with anti‐Xrn1 or anti‐PMscl75 antibodies. RNA affinity purification verified that these proteins associated specifically with ARE sequences in a TTP‐dependent manner. Using a competition binding assay, we found that the TTP‐containing complex bound with high affinity to short ARE sequences from GM‐CSF, IL‐3, TNF‐alpha, IL‐2, and c‐fos, but did not bind to a U‐rich sequence from c‐myc, a 22 nucleotide poly U sequence or a mutated GM‐CSF control sequence. High affinity binding by the TTP‐containing complex correlated with TTP‐dependent deadenylation and decay of capped, polyadenylated transcripts in a cell‐free mRNA decay assay, suggesting that the TTP‐containing complex was functional. These data support a model whereby TTP functions to enhance mRNA decay by recruiting components of the cellular mRNA decay machinery to the transcript. J. Cell. Biochem. 100: 1477–1492, 2007.

Tristetraprolin Mediates Interferon-γ mRNA Decay

Tristetraprolin (TTP) regulates expression at the level of mRNA decay of several cytokines, including the T cell-specific cytokine, interleukin-2. We performed experiments to determine whether another T cell-specific cytokine, interferon-γ (IFN-γ), is also regulated by TTP and found that T cell receptor-activated T cells from TTP knock-out mice overproduced IFN-γ mRNA and protein compared with activated T cells from wild-type mice. The half-life of IFN-γ mRNA was 23 min in anti-CD3-stimulated T cells from wild-type mice, whereas it was 51 min in anti-CD3-stimulated T cells from TTP knock-out mice, suggesting that the overexpression of IFN-γ mRNA in TTP knock-out mice was due to stabilization of IFN-γ mRNA. Insertion of a 70-nucleotide AU-rich sequence from the murine IFN-γ 3′-untranslated region, which contained a high affinity binding site for TTP, into the 3′-untranslated region of a β-globin reporter transcript conferred TTP-dependent destabilization on the β-globin transcript. Together these results suggest that TTP binds to a functional AU-rich element in the 3′-untranslated region of IFN-γ mRNA and mediates rapid degradation of the IFN-γ transcript. Thus, TTP plays an important role in turning off IFN-γ expression at the appropriate time during an immune response.

Efficacy of adjunctive sertraline for the treatment of HIV-associated cryptococcal meningitis: an open-label dose-ranging study

Background Cryptococcus is the most common cause of adult meningitis in Africa. We evaluated the activity of adjunctive sertraline, previously demonstrated to have in vitro and in vivo activity against Cryptococcus. Methods We enrolled 172 HIV-infected Ugandans with cryptococcal meningitis from August 2013 through August 2014 into an open-label dose-finding study to assess safety and microbiologic efficacy. Sertraline 100–400mg/day was added to standard therapy of amphotericin + fluconazole 800mg/day. We evaluated early fungicidal activity via Cryptococcus cerebrospinal fluid (CSF) clearance rate, sertraline pharmacokinetics, and in vitro susceptibility. Findings Participants receiving any sertraline dose averaged a CSF clearance rate of −0·37 (95%CI: −0·41, −0·33) colony forming units (CFU)/mL/day. Incidence of paradoxical immune reconstitution inflammatory syndrome (IRIS) was 5% (2/43) and relapse was 0% through 12-weeks. Sertraline reached steady state concentrations in plasma by day 7, with median steady-state concentrations of 201 ng/mL (IQR, 90–300; n=49) with 200mg/day and 399 ng/mL (IQR, 279–560; n=30) with 400mg/day. Plasma concentrations reached 83% of steady state levels by day 3. The median projected steady state brain tissue concentration at 200mg/day was 3·7 (IQR, 2·0–5·7) mcg/mL and 6·8 (IQR, 4·6–9·7) mcg/mL at 400mg/day. Minimum inhibitory concentrations were ≤2 mcg/mL for 27% (35/128), ≤4 mcg/mL for 84% (108/128), ≤6 mcg/mL for 91% (117/128), and ≤8 mcg/mL for 100% of 128 Cryptococcus isolates. Interpretation Sertraline had faster cryptococcal CSF clearance, decreased IRIS, and decreased relapse compared with historical experiences. Sertraline reaches therapeutic levels in a clinical setting. This inexpensive and off-patent oral medication is a promising adjunctive antifungal therapy. Funding National Institutes of Health, Grand Challenges Canada.

The Effect of Therapeutic Lumbar Punctures on Acute Mortality From Cryptococcal Meningitis

Intracranial pressure management with repeat lumbar puncture (LP) was investigated in patients with cryptococcal meningitis in sub-Saharan Africa. Conducting at least 1 additional LP soon after cryptococcal diagnosis was related to decreased risk of acute mortality regardless of initial pressure.

Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda

Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: cytomegalovirus (n=2), varicella zoster virus (n=2), human herpes virus 6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis.

Early ART After Cryptococcal Meningitis Is Associated With Cerebrospinal Fluid Pleocytosis and Macrophage Activation in a Multisite Randomized Trial

Introduction. Earlier antiretroviral therapy (ART) initiation in cryptococcal meningitis resulted in higher mortality compared with deferred ART initiation (1–2 weeks vs 5 weeks postmeningitis diagnosis). We hypothesized this was due to ART-associated immune pathology, without clinically recognized immune reconstitution inflammatory syndrome. Methods. Three macrophage activation markers and 19 cytokines/chemokines were measured from cryopreserved cerebrospinal fluid (CSF) and serum during the Cryptococcal Optimal ART Timing (COAT) trial. Comparisons were made between trial arms (early vs deferred) at 1, 8, 14, and 21 days following meningitis diagnosis. Results. More participants with early ART initiation had CSF white cell count (WCC) ≥5/µL at day 14 (58% vs 40%; P = .047), after a median of 6-days ART. Differences were mainly driven by participants with CSF WCC <5/µL at meningitis diagnosis: 28% (10/36) of such persons in the early ART group had CSF WCC ≥5/µL by day 14, compared with 0% (0/27) in the deferred arm (P = .002). Furthermore, Kampala participants (the largest site) receiving early ART had higher day-14 CSF levels of interleukin-13 (P = .04), sCD14 (P = .04), sCD163 (P = .02), and CCL3/MIP-1α (P = .02), suggesting increased macrophage/microglial activation. Conclusions. Early ART initiation in cryptococcal meningitis increased CSF cellular infiltrate, macrophage/microglial activation, and T helper 2 responses within the central nervous system. This suggests that increased mortality from early ART in the COAT trial was immunologically mediated.

Diagnostic accuracy of Xpert MTB/RIF Ultra for tuberculous meningitis in HIV-infected adults: a prospective cohort study

Summary Background WHO recommends Xpert MTB/RIF as initial diagnostic testing for tuberculous meningitis. However, diagnosis remains difficult, with Xpert sensitivity of about 50–70% and culture sensitivity of about 60%. We evaluated the diagnostic performance of the new Xpert MTB/RIF Ultra (Xpert Ultra) for tuberculous meningitis. Methods We prospectively obtained diagnostic cerebrospinal fluid (CSF) specimens during screening for a trial on the treatment of HIV-associated cryptococcal meningitis in Mbarara, Uganda. HIV-infected adults with suspected meningitis (eg, headache, nuchal rigidity, altered mental status) were screened consecutively at Mbarara Regional Referral Hospital. We centrifuged CSF, resuspended the pellet in 2 mL of CSF, and tested 0·5 mL with mycobacteria growth indicator tube culture, 1 mL with Xpert, and cryopreserved 0·5 mL, later tested with Xpert Ultra. We assessed diagnostic performance against uniform clinical case definition or a composite reference standard of any positive CSF tuberculous test. Findings From Feb 27, 2015, to Nov 7, 2016, we prospectively evaluated 129 HIV-infected adults with suspected meningitis for tuberculosis. 23 participants were classified as probable or definite tuberculous meningitis by uniform case definition, excluding Xpert Ultra results. Xpert Ultra sensitivity was 70% (95% CI 47–87; 16 of 23 cases) for probable or definite tuberculous meningitis compared with 43% (23–66; 10/23) for Xpert and 43% (23–66; 10/23) for culture. With composite standard, we detected tuberculous meningitis in 22 (17%) of 129 participants. Xpert Ultra had 95% sensitivity (95% CI 77–99; 21 of 22 cases) for tuberculous meningitis, which was higher than either Xpert (45% [24–68]; 10/22; p=0·0010) or culture (45% [24–68]; 10/22; p=0·0034). Of 21 participants positive by Xpert Ultra, 13 were positive by culture, Xpert, or both, and eight were only positive by Xpert Ultra. Of those eight, three were categorised as probable tuberculous meningitis, three as possible tuberculous meningitis, and two as not tuberculous meningitis. Testing 6 mL or more of CSF was associated with more frequent detection of tuberculosis than with less than 6 mL (26% vs 7%; p=0·014). Interpretation Xpert Ultra detected significantly more tuberculous meningitis than did either Xpert or culture. WHO now recommends the use of Xpert Ultra as the initial diagnostic test for suspected tuberculous meningitis. Funding National Institute of Neurologic Diseases and Stroke, Fogarty International Center, National Institute of Allergy and Infectious Disease, UK Medical Research Council/DfID/Wellcome Trust Global Health Trials, Doris Duke Charitable Foundation.

Evaluation of Fingerstick Cryptococcal Antigen Lateral Flow Assay in HIV-Infected Persons: A Diagnostic Accuracy Study

When testing 207 people with suspected meningitis by fingerstick with the cryptococcal antigen (CRAG) lateral flow assay, there was 100% agreement with serum or plasma CRAG testing. In 5% of participants, fingerstick testing detected cryptococcal antigenemia in peripheral blood.