AbstractObjectives

: Cytomegalovirus (CMV) viremia is associated with increased mortality in persons with HIV. We previously demonstrated that CMV viremia was a risk factor for 10-week mortality in antiretroviral therapy (ART)-naïve persons with cryptococcal meningitis. We investigated whether similar observations existed over a broader cohort of HIV-associated meningitis at 18 weeks.

Methods

: We prospectively enrolled Ugandans with cryptococcal or tuberculous (TB) meningitis into clinical trials during 2015–2019. We quantified CMV DNA concentrations from stored baseline plasma or serum samples from 340 participants. We compared 18-week survival between those with and without CMV viremia.

Results

: We included 308 persons with cryptococcal meningitis and 32 with TB meningitis, of whom 121 (36%) had detectable CMV DNA. Baseline CD4+ T cell counts (14 vs. 24 cells/µL; P=0.07) and antiretroviral exposure (47% vs. 45%; P=0.68) did not differ between CMV viremic and non-viremic persons, respectively. The 18-week mortality was 50% (61/121) in those with CMV viremia versus 34% (74/219) in those without (P=0.003). Any detectable CMV viremia (aHR=1.60; 95%CI, 1.13–2.25; P=0.008) and greater viral load (aHR=1.22 per log10 IU/mL increase; 95%CI, 1.09–1.35; P<0.001) were positively associated with all-cause mortality through 18 weeks.

Conclusions

: CMV viremia at baseline was associated with a higher risk of death at 18 weeks among persons with HIV-associated cryptococcal or TB meningitis, and the risk increased as the CMV viral load increased. Further investigation is warranted to determine if CMV is a modifiable risk contributing to deaths in HIV-associated meningitis, versus its presence representing a biomarker of immune dysfunction.

Key words

Introduction

Cryptococcus neoformans and Mycobacterium tuberculosis are common causes of HIV-associated meningitis and result in high mortality in persons with advanced HIV disease, particularly in sub-Saharan Africa (Rajasingham et al., 2017; Ellis et al., 2019; Tenforde et al., 2020). Interest exists in identifying modifiable risk factors contributing to the high mortality observed in advanced HIV. One underexplored area is the impact that frequently undiagnosed concomitant opportunistic infections may have on persons with HIV-associated meningitis.

Cytomegalovirus (CMV) is a human herpesvirus that commonly causes life-long latent infection but may reactivate in immunocompromised persons (Springer and Weinberg 2004; Gianella and Letendre 2016). CMV IgG seropositivity is typically >90% in adult South American (Souza et al., 2010; de Matos et al., 2011; Tuon et al., 2019), Asian (Lim et al., 2013; Wang et al., 2017; Choi et al., 2021), and African (Compston et al., 2009; Gronborg et al., 2017) populations; reaching essentially 100% in Africans with HIV-associated cryptococcal meningitis (Skipper et al., 2020). The presence of detectable CMV viremia increases with decreasing CD4+ T cell count, with reported prevalence ranging from 23–55% in HIV-positive persons with CD4+ T cell counts ≤100 cells/μL (Micol et al., 2009; Fielding et al., 2011; Brantsaeter et al., 2012; Durier et al., 2013; Moore et al., 2019; Jabbari et al., 2021). In our 2010–2013 Uganda and South Africa study, we reported that 52% of antiretroviral (ART) naïve adults with first-episode cryptococcal meningitis (median CD4 = 19 cells/μL) had detectable CMV DNA (median viral load = 498 IU/mL [interquartile range, 259–2390]) in plasma (Skipper et al., 2020). We demonstrated CMV viremia is associated with a ∼2-fold greater mortality rate (Hazard Ratio = 2.19; 95%CI, 1.07–4.49) in ART naïve persons with first-episode cryptococcal meningitis compared to those without detectable CMV viremia (Skipper et al., 2020). Detectable CMV viremia has been repeatedly demonstrated as a risk factor for mortality in HIV-positive persons across multiple continents and eras of antiretroviral (ART) use (Spector et al., 1998; Deayton et al., 2004; Fielding et al., 2011; Durier et al., 2013). However, it is unknown whether CMV is driving this risk or rather serves as a marker of a dysfunctional immune response to the primary opportunistic infection.

We designed this study to validate our prior findings in a larger population, while expanding our cohort to include: (a) ART-experienced patients with cryptococcal meningitis; and (b) ART-naïve and ART-experienced patients with tuberculous (TB) meningitis. Similar to cryptococcal meningitis, TB meningitis predominantly affects persons with low CD4+ T cell counts, though can occur at any CD4 count and in HIV-negative individuals. High levels of CMV IgG antibody are associated with a 3.4-fold increased risk of developing tuberculosis (Stockdale et al., 2020). Additionally, data from South Africa demonstrated a trend toward increased mortality in HIV-positive persons with non-central nervous system tuberculosis who also had CMV viremia, particularly in older patients (Ward et al., 2016). Future clinical trials of CMV antivirals may provide insight into whether CMV viremia is a modifiable risk factor in these patient populations.

Methods

Study Population and Setting

We retrospectively quantified CMV DNA concentrations from participants with HIV-associated cryptococcal or TB meningitis previously enrolled into our prospective longitudinal cohort, which spanned the time of two clinical trials. The Adjunctive Sertraline for the Treatment of HIV-Associated Cryptococcal Meningitis (ASTRO-CM) trial (NCT01802385) was a randomized clinical trial testing whether sertraline added to standard-of-care cryptococcal meningitis treatment improved survival (Rhein et al., 2019). The ASTRO-CM trial enrolled participants with cryptococcal meningitis from Mulago and Mbarara Hospitals in Uganda from March 2015 to May 2017. Diagnosis was by cryptococcal antigen lateral flow assay (IMMY, Norman, OK, USA) (Boulware et al., 2014). Induction therapy consisted of amphotericin B deoxycholate (0.7-1.0 mg/kg/day) plus fluconazole (800 mg/day) for up to 2 weeks, followed by fluconazole consolidation and maintenance therapy. Participants were randomized to additionally receive sertraline 400 mg daily for 2 weeks followed by 200 mg daily for an additional 12 weeks, or matching placebo. All participants received supportive care (electrolyte monitoring/replacement) and therapeutic lumbar punctures to control elevated intracranial pressure as needed. Survival at 18 weeks was the primary endpoint of the ASTRO-CM trial. The study demonstrated no survival difference between the adjunctive sertraline and placebo groups (Rhein et al., 2019).

The High Dose Oral and Intravenous Rifampicin for Improved Survival of Adult Tuberculous Meningitis (RifT) trial (ISRCTN42218549) evaluated whether high dose rifampicin resulted in higher CSF drug concentrations compared to standard-of-care dosing in persons with TB meningitis (Cresswell et al., 2021). The trial enrolled participants with suspected or confirmed TB meningitis from Mulago and Mbarara Hospitals in Uganda from January to December 2019. All participants received daily oral isoniazid (5 mg/kg/day), pyrazinamide (25 mg/kg/day), ethambutol (20 mg/kg/day), and dexamethasone (0.4 mg/kg/day, followed by an 8-week taper). Participants were randomized to receive standard dose oral rifampicin (10 mg/kg/day), high dose oral rifampicin (35 mg/kg/day), or high dose intravenous rifampicin (20 mg/kg/day) in a 1:1:1 fashion. Survival at 24 weeks was a secondary outcome for the RifT trial. Survival did not statistically differ, though this pharmacokinetic trial was not powered to determine survival difference between the three groups (Cresswell et al., 2021).

Participants enrolled into the ASTRO-CM trial had stored baseline plasma available for CMV DNA quantification, while those enrolled into the RifT trial had stored baseline serum available. We shipped all available frozen, stored samples from Uganda to Minneapolis in February 2020.

DNA Extraction and Polymerase Chain Reaction (PCR)

We performed DNA extraction from 200 µL of plasma or serum using the QIAcube HT (QIAGEN, Hilden, Germany) and QIAamp 96 Virus DNA QIAcube kit according to manufacturer's instructions. Multiplex qPCR was performed using the LightCycler 480 System (Roche, Basel, Switzerland) and UL83 targeted primers (Dollard et al., 2021). Detailed PCR methods can be found in the Supplemental Methods section. Laboratory staff processed de-identified samples and were blinded to participant data and outcome. CMV DNA quantification in copies/mL values were converted to international units (IU)/mL using the WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques (NIBSC code 09/162) as controls for CMV viral load.

Statistical Analysis

We defined CMV viremia as any quantity of CMV DNA in plasma or serum above the threshold of PCR detection, which was 88.5 IU/mL for our assay (Levi et al., 2022). For this study, we considered any sample within 14 days of meningitis diagnosis as a baseline sample. Baseline characteristics were compared by CMV viremia status, CMV viral load grouping, and meningitis subgroup using chi-square or Kruskal-Wallis testing. The primary outcome in our analysis was 18-week all-cause mortality in those with HIV-associated cryptococcal or TB meningitis, which we will hereafter refer to as HIV-associated meningitis. Survival was calculated in days from the date of meningitis diagnosis until either death, loss to follow-up, or 18-weeks. We chose 18-weeks of follow up as 95% of the 5-year mortality occurs within the first 18-weeks after cryptococcal meningitis diagnosis (Butler et al., 2012). We analyzed the impact of CMV viremia as both a binary variable (present versus absent) and as a continuous variable using log10-transformed CMV viral load in our survival models. We additionally considered categorical variables of: 1) no CMV viremia; 2) CMV DNA concentrations 5000 IU/mL (high-grade viremia) to further assess how the magnitude of viremia impacted mortality at clinically useful cutoffs. Univariate Cox proportional hazard models evaluated risk factors for 18-week mortality. We reported hazard ratios, 95% confidence intervals (95% CI), and P-values for each analysis. Seven participants were lost to follow up and were right-hand censored before 18 weeks in the time-to-event model. For eight participants missing CD4 values, we imputed the median CD4 value for the type of meningitis. We then performed multivariate adjustment in two different proportional hazards models. The goal of the first model set (Table 2, Multivariate Models #1 & 2) was to maintain all 340 participants and adjust for what we considered the two most important potential confounders, CD4+ T cell count and a Glasgow Coma Scale (GCS) score of + T cell count is important because of how it is traditionally correlated with the risk of the reactivation of CMV, and GCS score

Ethical approval

The parent trials received institutional review board (IRB) approval from the appropriate US, UK, and Ugandan institutions, including the Uganda National Drug Authority, and Uganda National Council of Science and Technology. All trial participants provided written informed consent for the storage and future testing of samples for research purposes.

Results

We included 308 persons with cryptococcal meningitis and 32 persons with TB meningitis into this CMV substudy, for a total analysis cohort of 340 participants. The study cohort comprised 61% men with a median age of 35 years (interquartile range [IQR], 30-40). The median CD4+ T cell count was 14 cells/µL (IQR, 6–53) for CMV viremic persons, versus 24 cells/µL (IQR, 8–61) in non-viremic persons (P=0.07). Altogether, 121 (36%) had detectable CMV viremia at baseline, with the median CMV viral load 452 IU/mL (IQR, 142–2450; range, 119–10 million). We compared baseline characteristics by CMV viremia status in Table 1. Persons with CMV viremia were noted to have lower hemoglobin values and were less likely to mount a white blood cell response in the CSF. Most baseline samples tested for CMV DNA were collected within 3 days from meningitis diagnosis (91% [309/340]), with 279 (82%) collected the same day as diagnosis.

Table 1Baseline Characteristics by CMV Viremia Status

Data are N (%) or median [interquartile range].

Overall, mortality at 18 weeks was 50% (61/121) in those with CMV viremia versus 34% (74/219) in those without CMV viremia (P=0.003) (Figure 1). In a univariate proportional hazards model, the presence of CMV viremia demonstrated a two-thirds greater risk (Hazard Ratio = 1.66; 95%CI, 1.18–2.33; P=0.003) for death through 18 weeks (Table 2). When assessing the impact of greater CMV viral load, we found a hazard ratio of 1.24 (95%CI, 1.11–1.38; P10 IU/mL unit increase in CMV DNA concentration. Thus, both the presence of any detectable CMV DNA and increasing levels of CMV DNA at baseline were both associated with higher 18-week mortality before adjustment in HIV-associated meningitis.

Figure 1Survival Curve by CMV Viremia Status Within 18 Weeks

Show full captionFigure 1 demonstrates a Kaplan Meier curve for survival at 18 weeks. Persons with CMV viremia were more likely to die at 18 weeks as compared to persons without CMV viremia. Seven participants were right-hand censored before 18 weeks for transfer of care. This figure does not include any multivariate adjustments. P-value calculated by log rank testing.

Table 2Mortality Within 18 Weeks by Meningitis Cohort in Cox Regression Model

The median CMV DNA concentration was 2.66 (IQR, 2.15, 3.39; maximum:7.00) log10(IU/mL). All persons with TB meningitis and GCS <15 died, leading to a hazard ratio that approaches infinity; P-values are not valid in such situations. The median CD4 count for the cryptococcal meningitis group was 16 cells/µL (IQR, 7, 49) and the TB meningitis group was 107 cells/µL (IQR, 45, 273).

Multivariable Models #1 and #2 adjust for: age, sex, CD4+ T cell count per 10 cells/µL, and Glasgow coma scale score <15. CMV log10 increase (as a continuous variable) had a similar but marginally better relative model of fit by Akaike testing compared to CMV viremia present (as a categorical variable) (AIC=1491 vs 1496, respectively). Supplemental Table 3 includes a secondary model with (3a) univariate hazards for all considered co-variates, and (3b) CMV viremia hazard ratios after adjusting for all considered co-variates, at the expense of case exclusion secondary to missing data.

Abbreviations: 95% CI, 95% confidence interval; CMV, cytomegalovirus; IQR, interquartile range; IU/mL, international units per milliliter.

We further stratified CMV viral load into low-grade viremia (5000 IU/mL) groups to see how mortality associated with the magnitude of CMV viremia at clinically useful thresholds. Of the 121 participants with CMV viremia, 76 (63%) had low CMV viral loads (median 188 IU/mL [IQR, 129–354]), 21 (17%) had moderate CMV viral loads (median 1,750 IU/mL [1,200–2,740]), and 24 (20%) had high CMV viral loads (median 9,970 IU/mL [6,750–62,100]). Baseline characteristics were mostly similar amongst the viral load groups, although a non-significant trend of declining CD4+ T cell count was noted as CMV viral load increased (Supplemental Table 1). Mortality was highest in the high CMV viral load group (71% [17/24]) and decreased with declining viral load grouping (Figure 2).

Figure 2Death Within 18 Weeks by CMV Viral Load Groups

Show full caption

Figure demonstrating the proportion of participants who died by 18 weeks grouped by level of CMV viral load, in international units/milliliter (IU/mL). Chi-Square P-value of 0.001 when comparing across all groups.

We analyzed whether known risk factors or possible confounders other than CMV viremia were contributing to 18-week mortality (Table 2). In our univariate analyses, CD4+ T cell count per 10 cells/μL increase (Hazard Ratio = 0.98; 95%CI, 0.96–1.01; P=0.15) was not significantly associated with mortality while GCS score + T cell count, and GCS score 10 increase in IU/mL; 95%CI, 1.09–1.35; PTable 2). In our secondary adjusted model (N=279) considering numerous additional co-variates, results were similar (Supplemental Table 3b).We conducted a subgroup analysis of mortality risk for persons with cryptococcal meningitis and TB meningitis, separately. Baseline characteristics by meningitis subgroup are list in Supplemental Table 2. Of the 308 persons with cryptococcal meningitis, 55 (50%) died in the CMV viremia group vs 73 (37%) in the non-viremic group (P=0.03). In the 32 persons with TB meningitis, 6 (60%) died in the CMV viremia group vs 1 (5%) in the non-viremic group (P=0.001). The categorical presence of CMV viremia was significantly associated with 18-week mortality for both cryptococcal meningitis (Hazard Ratio = 1.48; 95%CI, 1.04–2.09; P=0.03) and TB meningitis (Hazard Ratio = 17.1; 95%CI, 2.04–142.6; P=0.009) (Table 2). Similarly, using CMV log10 IU/mL as a continuous variable in our survival model, we found significant mortality associations for cryptococcal meningitis (Hazard Ratio = 1.19; 95%CI, 1.07–1.33; P=0.002), as well as TB meningitis (Hazard Ratio = 2.59; 95%CI, 1.45–4.63; P=0.002). The primary adjusted model set (N=340) remained significant for all CMV variables by subgroup (Table 2, Multivariate Models #1 & 2). The secondary full co-variate model set (N=279) resulted in an invalid analysis for TB meningitis due to case exclusions, while cryptococcal meningitis saw CMV log10 IU/mL remain statistically significant (P=0.02), but the categorical presence of CMV did not (P=0.11) (Supplemental Table 2b, Multivariate Models #3 &4).Lastly, we investigated whether baseline ART status affected either the probability of having CMV viremia or survival outcome. Detectable CMV viremia occurred in 37% (57/155) of those receiving ART at baseline compared with 35% (64/185) not receiving ART at baseline. Among those 154 with known timing of initiation of ART, 45 had initiated ART within 30 days, and 47% (21/45) of those were CMV viremic. Of the 109 participants who had been on ART for more than 30 days, 32% (35/109) of those were CMV viremic (p=0.09). The trend for increased mortality with CMV viremia was consistent in both subgroups of participants who were ART naïve (52% vs 38%; P=0.08) and those who were ART experienced (49% vs 29%; P=0.015) respectively (Table 3).

Table 3Contingency Tables of Antiretroviral Exposure at Baseline by CMV Viremia Status

Discussion

We demonstrate that CMV viremia is associated with increased all-cause mortality among persons with HIV-associated cryptococcal and TB meningitis. Compared to our previous study (Skipper et al., 2020), we expanded our cohort to include both ART-experienced persons and those with TB meningitis. Any detectable CMV viremia was associated with ∼60% greater risk of death at 18-weeks compared to non-viremic persons in our expanded cohort of HIV-associated meningitis. We found the magnitude of viremia was positively associated with mortality when either CMV DNA concentrations were log10 transformed as a continuous variable in our survival model, or when categorically grouped by clinical cutoffs. Further, CMV viremia remained significantly associated with mortality after adjustment for age, sex, CD4+ T cell count, and baseline GCS score. Taken as a whole, mounting evidence suggests that CMV viremia poses a notable risk for increased mortality in persons with HIV-associated meningitis—again raising the question of cause vs. effect—is CMV a bystander signaling worsened immune function, or is CMV replication itself driving mortality in part? We continue to hypothesize that ongoing viral replication in persistent infection produce type I interferons which may suppress effective type-1 T helper (Th1) CD4 responses necessary to clear intracellular pathogens such as Cryptococcus and M. tuberculosis, as one plausible mechanism (Osokine et al., 2014). A second mechanism of interest involves the observed down-regulation of major histocompatibility complex (MHC) class-II proteins during CMV reactivation, thereby impairing CD4+ T cell function (Chevalier et al., 2002; Jenkins et al., 2008). We are currently designing future experiments to test these hypotheses.

An important new observation in this study is the association of the magnitude of CMV viremia (i.e. greater baseline viral load) with higher mortality. We found that 18-week mortality exceeded 50% in participants with CMV DNA concentrations of >1000 IU/mL, rising to 71% in those with >5000 IU/mL. Similarly, every log10 increase in CMV DNA IU/mL increased mortality ∼1.2-fold. This data suggests that persons with high viral loads may benefit from anti-CMV treatment, and that reduction of viral load below a critical level might ameliorate mortality risk. Our prior study (Skipper et al., 2020) failed to find a “dose-response” relationship between CMV magnitude and mortality, but this may be a consequence of the smaller sample size and fewer cases with viral loads >1000 IU/mL. Importantly, the association between the magnitude of CMV viremia and mortality held true across all adjusted models we tested.

When we analyzed 18-week mortality by meningitis subgroup, we found that the presence of CMV viremia was associated with mortality (HR=1.48 [1.04–2.09]) in persons with cryptococcal meningitis, but to a lesser degree than our prior cohort (HR=2.19 [1.07–4.49]). After adjustment, the association remained significant in our primary multivariate model set (N=340; HR=1.43 [1.00–2.04]), but loss statistical significance in the secondary full co-variate model set (N=279; HR=1.38 [0.93–2.04]). We believe the smaller sample size in the secondary model set reduced the power of this model to detect statistically significant differences, and we are reassured that the hazard ratio is similar. However, we acknowledge that the relationship between CMV and mortality in persons with advanced HIV disease is likely complex and may be confounded by complicated interactions or factors not included in our models, obfuscating the precise risk that the presence of CMV viremia alone imparts on mortality. An alternative interpretation of our data could be that higher CMV viral load is associated with increased mortality in persons with cryptococcal meningitis, while lower viral loads may not be associated with the increased risk.

The large hazard ratio (17-fold) seen for detectable CMV viremia in persons with TB meningitis is striking but should be interpreted with caution, given the relatively small sample size and wide 95% confidence interval. Still, given previously published studies demonstrating CMV as a potential risk factor for non-central nervous system TB, combined with the biological plausibility of CMV-mediated impairment of the type 1 helper T cell response critical to controlling TB, it remains an intriguing finding worthy of additional study (Engstrand et al., 2003; Essa et al., 2009).

Another interesting observation is that CMV viremia was associated with lower hemoglobin values and reduced white blood cell response in the CSF. In in vitro models, CMV can infect bone marrow progenitor cells and disrupt hematopoiesis (Sing and Ruscetti 1990). Anemia and leukopenia are known clinical complications of CMV disease. Less is known about how CMV might affect white blood cell response in the cerebrospinal fluid. It is possible that CMV may be implicated in a multifactorial process affecting both the quality of the host's immune response and the quantity of critical blood cellular components. While CMV viremia might be associated with lower hemoglobin and reduced CSF white blood cell response, neither variable appears to significantly impact mortality alone (Supplemental Table 3a). We advocate for the inclusion of these variables in future CMV studies to further disentangle these complex interactions.

One key difference in the current cryptococcal meningitis cohort is that 45% of participants were ART experienced at baseline (persisting with advanced disease by either a failing ART regimen, or having only recently initiated ART after their HIV diagnosis), whereas our prior cryptococcal meningitis cohort was entirely ART naïve at baseline, with half initiating early ART within 1-2 weeks after meningitis diagnosis (Boulware et al., 2014; Skipper et al., 2020). It is unknown how ART exposure effects CMV viremia in persons who remain significantly CD4+ T cell deficient. Our analysis demonstrates similar rates of detectable CMV in ART experienced and ART naïve participants, and those that started ART recently (i.e. effective HIV treatment) appeared to have a similar frequency of viremia (47%) compared those who had been on ART longer (i.e. likely failing regimen) (32%). Similarly, CD4+ T cell counts were not notably higher in CMV non-viremic persons, as one might expect. Interestingly, we noted mildly worse survival in ART-experienced persons with CMV viremia compared to ART-naïve viremic persons in our post-hoc analysis—invoking a possible detrimental immunologic contribution associated with previous ART exposure. CMV-associated immune reconstitution inflammatory syndrome (IRIS) has been described in the literature, particularly as relates to ocular involvement, but is rarely identified in our study setting (Müller et al., 2010; Levi et al., 2022). Prospectively measuring CMV DNA longitudinally over time and performing serial retinal exams, including after ART initiation or switch in failing patients, would provide better insight into the impact of early ART on CMV viremia—a study we are currently planning. However, we do continue to advocate that effective ART remains critical to mitigate morbidity and mortality from CMV disease and advanced HIV.

Ultimately, the most impactful question is whether CMV is a modifiable risk factor in this population. CMV disease is associated with graft loss and increased mortality in solid organ transplant recipients (Hakimi et al., 2017; McBride et al., 2019), and treating with anti-CMV therapy has been demonstrated to mitigate these poor outcomes (Dunn et al., 1991; Asberg et al., 2007). Transplant recipients also benefit from strategies of prophylaxis or preemptive therapy (initiating treatment based on a CMV DNA threshold) prior to the development of CMV disease (Owers et al., 2013; Witzke et al., 2018). Observations of the benefit of prophylactic or pre-emptive anti-CMV therapy in the transplant population raise the question of whether a similar benefit could be demonstrated in the advanced HIV population. Essentially all persons with HIV-associated meningitis in sub-Saharan Africa are CMV seropositive, and thus at risk for CMV reactivation (Skipper et al., 2020). Given the high mortality rate of HIV-associated meningitis (40% [135/340] in this study), addressing any modifiable risk factor may yield a real survival benefit. A randomized clinical trial testing anti-CMV therapy in persons with HIV-associated meningitis would help determine if CMV is indeed a modifiable risk factor in a sub-population of advanced HIV disease.

Our study is limited as a post-hoc analysis of participants enrolled into prospective randomized trials; although importantly randomization to intervention was not different between CMV viremic and non-viremic persons. Additionally, our proportional hazards model did not find a mortality association by intervention arm, similar to the parent trial findings (Rhein et al., 2019; Cresswell et al., 2021). We do not have CMV antibody data on this current cohort, but our prior study demonstrated that all 111 persons with cryptococcal meningitis were CMV IgG positive (Skipper et al., 2020). CMV seropositivity may be different in other regions of the world, impacting the generalizability of this study. CMV viremia was only determined at baseline, and the dynamics of the viremia over time would be a future area for exploration. Despite CMV DNA concentrations being measured using the same lab and instruments as previously, inter-assay variability may still occur at the lower limit of detection. The clinical relevance of low-level CMV viremia in the setting of HIV-associated meningitis remains unclear. We limited the primary multivariate model to CD4+ T cell count and GCS score (in addition to age and sex) to balance what we felt were the most influential potential confounders while maintaining our full sample size. For transparency and to improve data interpretability, we have additionally included a secondary supplemental model that adjusts for a full array of co-variates, with the recognition that 61 participants are excluded due to missing data. Unfortunately we do not have consistent HIV viral load information from our study cohorts, which would be valuable to include in a future multivariate model. CMV appears as a particularly strong risk in TB meningitis, which influences the overall model, though the small sample size and wide confidence intervals suggest the hazard ratio could change with a larger study population. We also note these findings only apply to HIV-associated cryptococcal and TB meningitis, limiting the generalizability of the findings to either persons with HIV-associated meningitis of a different etiology or HIV-negative meningitis. Lastly, this study did not systematically investigate for CMV end-organ disease, limiting the ability to determine how primary CMV disease might contribute to all-cause mortality.

In conclusion, we demonstrate that detectable CMV viremia is associated with an ∼60% greater risk of death in persons with HIV-associated cryptococcal or TB meningitis. The mechanisms driving the increased risk of death are unclear, though the positive association between the magnitude of CMV viral load and mortality suggests that a greater CMV burden is detrimental, perhaps through CMV-specific modulation of the immune system. On the contrary, CMV reactivation may simply reflect an immune state less equipped to handle the primary opportunistic infection, thus serving as a marker of heightened immune dysfunction rather than a cause. A randomized clinical trial is needed to determine if preventing or treating CMV viremia in persons with HIV-associated meningitis can reduce mortality and improve survival in this vulnerable population.

Funding: This research was made possible by the National Institutes of Health's National Center for Advancing Translational Sciences (KL2TR002492 and UL1TR002494); National Institute of Allergy and Infectious Diseases (T32AI055433, U01AI089244); Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD079918); National Institute of Neurologic Disorders and Stroke (R01NS086312); and the Fogarty International Center (K01TW010268, D43TW009345). FVC is supported by a Wellcome PhD Fellowship (210772/Z/18/Z).

Potential Conflicts of Interest: The authors have no conflicts of interests to declare.

Acknowledgements: We are appreciative of the study teams and hospital staff working continuously to provide compassionate care to the participants and patients at Mulago National Referral Hospital.

ASTRO-CM Study team: Joshua Rhein, Kathy Huppler Hullsiek, Lillian Tugume, Edwin Nuwagira, Edward Mpoza, Emily E Evans, Reuben Kiggundu, Katelyn A Pastick, Kenneth Ssebambulidde, Andrew Akampurira, Darlisha A Williams, Ananta S Bangdiwala, Mahsa Abassi, Abdu K Musubire, Melanie R Nicol, Conrad Muzoora, David B Meya, David R Boulware, Jane Francis Ndyetukira, Cynthia Ahimbisibwe, Florence Kugonza, Carolyne Namuju, Alisat Sadiq, Alice Namudde, James Mwesigye, Kiiza K Tadeo, Paul Kirumira, Michael Okirwoth, Tonny Luggya, Julian Kaboggoza, Eva Laker, Leo Atwine, Davis Muganzi, Stewart Walukaga, Bilal Jawed, Matthew Merry, Anna Stadelman, Nicole Stephens, Andrew G Flynn, Ayako W Fujita, Richard Kwizera, Liliane Mukaremera, Sarah M Lofgren, Fiona V Cresswell, Bozena M Morawski, Nathan C Bahr, Kirsten Nielsen

RIFT Study team: Jane Frances, Florence Kigundu, Cythia Ahimbisibwe, Carol Karuganda, Alice Namudde, Kiiza Tadeo Kandole, Alisat Sadiq, Mable Kabahubya, Edward Mpoza, Gavin Stead, Samuel Jjunju, Edwin Nuwagira, Nathan Bahr, Joshua Rhein, Darlisha Williams, Rhona Muyise, Eva Laker

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Tabled 1

Mortality by 18 weeks was 49% in ART-experienced, CMV viremic participants versus 29% ART-experienced, CMV non-viremic participants (P=0.01).

Appendix. Supplementary materialsArticle InfoPublication History

Accepted: July 11, 2022

Received in revised form: June 22, 2022

Received: March 7, 2022

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DOI: https://doi.org/10.1016/j.ijid.2022.07.035

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