Incidence, presentation and outcome of toxoplasmosis in HIV infected in the combination antiretroviral therapy era

Accepted Manuscript Incidence, Presentation and Outcome of Toxoplasmosis in HIV infected in the Combination Antiretroviral Therapy era Raquel Martin-Iguacel, Magnus Glindvad Ahlström, Madeleine Touma, Frederik Neess Engsig, Nina Breinholt Stærke, Mette Stærkind, Niels Obel, Line D. Rasmussen PII:

S0163-4453(17)30166-4

DOI:

10.1016/j.jinf.2017.05.018

Reference:

YJINF 3937

To appear in:

Journal of Infection

Received Date: 5 January 2017 Revised Date:

10 May 2017

Accepted Date: 26 May 2017

Please cite this article as: Martin-Iguacel R, Ahlström MG, Touma M, Engsig FN, Stærke NB, Stærkind M, Obel N, Rasmussen LD, Incidence, Presentation and Outcome of Toxoplasmosis in HIV infected in the Combination Antiretroviral Therapy era, Journal of Infection (2017), doi: 10.1016/j.jinf.2017.05.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Incidence, Presentation and Outcome of Toxoplasmosis

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in HIV infected in the Combination Antiretroviral

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Therapy era

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Raquel Martin-Iguacela, Magnus Glindvad Ahlströmb, Madeleine Toumab, Frederik Neess Engsigc, Nina Breinholt

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Stærked, Mette Stærkinde, Niels Obelb, Line D. Rasmussena.

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a Department

of infectious Diseases, Odense University Hospital, Søndre Boulevard 29, 5000 Odense C, Denmark

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b Department

of Infectious Diseases, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark

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c

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Denmark d Department

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Denmark

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e Department

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Department of infectious Diseases, Copenhagen University Hospital, Kettegård Alle 30, 2650 Hvidovre,

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of infectious Diseases, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus,

of infectious Diseases, Aalborg University Hospital, Hobrovej 18-22, 9100 Aalborg, Denmark

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Word count: manuscript: 3402; abstract: 200

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Running head: Cerebral toxoplasmosis in the cART-era

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Corresponding author:

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Raquel Martin-Iguacel, MD

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Department of Infectious Diseases

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Odense University Hospital

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Sdr. Boulevard 29

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5000 Odense C, Denmark

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Tel. +45 23604760

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Mail: [email protected]

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Key words: Cerebral toxoplasmosis, combination antiretroviral therapy, HIV, opportunistic

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infections.

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ABSTRACT

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Background:

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HIV-associated incidence and prognosis of cerebral toxoplasmosis (CTX) is not well

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established during later years.

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Methods:

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From the Danish HIV Cohort Study, we identified 6,325 HIV-infected individuals. We assessed

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incidence, mortality, predictive and prognostic factors of CTX during the pre-combination

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antiretroviral therapy (pre-cART; 1995-1996) and cART-era (1997-2014). Adjusted incidence

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rate ratios (aIRR), mortality rate ratios (aMRR) and 95% confidence intervals (CI) were

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assessed using Poisson regression analysis.

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Results:

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CTX IR was 1.17/1000 PYR (95% CI 0.93-1.47). We observed no change in CTX-risk in the first

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year after HIV-diagnosis, but a substantial reduction in mortality in the first 3 months after

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CTX diagnosis when comparing the cART-era to the pre-cART-era; {(aIRR: 0.79; 95% CI: 0.37-

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1.72) (aMRR: 0.15; 95% CI: 0.06-0.38)}. For individuals surviving the first year after HIV-

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diagnosis or the first 3 months after CTX-diagnosis, IRR and MRR had declined to minimal

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levels {(aIRR: 0.06; 95% CI: 0.03-0.10); (aMRR: 0.02; 95% CI: 0.01-0.05)}. Three years after

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CTX-diagnosis 30% of the patients still had neurological deficits.

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Conclusion:

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Although, CTX remains an important cause of morbidity and mortality in the cART-era, with

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high prevalence of neurological sequelae, incidence and mortality has largely declined,

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especially among those surviving the first year after diagnosis.

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HIGHLIGHTS •

Data from the Danish HIV Cohort Study - a nationwide population-based cohort study.

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Incidence and prognosis of cerebral toxoplasmosis (CTX).

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Declining incidence and mortality of CTX in the cART-era after surviving the 1st year.

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CTX remains an important cause of morbidity and mortality in late presenters.

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CTX has an important impact in the patient’s later well being and quality of life.

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INTRODUCTION

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Cerebral toxoplasmosis (CTX) is the most common of the opportunistic infections (OI) in the

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central nervous system (CNS) of HIV-infected patients. The reported incidence rate (IR) of

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CTX range from 2.0 to 43.6 per 1,000 person-years of follow-up (PYR) and during the pre-

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combination antiretroviral therapy (cART) era the prognosis was poor (1-3). Since the

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introduction of cART, the overall IR of OI and the post-OI mortality rate (MR) have declined

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dramatically (3-9). Still, incidence and mortality of CTX vary considerably between studies

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probably due to differences in sociodemographic characteristics, access to care and late

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presentation of HIV-infection of the different study populations. As CTX still remains an

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important cause of morbidity and mortality in HIV-infected patients (4,10-12), it seems

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important to further investigate the incidence, presenting symptoms, risk factors and

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prognosis of HIV-associated CTX as of today.

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We conducted a cohort study to assess the risk of CTX and associated mortality in HIV-

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infected patients during the pre-cART (1995-1996) and cART-era (1997-2014), and assessed

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the associated predictive and prognostic factors. We further described the presenting

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characteristics and the clinical course of patients with CTX.

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METHODS

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In a Danish nationwide population-based cohort of HIV-1-infected individuals, we estimated

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the incidence, clinical presentation, and prognosis of CTX. In the first part of the study, the

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outcome was time to CTX and in the second part of the study, the population was all patients

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diagnosed with CTX, and the outcomes were neurological symptoms and mortality.

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Setting

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As of 1 January 2015, Denmark had a population of 5.7 million with an estimated HIV

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prevalence among the adult population of 0.1% (13). In the study period HIV-infected patients

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have been followed at established intervals of 12-24 weeks in outpatient clinics in one of the

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8-10 specialized infectious diseases departments in Denmark. The Danish healthcare system

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is universal tax-funded and antiretroviral therapy is provided free-of-charge. Antiretroviral

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therapy is prescribed according to national guidelines(14).

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Study period

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From 1 January 1995 through 1 January 2015.

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Data sources

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We used the unique 10-digit personal identification number assigned to all individuals in

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Denmark at birth or upon immigration to track individuals in the following national

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healthcare registries:

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The Danish HIV Cohort Study (DHCS)

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DHCS is an ongoing nationwide, prospective, population-based cohort study of all HIV-

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infected patients treated at Danish hospitals since 1 January 1995, which has been described

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in detail elsewhere(15). DHCS is consecutively enrolling patients newly diagnosed with HIV

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and immigrants with HIV-infection. The collected data is updated yearly and include

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demographics, date of HIV-infection, AIDS-defining events, cause and date of death, cART,

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CD4+ T-cell count and HIV RNA measurements.

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The Danish Civil Registration System (DCRS)

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DCRS, established in 1968, is a national registry, which stores information on vital status,

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residency, and migration for all Danish resident (16).

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Study population

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In the first part of the study we included all HIV-infected patients from DHCS who were ≥16

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years, alive and living in Denmark at study inclusion, followed for HIV in Denmark between

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1995 and 1 January 2015, and were not diagnosed with CTX prior to inclusion. In the second

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part of the study we included all patients diagnosed with CTX.

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Outcomes

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In the first part of the study outcome was time to CTX diagnosis.

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In the second part of the study outcomes were neurological symptoms and time to all-cause

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mortality.

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All patients diagnosed with CTX were identified from DHCS. Trained medical physicians

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subsequently reviewed the medical files of all CTX patients in order to validate the diagnosis

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and further register information regarding neurological symptoms, laboratory results and

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radiological findings at CTX diagnosis. A diagnosis was established according to the 1993

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classification of Center for Disease Control and Prevention and the EuroSIDA

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definition(17,18). Definitive diagnosis was defined as clinical and neuroradiological findings

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compatible with CTX and detection of Toxoplasma gondii by neuropathology (biopsy or

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autopsy) and/or in spinal fluid by polymerase chain reaction (PCR). Presumptive was defined

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as clinical and neuroradiological findings compatible with CTX and favourable response to

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CTX therapy and/or positive toxoplasma serology. We included definitive as well as

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presumptive outcomes in our analyses.

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Clinical symptoms were coded as absent if they were not described in the patient files. If the

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patients temperature at CTX diagnosis was not registered, it was assumed to be <

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37.5°Celcius. Neurological deficits were assessed four months and three years after the CTX

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diagnosis. Neurological conditions were registered as resolved, improved, stable or

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deteriorated compared to the initial presentation.

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Criteria for stopping secondary prophylaxis was according to HIV guidelines, being done in

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patients successfully treated for CTX, and experiencing an increase in their CD4+ cell count to

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>200 cells/μL after cART sustained for more than 6 months (19).

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Covariates

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The following clinical relevant explanatory variables were included in the final multivariate

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model: age at study inclusion, sex, race (Caucasian vs. non-Caucasian), reporting injection

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drug use as route of HIV-transmission, baseline toxoplasmosis serology (negative, positive or

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missing), and CD4+ T-cell count at study inclusion (< 100, ≥ 100-199 and ≥ 200 cells/μL, or <

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50 vs. ≥50-99 and ≥ 100 cells/μL when assessing risk of CTX and death respectively), and

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cART prior to study inclusion. In a sensitivity analysis, the multivariate model (i.e. predictive

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and prognostic model) was based on statistically significant variables guided by forwards and

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backwards selection.

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Statistical analysis

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Risk of CTX after HIV diagnosis

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Study inclusion was defined as the date of HIV diagnosis, date of immigration to Denmark or

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January 1, 1995, whichever was more recent. We computed time from study inclusion until

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date of CTX, death, emigration, loss to follow-up or 1 January 2015, whichever occurred first.

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We calculated crude IR per 1,000 PYR and 95% confidence intervals (CI) as a measure of the

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absolute risk of disease. CTX IR was further assessed by calendar time (1995-1996 and 1997-

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2014; time-updated) and years after study inclusion (0-1 and >1 year; time-updated). As the

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effect of the one period was affected by the status of the other period (p < 0.0001), an

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interaction term between these time periods was included in the model. To control for

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confounders and further assess predictive factors of CTX, we used Poisson regression analysis

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including covariates as described above.

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Risk of mortality after CTX diagnosis

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We computed time from CTX diagnosis to date of death, emigration, loss to follow-up or 1

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January 2015, which ever occurred first.

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We calculated crude MR and 95% CI. Kaplan-Meier analysis was used to assess survival

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according to calendar time of CTX diagnosis (1995-1996 and 1997-2014). Mortality after CTX

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diagnosis was further assessed by the time-updated variables calendar time and time after

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CTX (0-3 months and >3 months; time-updated) as mentioned above. To control for

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confounding and further assess prognostic factors for mortality, we used Poisson regression

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analyses including the above-mentioned covariates. Causes of death were classified as 1) CTX-

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related, 2) HIV associated, not CTX-related 3) not HIV-associated, not CTX-related.

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In a sensitivity analysis, 1) CTX IR and associated mortality was assessed by more split in

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calendar time (pre-cART:1995-1996, early cART:1997-2005, late cART: 2006-2014; time-

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updated), 2) Kaplan meier curves were presented according to time of inclusion (pre- early or

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late cART-era) and 3) prognostic factors were assessed before and after 1 year from CTX

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(appendix 1).

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STATA software (version 14) was used for data analysis.

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The study was approved by the Danish Data Protection Agency (journal no 2008-41-1781).

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Ethics approval and individual consent are not required by Danish legislation governing this

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type of research on HIV-infected individuals.

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RESULTS

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A total of 6,325 HIV-infected patients, who fulfilled the inclusion criteria, were identified from

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DHCS, giving rise to 61,698 PYR (median follow-up time: 9 years; interquartile range, IQR 4-

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16). Seventy nine percent of the patients were Caucasian, 76.1% were males and the median

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age at index date was 37 years (IQR, 30-45 years).

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During the study period, 72 patients were diagnosed with CTX (IR: 1.17; 95% CI 0.93-1.47), of

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whom the majority (56.9%) were diagnosed with HIV before 1995. All CTX patients had

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advanced HIV-disease (table 1). In the cART-era, CTX led to the HIV diagnosis in 30% of the

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patients (12/40) and was the first AIDS-defining event in 67.5% of patients (27/40)

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compared to 3.1% (1/32) and 40.6%, (13/32), respectively in the pre-cART era. Additional

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characteristics are provided in table 1.

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Incidence and predictive factors associated with CTX

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From the pre-cART to the cART-era we observed an unchanged risk of CTX during the first

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year after study inclusion (i.e. HIV diagnosis) (aIRR: 0.82; 95%CI: 0.42-1.59). In contrast, a

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substantial reduction in risk of CTX was observed in the subsequent years during the cART-

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era (aIRR: 0.06; 0.03-0.10)(table 2). Similar results were found when assessing the CTX risk

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in a sensitivity analysis by pre-, early and late-cART (1995-1996, 1997-2005, 2006-2014)

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(table 1, appendix 1). Apart from these time-periods, a higher risk of CTX was significantly

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associated with a low CD4+ cell count (< 100 cells/μL), high VL (VL ≥100.000 c/mL) and

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positive toxoplasma serology (table 2). Using backward selection to model the multivariate

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regression model did not change the estimated predictors substantially.

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Presenting symptoms and treatment in patients diagnosed with CTX

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Headache (37.5%), cognitive deficit (41.7%), limb paresis (36.1%) and fever (75.9% > 37.5°C

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and 31% > 38.5°C) were the most common symptoms at presentation (table 3). Coordination

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and speech disturbances, facial palsy and seizures were seen in almost 1/3 of the patients.

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Signs of systemic inflammation and increased levels of protein in the cerebrospinal fluid were

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common (table 4).

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Sulfadiazine/pyrimethamine was the most common induction regimen (87.5%) followed by

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clindamycin/pyrimethamine (29.2%)(table 5). Adjunctive steroids were prescribed in 55.6%

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of patients. Treatment modification during induction therapy was common (25%), mostly due

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with sulfadiazine/pyrimethamine and the median duration was 0.7 years (IQR 0.4-1.7)(table

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5). We did not observe any recurrences after stopping secondary prophylaxis in the study

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period.

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A considerable immunological restoration was observed after cART initiation with a median

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CD4+ cell count of 320 (160-398) 3 years after CTX diagnosis. Additional results are provided

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in table 3-5.

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Neurological outcome

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Four months after CTX, 61.2% of the patients experienced an improvement of their

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neurological symptoms and 18.4% experienced a complete resolution of their deficits. Three

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years after CTX, further improvement was observed, with 45.5% experiencing additional

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improvement of the neurological symptoms and 30% reporting a complete resolution of

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symptoms (table 3). Most patients had regression of cerebral lesions at control neuroimaging;

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however, rescanning was not systematically performed during follow-up. Additional results

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are provided in table 3.

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Mortality

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Forty-two patients (58.3%) diagnosed with CTX died during the study period of whom 30

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(71%) died within the first year and 21 (50%) within the first 90 days after the CTX diagnosis

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(data not shown).

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Kaplan-Meier survival curves for post-CTX mortality stratified by calendar time of CTX

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diagnosis are shown in figure 1 (dividing the cART-era in early-cART and late-cART in figure

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1, appendix 1).

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We observed a substantial reduction in post CTX mortality with later calendar periods (1995-

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1996 vs. 1997-2014) during both the first 3 months (aMRR: 0.24; 0.08-0.71) and the

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subsequent period (aMMR: 0.02; 0.007-0.04). In a sensitivity analysis, in which risk of

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mortality 1) before and after 3 months and 2) before and after 1 year from CTX diagnosis was

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assessed according to pre-, early and late-cART periods (1995-1996, 1997-2005, 2006-2014),

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almost similar results were observed (table 2, 3 and 4, appendix 1). And, during 1997-2014,

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no statistically significant difference in risk death was observed between patients who had

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year 1996 (aMRR: 0.29; 0.07-1.17).

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When evaluating prognostic factors apart from calendar time, injection drug use and no

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exposure to cART before CTX diagnosis were associated with a statistically significant higher

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risk of mortality in univariate models. However, none of these variables remained significant

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in the multivariate analyses. Similar results were observed when using backwards selection to

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model the multivariate regression model.

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Causes of death were as following: 21 deaths were related to CTX, 6 to HIV-infection (1 Kaposi

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sarcoma, 2 lymphoma, 1 AIDS dementia, 1 progressive multifocal leukoencephalopathy, 3

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HIV-wasting), 4 not related to HIV (1 aspiration pneumonia, 1 traffic accident, 1 pulmonary

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embolism and 1 cerebral haemorrhage) and 11 due to unknown aetiology. All patients who

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died within 30 days of CTX diagnosis died of CTX related causes.

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DISCUSSION

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In this Danish population-based cohort study we found a substantial reduction in risk of CTX

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from the pre-cART (1995-1996) to the cART-era (1997-2014) after the first year of HIV

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diagnosis (aIRR: 0.06; 95% CI: 0.03-0.10)). During the same calendar years, post-CTX

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mortality also declined with a 76% reduction in the first 3 months after CTX diagnosis (aMRR:

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0.24; 95%CI: 0.08-0.71) and a 98% reduction in the following years (aMRR: 0.002; 95%CI:

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0.007-0.04).

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The observed reduction in CTX and post-CTX mortality rates probably reflect the coverage of

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cART after HIV diagnosis, the availability of more potent and better-tolerated cART regimens

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resulting in an improved adherence, as well as routine use of antimicrobial prophylaxis. This

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temporal trend is in agreement with finding from other cohorts (2-6,8,20,21). First, the

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multicenter UK Collaborative HIV Cohort (CHIC) study (2), that included 30,954 patients

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between 1996 and 2007, observed a reduction in incidence of CTX from 3.2 (95% CI: 2.4-4.1)

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to 0.4 per 1000 PYR (95% CI:0.3-0.7) in the period 1996-1997 to 2006-2007 (crude IRR 0.13).

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In accordance with our study, the largest mortality was observed during the first year after

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CTX diagnosis (estimated from their Kaplan Meier curve); however, calendar time specific MR

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and MRR were not accounted for. The HIV outpatient study (HOPS) (4), that included 8,070

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patients , observed a reduction in risk of CTX from 4.1 (95% CI: 2.7-5.9) to 0.5 per 1000 PYR

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(95 CI:0.2-1.0) during the period 1994-1997 to 2003-2007 (crude IRR 0.12). Lastly, a

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Brazilian study (3), including 3,378 HIV-infected patients from 1987 to 2012 reported a

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marked decrease in CTX risk from 43.6 to 4.0 per 1000 PYR (95% CI not detailed) during the

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very early pre-cART-era 1987-1990 to the late cART-era 2009-2012 (crude IRR 0.09). None of

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these studies (2-4) stratified their results by time after inclusion (≤1year vs. > 1 year) or

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reported estimates of MR and MRR. The reported reduction in risk of CTX from the pre-cART

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to the cART-era varies between studies, which may be explained by sociodemographic

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differences of the study populations.

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In our study, CTX led to the HIV diagnosis in 30% of cases and was the first AIDS-defining

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event in 67.5% of patients during the cART-era. As expected and observed in other studies

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(22), we found that CTX was significantly associated with a low CD4+ cell count and a high VL

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at study entry. In accordance with other studies from the cART-era, less than 50% of our

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the results of the START study (25), proving the benefits of early cART initiation in reducing

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risk of AIDS and non-AIDS events, treatment practice has been largely changed resulting in

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cART initiation shortly after HIV diagnosis. We expect that this will have some impact on the

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risk of CTX and CTX associated mortality. However, late presenters and non-adherence to

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cART still represents the largest challenge in industrialized countries (11,23,24,26). A recent

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large U.S. study (evaluating 239,147 HIV-associated hospitalizations) (21) reported an

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important reduction in CTX hospitalizations after cART introduction (1993 vs 2001) (Odds

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ratio 0.82, p<.0001); however, only a small reduction in CTX hospitalizations was described

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during later years (2002-2008) (OR 0.96, 0<.0001). We observed a similar trend (results not

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shown), which is probably related to lack of substantial reductions in the number of late

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presenters and patients non-adherent to cART.

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During the cART-era, 66.7% (8/12) of patients diagnosed with HIV at CTX diagnosis started

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cART within 3 weeks (results not shown) which is according to current guidelines. Currently

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there are no specific studies investigating the optimal time for initiation of cART in patients

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with CTX. However, cART initiation within 2 weeks of several OI diagnosis has previously

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been proven beneficial with reduced risk of AIDS progression and death(19,27).

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The main neurological symptoms observed at presentation of CTX are consistent with

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previous reports and include cognitive deficits, headache, limb paresis, speech disturbances,

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coordination disturbance and seizures (28). The majority of our study population (95.7%)

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had positive toxoplasma IgG titers whereas less than 10% presented positive IgM titers. This

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is in line with other studies (23,28), representing the fact that CTX primarily is due to

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reactivation of latent toxoplasma infection acquired earlier in life. Interestingly, we found a

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much lower sensitivity of PCR for toxoplasma in CSF (8.7%) than reported in other studies

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(70-100%) (28-30). However, the number of PCR tests conducted was low and the majority of

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the negative PCR results were performed before year 2000. As all the positive results were

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performed in more recent years, we presume that this is a result of newer and more sensitive

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PCR techniques, as described in other studies (31). In our study, CTX diagnosis was mainly

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based on suggestive neuroimaging and clinical symptoms (table 4). The localization of the

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lesions in the CNS reflected previous reports, with focal or multifocal lesions being more

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commonly localized in the cortex and white matter, and in the basal ganglia (23,32,33).

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Complete neurological recovery was observed in less than 20% after 4 months and in

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approximately 30% after 3 years of follow-up (table 3) with no differences between the pre-

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cART and cART-era (data not shown). Similar findings have been described by others (28,34).

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Although, CTX was highly associated with advanced immunosuppression, cART initiation

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following CTX diagnosis lead to a substantial improvement of the immunological status in

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those individuals who were alive after 3 years of follow-up. Furthermore, post-CTX survival

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improved largely from the pre-cART-era to the cART-era. In the cART-era the post-CTX

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mortality after the first year of CTX had declined to almost negligible levels. As illustrated in

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table 6, this indicates, that the risk of death of a patient who was diagnosed with CTX in the

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pre-cART-era, whom is compliant and still alive as of today, has almost the same prognosis as

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those diagnosed with CTX in the cART-era. Although, no statistical significant prognostic

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factors were detected in the multivariable analysis beside the time periods, and as the

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standard therapy for CTX has not changed over time, these findings indicate that cART is

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indeed a critical factor for the improved survival.

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The strengths of our study include the use of a population-based, nationwide HIV cohort with

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a high coverage, long observation time and almost complete follow-up. We had full access to

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Danish registries of high quality providing data on all individuals concerning vital status,

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residency and migration. This allowed us to estimate longitudinal trends concerning risk of

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CTX and subsequent mortality in the pre-cART and well-established cART-era up to recent

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time. We are not aware of other studies with a similar design.

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Our study has some limitations. Assessment of neurological deficits and performance was

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establish by review of medical files and not by standardized scoring systems. As this may have

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led to inaccuracy, the presence of neurological deficits after CTX may have been slightly

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underestimated. However, any lack of registration is unlikely to be associated with time after

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CTX diagnosis or calendar time. Most CTX cases were based on a presumptive rather than

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definitive diagnosis. In 6 cases the toxoplasma serology was negative or unknown. In these

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cases the presumptive diagnosis was based on clinical and radiological evidence of CTX and

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favourable response to therapy. Although, a positive serology supports the CTX diagnosis it is

14

ACCEPTED MANUSCRIPT 374

neither a requirement in the CDC (17) or the Eurosida definition (18), nor does a negative

375

serology exclude CTX . Some studies (35) have reported a correlation between serum T.

376

gondii IgG levels and the risk of development of CTX. In our study we no not dispose of

377

quantitative measurements of IgG levels why we cannot add information in this regard.

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In conclusion, CTX still remains an important cause of morbidity and mortality among HIV-

380

infected patients with advanced immunosuppression. However, the incidence of CTX and post

381

CTX mortality has declined substantially during the cART-era, especially when surviving the

382

first year of HIV-infection and CTX. As a result, individuals diagnosed with HIV or CTX during

383

the pre-cART-era can be assured a low risk of CTX or post CTX mortality when compliant to

384

cART. Hence, early diagnosis of HIV and cART initiation remains paramount.

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ACCEPTED MANUSCRIPT 387

Acknowledgments:

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We are grateful to the staff of our clinical departments for their continuous support and

389

enthusiasm.

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Centers in the Danish HIV Cohort Study:

392

Departments of Infectious Diseases at Copenhagen University Hospitals, Rigshospitalet (J

393

Gerstoft, N Obel) and Hvidovre (G Kronborg), Odense University Hospital (C Pedersen),

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Aarhus University Hospitals, Skejby (CS Larsen) and Aalborg (G Pedersen), Herning Hospital

395

(AL Laursen), Hillerød Hospital (L Nielsen) and Kolding Hospital (J Jensen).

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Contributors:

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Conception and design: Martin-Iguacel R, Ahlström MG, Touma M, Engsig FN, Rasmussen LD,

399

Obel N.

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Analysis of the data: Martin-Iguacel R, Rasmussen LD

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Statistical expertise: Martin-Iguacel R, Rasmussen LD, Obel N

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Interpretation of the data: Martin-Iguacel R, Rasmussen LD, Obel N.

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Drafting of the article: Martin-Iguacel R, Rasmussen LD, Obel N.

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Critical revision of the article for important intellectual content: Martin-Iguacel, Ahlström MG,

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Touma M, Engsig FN, Stærke NB Rasmussen LD, Obel N.

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Final approval of the article: Martin-Iguacel, Ahlström MG, Touma M, Engsig FN, Stærke NB,

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Stærkind M, Obel N, Rasmussen LD.

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Provision of study materials or patients: Martin-Iguacel, Ahlström MG, Touma M, Engsig FN,

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Stærke NB, Stærkind M, Rasmussen LD, Obel N.

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Obtaining of funding: Obel N.

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Administrative, technical, or logistic support: Martin-Iguacel R, Rasmussen LD, Obel N.

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Collection and assembly of data: Martin-Iguacel, Ahlström MG, Touma M, Engsig FN, Stærke

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NB, Stærkind M, Rasmussen LD, Obel N.

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414 415

Conflict of interest:

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ACCEPTED MANUSCRIPT Obel N has received funding from Roche, Bristol-Myers Squibb, Merck Sharp & Dohme,

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GlaxoSmithKline, Abott, Boehringer Ingelheim, Janssen-Cilag and Swedish Orphan Drugs.

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Engsig FN has participated in a Hepatology Preceptor sponsored by Abbvie.

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For the remaining authors none were declared.

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Funding:

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This work was supported by Preben og Anna Simonsens Fond, NOVO Nordisk Foundation,

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The Danish AIDS Foundation and The Augustinus Foundation. The study was investigator-

423

driven and thus independent of any pharmaceutical company. The funding sources were not

424

involved in study design, data collection, analyses, report writing, or decision to submit the

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paper. The authors had full access to all data in the study and the responsibility for the

426

decision to submit for publication was shared between all authors.

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17

ACCEPTED MANUSCRIPT Table 1. Demographics and HIV-related characteristics of the study patients and the patients who developed cerebral toxoplasmosis (CTX) diagnosed during the study period HIV-infected individuals (n=6,325) 4,817 (76.1) 37 (31-45) 5,001 (79.0)

HIV-infected individuals with CTX diagnosis (n=72) 53 (73.6) 36 (32-47) 55 (76.4)

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Male, n (%) Age at baseline, median years (IQR) Caucasian, n (%) Infection mode: MSM, n (%) 2,936 (46.8) 30 (41.7) Heterosexually infected, n (%) 2,353 (37.4) 31 (43.1) IDU, n (%) 612 (9.7) 7 (9.7) HCV, n (%) 921 (14.6) 7 (9.7) HIV diagnosis before 1995, n (%) 2,023 (32.0) 41 (56.9) AIDS before inclusion, n (%) 533 (8.4) 17 (23.6) On cART before inclusion, n (%) 276 (4.4) 0 CD4+ cell count at study inclusion, median 300(117-497) 61 (20-194) cells/μL (IQR) VL at study inclusion, median log10 copies/mL 4.6 (3.7-5.3) 5.3 (4.5-5.9) (IQR) PYR, n (%) 61,698 160 Median observation time (IQR) 8.9 (3.7-15.7) 0.9 (0.2-3.4) Emigration during the study period, n (%) 307 (4.9) 0 Loss to follow, n (%) 29 (0.5) 0 Death, n (%) 1,556 (24.6) 42 (58.3) CTX, n (%) 72 (1.0) 72 (100) Positive Toxoplasma gondii IgG 2076 (32.8) a 66/69 (95.6%)b Age at CTX diagnosis, n (%) 40 (33-48) Median time from HIV to CTX diagnosis, years . 4.4 (0.5-9.6) (IQR) AIDS before CTX diagnosis (%) 32 (44.4) + CD4 cell count <200 cells/μL at CTX . 67 (90.3) diagnosis, n (%) Virological suppression at CTX diagnosis 0/36c On cART at CTX diagnosis, n (%) . 22 (31.0) Abbreviations: CTX, cerebral toxoplasmosis; MSM, men who have sex with men; IDU, injection drug use; IDU, injection drug use; HCV, hepatitis C virus, HIV, human immunodeficiency virus; AIDS, Acquired Immune Deficiency Syndrome; cART, combination antiretroviral therapy; VL, viral load; PYR, person-years at risk; IQR, Interquartile Range. a Missing

data on CTX serology in 632 patients. . bMissing data on CTX serology in 3 patients at CTX diagnosis. Missing data on VL at CTX in 36 patients. c

18

ACCEPTED MANUSCRIPT Table 2. Predictive factors for cerebral toxoplasmosis in HIV-infected individuals

-

aIRR (95% CI) -

20

2,156

9.28 (5.98-14.38)

Ref (1)

Ref (1)

12

1,708

7.03 (3.99-12.37)

0.76 (0.37-1.55)

0.89 (0.43-1.82)a

19

3,838

4.95 (3.16-7.76)

0.53 (0.28-1.00)

0.82 (0.42-1.59)a

21

53,996

0.39 (0.25-0.60)

19 53

15,945 45,752

1.19 (0.76-1.87) 1.16 (0.89-1.52)

55 17

48,715 12,982

65 7

56,104 5,593

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IRR (95% CI)

61,698

IR per 1,000 PYR (95% CI) 1.17 (0.93-1.47)

0.04 (0.02-0.08)

0.06 (0.03-0.10)a

Ref (1) 0.97 (0.58-1.64) 1.02 (1.00-1.04)

Ref (1) 0.81 (0.45-1.45)a 0.99 (0.97-1.02)a

1.13 (0.87-1.47) 1.31 (0.81-2.11)

Ref (1) 1.16 (0.67-2.00)

Ref (1) 1.17 (0.62-2.21)a

1.16 (0.91-1.48) 1.25 (0.60-2.63)

Ref (1) 1.08 (0.50-2.36)

Ref (1) 1.09 (0.49-2.44)a Ref (1) 38.35 (12.03122.32) 4.76 (0.95-23.83)a

3 66

36,031 20,785

0.1 (0.03-0.3) 3 (2-4)

3

4,880

0.6 (0.2-2)

Ref (1) 38.14 (11.99121.29) 7.38 (1.49-36.58)

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Missing CD4+ cell count at baseline CD4+cell count ≥ 200 cells/μL CD4+ cell count <200, ≥ 100 cells/μL CD4+ cell count <100

PYR

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Total Calendar time (timeupdated) Calendar time 1995-1996: -First year after inclusion in the study -Beyond first year after inclusion in the study Calendar time 1997-2014: -First year after inclusion in the study -Beyond first year after inclusion in the study Gender Female Male Ageb Race Caucasian Non-Caucasian Injection drug use No injection drug use Injection drug use Toxoplasma serology Negative Positive

CTX events 72

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Category

45,027

0.58 (0.39-0.85)

Ref (1)

Ref (1)

9

7,461

1.21 (0.63-2.32)

2.09 (0.98-4.46)

1.90 (0.89-4.07)a

37

9,210

4.02 (2.91-5.54)

6.96 (4.21-11.49)

5.65 (3.36-9.51)a

9

22,154.2

Ref (1)

Ref (1)

3.92 (1.95-7.89)

2.90 (1.38-6.09)a

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VL at baseline

VL < 100.000 c/mL

63 VL >=100.000 c/mL

0.41 (0.21-0.78) 39,543.3 1.59 (1.24-2.04)

Subanalysis Calendar time 1997-2014 and beyond 1 year after inclusion in the study

19

ACCEPTED MANUSCRIPT

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stratified by: Inclusion < 1997 15 26,642 0.56 (0.34-0.93) Ref (1) Ref (1) Inclusion >= 1997 6 27,354 0.22 (0.10-0.49) 0.39 (0.15-1.00) 0.57 (0.18-1.82)c Abbreviations: CTX, cerebral toxoplasmosis; PYR, person-years at risk; IR, incidence rates; IRR, incidence rate ratio; aIRR, adjusted incidence rate ratio; CI, confidence interval; VL, viral load; IDU, injection drug use; AIDS, Acquired immunodeficiency syndrome. a Adjusted for the following baseline variables: gender, age, race, injection drug use, toxoplasma serology, calendar time, CD4+ cell count and viral load. b Age, continuous variable, per 1 year c Adjusted for the following baseline variables: gender, age, race, injection drug use, toxoplasma serology, calendar time, CD4+ cell count and viral load.

27 (38) 14 (19) 30 (42) 21 (29) 22 (31) 14 (19) 19 (26) 26 (36) 5 (7) 22 (31) 6 (8)

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Neurological symptoms Headache Nausea and vomiting Cognitive deficit Coordination disturbance Speech disturbance Visual impairment Facial palsy Limb paresis Sensory affection Seizures Need help in everyday life Status at follow-up Progression of neurological symptoms Unchanged neurological symptoms Improvement of neurological symptoms Resolution of neurological symptoms

Follow-up 4 months (N=49) 3 years (N=33) n (%) n (%)

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Table 3. Neurological symptoms of cerebral toxoplasmosis in HIV-infected individuals at primary presentation, at first follow-up visit after 4 months, and after 3 years

3 (6) 2 (4) 8 (16) 4 (8) 6 (12) 3 (6) 3 (6) 13 (27) 2 (4) 5 (10) 6 (12)

0 (0) 1 (3) 3 (9) 0 (0) 1 (3) 0 (0) 0 (0) 3 (9) 1 (3) 3 (9) 1 (3)

6 (12)

1 (3)

2 (4) 30 (61)

3 (9) 15 (46)

9 (18)

10 (30)

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ACCEPTED MANUSCRIPT Table 4. Clinical data and laboratory results at presentation of HIV-infected individuals with cerebral toxoplasmosis.

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Category, parameter Proportion (%) Clinical and paraclinical signs at presentation Severely impaired GCS 5/72 (7) Moderately impaired GCS 8/72 (11) Mildly impaired GCS 59/72 (82) Fever > 37.5°C 22/29 (76) Fever > 38.5°C 9/29 (31) Elevated CRP or sedimentation rate 32/51 (63) Leukocytosis in peripheral blood (>9 x 109 cells/L) 10/66 (15) CSF pleocytosis (>10 cells/μL) 10/45 (22) Elevated CSF protein level (>0.5 g/L) 33/44 (75) Abnormal glucose level in CSF (< 2 mmol/L) 2/44 (5) Results of diagnostic test for CTX Positive IgG titers for toxoplasma 59/66 (89) Positive IgM titers for toxoplasma 5/56 (9) CTX diagnosed by suggestive MRI/CT-scans and 66/72 (92) clinical symptoms CTX diagnosed by PCR in CSF 4/23 (17) CTX diagnosed by brain biopsy 3/5 (60) CTX diagnosed at autopsy 1/1 (100)a Diagnosis Definitive diagnosisb 8/72 (11) Presumptive diagnosis with positive toxoplasma 66/72 (92) serologyc Presumptive diagnosis with negative toxoplasma 3/72 (4) serologyd Presumptive diagnosis with unknown toxoplasma 3/72 (4) serologyd Abbreviations: CRP: c reactive protein; CSF cerebrospinal fluid; CTX cerebral toxoplasmosis a Autopsy might have been performed in more patients; however, the results of the autopsy is generally not presented in the patient files b Definitive diagnosis was based on clinical and neuroradiological findings compatible with CTX and detection of Toxoplasma gondii by neuropathology or by polymerase chain reaction (PCR). c Presumptive diagnosis was based on clinical and neuroradiological findings compatible with CTX, positive toxoplasma serology and favourable response to CTX. d In the 3 cases where the serology was negative and in the 3 cases where serology was not available, presumptive diagnosis was based on clinical and radiological evidence of CTX and favourable response to therapy.

21

ACCEPTED MANUSCRIPT Table 5. Treatment information related to the initial therapy, secondary prophylaxis and immunological status during follow-up in HIV-infected individuals with cerebral toxoplasmosis.

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Category, parameter Proportion (%) Regimens used as induction therapy Sulfadiazine, pyrimethamine 63/72 (88) Clindamycin, pyrimethamine 21/72 (29) Other 6/72 (8) Adjunctive steroid 40/72 (56) Treatment modification during the first regimen of induction therapy due to side effects Discontinuation of sulfadiazine 15/63 (24)a Discontinuation of clindamycin 3/7 (43)b Secondary prophylaxis after induction therapy Sulfadiazine, pyrimethamine 34/72 (47) Clindamycin, pyrimethamine 9/72 (13) Clarithromycin 4/72 (6) Other 13/72 (18) Duration of secondary prophylaxis (median year, IQR ) 0.7 (0.4-1.7) Cessation of secondary prophylaxis due to immunological restoration on 22/77 (31) cART Immunological response during follow-up after initiation of cART CD4+ cell count at presentation with CTX, median cells/μL (IQR) 42 (10-80) CD4+ cell count at first follow-up after 4 months, median cells/μL (IQR) 110 (30-190) CD4+ cell count at first follow-up after 3 years, median cells/μL (IQR) 320 (160-398) a Of the 63 patients initiating induction therapy with sulfadiazine/pyrimethamine, 48 patients completed the induction therapy without treatment modification. b Of the 7 patients initiating induction therapy with clindamycin/pyrimethamine, 4 patients completed the induction therapy without treatment modification.

22

ACCEPTED MANUSCRIPT Table 6. Prognostic factors for death in HIV-infected individuals diagnosed with cerebral toxoplasmosis. Death

PYR

42

388

MR (95% CI) per 1000 PYR 108 (80-146)

16

5

2,970 (1,820-4,849)

8

7

31,143 (571-2,285)

5

9

542 (226-1302)

0.18 (0.07-0.50)

0.24 (0.08-0.71)a

13

367

35 (21-61)

0.01 (0.006-0.02)

0.02 (0.0070.04)a

8 34

100 289

80 (40-160) 118 (84-165)

Ref (1) 1.47 (0.68-3.17) 1.04 (1.01-1.07)

Ref (1) 0.94 (0.40-2.20)a 1.03 (1.00-1.06)a

37 5

306 83

121 (88-167) 60 (25-145)

Ref (1) 0.50 (0.20-1.27)

Ref (1) 0.67 (0.24-1.92)a

35 7

374 14

94 (67-130) 494 (235-1036)

Ref (1) 5.28 (2.34-11.88)

Ref (1) 1.86 (0.77-4.47)a

6

116

52 (23-115)

Ref (1)

9

107

84 (44-162)

0.95 (0.31-2.93)a

27

165

163 (112-238)

Ref (1) 1.63 (0.58-4.58) 3.16 (1.31-7.66)

34 8

219 170

155 (111-218) 47 (24-94)

Ref (1) 0.30 (0.14-0.66)

Ref (1) 0.91 (0.36-2.29)a

SUBANALYSIS Calendar time 1997-2014 and months >3 after CTX stratified by: CTX < 1997

7

170

41 (20-86)

Ref (1)

CTX >= 1997

3

169

18 (6-55)

0.29 (0.08-1.07)

Ref (1) 0.29 (0.071.17)b

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MRR (95% CI)

Ref (1)

0.38 (0.16-0.90)

0.34 (0.14-0.82)a

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Ref (1)

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Mortality overall Calendar time (time-updated) Calendar time 1995-1996: -First 3 months after CTX diagnosis -Beyond the first 3 months after CTX diagnosis Calendar time 1997-2014 -First 3 months after CTX diagnosis -Beyond the first 3 months after CTX diagnosis Gender Female Male Age at CTX Race Caucasian Non-Caucasian IDU/no IDU No IDU IDU CD4+ count at CTX diagnosis CD4+ cell count ≥ 100 cells/μL CD4+ cell count <100, ≥50 cells/μL CD4+ cell count <50 cells/μL cART before CTX No Yes

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1.27 (0.48-3.38)

Abbreviations: PYR, person-years at risk; MR: Mortality Rate, MRR Crude mortality rate ratio, aMRR: adjusted Mortality Rate Ratio; cART, combination antiretroviral therapy; IDU, injection drug use; AIDS, Acquired immunodeficiency syndrome.

23

ACCEPTED MANUSCRIPT a Adjusted

for the following baseline variables: gender, age (continuous variable, per 1 year), race, injection drug use, CD4 and cART before CTX. b Adjusted for the following baseline variables: gender, age (continuous variable, per 1 year) race, injection drug use, CD4 and cART before CTX.

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Figure 1. Kaplan-Meier curves for overall survival of HIV-infected individuals with cerebral toxoplasmosis (CTX) by calendar time of CTX diagnosis (1995-1996 (solid line), 1997-2014

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(striped).

Log rank test 18.70 p<0.00001

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