Sequelae and mortality in patients with HIV/AIDS and Progressive Multifocal Leukoencephalopathy: Systematic review and case series in the Brazilian Amazon

Background Progressive Multifocal Leukoencephalopathy (PML) is an opportunistic neurological disease that mainly affects individuals with HIV/AIDS and has high morbidity and mortality, due to its demyelinating characteristic. This co-infection has been reported since the begging of HIV/Aids epidemic with increasing unfavorable outcomes, however, factors associated to sequelae and death are greatly unknown. In this study we aimed to understand factors associated with the main outcomes of individuals diagnosed with PML and HIV/AIDS, in addition to reporting the characteristics of patients presenting to a referral center in infectious diseases in the Brazilian Amazon. Methods A systematic review was performed until July 2022, following the PRISMA guidelines, at Medline/Pubmed, Web of Science, Lilacs and Scielo databases using combinations of HIV, Aids, JC Virus and Progressive Multifocal Leukoencephalopathy, with no restriction to publication date. Additional cases, meeting the eligibility criteria, were added from our hospital database, which consisted of patients presenting PML/HIV between 2010 and 2022. A meta-analysis aiming to explore factors associated to sequelae and death was performed. Baseline characteristics were described using mean and standard deviation, or median and interquartile range when appropriate; multivariate analysis was performed to study factors associated to death and sequelae outcomes. Results Eighteen patients were diagnosed between 2010 and 2022, of these, 10 had positive PCR for JC virus. In the Systematic Review, 216 studies yielded 235 confirmed cases of co-infection. A total of 245 were included for analysis. The rates of death and sequelae were, respectively, 47.1% (114/242) and 41.2% (54/131). The use of antiretroviral therapy was more associated with a lower chance of death (OR 0.30, 95% CI: 0.11-0.83), while muscle weakness (OR 4.82, 95% CI: 2.07-11.21) and muscle spasms (OR 6.12, 95% CI: 1.05-35.76) were associated with greater chances of sequelae. Conclusion Those on antiretroviral therapy appear to be less likely to die, and among those who survive, those who have muscle weakness as a symptom on admission are more likely to develop sequelae. Adherence to ART, as well as a comprehensive clinical evaluation and follow-up may help to improve clinical outcomes and awareness of morbidities.

Despite being a rare disease, PML is of great clinical importance mainly due to the lack of a specific treatment, poor prognosis, and unfavorable outcomes (2,3,14). This is related to the central nervous system (CNS) tropism, more specifically to oligodendrocytes, cells responsible for the formation and maintenance of the myelin sheaths (3,7). Without intervention, permanent sequelae and death are common events (9). Clinically, PML frequently manifests motor symptoms, including monoparesis, hemiparesis, ataxia, and occasionally, extrapyramidal manifestations such as tremors, walk disturbances, spasms, and involuntary movements (12). PML can be diagnosed using molecular methods for detecting JCV, imaging, and/ or brain biopsy, which implies that the disease has an easier diagnosis (and consequently higher prevalence) in higher-income countries since it affords the necessary resources (6,(18)(19)(20).
The number of new cases of HIV/Aids has been rising in the last years, especially in the western Brazilian Amazon, with several cities above the Brazilian Aids detection rate (21). Despite its importance, evidence on HIV/JCV co-infection is still scarce in low and middleincome countries, like Brazil. Also, the literature, which is mostly based on case reports and case series, lacks evidence regarding the association of risk factors to important clinical outcomes, such as death and sequelae (22)(23)(24). The hypothesis of this study is that clinical factors at hospital admission may be predictors of death and sequelae in this population. We performed a systematic review of the literature available for HIV/ JCV co-infection and report a series of cases of this co-infection in patients managed at a reference center for the treatment of infectious diseases in the Western Brazilian Amazon. Finally, we present a metanalysis aiming to study risk factors associated with death and sequelae in this population. Moreover, to our knowledge, this is the first study aiming to evaluate risk factors associated with both these outcomes on individuals with HIV/JCV co-infection.

Ethical considerations
FMT-HVD Ethics Review Board approved this study per the guidelines and standards for regulating research on human subjects established in Resolution 466/12, of the National Health Council of the Brazilian Ministry of Health (CAAE 89665118.6.0000.0005). A waiver of informed consent was obtained due to the retrospective nature of the study. Patient anonymity was preserved throughout data extraction and analysis.

Case series
All electronic medical records (EMR) of patients treated at Fundacão de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD), between January 2010 and July 2022, were screened based on diagnoses. EMRs were carefully evaluated and those containing information from patients diagnosed with PML and HIV were eligible. Patients with confirmed HIV infection and diagnosis of PML were included. Those without a diagnosis of the diseases of interest or with insufficient records in the database were discarded. FMT-HVD is a tertiary-care referral hospital for infectious diseases located in Manaus, Western Brazilian Amazon, which receives patients seeking medical care as well as those referred from public and private healthcare units in surrounding localities. FMT-HVD adopts all Brazilian guidelines for the management of sexually transmitted infections and HIV infection (25), since it belongs to the Brazilian Unified Health Care System (SUS). HIV infection was previously determined by two positive rapid diagnostic tests (Abbott, Chicago, United States and Bio-Manguinhos, Fiocruz, Rio de Janeiro, Brazil) and confirmed by an immunoassay test, as defined by the Brazil Ministry of Health (26). HIV diagnosis, treatment, and follow-up are free of charge to all Brazilians and foreigners. The diagnosis of PML was performed using laboratory confirmation when available. In those cases where this confirmation was not possible/available, the physicians performed the diagnosis following clinical and imaging evaluations accompanied by differential diagnosis. These followed the Brazilian Guidelines for HIV and Coinfections management (25). All physicians are trained and highly experienced in tropical medicine. Before the inclusion of participants in this study, the medical charts were reviewed by experienced physicians who authored this manuscript (MAA and ML). Only those with PCR confirmation for JCV were included in the metanalysis. Data on demographics, clinical, radiological, and/or laboratory diagnosis, length of hospitalization, and any supportive procedures and outcome (sequelae and death) described in the electronic medical records were retrieved from Individual EMRs and sent anonymously to the database. Sequelae were only considered present when there was a detailed description of their characteristics on the medical charts. Muscle weakness was assessed through the capacity of contraction against applied manual resistance, asthenia was described as a feeling of extreme tiredness or generalized lack of physical energy, and spams were described as involuntary contractions, all assessed at hospital admission. Data collection was carried out by two independent researchers in an anonymous manner. Any disagreements were resolved by consensus.

Systematic review and meta-analysis
An Individual participant data (IPD) systematic review of HIV/ AIDS-associated PML was performed in accordance with the Preferred reporting items for a systematic review and meta-analysis of individual participant data (PRISMA-IPD) (27). Studies reporting cases of coinfection were systematically identified from multiple electronic databases (Medline/PubMed, Web of Science, Lilacs, and Scielo), using the following Keywords in the Search strategy: (Hiv AND Progressive Multifocal Leukoencephalopathy) OR (Aids AND Progressive Multifocal Leukoencephalopathy) OR (Hiv AND JC virus) OR (Aids AND JC virus) in English, Portuguese and Spanish. The last search was performed in July 2022, considering all publications, with no restrictions on the start date. All cases included in the systematic review have laboratory confirmation of HIV/JCV co-infection. Suspected or inconclusive cases were discarded. Studies identified by searching the bibliographic references of the articles were also included. Duplicate articles were removed. Additional studies titles and abstracts were reviewed to confirm the inclusion criteria. Included studies were assessed for eligibility by a full-text review ( Figure 1). Two independent authors of the study conducted the systematic review process. Disagreements were resolved by consensus. The following data were retrieved: author, year of publication, country, demographic, clinical, and laboratory data. To compose the meta-analysis, we considered all cases reported in the literature identified through the systematic review plus the cases seen at the FMT-HVD ( Figure 1). We considered death and sequelae as the main outcomes, which should be clearly stated in the description of the original articles or in the electronic medical record.

Statistical analysis
The baseline characteristics of individuals were summarized as medians with interquartile range (IQR) or means with standard deviation (SD). The proportions between the groups (sequelae x no sequelae/deaths x non-deaths) were compared using the chi-square or Fisher test for categorical variables. Mean and standard deviation, as well as median and interquartile range, were calculated for continuous variables and analyzed using ANOVA or Wilcoxon test, respectively. Those presenting inclusive data regarding clinical outcomes (sequelae and/or death) were excluded from the analysis. To analyze the factors associated with sequelae and death, univariate and multivariate logistic regression models were applied, using the automatic backward stepwise method. A significance level of p<0.2 in the univariate analysis was considered to compose the multivariate analysis. Statistical significance was considered if p<0.05 in the final multivariate model. All analyzes were performed using the statistical package Stata (v.17).  Table 1 shows a summary of HIV/AIDS and PML patients. Nine (50%) were men. The mean age was 35,2 ( ± 10.7 years), with the youngest patient being 21 (case 7). All had a previous diagnosis of HIV. 61% (11/18) were on regular ART use. Neurotoxoplasmosis (44.4%) and tuberculosis (27.7%) were the most evidenced infectious comorbidities. The mean hospitalization time was 69.2 days. Lumbar puncture for cytochemical analysis was performed in 16 patients (88.9%). Mean glucose and protein in CSF were 57.3 (2-138) and 114.4 (14-831), respectively. Sequelae were evidenced in 10 (55%) patients, and they were mainly related to movement/locomotion. Lesions such as cerebral abscesses/edema and ventricular lesions were less present, and all brain regions were affected (Table 1). Figure 2 shows the magnetic resonance images of case 7, where we can observe the areas of lesion in the white matter (indicated by the arrows): multifocal lesions with emphasis on the involvement of the U fibers and the cerebellar peduncle. In Figure 3, we observe the magnetic resonance images of case 8, where we were also able to identify multifocal lesions in the white matter, with emphasis on the frontal cortico-subcortical involvement, which worsens and extends to the left, reaching the temporal region.

Systematic review
The original search yielded 3498 studies. After removing duplicates, applying criteria for study inclusion, and searching for references, 216 studies were considered eligible, covering 235 reported cases ( Figure 1). The highest number of related cases and studies was found in high-income countries (78.7%). Supplementary Table 1 describes the characteristics of included patients. The average age was 38.6 (28-42). Most cases were reported in men (78.2%). Upon admission to the health service, the most frequently reported symptoms were limb paresis (52.3%), aphasia (50.2%), ataxia (41.3%), visual changes (31%), cranial nerve palsy (30.2%), changes in consciousness (28%) and muscle weakness (26.8%). A total of 66.6% (55/77) were ART users. Abnormal CT and MRI findings were evidenced in 77.2% (78/101) and 99% (212/214), respectively. In the MRI reports, white matter lesions were the most common finding (90.1%), followed by cerebellar (46.4%) and ventricular (15%) lesions, and the most reported affected brain regions were the parietal and frontal lobes, with frequency of 33% and 29.6%, respectively. None of the patients were using immunomodulatory drugs or underwent any complementary drug therapy. As for the outcomes, 45.9% of the individuals died, and, among the survivors, 38.8% had sequelae, with limb paresis and communication disorders being the most frequent.

Study outcomes
The characteristics of the total study population (systematic review + case series), according to the outcomes, are shown in Table 2. Muscle weakness (OR 4.82, 95% CI: 2.07-11.21, p=0.001) was considered associated with sequelae on follow-up, while muscle

Discussion
Opportunistic infections represent the major cause of mortality in individuals living with HIV/AIDS. In addition, sequelae that persist after treatment are important outcomes of opportunistic CNS infections (43,44). PML stands out with expressive clinical relevance for its lethality and sequelae (2), and although relevant, only 235 cases of HIV/JCV patients are reported in the literature until July 2022, which may be related to the rarity of the disease or the difficulty of timely and/or appropriate diagnosis. Our results showed that muscle weakness and spasms were associated with sequelae in the study population, evidencing that our initial hypothesis did not was null, while the use of ART, as expected and delimited in the literature, showed a lower association with chances of death.
Through our systematic review, we were able to observe that reports of co-infection are scarce in lower-middle and upper-middle income countries (about 22%), such as Brazil. It is important to consider that in low-income countries there is no report of co-infection in the literature, although in these countries HIV infection is highly prevalent (28, 45,46). PML might be more frequently diagnosed in developing countries where modern diagnostic procedures are more frequently employed (18)(19)(20)24). PML has been highly associated with HIV and at the height of the AIDS pandemic was present in about 5% of people living with HIV (29). In the pre-ART era, PML affected about 3% to 9% of patients living with HIV infection and was a cause of up to 18% of death (30). Currently, however, the incidence of PML has decreased and life-year rates in patients with HIV and PML have increased, mainly due to antiretroviral therapies, PML is still the second most common cause (14%) of all AIDS-related deaths (31). A hospitalbased study recently carried out in São Paulo showed PML in 6% of patients with HIV, a rate similar to the one found in developed countries in the pre-ART era (24). In this study, we showed a low prevalence (1.23%) of HIV and JCV co-infection, 46,8% of the patients died and important sequelae were evidenced. Demographic, clinical and laboratory characteristics presented here are similar to recent reports in different countries (10,24,32,33). Also, the most frequent symptoms presented at admission (limb paresis, aphasia, ataxia, visual changes, cranial nerve palsy, and muscle weakness) are symptoms common to PML (29,34). PML usually develops in patients with CD4+ lymphocytes < 200 cells/mm3 and can occur in patients receiving ART (35). In our case Magnetic resonance imaging findings of case 7: Areas with increased signal and hyperintensity were observed on T2-weighted and FLAIR (Fluid-Attenuated Inversion Recovery) sequences, without contrast enhancement, showing asymmetric, multilobar, and multifocal white matter lesions (A, B) involving U-shaped fibers (C) and affecting the medial cerebellar peduncles bilaterally (D). series, 72,2% (13/18) were immunosuppressed, 33% (6/18) were severely immunosuppressed (CD4+ < 100 cells/mm3), and 61% (11/18) were on regular ART use. These findings differ little from what has been shown in a series of 20 PML/HIV cases in Portugal, where patients were also suppressed and on irregular use of ART, evidencing a high death rate (81%) (36). Here, a lower death rate was evidenced both in our case series (44%) and in our systematic review (47%).
The understanding of the factors associated with PML survival remains lacking (37). Older patients at the onset of PML are significantly different between survivors and non-survivors (38). However, age was not a significant factor for death outcomes in this study, and the use of ART showed a tendency to be an important factor in reducing them. It is known that the treatment of HIV patients with continuous ART and obtaining undetectable levels of viral load are good predictors of survival (14,15,39,40). Thus, the therapeutic approach may include initiating ART in untreated patients or optimizing previous ART regimens (36). Some factors were associated with an improved outcome of PML. Better chances of survival have been correlated with (i) higher CD4+ cell counts at disease onset, (ii) immunological responsiveness, (iii) particularly an improvement in CD4 lymphocyte counts and (iv) reduced JCV load in the CSF (36,41). Baseline CD4+ count is considered a prognostically significant variable. One study showed that patients with a CD4+ cell count <100 cells/mL have a 2.8 more chance of death (42). In addition, patients harboring JCV-specific CD8+ CD8 + T lymphocytes (CTL) have a significantly increased survival, however, the beneficial effect of CTL is predominant in individuals whose immunosuppression may be reversible, such as in HIV+ patients treated with ART (37), which again evidences the role of therapy in the best outcome associated with survival.
The use of ART and increased survival in people living with HIV and PML has made PML a chronic disease rather than a fatal disease, for this reason, it is important to understand the clinical course of survivors (47). People who survive PML may develop neurological sequelae due to patterns of demyelination in the CNS (48). In our study, 42.8% of the individuals who survived PML had sequelae, the main ones being related to muscle health, movement, locomotion, and communication. Sequelae related to visual disturbances, memory and cognitive functions were also evidenced in our systematic review, although it was observed in only 1 of the 18 cases presented in the case series, although the different regions of the brain that are related to these functions have been shown to be injured. Out of the 18 cases reported, we had access to MRI images of two of them, cases 7 and 8 (Figures 2, 3). In these cases, we observe the multifocal demyelinating lesions in the white matter, which are the main characteristic of PML (49,50). The main affected regions visualized presented similarity with findings from other studies, with the involvement of U fibers, lesions in the cerebellar peduncle, and lesions with cortical and subcortical involvement (51)(52)(53)(54). In an appropriate clinical context, brain imaging can strongly support the diagnosis of PML (49). Since the 1990s, pioneering studies involving patients with PML have already established that cranial MRI is much more sensitive for identifying white matter lesions than methods such as computed tomography (55,56). In the cases presented here, the lesions were visualized in a hyperintense manner on T2 FLAIR, which is the most sensitive modality for visualizing these lesions (49).
In general, permanent neurological deficits reflect the functional region injured in the infection, interestingly, lesions that affect the cerebellum are associated with ataxia and incoordination of gait, which makes independent living troublesome (47). In addition to white matter lesions, cerebellar lesions, ventricular injury, edema, inflammation, thalamus and corpus callosum injury were frequently evidenced in our study. In the classical form widely described in the HIV population, PML manifests as white matter lesions affecting subcortical U-shaped fibers with a decreased signal on T1-weighted sequences and an increased signal on T2-weighted sequences (57). Contrast enhancement (CE) of PML lesions on CT or MRI is associated with a favorable prognosis if the patient survives (37). Neurodegenerative changes with focal atrophy (including ex vacuo dilatation of CSF spaces) can develop over weeks to months (29). These characteristic findings of PML are also important to differentiate these cases from diseases with similar involvement, such as HIV-associated encephalopathies, characterized by encephalitis and encephalopathies that present cerebral atrophy as the most common finding on magnetic resonance imaging and are reported as lesions that can evolve into the "Dementia Complex Associated with HIV/AIDS", with symptoms predominantly focused on aspects cognitive, behavioral and memory disorders and, together with Asymptomatic Neurocognitive Disorder (ANI) and Mild Neurocognitive Disorder (DMN) make up the set of diseases called HAND (HIV-Associated Neurocognitive Disorders) (58,59).
Important neurological sequelae are reported in HIV-PML patients, even though the average survival time has increased for people leaving with HIV since ART (36). However, the factors associated with sequelae are poorly known. In the present study, muscle weakness was significantly associated with the presence of sequelae, with high OR (5.72), with muscle spasms tending to be associated with sequelae. Muscle symptoms have been cited in studies on PML as a prevalent symptom (33, 50, 60). Aspects of muscle tissue health are involved with limb paresis, the most reported sequelae in the studies from our systematic review. Limb paresis is closely associated with peripheral neuropathy. PML and peripheral neuropathy have been described in a man who presented with limb weakness and numbness (61). Spinal cord injuries, such as those caused by JCV, can cause muscle denervation and consequent muscle impairments (62, 63). Muscle health may also be linked to weight loss (and loss of muscle tissue) associated with       HIV, which is established in the literature as one of the presentations of people living with HIV (64,65). The time of disease progression and the patient's time in bed can also be related to muscle deficits. Most critically ill patients lose muscle because of an inability to maintain protein synthesis rates and showing a decline in muscle health, starting to present muscle weakness, spasms, paresthesias and, in more severe cases, plegia (66)(67)(68). The management of critically ill patients in the ICU alone indicates an important risk factor for sequelae regardless of the underlying disease. Neuromuscular weakness and impairments in physical function and quality of life abnormalities are common and can be long-lasting (69)(70)(71)(72)(73)(74)(75).

HIV/AIDS
In cases of neurological sequelae, rehabilitation through a multidisciplinary approach, including physical therapy, should be prioritized, aiming to minimize, or even revert, possible disabilities caused by disease; moreover, interventions leading to an individual's functional capacity and general quality of life are of great importance (76)(77)(78). Physical therapy is essential in cases of demyelinating/opportunistic diseases, easing the progression of sequelae and preserving the musculoskeletal tissue in people living with HIV/AIDS (79). Demyelinating opportunistic diseases present a similar spectrum of symptoms that mainly involve motor deficits such as weakness, contractures, gait and balance disorders, among others (24,56,(78)(79)(80). Several studies have already shown the improvement of balance deficits, strength gain, and maintenance of muscle tissue and general functional performance through rehabilitation by a multidisciplinary team (81,82). Even though the importance of physical therapy in such cases is well known, studies included in this systematic review rarely mentioned any rehabilitation approach. This should be a priority for future studies to better comprehend post-acute disease scenarios.
This study had several limitations. Its retrospective nature of case reports accounts for incomplete data available in patient's medical charts, mostly due to the absence of detailed targeted investigation, including proper registration of imaging, laboratory studies, adherence to ART therapy, survival time, and greater details about the sequelae and the persistence time in survivors. Patient admission and clinical management were made at the physician's discretion and may have resulted in classification bias. Of the cases presented, 8 were diagnosed through medical evaluation of clinical presentation, specific findings in imaging tests, and ruling out other diagnostic possibilities, in the absence of confirmation by PCR, biopsy, or autopsy. Regarding the systematic review, prevalence studies and case reports or case series exploring clinical outcomes may underestimate the HIV-PML co-infection and may have influenced the associations of clinical aspects and relevant outcomes, such as sequelae and deaths. The existence of coinfections such as neurotoxoplasmosis (17 cases out of 250), which also favor the appearance of symptoms such as weakness and muscle spasms, is also a limitation since the likelihood to associate these symptoms to one or another is very limited when both are present. In addition, the absence of systematic screening for PML, in general, due to the lack of more sensitive techniques, such as the evidence of JCV in CSF by PCR in low and middle-income countries may reduce the evidence of the high prevalence of this co-infection. In these countries, the diagnosis performed at the discretion of a clinician upon clinical suspicion can impair the rapid and adequate management and treatment which can culminate in unfavorable outcomes. Even so, our findings elucidate aspects not previously addressed about predictive factors of outcomes and death and sequelae of co-infection.
In conclusion, people living with HIV coinfected with JCV using antiretroviral treatment may be less likely to die. Among those who survive, those who have muscle weakness as a symptom on hospital admission are more likely to have sequelae. These findings may contribute to the implementation of a more detailed clinical evaluation, mainly on the muscular aspects of individuals admitted to health units, helping to improve the treatment and specific management of this population to avoid or minimize its sequelae. In addition, our results reinforce the importance of ART by the population living with HIV and/or the regularization of failed schemes, since it is a key factor for better clinical outcomes. In this regard, physical therapy could optimize patient management. Also, the awareness of the occurrence of these important clinical signs may perhaps influence the reduction of mortality rates and long-term morbidities.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Author contributions
CB, DB-D-S, FV, GA, JV, ML, NC-V and PD-T participated in study conception and design. JV and PD-T performed the systematic review. JV, NC-V, FB, SP, EM, MA, SM, EF, VA, RM and MB were responsible for collecting and reviewing patient data from the FMT-HVD. JV was responsible for the manuscript writing. FV and DB-D-S critically reviewed the manuscript. MR and VS were responsible for the statistical analysis. All authors read and approved the final manuscript.

Funding
This study was funded by the Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/PROAP 1247/2022. JV received a scholarship from FAPEAM (Fundação de Amparo à Pesquisa do Estado do Amazonas) for the master's degree. MVGL is a fellow from CNPq. DB-D-S was supported by FAPEAM (National Visiting Researcher Scholarship). Funding was also obtained from FAPEAM (FAPEAM/PPP-CNPq, 355 EDITAL N. 016/ 2014 and FAPEAM/PCTI/EMERGESAÚDE/AM, EDITAL N. 006/2020). The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all data in the study and had final responsibility for submitting it for publication.