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We detected hepatitis B virus (HBV) DNA in the cerebrospinal fluid (CSF) of 26 adolescents co-infected with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) with neurological disease and studied compartmentalization of HBV in the CSF. More than half of the subjects with positive HBV DNA plasma also had CSF positive for HBV. CSF HBV DNA was found in subjects with preserved blood-brain barrier integrity. In a subgroup of these subjects, compartmentalized evolution of HBV was demonstrated by distinct profiles of resistance mutations. Future studies are warranted to determine the clinical significance of HBV presence in the CSF and its contribution to HIV-associated neurological disease.

Cytomegalovirus infections and reactivations are more frequent in people living with HIV (PLWH) and have been associated with increased risk of HIV progression and immunosenescence. We explored the impact of combination antiretroviral therapy (cART) on latent CMV infection in 225 young adults parenterally infected with HIV during childhood. Anti-CMV IgG antibodies were present in 93.7% of participants, with lower levels correlating with longer cART exposure and better immunologic parameters. Patients with immunological treatment success (CD4 > 350 cells/mL) had significantly lower CMV IgG titers compared to those with suboptimal immune response to cART. In total, 78% of the tested patients had robust CMV-specific T-cell responses, measured by an IFN-γ release assay. A good immune response to treatment was significantly associated with CMV-specific cellular immunity: IFN-γ level was positively correlated with CD4 and CD8-T cell counts. No differences were observed between patients with suppressed/non-suppressed HIV viremia in terms of CMV humoral and cellular immune response. CMV DNA was detected in only 17% of participants, with lower levels among those with cART-induced immune recovery. The successful antiretroviral treatment with subsequent immunologic reconstitution may lead to restoration of CMV-specific immune responses and effective control of latent infection, limiting episodes of CMV reactivation in HIV-positive individuals.

The aim of the study was to evaluate the incidence of brain opportunistic pathologies and survival in patients living with HIV from a Romanian tertiary center. A 15-year prospective observational study of brain opportunistic infections diagnosed in HIV-infected patients was performed at Victor Babes Hospital, Bucharest, between January 2006 and December 2021. Characteristics and survival were compared related to modes of HIV acquisition and type of opportunistic infection. A total of 320 patients were diagnosed with 342 brain opportunistic infections (incidence 9.79 per 1000 person-years), 60.2% males with median age at diagnosis of 31 years (IQR 25, 40). Median CD4 cell count and VL were 36/μL (IQR 14, 96) and 5.1 log 10 copies/mL (IQR 4, 5.7) respectively. The routes of HIV acquisition were heterosexual (52.6%), parenteral route in early childhood (31.6%), injecting drug use (12.9%), men having sex with men (1.8%), and vertical (1.2%). The most common brain infections were progressive multifocal leukoencephalopathy (31.3%), cerebral toxoplasmosis (26.9%), tuberculous meningitis (19.3%), and cryptococcal meningitis (16.7%). Patients infected by parenteral mode in early childhood were younger at diagnosis of both opportunistic infection and HIV (p < 0.001 and p < 0.001, respectively), developed more frequently PML (p < 0.001), and had the lowest early (p = 0.002) and late (p = 0.019) mortality rates. Risk factors for shorter survival were age > 30 years (p = 0.001), injecting drug use (p = 0.003), CD4 +  < 100/μL (p = 0.007), and VL > 5 log 10 copies/mL at diagnosis (p < 0.001). The incidence and mortality rate of brain opportunistic infections were high and did not decrease significantly during the study period, due to late presentation or non-adherence to ART., (© 2023. The Author(s) under exclusive licence to The Journal of NeuroVirology, Inc.)

Introduction: An unusual cluster of myoclonic epilepsy was observed in a Romanian pediatric HIV cohort concurrent with measles outbreaks. We describe this particular form of subacute measles encephalitis (SME) in a group of HIV-infected children and adolescents with severe immunosuppression., Methods: This is a single-center study, starting in 1997 and covering 4 measles outbreaks in Romania. The presumptive diagnosis of subacute myoclonic measles encephalitis (SMME) was based on: (1) epidemiological data, previous measles episode or presumed contact with measles virus (MV), (2) clinical presentation with initial localized myoclonic jerks with rapid extension and subsequent motor deficit with preserved mental status, and (3) neuroimaging studies revealing cortical gray matter lesions. Definitive diagnosis was based on a neuropathological exam and immunohistochemistry of brain tissues, and measles RNA detection in the cerebrospinal fluid (CSF)., Results: Thirty-six patients were diagnosed with a particular form of SME during consecutive measles outbreaks in Romania: 1996-1998 (22); 2005-2008 (12); 2010-2011 (1) and 2016-2018 (1). Most children were born in the late 80s and had parenterally acquired HIV infection in early childhood. Before the episode of SMME, 11 patients had confirmed measles, while the rest, without typical rash, had a respiratory tract infection and/or presumed previous measles contact. In all patients, the clinical onset was sudden, with unilateral myoclonus. MRI findings revealed mainly focal cortical gray matter lesions. Neurologic symptoms progressed rapidly to coma and death in most patients. Three patients survived SMME, they had higher CD4 count at onset, slower progression of neurological symptoms, and benefit of immune recovery with cART. Immunocytochemistry studies revealed MV in the brain with a pattern suggesting an ascending viral neural infection. MV was isolated from CSF in 7 out of 8 patients. Sequence analysis of MV RNA from both nasopharyngeal swabs and CSF was available for one patient with similar N-450 strain characteristics., Conclusion: During an outbreak of measles, neurological manifestations, especially myoclonus in immunosuppressed patients, can be related to measles even in the absence of an acute episode. This particular form of subacute myoclonic measles encephalitis is an opportunistic fatal disease. Immune recovery due to effective antiretroviral treatment might increase survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ene, Duiculescu, Radoi, Lazar, Tardei, Ungureanu, Ruta, Vinters, Letendre, Grant, Ellis and Achim.)

We describe a series of severe neuroinvasive infections caused by Toscana virus, identified by real-time reverse transcription PCR testing, in 8 hospitalized patients in Bucharest, Romania, during the summer seasons of 2017 and 2018. Of 8 patients, 5 died. Sequencing showed that the circulating virus belonged to lineage A.

Background: In Romania, after a major outbreak in 1996, West Nile neuroinvasive disease (WNND) was reported only in a limited number of cases annually. During 2016-2017, a significant increase in the number of WNND cases was reported at the national level, associated with high mortality rates., Methods: A retrospective analysis of all cases confirmed with WNND, hospitalized during 2012-2017 in a single tertiary facility from Bucharest was performed in order to determine the annual prevalence and mortality rate and the risk factors associated with a severe outcome., Results: 47 cases were confirmed as WNND. The mortality rate was 25.5%, all death occurred during 2016-2017. Coma, confusion, obtundation, sleepiness and depressed deep tendon reflexes were symptoms predicting a severe outcome. In a univariate analysis age (p < 0.001), associated cancers (p = 0.012) and low levels of chloride in the CSF (p = 0.008) were risk factors for mortality. In a multinomial logistic analysis, age older than 75 years remained the only independent predictor of death in WNND., Conclusions: The increase in both the number and the mortality rate of WNND cases suggest a changing pattern of WNV infection in Romania. Public health authorities and clinicians should be aware of the risk of severe WNV infection in travelers returning from Romania., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We characterized influenza B virus-related neurologic manifestations in an unusually high number of hospitalized adults at a tertiary care facility in Romania during the 2014-15 influenza epidemic season. Of 32 patients with a confirmed laboratory diagnosis of influenza B virus infection, neurologic complications developed in 7 adults (median age 31 years). These complications were clinically diagnosed as confirmed encephalitis (4 patients), possible encephalitis (2 patients), and cerebellar ataxia (1 patient). Two of the patients died. Virus sequencing identified influenza virus B (Yam)-lineage clade 3, which is representative of the B/Phuket/3073/2013 strain, in 4 patients. None of the patients had been vaccinated against influenza. These results suggest that influenza B virus can cause a severe clinical course and should be considered as an etiologic factor for encephalitis.

Introduction: In the last years, we observed an alarming increase in the number of newly diagnosed HIV infected intravenous drug users (IDUs) co-infected with hepatitis viruses or with severe bacterial infections. The aim of our study was to assess the incidence, the demographic and clinical characteristics of IDUs diagnosed with HIV, HCV and tuberculosis (TB)., Materials and Methods: Prospective study on HIV infected IDUs with HCV and TB admitted in a single centre between January 2009 and April 2014. Data were compared to a group of HIV infected IDUs without TB. Statistical analysis was performed using Graphpad Prism 4.01., Results: Out of 450 HIV infected IDUs, 134 (29.7%) were diagnosed with HIV, HCV and TB. TB incidence among IDUs increases from 0% in 2009 to 30.2% in 2013. The TB coinfected patients had a mean age at diagnosis of 30 [15-56] years; were in majority males, 106 (84.4%); from urban areas, 120 (89.5%); and had significantly lower education level (85% vs 68.3%, p<0.0001) and higher rates of unemployment (80% vs 55%, p<0.0001) than those without TB. The median CD4 cell count was lower in the TB versus non TB IDUs (143 vs 472/mm(3), p<0.0001). TB infected IDUs tend to be more frequently late presenters (59.7 vs 24.6, p<0.0001) and to have advanced HIV disease (47.7 vs 7.59%, p<0.0001) than those without TB. TB cultures were positive in 64 (47.7%) patients, 3 (2.2%) had multidrug resistant TB and 2 (1.5%) had extended drug resistance. Disseminated and/or extrapulmonary TB was diagnosed in 51 patients (38%). The overall mortality rate was higher in TB compared to non TB IDUs (19.4% vs 8.2%, p=0.0007), disseminated TB being associated with the most severe immunosuppression (median CD4 cell count 42/mm(3)) and the highest mortality rate (27.4%)., Conclusions: The incidence of TB in HIV/HCV coinfected IDUs was high and rose over the time. TB infection was more frequent in patients with severe immunosuppression and the mortality rate was higher in IDUs with disseminated and/or extrapulmonary disease. IDUs are important candidates for acquiring and transmitting HIV infection, viral hepatitis and TB, being difficult to control due to their high-risk behaviours. Strengthening of HIV transmission prevention strategies, particularly in identified risk groups, is mandatory.

In the late 1980s an HIV-1 epidemic emerged in Romania that was dominated by subtype F1. The main route of infection is believed to be parenteral transmission in children. We sequenced partial pol coding regions of 70 subtype F1 samples from children and adolescents from the PENTA-EPPICC network of which 67 were from Romania. Phylogenetic reconstruction using the sequences and other publically available global subtype F sequences showed that 79% of Romanian F1 sequences formed a statistically robust monophyletic cluster. The monophyletic cluster was epidemiologically linked to parenteral transmission in children. Coalescent-based analysis dated the origins of the parenteral epidemic to 1983 [1981-1987; 95% HPD]. The analysis also shows that the epidemic's effective population size has remained fairly constant since the early 1990s suggesting limited onward spread of the virus within the population. Furthermore, phylogeographic analysis suggests that the root location of the parenteral epidemic was Bucharest.

Background: During the late 1980s and early 1990s, an estimated 10,000 Romanian children were infected with HIV-1 subtype F nosocomially through contaminated needles and blood transfusions. However, the geographic source and origins of this epidemic remain unclear., Methods: Here we used phylogenetic inference and "relaxed" molecular clock dating analysis to further characterize the Romanian HIV-1 subtype F epidemic., Results: These analyses revealed a major lineage of Romanian HIV sequences consisting nearly entirely of virus sampled from adolescents and children and a distinct cluster that included a much higher ratio of adult sequences. Divergence time estimates inferred the time of most recent common ancestor of subtype F1 sequences to be 1973 (1966-1980) and for all Angolan sequences to 1975 (1968-1980). The most common ancestor of the Romanian sequences was dated to 1978 (1972-1983) with pediatric and adolescent sequences interspersed throughout the lineage. The phylogenetic structure of the entire subtype F epidemic suggests that multiple introductions of subtype F into Romania occurred either from the Angolan epidemic or from more distant ancestors. Since the historical records note that the Romanian pediatric epidemic did not begin until the late 1980s, the inferred time of most recent common ancestor of the Romanian lineage of 1978 suggests that there were multiple introductions of subtype F occurred into the pediatric population from HIV already circulating in Romania., Conclusions: Analysis of the subtype F HIV-1 epidemic in an historical context allows for a deeper appreciation of how the HIV pandemic has been influenced by socio-political events., (Copyright © 2011 Elsevier B.V. All rights reserved.)

CD4+ T helper (Th) cells have been divided into different subsets as defined by their cytokine products and functions after their activation. CD4+ T cell subsets are continuously discovered and until now Th1, Th2, Th9, Th17, and regulatory T (Treg) cells have been almost unanimously recognized but yet not completely characterized. The selective production of cytokines by each of the subsets is probably the master key of the mechanisms of immune regulation. The cytokine milieu is extremely important on deciding the fate of T cells. Generally, more than one cytokine is needed for differentiating to a particular lineage and just recently it was shown that this status quo of commitment could be challenged. It is well known that cytokines bind to Type I/II cytokine receptors signaling via Janus kinases (JAKs) followed by activation of Signal Transducer and Activator of Transcription (STAT). STAT molecules work together with other transcription factors (Foxp3, RORgammat and RORalpha, T-bet, GATA3, Runx 1, NFAT, etc.) also controlled by cytokines, in modulating the Th phenotype and functions. In this review, we analyze the plasticity of Treg population focusing on the most recent discoveries on how microenvironmental cytokines refine/modify Treg phenotype and function, thus changing their fate.

Objective: To examine and compare the responses of hemodialysis (HD) patients to Mycobacterium tuberculosis antigens by using the tuberculin skin test (TST) and an interferon gamma assay (IFN-TB), and to investigate the relationship between T cells subpopulations and tests results. Observational, prospective, diagnostic study conducted in a HD center in a country with high prevalence of tuberculosis., Patients: 195 patients on maintenance HD who consented to participate in this study; 187 (6 were excluded for refusing TST and 2 for indeterminate responses to IFN-TB) were HIV negative, vaccinated with the Bacille Calmette-Guerin vaccine, and without any signs of active tuberculosis, were selected., Methods: Similar to the Mantoux method, 10 IU tuberculin was used for the TST. An IFN-gamma assay specific for Mycobacterium tuberculosis antigens and phytohemagglutinin was carried out. Flow cytometry analysis of peripheral lymphocytes was also performed., Results: TST and IFN-TB results were found to be positive (44% and 53%, respectively) or negative (32% and 47%, respectively) in similar proportions. Results were in agreement in 71% of positive and 58% of negative tests. IFN-gamma levels were found to be higher in patients with a positive TST. All cell counts and CD4/CD8 were found to be higher in TST-positive patients, whereas only total lymphocytes count and CD4/CD8 were reported to be high in IFN-TB-positive patients. A model of multivariable linear regression including cell counts explained 16% of the mitogen-induced IFN-gamma production (F = 5,11; P = .0003). The majority of subjects with positive tests were younger, in most cases male, belonged to the Roma ethnic group, had a shorter HD vintage, and a better nutritional status., Conclusions: TST and IFN-gamma production stimulated by Mycobacterium tuberculosis antigens rely on patient's immune status, which could be influenced by either individual (age, gender), dialysis-related (HD vintage), or nutritional factors. In addition, the diagnostic utility for tuberculosis is similar and moderate in HD patients., (Copyright 2010 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

We investigated the phagocytic function of monocytes in 7- to 10-year-old children horizontally infected with human immunodeficiency virus type 1 (HIV-1) in comparison to that in healthy sex- and age-matched controls. CR3-mediated phagocytosis was increased in patients with HIV-associated pulmonary tuberculosis, independently of CD4 counts and p24 antigenemia.

After an epidemic of West Nile (WN) virus neurologic infections in southeastern Romania in 1996, human and animal surveillance were established to monitor continued transmission of the virus. During 1997 and 1998, neurologic infections were diagnosed serologically as WN encephalitis in 12 of 322 patients in 19 southeastern districts and in 1 of 75 Bucharest patients. In addition, amid a countrywide epidemic of measles, the etiology of the febrile exanthem in 2 of 180 investigated cases was determined serologically to be WN fever; 1 case was complicated by hepatitis. Sentinel chickens placed in Bucharest seroconverted to WN virus during the summer months, indicating their potential value in monitoring transmission. The continued occurrence of sporadic WN infections in southeastern Romania in consecutive years after the 1996 epidemic is consistent with local enzootic transmission of the virus.

Several intracellular pathogens exploit macrophages as a niche for survival and replication. The success of this strategy requires the subversion or the avoidance of microbicidal functions of macrophages. Coxiella burnetii, the agent of Q fever, is a strictly intracellular bacterium that multiplies in myeloid cells. The survival of C. burnetii may depend on the selective use of macrophage receptors. Virulent C. burnetii organisms were poorly internalized but survived successfully in human monocytes, whereas avirulent variants were efficiently phagocytosed but were also rapidly eliminated. The uptake of avirulent organisms was mediated by leukocyte response integrin (alphavbeta3 integrin) and CR3 (alphaMbeta2 integrin), as demonstrated by using specific Abs and RGD sequence-containing peptides. The phagocytic efficiency of CR3 depends on its activation via alphavbeta3 integrin and integrin-associated protein. Indeed, CR3-mediated phagocytosis of avirulent C. burnetii was abrogated in macrophages from integrin-associated protein-/- mice. In contrast, the internalization of virulent C. burnetii organisms involved the engagement of alphavbeta3 integrin but not that of CR3. The pretreatment of monocytes with virulent C. burnetii organisms prevented the CR3-mediated phagocytosis of zymosan particles and CR3 activation assessed by the expression of the 24 neo-epitope. We conclude that the virulence of C. burnetii is associated with the engagement of alphavbeta3 integrin and the impairment of CR3 activity, which probably results from uncoupling alphavbeta3 integrin from integrin-associated protein. This study describes a strategy not previously reported of phagocytosis modulation by intracellular pathogens.