Brain opportunistic infections and tumors in people living with HIV - still a challenge in efficient antiretroviral therapy era.

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 log10 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 log10 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.

Subacute myoclonic measles encephalitis - An opportunistic HIV-associated infection.

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.

Emergence of Toscana Virus, Romania, 2017-2018.

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.

Re-emergence of severe West Nile virus neuroinvasive disease in humans in Romania, 2012 to 2017-implications for travel medicine.

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.

Neurologic Complications of Influenza B Virus Infection in Adults, Romania.

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.

GENOMIC ANALYSIS OF HEPATITIS B VIRUS STRAINS INFECTING ROMANIAN PATIENTS.

Chronic hepatitis B is widespread and represents an important cause of morbidity and mortality due to the evolution to cirrhosis and hepatocellular carcinoma. This study was designed to improve the national laboratory surveillance of hepatitis B virus (HBV) infection, focusing on genomic analysis of isolates from Romanian patients. Sera from ten patients with HBV were collected and analyzed. Phylogenetic analysis was conducted on a DNA fragment spanning almost the entire genome. The occurrence of mutations was assessed for each open reading frame in the viral genome. Phylogenetic analysis revealed five isolates belonging to genotype A (subgenotype A2) and other five clustering with genotype D strains (subgenotype D1). Two patients treated with lamivudine were found to carry isolates harboring rtM204V lamivudine resistance mutation. An HBV isolate displaying a lamivudine complex resistance pattern, rtM204I in conjunction with rtL180M and rtA200V, was found in a lamivudine naïve patient. All samples harbored sA105P substitution, usually found in HBIg therapy escape isolates. Three of the studied strains were simultaneously displaying T1753, T1762 and A1764 mutations which in vitro induce enhanced genome replication and reduction of HBeAg expression. The sequence obtained from a patient with decompensated liver cirrhosis presents a novel type of insertion consisting of nine nucleotides between positions 260 and 261 in the X gene. Despite the small number of samples, our findings suggest the need to determine the drug resistance pattern for each patient before taking a therapeutic decision and also highlight the necessity of knowing the real level of drug resistance among HBV strains circulating in Romania.

Alarming increase in tuberculosis and hepatitis C virus (HCV) among HIV infected intravenous drug users.

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.

Phylodynamic and phylogeographic patterns of the HIV type 1 subtype F1 parenteral epidemic in Romania.

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.

Using phylogeography to characterize the origins of the HIV-1 subtype F epidemic in Romania.

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.

Tuberculin skin test, interferon-gamma assay, and T cells subpopulations in hemodialysis patients.

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.

Plasticity of regulatory T cells under cytokine pressure.

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.

Comparative methods for genotyping hepatitis C virus isolates from Romania.

Accurate genotyping of hepatitis C virus (HCV) has clinical implications for treatment orientation and epidemiological impact in tracing the contamination sources. The aim of the study was to compare a genotyping assay by restriction fragment length polymorphism (RFLP) in the HCV 5'untranslated region (5'UTR) with sequencing in the 5'untranslated and NS5B regions. One hundred and three samples, collected between 2004 and 2006 from chronically infected patients with HCV, were tested with the 5'UTR and NS5B protocols. Of the total number of the samples tested by the 5'UTR-RFLP assay (n=103) the HCV subtype could be inferred by this method for 92 samples, by 5'UTR sequencing for 16 samples out of 23 tested (n=23) and by using the NS5B sequencing for all the samples tested (n=34). Our results showed that the HCV genotype distribution in Romania is: 1b--86.4%, 1a--10.7% and 4a--2.9%. In conclusion, RFLP screening in the 5'UTR is a convenient method for HCV genotyping and discrimination between 1b and non-1b genotypes but has a poor resolving power for subtyping and evaluation of the transmission routes. Sequencing in NS5B region is more adapted than RFLP and sequencing in 5'UTR for subtyping and epidemiological investigation.