Discovery of 2,9-diaryl-6-carbamoylpurines as a novel class of antitubercular agents.

A series of novel 9-alkyl/aryl-2-aryl-6-carbamoylpurines were synthesized, and their activity against Mycobacterium tuberculosis strain H37Rv was assessed. The SAR analysis on the first set of derivatives, with an alkyl or aryl unit at N-9 and a phenolic unit at C-2, showed that the activity depends on the purine ring substituents at N-9 and C-2. A phenyl group at N-9 combined with a 3-hydroxyphenyl or 4-hydroxyphenyl at C-2 improve the activity. The most active compound of this set has a phenyl group at N-9 and a 4-hydroxyphenyl group at C-2, displaying an IC90 = 1.2 µg/mL and a selectivity index higher than 25.5. This compound served as a Hit to design the second set of derivatives. A phenyl group at N-9 was maintained, and the group at C-2 was diversified. The SAR analysis showed that the aryl unit at C-2 must have an oxygen or nitrogen atom bonded in the para position. A proton, a small alkyl or a substituted aryl group may also be bonded to the oxygen. The compound with the 4-methoxyphenyl group at C-2, 1Bd, exhibits the highest activity with an IC90 < 0.19 µg/mL. This compound is highly potent against M. tuberculosis strain H37Rv and non-toxic for VERO mammalian cells with an SI > 153.8. Compound 1Bd was also non-cytotoxic against primary macrophage cultures at IC90, 2xIC90, and 10xIC90 and significantly reduced the bacterial load in M. tuberculosis-infected macrophages at the same concentrations. Compound 1Bd showed a favorable pharmacokinetic profile when administered orally, with major lung and liver accumulation. In vivo antimycobacterial efficacy of 1Bd was tested at 25 mg/kg. At the tested regimen, a decrease in bacterial burden was observed in the liver. Optimization of the treatment regimen should be performed to fully potentiate the in vivo efficacy of our lead molecule, particularly in the lung, the main target organ of M. tuberculosis.

HTLV-1 p12 modulates the levels of prion protein (PrPC) in CD4+ T cells.

Introduction: Infection with human T cell lymphotropic virus type 1 (HTLV-1) is endemic in Brazil and is linked with pro-inflammatory conditions including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neuroinflammatory incapacitating disease that culminates in loss of motor functions. The mechanisms underlying the onset and progression of HAM/TSP are incompletely understood. Previous studies have demonstrated that inflammation and infectious agents can affect the expression of cellular prion protein (PrPC) in immune cells. Methods: Here, we investigated whether HTLV-1 infection affected PrPC content in cell lines and primary CD4+cells in vitro using flow cytometry and western blot assays. Results: We found that HTLV-1 infection decreased the expression levels of PrPC and HTLV-1 Orf I encoded p12, an endoplasmic reticulum resident protein also known to affect post-transcriptionally cellular proteins such as MHC-class I and the IL-2 receptor. In addition, we observed a reduced percentage of CD4+ T cells from infected individuals expressing PrPC, which was reflected by IFN type II but not IL-17 expression. Discussion: These results suggested that PrPC downregulation, linked to both HTLV-1 p12 and IFN-γ expression in CD4+ cells, may play a role in the neuropathogenesis of HTLV-1 infection.

Mitochondria and the cell cycle in budding yeast.

As centers for energy production and essential biosynthetic activities, mitochondria are vital for cell growth and proliferation. Accumulating evidence suggests an integrated regulation of these organelles and the nuclear cell cycle in distinct organisms. In budding yeast, a well-established example of this coregulation is the coordinated movement and positional control of mitochondria during the different phases of the cell cycle. The molecular determinants involved in the inheritance of the fittest mitochondria by the bud also seem to be cell cycle-regulated. In turn, loss of mtDNA or defects in mitochondrial structure or inheritance often lead to a cell cycle delay or arrest, indicating that mitochondrial function can also regulate cell cycle progression, possibly through the activation of cell cycle checkpoints. The up-regulation of mitochondrial respiration at G2/M, presumably to fulfil energetic requirements for progression at this phase, also supports a mitochondria-cell cycle interplay. Cell cycle-linked mitochondrial regulation is accomplished at the transcription level and through post-translational modifications, predominantly protein phosphorylation. Here, we address mitochondria-cell cycle interactions in the yeast Saccharomyces cerevisiae and discuss future challenges in the field.

The APC/C Activator Cdh1p Plays a Role in Mitochondrial Metabolic Remodelling in Yeast.

Cdh1p is one of the two substrate adaptor proteins of the anaphase promoting complex/cyclosome (APC/C), a ubiquitin ligase that regulates proteolysis during cell cycle. In this work, using a proteomic approach, we found 135 mitochondrial proteins whose abundance was significantly altered in the cdh1Δ mutant, with 43 up-regulated proteins and 92 down-regulated proteins. The group of significantly up-regulated proteins included subunits of the mitochondrial respiratory chain, enzymes from the tricarboxylic acid cycle and regulators of mitochondrial organization, suggesting a metabolic remodelling towards an increase in mitochondrial respiration. In accordance, mitochondrial oxygen consumption and Cytochrome c oxidase activity increased in Cdh1p-deficient cells. These effects seem to be mediated by the transcriptional activator Yap1p, a major regulator of the yeast oxidative stress response. YAP1 deletion suppressed the increased Cyc1p levels and mitochondrial respiration in cdh1Δ cells. In agreement, Yap1p is transcriptionally more active in cdh1Δ cells and responsible for the higher oxidative stress tolerance of cdh1Δ mutant cells. Overall, our results unveil a new role for APC/C-Cdh1p in the regulation of the mitochondrial metabolic remodelling through Yap1p activity.

Mitochondrial respiration promotes Cdc37-dependent stability of the Cdk1 homolog Cdc28.

Cdc28, the homolog of mammalian Cdk1, is a conserved key regulatory kinase for all major cell cycle transitions in yeast. We have found that defects in mitochondrial respiration (including deletion of ATP2, an ATP synthase subunit) inhibit growth of cells carrying a degron allele of Cdc28 (cdc28td) or Cdc28 temperature-sensitive mutations (cdc28-1 and cdc28-1N) at semi-permissive temperatures. Loss of cell proliferation in the atp2Δcdc28td double mutant is associated with aggravated cell cycle arrest and mitochondrial dysfunction, including mitochondrial hyperpolarization and fragmentation. Unexpectedly, in mutants defective in mitochondrial respiration, steady-state protein levels of mutant cdc28 are strongly reduced, accounting for the aggravated growth defects. Stability of Cdc28 is promoted by the Hsp90-Cdc37 chaperone complex. Our results show that atp2Δcdc28td double-mutant cells, but not single mutants, are sensitive to chemical inhibition of the Hsp90-Cdc37 complex, and exhibit reduced levels of additional Hsp90-Cdc37 client kinases, suggesting an inhibition of this complex. In agreement, overexpression of CDC37 improved atp2Δcdc28td cell growth and Cdc28 levels. Overall, our study shows that simultaneous disturbance of mitochondrial respiration and Cdc28 activity reduces the capacity of Cdc37 to chaperone client kinases, leading to growth arrest.

Lessons from the Cerebrospinal Fluid Analysis of HTLV-1-Infected Individuals: Biomarkers of Inflammation for HAM/TSP Development.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease that leads to motor impairment due to a chronic inflammatory process in the central nervous system (CNS). However, the HAM/TSP pathogenesis is not completely clear, and biomarkers to define the disease prognosis are still necessary. Thus, we aimed to identify biomarkers for HAM/TSP and potential mechanisms involved in disease development. To that end, the concentrations of VILIP-1, BDNF, VEGF, ß-NGF, TGF-ß1, fractalkine/CX3CL1, IL-6, IL-18, and TNF-α, and the soluble forms of TREM-1, TREM-2, and RAGE, were assessed using a multiplex bead-based immunoassay in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n = 20), asymptomatic HTLV-1 carriers (AC) (n = 13), and HTLV-1-seronegative individuals (n = 9), with the results analyzed according to the speed of HAM/TSP progression. HAM/TSP patients had elevated fractalkine in the serum but not in the CSF, particularly those with low neuroinflammatory activity (CSF/serum ratio of neopterin <1 and of CXCL10 < 2). HAM/TSP patients with normal CSF levels of neurofilament light chain (NfL) showed elevated ß-NGF in serum, and serum BDNF levels were increased in HTLV-1-infected individuals, particularly in HTLV-1 AC. Both HTLV-1 AC and HAM/TSP patients had lower TGF-ß1 levels in CSF compared to uninfected individuals, and HAM/TSP patients with active CNS inflammation showed higher CSF levels of IL-18, which correlated with markers of inflammation, neuronal death, and blood−brain-barrier permeability. Although none of the factors evaluated were associated with the speed of HAM/TSP progression, reduced TGF-ß1 levels in CSF suggest that suppressive responses to control subclinical and/or active neurodegeneration are impaired, while increased CSF IL-18 indicates the involvement of inflammasome-mediated mechanisms in HAM/TSP development.

Chitotriosidase 1 in the cerebrospinal fluid as a putative biomarker for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) progression.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurodegenerative disease that affects motor, urinary, intestinal, and sensory functions. Typically, HAM/TSP is slowly progressive, but it may vary from limited motor disability after decades (very slow progression) to loss of motor function in a few years from disease onset (rapid). In this study, we aimed to identify prognostic biomarkers for HAM/TSP to support patient management. Thus, proteomic analysis of the cerebrospinal fluid (CSF) was performed with samples from HTLV-1 asymptomatic carriers (AC) (n=13) and HAM/TSP patients (n=21) with rapid, typical, and very slow progression using quantitative label-free liquid chromatography/tandem mass spectrometry. Enrichment analyses were also carried out to identify key biological processes associated with distinct neurological conditions in HTLV-1 infection. Candidate biomarkers were validated by ELISA in paired CSF and serum samples, and samples from HTLV-1-seronegative individuals (n=9) were used as controls. CSF analysis identified 602 proteins. Leukocyte/cell activation, immune response processes and neurodegeneration pathways were enriched in rapid progressors. Conversely, HTLV-1 AC and HAM/TSP patients with typical and very slow progression had enriched processes for nervous system development. Differential expression analysis showed that soluble vascular cell adhesion molecule 1 (sVCAM-1), chitotriosidase 1 (CHIT1), and cathepsin C (CTSC) were upregulated in HAM/TSP. However, only CHIT1 was significantly elevated after validation, particularly in HAM/TSP rapid progressors. In contrast, none of these biomarkers were altered in serum. Additionally, CSF CHIT1 levels in HAM/TSP patients positively correlated with the speed of HAM/TSP progression, defined as points in the IPEC-2 HAM/TSP disability scale per year of disease, and with CSF levels of phosphorylated neurofilament heavy chain, neopterin, CXCL5, CXCL10, and CXCL11. In conclusion, higher CSF levels of CHIT1 were associated with HAM/TSP rapid progression and correlated with other biomarkers of neuroinflammation and neurodegeneration. Therefore, we propose CHIT1 as an additional or alternative CSF biomarker to identify HAM/TSP patients with a worse prognosis.

Phosphoregulation of the ATP synthase beta subunit stimulates mitochondrial activity for G2/M progression.

Mitochondrial ATP synthase is a multifunctional enzyme complex involved in ATP production. We previously reported that the ATP synthase catalytic beta subunit (Atp2p in yeast) is regulated by the 2A-like protein phosphatase Sit4p, which targets Atp2p at T124/T317 impacting on ATP synthase levels and mitochondrial respiration. Here we report that Atp2-T124/T317 is also potentially regulated by Cdc5p, a polo-like mitotic kinase. Since both Cdc5p and Sit4p have established roles in cell cycle regulation, we investigated whether Atp2-T124/T317 phosphorylation was cell cycle-related. We present evidence that Atp2p levels and phosphorylation vary during cell cycle progression, with an increase at G2/M phase. Atp2-T124/T317 phosphorylation stimulates mitochondrial membrane potential, respiration and ATP levels at G2/M phase, indicating that dynamic Atp2p phosphorylation contributes to mitochondrial activity at this specific cell cycle phase. Preventing Atp2p phosphorylation delays G2/M to G1 transition, suggesting that enhanced bioenergetics at G2/M may help meet the energetic demands of cell cycle progression. However, mimicking constitutive T124/T317 phosphorylation or overexpressing Atp2p leads to mitochondrial DNA instability, indicating that reversible Atp2p phosphorylation is critical for homeostasis. These results indicate that transient phosphorylation of Atp2p, a protein at the core of the ATP production machinery, impacts on mitochondrial bioenergetics and supports cell cycle progression at G2/M.

Following the Clues: Usefulness of Biomarkers of Neuroinflammation and Neurodegeneration in the Investigation of HTLV-1-Associated Myelopathy Progression.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease due to axonal damage of the corticospinal secondary to an inflammatory response against infected T-cells. In the present work, we aimed to evaluate biomarkers of neurodegeneration and neuroinflammation in the definition of HAM/TSP prognosis. Neurofilament light (NfL) and phosphorylated heavy (pNfH) chains, total Tau protein, cellular prion protein (PrPc), inflammatory chemokines, and neopterin were quantified in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n=21), HTLV-1 asymptomatic carriers (AC) (n=13), and HTLV-1 seronegative individuals with non-inflammatory non-degenerative neurological disease (normal-pressure hydrocephalus) (n=9) as a control group. HTLV-1 proviral load in peripheral blood mononuclear cells and the expression of chemokine receptors CCR4, CCR5, and CXCR3 in infected CD4+ T-cells (HTLV-1 Tax+ cells) were also assessed. CSF levels of Tau, NfL, and pNfH were similar between groups, but PrPc and neopterin were elevated in HAM/TSP patients. Most individuals in the control group and all HTLV-1 AC had CSF/serum neopterin ratio < 1.0, and two-thirds of HAM/TSP patients had ratio values > 1.0, which positively correlated with the speed of disease progression and pNfH levels, indicating active neuroinflammation. HAM/TSP patients showed high serum levels of CXCR3-binding chemokines (CXCL9, CXCL10, and CXCL11) and elevated CSF levels of CCL2, CCL3, CCL4, CCL17, CXCL5, CXCL10, and CXCL11. Indeed, CXCL10 concentration in CSF of HAM/TSP patients was 5.8-fold and 8.7-fold higher in than in HTLV-1 AC and controls, respectively, and correlated with CSF cell counts. HAM/TSP patients with typical/rapid disease progression had CSF/serum CXCL10 ratio > 1.0 and a higher frequency of CXCR3+Tax+CD4+ T-cells in blood, which indicated a positive gradient for the migration of infected cells and infiltration into the central nervous system. In conclusion, the slow progression of HAM/TSP abrogates the usefulness of biomarkers of neuronal injury for the disease prognosis. Thus, markers of inflammation provide stronger evidence for HAM/TSP progression, particularly the CSF/serum neopterin ratio, which may contribute to overcome differences between laboratory assays.

Polymorphisms in HTLV-1 Tax-responsive elements in HTLV-1-associated myelopathy/tropical spastic paraparesis patients are associated with reduced proviral load but not with disease progression.

Human T-lymphotropic virus type 1 (HTLV-1) provirus expression is mainly directed by Tax-responsive elements (TRE) within the long terminal repeats (LTR). Mutations in TRE can reduce provirus expression and since a high proviral load (PVL) is a risk factor for the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we evaluated polymorphisms in the 5' LTR and the association with PVL and disease progression. HTLV-1 LTR and tax sequences derived from asymptomatic carriers (AC) and HAM/TSP patients followed in a longitudinal study were analysed according to PVL and clinical severity. Individuals infected with HTLV-1 presenting the canonical TRE, considering strain ATK-1 as the consensus, displayed sustained higher PVL. By contrast, an LTR A125G mutation in TRE was associated with slightly reduced PVL only in HAM/TSP patients, although it did not influence the speed of disease progression. Moreover, this polymorphism was frequent in Latin American strains of the HTLV-1 Cosmopolitan Transcontinental subtype. Therefore, polymorphisms in the 5' TRE of HTLV-1 may represent one of the factors influencing PVL in HAM/TSP patients, especially in the Latin American population. Indeed, higher PVL in the peripheral blood has been associated with an increased inflammatory activity in the spinal cord and to a poorer prognosis in HAM/TSP. However, this event was not associated with TRE polymorphisms.

Inflammatory Cytokine Patterns Associated with Neurological Diseases in Coronavirus Disease 2019.

Patients with coronavirus disease 2019 (COVID-19) can present with distinct neurological manifestations. This study shows that inflammatory neurological diseases were associated with increased levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12, chemokine (C-X-C motif) ligand 8 (CXCL8), and CXCL10 in the cerebrospinal fluid. Conversely, encephalopathy was associated with high serum levels of IL-6, CXCL8, and active tumor growth factor ß1. Inflammatory syndromes of the central nervous system in COVID-19 can appear early, as a parainfectious process without significant systemic involvement, or without direct evidence of severe acute respiratory syndrome coronavirus 2 neuroinvasion. At the same time, encephalopathy is mainly influenced by peripheral events, including inflammatory cytokines. ANN NEUROL 2021;89:1041-1045.

Cerebrospinal fluid findings in neurological diseases associated with COVID-19 and insights into mechanisms of disease development.

OBJECTIVES: To analyze the cerebrospinal fluid (CSF) of patients with SARS-CoV-2 infection and neurological manifestations to provide evidence for the understanding of mechanisms associated with central nervous system (CNS) involvement in COVID-19. METHODS: Patients (n = 58) were grouped according to their main neurological presentation: headache (n = 14); encephalopathy (n = 24); inflammatory neurological diseases, including meningoencephalitis (n = 4), acute myelitis (n = 3), meningitis (n = 2), acute disseminated encephalomyelitis (ADEM) (n = 2), encephalitis (n = 2), and neuromyelitis optica (n = 1); and Guillain-Barré syndrome (n = 6). Data regarding age, sex, cerebrovascular disease, and intracranial pressure were evaluated in combination with CSF profiles defined by cell counts, total protein and glucose levels, concentration of total Tau and neurofilament light chain (NfL) proteins, oligoclonal band patterns, and detection of SARS-CoV-2 RNA. RESULTS: CSF of patients with inflammatory neurological diseases was characterized by pleocytosis and elevated total protein and NfL levels. Patients with encephalopathy were mostly older men (mean age of 61.0 ± 17.6 years) with evidence of cerebrovascular disease. SARS-CoV-2 RNA in CSF was detected in 2 of 58 cases: a patient with refractory headache, and another patient who developed ADEM four days after onset of COVID-19 symptoms. Three patients presented intrathecal IgG synthesis, and four had identical oligoclonal bands in CSF and serum, indicating systemic inflammation. CONCLUSION: Patients with neurological manifestations associated with COVID-19 had diverse CSF profiles, even within the same clinical condition. Our findings indicate a possible contribution of viral replication on triggering CNS infiltration by immune cells and the subsequent inflammation promoting neuronal injury.

Peripheral facial nerve palsy associated with COVID-19.

COVID-19 pandemic revealed several neurological syndromes related to this infection. We describe the clinical, laboratory, and radiological features of eight patients with COVID-19 who developed peripheral facial palsy during infection. In three patients, facial palsy was the first symptom. Nerve damage resulted in mild dysfunction in five patients and moderate in three. SARS-Cov-2 was not detected in CSF by PCR in any of the samples. Seven out of eight patients were treated with steroids and all patients have complete or partial recovery of the symptoms. Peripheral facial palsy should be added to the spectrum of neurological manifestations associated with COVID-19.

Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Accurate Confirmatory Diagnosis of HTLV-1/2 Infection.

Laboratory diagnosis of human T-lymphotropic viruses (HTLV) 1 and 2 infection is performed by serological screening and further confirmation with serological or molecular assays. Thus, we developed a loop-mediated isothermal nucleic acid amplification (LAMP) assay for the detection of HTLV-1/2 in blood samples. The sensitivity and accuracy of HTLV-1/2 LAMP were defined with DNA samples from individuals infected with HTLV-1 (n = 125), HTLV-2 (n = 19), and coinfected with HIV (n = 82), and compared with real-time polymerase chain reaction (qPCR) and PCR-restriction fragment length polymorphism (RFLP). The overall accuracy of HTLV-1/2 LAMP (95% CI 74.8-85.5%) was slightly superior to qPCR (95% CI 69.5-81.1%) and similar to PCR-RFLP (95% CI 79.5-89.3%). The sensitivity of LAMP was greater for HTLV-1 (95% CI 83.2-93.4%) than for HTLV-2 (95% CI 43.2-70.8%). This was also observed in qPCR and PCR-RFLP, which was associated with the commonly lower HTLV-2 proviral load. All molecular assays tested showed better results with samples from HTLV-1/2 mono-infected individuals compared with HIV-coinfected patients, who present lower CD4 T-cell counts. In conclusion, HTLV-1/2 LAMP had similar to superior performance than PCR-based assays, and therefore may represent an attractive alternative for HTLV-1/2 diagnosis due to reduced working time and costs, and the simple infrastructure needed.

Smell dysfunction in COVID-19 patients: More than a yes-no question.

Anosmia has been recognized as a prevalent and early symptom by many COVID-19 patients. However, most researchers have recorded smell dysfunction solely as present or absent and based on subjective evaluation by patients. We described the results of 57 consecutive COVID-19 patients seen at FIOCRUZ, Rio de Janeiro, Brazil, from April to May 2020. Data about the presence of smell loss, the onset of smell loss and other COVID-19 symptoms such as ageusia and nasal congestion or rhinorrhea were recorded. All patients at the initial consultation and 34 healthy controls underwent the Q-SIT, which is a quick disposable three-item smell identification test, by a trained physician. We compared three groups: healthy controls, COVID+ patients with reported smell loss (COVID w/ SL) and COVID+ patients without smell loss (COVID+ w/o SL). The mean age of patients was 41.4 years (SD ± 10.4), and 54.4% were women. Smell loss was reported by 40.4% of COVID-19 patients. We observed a gradual effect with higher Q-SIT scores in healthy controls, followed by COVID+ w/o SL and COVID+ w/ SL (medians = 3, 2 and 0; respectively, p < 0.001). Anosmia or severe microsmia (Q-SIT≤1) was present in 11.1% (CI: 3.1%-26.1%) of controls, 32.4% (CI: 17.4%-50.5%) of COVID-19 w/o SL and 87% (CI: 66.4%-97.2%) of COVID+ w/ SL (p < 0.001). This study provides evidence that olfactory dysfunction in COVID-19 is common and more prevalent than what is perceived by patients. Q-SIT is a quick and reliable screening test for the detection of smell dysfunction during the pandemics.

Patients with COVID-19 and neurological manifestations show undetectable SARS-CoV-2 RNA levels in the cerebrospinal fluid.

We report that patients with COVID-19 displaying distinct neurological disorders have undetectable or extremely low levels of SARS-CoV-2 RNA in the cerebrospinal fluid, indicating that viral clearance precede the neurological involvement. This finding points to the need for the development of more sensitive molecular tests and the investigation of other neurotropic pathogens to exclude concurrent neuroinfection.

Loop-mediated isothermal amplification (LAMP) assay for rapid and accurate confirmatory diagnosis of HTLV-1/2 infection

Laboratory diagnosis of human T-lymphotropic viruses (HTLV) 1 and 2 infection is performed by serological screening and further confirmation with serological or molecular assays. Thus, we developed a loop-mediated isothermal nucleic acid amplification (LAMP) assay for the detection of HTLV-1/2 in blood samples. The sensitivity and accuracy of HTLV-1/2 LAMP were defined with DNA samples from individuals infected with HTLV-1 (n = 125), HTLV-2 (n = 19), and coinfected with HIV (n = 82), and compared with real-time polymerase chain reaction (qPCR) and PCR-restriction fragment length polymorphism (RFLP). The overall accuracy of HTLV-1/2 LAMP (95% CI 74.8­85.5%) was slightly superior to qPCR (95% CI 69.5­81.1%) and similar to PCR-RFLP (95% CI 79.5­89.3%). The sensitivity of LAMP was greater for HTLV-1 (95% CI 83.2­93.4%) than for HTLV-2 (95% CI 43.2­70.8%). This was also observed in qPCR and PCR-RFLP, which was associated with the commonly lower HTLV-2 proviral load. All molecular assays tested showed better results with samples from HTLV-1/2 mono-infected individuals compared with HIV-coinfected patients, who present lower CD4 T-cell counts. In conclusion, HTLV-1/2 LAMP had similar to superior performance than PCR-based assays, and therefore may represent an attractive alternative for HTLV-1/2 diagnosis due to reduced working time and costs, and the simple infrastructure needed.

Latin American Consensus Statement for the Use of Contrast-Enhanced Transcranial Ultrasound as a Diagnostic Test for Detection of Right-to-Left Shunt.

BACKGROUND: The role of patent foramen ovale is a field of debate and current publications have increasing controversies about the patients' management in young undetermined stroke. Work up with echocardiography and transcranial Doppler (TCD) can aid the decision with better anatomical and functional characterization of right-to-left shunt (RLS). Medical and interventional strategy may benefit from this information. SUMMARY: a group of experts from the Latin American participants of the Neurosonology Research Group (NSRG) of World Federation of Neurology created a task force to review literature and describe the better methodology of contrast TCD (c-TCD). All signatories of the present consensus statement have published at least one study on TCD as an author or co-author in an indexed journal. Two meetings were held while the consensus statement was being drafted, during which controversial issues were discussed and voted on by the statement signatories. The statement paper was reviewed and approved by the Executive Committee of the NSRG of the World Federation of Neurology. The main objective of this consensus statement is to establish a standardization of the c-TCD technique and its interpretation, in order to improve the informative quality of the method, resulting in expanding the application of TCD in the clinical setting. These recommendations optimize the comparison of different diagnostic methods and encourage the use of c-TCD for RLS screening and complementary diagnosis in multicenter studies.