Eugenio Ramírez

TIMPs and MMPs expression in CSF from patients with TSP/HAM

The tropical spastic paraparesis or human T-cell lymphotropic virus associated myelopathy (TSP/HAM), has been related with an overexpression of matrix metalloproteinases (MMPs), especially MMP-9. Initial studies of reverse zymography with cerebrospinal fluid (CSF) from TSP/HAM patients, and controls showed the presence of TIMPs, endogenous MMP inhibitors. We determined in CSF the levels of TIMPs by immunoanalysis in 25 patients with TSP/HAM, and compared with two groups: controls and patients with acute and subacute inflammatory neurological diseases. We found that TIMP-2, TIMP-3 and TIMP-4 levels were significantly higher than in controls in both TSP/HAM and inflammatory patients, while TIMP-1 was increased only in the inflammatory group. Levels of MMP-3 and MMP-9 from the two groups of patients showed a significant upregulation in CSF. In the CSF of around the 70% of TSP-HAM and inflammatory patients the presence MMP-9 was detected by zymography, but not in controls. MMP-2 was only overexpressed in the acute inflammatory group. The active form of MMP-2 was observed in both groups of patients with a similar high frequency (60%). MMPs overexpressions are independent of the evolution time of the disease in TSP/HAM. The chronic overexpression of these extracelullar matrix proteins detected in CSF of TSP/HAM should be indirectly produced by secreted viral proteins being responsible for the progression of this disease, accounting for the observed differences with acute inflammatory patients. Our results support the existence of an imbalance between MMPs and their endogenous tissue inhibitors, which could be a pathogenic factor in the chronicity of TSP/HAM.

Complete sequence of the genome of the human isolate of Andes virus CHI-7913: comparative sequence and protein structure analysis

We report here the complete genomic sequence of the Chilean human isolate of Andes virus CHI-7913. The S, M, and L genome segment sequences of this isolate are 1,802, 3,641 and 6,466 bases in length, with an overall GC content of 38.7%. These genome segments code for a nucleocapsid protein of 428 amino acids, a glycoprotein precursor protein of 1,138 amino acids and a RNA-dependent RNA polymerase of 2,152 amino acids. In addition, the genome also has other ORFs coding for putative proteins of 34 to 103 amino acids. The encoded proteins have greater than 98% overall similarity with the proteins of Andes virus isolates AH-1 and Chile R123. Among other sequenced Hantavirus, CHI-7913 is more closely related to Sin Nombre virus, with an overall protein similarity of 92%. The characteristics of the encoded proteins of this isolate, such as hydrophobic domains, glycosylation sites, and conserved amino acid motifs shared with other Hantavirus and other members of the Bunyaviridae family, are identified and discussed.

Identificación de subtipos B y F de VIH-1 en pacientes chilenos

BACKGROUND Type I human immunodeficiency virus (HIV) is characterized by a great genetic variability. There are three groups of virus throughout the world: O, N and M. Group M is responsible for AIDS pandemic and is subdivided in 9 genetic subtypes. Most viral strains in South America are subtype B. AIM To determine the frequency of HIV subtypes in Chilean patients. MATERIAL AND METHODS Genetic analysis of C2-V3-C3 region of the gene env in HIV strains coming from 77 Chilean subjects infected by different means. DNA heteroduplex mobility assay was used to determine HIV subtypes. RESULTS Sixty eight cases were infected with subtype B (88.3%) and nine cases were infected with subtype F (11.7%). CONCLUSIONS Subtype B is the predominant HIV in Chile, but subtype F is also present.

Proteína Tax (HTLV-I), probable factor patogénico y marcador del síndrome sicca que se asocia a HAM/TSP

Human T-cell lymphotropic virus type I (HTLV-I) isa retrovirus that influences cellular metabolism modifying biological responses. This results inoncogenic, degenerative or inflammatory changes. The myelopathy associated to HTLV-I ortropical spastic paraparesia (HAM/TSP) is a mainly degenerative response to the virus infection.On the other hand, Sjogren syndrome has an inflammatory appearance. Theimmunohistochemical study of CD-4, CD-8 and CD45 lymphocytes, metalloproteinase MMP-9and viral Tax protein in pathological samples of salivary glands may help to differentiateprimary from viral Sicca syndrome.

Molecular and clinical effects of betamethasone in human t-cell lymphotropic virus type-i-associated myelopathy/tropical spastic paraparesis patients†

There is no effective therapy for human T‐cell lymphotropic virus type I (HTLV‐I)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). Glucocorticoids are effective to reduce the motor disability in these patients, but its role as anti‐spastic drugs is unknown. Here it is reported the use of corticosteroids in HAM/TSP. The goal was to find reliable molecular markers linked to treatment effectiveness. The clinical efficacy of corticosteroids was studied in 22 HAM/TSP. The treatment was a single dose of 7.0 mg of systemic betamethasone. Pre‐treatment samples were obtained immediately before steroid administration and post‐treatment samples were collected after 5 days. Neurological disability was evaluated by the Osames Motor Disability Scales. Relative levels of Tax, Foxp3, IL‐10, TGF‐β, CTLA‐4, and GITR mRNA were measured and the percentage of CD4+Foxp3+ and CD4+Tax+ populations was quantified in PBMCs by real‐time PCR and flow cytometry, respectively. The same parameters were studied in eight untreated carriers. Betamethasone treatment showed neurological improvement in 21 HAM/TSP patients, with one patient without response to treatment. This therapy was associated with a decrease in Tax mRNA load and CD4+Tax+ T cells in HAM/TSP. Simultaneously, an increase in Foxp3 mRNA and CD4+Foxp3+ T cell was detected in these patients. The other markers studied had no significant changes after treatment. Clinical improvement in betamethasone‐treated HAM/TSP was associated with an inverse relationship between a decrease in Tax and an increase in Foxp3 at the mRNA and protein levels. These results suggest that both Tax and Foxp3 may represent potential biomarkers for drug treatment assessments in HAM/TSP. J. Med. Virol. 83:1641–1649, 2011.

Detection and genotyping of HPV in urine samples from Chilean women attending primary health care centers

Cervical cancer is the second most common malignant neoplasm in women worldwide representing approximately 10% of all types of cancers. Triage of women through cervical cytology has been an important strategy for the surveillance and control of new cases of cervical cancer. However, in many regions around the world cervical cytology has a low coverage compared to developed countries. The molecular detection of HPV is the most effective method to increase the screening sensitivity of women at risk of developing cervical cancer. There are very few studies about the efficacy of urine testing for detection of HPV in women followed up in primary health care centers. Consequently, the efficacy of using urine HPV screening in these populations has not been addressed yet. Here, we compared the detection of HPV in simultaneous urine and cervical samples of women followed up in primary health care centers. Urine and cervical samples were analyzed in 543 women attending at primary health care centers. HPV was detected by real time PCR, and HPV typing performed by PCR–RLB. A general HPV concordance of 86.2% (κ = 0.72) was determined between urine and cervical samples. The concordance for HPV-16 and 18 was almost perfect (κ = 0.82) and strong (κ = 0.77), respectively. The sensitivity and specificity for all HPV genotypes in urine using cervical samples as reference were 82.1 and 93.7%, respectively. The results showed that urine is a good alternative as clinical sample for HPV screening in women attending primary health care centers. Therefore, urine should be used as an alternative sample for increasing triage coverage either in refractory women participating in Pap surveillance programs or when cervical samples are not available.

Neuropathological Study of Acute Myelopathy and Encephalopathy Associated to HTLV-I

Background a chronic and progressive parapares is without remission silent on MRI is the ordinary neurological presentation of HTLV-I, expression of a central axonopathy product of axoplasmic transport alterations. Infrequently acute forms like “T2 hyperintense acute myelopathy on MRI” have been reported. The Purpose of this presentation will be to describe the histopathological expression of this acute form and look for their pathogenesis. Patient and method: A 60 years old woman carrier of refractory anaemia and subjected to multiple transfusions, positive for HTLV-I. She developed a severe cognitive impairment and paraplegia set up in four weeks. MRI showed T2 hyper intense lesions in white matter of brain hemispheres and in cervico-thoraxic segments of the spinal cord. She died suddenly by myocardial infarction. The neuropathological studies showed white matter necrosis insymmetricfrontal areas, conservation of cortical structures and U fibbers; necrosis in bothlateral tracts of the spinal cord (T2T4) without gray substance damage; the microvascular walls of these areas immuno-stained with anti-Tax, expressed Taxprotein suggesting HTLV-I infection. Conclusions: This necrotizing leukopathy in absence of a primary inflammation would be the expression of HTLV-I endothelial cells infection in specific white matter areas, changing of the blood-brain barrier permeability.

Inicio del brote de Influenza A (H1N1) pandémica en Chile: caracterización genética de los primeros casos detectados

BACKGROUND Following the announcement of the Influenza A(H1N1) pandemic by the World Health Organization in April 2009, a surveillance program was carried out in Chile to detect the introduction of the virus in the country and to monitor its propagation and impact. AIM To describe the onset of the outbreak and the genetic characterization of the pandemic H1N1 influenza virus in the first detected cases in Chile. MATERIAL AND METHODS Analysis of18 clinical samples coming from suspicious patients, received in a National Reference Laboratory. RNA reverse transcription and real time influenza gene DNA amplification was carried out in a 7500 Fast and Step One Real Time PCR Systems of Applied Biosystems and MxPro-Mx3000P thermocycler from Stratagene. Super Script III Platinum One-Step Quantitative RT-PCR was used. RESULTS The virus was first detected in three persons returning from the Dominican Republic via Panamá and a child from the east zone of Santiago. Genetic characterization of the virus showed that the child was infected by a different variant of the pandemic virus than the three persons returning from the Caribbean. CONCLUSIONS The onset of the Influenza outbreak in Chile apparently carne from two different epidemiological groups. The spread of the virus detected in the voyagers was limited immediately However the virus of the fourth case was found in different regions of Chile.

Estudio clínico y determinación de la parasitemia en un grupo de pacientes infectados por Trypanosoma cruzi de la región de Atacama, Chile

BACKGROUND: Trypanosoma cruzi infection is endemic in Northern/Central Chile. AIM: To perform a clinical assessment of patients infected with Trypanosoma cruzi. PATIENTS AND METHODS: Two hundred sixty three subjects with a positive serology for Trypanosoma cruzi, were invited by mail to a clinical assessment in a Regional Hospital. In a subsample of these, a polymerase chain reaction for Trypanosoma cruzi, was done. RESULTS: Of all the invited subjects, 183 responded and were assessed at the hospital. Of these, 60 had cardiac affections, 52 had colon problems and 17, esophageal disease. Seventy four were asymptomatic. Of the 64 patients in whom polymerase chain reaction was done, 35 had a positive result. CONCLUSIONS: A high percentage of subjects infected with Trypanosoma cruzi, had clinical consequences of the infection. Polymerase chain reaction showed persistency of the parasite in more than half of the infected patients.