Multi-disciplinary team of human herpes virus-6B encephalitis after liver transplantation / 器官移植

Objective To evaluate the role of multi-disciplinary team (MDT) in improving the diagnosis and treatment of human herpes virus-6B (HHV-6B) encephalitis after liver transplantation. Methods MDT consultation was delivered for one rare case of HHV-6B encephalitis after liver transplantation to establish an effective individualized treatment regime. Results On the 16 d after liver transplantation, the patient developed headache, and suddenly presented with unresponsiveness, unconsciousness, coma complicated with involuntary limb twitching on the 18 d. Blood ammonia level was increased. Brain CT scan showed cerebral ischemic changes. Electroencephalography prompted the epileptic seizure. After MDT consultation, the possibility of nervous system infection after liver transplantation was considered, and medication therapy was given to control the epileptic seizure. Cerebrospinal fluid examination via lumbar puncture hinted increased intracranial pressure. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) of the cerebrospinal fluid demonstrated that the patient was tested positive for HHV-6B nucleic acid, which confirmed the diagnosis of HHV-6B encephalitis. The immunosuppressant regime was adjusted, intravenous ganciclovir was given for antiviral treatment, and active interventions were delivered to prevent and treat relevant complications. Epileptic seizure disappeared after 4 d, and neurological symptoms were significantly alleviated after 2 weeks. After 4-week antiviral treatment, the patient was tested negative for virology testing, and the neurological function was restored to normal. Conclusions HHV-6B encephalitis rarely occurs after adult liver transplantation, which is primarily associated with the virus reactivation after use of immunosuppressant. MDT pattern may be employed to deepen the understanding of the patient's condition, formulate more effective individualized treatment regime, and enhance the clinical efficacy and safety.

Human herpes virus-8-negative kaposiform sarcoma in a HIV-positive male: Novel variant

Human herpes virus (HHV)-8 is associated with causation of Kaposi's sarcoma (KS). An HIV-positive male with multiple nodules on the body was clinically and histopathologically diagnosed as a case of KS; however, immunohistochemistry was negative for HHV-8. This peculiar scenario of HHV-8-negative tumor with typical clinical and histopathologic findings was labeled as “Atypical spindled endothelial proliferation suspicious of Kaposi sarcoma”, hitherto unreported novel entity

Diagnosis of elephantiasis nostras verrucosa as a clinical mani-festation of Kaposi's sarcoma / Diagnóstico de elefantiasis nostras verrucosa como mani-festación clínica del Sarcoma de Kaposi

Elephantiasis nostras verrucosa, a rare manifestation of Kaposi's sarcoma, is a progressive cutaneous hypertrophy caused by chronic non-filarial lymphedema secondary to obstruction of the lymphatic system that can lead to severe disfigurement of parts of the body that have gravity-dependent blood flow, due to edema, fibrosis, and hyperkeratosis, especially lower extremities. Among the various conditions that can induce chronic lymphedema are tumors, trauma, radiotherapy, obesity, hypothyroidism, chronic venous stasis, and AIDS-related Kaposi's sarcoma. Kaposi's sarcoma is a vascular tumor associated with the presence of human gammaherpesvirus 8 that is predominantly cutaneous, locally aggressive, with metastasis, and is associated with the production of factors that favor inflammation, lymphatic obstruction, and lymphedema.

Encefalitis por virus herpes humano tipo 6 en una adolescente con lupus eritematoso sistémico. Primer caso reportado en el ecuador / First case reported in ecuador: human herpes virus type 6 encephalitis in adolescents with systemic lupus erythematosus. First case reported in ecuador

Se describe el caso de una paciente de catorce años con Lupus Eritematoso Sistémico (LES) con índice de actividad severo y en tratamiento inmunosupresor. Acudió al servicio de Urgencias Pediátricas del Hospital de Especialidades Carlos Andrade Marín, por presentar fiebre, cefalea, náuseas, alucinaciones auditivas y paresia en extremidades inferiores. Se realizó estudio de líquido cefalorraquídeo por Reacción en Cadena de la Polimerasa, en el cual se detectó la presencia de Virus Herpes Humano tipo 6 o también llamado Roseolovirus. Se inició ganciclovir con respuesta clínica favorable en 72 horas. Conclusiones: Se debe considerar la presencia de encefalitis por HHV-6 en pacientes inmunocomprometidos con encefalopatía y el uso de ganciclovir como terapia dirigida.

The case of a fourteen-year-old patient with Systemic Lupus Erythematosus with severe activity index and immunosuppressive treatment is described. He went to the Pediatric Emergency Department of the Carlos Andrade Marín Specialty Hospital, for presenting fever, headache, nausea, auditory hallucinations and paresis in the lower extremities. Cerebrospinal fluid was studied by Polymerase Chain Reaction, in which the presence of Human Herpes Virus type 6 or also called Roseolovirus was detected. Ganciclovir was started with a favorable clinical response in 72 hours. Conclusions: The presence of HHV-6 encephalitis should be considered in immunocompromised patients with encephalopathy and use of ganciclovir as directed therapy.

Sarcoma de kaposi en pacientes vih: a proposito de dos casos / Kaposi´s sarcoma in hiv patients: regarding two case report

RESUMEN: El Sarcoma de Kaposi es unaneoplasia de origen vascular,asociado obligadamente al virus Herpes Humano tipo 8. Presenta manifestaciones cutáneas en primer lugar, mucosas, ganglionares y viscerales. Puede evolucionar de forma leve con lesiones cutáneas, hasta casos fulminantes con compromiso sistémico, en caso de no realizar tratamiento. Existen múltiples opciones terapéuticas, las cuales se definen según el compromiso de la enfermedad y la afectación del paciente por su patología de base.El diagnóstico se basa en la sospecha clínica y se confirma con una biopsia histopatológica. El sarcoma de Kaposi corresponde a la neoplasia más frecuente en los pacientes con VIHse encuentraprincipalmente en hombres homosexuales. En este artículo se presentan dos pacientes con VIH con diagnóstico de Sarcoma de Kaposi y se realiza una breve revisión de la bibliografía.

ABSTRACT: Kaposi´s sarcoma is a vascular origin neoplasm, obligatory associated with Human Herpes Virus 8. It presents cutaneous manifestations at a first place, mucous, ganglionic and viscerals. It may present a mild presentation with cutaneous manifestations, up to fulminant cases with systemicinvolvement,in case of not being treated. There are a number of therapeutic options, defined by the state of the illness and the involvement of the patient due to the primary pathology. The diagnosis is based on the clinical suspicion and it is confirmed by a histopathological biopsy. Kaposi´s sarcomais the most frequent neoplasm in patients with HIV, being more frequently in homosexual men. In this article two HIV patients with Kaposi´s sarcoma diagnosis are exposed and a brief revision of the literature is done.

Risk factors and clinical features of septic patients with human herpes viruses' nucleic acid detected positive in blood / 中华急诊医学杂志

Objective To measure the reads numbers of Human Herpes Virus in blood sample from patients with sepsis by using Next Generation sequencing (NGS) and explore the relationship between read number of virus and the severity, prognosis, immune status of septic patients.Methods Blood sample and clinical information from 150 patients with sepsis were enrolled in this study. All patients' blood samples were sent to perform NGS pathogenic test. According to the results of NGS, septic patients were divided into HHV-detected group and HHV-undetected group. Besides, patients were scored with Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation Ⅱ(APACHE Ⅱ ) on the day of blood collection. The counts of total leukocytes, lymphocytes and the levels of cytokines were also measured. Results 51.3 percent of septic patients were detected with HHV nucleic acid. The APACHE Ⅱ and SOFA scores were significantly higher in HHV-detected patients compared with patients in HHV-undetected group. Besides, patients who had a higher SOFA score might lead to a higher detection rate of HHV. Moreover, the 28-day and 90-day mortality rates were higher in detected group (P< 0.01). The detection of HHV nucleic acid was positively correlated with a high 90-day mortality rate (P= 0.0056). One-way analysis of variance revealed that the counts of total lymphocyte and different types of lymphocyte (CD19+B、CD4+T、CD8+T、CD56+ lymphocyte) were significantly less in detected group than that in undetected group. Furthermore, both the levels of pro-inflammatory cytokines (TNF-α、IL-2R、IL-6、IL-8) and anti-inflammatory cytokines (IL-10) in detected group were significantly higher than those in undetected group. Gender, age, APACHE Ⅱ , SOFA, IL-2R, IL-10, CD19+B lymphocyte and T cells, were still significant even after multivariate logistic analyses. Conclusions The detection rate of HHV nucleic acid in patients with sepsis was high. The detection of HHV was a high-risk factor of death in patients with sepsis. The cut-off value which is more than 100 had a significant clinical value. The infection of HHV could be conducted by dysfunction of immunity.

Eyelid Kaposi Sarcoma in an HIV-negative Patient

Kaposi sarcoma (KS) is a low-grade, multicentric vascular neoplasm. Most commonly, it involves the skin, but it can occur at any site on the body. The cutaneous lesions are often located on the lower legs, genitalia, oral mucosa, and face. KS is categorized in four different types: classic, endemic, epidemic or AIDS associated, and transplantation associated. We report a case of HIV-negative, classic KS located on the eyelid. The eyelid lesion was completely excised, and after a 1-year follow-up, no recurrences were observed. Ocular involvement by KS in a patient who is serologically negative for HIV is extremely rare.

Treatment of multicentric castleman’s disease: Research advances / 国际药学研究杂志

Castleman’s disease (CD) is a rare lymphoproliferative disorder which comprises two distinct clinical subtypes: unicentric CD (UCD) and multicentric CD (MCD). Unlike UCD, which is a curable disease with surgical resection, MCD is a disease with systemic symptoms and high mortality rate (the 5-year mortality rate could be as high as 35%). There is no standard of care for MCD. Current treatment options include observation, glucocorticoids, chemotherapy (with or without rituximab) and immunomodulatory agents. Progress have been made during the recent years, interleukin-6 (IL-6) and human herpes virus 8 (HHV-8) have been recognized as key factors involved in the pathogenesis of MCD. Accordingly, drugs targeting these factors (especially IL-6) have been developed to treat MCD. In this paper, we try to review the current treatment options and new emerging therapies for MCD which might help Chinese clinicians to learn more about this rare disease.

Mucocutaneous Kaposi's sarcoma in an HIV-positive patient: diagnosis and treatment / Sarcoma de Kaposi mucocutâneo em paciente HIV positivo: diagnóstico e tratamento

ABSTRACT Kaposi's sarcoma (KS) is an endothelial neoplasia caused by infection with the human herpesvirus 8 (HHV-8), and the type associated with the human immunodeficiency virus (HIV) is considered the most aggressive and frequent. This paper reports a case of mucocutaneous KS in a patient not formerly aware of being an HIV bearer. A 38-year-old male patient has sought treatment with multiple oral lesions and one in the skin. Serology was positive for HIV and incisional biopsy diagnosed KS. After 11 months of chemotherapy and antiretroviral therapy, there was complete remission of the skin KS and partial remission of oral lesions.

RESUMO Sarcoma de Kaposi (SK) é uma neoplasia endotelial causada pelo herpes vírus humano tipo 8 (HHV-8), e o tipo associado ao vírus da imunodeficiência humana (HIV) é considerado o mais agressivo e frequente. Relata-se um caso de SK mucocutâneo em indivíduo não anteriormente ciente de ser portador de HIV. Paciente do sexo masculino, 38 anos, procurou atendimento com queixa de múltiplas lesões orais e uma lesão em pele. A sorologia foi positiva para HIV, e a biópsia incisional das lesões teve como diagnóstico SK. Após 11 meses de terapia antirretroviral e quimioterapia, houve remissão completa do SK cutâneo e parcial das lesões orais.

Infección por virus herpes humano 6 en un paciente inmunocompetente con síndrome DRESS secundario a carbamazepina / Human herpes virus 6 infection in an inmunocompetent patient with carbamazepine-induced DRESS syndrome

El síndrome DRESS (drug reaction with eosinophilia and systemic symptoms) constituye una reacción adversa a fármacos, potencialmente mortal, caracterizada por una erupción cutánea polimorfa asociada a fiebre, linfadeno-patías y compromiso multiorgánico con eosinofilia. Presentamos el caso clínico de un hombre inmunocompetente con un síndrome DRESS secundario a carbamazepina que cursó concomitantemente con una meningoencefalitis por virus herpes humano 6 (VHH-6). El rol patogénico del VHH-6 en el síndrome DRESS sigue siendo controversial; sin embargo, dada la importancia diagnóstica y eventualmente pronóstica de la infección por VHH-6, su tamizaje sería recomendable dentro del estudio de estos pacientes.

DRESS syndrome (drug reaction with eosinophilia and systemic symptoms) is an adverse life-threatening drug reaction characterized by a polymorphous rash associated with fever, lymphadenopathy and multiorgan involvement with eosinophilia. We present the case of an immunocompetent man with DRESS syndrome secondary to carbamazepine, that developed concomitantly meningoencephalitis caused by human herpes virus 6 (HHV-6), and a review of literature. The pathogenic role of HHV-6 in DRESS syndrome remains controversial. Given the diagnostic and possibly prognostic significance of HHV-6, the screening seems to be a good measure to use in the clinical management of these patients.

Human Herpes Virus 6 Encephalitis Following Bone Marrow Transplantation with Uncommon Magnetic Resonance Imaging Findings

BACKGROUND: Human Herpes Virus 6 (HHV6) is commonly associated with encephalitis following bone marrow transplantation. However, hippocampal atrophy and global hypometabolism are rare findings in HHV6 encephalitis. CASE REPORT: A 41-year-old right-handed woman with acute lymphoblastic leukemia presented with fever and mental changes 2 weeks after receiving a sibling bone marrow transplant. The patient's cerebrospinal fluid (CSF) was positive for HHV-6 deoxyribonucleic acid (DNA), but was negative for other viral DNA. Brain magnetic resonance imaging revealed atrophic changes in bilateral medial temporal lobes. Following 4 weeks of ganciclovir therapy, a CSF exam was negative for HHV-6 DNA and the patient's neurological symptoms partially improved. However, she was disoriented and had severe retrograde and anterograde amnesia. 18F-fluorodeoxyglucose-positron emission tomography indicated global hypometabolism in the medial temporal lobes and the fronto-parietal cortices. CONCLUSIONS: This is a rare and unusual case of hippocampal atrophy in the acute stage of HHV6 encephalitis. Our imaging findings may reflect the chronic indolent course of HHV6 encephalitis.

Extracavitary primary effusion lymphoma: A case report from India.

We present a case of extracavitary primary effusion lymphoma presenting, as jejunal polyps in a 38-year-old man. This is the first report of this entity from India. Although rare in our country, the diagnosis should be suspected in cases of CD20 negative large cell lymphoma with plasmablastic or immunoblastic differentiation in seropositive patients. Immunostaining for latency-associated nuclear antigen-1 and in situ hybridization for Epstein-Barr virus-associated RNA will confirm the diagnosis.

Seroprevalence of Human Herpes Virus 8 among Blood Donors in National Blood Centre, Kuala Lumpur, Malaysia.

Aims: In South East Asia, there is no regional or local HHV-8 seroprevalence data on blood donors. Thus this study was aimed to determine the seroprevalence of HHV-8 among blood donors in National Blood Centre, Kuala Lumpur (NBCKL) and to test its association with donor socio demographic and transfusion transmitted infection (TTI) seropositivity. Study Design: A cross sectional study. Place and Duration of Study: National Blood Centre, Kuala Lumpur (NBCKL). Duration of the study from January 2008 to June 2009. Methodology: A total of 761 serum samples were collected of which 670 from blood donors who were non-reactive for TTIs while 91 were from blood donors who were reactive for TTIs were tested for HHV 8 using BIOTRIN HHV-8IgG EIA kit and BIOTRIN HHV-8IgG Immuno fluorescent assay (IFA). Results: The HHV-8 seroprevalence among blood donors in NBCKL was 1.3% (10/761) of which 0.9% (6/670) among healthy blood donors and 4.4% (4/91) among TTI seropositive donors. TTI seropositivity (p=0.023) and gender (p=0.018) shows a significant risk factors contributed to HHV-8 seropositivity. Human Immunodeficiency Virus (HIV) and Hepatitis C were associated with an increased risk of HHV-8 seropositivity (OR 6.8; 95% CI, 0 to 0.2 and OR 10.0; 95% CI, 0.1 to 0.4 respectively). Conclusion: HHV-8 has a low seroprevalance among blood donors in the NBCKL with a male predominance. A donor with seropositivity for TTI, is associated with a higher risk HHV-8 seropositivity.

Sociodemographic and Clinical Factors Associated with Seroprevalence of HSV2 Infection among Antenatal Clinic Attendees in Benin.

Background: Knowledge of the Sociodemoraphic factors associated with HSV-2 seroprevalence and identification of the associated patient-recognizable clinical features will enable informed preventive public health interventions. There’s yet no documented data on the clinical and sociodemographic factors associated with HSV 2 Seroprevalence among pregnant women in Nigeria. Objectives: To identify sociodemographic and clinical correlates of HSV-2 seroprevalence among pregnant women attending ante-natal clinics in Benin, Nigeria. Study Location, Design and Duration: All the participants were prospectively recruited from the two major hospitals in Benin: University of Benin Teaching Hospital and Central Hospital, Benin. The cross-sectional study took place between November 2011 and June 2012. Methodology: Participants were recruited on booking. Data on their sociodemographic profiles, clinical history and obstetric characteristics were obtained by the use of structured questionnaires and hospital case records. Their blood samples were also promptly collected on recruition. Each participant’s serum was analyzed for HSV-2 IgG antibodies by gG-based type-specific ELISA. Counselling and testing for HIV were also carried out. Data analysis was done using SPSS version 16. Results: The average age of the 674 enrolled participants was 30.6±5.2 years and most of them were married and had complete secondary education. Seroprevalence of HSV-2 was 46.3%. Factors that was significantly associated with HSV-2 seropositivity included age, level of education, parity, HIV seropositivity and positive history of sexually transmitted infections. The HSV-2-infected were also significantly more likely to recall episodes of genital rashes (occurrence of rashes of any type in the external genitalia), vaginal discharge and urethral discharge. Conclusion: Risk factors for HSV-2 infection among pregnant women could include increasing age, higher parity, education below secondary level, HIV-positive status, and positive history of sexually transmitted infections. History of genital rashes, vaginal discharge and urethral discharge syndromes were associated with HSV-2 infection.

Polymorphisms in Interleukin-2 and Interleukin- 7 Receptor α-Chain Genes and Human Herpes Virus-6 as Risk Factors for Multiple Sclerosis.

Aim: The aim of this study was to examine whether a Human herpes virus-6 (HHV-6) infection increases the risk of MS in individuals harboring particular cytokine receptor α- chain gene alleles. Study Design: MS patients and controls were assessed for HHV-6 DNA and for single nucleotide polymorphisms (SNPs) in their IL7RA and IL2RA genes. Place and Duration of Study: The study was carried out at the Department of Experimental Pathology, Microbiology and Immunology, American University of Beirut, between March 2011 and March 2013. Methodology: Blood samples from 100 MS patients and 100 controls were investigated for the presence of HHV-6 by nested PCR. Single nucleotide polymorphisms (SNPs) in the IL7RA and IL2RA genes were examined by restriction fragment length polymorphism. Results: HHV-6 was detected in 58% of MS patients and 32% of controls (OR = 2.935, 95% CI = 1.582-5.463, p=0.000). We did not detect a statistically significant correlation between MS and the studied rs2104286, rs12722489 SNPs in the IL2RA gene and rs6897932 SNP in the IL7RA gene. Concomitant presence of rs2104286 and HHV-6 was detected in 56% of patients and 30% of controls (OR=2.970, 95% CI=1.594-5.53, P=0.000). Similarly, rs6897932 and HHV-6 were observed in 57% of patients and 28% of controls (OR=3.409, 95% CI= 1.815-6.428, P=0.000). Therefore, double positivity moderately increased the risk of MS compared to either factor alone. HHV-6 and rs12722489 double positivity did not increase the risk of MS. Conclusion: HHV-6 infections may enhance the risk of MS in subjects with particular genetic determinants.

Current Concepts in Primary Effusion Lymphoma and Other Effusion-Based Lymphomas

Primary effusion lymphoma (PEL) is a human herpes virus 8 (HHV8)-positive large B-cell neoplasm that presents as an effusion with no detectable tumor in individuals with human immunodeficiency virus infection or other immune deficiencies. PEL is an aggressive neoplasm with a poor prognosis. PEL cells show diverse morphologies, ranging from immunoblastic or plasmablastic to anaplastic. The immunophenotype of PEL is distinct, but its lineage can be misdiagnosed if not assessed thoroughly. PEL cells usually express CD45, lack B- and T-cell-associated antigens, and characteristically express lymphocyte activation antigens and plasma cell-associated antigens. Diagnosis of PEL often requires the demonstration of a B-cell genotype. HHV8 must be detected in cells to diagnose PEL. In most cases, PEL cells also harbor the Epstein-Barr virus (EBV) genome. Similar conditions associated with HHV8 but not effusion-based are called "extracavitary PELs." PELs should be differentiated from HHV8-negative, EBV-positive, body cavity-based lymphomas in patients with long-standing chronic inflammation; the latter can occur in tuberculous pleuritis, artificial pneumothorax, chronic liver disease and various other conditions. Despite their morphological similarity, these various lymphomas require different therapeutic strategies and have different prognostic implications. Correct diagnosis is essential to manage and predict the outcome of patients with PEL and related disorders.

Simultaneous detection of four types of human herpes virus by AllGlo quadruple fluorescence quantitatve PCR / 中华微生物学和免疫学杂志

ObjectiveTo optimize the condition of multiple fluorescence quantitative PCR and establish a new assay of four human herpes virus (HHV) detected by AllGlo quadruple fluorescence quantitative PCR.MethodsFour HHV including HSV-1,HSV-2,EBV and CMV were identified by sequence analysing the qualitative PCR production.Furthermore,they were quantitatively detected by AllGlo and TaqMan multiple fluorescence quantitative PCR respectively.ResultsBoth the positive rate and specificity of AllGlo and TaqMan in detecting single HHV achieved 100％.And AllGlo single fluorescence quantitative PCR prevailed over TaqMan's by Ct of 1-3.Four HHV can be simultaneously detected by AllGlo quadruple fluorescence quantitative PCR,comparing to the only two by TaqMan.ConclusionAllGlo fluorescence quantitative PCR assay allows a higher throughput,sensitivity and specificity than TaqMan in detection and thus provides a board prospect.

Drug Rash with Eosinophilia and Systemic Symptoms Syndrome in a Patient on Sulfasalazine for Ulcerative Colitis

Drug rash with eosinophilia and systemic symptoms (DRESS) is a life-threatening systemic drug reaction characterized by fever, rash, hematological abnormalities, lymphadenopathy, and multiple internal organ involvement. Unfortunately, a long latency period as well as clinicians' unawareness of the disease entity often results in a delay of prompt diagnosis and treatment in clinical practice. A search of the literature revealed only few reports on DRESS in patients with inflammatory bowel diseases. The pathogenesis of the disease is not clearly understood, although several possible mechanisms, such as drug detoxification, slow acetylation, and reactivation of human herpes viruses, have been proposed in its development. Here, we present a rare case of DRESS associated with viral reactivation and defects in drug metabolism in a 22-year-old man who had been on sulfasalazine for 6 weeks to treat ulcerative colitis.

Study on fast diagnostics of activated infection of human herpes virus type 6 in infants with fever and rash / 中华微生物学和免疫学杂志

Objective To explore the fast diagnostics of activated infection of human herpes virus type 6 (HHV-6) with IEA/ex3( an antibody specific for an immediate early protein encoded by variant B HHV-6 U90 gene)in infants with fever and rash . Methods To collect the 55 samples of peripheral blood mononuclear cells (PBMCs) from clinical infants whose having a fever and rash, age are below 2 years old.The immediate early antigen (IEA) of HHV-6 were analyzed with IEA/ex3 in PBMCs by indirect immunofluorescence then using confocal microscope to observe, and using fluoromicroscope to observe after the PBMCs of 44 samples were incubated with cord blood cell at 37℃ in a humidified 5% CO2 atmosphere for 48 hours. The virus was isolated from the PBMCs. Results The conformation rate of the HHV-6 IEA using confocal microscope to observe and virus isolation was 92.73%, sensitivity was 90.32%, specificity was 95.83% and it was respectively 87.50%, 77.27%, 100% using fluoromicroscope to observe after the PBMCs were incubated for 48 hours. Conclusion The study suggested that detection of HHV-6B IEA in PBMCs using confocal microscope to observe after the PBMCs were incubated for 48 hours can fast diagnose HHV-6 activated infection of infant with fever and rash than technique of virus isolation.

Normalización de un sistema de reacción en cadena de la polimerasa en tiempo real para la cuantificación del herpesvirus humano 8 / Standardization of a real-time polymerase chain reaction system for quantification of human herpesvirus 8

OBJETIVO: normalizar un sistema de reacción en cadena de la polimerasa en tiempo real para determinar la carga viral del herpesvirus humano 8, en diferentes muestras clínicas de pacientes en los que se sospeche la infección por este agente. MÉTODOS: se evaluaron 3 de los métodos reportados internacionalmente para obtener ADN estándar en la construcción de curvas externas estándar, que permiten determinar el número de copias de ADN diana en la muestra problema. RESULTADOS: se obtuvieron 3 ADN estándar a partir del clonaje de un fragmento del gen ORF26 del herpesvirus humano 8 en un vector (ADN plasmídico), con la utilización de productos purificados de reacción en cadena de la polimerasa y el empleo de ADN genómico de la línea celular BCBL. Se pudieron construir las curvas patrón a partir de cada uno de los ADN estándar obtenidos, los que mostraron una fuerte correlación lineal (r= -1) y valores muy bajos de error a lo largo de 6 magnitudes de concentración de ADN diana. El límite inferior de detección a partir del ADN plasmídico y de los productos de reacción en cadena de la polimerasa fue de hasta 100 copias, mientras que con el ADN genómico fue de hasta 10 copias; este último sistema resultó el más sensible. CONCLUSIONES: la reacción en cadena de la polimerasa en tiempo real normalizada a partir de los 3 ADN estándar probó ser un sistema rápido, específico y altamente sensible que permitirá un mejor diagnóstico y además desarrollar estudios sobre la patogenia de la infección por el herpesvirus humano 8 en Cuba.

OBJECTIVE: to standardize a real-time polymerase chain reaction system to determine the human herpes virus 8 viral load in several samples from patients suspected of this type of infection. METHODS: three internationally known methods were evaluated to obtain standard DNA in standard external curve constructions, which allow determining the number of target DNA copies in the suspected samples. RESULTS: three standards DNA were obtained from cloning ORF26 gene fragment of human herpesvirus 8 in a vector (plasmid DNA), with the use of purified polymerase chain reaction products and of genomic DNA of BCBL cell lines. The pattern curves were constructed on the basis of each of the resulting standard DNA, which showed strong linear correlation (r= -1) and very low error values throughout 6 target DNA concentrations. The lower detection limit based on plasmid DNA and the polymerase chain reaction products was 100 copies, whereas that obtained with genomic DNA reached up to 10 copies; this last system turned to be the most susceptible. CONCLUSIONS: real-time polymerase chain reaction system, standardized for the three standard DNA proved to be a rapid, specific and highly sensitive system for better diagnosis, and for the development of studies on the pathogenesis of human herpesvirus 8 infection in Cuba.