Development and validation of a simple and rapid way to generate low volume of plasma to be used in point-of-care HIV virus load technologies

ABSTRACT A new point-of-care HIV viral load, mPIMA HIV-1/2 VL, Abbott, USA, has been recently developed. This point-of-care viral load requires no skilled person to run and uses a small plasma volume (50 µL). However, obtaining 50 µL of plasma can be a challenge in limited resource settings. We validated a simple and easy method to obtain enough amount of plasma to run a point-of-care viral load. The study utilized 149 specimens from patients failing antiretroviral therapy. At least 250 µL of whole blood was collected in a microtube/EDTA from fingerstick (fs-plasma) and immediately centrifuged. Parallel collection of venous blood to obtain plasma (vp-plasma) was used to compare performance in a point-of-care viral load assay and in methodology used in centralized laboratories Abbott M2000, Abbott, USA. The procedure for plasma collection takes less than 10 min and in 94% of the cases only one fingerstick was sufficient to collect at least 250 µL of blood. The Pearson correlation coefficient value for vp-plasma versus fs-plasma ran on mPIMA was 0.990. The Bland-Altman mean difference (md) for this comparison were virtually zero (md = −0.001) with limits of agreement between −0.225 and 0.223. In addition, the Pearson correlation coefficient value for fs-plasma in mPIMA versus vp-plasma in Abbott M2000 was 0.948 for values above the mPIMA limit of quantification (LoQ; from 800 to 1,000,000 copies/mL). These results validate this simple plasma isolation method capable to be implemented in low resource countries where point-of-care decentralization is deeply needed.

One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil

ABSTRACT The Brazilian Public Health Service provides freely αPEG-IFN to treat patients infected with HCV. The primary goal of HCV therapy is the long-term elimination of HCV from the blood to reduce the risk of HCV associated complications and death. Patient viremia affects the treatment duration and response, thus influencing clinical decisions. We developed a high-throughput method to perform the quantification of RNA hepatitis C virus (HCV) virus load in plasma samples to monitor patients under treatment. The method is based on a duplex detection, in a one-step real-time RT-PCR assay and it has been validated according to the rules established by the official Brazilian regulatory agency (ANVISA). This new method was compared to a commercial kit (Cobas/Taqman HCV Test v2.0 - Roche), showing virus load results with significant correlation between them (p= 0,012) using commercial and clinical panels. In addition, 611 samples from patients treated with peguilated alfa-interferon (αPEG-IFN) from different regions of Brazil were analyzed. Our one-step real-time RT-PCR assay demonstrated good performance in viral load measurement and in treatment course monitoring, with acceptable sensitivity and specificity values.

Optimización de recursos para determinar la carga viral de HIV-1 en un país con pocos recursos / Optimizing resources to reduce costs to determine HIV viral load in limited resources settings

Resumen Introducción. Las metas globales para controlar la epidemia de HIV contemplan que la carga viral sea indetectable en 90 % de las personas en tratamiento. El costo de la medición de la carga viral en lotes de muestras puede reducirse y, así, aumentar la cobertura cuando los recursos son limitados; sin embargo, su eficacia disminuye al aumentar la prevalencia del fracaso del tratamiento antirretroviral. Objetivo. Evaluar estrategias para disminuir la proporción de pacientes con fracaso del tratamiento antirretroviral en los lotes de muestras y, de esta manera, aumentar el ahorro en las pruebas de carga viral. Materiales y métodos. Las estrategias evaluadas fueron: a) la organización de los lotes de muestras según el esquema de tratamiento antirretroviral, y b) la exclusión de aquellos pacientes con antecedente reciente de fracaso del tratamiento antirretroviral, aquellos con menos de 12 meses de tratamiento antirretroviral y aquellos sin tratamiento antirretroviral previo. Los resultados de los lotes se compararon con los resultados individuales. Resultados. El valor diagnóstico negativo fue similar para los pacientes con esquema de primera línea (100,0 %; IC95% 99,5-100,0) o de segunda línea de tratamiento (99,4 %; IC95% 96,9-99,9). La incidencia del fracaso del tratamiento antirretroviral fue menor en los pacientes con tratamiento de primera línea (p<0,01), lo cual permitió un mayor ahorro en las pruebas de laboratorio en este grupo (74,0 %; IC95% 71,0-76,7) que en los pacientes con tratamiento de segunda línea (50,9 %; IC95% 44,4-57,3) (p<0,01). Conclusión. La selección de las muestras que se incluyeron en los lotes para determinar la carga viral del HIV según el tipo de esquema de tratamiento, permitió maximizar el porcentaje de ahorro en pruebas de laboratorio.

Abstract Introduction: HIV viral load testing is a key factor to evaluate the accomplishment of the UNAIDS target of 90% of viral suppression among people receiving antiretroviral therapy. Pooled samples are a potentially accurate and economic approach in resource-constrained settings, but efficiency can be negatively affected by high prevalence rates of virological failure. Objective: Strategies were assessed to increase the relative efficiency of pooled HIV viral load testing in resource-constrained settings. Materials and methods: We evaluated two strategies: a) plasma samples were not included in pools if patients had <12 months on antiretroviral therapy, patients had previous viral load >1,000 copies/ml, or were antiretroviral therapy naïve patients, and b) plasma pools were organized separately for first and second-line antiretroviral therapy regimens. Individual viral load tests were used to compare pooled results. Results: Negative predictive values were similar for patients on first (100.0%; 95% CI 99.5 to 100.0) and second-line antiretroviral therapy regimens (99.4%; 95% CI 96.9 to 99.9). However, the incidence of virological failure among individuals on first-line antiretroviral therapy was lower than second-line antiretroviral therapypatients (p <0.01), resulting in greater savings in laboratory tests in patients on first-line antiretroviral therapy (74.0%; 95% CI 71.0 to 76.7) compared with the group of patients on second-line antiretroviral therapy (50.9%; 95% CI 44.4 to 57.3) (p<0.01). Conclusion: Selecting the samples to be included in the pools and selecting the pools according to ART regimens are criteria that could lead to decreased spending on laboratory tests for HIV viral load determination in resource-constrained settings.

Infección por citomegalovirus en receptores de trasplante alogénico de células progenitoras hematopoyéticas: terapia temprana basada en el estudio de la antigenemia y la carga viral en sangre / Cytomegalovirus infection in allogeneic hemapotopoietic stem-cell transplantation: early treatment based on the study of antigenemia assay and viral load determination in blood

La prevención de la enfermedad por CMV en los receptores de trasplante de células progenitoras hematopoyéticas se basa en la terapia temprana de la reactivación viral. La Antigenemia y el estudio de la carga viral por PCR son las dos técnicas diagnósticas actualmente vigentes. Se siguió una cohorte de 35 pacientes con estudios semanales con ambos métodos desde la recuperación de la neutropenia hasta el día 100 días postrasplante. Se inició tratamiento empírico con antivirales (Ganciclovir o Foscarnet) con un resultado positivo (antigenemia > 1 cel/200.000 o carga viral > 500 copias / ml) y se mantuvo 3-6 semanas. La serología previa fue positiva en R y D en 66% de los casos, en R o D en 20%, negativa en 3% y no evaluable en 11%. Se detectó infección por CMV en el 50% de los pacientes. En 15 ptes el diagnóstico fue por PCR, en 2 ambas pruebas fueron positivas y en uno solo la antigenemia. Un paciente presentó neumonía por CMV y falleció dentro de los 100 días de seguimiento. En 11,4% de los casos se detectó reactivación viral luego de los 100 días y dos ptes presentaron neumonía por CMV tardía que fue causa de muerte. Conclusión: Con los umbrales utilizados la carga viral precedió a la antigenemia en el diagnóstico de reactivación de CMV. La terapia temprana previene la enfermedad temprana por CMV en la mayoría de los casos pero la enfermedad tardía es un problema pendiente de resolución (AU)

Prevention of CMV disease in hematopoietic stem-cell transplantation recipients is based on the early management of viral reactivation. Antigenemia assay and PCR viral load detection are the current diagnostic techniques of choice. We followed a cohort of 35 patients with weekly studies using both methods from recovery from neutropenia to day 100 post-transplant. Empirical viral treatment (Ganciclovir or Foscarnet) was started after a positive result (antigenemia > 1 cell/200,000 or viral load > 500 copies / ml) and maintained for 3-6 weeks. Previous serology was positive in R and D in 66% of the cases, in R or D in 20%, negative in 3%, and not evaluable in 11%. CMV infection was detected in 50% of the patients. In 15 patients the diagnosis was made using PCR, in 2 both tests were positive, and in one only the antigenemia assay was positive. One patient presented with pneumonia due to CMV and died within the 100 days of follow-up. In 11.4% of the cases viral activation was detected after 100 days and two patients developed late pneumonia due to CMV and consequently died. Conclusion: Using these thresholds viral load detection preceded antigenemia assay in the diagnosis of CMV reactivation. Early treatment prevents early disease due to CMV in the majority of cases, but late disease remains a problem to be solved (AU)

Implementación del ensayo de carga viral COBAS Taqman HIV-1 Test, v1. 0, para el diagnóstico de la infección congénita por HIV-1 / Implementation of the viral load assay COBAS Taqman HIV-1 Test, v1.0, for the diagnosis of congenital HIV-1 infection

La transmisión vertical es la principal vía de contagio del HIV en la edad pediátrica. El diagnóstico de la infección congénita antes de los 18meses se realiza mediante ensayos virológicos: detección de genoma viral como ARN plasmático y ADN proviral. La sensibilidad de estos ensayos varía según la edad del niño, con valores de especificidad mayores al 95%. El objetivo de este trabajo fue evaluar el desempeño del ensayo de carga viral (CV) COBAS Taqman HIV-1 Test, v1.0 (Roche), y su concordancia con una PCR múltiple anidada in-house para la detección del ADN proviral. De 341 muestras procesadas, 15 resultaron positivas y 326 negativas por ambas metodologías. Para la metodología de CV, la sensibilidad general fue del 88,2% y la especificidad del 100%. Nuestros resultados indican que la metodología de CV evaluada puede utilizarse como técnica alternativa para el diagnóstico de infección congénita por HIV

Vertical transmission is the main route of HIV infection in childhood. Because of the persistence of maternal HIV antibodies, virologic assays that directly detect HIV are required to diagnose HIV infection in infants younger than 18months of age. The sensitivity of HIV RNA/DNA assays increases as the child becomes older. These tests have specificity values greater than 95%. The aim of this study was to evaluate the performance of the COBAS Taqman HIV-1 Test, v1.0 assay (Roche) and its concordance with a Multiplex Nested-PCR. Of 341 samples processed, 15 were positive and 326 negative by both methods. Sensitivity and specificity overall values for the viral load assay were 88.2% and 100%, respectively. Our results indicate that the COBAS Taqman assay evaluated could be used as an alternative method to diagnose HIV congenital infection

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

Padronização da PCR em Tempo Real para o Diagnóstico da Hepatite B / Standardization of real-time PCR for the hepatitis B diagnosis

A hepatite B é uma doença de interesse em saúde pública, acometendo cerca de 2 bilhões de pessoas no mundo, das quais aproximadamente 350 milhões tornam-se portadoras crônicas. Os ensaios quantitativos de detecção do DNA viral são empregados para avaliar a infectividade do vírus e para o monitoramento do tratamento, sendo importante para avaliar a progressão da doença. O objetivo deste estudo foi padronizar a técnica de amplificação quantitativa em tempo real (PCR em tempo real) do DNA do vírus da Hepatite B e comparar a técnica com métodos comerciais (COBAS AmpliPrep/COBAS TaqMan® e COBAS AmpliPrep/COBAS TaqMan®). A amostragem foi constituída por 397 amostras de soro ou plasma recebidas e estocadas no laboratório de hepatites virais do Centro de Virologia do Instituto Adolfo Lutz, no período de 2009 a 2011. Do total de amostras, 345 foram utilizadas para a comparação com os testes comerciais disponíveis e 52 amostras foram utilizadas para o teste de especificidade, sendo 27 amostras HIV positivas e 25 amostras HCV positivas, todas com AgHBS e Anti-HBc total não reagentes. Estas amostras foram inicialmente analisadas por testes comerciais e posteriormente pela técnica padronizada. Para o controle da quantificação, uma curva padrão foi construída em todas as reações, utilizando-se padrões internacionais produzidos pela Organização Mundial de Saúde, em diluições seriadas. Para a extração do DNA das amostras e padrão, foi utilizado kit comercial (QIAGEN®), seguindo os procedimentos descritos pelo fabricante. Os testes foram realizados empregando o método do TaqMan – PCR, em um equipamento ABI 7300 (Applied Biosystems, Foster City, CA). Os “primers” e sondas utilizadas para a padronização foram selecionados a partir da literatura e os que apresentaram melhores resultados nos testes iniciais foram escolhidos para o estudo. A técnica de sequenciamento foi realizada nas amostras com carga viral quantificável...

Hepatitis B is a disease of public health concern. Worldwide, it affects approximately 2 billion people, of whom approximately 350 million become chronic carriers. Quantitative assays for the detection of viral DNA are used to evaluate the infectivity of the virus and to monitor treatment, which is crucial to evaluate the progression of the disease. The aim of this study was to standardize real-time (real-time PCR) quantitative amplification of the DNA of hepatitis B and to compare this technique with commercial methods (COBAS AmpliPrep / COBAS ® TaqMan and COBAS AmpliPrep / COBAS TaqMan ®). The sample consisted of 397 serum or plasma samples received and stored at the laboratory of viral hepatitis from the Center of Virology, Instituto Adolfo Lutz, from 2009 to 2011. Of the total samples, 345 were used for comparison with the available commercial tests and 52 samples were used for the specificity test. Of these, 27 were HIV positive and 25, HCV positive, and all were HBsAg and anti- HBc nonreactive. These samples were initially analyzed by commercial tests and later by the standardized technique. To control the quantification, a standard curve was constructed in all reactions using international standards established by the World Health Organization, in serial dilutions. For the extraction of DNA samples and standard, a commercial kit used was (QIAGEN ®) according to the manufacturer’s instructions. The tests were performed using the TaqMan method - ABI 7300 PCR system (Applied Biosystems, Foster City, CA). Primers and probes used for standardization were selected from the literature and those presenting better results in initial tests were chosen for the study. Sequencing technique was performed on samples with measurable viral load. The standardized real-time PCR technique yielded satisfactory results...

Optimization of one-step real-time PCR for the X-tail target of HCV as a diagnostic test

Infection with hepatitis C virus (HCV) is a global public health issue. The bloodborne nature of HCV transmission poses a substantial risk to healthcare workers, due to occupational exposure to needlestick injuries and blood and other body fluids containing the virus. Undiagnosed HCV infection, including in healthcare workers, represents a growing problem worldwide as the infected population ages, and HCV-related mortality and morbidity is expected to rise substantially over the coming decades. Consequently, diagnostic tests for HCV play an important role in this scenario. The aim of this study was to standardize a one-step RT-PCR assay for detection of HCV. The test demonstrated reproducibility, sensibility (100%), and the limit of detection was set at 100IU/mL. Our study indicates that this assay can be used as a diagnostic tool to follow up healthcare workers after occupational exposure

Low-cost assays for monitoring HIV infected individuals in resource-limited settings.

Use of a combination of CD4 counts and HIV viral load testing in the management of antiretroviral therapy (ART) provides higher prognostic estimation of the risk of disease progression than does the use of either test alone. The standard methods to monitor HIV infection are flow cytometry based for CD4+ T cell count and molecular assays to quantify plasma viral load of HIV. Commercial assays have been routinely used in developed countries to monitor ART. However, these assays require expensive equipment and reagents, well trained operators, and established laboratory infrastructure. These requirements restrict their use in resource-limited settings where people are most afflicted with the HIV-1 epidemic. With the advent of low-cost and/or low-tech alternatives, the possibility of implementing CD4 count and viral load testing in the management of ART in resource-limited settings is increasing. However, an appropriate validation should have been done before putting them to use for patient testing.

Current practices in laboratory monitoring of HIV infection.

After a diagnosis of HIV infection is made, the patient needs to be monitored using both clinical assessment and laboratory markers. HIV/AIDS monitoring is essential in guiding when to recommend initiation of therapy. Clinical monitoring will include staging of the HIV/AIDS disease using either the presence or absence of HIV-related signs and symptoms using the WHO staging system. Various laboratory methods can be used to monitor the disease progression and to guide whether the patient will need antiretroviral therapy or not. Laboratory monitoring for patients who are not on drugs is done to provide information about the stage of illness; to enable the clinician to make decisions on treatment and to give information on prognosis of the patient. Patients on drugs are monitored to assess their response to treatment with antiretroviral drugs and to detect any possible toxicity and improvement associated with the antiretroviral drugs.

Performance Evaluation of Abbott RealTime HBV Quantification Kit for HBV Viral Load by Real-Time PCR / 대한진단검사의학회지

BACKGROUND: Hepatitis B virus (HBV) DNA quantification is necessary for starting and monitoring of antiviral therapy in patients with chronic hepatitis B. This study was intended to assess the clinical performance of Abbott RealTime HBV Quantification kit (Abbott Laboratories, USA). METHODS: The performance was evaluated in terms of precision, linearity, detection sensitivity, cross-reactivity, and carry-over. A correlation with the Real-Q HBV Quantification kit (BioSewoom Inc., Korea) was also examined using serum samples from 64 patients diagnosed with chronic hepatitis B and underwent lamivudine therapy in Asan Medical Center. We verified the trueness of the system by comparing the outputs with the assigned values of the BBI panel (BBI Diagnostics, USA). RESULTS: Within-run and between-run coefficients of variation (CV) were 3.56-4.71% and 3.03-4.98%, respectively. Linearity was manifested ranging from 53 to 10(9) copies/mL and the detection sensitivity was verified to be 51 copies/mL. None of hepatitis C virus showed cross-reactivity. No cross-contamination occurred when negative and positive samples were alternatively placed in a row. It showed a good correlation with the Real-Q HBV (r2=0.9609) and the test results for the BBI panel were also well agreed to the assigned values (r2=0.9933). CONCLUSIONS: The performance of Abbott RealTime HBV Quantification kit was excellent; thus, it should be widely used in starting and monitoring of antiviral therapy in Korean patients with chronic hepatitis B.

Despistaje de infección por citomegalovirus en niños VIH positivos mediante PCR: relación con serología y evolución clínica / PCR screening for cytomegalovirus infection in HIV-positive children: relationship between serology and clinical evolution

En pacientes VIH positivos es fundamental diagnósticar infección por CMV. La relación entre serología, detección viral y evolución clínica no está plenamente establecida. Detectar la presencia de CMV por PCR en sangre periférica en pacientes pediátricos, sin síntomas de infección, VIH positivos; relacionar estos resultados con la serología y la evolución clínica durante un año de seguimiento. Criterios de inclusión: Niños menores de 12 años, ambos sexos, infección diagnosticada por VIH, recibiendo terapia antirretroviral altamente efectiva (TARAE), consentimiento informado por escrito, firmado. La serología anti CMV se realizó mediante el método ELISA, la semicuantificación de CMV en sangre periférica, mediante PCR, y el inmunofenotipaje por citometría de flujo. Aquellos niños con carga viral inicial detectable para CMV fueron evaluados un año después (valoración cualitativa y oftalmológica). Correlación de Pearson, t Student. Se estudiaron 23 niños, 17 menores de 6 años; 21 de ellos (82.6 por ciento): lgG CMV +. Dos pacientes (8.7 por ciento): lgM CMV+ y promedio de carga viral: 11920 VID, dos IgM-IgG+, promedio de carga de 23129 VID; el resto negativos; todos con linfocitos CD4+ por encima del 25 por ciento. 50 por ciento de los niños con carga viral negativa para CMV, con contajes de CD4+ inferiores al 25 por ciento. El análisis de correlación de Pearson no mostró correlación entre la carga viral del VIH y los valores de VID para CMV (R²=0.13). Linfocitos CD8+: 32,3 ± 6,8 por ciento en los pacientes con carga para CMV, estadísticamente inferior al promedio del grupo sin cargas virales CMV: 49,1 ± 8,8. (t Student= 3,508; g.l: 17. P<0,003). Ningún niño presentó evidencia de enfermedad órgano específica (EOE), incluyendo retinitis. Independientemente de la presencia o no de IgM positiva para CMV, 4 pacientes tuvieron carga viral detectable. No hay correlación entre las cargas virales de ambos virus. Ningún niño con carga viral detectable para CMV...

In HIV-positive patients the diagnosis of CMV infection is essential. The relationship between serology, viral detection and clinical evolution has not been fully established. To detect the presence of CMV in peripheral blood by PCR testing in HIV positive pediatric patients with no infection symptoms, and to relate the results with serology and clinical evolution during a year follow up. Inclusion criteria: Children under twelve years of age, both genders, with a diagnosis of HIV infection, and receiving HAART therapy, written consent signed by parents. Anti CMV serology was performed by ELISA, semi-quantification of CMV in peripheral blood by PCR and immunophenotyping by flow-cytometry. Children with detectable initial viral load for CMV were submitted to aqualitative and ophtalmologic assessment one year later. Statistics: Pearson’s Correlation, Student’s t. 23 children, both genders, 17 under 6 years of age; 21 (82.6%): IgG CMV+. Two patients (8.7%): IgM CMV+ and viral load. 11920 IDV, two with IgM- IgG+, viral load average: 23129 IDV. The rest of the children were negative, all with lymphocytes CD4+ above 25%. 50% of the children had a negative viral load for CMV with CD4+ counts under 25%. There was no correlation between the HIV viral load and IDV values for CMV (r2=0.13). Lymphocytes CD8+32.3+ 6.8% in patients with viral load for CMV.This is statistically lower than the average for the group without viral loads CMV: 49.1 + 8.8 (Student’s t= 3.508; g.l: 17. p<0.003). No child showed evidence of specific organ disease (SOD), including retinitis. Regardless of serology for IgM, 4 patients had detectable viral loads. There was no correlation between the viral loads of the two viruses. No child with detectable viral load for CMV developed a specific organ disease, probably due to a highly efficient antiretroviral treatment. Viral load quantification for CMV in HIV + patients is recommended, regardless of specific IgM result.

Evaluation of BiosewoomTM Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus Viral Load Measure / 대한진단검사의학회지

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.

Evaluation of BiosewoomTM Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus Viral Load Measure / 대한진단검사의학회지

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.

Carga proviral do HTLV-1 e HTLV-2: um método simples através da PCR quantitativa em tempo real / HTLV-1 and HTLV-2 proviral load: a simple method using quantitative real-time PCR

Os vírus linfotrópicos de células T humanas, quando integrados ao genoma da célula hospedeira, provírus, têm como marcador de replicação seu DNA proviral. A carga proviral parece ser um importante fator no desenvolvimento de patologias associadas a estes retrovírus. Neste estudo foi desenvolvida uma metodologia para quantificação absoluta da carga proviral dos HTLV-1 e HTLV-2 através da PCR em tempo real. Cinqüenta e três amostras de doadores de sangue com teste de ELISA reagente foram submetidas à metodologia, que utilizou o sistema TaqMan® para três seqüências alvo: HTLV-1, HTLV-2 e albumina. A quantificação proviral absoluta foi determinada através da proporção relativa entre o genoma do HTLV e o genoma da célula hospedeira, levando em consideração o número de leucócitos. O método apresentado é sensível (215 cópias/mL), prático e simples para quantificação proviral, além de eficiente e adequado para confirmação e discriminação da infecção pelos tipos virais.

When the human T cell lymphotropic virus (HTLV) is integrated with the host cell genome (provirus), its proviral DNA is a replication marker. Proviral load appears to be an important factor in the development of diseases related to these retroviruses. In this study, a methodology for absolute quantification of the HTLV-1 and HTLV-2 proviral load using real-time PCR was developed. Fifty-three blood donor samples with positive ELISA test result were subjected to this methodology, which utilized the TaqMan® system for three target sequences: HTLV-1, HTLV-2 and albumin. The absolute proviral load was quantified using the relative ratio between the HTLV genome and the host cell genome, taking into consideration the white blood cell count. The method presented is sensitive (215 copies/ml), practical and simple for proviral quantification, and is efficient and appropriate for confirming and discriminating infections according to viral type.

La carga viral y células TCD4+ en pacientes infectados con el virus de inmunodeficiencia humana (VIH) coinfectados con sífilis / The viral load and TCD4+ cells in human immunodeficiency virus (HIV) infected patients coinfected with syphilis

La coinfección con sífilis en el paciente infectado con VIH-1 ha aumentado en los últimos años. La sífilis se ha asociado con activación inmunológica, pero el efecto de la misma sobre los parámetros inmunovirológicos en esta población aún son controversiales. El objetivo es evaluar el efecto de la sífilis en el contaje de células TCD4+ y la carga viral del paciente VIH+. Se realizó un estudio retrospectivo, multicéntrico, de casos y controles, extrayendo de las historias clínicas de los pacientes VIH+ que asistieron a control en los últimos 10 años, los reportes de contaje de células TCD4+ y carga viral, antes, durante y después del diagnóstico de sífilis para compararlos entre sí y con un grupo control. 48 pacientes VIH+ diagnosticados con sífilis conformaron el grupo de estudio y 56 sin sífilis, el grupo control. 14 (29%) pacientes con sífilis secundaria, 33 (69%) latente y 1 (2%) primaria. 38 (80%) recibían TARV en el momento de la sífilis. 24 (70%) elevaron sus valores de TCD4+ durante la enfermedad y 27(59%) posterior a ella, con una media de elevación de 19,41 celulas x mm³ (p=0,43) y 23,74 células x mm³ (p=0,28) respectivamente. Las determinaciones de carga viral se elevaron durante la enfermedad en 8 (38%) pacientes con una media de elevación de 64688 copias ARN-VIH/ml (4,96 log10) (p=0,03), y disminuyeron en 13 (45%) con una media de -1163 copias de ARN/ml (3,06 log10) (p=0,99) posteriormente. La sífilis en el paciente VIH+ estuvo asociada a elevaciones significativas en la carga viral y a cambios no significativos en el contaje de células TCD4+ durante la enfermedad.

Syphilis co-infection in HIV-1 patients has increased in recent years. Syphilis has been linked to immunoactivation, however, its effect on immunovirological parameters in this population is still controversial. Objective evaluate the effect of syphilis on the TCD4+ cell count andviral load of the HIV+ patient. A retrospective, multicenter case-control study was conducted by extracting the TCD4+ cell counts and viral load before, during, and after diagnosis of syphilis, from the clinical records of HIV+ patients who attended check-ups in the past 10 years, in order to compare them among themselves and against a control group. Seroprevalence of HIV/Syphilis co-infection was 18%. 48 HIV+ patients diagnosed with syphilis formed the study group, while 56, without syphilis, the control group. 14 (29%) had secondary, 33 (69%) latent and 1 (2%) primary syphilis. 38 (80%) received ART at the time of their syphilis. Of 24 (70%) the TCD4+ values increased during illness, and of 27 (59%) they increased subsequently, with a mean increase of 19.41 cell/mm³ (p=0.43) and 23.74 cell/mm³ (p=0.28) respectively. Measurements of the viral load increased in 8 (38%) patients during the disease with a mean increase of 64,688 HIV RNA copies/ml (4.96 log10) (p=0.03); and in 13 they decreased subsequently (45%) with a mean of -1,163 RNA copies/ml (3.06 log10) (p=0.99). Syphilis in the HIV+ patient was linked to significant increases in the viral load and to non-significant changes in the TCD4+ cell count during the disease.

Doenças sexualmente transmissíveis na AIDS: estudo com base no relato dos pacientes que se submeteram a exames de carga viral e CD4 no Hospital Universitário Antonio Pedro / Sexually transmitted diseases in AIDS: a study based on the reports of patients who underwent tests of viral load and CD4 at the University Hospital Antonio Pedro

O estudo das doenças sexualmente transmissiveis (DST) e a aplicação de medidas para o seu controle sãoi fundamentais na prevenção da AIDS, pois elas tem importância crucial na sua disseminação...Da mesma forma, oito pacientes continuaram a fazer uso de drogas após saberem-se infectados por HIV.

Candida spp en pacientes VIH/SIDA / Candida spp in patient VIH/SIDA

La candidiasis es la infección fúngica más frecuente en pacientes VIH seropositivos y con SIDA, siendo C. albicans la especie que predomina. En estos pacientes, la candidiasis se manifiesta más comúnmente como orofaringitis, con una incidencia que oscila entre 45 por ciento y 95 por ciento de todos los casos. En Venezuela son pocos los reportes que existen acerca de esta entidad en pacientes VIH/SIDA, por lo cual uno de los objetivos de esta investigación fue evaluar este aspecto. Se estudió una cohorte de 402 pacientes con VIH/SIDA con o sin orofaringitis y con o sin tratamiento antifúngico. Las especies de Candida más importantes aisladas fueron: C. albicans (80,41 por ciento), C. tropicalis (8,78 por ciento), C. glabrata (4,90 por ciento), C. dubliniensis (3,26 por ciento). Se evaluó la susceptibilidad de los diferentes aislados a las drogas antifúngicas con el método de la NCCLS y el método de los pozos de difusión. El 37,5 por ciento C. albicans provenientes de pacientes con tratamiento antimicótico fueron resistentes al fluconazol (resistencia secundaria) y 23,95 por ciento mostraron también resistencia al itraconazol (resistencia cruzada). Los ailados obtenidos se tipificaron a través de la reacción en cadena de la polimera (RAPD) y se estableció la que la misma representa una técnica útil para diferenciar Candida spp. En esta cohorte de pacientes evaluados, las características clínicas y demográficas son similares a las reportadas mundialmente, y que desde la introducción del TARGA, la frecuencia de las infecciones por Candida spp disminuyó. Hasta la presente fecha, en Venezuela no se había reportado el hallazgo de C. dubliniensis (3,26 por ciento).

Quantification of HIV-1 RNA load by one-tube-one-step RT PCR and real time PCR assay with TaqMan probe.

OBJECTIVES: To develop a less expensive assay to calculate HIV-1 viral load for use in resource-limited countries. MATERIAL AND METHOD: An In-house One-tube-one-step Viral Load Assay (IOVA) was developed by using real-time PCR-based with TaqMan probe. Primers and probe were designed from the conserved region of sequences from all HIV subtypes. A standard curve was generated from reference virus in various dilutions. IOVA was applied on 105 HIV-positive and 25 HIV-negative samples and compared with the results from ROCHE AMPLICLOR. RESULTS: IOVA measured HIV RNA in the samples ranging from 125 to 2 x 10(6) copies/mL. The coefficient of variation of intra- and inter-assay ranged from 0.68% to 7.89%. The sensitivity, specificity, positive and negative predictive values were 92%, 100%, 100% and 79.5% respectively. The parallel quantitative analysis showed high correlation (r=0.95) between IOVA and AMPLICOR. CONCLUSION: A new HIV-1 viral load assay was developed and validated. It was reliable and less expensive.

HIV-1 viral load: comparative evaluation of three commercially available assays in Argentina

Viral load (HIV-RNA copies per milliliter of plasma) has good correlation to prognosis considering progression to AIDS. The evaluation of commercial kits to measure viral load has become a need to find the most specific, sensitive and reproducible procedure to follow up HIV-infected patients. Hereby, a comparative analysis was done by using three different assays available in Argentina for quantitation of HIV-RNA in plasma. A plasma panel: 20 from HIV-1 infected individuals (9 asymptomatic and 11 symptomatic) and 9 from HIV-1 seronegative individuals was studied. Samples were run by Amplicor HIV-1 Monitor (Roche Diagnostic System, USA) Quantiplex HIV-1 RNA 2.0 Assay (Chiron Corporation, USA) and NASBA HIV-1 RNA QT (Organon Teknika, Holland). RNA was extracted from 0.2 ml of plasma for Amplicor, 0.1 ml and 1 ml of plasma for NASBA and, duplicates of 1 ml of plasma was centrifuged and pellet was used for bDNA assay no RNA extraction step. For a given specimen, a log difference of <0.5 between assays was considered as concordant result. All seronegative samples were bellow the detection limit of all assays (Amplicor 200 c/ml, NASBA 400 c/ml and Quantiplex (bDNA) 500 c/ml). Two samples from asymptomatic patients were not detectable by NASBA (Sensitivity: 90 per cent) Sensitivity was increased to 100 per cent by using 1 ml of plasma. All samples were detectable by the other assays (sensitivity: 100 per cent). For NASBA-bDNA, 74 per cent samples were concordant, 35 per cent for Amplicor-bDNA and 53 per cent for NASBA-Amplicor. By using 1 ml of plasma from asymptomatic patients, concordance was 65 per cent for NASBA-bDNA and 60 per cent for NASBA Amplicor. Comparing samples from asymptomatic patients, only 22 per cent was concordant in both cases. Reproducibility of NASBA was low (33 per cent, with differences lower than 0.5 Log) when 0.1 and 1 ml were used. Due to the levels of concordance of these results, it would be suggested to use always the same technique to follow up HIV-1 infection. The reproducibility of the assays should be tested by every laboratory and for every technician in charge of the assay in order to have confidence in the results specially to follow up HIV-infected patients or to monitor anti-viral therapies.