Discrepant results in the interpretation of HIV-1 drug-resistance genotypic data among widely used algorithms.

OBJECTIVES: The aim of this study was to assess the concordance on the interpretation of HIV-1 drug-resistance genotypic data by three widely used algorithms: Stanford University Database (SU), TruGene (Visible Genetics, Canada) (VG) and VirtualPhenotype (Virco, Belgium) (VP). METHODS: Genotypic data from 293 HIV-1-infected individuals with treatment failure was interpreted for 14 antiretroviral drugs by the three algorithms. RESULTS: Complete concordant results among the three systems for all the drugs studied were found in 40/293 (13.7%) samples. Low concordance in the interpretation was observed for most nucleoside reverse transcriptase inhibitors (NRTIs), while results agreed highly for all nonnucleoside reverse transcriptase inhibitors (NNRTIs) and most protease inhibitors (PIs). In pair-wise comparisons, discordant interpretations between SU and VP were found in over 50% of the samples for didanosine, zalcitabine, stavudine and abacavir, and the level of disagreement between VG and VP exceeded 40% for the same drugs. Major discrepancies (high-level resistance interpretation by one algorithm with sensitive interpretation by another) were observed between VG and VP in over 10% of the cases for didanosine, zalcitabine, stavudine and abacavir. On the other hand, the three algorithms had concordant results for lamivudine in over 90% of the cases. CONCLUSIONS: This work demonstrates the great level of discordance in the interpretation of genotyping results among algorithms, clearly showing the necessity for clinical validation. Moreover, these results suggest that a joint effort from the scientific community as well as national and international HIV societies is needed to achieve a consensus for the interpretation of genotypic data.

Resistance profiles to antiretroviral drugs in HIV-1 drug-naive patients in Argentina.

The drug resistance profile of treatment-naive HIV-infected individuals living in Buenos Aires, Argentina, was studied. Samples taken from 94 drug-naive individuals with established HIV infection and 13 patients with primary HIV infection were assessed by nucleotide sequencing and LIPA. The prevalence of drug-associated primary mutations in individuals with established infection was very low. In the viral protease region, 1/86 (1.2%) individuals carried the D30N mutation, whereas 1/85 (1.2%) had the M41L mutation in the reverse transcriptase (RT) region. Secondary mutations in both the protease and RT regions were found in almost 90% of the individuals. In individuals with primary infection, primary mutations were detected in 2/13 (15.4%) patients, one of them carrying M461 mutation in the protease while the other patient had a mutation at codon 184 of the RT. In accordance with current drug resistance testing guidelines, the results of this study suggest that susceptibility tests need not be performed at this time prior to initiation of antiretroviral therapy in HIV-1-infected people in Argentina. However, the public health implications of this subject warrant follow-up studies that will examine a larger number of drug-naive patients, not only in Buenos Aires but also in other major Argentinian cities and in rural areas.

Cytotoxic and DNA interaction activities of extracts from medicinal plants used in Argentina.

Eight crude extracts from seven Argentine plants with cancer-related ethnobotanical uses have been subjected to a bioscreening study to detect cytotoxic activity. The plants studied were: Aristolochia triangularis, Baccharis grisebachii, Bolax gummifera, Eupatorium hecatanthum, Erythrina crista-galli, Pterocaulon polystachium and Salpichroa origanifolia. Crown gall tumour inhibition, DNA interaction and cytotoxicity towards KB cells were assayed using the potato disc, the DNA-methyl green (DNA-MG) and the KB cells cytotoxicity bioassays respectively. The results obtained indicate that A. triangularis (ED50=47 microg/ml), B. gummifera (ED50=32 microg/ml) and E. hecatanthum (ED50=35 microg/ml) contained cytotoxic compounds against KB cells. All of the plants studied inhibited the growth of crown gall tumours, showing correlation between the experimental data and the uses reported for these plants. Moreover, the results obtained for the extracts of E. hecatanthum and P. polystachium indicate the presence of compounds that interact with DNA (48 and 22% of absorbance decrease, respectively). The results obtained suggest that cytotoxicity could play an important role in the activities claimed for the plants under study.

Regression of AIDS-related Kaposi's sarcoma following combined antiretroviral treatment.

The aim of the study was to assess regression of Kaposi's sarcoma (KS) in AIDS patients in Argentina. Eighteen male AIDS patients with human immunodeficiency virus (HIV)-associated Kaposi's sarcoma at different clinical stages received KS specific treatment and/or anti-retroviral therapy. Triple anti-retroviral therapy was given to most of the patients with the exception of four who received zidovudine (ZDV) in combination with another nucleoside analogue but no protease inhibitors. Plasma viral load and CD4+ T lymphocyte number were measured in two blood samples (before and after treatment). Complete remission was found in all patients (five) at KS stage I, three out of eight patients at stage II but in none at stages III and IV. Two out of three patients at KS stage IV did not respond to treatments at all. Three patients at KS stages I and II showed complete remission of sarcoma with only anti-retroviral therapy suggesting that anti-retroviral therapy and non-KS specific chemotherapy can successfully control KS.

Regression of AIDS-related Kaposis sarcoma following combined antiretroviral treatment.

The aim of the study was to assess regression of Kaposis sarcoma (KS) in AIDS patients in Argentina. Eighteen male AIDS patients with human immunodeficiency virus (HIV)-associated Kaposis sarcoma at different clinical stages received KS specific treatment and/or anti-retroviral therapy. Triple anti-retroviral therapy was given to most of the patients with the exception of four who received zidovudine (ZDV) in combination with another nucleoside analogue but no protease inhibitors. Plasma viral load and CD4+ T lymphocyte number were measured in two blood samples (before and after treatment). Complete remission was found in all patients (five) at KS stage I, three out of eight patients at stage II but in none at stages III and IV. Two out of three patients at KS stage IV did not respond to treatments at all. Three patients at KS stages I and II showed complete remission of sarcoma with only anti-retroviral therapy suggesting that anti-retroviral therapy and non-KS specific chemotherapy can successfully control KS.

[Evaluation of indirect immunofluorescence as a supplementary test for the diagnosis of HIV-1 infection]. / Evaluación de la inmunofluorescencia indirecta como prueba suplementaria para el diagnóstico de la infección por HIV-1.

In order to be used as an alternative or complementary test to confirm HIV-1 infection, the efficiency of indirect immunofluorescence assay (IFA) was compared with Western blot (WB) in 362 samples from persons with high and low risk behaviour. A panel of sera with 220 WB positive, 122 WB negative and 20 WB indeterminate sera were tested by an "in house" IFA. The sensitivity of IFA was found to be 98.63% and the specificity 98.36%. Therefore, IFA appeared to be an efficient alternative method to WB, since the cost of testing by IFA is less than 10% of WB testing. We observed a direct relationship between WB protein reactivity and IFA results. In 15 samples with coincident indeterminate results for WB and IFA, antibody reactivity to p24 and gp160 presented the highest frequency. On the other hand, antibodies to viral glycoproteins were always present in IFA weak positive samples, showing their high predictive value.

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 for 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%) Sensitivity was increased to 100% by using 1 ml of plasma. All samples were detectable by the other assays (sensitivity: 100%). For NASBA-bDNA, 74% samples were concordant, 35% for Amplicor-bDNA and 53% for NASBA-Amplicor. By using 1 ml of plasma from asymptomatic patients, concordance was 65% for NASBA-bDNA and 60% for NASBA Amplicor. Comparing samples from asymptomatic patients, only 22% was concordant in both cases. Reproducibility of NASBA was low (33% 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.

[Detection of the viral load and its use as a virological marker in the follow-up of HIV-1-positive patients]. / Detección de la carga viral y su uso como marcador virológico en el seguimiento de pacientes HIV-1 positivos.

The evaluation of viral load as virological marker and its clinical and immunological correlation are presented. The first viral load studies were performed during 1996 at the National Reference Center for AIDS in Argentina in HIV-1 positive patients derived from different Hospitals in Buenos Aires. The study included 216 HIV-1 positive patients, 49 females and 167 males. Plasma was used for evaluating viral load and a second sample was obtained in 25 of the 216 patients for their monitoring. Viral load was performed using bDNA technique (Quantiplex HIV RNA assay 2.0, Chiron Corporation, USA). Other parameters such as CD4 count determined by flow cytometry and clinical stages according to CDC classification were obtained in order to correlate clinical and immunological status of the patients. When CD4 count was compared with viral load, the results showed a trend of viral RNA increase in plasma along with a decrease in CD4+ lymphocytes. This trend was also observed to correlate with the progression to AIDS disease. In all groups of patients, considering either CD4 counts or clinical status, ranges of viral load values were broad. Thus, as shown by percentiles 25 and 75, patients with CD4 counts < 200/ml, presented viral load values between 18,395 c/ml to 215,425 c/ml and patients with > 200/ml viral RNA showed values from < 10,000 to 35,180 c/ml. Patients with CDC's A and B stages presented values from < 10,000 to 45,160 c/ml and 87,000 c/ml respectively, while patients classified as C had 10,582 to 215,000 c/ml. Results of two consecutive samples in the 25 patients showed the usefulness of this technique for monitoring antiretroviral therapy. Nevertheless, despite the tendency of viral load to increase along with the progression of the disease, the broad range of values suggested the importance of using both virological and immunological parameters for the management of HIV infected patients.

Evaluación de la inmunofluorescencia indirecta como prueba suplementaria para el diagnóstico de la infección por HIV-1 / Evaluation of indirect inmunofluorescence as a confirmatory test for the diagnosis of HIV-1 infection

Se comparó la eficiencia diagnóstica de la inmunofluorescencia indirecta (IFI) como método confirmatorio de la infección por HIV-1 en muestras de suero de 362 personas con conductas de alto y bajo riesgo. El panel compuesto por 220 positivos, 122 negativos y 20 indeterminados por Western blot (WB) fue ensayado por una técnica de IFI desarrollada en nuestro laboratorio. La sensibilidad calculada fue 98,63 por ciento y la espicificidad 98,36 por ciento, indicando que la IFI es un método alternativo para la confirmación de la presencia de anticuerpos contra el HIV-1. Dado que su costo es menor que el 10 por ciento comparado con el del WB, se justifica su introducción en el algoritmo de diagnóstico serológico de HIV-1. Se observó también una relación directa entre la reactividad de las proteínas del WB y los resultados de IFI. En 15 muestras con resultado indeterminado por WB e inespecífico por IFI, las bandas más observadas fueron la p24 seguida de la gp160; por otro lado los anticuerpos contra las glicoproteínas virales son los que presentan mayor frecuencia en las muestras positivas débiles, demostrando su alto valor predictivo

Evaluación de la inmunofluorescencia indirecta como prueba suplementaria para el diagnóstico de la infección por HIV-1 / Evaluation of indirect inmunofluorescence as a confirmatory test for the diagnosis of HIV-1 infection

Se comparó la eficiencia diagnóstica de la inmunofluorescencia indirecta (IFI) como método confirmatorio de la infección por HIV-1 en muestras de suero de 362 personas con conductas de alto y bajo riesgo. El panel compuesto por 220 positivos, 122 negativos y 20 indeterminados por Western blot (WB) fue ensayado por una técnica de IFI desarrollada en nuestro laboratorio. La sensibilidad calculada fue 98,63 por ciento y la espicificidad 98,36 por ciento, indicando que la IFI es un método alternativo para la confirmación de la presencia de anticuerpos contra el HIV-1. Dado que su costo es menor que el 10 por ciento comparado con el del WB, se justifica su introducción en el algoritmo de diagnóstico serológico de HIV-1. Se observó también una relación directa entre la reactividad de las proteínas del WB y los resultados de IFI. En 15 muestras con resultado indeterminado por WB e inespecífico por IFI, las bandas más observadas fueron la p24 seguida de la gp160; por otro lado los anticuerpos contra las glicoproteínas virales son los que presentan mayor frecuencia en las muestras positivas débiles, demostrando su alto valor predictivo (AU)

Detección de la carga viral y su uso como marcador virológico en el seguimiento de pacientes HIV-1 positivos / Viral load detection and its role as virological marker for the monitoring of HIV-1 positive patients

Se realizaron los primeros estudios de carga viral en pacientes HIV-1 positivos provenientes de diferentes instituciones asistenciales de la Ciudad de Buenos Aires. Se evaluó la carga viral como marcador virológico y su correlación con la clínica y el recuento de los linfocitos CD4+ para 216 pacientes HIV-1 positivos. La técnica utilizada fue bDNA (Quantiplex HIV RNA 2.0 assay, Chiron Corporation). Se observó una tendencia al aumento de la carga viral en los pacientes con menor cantidad de linfocitos CD4+ y en los estadíos clínicos con sintomatología. En pacientes que no recibieron ninguna terapia antirretroviral se encontraron valores desde < 10000 copias de ARN viral/ml de plasma hasta 48995 c/ml. En aquéllos que recibieron terapia antirretroviral se observó mayor variación en los valores de la carga viral como lo mostró un rango de < 10000 c/ml hasta 96605 c/ml. Se obtuvieron muestras consecutivas en 25 pacientes y se observaron diferencias entre ambas muestras que permitieron corroborar la utilidad de la técnica en el seguimiento de los pacientes infectados con HIV

Detección de la carga viral y su uso como marcador virológico en el seguimiento de pacientes HIV-1 positivos / Viral load detection and its role as virological marker for the monitoring of HIV-1 positive patients

Se realizaron los primeros estudios de carga viral en pacientes HIV-1 positivos provenientes de diferentes instituciones asistenciales de la Ciudad de Buenos Aires. Se evaluó la carga viral como marcador virológico y su correlación con la clínica y el recuento de los linfocitos CD4+ para 216 pacientes HIV-1 positivos. La técnica utilizada fue bDNA (Quantiplex HIV RNA 2.0 assay, Chiron Corporation). Se observó una tendencia al aumento de la carga viral en los pacientes con menor cantidad de linfocitos CD4+ y en los estadíos clínicos con sintomatología. En pacientes que no recibieron ninguna terapia antirretroviral se encontraron valores desde < 10000 copias de ARN viral/ml de plasma hasta 48995 c/ml. En aquéllos que recibieron terapia antirretroviral se observó mayor variación en los valores de la carga viral como lo mostró un rango de < 10000 c/ml hasta 96605 c/ml. Se obtuvieron muestras consecutivas en 25 pacientes y se observaron diferencias entre ambas muestras que permitieron corroborar la utilidad de la técnica en el seguimiento de los pacientes infectados con HIV (AU)

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.

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. (AU)

Retrospective study of children born to HIV-1-infected mothers in a pediatric hospital in Argentina.

The aim of this retrospective study, which included 103 children born to human immunodeficiency virus type 1 (HIV-1)- infected mothers, is to initiate a database on HIV-infected children, which has to date been unavailable in Argentina. All HIV-1 seropositive children admitted to the Pedro de Elizalde Children's Hospital in Buenos Aires from March 1, 1987, to December 31, 1992, were enrolled in this study. The number of patients enrolled dramatically increased each year during the period of study. Of the 60 infected children, 22 (36.66%) have died with a clinical diagnosis of HIV-1 infection; in 10 of those children HIV infection was also confirmed by P24 antigenemia and/or polymerase chain reaction (PCR): 20 qualified for the Centers for Disease Control and Prevention (CDC) P2D class (P2D1 = 7, P2D2 = 10, P2D3 = 3), 1 for P2C, and 1 for P2A, whose cause of death was pneumonia. The mean age of death was 14.8 months, 18 (82%) died before 18 months. When immunoglobulin G (IgG), IgM, and IgA levels were determined according to age and clinical status, significant differences (P < 0.005) were observed when both asymptomatic and symptomatic infected children (P1, P2) were compared with noninfected children (P3). A significant difference was also obtained between those children who qualified for P2 classification prior to 12 months of age who died early (at or prior to 25 months) and those who reached stage P2 after 12 months of age and have survived to date (X2 = 24.73, p < 0.0001; RR = 5.83, 2.52 < RR < 13.49).