Kidney graft loss associated with JC polyomavirus nephropathy.

This is the first case of documented treatment failure of JCV nephritis, despite a reduction of immunosuppressive agents and the use of antiviral therapy. We consistently detected high levels of JCV viremia, which correlated with the progressive deterioration of the graft and caused the final loss of the kidney, suggesting that JCV plays an etiological role in the onset of severe nephropathy in kidney transplant patients.

Rapid and accurate quantification of different HCV genotypes by LightCycler Real Time PCR and direct sequencing of HCV amplicons.

Follow-up of chronically infected HCV patients is the primary clinical goal in therapy administration. In the absence of an HCV vaccine, the timely monitoring of HCV viral load combined with the information of the viral genotype could contribute to patient disease management. A LightCycler Real Time RT-PCR assay was developed and optimized allowing rapid and accurate quantification of HCV RNA over an extended dynamic range using a single human reference standard. A total of 5,096 plasma samples, collected over almost 5 years, were tested and HCV RNA was quantified in 2,435 samples with levels ranging from 5.7x10(1) to 2.52x10(9) IU/ml. The precision and reproducibility of the test are documented by various inter-assay parameters of the reference standard obtained in 409 RT-PCR runs. This Real Time RT-PCR protocol uses the LightCycler cDNA amplicons for direct sequence analysis and reduces the sequencing time to approximately 3 hours. Nearly all HCV genotypes were identified. Viral sequences showed a similarity level close to 100%, independently from the viral load, while the LightCycler melting temperature analysis did not correlate with HCV genotypes. All this makes the LightCycler Real Time RT-PCR protocol a suitable tool for the diagnosis and monitoring of HCV infections.

Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent.

Epigallocatechin-3-gallate (EGCG), one of the components of green tea, has been suggested to have antiviral activity. To determine the effects of EGCG on HIV infection, peripheral blood lymphocytes were incubated with either LAI/IIIB or Bal HIV strains and increasing concentrations of EGCG. EGCG strongly inhibited the replication of both virus strains as determined by reverse transcriptase and p24 assays on the cell supernatants.

[Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA]. / Marcadores virológicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV-2LTR y ARN de HIV.

Highly active antiretroviral therapy (HAART) induces a persistent reduction of the plasmatic viremia, contributing to decrease mortality and morbidity of infected people with human immunodeficiency virus (HIV). Thus, viral load (VL) is the reference method to evaluate therapy effectiveness. However, even in the presence of efficient HAART viral eradication was yet not achieved. In this study, we analyzed the presence of total HIV DNA (T-HIV DNA), non-integrated DNA with 2LTR (2LTR-HIV DNA) and HIV RNA in a group of 55 HIV-positive subjects from Rosario City, with different clinical stages, with and without HAART. All markers were evaluated by PCR assays optimized in our laboratory that included colorimetric detection in microplate. HIV RNA clinical sensitivity was compared with a reference test, bDNA, resulting in 74% and 64% respectively, with an 85% of agreement. Thus, our HIV RNA assay could be used to monitor patients under HAART and at risk of infection. The 2LTR-HIV DNA was 54% positive although it was absent in patients with high VL. This marker was considered a labile product therefore its presence was associated with recent infection. However, current evidences question its stability. Thus, its clinical significance should be reconsidered. The absence of 2LTR-HIV DNA in patients with detectable VL may relate to the heterogeneity of the sequence used for its detection. T-HIV DNA was present in 100% of the samples and could be a relevant remission marker when therapies that effectively eradicate the infection became available.

A fatal case of streptococcal and meningococcal meningitis in a 2-years-old child occurring as Waterhouse-Friderichsen Syndrome.

We report a fatal case of streptococcal and meningococcal meningitis in a previously healthy 2-year-old child, a simultaneous co-infection of both pathogens that is poorly reported in the reviewed literature. The lack of a clinical diagnosis in addition to the medico-legal aspects arising from possible professional liability for the emergency service doctor who had failed to recognize the child's symptoms led to a forensic autopsy within 48 h after the death. After external and internal examination, Waterhouse-Friderichsen Syndrome (WFS) was suspected. Consequently, cerebrospinal fluid, whole blood, nasal and pharyngeal swab and pleural liquid samples were selected and collected for microbiological studies. All tested samples resulted Neisseria meningitidis DNA and Streptococcus pneumoniae DNA positive. The NM genotyping Real-Time PCR resulted positive for NM serotype C. Microscopic histological study confirmed these findings. We underline that when a patient presents fever and petechiae (50-60% of patients), WFS must be considered, even when the patient has a non-toxic appearance. Due to its rapid progression and often devastating consequences, therapy should be started as soon as WFS is suspected. Emphasis should also be placed on the importance of public education programs and on broadening protection against meningitis through new vaccines. In such cases, from a forensic point of view, there is a strong need for a robust, multidisciplinary approach in order to reach the correct post-mortem diagnosis.

Decay of human immunodeficiency virus type 1 unintegrated DNA containing two long terminal repeats in infected individuals after 3 to 8 years of sustained control of viremia.

Covert human immunodeficiency virus (HIV) replication was ongoing during the first 3 years of aviremia in 22 patients, as determined by detection of DNA containing two long terminal repeats (2 LTR DNA). Although total HIV DNA was detected in 60 2 LTR DNA-negative samples, the absence of 2 LTR DNA in 90% of patients following 7 to 8 years of highly active antiretroviral therapy suggests suppression of cryptic viral replication.

Evaluation of an ultrasensitive p24 antigen assay as a potential alternative to human immunodeficiency virus type 1 RNA viral load assay in resource-limited settings.

An inexpensive enzyme-linked immunosorbent assay method for human immunodeficiency virus type 1 quantitation, ultrasensitive p24 antigen assay (Up24), was compared with RNA viral load assay (VL). Up24 had 100% sensitivity of detection at a viral load of >/=30,000, with sensitivity of 46.4% at a viral load of <30,000 (232 specimens from 65 seropositive subjects). The assay was highly reproducible, with excellent correlation between duplicates and among three laboratories.

Comparison of two human immunodeficiency virus (HIV) RNA surrogate assays to the standard HIV RNA assay.

Human immunodeficiency virus (HIV) RNA testing is the gold standard for monitoring antiretroviral therapy in HIV-infected patients. However, equipment and reagent costs preclude widespread use of the assay in resource-limited settings. The Perkin-Elmer Ultrasensitive p24 assay and the Cavidi Exavir Load assay both offer potentially simpler, less costly technologies for monitoring viral load. These assays were compared to the Roche Amplicor HIV-1 Monitor Test, v1.5, using panels of clinical samples (subtype B) from HIV-positive subjects and HIV-spiked samples (subtypes A, C, D, CRF_01AE, CRF_02AG, and F). The Ultrasensitive p24 assay detected 100% of the spiked samples with virus loads of >250,000 copies/ml and 61% of the clinical samples with virus loads of 219 to 288,850 copies/ml. Detection rates were improved substantially if an external lysis buffer was added to the procedure. The Cavidi assay detected 54 to 100% of spiked samples with virus loads >10,000 copies/ml and 68% of the clinical samples. These detection rates were also greatly improved with a newly implemented version of this kit. Coefficients of variation demonstrate good reproducibility for each of these kits. The results from the Cavidi v1.0, Cavidi v2.0, and Perkin-Elmer, and the Perkin-Elmer Plus external buffers all correlated well with the results from the Roche Monitor Test (r = 0.83 to 0.96, r = 0.84 to 0.99, r = 0.58 to 0.67, and r = 0.59 to 0.95, respectively). Thus, the use of these two assays for monitoring patients, together with less-frequent confirmation testing, offers a feasible alternative to frequent HIV RNA testing in resource-limited settings.

Marcadores virologicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV-2LTR y ARN de HIV / Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA

La terapia antirretrovial de alta eficacia (TAAE) induce una reducción marcada y persisatente de la viremia plasmática, contribuvendo a disminuir la mortalidad de los pacientes HIV-positivos. Así, la carga viral (CV) es el método de referencia para evaluar la eficacia terapéutica. Sin embargo, aun en presencia de una TAAE eficiente no se ha logrado la erradicación viral. En este estudio analizamos la presencia del ADN total de HIV (ADN HIV-T), del ADN no integrado con 2LTR (ADN HIV-2LTR) y del ARN de HIV, en un grupo de 55 pacientes HIV-positivos en distintos estadios clínicos, con y sin TAAE, mediante ensayos de PCR con revelado colorimétrico en microplaca, optimizados en nuestro laboratorio. La sensibilidad clínica de ARN del HIV fue evaluada con el bDNA, resultando del 74% y del 64%, respectivamente, con una concordancia del 85%. Este ensayo podría utilizado en el seguimiento de pacientes bajo TAAE. EI ADN HIV-2LTR resultó positivo en el 54% aunque estuvo ausente en pacientes con elevada CV. Este marcador se considereba un producto lábil y su presencia se asociaba a infección reciente. Sin embargo, actuales evidencias ponen en discusión su estabilidad por lo que su significado clínico debe ser reconsiderado. La ausencia del ADN HIV-2LTR en pacientes con CV detectable puede relacionarse con la heterogeneidade de la secuencia utilizada para su detección. EI ADN HIV-T estuvo presente en el 100% de las muestras y resultaría relevante como marcador de remisión cuando se dispongan de terapias que efectivamente erradiquen la infección.