S. Delia

Multiplex polymerase chain reaction for the simultaneous detection and typing of polyomavirus JC, BK and SV40 DNA in clinical samples.

A novel multiplex nested PCR (nPCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK and SV40 in a single tube. In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different size for each related virus. The thermocycling parameters and concentration of each reaction component were optimised systematically to achieve optimal specificity and sensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was examined from AIDS patients with clinical and neuroradiological evidence of progressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by haemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease.

Risk of failure in patients with 215 HIV-1 revertants starting their first thymidine analog-containing highly active antiretroviral therapy.

Objective: To investigate the impact of 215 HIV-1 revertants on the risk of virological failure of the first thymidine analog-containing highly active antiretroviral therapy (HAART). Design: The study included 491 HIV-1 subjects of the Italian Cohort Naive for Antiretrovirals, 405 of whom received a genotypic assay before therapy and had a virological follow-up. Methods: Pre-treatment genotypic resistance was assessed by sequencing of the whole protease (PR) and reverse transcriptase (RT) region. Results: Three (3.2%) and 13 (3.3%) individuals with recent (n = 95) and chronic (n = 396) HIV-1 infection carried an HIV-1 strain with 215 revertants (215D/C/E/A/V), respectively. In contrast, nucleoside associated mutations were higher in the former (15.8%) compared with the latter group (6.8%) (P = 0.005). A multivariable regression model, considering pre-HAART viral load levels, use of saquinavir-hard gel as the only PI, use of zidovudine, number of other RT and PR mutations, indicated that patients carrying 215 revertants had an increased risk of virological failure compared with those not carrying such mutants (adjusted relative hazard = 2.97 95% confidence interval, 1.11–7.94, P = 0.03). Among patients with 215 revertants, who experienced virological failure, four out of seven showed the emergence of the 215Y resistant mutation. The probability of 215Y occurrence was different between patients carrying 215 revertants compared with those who did not carried these mutants (P = 0.006). Conclusions: HIV-1 215 revertants with an increased ability for selecting 215Y mutation are associated with a higher risk of virological failure and may lead to the appearance of virus carrying 215Y/F mutation in vivo. These findings suggest that 215 revertant viruses may compromise the efficacy of the first thymidine analog-containing regimen.

High Dose L-Carnitine Improves Immunologic and Metabolic Parameters in Aids Patients

Several reports indicate that systemic carnitine deficiency could occur in acquired immunodeficiency disease syndrome (AIDS), and that primary and secondary carnitine deficiency leads to critical metabolic dysfunctions. L-carnitine supplementation to peripheral blood mononuclear cells (PBMCs) of AIDS patients resulted in significant enhancement of the phytohemagglutinin (PHA)-driven proliferative response. High dose L-carnitine administration (6 gr per day for two weeks) to AIDS patients treated with zidovudine also led to increased PBMCs proliferation and reduced blood levels of triglycerides. In addition, a reduction of beta 2-microglobulin serum levels as well as circulating tumor necrosis factor (TNF)-alpha, mostly in patients exhibiting highly elevated levels, were found at the end of the treatment period. Our data suggest that in vivo L-carnitine could prove useful in ameliorating both the immune response and lipid metabolism in patients with AIDS, irrespective of initial serum carnitines levels. The mechanism(s) accounting for the observed results are currently not clear. Further studies are needed to confirm the hypothesis that L-carnitine affects the expression of HIV-induced cytokine.

Tumour necrosis factor (TNF-alpha) and neurological disorders in HIV infection.

Tumour necrosis factor (TNF-alpha) concentrations were determined in the CSF from 42 HIV-infected patients, with or without CNS involvement. In addition, 14 subjects with various neurological disorders but without HIV antibodies were included as controls. Raised CSF concentrations of TNF-alpha (greater than 40 ng/l) were detected both in patients with AIDS dementia complex (ADC) (6/9) and with CNS opportunistic infections (10/19) and, less commonly, in HIV infected subjects without CNS diseases (2/14) and in anti-HIV negative controls (1/14). The highest CSF concentrations of TNF-alpha (greater than 100 ng/l), however, were found in seven out of eight patients with cryptococcal meningitis. Although a role for TNF-alpha in demyelinating lesions associated with ADC has been suggested, our results indicate that a clear elevation of TNF-alpha in the CSF from HIV positive patients mostly occurs in acute inflammatory disorders, such as cryptococcal meningitis.

Cerebrospinal fluid myelin basic protein as predictive marker of demyelination in AIDS dementia complex

Myelin basic protein (MBP) was measured in cerebrospinal fluid (CSF) of patients with acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) in order to investigate the degree of white matter destruction. Results show that increased CSF levels of MBP were detected in all patients with severe ADC (10/10) and, less often, in subjects with mild (2/7) or moderate dementia (7/16). No evidence of MBP-elevated concentration was observed in 14 human immunodeficiency virus (HIV)-seropositive subjects without neurological disorders and in nine HIV-seronegative controls. Our findings suggest that the measurement of CSF MBP concentration may represent a predictive marker of myelin injury and neurologic damage during the course of ADC.

Identical Rearranged Forms of JC Polyomavirus Transcriptional Control Region in Plasma and Cerebrospinal Fluid of Acquired Immunodeficiency Syndrome Patients with Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by the human polyomavirus JC (JCV). JCV has a hypervariable noncoding transcriptional control region (TCR) that spans the origin of replication of the JCV genome through to the first ATG start codon for late gene transcription. The archetype form of TCR is frequently found in the urine and kidneys of healthy and immunocompromised subjects. However the rearranged forms, whose prototype is Mad-1, possibly generated by deletion and duplication of segments of the archetype sequence, are found in the brain and cerebrospinal fluid (CSF) of PML patients. In this study the authors compared JCVTCR detected in paired CSF, plasma, and urine samples of 11 acquired immunodeficiency syndrome (AIDS) patients affected by PML to try to determine where the rearranged JCV TCRs are selected. In one patient, it was also possible to amplify and sequence the TCR in the brain and lymphocytes. Moreover, in 5/11 patients, the CSF, plasma, and urine samples corresponding to 2 months after PML development were available; and in another patient, it was possible to sequence the TCR in plasma and lymphocytes sampled 8 months before the onset of PML. The presence of the same TCR sequences in all the CSF and plasma samples taken from individual patients could strengthen the hypothesis that the blood is a compartment where JCV may replicate and undergo rearrangement of the TCR. This further supports the hypothesis that JCV reaches the brain by a hematogenous route and indicates that the JCV TCR sequences detected in plasma could be used as an early marker of JCV pathogenicity before the clinical appearance of PML in immunocompromised patients.

Detection of cerebrospinal fluid antibodies against myelin basic protein in patients with AIDS dementia complex

We evaluated cerebrospinal fluid (CSF) antibody levels against a lipid-free, denatured form of myelin basic protein (LF-MBP) in 11 patients with AIDS dementia complex (ADC) by using an enzyme-linked immunosorbent assay (ELISA). In 9 out of 11 patients, anti-LF-MBP antibody levels were significantly higher than those observed both in 15 human immunodeficiency virus (HIV)-infected patients without neurological disorders and in 9 anti-HIV-negative subjects affected by other neurological diseases. Furthermore, we followed up anti-MBP levels in 5 out of the 11 ADC patients and detected a strict relationship with the encephalopathy progression. At the same time, with the aim to detect early demyelinating events we investigated CSF antibody levels against a lipid-bound, native-like form of MBP (LB-MBP). Results did not show any significant difference between LF-MBP and LB-MBP in terms of antibody reactivity. The detection of anti-MBP antibodies in CSF may provide the opportunity to assess a diagnostic tool for discovering demyelinating lesions in ADC patients.

Acute Cerebellitis Caused by Herpes Simplex Virus Type 1

Cerebellar disorders due to herpes simplex virus (HSV) infection are rare and always associated with herpes simplex encephalitis. We report 2 cases of severe primary acute cerebellitis caused by HSV type 1 that were identified by nested polymerase chain reaction performed on cerebrospinal fluid samples.

Cytomegalovirus encephalitis in two patients with AIDS receiving ganciclovir for cytomegalovirus retinitis

Cytomegalovirus (CMV) encephalitis has been reported with increasing frequency in patients with AIDS. Nevertheless, the management of CMV-related encephalitis appears to be problematic and data in the literature on the clinical efficacy of ganciclovir therapy is sparse and controversial. We describe two patients with AIDS who developed CMV encephalitis while receiving ganciclovir maintenance therapy for CMV retinitis. Moreover, there was no improvement in neurological status or virological and radiological response during a further induction course of ganciclovir. These observations suggest that the currently recommended therapeutic protocols with ganciclovir are not effective in the prevention and treatment of CMV encephalitis in patients with AIDS.

Defective production of interferon-γ and tumour necrosis factor-α by AIDS mononuclear cells after in vitro exposure to Rhodococcus equi

The production of interferon-γ and tumour necrosis factor-α was evaluated in the peripheral blood mononuclear cells (PBMCs) from healthy donors and AIDS patients after Rhodococcus equi infection in vitro. PBMCs from healthy donors secreted elevated levels of IFN-γ and TNF-α when challenged in vitro with killed R. equi, whereas the release of both cytokines was impaired in supernatant cultures from AIDS patients. We conclude that the failure of IFN-γ generation in AIDS patients in response to R. equi is not antigen-specific but it may reflect the global impairment of T-cell function. In such patients, however, the infection with R. equi, a facultative intracellular pathogen which survives and replicates within macrophages, may be responsible for the impairment in the TNF-α release, possibly enhancing the HIV-induced macrophage dysftmction.