Marinunzia Catucci

Antiretroviral Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Protease from Paired Cerebrospinal Fluid and Plasma Samples

Twenty-four adults infected with human immunodeficiency virus type 1 (HIV-1) with central nervous system symptoms were studied for antiretroviral resistance mutations in HIV-1 RNA obtained from paired cerebrospinal fluid (CSF) and plasma samples. Paired sequences were obtained from 21 and 13 patients for reverse transcriptase (RT) and for protease, respectively. Mutations conferring resistance to the RT inhibitors zidovudine, lamivudine, or nevirapine were detected in 14 patients, including 11 pretreated and 3 drug-naive subjects. The mutation patterns in the 2 compartments were different in most patients. Genotypic resistance to protease inhibitors was detected in both plasma and CSF from 1 patient treated with multiple protease inhibitors. However, accessory protease inhibitor resistance mutations at polymorphic sites were different in plasma and CSF in several patients. Partially independent evolution of viral quasispecies occurs in plasma and CSF, raising the possibility that compartmentalization of drug resistance may affect response to antiretroviral treatment.

Evaluation of the presence of 2-LTR HIV-1 unintegrated DNA as a simple molecular predictor of disease progression.

In a preliminary cross‐sectional analysis of 109 human immunodeficiency virus type 1 (HIV‐1)‐infected subjects the presence of 2‐long terminal repeat (LTR) unintegrated circular HIV‐1 DNA in peripheral blood mononuclear cells (PBMC) was found to be associated with both symptomatic infection (P = 0.0037) and low CD4 counts (P = 0.0004). To investigate the prognostic significance of the presence of 2‐LTR HIV‐1 DNA, a subset of 23 2‐LTR‐negative and 25 2‐LTR‐positive asymptomatic individuals were followed up for 12–24 months. The two groups did not differ in terms of baseline CD4 counts, zidovudine (ZDV) therapy, and duration of HIV‐1 infection. Longitudinal analysis of CD4 values did not indicate a significantly different CD4 outcome between the two groups. However, when only ZDV‐treated subjects were considered, a significant (P = 0.042) decrease in CD4 counts was found at month 24 with respect to baseline in 2‐LTR‐positive (n = 12) but not in 2‐LTR‐negative (n = 11) patients. Moreover, when >40% CD4 loss from baseline and/or development of CDC stage B or C symptoms were considered as indicators of disease progression, there was a significantly higher number of events in the whole 2‐LTR‐positive group than in the whole 2‐LTR‐negative group (P = 0.0197 at month 12, P = 0.0299 at month 18, P = 0.0373 at month 24). Thus, the presence of 2‐LTR‐HIV‐1 DNA in PBMC merits further investigation as a simple, qualitative, molecular predictor of disease progression and decreased response to antiretroviral therapy. J. Med. Virol. 52:20–25, 1997.

Antiretroviral therapy with protease inhibitors in human immunodeficiency virus type 1– and human herpesvirus 8–coinfected patients

Human herpesvirus 8 (HHV‐8) is believed to play a role in the pathogenesis of Kaposis sarcoma (KS) and possibly in other proliferative disorders often associated with human immunodeficiency virus type 1 (HIV‐1) infection. Recent case reports have indicated resolution of KS and clearance of HHV‐8 DNA from peripheral blood mononuclear cells (PBMC) in HIV‐1–infected subjects following highly effective antiretroviral therapy, including HIV‐1 protease inhibitors (PI), suggesting a possible activity for these compounds on HHV‐8 replication. In the present study, the time course of PBMC HHV‐8 DNA levels, plasma HIV‐1 RNA load, and CD4+ T‐cell counts were followed up in six coinfected subjects (four with and two without KS) under antiretroviral therapy with PI. A specific anti–HHV‐8 role for PI was not consistently found, since fluctuation of HHV‐8 viral load over time appeared to be independent of treatment. Nevertheless, our data support the hypothesis that KS patients may significantly benefit from PI therapy as an indirect consequence of partial restoration of immune functions following effective anti–HIV‐1 combination therapy. J. Med. Virol. 57:140–144, 1999.

Development and significance of the HIV-1 reverse transcriptase M184V mutation during combination therapy with lamivudine, zidovudine, and protease inhibitors.

To analyze the emergence and role of the lamivudine (3TC)-selected HIV-1 reverse transcriptase (RT) M184V mutation under triple therapy, we performed a retrospective study of 40 nucleoside RT inhibitor-pretreated and 16 drug-naive patients who were switched to combined treatment with zidovudine (ZDV) plus 3TC plus a protease inhibitor (PI). Plasma viral load and pol genotype were analyzed at baseline and after 24 and 48 weeks of combination therapy. Emergence of the M184V RT mutation at week 48 was detected in 3 of 16 (18.7%) initially drug-naive subjects as opposed to 21 of 40 (52.5%) ZDV-pretreated patients. Multivariate logistic analysis detected HIV-1 RNA load at week 24 as the best predictor of subsequent selection of the M184V mutant (p = .0121). Among ZDV-resistant study subjects at week 24 (n = 17), those with mutant RT M184V codon had a more favorable HIV-1 RNA slope than those with wild-type RT 184M codon (p = .0551). This trend was observed, although in a less evident manner, even in pretreated ZDV-sensitive patients. These findings suggest that development of the 3TC-resistance M184V mutation under triple therapy with 3TC, ZDV, and a PI may have unexpected beneficial effects in vivo in addition to those associated with resensitization of ZDV-resistant virus to ZDV.

Detection of human herpesviruses 6 and 7 in heart transplant recipients by a multiplex polymerase chain reaction method

In order to evaluate the possible reactivation of human herpesviruses 6 (HHV-6) and 7 (HHV-7) after heart transplantation, buffy-coat and plasma specimens from 21 transplant patients and 56 healthy blood donors were examined for HHV-6 and HHV-7 DNA by polymerase chain reaction. Human herpesvirus 6 and HHV-7 infection or reactivation has been suggested to play a role in cytomegalovirus disease progression in renal transplant recipients. In the present study, however, no significant difference in the prevalence of HHV-6 and HHV-7 was found between the immunosuppressed and the healthy population; moreover, no viral reactivation was found in the heart transplant recipients.

Long-read direct infrared sequencing of crude PCR products for prediction of resistance to HIV-1 reverse transcriptase and protease inhibitors

Patients infected with human irnmunodeficiency virus type 1 (H1V-1) are being treated with a number of different combinations of antiretroviral compounds that target the essential viral enzymes reverse transcriptase and protease. Different sets of HIV-1 mutations that confer drug resistance have been well defined; they allow reasonabe prediction of the drug sensitivity pattern from analysis of the HIV-1 genotype in vivo. Since periodical monitoring of genotypic resistance is expected to improve clinical management in a large number of infected patients, practical and cost-effective methods are highly desirable to set at least medium-scale sequencing in clinical diagnostic settings. We present a complete protocol for direct sequencing of HIV-1 reverse transcriptase and protease-coding regions. Features making the system amenable to routine clinical use include:1.Highly robust presequencing steps (plasma RNA extraction, reverse transcription, and nested PCR);2.Direct use of the crude unpurified PCR product as the sequencing template; and3.Use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.

Analysis of the HIV-1 nef gene in five intravenous drug users with long-term nonprogressive HIV-1 infection in Italy.

Great variability in the course of human immunodeficiency virus type 1 (HIV‐1) infection results from a complex interplay between host and virus factors. Some of the patients with prolonged nonprogressive infection have been reported to harbor virus variants with gross deletions in the accessory nef gene that has been implicated in in vivo pathogenicity in simian and mouse models. To investigate the role of nef‐deleted HIV‐1 in long‐term nonprogressor (LTNP) drug addicts in Italy the nef sequence from proviral DNA was analyzed from five LTNPs and five rapid progressor controls. Only small (2–12 amino acids) in‐frame deletions and insertions were detected in the N‐terminal polymorphic and variable regions obtained from three LTNPs and one rapid progressor. There was no evidence of premature termination of the Nef protein and all of the identified functional motifs were well conserved in both groups. Phylogenetic analysis showed interdigitation of nef sequences obtained from LTNPs and rapid progressors. The nef sequence of one LTNP, however, diverged significantly from those of the other patients. Availability of two additional blood DNA samples obtained previously from this subject allowed to detect evolution of nef at 14–17 years of HIV‐1 infection, including progressive deletions. Although alterations of nef may be relatively frequent and continue to evolve in LTNPs, this study of a small number of patients does not indicate that gross deletions or loss of functional motifs play a major role in delaying or halting disease progression in infected drug abusers in Italy. J. Med. Virol. 60:294–299, 2000.

Evaluation of Cell-Free and Cell-Associated Peripheral Blood Human Immunodeficiency Virus Type 1 RNA Response to Antiretroviral Therapy

Plasma human immunodeficiency virus (HIV) type 1 RNA load is the reference marker for response to antiretroviral therapy. To compare peripheral blood mononuclear cell (PBMC)-associated and plasma HIV-1 RNA response to treatment, HIV-1 RNA was quantified by reverse transcription-competitive polymerase chain reaction in 20 patients at 0, 12, and 24 weeks following addition of saquinavir to their treatment regimens. HIV-1 RNA was undetectable in 15 plasma samples but in only 2 PBMC samples (P=.002) and CD4 cell counts correlated more with PBMC than with plasma HIV-1 RNA load. Changes in HIV-1 RNA load in PBMC and in plasma were correlated, and the decrease was higher in plasma than in PBMC at weeks 12 (P=.002) and 24 (P=.017). Moreover, PBMC, but not plasma HIV-1 load, at week 12 was predictive of HIV-1 RNA levels at week 24 in both plasma (P=.004) and PBMC (P<. 001). Thus, measurement of PBMC HIV-1 RNA may be useful during antiretroviral therapy.

Zidovudine resistance mutations and human immunodeficiency virus type 1 DNA burden: Longitudinal evaluation of six patients under treatment

SummaryZidovudine (ZDV) is by far the most widely used drug to counteract human immunodeficiency virus type 1 (HIV-1) infection, both in monotherapy and in combination therapy regimens. However, the majority of patients under prolonged ZDV therapy have been shown to harbour HIV-1 mutant genomes displaying reduced sensitivity to the drugin vitro. In order to investigate the pathogenic role ofin vitro resistance to ZDV, six HIV-1-infected ZDV-treated subjects were evaluated longitudinally (mean follow-up 28.5 months, range 12–39 months) for HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) and for the presence of HIV-1pol gene mutations responsible for ZDV resistance. Quantitation of HIV-1 DNA was performed by competitive polymerase chain reaction (cPCR) and thepol genotype was determined by direct sequencing of PCR products. All of the six patients developed one or more of the HIV-1pol mutations known to confer resistance to ZDVin vitro (Met41→Leu, Asp67→Asn, Lys70→Arg, Thr215→Phe/Tyr, Lys219→Gln/Glu). A temporal association was found between HIV-1 DNA burden and the level of ZDV resistance, as predicted on the basis of thepol genotype (genotypic resistance). Both virus load and ZDV resistance were inversely correlated with CD4+ cell counts. These results are compatible with a directin vivo pathogenetic role forpol gene mutations shown to be involved in resistance to ZDVin vitro. Monitoring the degree of genotypic resistance to ZDV and to other antiretroviral drugs should be considered in designing protocols for the management of treated patients.ZusammenfassungZidovudin (ZDV) ist das Medikament, das weitaus am häufigsten in Monotherapie oder Kombinationstherapie zur Behandlung von Infektionen durch HIV-1 eingesetzt wird. Unter Langzeitbehandlung mit ZDV wurden jedoch bei der Mehrzahl der Patienten HIV-1-Mutanten mit reduzierterIn-vitro-Empfindlichkeit gegen das Medikament nachgewiesen. Um die pathogene Bedeutung derIn vitro-Resistenz gegen ZDV zu untersuchen, wurden die HIV-1 DNA-Last in mononukleären Zellen des peripheren Blutes und das Vorliegen von für die ZDV-Resistenz verantwortlichen HIV-1pol-Genmutationen bei sechs HIV-infizierten Patienten unter ZDV-Therapie in einer Längsschnittstudie (mit mittlerer Beobachtungszeit von 28.5 Monaten, Bereich 12–39 Monate) gemessen. Die quantitative Bestimmung der HIV-1 DNA erfolgte durch kompetitive Polymerasekettenreaktion (cPCR). Derpol-Genotyp wurde durch direkte Sequenzierung der PCR-Produkte ermittelt. Alle sechs Patienten entwickelten eine oder mehrere der HIV-1 Mutationen, deren Assoziation mitIn vitro-Resistenz gegen ZDV bekannt ist (Met41→Leu, Asp67→Asn, Lys70→Arg, Thr215→Phe/Tyr, Lys219→Gln/Glu). Zwischen dem auf der Basis derpol-Genotypen (Genotypresistenz) vorhergesagten Spiegel der ZDV-Resistenz und der HIV-1 DNA-Last fand sich eine zeitliche Assoziation. Sowohl die Viruslast wie auch die ZDV-Resistenz standen in umgekehrter Beziehung zu den CD4+-Zellzahlen. Diese Ergebnisse sind mit direkten pathogenenin vivo-Effekten der für die ZDV-Resistenzin vitro verantwortlichenpol-Genmutationen vereinbar. Das Monitoring des Ausmaßes genotypischer Resistenz gegen ZDV und andere antiretrovirale Substanzen sollte bei der Entwicklung von Behandlungsprotokollen berücksichtigt werden.

Increased reliability of selective PCR by using additionally mutated primers and a commercialTaq DNA polymerase enhancer

A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3′ end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selectivePCR.