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Biomedical subjects

M Nijhuis

Publications and source records attributed to M Nijhuis.

At least 19 recordsLinked to original sources
PubMed2005

A novel real-time PCR assay to determine relative replication capacity for HIV-1 protease variants and/or reverse transcriptase variants.

The emergence of drug-resistant viruses is a major issue in the treatment of HIV-1 infections. Quite often these drug-resistant viruses have a reduced replication capacity. A novel assay was developed to study the impact of mutations selected during therapy on viral replication capacity. Two HIV-1 HXB2 reference clones were constructed for this assay based on viral competition experiments, which are identical except for the presence of two silent nucleotide changes in p24 in one of the two clones. Within these two reference clones, three different contiguous deletions were constructed: (I) the C-terminus of Gag and protease, (II) the N-terminus of RT and (III) the C-terminus of Gag and protease together with the N-terminus of RT. Using these reference clones, recombinant viruses were created and viral competition experiments were performed. The proportion of each virus during the competition experiments was determined with a real-time PCR assay based on the two silent nucleotide changes in p24 in one of the two reference clones. With this novel assay it was possible to detect accurately differences in replication capacity due to mutations in the C-terminus of Gag and protease and/or the N-terminus of RT.

Evaluation Studies as Topic↗
PubMed2003

Diagnosis of enterovirus infection in the first 2 months of life by real-time polymerase chain reaction.

During summer and fall, enterovirus infections are responsible for a considerable proportion of hospitalizations of young infants. We prospectively studied the incidence of enterovirus infections via real-time polymerase chain reaction (PCR) in blood, feces, and cerebrospinal fluid samples from infants <or=60 days old who had received a clinical diagnosis of sepsis. Forty-five patients were included: 19 were admitted to the pediatric wards of 2 general hospitals, and 26 had been hospitalized since birth in the neonatal intensive care unit (NICU) of a tertiary care hospital. None of the NICU patients developed enteroviral disease. In contrast, an enterovirus was detected in 11 (58%) of the patients admitted to the 2 general hospitals, 10 of whom (53%) showed evidence of systemic infection. Enterovirus infections are an important cause of sepsis in infants admitted to the hospital. Real-time PCR in serum was a rapid and sensitive method for diagnosis of enterovirus infection.

Enterovirus↗
PubMed2003

Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults.

Respiratory syncytial virus (RSV) accounts for the majority of respiratory virus infections, producing high mortality rates in immunocompromised patients with hematologic malignancies. The available methods for the rapid detection of RSV by antigen detection or PCR either lack sensitivity, require complex laboratory manipulation, or have not been evaluated in this patient population. To assess the applicability of a TaqMan-based real-time PCR technique for the detection of RSV A and B in immunocompromised adults, we developed a rapid, sensitive detection method that simultaneously detects RSV A and B and can be applied in routine diagnostics. The specificity of the assay was assessed using a panel of reference strains of other respiratory viruses and RSV. Electron microscopy-counted stocks of RSV A and B were used to develop a quantitative PCR format. Eleven copies of viral RNA could be detected for RSV A strain Long, and 14 copies could be detected for RSV B strain 9320, corresponding to 50% tissue culture infective doses of 0.86 and 0.34, respectively. The assay was evaluated on 411 combined nose and throat swabs derived from immunocompromised adults with or without signs of respiratory tract infection. The diagnostic efficacy of the TaqMan PCR determined on the clinical samples showed that this real-time PCR technique was substantially more sensitive than the combination of conventional viral culture and shell vial culture. None of the clinical specimens derived from patients without signs of respiratory illness were found to be positive for RSV by real-time TaqMan PCR.

Adult↗
PubMed2001

Implications of antiretroviral resistance on viral fitness.

Treatment of HIV infected patients with antiretroviral drugs often results in the emergence of virus variants with reduced sensitivity to these drugs. However, the viral load often remains partially suppressed below pretherapy levels, which might be explained by a reduced fitness of the drug resistant viral population. This review describes the effects of antiretroviral resistance development on the fitness of the viral population and its clinical implications.

Anti-HIV Agents↗
PubMed2001

Simultaneous detection of influenza viruses A and B using real-time quantitative PCR.

Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.

Follow-Up Studies↗
PubMed2001

Clinical diagnosis of influenza virus infection: evaluation of diagnostic tools in general practice.

BACKGROUND: With the development of new antiviral agents for influenza, the urge for rapid and reliable diagnosis of influenza becomes increasingly important. Respiratory virus infections are difficult to distinguish on clinical grounds. General practitioners (GPs) however, still depend on their clinical judgement. AIM: To evaluate the importance of clinical symptoms in the diagnosis of influenza virus infection. DESIGN OF STUDY: A multicentre questionnaire study. SETTING: Eighty-one patients from 14 general practices. METHOD: Patients with fever and at least one constitutional symptom and one respiratory symptom were included. A questionnaire with the medical history and clinical symptoms was completed and a combined nose-throat swab was taken. Virus culture, rapid culture, and polymerase chain reaction (PCR) amplification were performed on each specimen. Multivariate analysis was used to obtain the best predictive model. RESULTS: By using PCR, an increase was seen in the detection of the viral pathogens compared with the results of culture. In 42 out of 81 patients PCR was positive for influenza. A positive predictive value (PPV) of 75% was observed for the combination of headache at onset, feverishness at onset, cough, and vaccination status during the period of increase influenza activity. Criteria used by the ICHPPC-2 resulted in a PPV of 54%. The PPV for diagnosis made by the GP was 76%. CONCLUSION: Although influenza is difficult to diagnose on clinical grounds, the GPs in this study were able to diagnose influenza as such more accurately on their judgement than by the other criteria.

Adult↗
PubMed2000

Methods for investigation of the relationship between drug-susceptibility phenotype and human immunodeficiency virus type 1 genotype with applications to AIDS clinical trials group 333.

Use of human immunodeficiency virus (HIV) drug-resistance testing in therapeutic decision making may be aided by understanding the relationship between results of genotypic and drug-susceptibility phenotypic assays. We investigated this relationship by applying 3 different statistical methods-cluster analysis, recursive partitioning, and linear discriminant analysis-to results for 72 patients followed in the Adult AIDS Clinical Trials Group (ACTG) protocol 333. ACTG 333 was a multicenter, randomized trial comparing 2 formulations of saquinavir (SQV) to indinavir (IDV) in patients with extensive hard-gel SQV experience. Data include protease amino acid sequences and 50% inhibitory concentrations for SQV and IDV at baseline. The 3 methods give similar results showing the association of mutations at codons 10, 63, 71, and 90 with in vitro resistance to IDV and SQV. Recursive partitioning is especially useful because it can identify interactions among mutations at different codons and accommodates many types of data as well as missing observations.

Adult↗
PubMed2000

Evolution of lamivudine resistance in human immunodeficiency virus type 1-infected individuals: the relative roles of drift and selection.

Human immunodeficiency virus type 1 (HIV-1) rapidly develops resistance to lamivudine during monotherapy, typically resulting in the appearance at position 184 in reverse transcriptase (RT) of isoleucine instead of the wild-type methionine (M184I) early in therapy, which is later replaced by valine (M184V). M184V reduces viral susceptibility to drug in vitro by approximately 100-fold, but also results in a lower processivity of RT. We show that a drop in absolute viral fitness associated with the outgrowth of M184V results in a drop in viral load only in individuals with high CD4(+) counts, from whom we estimate the relative fitness of M184V in the presence of drug to be approximately 10% of that of the wild type prior to therapy. The timing of emergence of the M184V mutant varies widely between infected individuals. From analysis of the frequency of M184I and M184V mutants determined at multiple time points in seven individuals during lamivudine therapy, we estimated the fitness advantage of M184V over M184I during therapy to be approximately 23% on average. We have also estimated the average ratio of the frequencies of the two mutants prior to therapy to be 0. 2:1, with a range from 0.12:1 to 0.33:1. We have found that the differences between individuals in the rate of evolution of lamivudine resistance arise due to genetic drift affecting the relative frequency of M184I and M184V prior to therapy. These results show that stochastic effects can be significant in HIV evolution, even when there is large fitness difference between mutant and wild-type variants.

Anti-HIV Agents↗
PubMed1999

Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy.

OBJECTIVE: It is thought as a consequence of continuous replication, HIV-1 has acquired an optimal fitness state and that suboptimal antiretroviral therapy selects for drug resistant variants which show impaired fitness in the absence of the drug. In this paper we studied the evolution and fitness of viral populations appearing in a patient who received protease monotherapy. METHODS: Two factors contributing to fitness, drug resistance and protease catalytic activity, were studied at the enzymatic and virological level. RESULTS: The first drug resistant viral variants that were selected in vivo harboured one to three protease substitutions. These mutants showed reduced protease activity and consequently a reduction in viral replication capacity. During continued in vivo replication of these viruses in the presence of the drug, novel variants harbouring additional substitutions in the viral protease appeared. These variants did not display any further increase in drug resistance but demonstrated clearly increased protease activity. Consequently the replication capacity of these viruses was raised to a level at which they replicated better than the original wild-type virus. CONCLUSION: This study indicates that the viral population in the patient does not have to represent the fittest possible variants, and thus antiretroviral therapy may drive the viral population first through a lower fitness level and then to a higher fitness level.

Amino Acid Sequence↗
PubMed1998

Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy.

It has long been assumed that HIV-1 evolution is best described by deterministic evolutionary models because of the large population size. Recently, however, it was suggested that the effective population size (Ne) may be rather small, thereby allowing chance to influence evolution, a situation best described by a stochastic evolutionary model. To gain experimental evidence supporting one of the evolutionary models, we investigated whether the development of resistance to the protease inhibitor ritonavir affected the evolution of the env gene. Sequential serum samples from five patients treated with ritonavir were used for analysis of the protease gene and the V3 domain of the env gene. Multiple reverse transcription-PCR products were cloned, sequenced, and used to construct phylogenetic trees and to calculate the genetic variation and Ne. Genotypic resistance to ritonavir developed in all five patients, but each patient displayed a unique combination of mutations, indicating a stochastic element in the development of ritonavir resistance. Furthermore, development of resistance induced clear bottleneck effects in the env gene. The mean intrasample genetic variation, which ranged from 1.2% to 5.7% before treatment, decreased significantly (P < 0.025) during treatment. In agreement with these findings, Ne was estimated to be very small (500-15,000) compared with the total HIV-1 RNA copy number. This study combines three independent observations, strong population bottlenecking, small Ne, and selection of different combinations of protease-resistance mutations, all of which indicate that HIV-1 evolution is best described by a stochastic evolutionary model.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Efficacy of adding indinavir to previous reverse transcriptase nucleoside analogues in relation to genotypic and phenotypic resistance development in advanced HIV-1-infected patients.

We assessed the efficacy of adding indinavir in patients with advanced HIV-1 infection, who were previously exposed to different reverse transcriptase (RT) nucleoside analogues. Twenty-five patients with an initial median CD4 cell count of 20 cells/mm3 (range, 0-80 cells/mm3) were treated with indinavir (800 mg three times per day) for 24 weeks. The median initial viral load was 5.4 log (range, 3.6-6.7 log). Of these patients, 56% (14 of 25) had an initial decrease in viral load of >1 log and sustained response of >0.5 log of HIV-1 RNA from baseline. Twelve of these 14 responder patients (85%) showed a sustained RNA response undetectable by NASBA assay, and no genotypic changes in protease were detected at week 24. In those with a temporary or absent response to indinavir, either resistant viruses or lack of compliance was observed. In compliant patients (15 of 16), relatively small increases in 50% inhibitory concentration (IC50) to indinavir and only two to three amino acid changes were sufficient to produce treatment failure. Phenotypic drug-resistant assays at 24 weeks revealed cross-resistance to ritonavir in all the patient isolates and to saquinavir in one third of the isolates. We observed an initial and persistent response to the addition of indinavir in patients with advanced disease and prolonged antiretroviral treatment. Therapy failure, as defined by increases in viral RNA, was associated with either lack of compliance or the development of low level indinavir-resistant virus. Clinical studies need to be designed to determine to what extent these viruses may respond to other protease inhibitors.

Anti-HIV Agents↗
PubMed1998

Efficient inhibition of both syncytium-inducing and non-syncytium-inducing wild-type HIV-1 by lamivudine in vivo.

OBJECTIVE: To determine the effect of lamivudine (3TC) on syncytium-inducing (SI) and non-SI (NSI) HIV-1 populations in vivo. DESIGN: Responses in virus load and 3TC resistance in 40 3TC-treated subjects were analysed in relation to the presence or absence of SI HIV-1 variants. METHODS: Peripheral blood samples were collected at regular intervals from 40 HIV-1-infected subjects during 3TC treatment. Virus isolates obtained at the start of treatment were typed for SI-capacity by coculture with MT-2 cells. Changes in levels of viral RNA in plasma were determined by quantitative reverse transcriptase PCR. The relative amount of wild-type and mutant virus at codon 184, associated with HIV resistance to 3TC, was determined using a primer-guided nucleotide incorporation assay after amplification of part of the reverse transcriptase gene. In five subjects the frequency of productively infected CD4+ cells with SI or NSI variants was determined in relation to codon 184 genotype. RESULTS: Twenty-six subjects harboured only NSI variants at baseline, whereas 14 subjects also harboured SI variants. Although baseline plasma viral RNA load and CD4 cell counts were different between the two groups, no differences in the response to 3TC therapy were observed for these parameters. In-depth analysis of five subjects showed that the kinetics of virus load changes and emergence of 3TC resistance mutations were similar in plasma and cells, and comparable for the SI and NSI populations present in one individual. CONCLUSIONS: These data show that, in contrast to didanosine and zidovudine, the pressure exerted by 3TC is similar for SI and NSI M184 populations.

Anti-HIV Agents↗
PubMed1997

Lamivudine-resistant human immunodeficiency virus type 1 variants (184V) require multiple amino acid changes to become co-resistant to zidovudine in vivo.

Exposure of human immunodeficiency virus to the nucleoside analogue lamivudine (3TC) rapidly selects for resistant variants with a valine at codon 184 (M184V) in the catalytic site of reverse transcriptase. In vitro, 184V demonstrated increased enzyme fidelity and suppressed zidovudine resistance. Clinical trials demonstrated that 3TC-zidovudine combination therapy results in a strong and sustained antiviral response. To investigate the role of 184V on in vivo virus evolution, the effect of zidovudine addition in 3TC-pretreated patients harboring 184V was studied. In vivo, no significant change in fidelity was observed with 184V, shown by generation of the classical pattern of zidovudine mutations. Of interest, in contrast to zidovudine monotherapy, in which just one substitution is sufficient for in vivo development of significant zidovudine resistance, multiple substitutions are required for the same level of zidovudine resistance in strains harboring 184V. This need for multiple substitutions may be one of the mechanisms explaining the sustained antiretroviral response of the 3TC-zidovudine combination.

Amino Acid Sequence↗
PubMed1997

Results of long-term follow-up of HIV-infected patients treated with lamivudine monotherapy, followed by a combination of lamivudine and zidovudine.

The objective of this study was to determine the efficacy and safety of adding zidovudine to continuous treatment with lamivudine in symptomatic human immunodeficiency virus type 1 (HIV-1)-infected patients. Forty patients were monitored throughout lamivudine monotherapy and subsequent combination therapy with lamivudine and zidovudine, which was initiated because of disease progression, declining CD4 cell counts or prolonged use of lamivudine. Eleven of these patients had been treated with zidovudine before the start of the study. The median CD4 cell count at the start of lamivudine monotherapy was 200 x 10(6) cells/l. After a median interval of 69 weeks (range 23-102 weeks), the median CD4 cell count had dropped to 110 x 10(6) cells/l. Initial improvements in all laboratory markers for antiretroviral efficacy were observed after the addition of zidovudine. The median CD4 cell count remained 18% above baseline after 48 weeks of treatment with lamivudine and zidovudine, however plasma HTV-1 RNA load and CD4 cell counts returned towards baseline during prolonged treatment in most patients. The combination was well tolerated, although anaemia was observed in nine patients. Repeated measures analysis of variance suggested a superior effect of lamivudine monotherapy in patients who had previously used zidovudine. In conclusion, zidovudine was found to be effective in patients who have been treated with lamivudine. The study stresses the need to further define the mechanisms underlying this prolonged antiviral effect.

Anti-HIV Agents↗
PubMed1996

Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild-type and 3TC-resistant HIV-1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild-type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC-resistant RT mutants produce significantly shorter cDNA molecules than the wild-type enzyme [Met (wt)>Val>Ile], indicating that 3TC-resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC-resistant HIV-1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC-treated patients that develop drug-resistant HIV-1 variants, this may be of considerable clinical benefit.

Antiviral Agents↗
PubMed1996

Human immunodeficiency virus type 1 drug susceptibility determination by using recombinant viruses generated from patient sera tested in a cell-killing assay.

A simple approach for the determination of drug susceptibilities by using human immunodeficiency virus type 1 (HIV-1) RNA from the sera of patients is described. HIV-1 RNA was extracted from patient sera, and the 5' part of the reverse transcriptase (RT) gene was transcribed into DNA and amplified in a nested PCR. The amplified fragment covers the 3' part of the protease gene and amino acids 1 to 304 of the RT gene. This fragment can be introduced through homologous recombination, as described previously, into a novel HIV-1 reference strain (pHXB2 delta 2-261RT) from which amino acids 2 to 261 of RT have been deleted. The resulting recombinant virus expresses all properties of the HXB2 reference strain except for those encoded by the introduced part of the patient RT gene. Recombinant viruses were subsequently tested for drug susceptibility in a microtiter format killing assay [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay] as well as in the standard HeLa CD4+ plaque reduction assay. Similar susceptibility profiles were obtained by each assay with recombinant viruses derived from patients receiving alternating nevirapine and zidovudine treatment or lamivudine-zidovudine combination therapy. In conclusion, this approach enables high-through-put determination of the drug susceptibilities of serum RNA-derived RT genes, independent of the patient's viral background, and generates the possibility of relating changes in susceptibility to changes in viral genotypes.

Anti-HIV Agents↗