Increase of Diversity of Mumps Virus Genotype G SH Variants Circulating Among a Highly Immunized Population: Spain, 2007-2019.

MuV caused three epidemic waves in Spain since genotype G emerged in 2005, despite high vaccination coverage. SH gene sequencing according to WHO protocols allowed the identification of seven relevant variants and 88 haplotypes. While the originally imported MuVi/Sheffield.GBR/1.05/-variant prevailed during the first two waves, it was subsequently replaced by other variants originated by either local evolution or importation, according to the additional analysis of hypervariable NCRs. The time of emergence of the MRCA of each MuV variant clade was concordant with the data of the earliest sequence. The analysis of Shannon entropy showed an accumulation of variability on six particular positions as the cause of the increase on the number of circulating SH variants. Consequently, SH gene sequencing needs to be complemented with other more variable markers for mumps surveillance immediately after the emergence of a new genotype, but the subsequent emergence of new SH variants turns it unnecessary.

In vitro analysis of synergism and antagonism of different nucleoside/nucleotide analogue combinations on the inhibition of human immunodeficiency virus type 1 replication.

In this study we have developed an in vitro system to evaluate the combined effect of two NRTIs on HIV replication and to assess their antagonism or synergy. Synergy or antagonism effect was determined in peripheral blood mononuclear cells (PBMCs) to approach a more physiological model than T-cell lines. PBMCs were infected with a full-length HIV-1 clone carrying the luciferase gene as a reporter. The following combinations were investigated: zidovudine+stavudine (ZDV + d4T), lamivudine + abacavir (3TC + ABC), lamivudine + didanosine (3TC + ddI), lamivudine + stavudine (3TC + d4T), tenofovir + stavudine (TDF + d4T), tenofovir + didanosine (TDF + ddI), tenofovir + abacavir (TDF + ABC), tenofovir + lamivudine (TDF + 3TC), tenofovir + zidovudine (TDF + ZDV), stavudine + didanosine (d4T + ddI), zidovudine + lamivudine (ZDV + 3TC), abacavir + didanosine (ABC + ddI), zidovudine + didanosine (ZDV + ddI), and abacavir + stavudine (ABC + d4T). The effect of combining two drugs was evaluated with a quantitative method based on the median-effect principle of Chou and Talalay. A synergistic effect was observed with combinations containing TDF and ZDV or d4T, d4T and ddI and ZDV plus 3TC. In contrast, combinations including TDF + ddI, 3TC + ddI, ABC + ddI, and ZDV + ddI showed an antagonistic effect on the inhibition of viral replication at all levels of inhibition tested. Lower antagonistic effect was also found in drug combinations that included 3TC + ABC, 3TC + TDF, 3TC + d4T, and TDF + ABC. In conclusion, the method developed allows to measure in vitro the effect of different combinations of two NRTIs on HIV replication. The results suggest that combined therapy including TDF with thymidine analogues may be considered for future therapeutic options in contrast to clearly antagonistic combinations such us TDF plus ddI or 3TC plus ddI, that would explain virological failure in clinical studies when these combinations were used.

A new strategy based on recombinant viruses for assessing the replication capacity of HIV-1.

OBJECTIVE: In heavily pretreated patients, resistance mutations arise in both protease (PR) and reverse transcriptase (RT) sequences; however, the relative impact of PR and RT mutations on viral fitness cannot be evaluated with the majority of systems. To address this issue we have developed a model based on recombinant viruses (RVs) that allows the analysis of the replication capacity (RC) of viral populations in which PR and RT are cloned either in combination or separately. METHODS: RVs were generated for full-length polymerase (pol) gene, PR or RT sequences from nine naïve and 14 heavily pretreated HIV-infected patients in therapeutic failure. The relative RC was assessed by comparing luciferase activity between mutant RV and wild-type (wt) isolates. RESULTS: A strong decrease (>60%) in the RC of the pol RV population was observed in the 14 heavily pretreated patients as compared with the wt RVs. The analysis of PR and RT RVs from these patients showed that the decrease in RC was mainly attributable to PR sequences in three of these 14 patients and to RT sequences in seven of these patients. In the four remaining patients, PR and RT sequences independently reduced the RC of the RVs to similar extents. CONCLUSIONS: Different patterns of mutations in either PR or RT have a strong impact on RC in highly experienced HIV-infected patients.

A new strategy based on recombinant viruses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1.

The emergence of drug-resistant variants during antiretroviral therapy is a serious obstacle to sustained suppression of the human immunodeficiency virus type 1 (HIV-1). For that reason, resistance assays are essential to guide clinicians in the selection of optimal treatment regimens. Genotypic assays are less expensive and results are available faster than phenotypic assays. However, in heavily experienced patients with multiple treatment failures interpretation of complex mutation patterns remains difficult, and in these cases phenotypic assays are recommended. This report describes a novel recombinant virus assay where protease (PR) and reverse transcriptase (RT) sequences derived from the plasma isolated from patients are introduced into the back-bone of an HIV molecular clone that expresses Renilla luciferase protein in the place of nef gene. All drug resistance profiles analyzed correlate with previously reported data and showed high reproducibility. This assay, in addition to a fast (completed in 10 days), precise, reproducible and automated method, presents several advantages as compared to other phenotypic assays. The system described below allows the generation of recombinant viruses with multiples cycles of replication carrying a reporter gene in their genomes. These features increase the sensitivity of the test, an important aspect to be considered in the evaluation of less fit viral isolates. In conclusion, the assay permits the quantitation of the level of resistance of clinical HIV-1 isolates to PR and RT inhibitors.

Management of hepatitis C in HIV-infected persons.

The life expectancy of HIV-infected persons has extended significantly since the introduction of highly active antiretroviral therapies. Although classical opportunistic infections are now rarely seen, the toxicity of antiretroviral drugs as well as liver disease caused by hepatitis viruses represent an increasing cause of morbidity and mortality among HIV-positive persons. Since the rate of hepatitis C virus (HCV) infection is high among HIV carriers (up to 75% among intravenous drug users), HCV/HIV coinfection is widely prevalent. Predisposing liver damage favors a higher rate of hepatotoxicity of antiretroviral drugs, which can limit the benefit of HIV treatment in some individuals. Overall, severe hepatotoxicity appears in around 10% of subjects who began triple combinations including either protease inhibitors or non-nucleosides. The progression to cirrhosis seems to occur faster in the setting of HIV infection, and conversely recent data demonstrate that HCV infection can accelerate the progression to AIDS in HIV-positive persons. Although clinicians have been reluctant to treat hepatitis C in HIV-infected people, this therapeutic nihilism is unwarranted. The availability of new more successful regimens to treat hepatitis C, in particular using the new pegylated forms of interferon in combination with ribavirin, open new hopes for the care of HIV-HCV-coinfected persons.

Primary genotypic and phenotypic HIV-1 drug resistance in recent seroconverters in Madrid.

OBJECTIVE: Transmission of drug-resistant HIV-1 strains is increasing with widespread use of antiretroviral drugs in developed countries. This study examined the prevalence of resistant viruses in recent seroconverters in Madrid, Spain. DESIGN: HIV isolates from 30 consecutive participants with positive or indeterminate HIV antibody test results and a negative test result at a mean of 6.6 months earlier were examined for HIV drug resistance. All study subjects admitted to having very recently engaged in high-risk practices. All were therapeutically naive and were recruited between 1997 and 1999 in a referring health care facility for sexually transmitted diseases. METHODS: Population-based sequencing of the viral reverse transcriptase (RT) and protease (PR) regions derived from plasma viral RNA was performed. Phenotypic resistance was assessed by a recombinant virus assay. RESULTS: Overall prevalence of genotypes associated with reduced susceptibility was 26.7% (8 of 30 participants). Resistance mutations were seen against nucleoside analogues in 7 (23.3%), nonnucleoside reverse transcriptase inhibitors in 1 (3.3%), and protease inhibitors in 2 (6.7%). Zidovudine-resistance mutations M41L and/or T215Y were the commonest, found in 20% (6 of 30 participants). Resistance mutations to at least two antiretroviral families (multidrug-resistance) were detected in 2 (6.7%) study subjects. A median infectious dose (IC50) increase of fourfold for any drug was found in 7 patients, and in 2 was > tenfold for zidovudine (genotype M41L + T215Y) and lamivudine (genotype M184V), respectively. CONCLUSIONS: Drug-resistant HIV variants were present in over one quarter of individuals recently diagnosed as infected in Madrid, Spain. Therefore, resistance testing at baseline should be considered for the optimal design of first-line antiretroviral combinations.

Primary HIV-1 drug resistance in Spain before and after the introduction of protease inhibitors.

The prevalence of genotypic resistance to nucleoside analogues was examined in 150 naive individuals with chronic HIV-1 infection acquired before June 1996, when protease inhibitors began to be used in Spain, and in a group of 52 recent seroconverters infected since 1997. Overall, the prevalence of subjects carrying nucleoside-resistant genotypes was similar in both groups (12.6% and 15.4%, respectively). A polymorphism at codon 70 accounted for more than half of cases (63.1%) in naive individuals infected before protease inhibitors became available, in contrast most recent seroconverters carried genotypes with one or more key nucleoside resistance mutations, mainly associated with lack of sensitivity to zidovudine or lamivudine (prevalence 9.6% and 5.9%, respectively). When only key mutations were considered in each of the two periods, the prevalence of nucleoside-resistant genotypes was increased significantly among recent seroconverters (13.5% vs. 4.7%; P<0.05). The relatively high rate of resistance to nucleosides in Spain justifies the introduction of drug resistance testing in naive recent seroconverters before beginning antiretroviral therapy.

Prevalence and genetic heterogeneity of the reverse transcriptase T69S-S-X insertion in pretreated HIV-infected patients.

The emergence of resistance to antiretroviral drugs represents one of the main reasons for treatment failure in HIV-infected persons. Resistance to multiple nucleoside analogues may result from rearrangements in the HIV pol gene, in particular one insertion of two amino acids at position 69. Herein, we examined the prevalence of this resistant genotype and its genetic heterogeneity in a group of 475 healthy pretreated HIV-positive subjects in Spain. Only 4 (0.8%) carried the codon 69 insertion. It was always found coupled to the T69S mutation. The extra amino acids were S-S in 2 subjects and S-G in the other 2. The presence of the insert was seen only in subjects previously exposed to AZT monotherapy for at least 6 months.

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain. The ERASE-2 Study Group.

OBJECTIVE: To examine the prevalence of resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) in a representative HIV-1 population in Spain. METHODS: A cross-sectional study was conducted including 601 HIV-infected patients who attended 20 Spanish hospitals in June 1998. Drug resistant mutations were examined using hybridization line probe assays (LiPA). The 6 bp insert at position 69 and the codon 75 mutant were examined by sequencing analysis in specimens lacking reactivity to 69/70 and 74 bands on LiPA, respectively. RESULTS: Primary resistance to NRTI was recognized in nine out of 52 (17%) naive individuals, whereas primary resistance to PI was found in seven out of 126 (6%) PI-naïve patients. The codons most frequently involved in NRTI resistance were at positions 70 (66%), 184 (44%), 215 (33%), and 41 (11%), whereas the most common PI resistance mutation was at codon 82 (6/7 subjects). In pre-treated patients, the overall prevalence of resistant genotypes was 72.9% for NRTI and 27.2% for PI. The most frequent NRTI mutations occurred at codons 184 (38.5%), 215 (30.1%), and 41 (22.5%), whereas the most frequent PI mutations in pre-treated subjects were found at positions 82 (15.8%) and 84 (11.4%). Overall, patients who began triple combinations as initial therapy showed a lower number of key resistance mutations than those who began highly active antiretroviral therapy (HAART) after being exposed to NRTI for a period of time (mean number of mutations, 0.1 versus 1.8, P< 0.05). Codon 75 mutant was found in three out of 387 patients (0.7%), whereas no insertions at codon 69 were recognized. CONCLUSION: The prevalence of primary genotypic resistance to NRTI and PI in Spain was 17% and 6%, respectively. Zidovudine, lamivudine, indinavir and ritonavir were the drugs most frequently affected. These data support the use of resistance testing prior to the introduction of first-line antiretroviral therapies in Spain. Among pre-treated subjects, drug resistance genotypes were less prevalent in those who began HAART as initial therapy.

Efficacy and safety of didanosine and lamivudine both once daily plus indinavir in human immunodeficiency virus-infected patients.

BACKGROUND: The combination of didanosine (DDI) and lamivudine (3TC) has been not recommended in guidelines for the first-line treatment of human immunodeficiency virus (HIV) infection because two major considerations might reduce its efficacy. First, both drugs are non-thymidine nucleosides and might exert less antiviral activity on activated T cells. Second, the codon 184 mutation emerging under 3TC failure could confer cross-resistance to DDI. However, because the intracellular half-life of their active metabolites allows administration once daily, the resulting improvement in patient compliance should favor this combination over others. PATIENTS AND METHODS: We analyzed the virologic and immunologic outcome at 6 months in 46 naive HIV-infected patients (41% were intravenous drug users) who began a treatment of a triple combination of DDI, 3TC, and indinavir (IDV) with the schedule optimized to improve adherence. Both DDI and 3TC were administered once daily (450 mg and 300 mg, respectively) and IDV was taken twice daily (1,200 mg). Patients chose schedules at their convenience, according to their job timings and preferences. All had a plasma viral load (PVL) of more than 500 HIV-RNA copies/mL at the time they entered the study, and overall the mean PVL value was 54,201 copies/mL. RESULTS: Overall, nine patients did not complete the study period: two died of non-acquired immunodeficiency syndrome (AIDS)-related causes, three described severe gastrointestinal symptoms, one discontinued therapy voluntarily, and three were lost to follow-up. Among the remaining 37 patients, a PVL value of less than 500 copies/mL was reached by 31 (83.7%) patients, and values below 40 copies/mL were recorded in 71% (22/31) of them. Overall, a clinically significant increase in the CD4 cell count (more than 60 cells/microL) was seen in 70.2% (26/37) of patients. Treatment adherence, assessed using both self-reporting and the pill count method, was considered good (more than 90% of pills prescribed were taken) in all but four patients; three of them were among those six who did not reach undetectable PVL values at the sixth month. Mutations at codons 184, 74, 65, and 151, which confer resistance to 3TC and DDI, could be examined in 12 of the 15 patients having PVL values above 40 copies/mL at the sixth month. The codon 184 mutation emerged in 25%, meanwhile the codon 74 mutation only appeared in one patient; none carried the codon 151 mutant genotype. Nine months after beginning treatment, 29 (93.5%) of the 31 patients who reached less than 500 copies/mL at the sixth month still sustained PVL values below this threshold, and 25 (80.6%) had less than 40 copies/mL. CONCLUSION: The combination of DDI and 3TC both once daily plus IDV twice daily is well tolerated, seems to favor a good adherence, and shows significant antiviral activity.

Usefulness of genotypic analysis of resistance to nucleoside analogues in the clinical setting.

The introduction of drug resistance testing during clinical care of patients infected with the human immunodeficiency virus (HIV) is still a matter for discussion. Thirty-seven HIV-infected subjects who had positive plasma viraemia despite undergoing antiretroviral therapy were tested for the presence of genotypic mutations linked to resistance to nucleoside analogues. Thirty (81.1%) individuals showed one or more mutations conferring drug resistance. Eight harboured mutants at codons 215 and 41, which confer resistance to zidovudine and are associated with the worst prognosis. One individual carried the codon 69-SSS insertion, which confers multidrug resistance. Seven (18.9%) subjects did not show any mutation, but all of them failed to adhere to the treatment regimen and/or experienced diarrhea that likely caused malabsorption of the drugs. Clinicians referred to the resistance profile when they adjusted or prescribed subsequent combination regimens for their patients.

Evolution of genotypic resistance to nucleoside analogues in patients receiving protease inhibitor-containing regimens.

The prevalence of genotypic resistance to nucleoside analogues (NA) was examined using a line probe assay (LiPA, Innogenetics, Spain) and a point mutation assay to test for codon 151 polymorphism in plasma from 34 individuals who had been exposed to NA for longer than 1 year. The testing was repeated in the same population after 6 months of being on a new potent protease inhibitor (PI)-containing antiretroviral combination. Only nine (47%) of the 19 patients initially carrying the codon 41 mutation restored zidovudine wild-type (WT) virus population. Similarly, eight (33%) out of 24 carrying the codon 215 mutation restored the wild-type variant. Two subjects carrying codon 74 didanosine mutation reverted to wild-type genotype, as well as two (18%) out of 11 harbouring the codon 184 lamivudine-resistant variant. To conclude, the extent to which drug recycling might be of benefit in subjects showing a restoration of genotypic sensitivity to former drugs needs to be explored.

Different outcomes in patients achieving complete or partial viral load suppression on antiretroviral therapy.

BACKGROUND: Potent combination antiretroviral therapy can reduce HIV plasma viral load (VL) to levels below the detection limit for as long as 2 years or more. A VL <500 HIV RNA copies/mL was until recently considered a reasonable therapeutic goal. However, lower levels seem necessary if VL rebounds and development of drug resistance are to be avoided. PATIENTS AND METHODS: The clinical and virologic outcome at 1 year were prospectively examined in a group of 100 patients who began a triple combination antiretroviral therapy regimen consisting of stavudine (d4T), lamivudine (3TC), and indinavir (IDV). A modified ultrasensitive VL test with a detection limit of 40 copies/mL and a point mutation nested polymerase chain reaction (PCR) assay for detecting the codon 184 mutation conferring 3TC resistance were used for testing samples collected longitudinally from these individuals. RESULTS: Overall, VL values <40 copies/mL were reached in 45% and 32% of patients at nadir and at 12 months, respectively. More than half (24 of 45 persons) who achieved a level <40 copies/mL at nadir remained with undetectable VL at 1 year, whereas this occurred in only one fourth (7 of 28 persons) of those having levels of 40 to 500 copies/mL (P < .05). However, rebounds in VL to >500 copies/mL at 1 year were seen at similar rates (26.6% and 25%, respectively) in persons achieving either complete (<40 copies/mL) or partial (40-500 copies/mL) VL suppression at nadir. In contrast, the codon 184 mutation emerged more frequently at 1 year in patients whose VL remained between 40 and 500 copies/mL at nadir than in those who reached a level <40 copies/mL (30.7% versus 0%; P < .05). CONCLUSION: Plasma VL at nadir after beginning highly active antiretroviral therapy (HAART) predicts the 1-year outcome. The achievement of levels of viremia <40 copies/mL are desirable during antiretroviral therapy if prolonged benefit is to be obtained. Because more than two thirds of persons with residual viremia do not show drug resistance, intensification strategies should be investigated for those patients with a good virologic response but without complete suppression during the first 6 months on HAART.

Presence of genotypic resistance in nucleoside analogue-treated HIV-1-infected patients with undetectable viral load.

Patients harbouring drug-resistance viruses usually suffer a rise in serum viraemia after a variable period of time. We have investigated the relationship between the appearance of resistant genotypes and the viral load of each patient after treatment. Our objective was to assess the association between human immunodeficiency virus (HIV) RNA plasma levels and the number of drug resistance-associated point mutations after treatment. A total of 150 patients from three reference centres in Spain (Madrid, Barcelona and Seville) from a previous study (Erase Study) were included. Patients had at that time undergone antiretroviral treatment with nucleoside analogues for at least 1 year (zidovudine/didanosine; zidovudine/zalcitabine; zidovudine/zalcitabine/lamivudine; zidovudine/didanosine/lamivudine). In this study, plasma viraemia in these patients was quantified and a line probe assay was used to determine the genotype of the virus. Viral load was significantly higher in patients harbouring virus with more than three mutations than in those individuals who harboured wild-type strains (P < 0.05). Surprisingly, when patients with viral load < 500 copies/ml (13/150) were analysed, only two carried wild-type strains, whereas three had virus with more than three point mutations. The viral load of six samples was assayed using an ultrasensitive test (detection limit < 20 copies/ml). Of the three samples where viral load was < 20 copies/ml, one patient harboured wild-type virus, whereas two carried mutant virus strains. These results suggest that even in patients with undetectable viral loads by conventional methods, viral replication may continue and mutations develop. Therefore, standard values of plasma viraemia for measuring the effectiveness of the treatment should be reconsidered when patients are on antiviral regimens of just two or three nucleoside analogues.

Efficiency of drug resistance genotypic tests in specimens with low HIV viral load.

The early recognition of resistance to antiretroviral agents could allow a rapid switch in therapy and therefore avoid the accumulation of mutations and reduce the risk of cross-resistance. However, the efficiency of genotypic tests in specimens with low viral load (VL) is severely compromised since human immunodeficiency virus (HIV) RNA in these samples often goes unrecognized. The frequency of results provided by a line probe assay (LiPA, Murex), a commercially available drug resistance test and a home-made point mutation assay (PMA) for recognizing the codon 151 multidrug-resistance mutation was examined in 664 plasma samples stratified with respect to VL values. Overall, 421 (63%) samples could be interpreted by both LiPA and PMA. The sensitivity decreased as plasma VL lowered: 89% for samples with VL > 10,000 HIV RNA copies/ml, 77% for those with VL between 500 and 10,000 HIV RNA copies/ml and 37% for specimens with VL < 500 HIV RNA copies/ml. A good agreement existed comparing the sensitivity of the home-made PMA and LiPA. Although the former tends to produce more results, the difference did not achieve statistical significance. Our results support that new, more sensitive, HIV RNA extraction methods need to be implemented for the rapid recognition of drug-resistant mutants in patients experiencing an early rebound in plasma viraemia.