Minor Mutations Research Articles

Genetic variation of the HIV-1 integrase region in newly diagnosed anti-retroviral drug-naïve patients with HIV/AIDS in Korea

The survival time of HIV/AIDS patients in Korea has increased since HAART (highly active anti-retroviral therapy) was introduced. However, the occurrence of drug-resistant strains requires new anti-retroviral drugs, one of which, an integrase inhibitor (INI), was approved by the US Food and Drug Administration (FDA) in 2007. INIs have been used for therapy in many countries and are about to be employed in Korea. Therefore, it is important to identify basic mutant variants prior to the introduction of INIs in order to estimate their efficacy. To monitor potential drug-resistant INI mutations in Korean HIV/AIDS patients, the polymorphism of the int gene was investigated together with the pol gene using a genotypic assay for 75 randomly selected Korean HIV-1 patients newly diagnosed in 2007. The drug-resistant mutation sequences were analysed using the Stanford HIV DB and the International AIDS Society resistance testing-USA panel (IAS-USA). Seventy strains of Korean subtype B were compared with foreign subtype-B strains, and there were no significantly different variants of the int gene region in the study population. Major mutation sites in the integrase (E92Q, F121Y, G140A/S, Y143C/R, Q148H/R/K and N155H) were not detected, and only a few minor mutation sites (L74M, V151I, E157Q, V165I, I203M, S230N and D232N) were identified in 21 strains (28%). Resistance due to mutations in the pol gene was observed in a single strain (1.3%) resistant to protease inhibitors (PIs) and in four strains (5.3%) resistant to reverse transcriptase inhibitors (RTIs). In summary, this demonstrates that INIs will be susceptible to drug naïve HIV/AIDS patients in Korea.

Single Genome Sequencing of HIV-1 Gag and Protease Resistance Mutations at Virologic Failure during the Ok04 Trial of Simplified versus Standard Maintenance Therapy

Ritonavir-boosted lopinavir (LPV/RTV) alone has been evaluated as simplified maintenance therapy for HIV-1 infection, but there are concerns about greater potential for emergence of protease inhibitor (PI) resistance. The OK04 trial evaluated maintenance therapy with LPV/RTV alone versus standard therapy (ST) with two NRTIs plus LPV/RTV in 205 patients, of whom 15 had virological rebound by week 48 (11 versus 4 patients, respectively). We developed a single genome sequencing (SGS) assay of HIV-1 gag and protease to assess the emergence of low frequency drug-resistant variants during virological rebound. Plasma samples from 15 subjects at virological rebound were analysed by SGS of HIV-1 gag and protease genes. A total of 45 SGS sequences were planned per sample, providing 90% power to detect variants comprising >5% of the virus population. Overall, 521 single sequences obtained from 13 patients (range 4-48 sequences/patient) revealed similar frequencies of major protease resistance mutations in samples from the LPV/RTV alone (3/11) and ST (3/4) arms (P=0.10), with a median number of minor protease resistance mutations of 3.0 versus 3.5, respectively (P=0.23). Median number of gag PI resistance mutations were similar between the LPV/RTV alone and ST arms at cleavage sites (3.0 versus 2.5; P=0.83), non-cleavage sites (21 versus 16.5; P=0.71) and the transframe protein-p6 pol region cleavage sites (4.0 versus 3.0; P=0.6). Although more subjects with simplified maintenance therapy with LPV/RTV alone had virological rebound compared to the ST arm, this was not associated with more frequent emergence of variants encoding PI resistance mutations in gag or protease detected by SGS.

Locked nucleic acid probes for enhanced detection of FLT3 D835/I836, JAK2 V617F and NPM1 mutations

AimsDetecting low-level clinically significant cancer-relevant somatic mutations can be difficult. Several technologies exist for detecting minority mutations. One method is locked nucleic acid (LNA) PCR. In this study, LNA probes...

Prevalence and factors associated with darunavir resistance mutations in multi-experienced HIV-1-infected patients failing other protease inhibitors in a referral teaching center in Brazil

Information about resistance profile of darunavir (DRV) is scarce in Brazil. Our objectives were to estimate the prevalence of DRV resistance mutations in patients failing protease inhibitors (PI) and to identify factors associated with having more DRV resistance mutations. All HIV-infected patients failing PI-based regimens with genotyping performed between 2007 and 2008 in a referral teaching center in São Paulo, Brazil, were included. DRV-specific resistance mutations listed by December 2008 IAS-USA panel update were considered. Two Poisson regression models were constructed to assess factors related to the presence of more DRV resistance mutations. A total of 171 HIV-infected patients with available genotyping were included. The number of patients with lopinavir, saquinavir, and amprenavir used in previous regimen were 130 (76%), 83 (49%), and 35 (20%), respectively. The prevalence of major DRV resistance mutations was 50V: 5%; 54M: 1%; 76V: 4%; 84V: 15%. For minor mutations, the rates were 11I: 3%; 32I: 7%; 33F: 23%; 47V: 6%; 54L: 6%; 74P: 3%; 89V: 6%. Only 11 (6%) of the genotypes had > 3 DRV resistance mutations. In the clinical model, time of HIV infection of > 10 years and use of amprenavir were independently associated with having more DRV resistance mutations. In the genotyping-based model, only total number of PI resistance mutations was associated with our outcome. In conclusion, the prevalence of DRV mutations was low. Time of HIV infection, use of amprenavir and total number of PI resistance mutations were associated with having more DRV mutations.

Impact of Adding Enfuvirtide to the Predictive Value of the Darunavir Genotypic Resistance Score

Impact of Adding Enfuvirtide to the Predictive Value of the Darunavir Genotypic Resistance Score

Prevalence and factors associated with darunavir resistance mutations in multi-experienced HIV-1-infected patients failing other protease inhibitors in a referral teaching center in Brazil

Information about resistance profile of darunavir (DRV) is scarce in Brazil. Our objectives were to estimate the prevalence of DRV resistance mutations in patients failing protease inhibitors (PI) and to identify factors associated with having more DRV resistance mutations. All HIV-infected patients failing PI-based regimens with genotyping performed between 2007 and 2008 in a referral teaching center in São Paulo, Brazil, were included. DRV-specific resistance mutations listed by December 2008 IAS-USA panel update were considered. Two Poisson regression models were constructed to assess factors related to the presence of more DRV resistance mutations. A total of 171 HIV-infected patients with available genotyping were included. The number of patients with lopinavir, saquinavir, and amprenavir used in previous regimen were 130 (76%), 83 (49%), and 35 (20%), respectively. The prevalence of major DRV resistance mutations was 50V: 5%; 54M: 1%; 76V: 4%; 84V: 15%. For minor mutations, the rates were 11I: 3%; 32I: 7%; 33F: 23%; 47V: 6%; 54L: 6%; 74P: 3%; 89V: 6%. Only 11 (6%) of the genotypes had ≥ 3 DRV resistance mutations. In the clinical model, time of HIV infection of > 10 years and use of amprenavir were independently associated with having more DRV resistance mutations. In the genotyping-based model, only total number of PI resistance mutations was associated with our outcome. In conclusion, the prevalence of DRV mutations was low. Time of HIV infection, use of amprenavir and total number of PI resistance mutations were associated with having more DRV mutations.

Impact of HIV-1 Group O Genetic Diversity on Genotypic Resistance Interpretation by Algorithms Designed for HIV-1 Group M

HIV-1 group O (HIV-O) is characterized by a high genetic divergence from HIV-1 group M viruses. Little is known about the therapeutic impact of this diversity. The aim of this study was to assess in a large series of samples (1) the genotypic impact of natural polymorphism of the HIV-O reverse transcriptase and protease genes; and (2) the predictive value of resistance interpretation algorithms developed for HIV-1 group M when used for highly mutated HIV-O viruses. Sixty-eight antiretroviral-naive and 9 highly antiretroviral-experienced HIV-O-infected patients were included. The viruses were sequenced and resistance-associated mutations were identified using 3 different algorithms (Agence Nationale de Recherches sur le SIDA et les hépatites virales, Rega, Stanford). All HIV-O samples naturally exhibited the A98G and V179E resistance mutations in the reverse transcriptase region; 54% of samples presented the Y181C mutation, conferring resistance to nonnucleoside reverse transcriptase inhibitors. Twelve minor resistance mutations, present in more than 75% of the protease sequences, led to the different algorithms giving discrepant results for nelfinavir and saquinavir susceptibility. A marked virological response was observed in 8 of the 9 antiretroviral-experienced patients, despite the prediction of limited activity of the combination for 5 to 8 patients according to the algorithm used. The high level of natural polymorphism in HIV-O genes, and the important discrepancies between genotypic resistance interpretation and the virological response, emphasize the need for resistance algorithm rules better adapted to HIV-O.

Characterization of HIV‐1 from patients with virological failure to a boosted protease inhibitor regimen

The use of highly active antiretroviral treatment (HAART) regimens with unboosted protease inhibitors (PIs) has resulted in a high level of virological failure primarily due to the development of resistant virus. Current boosted PI regimens combine successfully low-dose ritonavir (r) with a second PI. The aim of the study was to estimate the proportion of patients, in a population based setting, who develop virological failure on a PI/r regimen. Through The Danish HIV Cohort Study 1,007 patients who received PI/r based treatment between 1995 and 2008 were identified. Twenty-three (2.3%) experienced virological failure, of whom 19 (83%) started PI/r treatment before 2001. Patients from Copenhagen (n=19) were selected to study the development of protease (PR) and gag cleavage site (CS) mutations during PI/r treatment and PI plasma levels at the time of virological failure. Three patients (16%) developed major PI resistance mutations. Mutations in the p7/p1 and p1/p6 gag CS only developed in patients with major or minor mutations in PR. Drug concentrations were low or undetectable in 10 out of the 19 patients. In total PR resistance mutations and low drug levels could account for 12 (63%) of the failure cases. In conclusion, virological failure to PI/r is a low and decreasing problem primarily caused by low plasma drug levels and to a lesser extent major PR mutations. Gag CS mutations did not contribute significantly to resistance development and virological failure.

Viral response in stable patients switching to fosamprenavir/ritonavir monotherapy (the FONT Study)

The aim of the study was to evaluate the efficacy of fosamprenavir/ritonavir (FPV/r) monotherapy in plasma and reservoirs in virologically suppressed patients. A 48-week, prospective, single-arm pilot trial was carried out (trial registration: ISRCTN78584791). Patients receiving triple therapy [FPV/r plus two nucleoside reverse transcriptase inhibitors (NRTIs) for at least the previous month], with viral load (VL) <40 HIV-1 RNA copies/mL and no previous virological failure (VF) on protease inhibitors (PIs), were included in the trial and received FPV/r monotherapy (700/100 mg/12 h). VL and FPV/r levels [by liquid chromatography-tandem mass spectrometry (LC/MS/MS); limit of detection (LOD) 0.5 ng/mL] in cerebrospinal fluid (CSF) were determined at week 24. VF was defined as VL >40 copies/mL in three consecutive samples or >500 copies/mL in two samples. Enrolment was prematurely stopped because of a high percentage of VF. Twenty patients (45% men; median age 43.5 years) were included in the trial. Nine patients (45%) presented therapeutic failure [seven (35%) had VF, and two discontinued therapy]. Resistance testing was available in five patients. One patient presented major PI mutations (54L, 32I and 47V) in addition to one minor mutation (13V), whereas two patients had minor PI mutations (10V+36I and 71T, respectively). The patient with major PI mutations switched from FPV/r to darunavir/r and VL was re-suppressed. In the other six patients with VF, VL was re-suppressed after the reintroduction of NRTIs. VL was <40 copies/mL in all CSF samples (n=10). Median amprenavir plasma levels were 2.5 μg/mL (range 0.7-8.6 μg/mL) at week 24 and 2.5 μg/mL (range 0.4-3.8 μg/mL) at VF. The CSF amprenavir concentration was 28.1 ng/mL (range 6.39-83.6 ng/mL), exceeding the reported 50% inhibitory concentration (IC(50) ) range for CSF in nine of 11 patients. The high percentage of patients with VF in our study suggests that the use of FPV/r in a simplification monotherapy strategy should be discouraged. Adequate amprenavir levels and undetectable VL in CSF were documented in all samples evaluated.

Genotypic prediction of resistant mutation in HIV-1 pol gene towards the antiretroviral drugs

Mutation in HIV-1 pol gene confers the resistance to antiretroviral drugs. Three nucleotide sequences of HIV-1 pol gene products were selected and HIV BLAST was performed. Amino acid positions of 30 sequences were compared with HXB2 reference strain. Two sequences of protease show the major mutation with mutations sites of I54V and V82A and 8 sequences shown other mutations. One sequence of integrase with minor mutation of E157Q and 9 sequences with other mutations were inferred. This study is useful in making more accurate prediction of results with better combination Highly Active Antiretroviral Therapy (HAART) and important mutations.

The HIV Type 1 Protease L10I Minor Mutation Decreases Replication Capacity and Confers Resistance to Protease Inhibitors

The effect of minor mutations in PR on treatment outcome has not been well established. We characterized the HIV protease minor mutations, L10I, compared to the minor mutation, L63P, and the major mutation D30N and their impact on viral fitness and resistance to protease inhibitors. Mutations were introduced individually and in combination by site-directed mutagenesis into the provirus pNL4.3ren and constructs used for replication capacity (RC) and resistance assays. A structure prediction of the protease carrying the L10I mutation was determined. The prevalence of the minor mutation L10I had a pattern similar to that found for major mutations D30N, with a low prevalence (4.9%) in naive patients and significantly higher prevalence in treated patients. Furthermore, viruses carrying the major mutation D30N or the minor mutation L10I showed a significant decrease in RC (p-value <0.05), whereas viruses carrying the minor mutation L63P had RC similar to wild-type virus. In addition, the L10I mutation conferred resistance to saquinavir, which was supported by the higher prevalence in the cohort of the L10I mutation among patients with SQV resistance. The molecular modeling suggests that L10I may affect the conformation of Leu-23, a critical residue in the substrate binding site. In conclusion, the L10I mutation impairs RC and confers resistance to SQV, similarly to other major mutations, which may be related with changes in the conformation in the protease binding site. The presence of this mutation in the genotype of HIV from patients should be taken into consideration when designing new optimize treatments.

Minority HIV mutation detection in dried blood spots indicates high specimen integrity and reveals hidden archived drug resistance

Background Dried blood spots (DBS) could serve as an attractive, cost-effective alternative to plasma for HIV drug resistance testing. Objectives To assess the utility and potential gain in genotypic information with sensitive testing of DBS compared to conventional bulk plasma genotyping, and examine the correlation of majority and minority-level resistance mutations in DBS with treatment history. Study design Evaluate nucleic acids from the DBS of 33 antiretroviral-experienced subtype B-infected subjects for minority M41L, K65R, K70R, K103N, Y181C, M184V, and T215Y/F mutations by real-time PCR. Compare minority resistance mutations in DBS with bulk genotypes from the same DBS cards and available plasma specimens. Results All but one (50/51, 98%) mutation from the original plasma bulk sequencing were still detectable in the DBS after three years of storage. The one mutation not identified in DBS was also no longer detectable by bulk sequencing. Furthermore, sensitive testing found 12 additional drug resistance mutations at minority levels in the DBS of 11 (33%) patients. Six minority mutations were in the RNA compartment and six were detected only in the DNA compartment. Resistance was detected in the DBS RNA compartment only in cases where the associated drug was in use within one year of sample collection. Conclusions Our ability to identify majority and additional minority-level resistance mutations demonstrated that DBS, if stored properly, is a high-integrity specimen type for conventional and sensitive drug resistance testing. Our data further support the global utility of DBS for drug resistance surveillance and clinical monitoring.

Relevance of Minor PIG-A Mutations In Acquired Aplastic Anemia and Myelodysplastic Syndromes

AbstractAbstract 4433 Introduction:Aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndromes (MDS) are acquired bone marrow failure diseases. About 15% AA patients eventually transform to classical PNH; however, MDS patients almost never evolve to classical PNH. PNH originates from a multipotent HSC that acquires a PIG-A mutation. PIG-A gene mutations lead to absence of glycosylphosphatidylinositol-anchor proteins (GPI-AP). In PNH, matching mutations can be found in myeloid, erythroid and lymphoid lineages. Roughly 1 in 50,000 (0.002%) colony-forming-cells (CFC) from healthy controls harbor PIG-A mutations. However, PIG-A mutations in healthy controls arise from progenitors rather than multipotent HSC; and are often transient. More than 50% AA patients and 10~30% MDS patients were reported to have small populations (0.01 to 10%) of PNH granulocytes. The objective of this study is to determine the origin of PIG-A mutations in patients with AA and MDS. Method:A prospective study of 3 AA patients with small PNH granulocyte populations and 17 MDS patients was performed. AA and MDS were diagnosed as per clinical criteria and WHO classification (2008) respectively. The median age for AA patients was 49 (range: 28~66). The median age for MDS patients was 66 (range: 48~79). Two AA patients were managed with supportive care, and 1 received HiCy. Five MDS patients were under observation; 3 each received Aranesp, Revlimid, 5-azacytidine, and MS-275/GM-CSF respectively. PNH granulocyte size was measured by flow cytometry. Bone marrow CD34+ cells were isolated via Ficoll-Paque separation and magnetic selection. Proaerolysin is a toxin that selectively kills GPI-AP intact cells but not PNH cells. PNH CFC assay was performed using proaerolysin-containing methylcellulose culture. T cell enrichment culture was performed using peripheral mononuclear cells, RPMI1640 medium with 10% AB serum, micro-beads coated with antibodies against CD2, CD3, and CD28, rIL-2, in the presence of 1nM proaerolysin. DNA was extracted from individual proaerolysin resistant CFC and T cell colonies and was PCR amplified and sequenced for PIG-A mutation analysis. PIG-A mutations were identified using forward/reverses primers. Result:Of 3 AA patients bearing a small PNH granulocyte population (range: 3.2~3.8%), median granulocyte PIG-A mutation frequency was 1.07% (range: 0.88~1.33%), median bone marrow cellularity was 10% (range: 5~20%), and median CD34+ cell was 0.05% (range: 0.019~0.09%). All AA patients had normal karyotypes. PIG-A mutation analysis on 2 of them showed that PIG-A mutations occurred at multipotent HSC level: 1 had a single base substitution mutation at bp549, switching a C to T in both CFU-Gs and T cells; and the other one had a 3 bps (GTC from bp776 to 778) deletion mutation in cells from both lineages. The third patient had a substitution mutation at bp1005 in CFCs, switching an A to T. Her T cell PIG-A mutation analysis is pending. All mutations were confirmed in 3 independent colonies from each lineage.Of 17 MDS patients, 14 had hypercellular bone marrow, 2 had normal cellularity, and 1 had hypocellularity. Seven had normal karyotypes, 9 had abnormal karyotypes. Only 3 patients (17.6%) had detectable PNH granulocyte populations (range: 0.1~3.2%). Studies on the patient bearing a 3.2% PNH granulocytes indicated that PIG-A mutation occurred at progenitor level and was transient. Proaerolysin-resistant CFC assay only grew 1 proaerolysin-resistant CFU-G in 10 plates with a granulocyte PIG-A mutation frequency of 0.03%. DNA sequence analysis of CFC from that colony demonstrated a PIG-A gene single base T deletion mutation at bp 210. However, no PNH T cells were detected via either flow cytometry or T cell enrichment culture. After 4 months of supportive care, he no longer had detectable PNH cells via either flow cytometry or proaerolysin-resistant CFC colony assay. Another patient, who initially bore a 1.5% PNH granulocyte population, also had his PNH granulocyte population disappeared after 2 months of therapy with 5-azacytidine. The 3rd patient is still on surveillance. No PNH T cells were detected in last 2 patients either. Conclusion:Similar to classical PNH, PIG-A mutations in AA are clonal and arise from multipotent HSC. However, PIG-A mutations in MDS arise from progenitor cells, which likely explains why PNH populations in MDS patients are transient and seldom evolve into classical PNH. Disclosures:No relevant conflicts of interest to declare.

Dynamics and Characteristics of BCR-ABL Multiple Mutations In Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia.

AbstractAbstract 3443BCR-ABL kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment.From January 2002 to June 2010 at Seoul St Mary's Hospital, 277 CML patients were screened for mutation analysis due to sign of resistance to tyrosine kinase inhibitors including imatinib, nilotinib, dasatinib or bosutinib. We found that 95 patients have point mutation in BCR-ABL kinase domain through direct sequencing or ASO-PCR. Among them, 17 patients showed multiple mutation containing more than one type of point mutations in BCR-ABL KD.We investigated the patients with multiple mutations to characterize its clinical relevance and dynamics. Once mutation found, follow-up samples from the corresponding patients were collected and analyzed prospectively, or mutation status was analyzed retrospectively with cryopreserved samples if they were available.Status of the patients with multiple mutation is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 17 patients. Cloning of cDNA region corresponding to BCR-ABL KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing.Multiple mutations of 88% patients (15 out of 17) existed compound mutation which means the individual mutant types are located on the same BCR-ABL molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL gene in patients with point mutation are relatively unstable.Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutation as treatment went on. Currently investigation of clinical relevance of compound mutation and other analyses are being carried on and more results will be provided in detail at the conference. Disclosures:No relevant conflicts of interest to declare.

The Usage of Codons Which are Similar to Stop Codons in the Genomes of Xylella fastidiosa and Xanthomonas citri

During the evolution of living organisms, a natural selection event occurs toward the optimization of their genomes regarding the usage of codons. During this process which is known as codon bias, a set of preferred codons is naturally defined in the genome of a given organism, since there are 61 possible codons (plus 3 stop codons) to 20 amino acids. Such event leads to optimization of metabolic cellular processes such as translational efficiency, RNA stability and energy saving. Although we know why, we do not know how exactly a set of preferred codons for each amino acid is defined for a given genome considering that the usage frequency of each synonymous codons is peculiar to each organism. In order to help answering this question, we analyzed the usage frequency of codons which are similar to stop codons, since a minor mutation on these codons may lead to a stop codon into the open reading frame compromising the protein expression as a result. We found a reduced use of those codons in Xanthomomas axonopodis pv. citri which presents an optimized genome regarding codon usage. On the other hand, such codons are more often used in Xylella fastidiosa, which does not seem to have established codon preferences as previously shown. Our results support that a set of preferred codons is not randomly selected and propose new ideas to the field warranting further experiments in this regard.

The Persistence of Influenza Infection

To the Editor: The report by Pinsky et al. (1) is interesting, but it raises some major questions. The finding of influenza virus in stool is not new (2). Of more interest is their statement regarding the prolonged shedding of influenza virus in the stool (for >2 months) and respiratory secretions (for >1.5 years). How frequently were respiratory samples collected and tested to confirm that the same virus was shed for these periods in these samples? Influenza virus, like most other acute respiratory viruses, typically does not cause long-term latent or persistent infections in humans. The authors need to exclude the possibility of frequent reinfection with contemporary circulating seasonal hemagglutinin 1 (H1) influenza viruses. However, they do not provide any data to this effect. Currently, with the wider availability and more stringent expectations of modern molecular techniques, such data might be obtained by collecting and sequencing several genes (ideally full genomes) from contemporary circulating seasonal H1 viruses and comparing them, phylogenetically, with the virus shed, contemporaneously, from the child, at monthly intervals, for example (if the child tolerates this testing). Even with this testing frequency, several influenza infection episodes may go undetected. Although the child’s virus, if it truly persists, may undergo some minor host-induced mutations, new infections with seasonal H1 viruses will likely demonstrate a greater, sudden sequence variability, which enables them to be relatively easily distinguished from the more minor, gradually accumulated mutations that can be seen in a persisting infection (3). Second, ribavirin is not recommended for treating influenza infection (4,5). Can the authors explain why this child was taking ribavirin for influenza infection, and how often was his condition treated with this drug during the 1.5 years when influenza H1 was shed? Was his treatment regimen eventually changed? Currently, the recommended treatment for influenza is with the neuraminidase inhibitors (oseltamivir, zanamivir), which have a much safer adverse effect profile, and their effectiveness has been shown to be cost-effective (5).

HIV-1 Integrase Sequence Variability in Antiretroviral Naïve Patients and in Triple-Class Experienced Patients Subsequently Treated with Raltegravir

Viruses were sequenced from 75 antiretroviral therapy (ARV)-naïve and from 75 ARV-treated patients who subsequently received a raltegravir-containing regimen. No major integrase inhibitor (INI)-resistance mutations were present in the 150 integrase (IN) sequences. Four ARV-naïve (5.3%) and two ARV-treated patients (2.7%) had one of the following minor INI-resistance mutations: L74M, E157Q, G163R, and R263K but there was no association between baseline raltegravir genotype and subsequent response to raltegravir treatment. We also combined our sequences with 4170 previously published group M IN sequences from INI-naïve patients to assess IN sequence variability and compared our findings with those of a study we performed in 2008 using data from 1563 patients. The number of polymorphic IN positions increased from 40% to 41% between the two studies. However, none of the major INI-resistance mutations was found to be polymorphic in either study and there were no significant changes in the prevalence of any of the minor INI-resistance mutations.

Drug Resistance Mutations in Drug-Naive HIV Type 1 Subtype C-Infected Individuals from Rural Malawi

In this preliminary study we show that in 2008, 3 years after antiretroviral therapy was introduced into the Karonga District, Malawi, a greater than expected number of drug-naive individuals have been infected with HIV-1 subtype C virus harboring major and minor drug resistance mutations (DRMs). From a sample size of 40 reverse transcriptase (RT) consensus sequences from drug-naive individuals we found five showing NRTI and four showing NNRTI mutations with one individual showing both. From 29 protease consensus sequences, again from drug-naive individuals, we found evidence of minor DRMs in three. Additional major and minor DRMs were found in clonal sequences from a number of individuals that were not present in the original consensus sequences. This clearly illustrates the importance of sequencing multiple HIV-1 variants from individuals to fully assess drug resistance.

Variation of the V3 loop tip motifs and primary drug resistance of HIV-1 strains

To study the characteristics of the variation of the V3 loop tip motifs and the drug resistance in the primary treatment patients. The partial region of the HIV-1 env and pol gene in 51 samples was amplified by nested polymerase chain reaction (PCR) ,purified products were cloned into the vectors, the obtained were analyzed by MEGA soft wares. The V3 loop tip motifs had four types in our study (GPGR, GPGQ, GPGK, GQGR); the study on the drug resistance in primary treatment patients, showed that there were not major resistance associated with PI, and the resistance were minor mutations in protease gene. In the RT region, there were nine resistance mutants were single NRTIs or NNRTIs. The GPGR which was the typical western V3 loop tip motifs attained to 44.44%. This results showed that the percentage of primary drug resistance was still low in our study region, suggesting no need for genotyping detection in blood donor patients before primary therapy.

Autocatalytic maturation, physical/chemical properties, and crystal structure of group N HIV‐1 protease: Relevance to drug resistance

The mature protease from Group N human immunodeficiency virus Type 1 (HIV-1) (PR1(N)) differs in 20 amino acids from the extensively studied Group M protease (PR1(M)) at positions corresponding to minor drug-resistance mutations (DRMs). The first crystal structure (1.09 Å resolution) of PR1(N) with the clinical inhibitor darunavir (DRV) reveals the same overall structure as PR1(M), but with a slightly larger inhibitor-binding cavity. Changes in the 10s loop and the flap hinge propagate to shift one flap away from the inhibitor, whereas L89F and substitutions in the 60s loop perturb inhibitor-binding residues 29-32. However, kinetic parameters of PR1(N) closely resemble those of PR1(M), and calorimetric results are consistent with similar binding affinities for DRV and two other clinical PIs, suggesting that minor DRMs coevolve to compensate for the detrimental effects of drug-specific major DRMs. A miniprecursor (TFR 1-61-PR1(N)) comprising the transframe region (TFR) fused to the N-terminus of PR1(N) undergoes autocatalytic cleavage at the TFR/PR1(N) site concomitant with the appearance of catalytic activity characteristic of the dimeric, mature enzyme. This cleavage is inhibited at an equimolar ratio of precursor to DRV (∼6 μM), which partially stabilizes the precursor dimer from a monomer. However, cleavage at L34/W35 within the TFR, which precedes the TFR 1-61/PR1(N) cleavage at pH ≤ 5, is only partially inhibited. Favorable properties of PR1(N) relative to PR1(M) include its suitability for column fractionation by size under native conditions and >10-fold higher dimer dissociation constant (150 nM). Exploiting these properties may facilitate testing of potential dimerization inhibitors that perturb early precursor processing steps.