Sequence analysis of genes associated with resistance to chloroquine and sulphadoxine pyrimethamine in P. falciparum and P. vivax isolates from the Bannu district of Pakistan

Plasmodium vivax and Plasmodium falciparum are becoming resistant to drugs including antifolates, sulphonamides and chloroquine. This study was focused at sequence analysis of resistant genes of these parasites against sulphadoxine–pyrimethamine and chloroquine, from Bannu, Pakistan. Known mutations were detected at codons 57, 58 and 117 of pvdhfr gene of P. vivax, while none of the isolates had any pvdhps mutation. Similarly P. falciparum isolates exhibited double 59R + 108N mutations in pfdhfr, and single 437G in pfdhps thus demonstrating the existance of triple mutant 59R + 108N + 437G haplotype in this region. The key chloroquine resistance mutation, 76T in pfcrt was observed in 100% of the P. falciparum isolates, with haplotype SVMNT which is also associated with resistance to amodiaquine. Some novel mutations were also observed in pvdhfr and pfdhfr genes.     

Combination Treatment with Hepatitis C Virus Protease and NS5A Inhibitors Is Effective against Recombinant Genotype 1a, 2a,and 3a Viruses

With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3′ untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ∼4 log₁₀ focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.     

Adapted J6/JFH1-Based Hepatitis C Virus Recombinants with Genotype-Specific NS4A Show Similar Efficacies against Lead Protease Inhibitors,Alpha Interferon,and a Putative NS4A Inhibitor

To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log₁₀ focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to alpha interferon 2b, and to the putative NS4A inhibitor ACH-806. The efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (in the NS3 protease), which have been suggested to mediate resistance to ACH-806 in replicons. Genotype 1a, 1b, and 2a recombinants showed viral spread under long-term treatment with ACH-806, without acquisition of resistance mutations in the NS3-NS4A region. Relatively high concentrations of ACH-806 inhibited viral assembly, but not replication, in a single-cycle production assay. The developed HCV culture systems will facilitate studies benefitting from expression of genotype-specific NS4A in a constant backbone in the context of the complete viral replication cycle, including functional studies and evaluations of the efficacy of antivirals.     

Temporal stability of objective structured clinical exams: a longitudinal study employing item response theory

Background

The objective structure clinical examination (OSCE) has been used since the early 1970s for assessing clinical competence. There are very few studies that have examined the psychometric stability of the stations that are used repeatedly with different samples. The purpose of the present study was to assess the stability of objective structured clinical exams (OSCEs) employing the same stations used over time but with a different sample of candidates, SPs, and examiners.

Methods

At Time 1, 191 candidates and at Time 2 (one year apart), 236 candidates participated in a 10-station OSCE; 6 of the same stations were used in both years. Generalizability analyses (Ep²) were conducted. Employing item response analyses, test characteristic curves (TCC) were derived for each of the 6 stations for a 2-parameter model. The TCCs were compared across the two years, Time 1 and 2.

Results

The Ep² of the OSCEs exceeded.70. Standardized thetas (θ) and discriminations were equivalent for the same station across the two year period indicating equivalent TCCs for a 2-parameter model.

Conclusion

The 6 OSCE stations used by the AIMG program over two years have adequate internal consistency reliability, stable generalizability (Ep²) and equivalent test characteristics. The process of assessment employed for IMG’s are stable OSCE stations that may be used several times over without compromising psychometric properties.With careful security, high-stakes OSCEs may use the same stations that have high internal consistency and generalizability repeatedly as the psychometric properties are stable over several years with different samples of candidates.

     

A large unilocular mucinous cystadenoma in third trimester of pregnancy

Ovarian malignancy is the second most common gynaecological malignancy diagnosed during pregnancy. A grand multipara, aged 30 years presented with gestational amenorrhea with abdominal discomfort and breathlessness for last 15 days. Sonographic examination demonstrated a huge, unilocular ovarian cyst and an alive fetus of about 30 weeks gestation. Intraoperative findings were huge left ovarian cyst (42x40x20 cm) with straw coloured mucinous fluid. Left salpingo-oophorectomy was performed followed by peritoneal washings and omental biopsy. Histopathology revealed mucinous cystadenomas with inflammatory changes in omentum and no malignant cells in peritoneal washings. She delivered vaginally a female baby of 3.5 kg at 38 weeks with good Apgar score.