Technological and cost comparison of cytochrome P450 2B6 (516G>T) genotyping methods in routine clinical practice

Pharmacogenetics requires robust and affordable tests to determine genetic variability. This study compares three genotyping methods: gene re-sequencing, real time polymerase chain reaction (PCR) allelic discrimination and PCR-RFLP for the detection of a genetic variation (516G>T) in the gene which codes for the enzyme, CYP2B6, the main enzyme in the metabolic pathway of the antiretroviral drug, efavirenz. The CYP2B6 (516G>T) variant has reduced metabolic capacity. Twenty (20) samples obtained from human immunodeficiency virus acquired immunodeficiency syndrome (HIV/AIDS) positive patients on an efavirenz containing regimen were used to establish whether these methods produce the same CYP2B6 genotype results on the same samples. Results were directly compared for concordance and revealed a 100% correlation with all three methods. Comparison for cost of equipment and reagents required for each method revealed an order of: sequencing > real time-PCR > PCR-RFLP. This study demonstrates the reproducibility of these three methods and provides an opportunity for the clinical applicability in routine clinical practice.Keywords: Polymorphism, drug response, efavirenz, Zimbabwe.African Journal of Biotechnology Vol. 12(19), pp. 2706-271

Identification and differentiation of Fusarium species using selected molecular methods

The taxonomy of Fusarium at the species level is based on morphological characteristics which include hyphae, conidia and microconidia. These features require expertise in taxonomy for an accurate and reliable diagnosis which is crucial as it aids in disease management and genetic diversity studies. This study aimed to develop alternative and/ or complementary taxonomic tools through the use of molecular based techniques. Initially, 113 morphologically identified Fusarium isolates were selected for polymerase chain reaction (PCR) amplification with a set of six inter-simple sequence repeat (ISSR) primers and four universally-primed (UP-PCR) primers to establish identifiable differences and similarities. Genetic variation was also assessed by amplification and sequencing of internally transcribed spacer (ITS) regions of rDNA from 13 isolates using Fusarium specific primers. ISSR amplifications employing four primers and UP-PCR analysis using one primer pair revealed scorable polymorphisms among an average of 86 isolates per primer. Some of the isolates did not yield amplifiable DNA with the selected primers used. The four ISSR primers yielded a total of 500 polymorphic bands and the UP-PCR primer pair revealed 126 polymorphic bands. Genetic similarities among the isolates were calculated using Jaccard’s coefficient while cluster analysis was used to generate dendrograms showing genetic relationships. The isolates were grouped according to similarity levels. Results obtained indicated a high degree of genetic variability in the genus Fusarium. High intraspecies diversity was observed in Fusarium oxysporum and Fusarium solani isolates. Some unexpected genetic similarities were observed among the isolates indicating non-agreement between morphological and molecular identification of the isolates. This suggests the need to use species-specific primers in further analyzing the revealed genetic relationships. Sequencing of the amplified ITS regions among the 13 Fusarium species revealed two groups with 85% genetic similarity. The ITS regions in the two groups showed a relative similarity ranging from 87 to 100%. A 100% genetic similarity was noted between two F. oxysporum isolates which indicated an agreement between morphological and molecular identification. Another 100% genetic similarity was noted among three species, F. moniliforme, F. pallidoroseum and F. lateritium suggesting that species designation can be unreliable if based on morphological data alone. Based on the overall results, the use of molecular methods constitutes an important complement of the morphological criteria needed to allow fungi to be more easily identifie

HIV-1 Genetic Diversity and Natural Polymorphisms of the Integrase Gene in Integrase Inhibitor-Naive Patients in Harare, Zimbabwe.

Previously used as part of salvage therapy, integrase strand transfer inhibitors (INSTIs) have become part of the preferred antiretroviral therapy (ART) first-line regimen in most low- to middle-income countries. With the extensive use of dolutegravir in first-line ART, drug resistance mutations to INSTIs are inevitable. Therefore, active monitoring and surveillance of INSTI drug resistance is required. The aim of this study was to evaluate the genetic diversity of the integrase gene and determine pretreatment INSTI resistance in Harare, Zimbabwe. Forty-four HIV-1 Integrase sequences from 65 were obtained from treatment-naive individuals using a custom genotyping method. Drug resistance mutations were determined using the Stanford HIV Drug Resistance Interpretation program. Viral subtyping was done by phylogenetic analysis and the REGA HIV subtyping tool determined recombinants. Natural polymorphisms were evaluated relative to the global subtype B and C consensus sequences. One hundred ninety-two sequences from the region were accessed from GenBank to assess differences between the Zimbabwean sequences and those from neighboring countries. No major INSTI resistance mutations were detected; however, the L74I polymorphism was detected in three sequences of the 44 (6.8%). There was little genetic variability in the Integrase gene, with a mean genetic distance range of 0.053015. The subtype C consensus was identical to the global subtype C consensus and varied from the global subtype B consensus at five major positions: T124A, V201I, T218I, D278A, and S283G. This study has provided baseline sequence data on the presence of HIV-1 subtype C Integrase gene drug resistance mutations from Harare, Zimbabwe