A New Variant of Mutational and Polymorphic Signatures in the ERG11 Gene of Fluconazole-Resistant Candida albicans.

Background: Resistance to antifungal drugs for treating Candida infections remains a major concern globally despite the range of medications available. Most of these drugs target key proteins essential to the life cycle of the organism. An enzyme essential for fungal cell membrane integrity, lanosterol 14-α demethylase (CYP51), is encoded by the ERG11 gene in Candida species. This enzyme is the target of azole-based drugs. The organism has, however, devised molecular adaptations to evade the activity of these drugs. Materials and Methods: Classical methods were employed to characterize clinical isolates sampled from women and dogs of reproductive age. For fluconazole efficacy studies, CLSI guidelines on drug susceptibility testing were used. To understand the susceptibility pattern, various molecular and structural analytic approaches, including sequencing, in silico site-directed mutagenesis, and protein-ligand profiling, were applied to the ERG11 gene and CYP51 protein sequences. Several platforms, comprising Clustal Omega, Pymol plugin manager, Pymol molecular visualizer, Chimera-curated Dynameomics rotamer library, protein-ligand interaction profiler, Charmm36 force field, GROMACS, Geneious, and Mega7, were employed for this analysis. Results: The following Candida species distribution was obtained: 37.84% C. albicans, 8.12% C. glabrata, 10.81% C. krusei, 5.41% C. tropicalis, and 37.84% of other unidentified Candida species. Two codons in the nucleotide sequence of the wild-type (CTC and CCA) coding for LEU-370 and PRO-375, respectively, were mutated to L370S and P375H in the resistant strain. The mutation stabilized the protein at the expense of the heme moiety. We found that the susceptible isolate from dogs (Can-iso-029/dog) is closely related to the most resistant isolate from humans. Conclusion: Taken together, our results showed new mutations in the heme-binding pocket of caCYP51 that explain the resistance to fluconazole exhibited by the Candida isolates. So far, the L370S and P375H resistance-linked mutations have not been previously reported.

PREVALENCE OF TUBERCULOSIS, DRUG-RESISTANT TUBERCULOSIS AND HIV/TB CO-INFECTION IN ENUGU, NIGERIA.

BACKGROUND: Tuberculosis (TB) remains a global public health problem, with developing countries bearing the highest burden. Nigeria is first in Africa and sixth in the world among the countries with the highest TB burden, but is among the 10 countries accounting for over 70% of the global gap in TB case detection and notification. Enugu State, Nigeria reportedly has a notification gap of almost 14,000 TB cases; a situation which must be addressed. MATERIALS AND METHODS: A total number of 868 individuals accessing DOTS services in designated centres within the six Local Government Areas (LGAs) of Enugu North geographical zone, was recruited into the study. The participants were screened for HIV seropositivity by standard protocols, while screening for TB and drug-resistant TB were conducted by a combination of Zhiel Neelsen staining and Nucleic Acid Amplification Test (Xpert® MTB/Rif). RESULTS: Of the 868 subjects that participated in the study, 176 (20.3%) were HIV seropositive. The highest prevalence (26.7%) of HIV was recorded in Udenu LGA, while the least (13.1%) was recorded in Nsukka LGA. Overall TB prevalence was found to be 22.1% and 21.3% by sputum-smear and NAAT, respectively. Uzo Uwani LGA recorded the highest prevalence of both TB (33.3%) and TB/HIV co-infection (16.7%), but the lowest prevalence of resistant TB. Nsukka LGA had the highest prevalence of resistant TB. CONCLUSION: Enugu North geographical zone, Nigeria, has a high prevalence of both HIV and TB, including resistant TB and there is need to increase monitoring of individuals resident in this region.

Rifampicin resistance patterns and dynamics of tuberculosis and drug-resistant tuberculosis in Enugu, South Eastern Nigeria.

INTRODUCTION: Tuberculosis (TB) continues to be a public health problem globally. The burden is further exacerbated in developing countries like Nigeria, by poor diagnosis, management and treatment, as well as rapid emergence of drug-resistant TB. This study was conducted to evaluate the prevalence of drug-resistant TB, determine the rpoB gene mutation patterns of Mycobacterium tuberculosis (MTB) and model the dynamics of multidrug resistant TB (MDR-TB) in Enugu, Nigeria. METHODOLOGY: A total of 868 samples, from patients accessing DOTS services in designated centres within the zone, were screened by sputum-smear microscopy, while 207 samples were screened by Nucleic Acid Amplification (Xpert® MTB/Rif) Test (NAAT). A deterministic model was formulated to study the transmission dynamics of TB and MDR-TB, using live data generated through epidemiological study. RESULTS: The results showed TB prevalence values of 22.1% and 21.3% by sputum-smear and NAAT assays, respectively. Analysis of the rifampicin resistance patterns showed the highest occurrence of mutations (50%) along codons 523 - 527. Factors such as combination therapy, multiple therapy and compliance to treatment had influence on both prevalence and development of TB drug resistance in the population. CONCLUSIONS: This first documentation of Rifampicin resistance patterns in MTB from Nigeria shows that a majority of rpoB gene mutations occurred along codons 523 to 527, contrary to the widely reported codon 531 mutation and that multiple interventions such as combination therapy, with good compliance to treatment are needed to reduce both prevalence and development of TB drug resistance in the population.

Rifampicin resistance patterns and dynamics of tuberculosis and drug-resistant tuberculosis in Enugu, South Eastern Nigeria

Introduction: Tuberculosis (TB) continues to be a public health problem globally. The burden is further exacerbated in developing countries like Nigeria, by poor diagnosis, management and treatment, as well as rapid emergence of drug-resistant TB. This study was conducted to evaluate the prevalence of drug-resistant TB, determine the rpoB gene mutation patterns of Mycobacterium tuberculosis (MTB) and model the dynamics of multidrug resistant TB (MDR-TB) in Enugu, Nigeria.Methodology: A total of 868 samples, from patients accessing DOTS services in designated centres within the zone, were screened by sputum-smear microscopy, while 207 samples were screened by Nucleic Acid Amplification (Xpert® MTB/Rif) Test (NAAT). A deterministic model was formulated to study the transmission dynamics of TB and MDR-TB, using live data generated through epidemiological study.Results: The results showed TB prevalence values of 22.1% and 21.3% by sputum-smear and NAAT assays, respectively. Analysis of the rifampicin resistance patterns showed the highest occurrence of mutations (50%) along codons 523 ­ 527. Factors such as combination therapy, multiple therapy and compliance to treatment had influence on both prevalence and development of TB drug resistance in the population.Conclusions: This first documentation of Rifampicin resistance patterns in MTB from Nigeria shows that a majority of rpoB gene mutations occurred along codons 523 to 527, contrary to the widely reported codon 531 mutation and that multiple interventions such as combination therapy, with good compliance to treatment are needed to reduce both prevalence and development of TB drug resistance in the population

Cryptosporidium species and subtypes in diarrheal children and HIV-infected persons in Ebonyi and Nsukka, Nigeria.

INTRODUCTION: Cryptosporidiosis is a common disease of children and immune-compromised persons. This study evaluated the diversity and distribution of Cryptosporidium species in diarrheal children and HIV-infected persons on highly active antiretroviral therapy (HAART) and those not on HAART. METHODOLOGY: A total of 394 fecal specimens were collected from patients attending clinics in Nsukka and Ebonyi, Nigeria. Detection and identification of Cryptosporidium species were conducted by PCR-RFLP of the small subunit (SSU) rRNA gene, whereas subtyping was done by sequence analysis of the 60 kDa glycoprotein (gp60) gene. RESULTS: Twenty-five (6.3%) specimens yielded four Cryptosporidium species, including C. hominis, C. parvum, C. felis, and C. viatorum. C. hominis was the most dominant species with 48.0% occurrence and three identified subtype families: Ia (six specimens), Ib (three specimens), Ie (two specimens), and one un-subtyped species. C. parvum had 44.0% occurrence and two subtype families: IIc (eight specimens) and IIe (three specimens), while C. felis and C. viatorum each had 4.0% occurrence. There were significant differences in Cryptosporidium species distribution between age groups in children and HIV-infected persons, between suburban and urban areas, and between low and high CD4+ cell counts in HIV-infected patients. There were no significant differences in infection rate and species distribution between HIV-infected patients on HAART and those not on HAART. CONCLUSIONS: The results from this study show that there is a high diversity of Cryptosporidium spp. in humans in Ebonyi and Nsukka, Nigeria, and that all the C. parvum subtypes identified are most likely anthroponotic in origin.