Helicobacter pylori Mutations Conferring Resistance to Fluoroquinolones and Clarithromycin among Dyspeptic Patients Attending a Tertiary Hospital, Tanzania.

Objectives. Helicobacter pylori (H. pylori) isolates resistant to clarithromycin and quinolones are increasing worldwide. Data regarding the magnitude of H. pylori resistance are limited in developing countries. Here, we report the prevalence of mutations conferring resistance to clarithromycin and fluoroquinolones among dyspeptic patients attending a tertiary hospital, Tanzania. Methods. Between August 2014 and August 2016, patients undergoing upper gastrointestinal endoscopy at the Bugando Medical Centre were enrolled. Biopsies were taken for polymerase chain reaction (PCR) and sequencing to detect mutations conferring resistance to clarithromycin and fluoroquinolones. Results. A total of 208 nonrepetitive biopsies were examined of which 188 (90.4%) tested positive for H. pylori specific 23S rRNA PCR. Clarithromycin resistance mutations were detected in 54/188 (28.7%) of patients tested. The most frequently detected mutation was A2143G (30) followed by A2142G (20). Out of 131 nonrepetitive biopsies tested for fluoroquinolones resistance mutations, 77/131 (58.8%) were positive, with N87I (20) mutation being the most frequently detected mutation followed by A92T mutation which was detected in 16 samples. Conclusion. A significant proportion of dyspeptic patients attending tertiary hospital in Tanzania are infected with H. pylori strains harbouring clarithromycin or fluoroquinolones resistance mutations. Detection of more than 50% of strains with fluoroquinolones resistance mutations makes the H. pylori second line treatment questionable in our setting. There is a need of surveillance of H. pylori resistance patterns in Tanzania to provide data that can guide empirical treatment to reduce associated morbidity of H. pylori infections. The correlation between A92T fluoroquinolone mutation and phenotypic resistance requires further investigations.

Progressive Dispersion of Azole Resistance in Aspergillus fumigatus: Fatal Invasive Aspergillosis in a Patient with Acute Myeloid Leukemia Infected with an A. fumigatus Strain with a cyp51A TR46 Y121F M172I T289A Allele.

Patients with hematologic malignancies as well as allogeneic hematopoietic stem cell transplantation (HSCT) patients are at high risk for invasive aspergillosis. Here, we report a culture- and autopsy-proven fatal invasive aspergillosis in an allogeneic HSTC patient which he developed despite posaconazole prophylaxis. The agent was determined to be an azole-resistant Aspergillus fumigatus strain bearing the cyp51A mutation combination TR46 Y121F M172I T289A. At increasing frequency, the azole resistance of A. fumigatus is being reported globally, limiting treatment options and complicating regimens.

Facultative sterol uptake in an ergosterol-deficient clinical isolate of Candida glabrata harboring a missense mutation in ERG11 and exhibiting cross-resistance to azoles and amphotericin B.

We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 µg ml(-1), respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium ((glc)YM). However, when grown on sterol-supplemented (glc)YM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ(7)-cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented (glc)YM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using (glc)YM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using (glc)YM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 µg AMB ml(-1), respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata.

Transferrin receptor induction in Toxoplasma gondii-infected HFF is associated with increased iron-responsive protein 1 activity and is mediated by secreted factors.

Infection with the apicomplexan parasite Toxoplasma gondii results in a significant alteration of the host-cell transcriptional profile. We have previously shown that the transferrin receptor (TfR) is specifically up-regulated in T. gondii-infected human fibroblasts but not in host cells infected with the bacterial pathogens Salmonella Typhimurium and Chlamydia trachomatis. In this report, we describe the prerequisites and physiological conditions that are associated with this pathogen-specific gene induction. Band-shift assays revealed that T. gondii infection resulted in increased activity in the iron response protein IRP1, which, in this state, stabilizes TfR mRNA from degradation. Although T. gondii depends on host-cell iron as demonstrated by sensitivity to deferoxamine, a parasite-induced iron starvation is not responsible for TfR up-regulation. The increased iron availability due to treatment with holotransferrin and FeNTA did not prevent TfR induction nor was the transferrin-independent iron-transporter NRAMP2 up-regulated in infected host cells. In addition, inhibition of parasite replication by drug treatment did not prevent TfR up-regulation. Instead, TfR induction was sensitive to cycloheximide and could be induced by treatment with conditioned media from infected human fibroblasts. Together our findings suggest that the T. gondii-specific TfR up-regulation is not due to a direct interaction of parasitic factors with the iron-uptake machinery of the host cell but is instead mediated indirectly as a result of secreted host cell- or parasite-derived factors.