Using (1,3)-ß-D-glucan concentrations in serum to monitor the response of azole therapy in patients with eumycetoma caused by Madurella mycetomatis.

INTRODUCTION: (1,3)-ß-D-glucan is a panfungal biomarker secreted by many fungi, including Madurella mycetomatis, the main causative agent of eumycetoma. Previously we demonstrated that (1,3)-ß-D-glucan was present in serum of patients with eumycetoma. However, the use of (1,3)-ß-D-glucan to monitor treatment responses in patients with eumycetoma has not been evaluated. MATERIALS AND METHODS: In this study, we measured (1,3)-ß-D-glucan concentrations in serum with the WAKO (1,3)-ß-D-glucan assay in 104 patients with eumycetoma treated with either 400 mg itraconazole daily, or 200 mg or 300 mg fosravuconazole weekly. Serial serum (1,3)-ß-D-glucan concentrations were measured at seven different timepoints. Any correlation between initial and final (1,3)-ß-D-glucan concentrations and clinical outcome was evaluated. RESULTS: The concentration of (1,3)-ß-D-glucan was obtained in a total of 654 serum samples. Before treatment, the average (1,3)-ß-D-glucan concentration was 22.86 pg/mL. During the first 6 months of treatment, this concentration remained stable. (1,3)-ß-D-glucan concentrations significantly dropped after surgery to 8.56 pg/mL. After treatment was stopped, there was clinical evidence of recurrence in 18 patients. Seven of these 18 patients had a (1,3)-ß-D-glucan concentration above the 5.5 pg/mL cut-off value for positivity, while in the remaining 11 patients, (1,3)-ß-D-glucan concentrations were below the cut-off value. This resulted in a sensitivity of 38.9% and specificity of 75.0%. A correlation between lesion size and (1,3)-ß-D-glucan concentration was noted. CONCLUSION: Although in general (1,3)-ß-D-glucan concentrations can be measured in the serum of patients with eumycetoma during treatment, a sharp decrease in ß-glucan concentration was only noted after surgery and not during or after antimicrobial treatment. (1,3)-ß-D-glucan concentrations were not predictive for recurrence and seem to have no value in determining treatment response to azoles in patients with eumycetoma.

Novel Compound MMV1804559 from the Global Health Priority Box Exhibits In Vitro and In Vivo Activity against Madurella mycetomatis.

OBJECTIVES: Eumycetoma is a neglected tropical disease (NTD) characterized by subcutaneous lesions and the formation of grains. Attempts to treat eumycetoma involve a combination of antifungal treatment and surgery, although the outcome is frequently disappointing. Therefore, there is a need to identify novel antifungal drugs to treat eumycetoma. In this respect, Medicines for Malaria Venture (MMV) has assembled libraries of compounds for researchers to use in drug discovery research against NTD. Therefore, we screened two MMVOpen compound libraries to identify novel leads for eumycetoma. METHODS: A total of 400 compounds from the COVID Box and the Global Health Priority Box were screened in vitro at 100 µM and 25 µM against the most common causative agents of eumycetoma, namely Madurella mycetomatis and Falciformispora senegalensis, and the resulting IC50 and MIC50 values were obtained. Compounds with an IC50 < 8 µM were identified for possible in vivo efficacy studies using an M. mycetomatis grain model in Galleria mellonella larvae. RESULTS: Out of the 400 compounds, 22 were able to inhibit both M. mycetomatis and F. senegalensis growth at 100 µM and 25 µM, with compounds MMV1593278, MMV020335, and MMV1804559 being selected for in vivo testing. Of these three, only the pyrazolopyrimidine derivative MMV1804559 was able to prolong the survival of M. mycetomatis-infected G. mellonella larvae. Furthermore, the grains in MMV1804559-treated larvae were significantly smaller compared to the PBS-treated group. CONCLUSION: MMV1804559 shows promising in vitro and in vivo activity against M. mycetomatis.

Integrated analysis of viral blips, residual viremia, and associated factors in people with HIV: Results from a retrospective cohort study.

The etiology of viral blips is not yet fully elucidated. One of the hypotheses is that blips reflect variations in residual viremia (RV) near the detectability threshold. In this study, we evaluated whether RV is associated with viral blips and which factors are associated with RV. All treatment regimens in 2010-2020 consisting of two nucleos(-t)ide reverse transcriptase inhibitors and one anchor (integrase strand transfer inhibitor [INSTI], non-nucleoside reverse transcriptase inhibitor [NNRTI], or protease inhibitor [PI]) in people with HIV (PWH) were evaluated for RV (detectable viremia <50 cp/mL) and blips (isolated viral loads [VLs] 50-499 cp/mL between measurements <50 cp/mL). All medical records were reviewed and regimens in which a VL ≥ 50 cp/mL was deemed to result from non-adherence (based on the documented conclusion by the treating physician) were excluded. Factors associated with blips and RV were identified using generalized linear mixed models. In total, 24 518 VLs from 1658 PWH were analyzed. VLs were measured during INSTI- (n = 5119; 20.9%), PI- (n = 8935; 36.4%), and NNRTI-use (n = 10 464; 42.7%). VLs were categorized as blips in 1.4% (n = 332). The 24,186 non-blip VLs were RNAneg (no RV) (n = 15 326; 63.4%), 1-19 cp/mL (n = 6318; 26.1%), 20-49 cp/mL (n = 1620; 6.7%), or <50 cp/mL with an unknown RV level (n = 922; 3.8%). In 193/1658 PWH (11.6%), the RV level was RNAneg in all VLs assessed. RV 1-19 cp/mL and 20-49 cp/mL (vs. RNAneg ) were significantly associated with subsequent viral blips (respective odds ratio 2.66 and 4.90 [95% confidence intervals: 1.98-3.58 and 3.41-7.04]). Zenith VL and use of PIs (vs. INSTIs/NNRTIs) were associated with higher RV and blip odds. This large cohort study showed that blips were associated with higher preceding RV. Both the anchor type and factors previously linked to the latent viral reservoir were associated with RV, suggesting blips having a multifactorial origin.

A Falciformispora senegalensis grain model in Galleria mellonella larvae.

Eumycetoma is a subcutaneous implantation mycosis often found in the foot. One of the hallmarks of eumycetoma is the formation of grains. These grains are either black or white, and the consistency and morphology differs per causative agent. The two most common causative agents of black-grain eumycetoma are Madurella mycetomatis and Falciformispora senegalensis. Since grains cannot be formed in vitro, in vivo models are needed to study grain formation. Here, we used the invertebrate Galleria mellonella to establish an in vivo grain model for F. senegalensis. Three different F. senegalensis strains were selected, and four different inocula were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 10 mg/larvae. Larval survival was monitored for 10 days. Grain formation was studied macroscopically and histologically. The efficacy of antifungal therapy was determined for itraconazole, amphotericin B, and terbinafine. A concentration of 10 mg F. senegalensis per larva was lethal for the majority of the larvae within 10 days. At this inoculum, grains were formed within 24 h after infection. The grains produced in the larvae resembled those formed in human patients. Amphotericin B given at 1 mg/kg 4 h, 28 h, and 52 h after infection prolonged larval survival. No enhanced survival was noted for itraconazole or terbinafine. In conclusion, we developed a F. senegalensis grain model in G. mellonella larvae in which grains were formed that were similar to those formed in patients. This model can be used to monitor grain formation over time and study antifungal efficacy.

Within eumycetoma lesions, the causative agents are embedded in grains. However, the grains differ per causative agent. In this study, we developed a grain model of Falciformispora senegalensis in the larvae of Galleria mellonella. This model can be used in the future to study the efficacy of novel antifungal agents.

The combination of manogepix and itraconazole is synergistic and inhibits the growth of Madurella mycetomatis in vitro but not in vivo.

Mycetoma is a neglected tropical disease commonly caused by the fungus Madurella mycetomatis. Standard treatment consists of extensive treatment with itraconazole in combination with surgical excision of the infected tissue, but has a low success rate. To improve treatment outcomes, novel treatment strategies are needed. Here, we determined the potential of manogepix, a novel antifungal agent that targets the GPI-anchor biosynthesis pathway by inhibition of the GWT1 enzyme. Manogepix was evaluated by determining the minimal inhibitory concentrations (MICs) according to the CLSI-based in vitro susceptibility assay for 22 M. mycetomatis strains and by in silico protein comparison of the target protein. The synergy between manogepix and itraconazole was determined using a checkerboard assay. The efficacy of clinically relevant dosages was assessed in an in vivo grain model in Galleria mellonella larvae. MICs for manogepix ranged from <0.008 to >8 mg/l and 16/22 M. mycetomatis strains had an MIC ≥4 mg/ml. Differences in MICs were not related to differences observed in the GWT1 protein sequence. For 70% of the tested isolates, synergism was found between manogepix and itraconazole in vitro. In vivo, enhanced survival was not observed upon admission of 8.6 mg/kg manogepix, nor in combination treatment with 5.7 mg/kg itraconazole. MICs of manogepix were high, but the in vitro antifungal activity of itraconazole was enhanced in combination therapy. However, no efficacy of manogepix was found in an in vivo grain model using clinically relevant dosages. Therefore, the therapeutic potential of manogepix in mycetoma caused by M. mycetomatis seems limited.

Treatment of Madurella mycetomatis-caused mycetoma consists of extensive exposure to antifungals and surgery. To improve therapy, we evaluated manogepix, a novel antifungal agent, as a therapeutic option against M. mycetomatis. Our findings suggest limited therapeutic potential for manogepix.

Using a Madurella mycetomatis-specific PCR on grains obtained via non-invasive fine-needle aspirated material is more accurate than cytology.

BACKGROUND: Eumycetoma is a chronic subcutaneous inflammatory fungal infection most often caused by the fungus Madurella mycetomatis. Using a species-specific PCR on DNA directly isolated from grains is currently the most reliable method for species identification. However, so far, PCR has been performed on grains obtained through deep-seated surgical biopsies, which are invasive procedures. Grains can also be obtained via ultrasound-guided fine-needle aspiration (US-FNA). Here we determined the diagnostic performance of species-specific PCRs performed on samples obtained through US-FNA. METHODS: From 63 patients, US-FNA was performed to obtain eumycetoma grains; 34 patients also underwent a deep-seated biopsy. From the grains, DNA was isolated, and one pan-fungal and two M. mycetomatis-specific PCRs were performed. The sensitivity and specificity were determined. RESULTS: Of the 63 patients who underwent US-FNA, 78% (49/63) had evidence of eumycetoma based on cytology and 93.7% (59/63) based on species-specific PCRs. In the 34 patients for whom surgical biopsies were performed as well, 31 patients had a positive PCR for M. mycetomatis when DNA was isolated from the deep-seated biopsy, and 30 had a positive PCR when DNA was obtained from the US-FNA material. This resulted in a 96.8% sensitivity, and 100% specificity with 97.1% diagnostic accuracy for PCR performed on US-FNA. CONCLUSION: PCR performed on the US-FNA material has a similar sensitivity and specificity as PCR performed on deep-seated biopsies. Therefore, when using PCR, a deep-seated biopsy may not be necessary to obtain grains.

Comparing the performance of the common used eumycetoma diagnostic tests.

OBJECTIVES: Mycetoma is a neglected tropical implantation disease caused by 70 different infectious agents. Identifying the causative organism to the species level is essential for appropriate patient management. Ultrasound, histopathology, culture and two species-specific PCRs are most the commonly used methods for species identification in endemic regions. The aim of this study was to compare the diagnostic performance of these commonly used assays using sequencing of barcoding genes as the gold standard. METHODS: This descriptive cross-sectional study was conducted at the Mycetoma Research Centre, University of Khartoum, Sudan. It included 222 patients suspected of fungal mycetoma caused by Madurella mycetomatis. RESULTS: 154 (69.3%) were correctly identified by ultrasound, histology, culture and both species-specific PCRs. In 60 patients, at least one of the diagnostic tests failed to identify M. mycetomatis. Five patients had no evidence of eumycetoma, and for three, only the ultrasound was indicative of mycetoma. The two species-specific PCRs were the most sensitive and specific methods, followed by culture and histology. Ultrasound was the least specific as it only allowed differentiation between actinomycetoma and eumycetoma. The time to result was 9.38 minutes for ultrasound, 3.76 hours for PCR, 8.5 days for histopathology and 21 days for grain culturing. CONCLUSION: Currently, PCR directly on DNA isolated from grains is the most rapid and reliable diagnostic tool to identify M. mycetomatis eumycetoma.

Wako ß-D-glucan assay can be used to measure serum ß-D-glucan in Sudanese patients to aid with diagnosis of eumycetoma caused by Madurella mycetomatis.

BACKGROUND: Eumycetoma is a neglected tropical infection of the subcutaneous tissue commonly caused by the fungus Madurella mycetomatis. Previously, we demonstrated that ß-D-glucan was present in the serum of eumycetoma patients. OBJECTIVE: To compare the performance of the recently approved easy-to-use Wako ß-D-glucan assay to that of the Fungitell assay in eumycetoma patients. METHODS: Using sera obtained from 41 eumycetoma, 12 actinomycetoma and 29 healthy endemic controls, we measured the ß-glucan serum concentrations using the Wako assay and compared the performance to that of the Fungitell assay. RESULTS: With the Fungitell assay, median ß-glucan serum concentrations of 208, 70 and 27 pg/ml were obtained for the 41 eumycetoma patients, the 12 actinomycetoma patients and the 29 healthy endemic controls, respectively. With the Wako assay these concentrations were 14.45, 11.57 and 2.5 pg/ml, respectively. We demonstrated that when using the optimized cut-off value (5.5 pg/ml) for the Wako assay, the Wako and Fungitell assays had comparable performance in terms of sensitivity and specificity. CONCLUSION: The Wako assay is comparable to the Fungitell assay for measurement of serum ß-glucan in mycetoma patients and hence can be used in combination with current diagnostic tools. However, this test should be used in combination with other tests to differentiate actinomycetoma from eumycetoma.

Significant Impact of Coronavirus Disease 2019 (COVID-19) on Human Immunodeficiency Virus (HIV) Care in Hospitals Affecting the First Pillar of the HIV Care Continuum.

During COVID-19 lockdown, the in-hospital number of HIV indicator conditions decreased disproportionally compared with other non-COVID-19 diseases, which was accompanied by reduced HIV testing rates, number and proportion of positive HIV tests, and new HIV referrals, with more late presentation after lockdown cessation, indicating a significantly impacted HIV care continuum.

Inhibiting DHN- and DOPA-melanin biosynthesis pathway increased the therapeutic value of itraconazole in Madurella mycetomatis infected Galleria mellonella.

Eumycetoma is a neglected tropical disease, and Madurella mycetomatis, the most common causative agent of this disease forms black grains in hosts. Melanin was discovered to be one of the constituents in grains. Melanins are hydrophobic, macromolecular pigments formed by oxidative polymerisation of phenolic or indolic compounds. M. mycetomatis was previously known to produce DHN-melanin and pyomelanin in vitro. These melanin was also discovered to decrease M. mycetomatis's susceptibility to antifungals itraconazole and ketoconazole in vitro. These findings, however, have not been confirmed in vivo. To discover the melanin biosynthesis pathways used by M. mycetomatis in vivo and to determine if inhibiting melanin production would increase M. mycetomatis's susceptibility to itraconazole, inhibitors targeting DHN-, DOPA- and pyomelanin were used. Treatment with DHN-melanin inhibitors tricyclazole, carpropamid, fenoxanil and DOPA-melanin inhibitor glyphosate in M. mycetomatis infected Galleria mellonella larvae resulted in presence of non-melanized grains. Our finding suggested that M. mycetomatis is able to produce DOPA-melanin in vivo. Inhibiting DHN-melanin with carpropamid in combination with the antifungal itraconazole also significantly increased larvae survival. Our results suggested that combination treatment of antifungals and melanin inhibitors can be an alternative treatment strategy that can be further explored. Since the common black-grain eumycetoma causing agents uses similar melanin biosynthesis pathways, this strategy may be applied to them and other eumycetoma causative agents. LAY SUMMARY: Melanin protects fungi from environmental stress and antifungals. We have discovered that Madurella mycetomatis produces DHN-, pyomelanin and DOPA-melanin in vivo. Inhibiting M. mycetomatis DHN-melanin biosynthesis increases therapeutic value of the antifungal itraconazole in vivo.

Madurella mycetomatis grains within a eumycetoma lesion are clonal.

Eumycetoma is a neglected tropical infection of the subcutaneous tissue, characterized by tumor-like lesions and most commonly caused by the fungus Madurella mycetomatis. In the tissue, M. mycetomatis organizes itself in grains, and within a single lesion, thousands of grains can be present. The current hypothesis is that all these grains originate from a single causative agent, however, this hypothesis was never proven. Here, we used our recently developed MmySTR assay, a highly discriminative typing method, to determine the genotypes of multiple grains within a single lesion. Multiple grains from surgical lesions obtained from 11 patients were isolated and genotyped using the MmySTR panel. Within a single lesion, all tested grains shared the same genotype. Only in one single grain from one patient, a difference of one repeat unit in one MmySTR marker was noted relative to the other grains from that patient. We conclude that within these lesions the grains originate from a single clone and that the inherent unstable nature of the microsatellite markers may lead to small genotypic differences. LAY ABSTRACT: In lesions of the implantation mycosis mycetoma many Madurella mycetomatis grains are noted. It was unknown if grains arose after implantation of a single isolate or a mixture of genetically diverse isolates. By typing the mycetoma grains we showed that all grains within a single lesion were clonal and originated from a single isolate.

Eumycetoma causative agents are inhibited in vitro by luliconazole, lanoconazole and ravuconazole.

INTRODUCTION: Eumycetoma is a subcutaneous mutilating disease that can be caused by many different fungi. Current treatment consists of prolonged itraconazole administration in combination with surgery. In many centres, due to their slow growth rate, the treatment for eumycetoma is often started before the causative agent is identified. This harbours the risk that the causative fungus is not susceptible to the given empirical therapy. In the open-source drug program MycetOS, ravuconazole and luliconazole were promising antifungal agents that were able to inhibit the growth of Madurella mycetomatis, the most common causative agent of mycetoma. However, it is currently not known whether these drugs inhibit the growth of other eumycetoma causative agents. MATERIALS AND METHODS: Here, we determined the in vitro activity of luliconazole, lanoconazole and ravuconazole against commonly encountered eumycetoma causative agents. MICs were determined for lanoconazole, luliconazole and ravuconazole against 37 fungal isolates which included Madurella species, Falciformispora senegalensis, Medicopsis romeroi and Trematosphaeria grisea and compared to those of itraconazole. RESULTS: Ravuconazole, luliconazole and lanoconazole showed high activity against all eumycetoma causative agents tested with median minimal inhibitory concentrations (MICs) ranging from 0.008-2 µg/ml, 0.001-0.064 µg/ml and 0.001-0.064 µg/ml, respectively. Even Ma. fahalii and Me. romeroi, which are not inhibited in growth by itraconazole at a concentration of 4 µg/ml, were inhibited by these azoles. CONCLUSION: The commonly encountered eumycetoma causative agents are inhibited by lanoconazole, luliconazole and ravuconazole. These drugs are promising candidates for further evaluation as potential treatment for eumycetoma.

Epidemiological cut-off values for itraconazole and ravuconazole for Madurella mycetomatis, the most common causative agent of mycetoma.

BACKGROUND: Eumycetoma is a neglected tropical disease. It is a chronic inflammatory subcutaneous infection characterised by painless swellings which produce grains. It is currently treated with a combination of itraconazole and surgery. In an ongoing clinical study, the efficacy of fosravuconazole, the prodrug of ravuconazole, is being investigated. For both itraconazole and ravuconazole, no clinical breakpoints or epidemiological cut-off values (ECV) to guide treatment are currently available. OBJECTIVE: To determine tentative ECVs for itraconazole and ravuconazole in Madurella mycetomatis, the main causative agent of eumycetoma. MATERIALS AND METHODS: Minimal inhibitory concentrations (MICs) for itraconazole and ravuconazole were determined in 131 genetically diverse clinical M. mycetomatis isolates with the modified CLSI M38 broth microdilution method. The MIC distributions were established and used to determine ECVs with the ECOFFinder software. CYP51A sequences were sequenced to determine whether mutations occurred in this azole target gene, and comparisons were made between the different CYP51A variants and the MIC distributions. RESULTS: The MICs ranged from 0.008 to 1 mg/L for itraconazole and from 0.002 to 0.125 mg/L for ravuconazole. The M. mycetomatis ECV for itraconazole was 1 mg/L and for ravuconazole 0.064 mg/L. In the wild-type population, two CYP51A variants were found for M. mycetomatis, which differed in one amino acid at position 499 (S499G). The MIC distributions for itraconazole and ravuconazole were similar between the two variants. No mutations linked to decreased susceptibility were found. CONCLUSION: The proposed M. mycetomatis ECV for itraconazole is 1 mg/L and for ravuconazole 0.064 mg/L.

Comparison of Disc Diffusion, Etest, and a Modified CLSI Broth Microdilution Method for In Vitro Susceptibility Testing of Itraconazole, Posaconazole, and Voriconazole against Madurella mycetomatis.

For many fungal infections, in vitro susceptibility testing is used to predict if an isolate is resistant or susceptible to the antifungal agent used to treat the infection. For Madurella mycetomatis, the main causative agent of mycetoma, in vitro susceptibility testing currently is not performed on a routine basis. The current in vitro susceptibility testing method is labor-intensive, and sonication must be done to generate a hyphal inoculum. For endpoint visualization, expensive viability dyes are needed. Here, we investigated if the currently used in vitro susceptibility method could be adapted to make it amendable for use in a routine setting which can be used in low-income countries, where mycetoma is endemic. First, we developed a methodology in which hyphal fragments can be generated without the need for sonication, by comparing different bead beating methodologies. Next, in vitro susceptibility was assessed using standard broth microdilution assays as well as disc diffusion, Etest, and VIPcheck methodologies. We demonstrate that after a hyphal suspension is generated by glass bead beating, disc diffusion, Etest, and VIPcheck can be used to determine susceptibility of Madurella mycetomatis to itraconazole, posaconazole, and voriconazole. The MICs found with Etest were comparable to those obtained with our modified CLSI-based broth microdilution in vitro susceptibility assay for itraconazole and posaconazole. Furthermore, we found an inverse relationship between the zones of inhibition and MICs obtained with the Etest and those obtained by the modified CLSI broth microdilution technique.

Rapid and Accurate Detection of Aminoglycoside-Modifying Enzymes and 16S rRNA Methyltransferases by Targeted Liquid Chromatography-Tandem Mass Spectrometry.

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in curbing drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside-modifying enzymes and 16S rRNA methyltransferases in Escherichia coli and Klebsiella pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, AAC(6')-Ib-cr, ANT(2″)-I, APH(3')-VI, ArmA, RmtB, RmtC, and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole-genome sequencing results. Of the 2,460 isolate and resistance mechanism combinations tested, 2,416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin, and amikacin in only the E. coli and K. pneumoniae isolates (n = 191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

A Short-Tandem-Repeat Assay (MmySTR) for Studying Genetic Variation in Madurella mycetomatis.

Madurella mycetomatis is the major causative agent of eumycetoma, a neglected tropical infection characterized by painless subcutaneous lesions, inflammation, and grains draining from multiple sinuses. To study the epidemiology of mycetoma, a robust discriminatory typing technique is needed. We describe the use of a short-tandem-repeat assay (MmySTR) for genotyping of M. mycetomatis isolates predominantly from Sudan. Eleven microsatellite markers (3 dinucleotides, 4 trinucleotide repeats, and 4 tetranucleotide repeats) were selected from the M. mycetomatis MM55 genome using the Tandem Repeats Finder software. PCR amplification primers were designed for each microsatellite marker using primer3 software and amplified in a multicolor multiplex PCR approach. To establish the extent of genetic variation within the population, a collection of 120 clinical isolates from different regions was genotyped with this assay. The 11 selected MmySTR markers showed a large genotypic heterogeneity. From a collection of 120 isolates, 108 different genotypes were obtained. Simpson's diversity index (D) value for individual markers ranged from 0.081 to 0.881, and the combined panel displayed an overall D value of 0.997. The MmySTR assay demonstrated high stability, reproducibility, and specificity. The MmySTR assay is a promising new typing technique that can be used to genotype isolates of M. mycetomatis Apart from the possible contribution of host factors, the genetic diversity observed among this group of isolates might contribute to the different clinical manifestations of mycetoma. We recommend that the MmySTR assay be used to establish a global reference database for future study of M. mycetomatis isolates.

Effective antimicrobial combination in vivo treatment predicted with microcalorimetry screening.

OBJECTIVES: The worldwide emergence of antibiotic resistance calls for effective exploitation of existing antibiotics. Antibiotic combinations with different modes of action can synergize for successful treatment. In the present study, we used microcalorimetry screening to identify synergistic combination treatments against clinical MDR isolates. The synergistic effects were validated in a murine infection model. METHODS: The synergy of meropenem combined with colistin, rifampicin or amikacin was tested on 12 isolates (1 Escherichia coli, 5 Klebsiella pneumoniae, 3 Pseudomonas aeruginosa and 3 Acinetobacter baumannii) in an isothermal microcalorimeter measuring metabolic activity. One A. baumannii strain was tested with two individual pairings of antibiotic combinations. The microcalorimetric data were used to predict in vivo efficacy in a murine peritonitis/sepsis model. NMRI mice were inoculated intraperitoneally and after 1 h treated with saline, drug X, drug Y or X+Y. Bacterial load was determined by cfu in peritoneal fluid and blood after 4 h. RESULTS: In vitro, of the 13 combinations tested on the 12 strains, 3 of them exhibited a synergistic reduction in MIC (23% n = 3/13), 5 showed an additive effect (38.5% n = 5/13) and 5 had indifferent or antagonistic effects (38.5% n = 5/13). There was a significant correlation (P = 0.024) between microcalorimetry-screening FIC index values and the log reduction in peritoneal fluid from mice that underwent combination treatment compared with the most effective mono treatment. No such correlation could be found between chequerboard and in vivo results (P = 0.16). CONCLUSIONS: These data support microcalorimetic metabolic readout to predict additive or synergistic effects of combination treatment of MDR infections within hours.

Transmission of NS5A-Inhibitor Resistance-Associated Substitutions Among Men Who Have Sex With Men Recently Infected with Hepatitis C Virus Genotype 1a.

The transmission of direct-acting antiviral resistance-associated substitutions (RAS) could hamper hepatitis C virus (HCV) cure rates and elimination efforts. A phylogenetic analysis of 87 men who have sex with men recently infected with HCV genotype 1a placed one-third (28/87) in a large cluster, in which 96% harbored NS5A M28V RAS.

Cervical cancer risk in women living with HIV across four continents: A multicohort study.

We compared invasive cervical cancer (ICC) incidence rates in Europe, South Africa, Latin and North America among women living with HIV who initiated antiretroviral therapy (ART) between 1996 and 2014. We analyzed cohort data from the International Epidemiology Databases to Evaluate AIDS (IeDEA) and the Collaboration of Observational HIV Epidemiological Research in Europe (COHERE) in EuroCoord. We used flexible parametric survival models to determine regional ICC rates and risk factors for incident ICC. We included 64,231 women from 45 countries. During 320,141 person-years (pys), 356 incident ICC cases were diagnosed (Europe 164, South Africa 156, North America 19 and Latin America 17). Raw ICC incidence rates per 100,000 pys were 447 in South Africa (95% confidence interval [CI]: 382-523), 136 in Latin America (95% CI: 85-219), 76 in North America (95% CI: 48-119) and 66 in Europe (95% CI: 57-77). Compared to European women ICC rates at 5 years after ART initiation were more than double in Latin America (adjusted hazard ratio [aHR]: 2.43, 95% CI: 1.27-4.68) and 11 times higher in South Africa (aHR: 10.66, 95% CI: 6.73-16.88), but similar in North America (aHR: 0.79, 95% CI: 0.37-1.71). Overall, ICC rates increased with age (>50 years vs. 16-30 years, aHR: 1.57, 95% CI: 1.03-2.40) and lower CD4 cell counts at ART initiation (per 100 cell/µl decrease, aHR: 1.25, 95% CI: 1.15-1.36). Improving access to early ART initiation and effective cervical cancer screening in women living with HIV should be key parts of global efforts to reduce cancer-related health inequities.

Inhibition of Fatty Acid Oxidation as a New Target To Treat Primary Amoebic Meningoencephalitis.

Primary amoebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoeba Naegleria fowleri The amoeba migrates along the olfactory nerve to the brain, resulting in seizures, coma, and, eventually, death. Previous research has shown that Naegleria gruberi, a close relative of N. fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several already-approved inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and miltefosine. Our data demonstrate that etomoxir, orlistat, perhexiline, thioridazine, and valproic acid inhibited growth of N. gruberi We then tested these compounds on N. fowleri and found etomoxir, perhexiline, and thioridazine to be effective growth inhibitors. Hence, not only are lipids the preferred food source for N. gruberi, but also oxidation of fatty acids seems to be essential for growth of N. fowleri Inhibition of fatty acid oxidation could result in new treatment options, as thioridazine inhibits N. fowleri growth in concentrations that can be reached at the site of infection. It could also potentiate currently used therapy, as checkerboard assays revealed synergy between miltefosine and etomoxir. Animal testing should be performed to confirm the added value of these inhibitors. Although the development of new drugs and randomized controlled trials for this rare disease are nearly impossible, inhibition of fatty acid oxidation seems a promising strategy as we showed effectivity of several drugs that are or have been in use and that thus could be repurposed to treat PAM in the future.