First comprehensive untargeted metabolomics study of suramin-treated Trypanosoma brucei: an integrated data analysis workflow from multifactor data modelling to functional analysis.

INTRODUCTION: Human African trypanosomiasis, commonly known as sleeping sickness, is a vector-borne parasitic disease prevalent in sub-Saharan Africa and transmitted by the tsetse fly. Suramin, a medication with a long history of clinical use, has demonstrated varied modes of action against Trypanosoma brucei. This study employs a comprehensive workflow to investigate the metabolic effects of suramin on T. brucei, utilizing a multimodal metabolomics approach. OBJECTIVES: The primary aim of this study is to comprehensively analyze the metabolic impact of suramin on T. brucei using a combined liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) approach. Statistical analyses, encompassing multivariate analysis and pathway enrichment analysis, are applied to elucidate significant variations and metabolic changes resulting from suramin treatment. METHODS: A detailed methodology involving the integration of high-resolution data from LC-MS and NMR techniques is presented. The study conducts a thorough analysis of metabolite profiles in both suramin-treated and control T. brucei brucei samples. Statistical techniques, including ANOVA-simultaneous component analysis (ASCA), principal component analysis (PCA), ANOVA 2 analysis, and bootstrap tests, are employed to discern the effects of suramin treatment on the metabolomics outcomes. RESULTS: Our investigation reveals substantial differences in metabolic profiles between the control and suramin-treated groups. ASCA and PCA analysis confirm distinct separation between these groups in both MS-negative and NMR analyses. Furthermore, ANOVA 2 analysis and bootstrap tests confirmed the significance of treatment, time, and interaction effects on the metabolomics outcomes. Functional analysis of the data from LC-MS highlighted the impact of treatment on amino-acid, and amino-sugar and nucleotide-sugar metabolism, while time effects were observed on carbon intermediary metabolism (notably glycolysis and di- and tricarboxylic acids of the succinate production pathway and tricarboxylic acid (TCA) cycle). CONCLUSION: Through the integration of LC-MS and NMR techniques coupled with advanced statistical analyses, this study identifies distinctive metabolic signatures and pathways associated with suramin treatment in T. brucei. These findings contribute to a deeper understanding of the pharmacological impact of suramin and have the potential to inform the development of more efficacious therapeutic strategies against African trypanosomiasis.

[Evaluation of the knowledge on alveolar echinococcosis among general practitioners in the province of Liege : impact of a formative intervention]. / Évaluation des connaissances de l'échinococcose alvéolaire réalisée auprès des médecins généralistes de la Province de Liège : impact d'une intervention formative.

Alveolar echinococcosis is an indigenouszoonosis caused by the growth of the larval stage of a small tapeworm, Echinococcus multilocularis. Despite a low incidence in Belgium, with about 10 cases on average recorded per year, this parasitosis poses a real public health problem because it often remains difficult to diagnose and is potentially fatal in the absence of treatment. General practitioners are on the frontline, but they do not always know enough about the disease, which causes a delay in the diagnosis and impacts the prognosis. The present study aims to assess the level of knowledge of alveolar echinococcosis among general practitioners in the province of Liège via a questionnaire, on the one hand, and to increase their level of knowledge via a formative intervention using a video capsule, on the other hand. We have performed a randomized controlled experimental study, which showed that general practitioners in the province of Liège have limited knowledge on alveolar echinococcosis. This mainly concerns symptomatology, diagnostic tools and treatment. The formative intervention carried out allowed increasing their level of knowledge about this disease.

L'échinococcose alvéolaire est une zoonose autochtone provoquée par le développement tissulaire de la larve d'un petit ténia, Echinoccocus multilocularis. Malgré une faible incidence en Belgique, avec une moyenne de 10 cas recensés par an, cette parasitose pose un réel problème de santé publique car elle reste souvent difficile à diagnostiquer et potentiellement mortelle en l'absence de traitement. Les médecins généralistes sont en première ligne, mais ils ne connaissent pas toujours suffisamment la maladie, ce qui retarde le diagnostic et compromet le pronostic. La présente étude visait à évaluer les connaissances à propos de l'échinococcose alvéolaire des médecins généralistes de la province de Liège via un questionnaire approprié et à accroître leur niveau de connaissance via une intervention formative sous forme de capsule vidéo. Nous avons réalisé une étude à visée expérimentale randomisée contrôlée qui a montré que les médecins généralistes de la province de Liège ont des connaissances partielles sur la symptomatologie, les outils de diagnostic et le traitement. L'intervention formative réalisée a permis d'augmenter leur niveau de connaissances à propos de cette maladie.

A Phylodynamic Workflow to Rapidly Gain Insights into the Dispersal History and Dynamics of SARS-CoV-2 Lineages.

Since the start of the COVID-19 pandemic, an unprecedented number of genomic sequences of SARS-CoV-2 have been generated and shared with the scientific community. The unparalleled volume of available genetic data presents a unique opportunity to gain real-time insights into the virus transmission during the pandemic, but also a daunting computational hurdle if analyzed with gold-standard phylogeographic approaches. To tackle this practical limitation, we here describe and apply a rapid analytical pipeline to analyze the spatiotemporal dispersal history and dynamics of SARS-CoV-2 lineages. As a proof of concept, we focus on the Belgian epidemic, which has had one of the highest spatial densities of available SARS-CoV-2 genomes. Our pipeline has the potential to be quickly applied to other countries or regions, with key benefits in complementing epidemiological analyses in assessing the impact of intervention measures or their progressive easement.

Biennial surveillance of Plasmodium falciparum anti-malarial drug resistance markers in Democratic Republic of Congo, 2017 and 2019.

BACKGROUND: Because of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)'s malaria treatment policy replaced CQ by sulfadoxine-pyrimethamine (SP) as first-line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin-based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti-malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti-malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC. METHODS: From July to November 2019, a cross-sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing. RESULTS: Out of 1087 enrolled patients, 906 (83.3%) were PCR-confirmed for P. falciparum. Like in the 2017-study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. However, non-synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019-studies. The overall prevalence of pfcrt-K76T mutation that confers CQ-resistance was 22.7% in 2019-study compared to 28.5% in 2017-study (p-value = 0.069), but there was high variability between sites in the two studies. Like in 2017-study, the pfcrt 72-76 SVMNT haplotype associated with resistance to amodiaquine was not detected. CONCLUSION: The study reported, within 2 years, the non-presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as-yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ-resistance marker was observed in the DRC, but this prevalence remained highly variable from region to region.

Cryptococcosis in the Democratic Republic of Congo from 1953 to 2021: A systematic review and meta-analysis.

Cryptococcosis is a common opportunistic infection associated with HIV/AIDS. The present review systematically describes the clinical and biological aspects of cryptococcosis in the Democratic Republic of Congo (DRC) and estimates its 2020 burden in people living with HIV (PLHIV). Following PRISMA guidelines, we searched online databases for records of cryptococcosis/Cryptococcus spp. in the DRC. Meta-analysis was then performed to estimate summary statistics and the corresponding 95% confidence intervals (CI). A total of 30 studies were included. These included 1,018 cryptococcosis patients, including 80.8% with neuromeningeal cryptococcosis (NMC) and predominantly immunocompromised due to HIV/AIDS (97.6%). The NMC mean prevalence was estimated at 9.63% (95% CI: 5.99-14.07). More than one in two patients (52.7%) under treatment died. Monotherapy with fluconazole was the main treatment administered (80.6%). Furthermore, we estimate that about 9,265 (95% CI: 5,763-13,537) PLHIV had cryptococcosis in 2020, in DRC; of which about 4,883 (95% CI: 3,037-7,134) would have died in the same year. Among isolates in all included studies, 74 strains have been characterised. Of these, 82.4% concerned Cryptococcus neoformans sensu lato (s.l) (exclusively of serotype A and mostly of molecular types VNI and VNII) and 17.6% concerned Cryptotoccus gattii s.l (belonging to serotype B/molecular type VGI). Cryptococcosis remains common with an unacceptably high mortality rate. A large number of PLHIV affected by and dying from cryptococcosis in 2020 demonstrates its heavy burden among the Congolese PLHIV. To mitigate this burden, it is important to improve the quality and accessibility of care for all PLHIV.

Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfk13-propeller, pfcrt and pfmdr1 genes in isolates from treatment failure patients in Democratic Republic of Congo, 2018-2019.

BACKGROUND: The national policy for malaria treatment of the Democratic Republic of Congo recommends two first-line artemisinin-based combinations for the treatment of uncomplicated malaria: artesunate-amodiaquine and artemether-lumefantrine. This study investigated the presence of markers associated with resistance to the current first-line artemisinin-based combination therapy (ACT) in isolates of Plasmodium falciparum from treatment failure patients in the Democratic Republic of Congo. METHODS: From November 2018 to November 2019, dried blood spots were taken from patients returning to health centres for fever within 28 days after an initial malaria treatment in six sentinel sites of the National Malaria Control Programme across Democratic Republic of Congo. The new episode of malaria was first detected by a rapid diagnostic test and then confirmed by a real-time PCR assay to define treatment failure. Fragments of interest in pfk13 and pfcrt genes were amplified by conventional PCR before sequencing and the Pfmdr1 gene copy number was determined by a TaqMan real-time PCR assay. RESULTS: Out of 474 enrolled patients, 364 (76.8%) were confirmed positive by PCR for a new episode of P. falciparum malaria, thus considered as treatment failure. Of the 325 P. falciparum isolates obtained from 364 P. falciparum-positive patients and successfully sequenced in the pfk13-propeller gene, 7 (2.2%) isolates carried non-synonymous mutations, among which 3 have been previously reported (N498I, N554K and A557S) and 4 had not yet been reported (F506L, E507V, D516E and G538S). Of the 335 isolates successfully sequenced in the pfcrt gene, 139 (41.5%) harboured the K76T mutation known to be associated with chloroquine resistance. The SVMNT haplotype associated with resistance to amodiaquine was not found. None of the isolates carried an increased copy number of the pfmdr1 gene among the 322 P. falciparum isolates successfully analysed. CONCLUSION: No molecular markers currently known to be associated with resistance to the first-line ACT in use were detected in isolates of P. falciparum from treatment failure patients. Regular monitoring through in vivo drug efficacy and molecular studies must continue to ensure the effectiveness of malaria treatment in Democratic Republic of Congo.

Care-seeking behaviour and socio-economic burden associated with uncomplicated malaria in the Democratic Republic of Congo.

BACKGROUND: This study aimed to estimate the socio-economic costs of uncomplicated malaria and to explore health care-seeking behaviours that are likely to influence these costs in the Democratic Republic of Congo (DRC), a country ranked worldwide as the second most affected by malaria. METHODS: In 2017, a cross-sectional survey included patients with uncomplicated malaria in 64 healthcare facilities from 10 sentinel sites of the National Malaria Control Programme (NMCP) in the DRC. A standard questionnaire was used to assess health care-seeking behaviours of patients. Health-related quality of life (HRQL) and disutility weights (DW) of illness were evaluated by using the EuroQol Group's descriptive system (EQ-5D-3L) and its visual analogue scale (EQ VAS). Malaria costs were estimated from a patient's perspective. Probabilistic sensitivity analyses (PSA) evaluated the uncertainty around the cost estimates. Generalized regression models were fitted to assess the effect of potential predictive factors on the time lost and the DW during illness. RESULTS: In total, 1080 patients (age: 13.1 ± 14 years; M/F ratio: 1.1) were included. The average total costs amounted to US$ 36.3 [95% CI 35.5-37.2] per malaria episode, including US$ 16.7 [95% CI 16.3-17.1] as direct costs and US$ 19.6 [95% CI 18.9-20.3] indirect costs. During care seeking, economically active patients and their relatives lost respectively 3.3 ± 1.8 and 3.4 ± 2.1 working days. This time loss occurred mostly at the pre-hospital stage and was the parameter associated the most with the uncertainty around malaria cost estimates. Patients self-rated an average 0.36 ± 0.2 DW and an average 0.62 ± 0.3 EQ-5D index score per episode. A lack of health insurance coverage (896 out of 1080; 82.9%) incurred substantially higher costs, lower quality of life, and heavier DW while leading to longer time lost during illness. Residing in rural areas incurred a disproportionally higher socioeconomic burden of uncomplicated malaria with longer time lost due to illness and limited access to health insurance mechanisms. CONCLUSION: Uncomplicated malaria is associated with high economic costs of care in the DRC. Efforts to reduce the cost-of-illness should target time lost at the pre-hospital stage and social disparities in the population, while reinforcing measures for malaria control in the country.

Comparison of clinical and biological characteristics of HIV-infected patients presenting Cryptococcus neoformans versus C. curvatus/C. laurentii meningitis.

BACKGROUND: Cryptococcal meningitis is mainly caused by Cryptococcus neoformans/C. gattii complex. We compared the clinical, biological, and antifungal susceptibility profiles of isolates from HIV-Infected Patients (HIVIP) with C. neoformans (Cn) versus C. curvatus/C. laurentii (Cc/Cl) meningitis. METHODS: Comparative analytical study were conducted. Apart from patients' clinical data, the following analysis were performed and the results were compared in both groups: biochemical examination, cryptococcal antigen test, India ink staining, and culture on Cerebral Spinal Fluid (CSF), strains identification by mass spectrometry, ITS sequencing, PCR serotyping and antifungal susceptibility. The main outcome variable was the "species of Cryptococcus identified", which was compared to other variables of the same type using the Pearson Chi-square test or the Fisher exact test. RESULTS: A total of 23 (79.3%) Cn meningitis cases versus 6 (20.7%) Cc/Cl meningitis were retained. Cn meningitis was more frequently associated with headache (100% vs 50%, p = 0.005) than Cc/Cl meningitis and meningeal signs were more frequent in Cn infected patients. Biologically, hypoglycorrhachia and low CD4 count were more observed in Cn group (90% vs 20% of patients, p = 0.01; 45.6 vs 129.8 cells/µL, p = 0.02, respectively). A higher proportion of Cn strains (91.3%) showed a low Minimum Inhibitory Concentration (MIC) (< 8 mg/L) for fluconazole compared to Cc/Cl strains (66.7%). Also, Cc/Cl strains resistant to 5-flucytosine and amphotericin B were found in 16.7% of cases for each of the two antifungal agents. Cryptococcus detection by routine analysis (India ink, culture, and antigens) was better for Cn samples than Cc/Cl. Except ITS sequencing, which identified all strains of both groups, mass spectrometry and serotyping PCR identified Cn strains better than Cc/Cl (100% vs 80%, p = 0.1; 100% vs 0%, p < 0.0001, respectively). After treatment with amphotericin B, 5-flucytosine, and fluconazole in both groups, the outcome was similar. CONCLUSIONS: Clinical presentation of Cn meningitis is certainly more severe than that of Cc/Cl meningitis, but Cc/Cl infection should be considered in the management of HIVIP with meningeal syndrome because of the diagnostic difficulty and the high MICs of antifungal agents required for the treatment of meningitis due to these cryptococcal species.

Clinical course and challenging management of early COVID-19 infection after heart transplantation: case report of two patients.

BACKGROUND: There are limited data on Coronavirus disease 2019 (COVID-19) in solid organ transplant patients, especially in heart transplant recipients, with only a few case reports and case series described so far. Heart transplant recipients may be at particular high risk due to their comorbidities and immunosuppressed state. CASE PRESENTATION: This report describes the clinical course and the challenging management of early COVID-19 infection in two heart transplant recipients who tested positive for the SARS-CoV-2 virus in the perioperative period of the transplant procedure. The two patients developed a severe form of the disease and ultimately died despite the initiation of an antiviral monotherapy with hydroxychloroquine coupled with the interruption of mycophenolate mofetil. CONCLUSIONS: These two cases illustrate the severity and poor prognosis of COVID-19 in the perioperative period of a heart transplant. Thorough screening of donors and recipients is mandatory, and the issue of asymptomatic carriers needs to be addressed.

Epidemiology of Dermatophytes in Belgium: A 5 Years' Survey.

Dermatophytes are among the most common fungal agents causing superficial skin infections worldwide. Epidemiology of these infections is evolving and variable in every country. This report presents the Belgian epidemiological data regarding the distribution of dermatophytes species isolated by the two national reference centers for mycosis during a period of 5 years (2012-2016). Trichophyton rubrum was the most frequently isolated species, considering all sampling sites (60.3% on average between 2012 and 2016). More precisely, this dermatophyte was the major agent of Tinea unguium and Tinea corporis during this period, followed by species of the Trichophyton mentagrophytes complex. Moreover, Microsporum audouinii was the main etiological agent of Tinea capitis (TC) with a frequency of 52.5% on average between 2012 and 2016. Other African dermatophytes species such as Trichophyton soudanense and Trichophyton violaceum were also agents of TC with a respective prevalence of 11.6% and 11.5% on average. This study highlights a different dermatophyte distribution in Belgium in comparison with other European countries.

A Recurrent Mutation at Position 26340 of SARS-CoV-2 Is Associated with Failure of the E Gene Quantitative Reverse Transcription-PCR Utilized in a Commercial Dual-Target Diagnostic Assay.

Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals while also clearing essential staff to continue to work. At the current time, a number of quantitative real-time PCR (qRT-PCR) assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains, it is prudent to monitor circulating viruses for variants that might compromise these assays. Here, we report the identification of a C-to-U transition at position 26340 of the SARS-CoV-2 genome that is associated with failure of the cobas SARS-CoV-2 E gene qRT-PCR in eight patients. As the cobas SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called SARS-CoV-2 positive. Whole-genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of single-nucleotide polymorphisms that might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.

Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes.

OBJECTIVES: Terbinafine resistance is increasingly reported in Trichophyton, rendering susceptibility testing particularly important in non-responding cases. We performed a multicentre evaluation of six EUCAST-based methods. METHODS: Ten laboratories susceptibility tested terbinafine, itraconazole, voriconazole and amorolfine against a blinded panel of 38 terbinafine WT and target gene mutant isolates. E.Def 9.3.1 modifications included: medium with/without addition of chloramphenicol and cycloheximide (CC), incubation at 25°C to 28°C for 5-7 days and three MIC endpoints [visually and spectrophotometrically (90%/50% inhibition)], generating 7829 MICs. Quality control (QC) strains were Aspergillus flavus ATCC 204304 and CNM-CM1813. Eyeball, ECOFFinder (where ECOFF stands for epidemiological cut-off) and derivatization WT upper limits (WT-ULs), very major errors (VMEs; mutants with MICs ≤WT-ULs) and major errors (MEs; WT isolates with MICs >WT-ULs) were determined. RESULTS: MICs fell within the QC ranges for ATCC 204304/CNM-CM1813 for 100%/96% (voriconazole) and 84%/84% (itraconazole), respectively. Terbinafine MICs fell within 0.25-1 mg/L for 96%/92%, suggesting high reproducibility. Across the six methods, the number of terbinafine MEs varied from 2 to 4 (2.6%-5.2%) for Trichophyton rubrum and from 0 to 2 (0%-2.0%) for Trichophyton interdigitale. Modes for WT and mutant populations were at least seven 2-fold dilutions apart in all cases. Excluding one I121M/V237I T. rubrum mutant and two mixed WT/mutant T. interdigitale specimens, the numbers of VMEs were as follows: T. rubrum: CC visual, 1/67 (1.5%); CC spectrophotometric 90% inhibition, 3/59 (5.1%); and CC spectrophotometric 50% inhibition, 1/67 (1.5%); and T. interdigitale: none. Voriconazole and amorolfine MICs were quite uniform, but trailing growth complicated determination of itraconazole visual and spectrophotometric 90% inhibition MIC. CONCLUSIONS: Although none of the laboratories was experienced in dermatophyte testing, error rates were low. We recommend the CC spectrophotometric 50% inhibition method and provide QC ranges and WT-ULs for WT/non-WT classification.

Economic costs analysis of uncomplicated malaria case management in the Peruvian Amazon.

BACKGROUND: Case management is one of the principal strategies for malaria control. This study aimed to estimate the economic costs of uncomplicated malaria case management and explore the influence of health-seeking behaviours on those costs. METHODS: A knowledge, attitudes and practices (KAP) survey was applied to 680 households of fifteen communities in Mazan-Loreto in March 2017, then a socio-economic survey was conducted in September 2017 among 161 individuals with confirmed uncomplicated malaria in the past 3 months. Total costs per episode were estimated from both provider (Ministry of Health, MoH) and patient perspectives. Direct costs were estimated using a standard costing estimation procedure, while the indirect costs considered the loss of incomes among patients, substitute labourers and companions due to illness in terms of the monthly minimum wage. Sensitivity analysis evaluated the uncertainty of the average cost per episode. RESULTS: The KAP survey showed that most individuals (79.3%) that had malaria went to a health facility for a diagnosis and treatment, 2.7% received those services from community health workers, and 8% went to a drugstore or were self-treated at home. The average total cost per episode in the Mazan district was US$ 161. The cost from the provider's perspective was US$ 30.85 per episode while from the patient's perspective the estimated cost was US$ 131 per episode. The average costs per Plasmodium falciparum episode (US$ 180) were higher than those per Plasmodium vivax episode (US$ 156) due to longer time lost from work by patients with P. falciparum infections (22.2 days) than by patients with P. vivax infections (17.0 days). The delayed malaria diagnosis (after 48 h of the onset of symptoms) was associated with the time lost from work due to illness (adjusted mean ratio 1.8; 95% CI 1.3, 2.6). The average cost per malaria episode was most sensitive to the uncertainty around the lost productivity cost due to malaria. CONCLUSIONS: Despite the provision of free malaria case management by MoH, there is delay in seeking care and the costs of uncomplicated malaria are mainly borne by the families. These costs are not well perceived by the society and the substantial financial impact of the disease can be frequently undervalued in public policy planning.

Molecular surveillance of anti-malarial drug resistance in Democratic Republic of Congo: high variability of chloroquinoresistance and lack of amodiaquinoresistance.

BACKGROUND: The loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as a first-line treatment for uncomplicated malaria in several endemic countries, such as the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72-76 on the same gene has been associated with resistance to amodiaquine (AQ). In the DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of this study was to determine the prevalence of K76T mutations in circulating strains of Plasmodium falciparum, 16 years after CQ withdrawal in the DRC and to investigate the presence of the SVMNT haplotype. METHODS: In 2017, ten geographical sites across the DRC were selected. Dried blood samples were collected from patients attending health centres. Malaria was first detected by a rapid diagnostic test (RDT) available on site (SD Bioline Malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72-76 was amplified by conventional PCR before sequencing. RESULTS: A total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 samples (28.5%; 95% CI 25.4%-31.9%), mainly (96%) with the CVIET haplotype. Prevalence of CQ resistance marker was unequally distributed across the country, ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected. CONCLUSION: Overall, the frequency of the P. falciparum CQ resistance marker has decreased significantly and no resistance marker to AQ was detected in the DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for continuous monitoring of the CQ resistance level for its prospective re-use in malaria management. The absence of the AQ resistance marker is in line with the use of this drug in the current DRC malaria treatment policy.

Comparison of practicability and effectiveness between unassisted HIV self-testing and directly assisted HIV self-testing in the Democratic Republic of the Congo: a randomized feasibility trial.

BACKGROUND: HIV self-testing (HIVST) can be performed using directly assisted and unassisted approaches in facilities or communities to reach different populations. The aim of this study was to compare the practicability and effectiveness of the two delivery approaches for HIVST, unassisted HIVST (UH) and directly assisted HIVST (DAH), in the field setting of Kisangani, the Democratic Republic of the Congo (DRC). METHODS: A randomized (1:1), non-blinded, non-inferiority trial using a blood-based and facility-based HIVST method was carried out in four facilities in Kisangani, the DRC, targeting populations at high risk for HIV infection. The primary outcome was the difference in the practicability of the HIV self-test between the two arms. Practicability was defined as successfully performing the test and correctly interpreting the result. Requests for assistance, positivity rate, linkage to care, and willingness to buy an HIV self-test kit constituted the secondary outcomes for HIVST effectiveness. The adjusted risk ratios (aRRs) were calculated using Poisson regression. RESULTS: The rate of successfully performing the test was same (93.2%) in the UH and DAH arms. The rate of correctly interpreting the results was 86.9% in the UH arm versus 93.2% in the DAH arm, for a difference of - 6.3%. After the follow-up 72 h later, participants in the UH arm had a significantly lower chance of correctly interpreting the test results than those in the DAH arm (aRR: 0.60; P = 0.019). Although the positivity rate was 3.4% among the participants in the DAH arm and 1.7% among those in the UH arm, no significant differences were found between the two arms in the positivity rate, requests for assistance, and linkage to care. Willingness to buy an HIV self-test was higher in the UH arm than in the DAH arm (92.3% versus 74.1%; aRR: 4.20; P < 0.001). CONCLUSION: The results of this study indicate that UH is as practicable and effective as DAH among individuals at high risk for HIV infection in Kisangani, the DRC. However, additional support tools need to be assessed to improve the interpretation of the self-test results when using the UH approach. TRIAL REGISTRATION: PACTR201904546865585. Registered 03 April 2019 - Retrospectively registered, https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=6032.

Evaluation of the new Id-Fungi plates from Conidia for MALDI-TOF MS identification of filamentous fungi and comparison with conventional methods as identification tool for dermatophytes from nails, hair and skin samples.

OBJECTIVES: We first compare the efficiency of mould/dermatophyte identification by MALDI-TOF MS using a new medium called Id-Fungi plates (IDFP) from Conidia® and two different databases. For the second purpose, we evaluated a new version of the medium supplemented with cycloheximide, Id-Fungi plates Plus (IDFPC) for the direct inoculation of nails, hair and skin samples and compared the efficiency of MALDI-TOF MS identification of dermatophytes to classical methods based on culture and microscopy. METHODS: A total of 71 strains have been cultured IDFP and Sabouraud gentamicin plates (SGC2) and were identified by MALDI-TOF MS. For the evaluation of the combination IDFPC/ MALDI-TOF MS as a method of identification for dermatophytes, 428 samples of hair nails and skin were cultivated in parallel on IDFPC and Sabouraud + cycloheximide medium (SAB-ACTI). RESULTS: For Aspergillus sp and non-Aspergillus moulds, the best performances were obtained on IDFP after maximum 48-h growth, following protein extraction. For dermatophytes, the best condition was using the IDFP at 72 hours, after extended direct deposit. Regarding the direct inoculation of nails, hair skin on IDFPC, 129/428 (30.1%) showed a positive culture against 150/428 (35%) on SAB-ACTI medium. Among the 129 positive strains, the identification by MALDI-TOF MS was correct for 92/129 (71.4%). CONCLUSION: The IDFP allows the generation of better spectra by MALDI-TOF MS compared to SGC2. It facilitates sampling and deposit. Regarding the use of IDFPC, this medium seems less sensitive than SAB-ACTI but among positive strains, the rate of correct identification by MALDI-TOF MS is satisfactory.

Clinical evaluation of the DermaGenius® Nail real-time PCR assay for the detection of dermatophytes and Candida albicans in nails.

Onychomycosis represents one of the most frequent mycoses in the world. Causative agents are mainly dermatophytes, but yeasts and nondermatophyte moulds can also be involved. Conventional diagnostic methods include direct microscopy (or histology) and culturing. However, molecular methods are becoming increasingly popular in this field. The DermaGenius® (DG) Nail multiplex assay (PathoNostics, The Netherlands) is a new commercial real-time polymerase chain reaction (PCR) kit, which can detect Trichophyton rubrum, Trichophyton interdigitale and Candida albicans directly in nails. The present study is a retrospective evaluation of the kit applied to 138 finger and toenail clippings in comparison to histology and culture methods. The sensitivity and specificity of the PCR assay are 80% (76/95) and 74.4% (32/43), respectively, when histology and culture are used as reference to define onychomycosis. DG performance is not different from histology combined with culture (P = .11) but the best diagnostic efficacy (88.4%, 122/138) is obtained by the combination of histology and DG. In conclusion, this study emphasizes the clinical usefulness of the DG in diagnostics. The high specificity of this test guarantees a better identification compared to culture that can lead to dermatophyte misidentifications. It is a reliable PCR assay that shortens the time to diagnosis and can unmask the presence of nongrowing fungal pathogens in nails.

Recent uptake of intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine is associated with increased prevalence of Pfdhfr mutations in Bobo-Dioulasso, Burkina Faso.

BACKGROUND: The impact of sulfadoxine-pyrimethamine (SP) used as intermittent preventive treatment during pregnancy (IPTp-SP) on mutant parasite selection has been poorly documented in Burkina Faso. This study sought first to explore the relationship between IPTp-SP and the presence of mutant parasites. Second, to assess the relationship between the mutant parasites and adverse pregnancy outcomes. METHODS: From September to December 2010, dried blood spots (DBS) were collected during antenatal care visits and at delivery from 109 pregnant women with microscopically confirmed falciparum malaria infection. DBS were analysed by PCR-restriction fragment length polymorphism (PCR-RFLP) for the polymorphisms at codons 51, 59, 108, and 164 of the Pfdhfr gene and codons 437 and 540 in the Pfdhps gene. RESULTS: Both the Pfdhfr and Pfdhps genes were successfully genotyped in 92.7% (101/109) of the samples. The prevalence of Pfdhfr mutations N51I, C59R and S108N was 71.3, 42.6 and 64.4%, respectively. Overall, 80.2% (81/101) of samples carried the Pfdhps A437G mutation. None of the samples had the Pfdhfr I164L and the Pfdhps K540E mutations. The prevalence of the triple mutation N51I + C59R + S108N was 25.7% (26/101). The use of IPTp-SP was associated with a threefold increased odds of Pfdhfr C59R mutation [crude OR 3.29; 95% CI (1.44-7.50)]. Pregnant women with recent uptake of IPTp-SP were at higher odds of both the Pfdhfr C59R mutation [adjusted OR 4.26; 95% CI (1.64-11.07)] and the Pfdhfr intermediate-to-high resistance, i.e., ≥ 2 Pfdhfr mutations [adjusted OR 3.45; 95% CI (1.18-10.07)]. There was no statistically significant association between the presence of the Pfdhfr intermediate-to-high resistance and parasite densities or both maternal haemoglobin level and anaemia. CONCLUSION: The data indicate that despite the possibility that IPTp-SP contributes to the selection of resistant parasites, it did not potentiate pregnancy-associated malaria morbidity, suggesting the continuation of SP use as IPTp in Burkina Faso.

Recurrence of visceral and muco-cutaneous leishmaniasis in a patient under immunosuppressive therapy.

BACKGROUND: Leishmaniasis is a protozoan disease caused by parasites of the genus Leishmania, transmitted to humans by sandflies. The diagnosis of leishmaniasis is often challenging as it mimics many other infectious or malignant diseases. The disease can present in three ways: cutaneous, mucocutaneous, or visceral leishmaniasis, which rarely occur together or consecutively. CASE PRESENTATION: The patient was a 52 years old immunosuppressed Belgian woman with a long history of severe rheumatoid arthritis. She underwent bone marrow biopsy to explore thrombocytopenia. Diagnosis of visceral leishmaniasis was made by identification of Leishman Donovan (LD) bodies in macrophages. Treatment with liposomal amphotericin B was successful. She later developed cutaneous leishmaniasis treated with amphotericin B lipid complex. She next presented with relapsing cutaneous lesions followed by rapidly progressing lymphadenopathies. Biopsy confirmed the diagnosis of leishmaniasis. Treatments by miltefosine, amphotericin B, N-methyl-glucamine antimoniate were subsequently initiated. She later presented a recurrent bone marrow involvement treated with intramuscular paromomycin and miltefosine. She died two years later from leukemia. At the time of death, she presented with a mucosal destruction of the nose. A Leishmania-specific PCR (Polymerase Chain Reaction) identified L. infantum as etiological agent. CONCLUSIONS: Clinicians should be aware of the potential concomitant or sequential involvement of multiple anatomic localizations of Leishmania in immunosuppressed patients.