An Overview of 10 Years of Activity of a Molecular Laboratory for Buruli Ulcer Diagnosis at a Field Hospital in Benin.

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. Early diagnosis is crucial to prevent morbidity. In November 2012, a field laboratory fully equipped for the rapid on-site quantitative PCR (qPCR) diagnosis of M. ulcerans was established at the Buruli ulcer treatment center (CDTLUB) center in Pobè Benin, a region where BU is endemic. We describe its first 10 years of activity and its gradual evolution into an expert laboratory for BU diagnosis. From 2012 to 2022, the laboratory analyzed 3,018 samples from patients attending consultations for suspected BU at the CDTLUB in Pobè. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. Since 2019, the laboratory has also received and analyzed 570 samples from other centers. The laboratory confirmed the diagnosis of BU by qPCR for 39.7% samples: M. ulcerans DNA was detected in 34.7% of swabs, 47.2% of all fine needle aspiration samples (FNA) and 44.6% of all skin biopsy specimens. Positive Ziehl-Neelsen staining results were obtained for 19.0% samples. Bacterial load, estimated by qPCR, was significantly greater for the Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples, and detection rates were highest for FNA samples. Overall, 26.3% of the samples received from other centers were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado, Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success. Optimal patient care depends on the close proximity of a molecular biology structure to BU treatment centers. Finally, FNA should be promoted among caregivers. IMPORTANCE Here, we describe the first 10 years of activity at a field laboratory established at the Buruli ulcer treatment center (CDTLUB) in Pobè, Benin, a country in which Mycobacterium ulcerans is endemic. Between 2012 and 2022, the laboratory analyzed 3,018 samples from patients consulting the CDTLUB of Pobè with a suspected clinical BU. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. In total, 39.7% of samples tested positive by qPCR and 19.0% tested positive by Ziehl-Neelsen staining. Detection rates were highest for FNA samples, and the bacterial loads estimated by qPCR were significantly higher for Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples. Since 2019, the laboratory has also analyzed 570 samples received from outside the CDTLUB of Pobè, 26.3% of which were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado in Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success, with major benefits for both the medical staff and patients. Our findings illustrate that the usefulness and feasibility of having a diagnostic center in rural Africa, where the disease is endemic, is a key part of optimal patient care, and that FNA should be promoted to increase detection rates.

Commiphora wildii Merxm. Essential Oil: Natural Heptane Source and Co-Product Valorization.

As an alternative to fossil volatile hydrocarbon solvents used nowadays in perfumery, investigation on essential oil of Commiphora wildii Merxm. oleo gum resin as a source of heptane is reported here. Heptane, representing up to 30 wt-% of this oleo gum resin, was successfully isolated from the C. wildii essential oil, using an innovative double distillation process. Isolated heptane was then used as a solvent in order to extract some noble plants of perfumery. It was found that extracts obtained with this solvent were more promising in terms of sensory analysis than those obtained from fossil-based heptane. In addition, in order to valorize the essential oil depleted from heptane, chemical composition of this oil was found to obtain, and potential biological activity properties were studied. A total of 172 different compounds were identified by GC-MS in the remaining oil. In vitro tests-including hyaluronidase, tyrosinase, antioxidant, elastase and lipoxygenase, as well as inhibitory tests against two yeasts and 21 bacterial strains commonly found on the skin-were carried out. Overall, bioassays results suggest this heptane-depleted essential oil is a promising active ingredient for cosmetic applications.

Probabilistic chemotherapy in knee and hip replacement infection: the place of linezolid.

Prosthetic joint infection (PJI) can occur with a wide range of microorganisms and clinical features. After replacement surgery of prosthetic joint, prescription of probabilistic broad-spectrum antimicrobial therapy is usual, while awaiting microbial culture results. The aim of our study was to describe the antibiotic susceptibility of microorganisms isolated from hip and knee PJI. The data were collected to determine the best alternative to the usual combination of piperacillin-tazobactam (TZP) or cefotaxime (CTX) and vancomycin (VAN). Based on a French prospective, multicenter study, we analyzed microbiological susceptibility to antibiotics of 183 strains isolated from patients with confirmed hip or knee PJI. In vitro susceptibility was evaluated: TZP+VAN, TZP+linezolid (LZD), CTX+VAN, and CTX+LZD. We also analyzed resistance to different antibiotics commonly used as oral alternatives. Among the 183 patients with PJI, 62 (34%) had a total knee prosthesis, and 121 (66%) a hip prosthesis. The main identified bacteria were Staphylococcus aureus (32.2% of isolates), coagulase-negative staphylococci (27.3%), Enterobacteriaceae (14.2%), and Streptococcus (13.7%). Infections were polymicrobial for 28 (15.3%) patients. All combinations were highly effective: CTX+VAN, CTX+LZD, TZP+VAN, and TZP+LZD (93.4%, 94%, 98.4%, and 98.9% of all cases respectively). Use of LZD instead of VAN in combination with a broad-spectrum beta-lactam covers almost all of the bacteria isolated in PJI. This association should be considered in probabilistic chemotherapy, as it is particularly easy to use (oral administration and no vancomycin monitoring).

A case of Ureaplasma parvum meningitis in an adult after transphenoidal ablation of craniopharyngioma.

We report the case of a Ureaplasma parvum meningitis in an immunocompetent patient, 17 days after surgical ablation of a craniopharyngioma. Presence of U. parvum in the cerebrospinal fluid was assessed by 16S rDNA sequencing and U. parvum specific PCR. This article details a surprising complication in an adult of a transphenoidal surgery for ablation of a craniopharyngioma. This is the first case, to our knowledge, of U. parvum meningitis in an adult patient.

Staphylococcus saprophyticus: Which beta-lactam?

BACKGROUND: Staphylococcus saprophyticus is resistant to the drugs most often used for the empirical treatment of urinary tract infections (UTI). The adequacy of antimicrobial treatments prescribed for UTI due to S. saprophyticus is not usually questioned. This study described the epidemiology of such infections and assessed the susceptibility of S. saprophyticus to ceftriaxone and amoxicillin-clavulanic acid. METHODS: Methicillin-susceptible S. saprophyticus (MSSS) isolated from clinical samples between November 2014 and July 2016 were included. Clinical data were recorded. The minimum inhibitory concentrations (MICs) of amoxicillin-clavulanic acid and ceftriaxone were measured for these MSSS strains and for 17 randomly selected methicillin-susceptible Staphylococcus aureus (MSSA) strains. RESULTS: Of the S. saprophyticus isolates from urine, 59.5% were associated with a diagnosis of cystitis and 33.3% with pyelonephritis. Sixty percent of S. saprophyticus cystitis cases and 25% of pyelonephritis cases were given an inappropriate antibiotic regimen. The MICs of ceftriaxone ranged from 4 to >32µg/ml for MSSS, and from 1.5 to 4µg/ml for MSSA. CONCLUSIONS: Many UTIs were treated with an empirical antibiotic therapy that was ineffective for S. saprophyticus, revealing that S. saprophyticus is an aetiology that is insufficiently considered in UTI. High MICs for ceftriaxone in MSSS were observed, which raises questions about the use of this antibiotic in UTIs due to S. saprophyticus.

Vetiver Essential Oil in Cosmetics: What Is New?

Background: Vetiver is a key ingredient for the perfume industry nowadays. However, with the constant and rapid changes of personal tastes, this appeal could vanish and this sector could decline quite quickly. New dissemination paths need to be found to tap this valuable resource. Methods: In this way, its potential use in cosmetics either as an active ingredient per se (with cosmeceutical significance or presenting antimicrobial activity) has hence been explored in vitro. Results: In this contribution, we demonstrated that vetiver essential oil displays no particularly significant and innovative cosmetic potential value in formulations apart from its scent already largely exploited. However, evaluated against twenty bacterial strains and two Candida species using the in vitro microbroth dilution method, vetiver oil demonstrated notably some outstanding activities against Gram-positive strains and against one Candida glabrata strain. Conclusions: Based on these findings, vetiver essential oil appears to be an appropriate aspirant for the development of an antimicrobial agent for medicinal purposes and for the development of a cosmetic ingredient used for its scent and displaying antimicrobial activity as an added value.

Clinical Features of Spontaneous Partial Healing During Mycobacterium ulcerans Infection.

Background. Buruli ulcer, caused by Mycobacterium ulcerans, is a necrotizing skin disease leading to extensive cutaneous and subcutaneous destruction and functional limitations. Spontaneous healing in the absence of medical treatment occurs in rare cases, but this has not been well described in the literature. Methods. In a retrospective case study in an area of Benin where this disease is highly endemic, we selected 26 Buruli ulcer patients presenting features of spontaneous healing from a cohort of 545 Buruli ulcer patients treated between 2010 and 2013. Results. The 26 patients studied had a median age of 13.5 years and were predominantly male (1.4:1). Three groups of patients were defined on the basis of their spontaneous healing characteristics. The first group (12 patients) consisted of patients with an ulcer of more than 1 year's duration showing signs of healing. The second (13 patients) group contained patients with an active Buruli ulcer lesion some distance away from a first lesion that had healed spontaneously. Finally, the third group contained a single patient displaying complete healing of lesions from a nodule, without treatment and with no relapse. Conclusions. We defined several features of spontaneous healing in Buruli ulcer patients and highlighted the difficulties associated with diagnosis and medical management. Delays in consultation contributed to the high proportion of patients with permanent sequelae and a risk of squamous cell carcinoma. Early detection and antibiotic treatment are the best ways to reduce impairments.

Design and stability study of a paediatric oral solution of methotrexate 2 mg/ml.

Oral paediatric forms development by pharmaceutical industry is still insufficient. The present study was performed to propose an adapted and pleasant formulation of liquid oral formulation of MTX. The solution is composed of injectable methotrexate, water, Ora Sweet(®) and sodium bicarbonate. After 120 days storage, pH remained stable at about 8 in all formulations, insuring no risk of MTX precipitation. MTX content in solution formulation, determined by high performance liquid chromatography measurements, remained in the specifications of >90% of the initial concentration when stored at 4 and 25°C. Forced degradation of MTX by heat and acidic conditions allowed formation and detection of degradation products by the analytical method. Microbial study of the preparation shows that the solution remains in the specifications during all the storage, or after one sample each week during one month, eventually indicating the microbial properties are not affected by patient use. To conclude, we here propose a new MTX liquid formulation stable for at least 120 days.

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis.

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

Synthesis, crystal structure, characterization of zinc(II), cadmium(II) complexes with 3-thiophene aldehyde thiosemicarbazone (3TTSCH). Biological activities of 3TTSCH and its complexes.

The reaction of zinc(II) chloride, cadmium(II) chloride and bromide with 3-thiophene aldehyde thiosemicarbazone leads to the formation of a series of new complexes. They have been characterized by spectroscopic studies: infrared, (1)H NMR, and electronic spectra. The crystal structures of the compound [ZnCl(2)(3TTSCH)(2)] and [CdBr(2)(3TTSCH)(2)] have been determined by X-ray diffraction methods. For the complexes [ZnCl(2)(3TTSCH)(2)] and [CdBr(2)(3TTSCH)(2)], the central ion is coordinated through the sulfur, and for the complexes [CdCl(2)(3TTSCH)], [CdBr(2)(3TTSCH)] the ion is coordinated through the sulfur as well as azomethine nitrogen atom of the thiosemicarbazone. In addition, fungistatic and bacteriostatic activities of both ligand and complexes have been evaluated. Cadmium(II) complexes have shown the most significant activities.

The virulence variability of different Acinetobacter baumannii strains in experimental pneumonia.

OBJECTIVES: Our objective was to compare the virulence of 5 strains of Acinetobacter baumannii by using a mouse model of pneumonia. METHODS: Six-week old female C3H/HeN mice were used. The pneumonia was inducted by intra-tracheal inoculation of 5.10(6) bacteria. Spontaneous outcome was evaluated by mortality, mice weight variations, and a clinical score. Bacterial counts in lungs, spleen and blood, and inflammatory response in lungs (dosages of tumor necrosis factor-alpha and macrophage inflammatory protein-2) were also measured. Lastly, a histological examination of lungs was performed for 3 strains, giving a histological score. RESULTS: Global mortality varied from 13% to 79% (P<10(-4)). Bacterial counts in lungs within the 4 days following inoculation varied significantly according to different strains. The evolution curves of bacterial counts were also different. There was a significant correlation between the clinical score and mortality (P<0.05) but not between bacterial counts in lungs and mortality. The increase of pro-inflammatory mediator production in lungs and the histological score also varied according to strains. CONCLUSIONS: These results demonstrate the variability of the virulence between strains, and suggest that bacterial proliferation is not the only virulence factor responsible for the pathogenesis in A. baumannii pneumonia.

High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2' epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Lipid nanocapsules: ready-to-use nanovectors for the aerosol delivery of paclitaxel.

Aerosol drug delivery permits the development of dose-intensification strategies in severe, malignant lung diseases. The aim of the study was to demonstrate that the encapsulation of paclitaxel in lipid nanocapsules (LNCs), a novel drug nanocarrier for lipophilic components, allows one to provide pulmonary drug delivery of paclitaxel by nebulisation, thereby allowing preclinical and clinical studies. LNC dispersions are made into aerosols with commercial nebulisers. The structure, drug payload and cytotoxicity of nebulised LNCs were compared to fresh LNCs. The results demonstrated that LNC dispersions could be made into aerosols by using mesh nebulisers without altering the LNC structure. Only eFlow rapid-produced aerosols are compatible with human use: the mean duration to nebulise 3 ml of LNC dispersion is less than 9 min, with an aerosol mass median aerodynamic diameter equal to 2.7+/-0.1 microm and a fine-particle fraction (between 1.0 and 5.0 microm) of 81.5+/-3.1%. No modifications of drug payload or cytotoxicity effects of paclitaxel-loaded LNC (PTX-LNC) were observed. In order to carry out preclinical studies, a scaled-up LNC formulation protocol was used. Chemical parameters, such as acidity and osmolarity, were optimised, and a storage procedure for PTX-LNC batches was set-up. Animal studies are now needed to determine the tolerance and therapeutic potential of LNC dispersion aerosols.

DNA array analysis of Candida albicans gene expression in response to adherence to polystyrene.

Candidiasis is often initiated by the colonization of inert surfaces. In order to elucidate the mechanisms involved in this adherence process, DNA macroarrays were used to analyze the transcriptome of Candida albicans, the main causative agent of this mycoses, in a simple adherence model using germ tubes produced in polystyrene Petri dishes. Non-adherent germ tubes produced on glass surface were used as a control. Analysis of gene expression displayed 77 genes identified as statistically overexpressed in adherent germ tubes. Among these genes, some encoded enzymes participating in metabolism of lipids (such as LIP6), of proteins (such as SAP1) or of carbohydrates (like PGI1, PMI40 and PSA1. Some of these genes have already been reported as playing a role in pathogenesis of C. albicans. However, functions were unknown for a large part (45.5%) of the overexpressed genes which will be analyzed further in order to define their relationship with adherence.