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Bacterial components and cytokines induce IL-7 receptor (IL-7Rα) expression in monocytes. Aberrant low IL-7Rα expression of monocytes has been identified as a feature of tuberculosis immunopathology. Here, we investigated the mechanisms underlying IL-7Rα regulation of monocytes and tuberculosis serum effects on IL-7Rα expression. Serum samples from tuberculosis patients and healthy controls, cytokine candidates, and mycobacterial components were analyzed for in vitro effects on IL-7Rα expression of primary monocytes, monocyte-derived macrophages (MDM), and monocyte cell lines. IL-7Rα regulation during culture and the role of FoxO1 were characterized. In vitro activation-induced IL-7Rα expression in human monocytes and serum samples from tuberculosis patients boosted IL-7Rα expression. Although pathognomonic tuberculosis cytokines were not associated with serum effects, we identified cytokines (i.e., GM-CSF, IL-1β, TNF-α, IFN-γ) that induced IL-7Rα expression in monocytes and/or MDM comparable to mycobacterial components. Blocking of cytokine subsets (i.e., IL-1β/TNF-α in monocytes, GM-CSF in MDM) largely diminished IL-7Rα expression induced by mycobacterial components. Finally, we showed that in vitro-induced IL-7Rα expression was transient and dependent on constitutive FoxO1 expression in primary monocytes and monocyte cell lines. This study demonstrated the crucial roles of cytokines and constitutive FoxO1 expression for transient IL-7Rα expression in monocytes.

Background Buruli ulcer caused by Mycobacterium ulcerans is endemic in parts of West Africa and is most prevalent among the 5–15 years age group; Buruli ulcer is uncommon among neonates. The mode of transmission and incubation period of Buruli ulcer are unknown. We report two cases of confirmed Buruli ulcer in human immunodeficiency virus-unexposed, vaginally delivered term neonates in Ghana. Case presentation Patient 1: Two weeks after hospital delivery, a baby born to natives of the Ashanti ethnic group of Ghana was noticed by her mother to have a papule with associated edema on the right anterior chest wall and neck that later ulcerated. There was no restriction of neck movements. The diagnosis of Buruli ulcer was confirmed on the basis of a swab sample that had a positive polymerase chain reaction result for the IS2404 repeat sequence of M. ulcerans. Patient 2: This patient, from the Ashanti ethnic group in Ghana, had the mother noticing a swelling in the baby’s left gluteal region 4 days after birth. The lesion progressively increased in size to involve almost the entire left gluteal region. Around the same time, the mother noticed a second, smaller lesion on the forehead and left side of neck. The diagnosis of Buruli ulcer was confirmed by polymerase chain reaction when the child was aged 4 weeks. Both patients 1 and 2 were treated with oral rifampicin and clarithromycin at recommended doses for 8 weeks in addition to appropriate daily wound dressing, leading to complete healing. Our report details two cases of polymerase chain reaction-confirmed Buruli ulcer in children whose lesions appeared at ages 14 and 4 days, respectively. The mode of transmission of M. ulcerans infection is unknown, so the incubation period is difficult to estimate and is probably dependent on the infective dose and the age of exposure. In our study, lesions appeared 4 days after birth in patient 2. Unless the infection was acquired in utero, this would be the shortest incubation period ever recorded. Conclusions Buruli ulcer should be included in the differential diagnosis of neonates who present with characteristic lesions. The incubation period of Buruli ulcer in neonates is probably shorter than is reported for adults.

Summary Across the globe, the outbreak of the COVID-19 pandemic is causing distress with governments doing everything in their power to contain the spread of the novel coronavirus (SARS-CoV-2) to prevent morbidity and mortality. Actions are being implemented to keep health care systems from being overstretched and to curb the outbreak. Any policy responses aimed at slowing down the spread of the virus and mitigating its immediate effects on health care systems require a firm basis of information about the absolute number of currently infected people, growth rates, and locations/hotspots of infections. The only way to obtain this base of information is by conducting numerous tests in a targeted way. Currently, in Ghana, there is a centralized testing approach, that takes 4–5 days for samples to be shipped and tested at central reference laboratories with results communicated to the district, regional and national stakeholders. This delay in diagnosis increases the risk of ongoing transmission in communities and vulnerable institutions. We have validated, evaluated and deployed an innovative diagnostic tool on a mobile laboratory platform to accelerate the COVID-19 testing. A preliminary result of 74 samples from COVID-19 suspected cases has a positivity rate of 12% with a turn-around time of fewer than 3 hours from sample taking to reporting of results, significantly reducing the waiting time from days to hours, enabling expedient response by the health system for contact tracing to reduce transmission and additionally improving case management. Funding Test kits were provided by AngloGold Ashanti Obuasi Mine (AngloGold Ashanti Health Foundation). The American Leprosy Mission donated the PCR machine, and the mobile laboratory van was funded by the Embassy of the Kingdom of the Netherlands (EKN). AAS, YAA was supported by (PANDORA-ID-NET RIA2016E-1609) and ROP supported by EDCTP Senior Fellowship (TMA2016SF), both funded by the European and Developing Countries Clinical Trials Partnership (EDCTP2) programme which is supported under Horizon 2020, the European Union.

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 &deg, C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.

Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs as well as achieving the WHO 2012-2020 road map for the total eradication of schistosomiasis. Recombinase polymerase amplification (RPA) has emerged as a rapid and simple molecular tool adaptable for fewer resources with diagnostic accuracy similar to polymerase chain reaction (PCR). This rapid molecular assay employs the use of enzymes for the amplification of nucleic acid taget at a constant temperature. The aim of this study was to validate a real-time RPA assay targeting the Dra 1 repittitive sequence of Schistosoma (S.) haematobium and evaluate its use in urogenital schistosomiasis diagnosis. S. haematobium Dra 1 molecular DNA standard was applied to determine the assay's analytical sensitivity. DNA extracts of S. haematobium, other Schistosoma species, protozoa and bacteria species were used to determine the specificity of the RPA assay. Clinical performance of the assay was validated with a panel of 135 urine samples from volunteers of schistosomiasis endemic communities. The developed assay was evaluated with urine samples extracted by just boiling and with SpeedXtract® DNA extraction kit. A specific fragment of S. haematobium Dra 1 repetitive sequence was amplified within 15 minutes at a constant 42˚C using the developed S. haematobium RPA assay. The detection limit was 15 copies of Dra1 molecular DNA standard per reaction. There was no cross-reaction with other protozoan and bacterial species except Schistosoma species, S. mansoni and S. japonicum. Using 135 urine samples, Schistosoma RPA assay had a clinical sensitivity and specificity of 98.4% (95% CI, 91.6-100) and 100% (95% CI, 94.9-99) respectively when compared to S. haematobium Dra 1 qPCR assay. The diagnostic performance of S. haematobium real-time RPA assay was not affected by the use of crude DNA extracted samples. The S. haematobium RPA assay can serve as an alternative to PCR, especially in low resource settings.

Isothermal amplification techniques such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) for diagnosing Buruli ulcer, a necrotic skin disease caused by Mycobacterium ulcerans, have renewed hope for the molecular diagnosis of clinically suspected Buruli ulcer cases in endemic districts. If these techniques are applied at district-level hospitals or clinics, they will help facilitate early case detection with prompt treatment, thereby reducing disability and associated costs of disease management. The accuracy as well as the application of these molecular techniques at point of need is dependent on simple and fast DNA extraction. We have modified and tested a rapid extraction protocol for use with an already developed recombinase polymerase amplification assay. The entire procedure from &ldquo, sample in, extraction and DNA amplification&rdquo, was conducted in a mobile suitcase laboratory within 40 min. The DNA extraction procedure was performed within 15 min, with only two manipulation/pipetting steps needed. The diagnostic sensitivity and specificity of this extraction protocol together with M. ulcerans RPA in comparison with standard DNA extraction with real-time PCR was 87% (n = 26) and 100% (n = 13), respectively. We have established a simple, fast and efficient protocol for the extraction and detection of M. ulcerans DNA in clinical samples that is adaptable to field conditions.

Background Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. Methodology and principal findings A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84–100), whiles the sensitivity was 88% (95% CI, 77–95). Conclusion The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure., Author summary Current diagnostic methods to detect M. ulcerans suffer from delayed time-to-results in most endemic countries by the prolonged period of time for the shipment and storage of samples to a distant, centralized laboratory. The M. ulcerans recombinase polymerase amplification assay (Mu-RPA) is a new, rapid diagnostic test developed for the detection of M. ulcerans infection, known commonly as Buruli ulcer, a chronic, debilitating, necrotizing disease of the skin and soft tissues. This assay is suitable for use on a portable detection device, with the potential to be used for quick diagnosis at the point of need, providing timely results to health workers at Buruli ulcer treatment clinics.

Bacteria in biofilms are encased in an extracellular polymeric matrix that limits exposure of microbial cells to lethal doses of antimicrobial agents, leading to resistance. In Pseudomonas aeruginosa, biofilm formation is regulated by cell-to-cell communication, called quorum sensing. Quorum sensing facilitates a variety of bacterial physiological functions such as swarming motility and protease, pyoverdine, and pyocyanin productions. Peptide mix from the marine mollusc, Olivancillaria hiatula, has been studied for its antibiofilm activity against Pseudomonas aeruginosa. Microscopy and microtiter plate-based assays were used to evaluate biofilm inhibitory activities. Effect of the peptide mix on quorum sensing-mediated processes was also evaluated. Peptide mix proved to be a good antibiofilm agent, requiring less than 39 μg/mL to inhibit 50% biofilm formation. Micrographs obtained confirmed biofilm inhibition at 1/2 MIC whereas 2.5 mg/mL was required to degrade preformed biofilm. There was a marked attenuation in quorum sensing-mediated phenotypes as well. At 1/2 MIC of peptide, the expression of pyocyanin, pyoverdine, and protease was inhibited by 60%, 72%, and 54%, respectively. Additionally, swarming motility was repressed by peptide in a dose-dependent manner. These results suggest that the peptide mix from Olivancillaria hiatula probably inhibits biofilm formation by interfering with cell-to-cell communication in Pseudomonas aeruginosa.

The incidence of antimicrobial resistance among microbial communities is a major threat to global health care and security. Landfills, which are reservoirs for many pharmaceuticals, provide a conducive habitat for antimicrobial-resistant microbes and resistant gene transfer and are therefore a major contributor to the phenomenon of antimicrobial resistance. Hence, this study determined the levels of three widely used antibiotics, metronidazole, penicillin, and amoxicillin, and the occurrence of antimicrobial resistance amongst microbes in soil and leachate samples from active and abandoned landfill sites in Kumasi, Ghana. Soil samples were collected from one active and four abandoned landfills, while leachate specimen was collected only from the active landfill. Sonication and solid-phase extraction (SPE) were used for sample preparation, followed by analysis via an HPLC-PDA method. Isolation and characterization of bacteria were done using standard bacteriological techniques. Antibiotic susceptibility testing was determined following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotics were detected at very high concentrations in the specimen collected from both active and abandoned landfill sites. For leachate samples obtained from Dompoase, penicillin was present at the highest concentration (67.42 ± 5.35 μg/mL, p<0.05) followed by metronidazole (18.25 ± 7.92 μg/mL) and amoxicillin (10.96 ± 6.93 μg/mL). In general, the levels of antibiotics in soil samples were similar at both active and abandoned landfill sites. Nonetheless, as with leachates, penicillin levels were much higher (p<0.05) than levels of amoxicillin and metronidazole within any particular site. When screened against some antibiotics, Enterobacteriaceae and some Bacillus and Listeria species isolated from the soil and leachate samples proved to be resistant. The high levels of antibiotics coupled with the presence of resistant microbes at these landfills sites call for immediate measures to halt the disposal of pharmaceuticals in the environment so as to avert any possible public health setback.

This document describes the standard operating procedure for the application of the real time Mu-RPA assay with Exo probe system. The assay detects M. ulcerans DNA (IS2404) from clinical sample or culture suspension. The assay consists of recombinase polymerase amplification TwistDx Exo kit procedure. The reaction mix must be prepared in an environment free of DNA amplicons. Personal protective clothing (i.e. lab coats, gloves) must be used throughout the process.

BackgroundThere are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasises early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unreliable in inexperienced hands and complicated by infections that have similar presentations. This study was to develop and evaluate the use of recombinase polymerase amplification (RPA) assay for the detection of Mycobacterium ulcerans at the point of patient care.MethodsA specific fragment of IS2404 of M. ulcerans was amplified in 15 min at a constant temperature of 42°C, using the RPA assay and analysed on a portable fluorometre. The’method was tested for sensitivity and specificity with molecular standard of IS2404 DNA fragment, various M.’ulcerans strains, other mycobacteria and environmentally associated bacteria. Additionally, the assay performance as a diagnostic tool was tested with archived DNA from symptomatic patients. All results were compared with that of a highly sensitive IS2404 PCR.ResultsThe detection limit was 50 copies of IS2404 in 15 min using plasmid standard and 125 fg with genomic Mu DNA equivalent 25 genomic copies. The assay was highly specific in detecting all strains of M. ulcerans with no observed cross reactivity with other mycobacteria and common skin colonising bacteria. The clinical sensitivity and specificity of the BU-RPA assay using clinical samples was 86% and 100% respectively.ConclusionWe have developed a real-time isothermal RPA assay for the detection of M. ulcerans as a cheaper alternative to PCR. Combining this assay with a simple extraction protocol will maximise its use as point-of-care test for Buruli ulcer.