Bacteriophage therapy in musculoskeletal infections: from basic science to clinical application.

The treatment of musculoskeletal infections (MSIs), including periprosthetic joint infection (PJI) and fracture-related infection (FRI), is often complicated by biofilm-related challenges necessitating multiple revision surgeries and incurring substantial costs. The emergence of antimicrobial resistance (AMR) adds to the complexity of the problem, leading to increased morbidity and healthcare expenses. There is an urgent need for novel antibacterial strategies, with the World Health Organization endorsing non-traditional approaches like bacteriophage (phage) therapy. Phage therapy, involving the targeted application of lytic potent phages, shows promise in the treatment of MSIs. Although historical clinical trials and recent case studies present significant milestones in the evolution of phage therapy over the past century, challenges persist, including variability in study designs, administration protocols and phage selection. Efforts to enhance treatment efficacy consist of personalized phage therapy and combination with antibiotics. Future perspectives entail addressing regulatory barriers, standardizing treatment protocols, and conducting high-quality clinical trials to establish phage therapy's efficacy for the treatment of MSIs. Initiatives like the PHAGEFORCE study and the PHAGEinLYON Clinic programme aim to streamline phage therapy, facilitating personalized treatment approaches and systematic data collection to advance its clinical utility in these challenging infections.

A prospective multicentre evaluation of BioFire® Joint Infection Panel for the rapid microbiological documentation of acute arthritis.

OBJECTIVES: To assess the performance of the rapid syndromic BioFire® Joint Infection Panel (BF-JIP) to detect bacterial and fungal pathogens, as well as antibiotic resistance genes, directly in synovial fluid specimens collected from patients with acute arthritis. METHODS: The study was conducted in six French bacteriological laboratories. To assess the performances of BF-JIP, results were compared with those of synovial fluid 14-day culture and, in case of discrepancy, with those of complementary molecular methods and intraoperative samples. A total of 308 synovial fluid specimens were tested after collection from 308 adults and children presenting with clinical and biological suspicion of acute arthritis; patients presenting with acute periprosthetic joint infection were included according to the European Bone and Joint Infection Society 2021 criteria. RESULTS: Only one specimen failed (no result). On the basis of the consolidated data, the BF-JIP was concordant with the 14-day culture in 280 (91.2%) of the 307 specimens finally included in the study. The positive percentage agreement was 84.9% (95% CI, 78.8-89.8%) and the negative percentage agreement was 100% (95% CI, 97.2-100%). The positive predictive value was extremely high (100%; 95% CI, 97.6-100%), whereas the negative predictive value was lower (82.6%; 95% CI, 75.7-88.2%), partially explained by the missing target species in the panel. DISCUSSION: The BF-JIP showed high performances to detect pathogens involved in acute arthritis.

Case report: Continuous infusions of ceftazidime-avibactam and aztreonam in combination through elastomeric infusors for 12 weeks for the treatment of bone and joint infections due to metallo-ß-lactamase producing Enterobacterales.

Among carbapenem-resistant Enterobacterales, metallo-beta-lactamase producing strains represent a growing therapeutic challenge. While the association of aztreonam and ceftazidime-avibactam has been investigated in recent years for the treatment of infections involving these strains, little to no clinical data support the use of this association for the treatment of bone and joint infections. We report two cases of complex bone and joint infections involving metallo-beta-lactamase-producing Enterobacterales, successfully treated at our referral center with aztreonam and ceftazidime-avibactam for 12 weeks in continuous infusions through elastomeric infusors.

Phage Therapy against Staphylococcus aureus: Selection and Optimization of Production Protocols of Novel Broad-Spectrum Silviavirus Phages.

Background: Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus. Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 1011 PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages. Conclusions: In sum, we propose a quality-by-design approach for the amplification of broad-spectrum anti-S. aureus phages, facilitating the subsequent steps of the manufacturing process; namely, purification and quality control.

Improving the Diagnosis of Bacterial Infections: Evaluation of 16S rRNA Nanopore Metagenomics in Culture-Negative Samples.

While 16S rRNA PCR-Sanger sequencing has paved the way for the diagnosis of culture-negative bacterial infections, it does not provide the composition of polymicrobial infections. We aimed to evaluate the performance of the Nanopore-based 16S rRNA metagenomic approach, using both partial and full-length amplification of the gene, and to explore its feasibility and suitability as a routine diagnostic tool for bacterial infections in a clinical laboratory. Thirty-one culture-negative clinical samples from mono- and polymicrobial infections based on Sanger-sequencing results were sequenced on MinION using both the in-house partial amplification and the Nanopore dedicated kit for the full-length amplification of the 16S rRNA gene. Contamination, background noise definition, bacterial identification, and time-effectiveness issues were addressed. Cost optimization was also investigated with the miniaturized version of the flow cell (Flongle). The partial 16S approach had a greater sensitivity compared to the full-length kit that detected bacterial DNA in only 24/31 (77.4%) samples. Setting a threshold of 1% of total reads overcame the background noise issue and eased the interpretation of clinical samples. Results were obtained within 1 day, discriminated polymicrobial samples, and gave accurate bacterial identifications compared to Sanger-based results. We also found that multiplexing and using Flongle flow cells was a cost-effective option. The results confirm that Nanopore technology is user-friendly as well as cost- and time-effective. They also indicate that 16S rRNA targeted metagenomics is a suitable approach to be implemented for the routine diagnosis of culture-negative samples in clinical laboratories.

Heterogeneous vancomycin resistance in Staphylococcus aureus does not predict development of vancomycin resistance upon vancomycin pressure.

BACKGROUND: It is unclear whether Staphylococcus aureus with heterogeneous intermediate vancomycin resistance (hVISA) can develop vancomycin resistance faster than vancomycin-susceptible S. aureus (VSSA) strains. METHODS: We compared the kinetics of vancomycin MIC increase for 15 days of sustained in vitro vancomycin exposure for clinical hVISA (n = 12) and VSSA (n = 24) isolates, as well as for reference strains Mu3 (hVISA) and ATCC 29213 (VSSA). Clinical isolates were categorized as hVISA using the population analysis profile method. MICs were monitored for 15 days and the rate of MIC increase under exposure, for each strain, was evaluated in a linear regression model relative to time. RESULTS: All isolates acquired vancomycin resistance upon exposure. Vancomycin MICs increased faster for VSSA compared with hVISA isolates (P < 0.01). CONCLUSIONS: The hVISA phenotype does not correspond to an enhanced adaptation potential to in vitro vancomycin pressure.

Pharyngeal abscess complicated by Lemierre's syndrome: Identification of an atypical germ.

INTRODUCTION: Lemierre's syndrome is a rare but serious complication of an oral infection mostly related to Fusobacterium necrophorum. This condition combines jugular vein thrombosis and septic emboli to the lungs or other organs. CASE PRESENTATION: We report here an original case of a pharyngeal abscess complicated by Lemierre's syndrome in a young healthy male patient. Samples taken from the pus of the pharyngeal abscess showed the presence of Gardnerella vaginalis associated with Fusobacterium necrophorum. The patient was treated by draining the abscess, antibiotic therapy and preventive anticoagulation for 1 month. The evolution was good with a resolution of the thrombosis. CONCLUSIONS: This case highlights the need for bacterial identification to adapt antibiotic therapy in Lemierre's syndrome. It also shows the possibility of extragenital localization of Gardnerella vaginalis in a male patient having oral sex with women. In contrast to sexually transmitted infections such as syphilis and pharyngeal gonococcus, this oral localization of Gardnerella vaginalis has not been described previously in the literature.

Has doxycycline, in combination with anti-malarial drugs, a role to play in intermittent preventive treatment of Plasmodium falciparum malaria infection in pregnant women in Africa?

According to the World Health Organization (WHO), Plasmodium falciparum malaria during pregnancy is responsible for deleterious consequences for the mother and her child. The administration of intermittent preventive treatment (IPTp) with sulfadoxine-pyrimethamine (SP) at each antenatal care visit as early as 13 weeks of gestation until the time of delivery is a strategy that is currently recommended by WHO for the prevention of malaria in pregnancy. However, the emergence and the spread of the resistance to SP in Africa raise the question of the short-term effectiveness of the strategy. Dihydroartemisinin-piperaquine 120 mg/960 mg once a day for 3 consecutive days administered at least three times during the pregnancy might be an option for IPTp. The combination of 200 mg of doxycycline once a day for 3 consecutive days seems to be a good option to retard the emergence and the spread of resistance to artemisinin-based combination therapy (ACT) in Africa and improve the effectiveness of ACT in term of preterm births, neonatal morbidity and mortality. Contrary to preconceived ideas, scientific and medical data suggest that the risk of congenital malformations in the fetus or of tooth staining in infants whose mothers take doxycycline and hepatotoxicity during pregnancy is very low or non-existent. Additionally, the use of doxycycline during the first and second trimesters leads to an increase in gestational age at delivery, a decrease in the number of preterm births and a reduction in neonatal morbidity and mortality due to the beneficial antimicrobial activity of doxycycline against other infections during pregnancy. Furthermore, doxycycline has anti-malarial properties and is already recommended as prophylaxis for travellers and for treatment of falciparum malaria in combination with other anti-malarial drugs.

The end of a dogma: the safety of doxycycline use in young children for malaria treatment.

Anti-malarial drug resistance to chloroquine and sulfadoxine-pyrimethamine has spread from Southeast Asia to Africa. Furthermore, the recent emergence of resistance to artemisinin-based combination therapy (ACT) in Southeast Asia highlights the need to identify new anti-malarial drugs. Doxycycline is recommended for malaria chemoprophylaxis for travel in endemic areas, or in combination with the use of quinine for malaria treatment when ACT is unavailable or when the treatment of severe malaria with artesunate fails. However, doxycycline is not used in young children under 8 years of age due to its contraindication due to the risk of yellow tooth discolouration and dental enamel hypoplasia. Doxycycline was developed after tetracycline and was labelled with the same side-effects as the earlier tetracyclines. However, recent studies report little or no effects of doxycycline on tooth staining or dental enamel hypoplasia in children under 8 years of age. In the United States, the Centers for Disease Control and Prevention have recommended the use of doxycycline for the treatment of acute and chronic Q fever and tick-borne rickettsial diseases in young children. It is time to rehabilitate doxycycline and to recommend it for malaria treatment in children under 8 years of age.

Antibiotics in malaria therapy: which antibiotics except tetracyclines and macrolides may be used against malaria?

Malaria, a parasite vector-borne disease, is one of the most significant health threats in tropical regions, despite the availability of individual chemoprophylaxis. Malaria chemoprophylaxis and chemotherapy remain a major area of research, and new drug molecules are constantly being developed before drug-resistant parasites strains emerge. The use of anti-malarial drugs is challenged by contra-indications, the level of resistance of Plasmodium falciparum in endemic areas, clinical tolerance and financial cost. New therapeutic approaches are currently needed to fight against this disease. Some antibiotics that have shown potential effects on malaria parasite have been recently studied in vitro or in vivo intensively. Two families, tetracyclines and macrolides and their derivatives have been particularly studied in recent years. However, other less well-known have been tested or are being used for malaria treatment. Some of these belong to older families, such as quinolones, co-trimoxazole or fusidic acid, while others are new drug molecules such as tigecycline. These emerging antibiotics could be used to prevent malaria in the future. In this review, the authors overview the use of antibiotics for malaria treatment.

Decontamination of a field laboratory dedicated to Ebola virus-infected patients.

In 2015, the French Armed Forces deployed a biosafety level 3 (BSL3) field laboratory as a part of an Ebola treatment center in Guinea. When closing the center, laboratory decontamination operations were necessary. We present the decontamination protocols applied for the BSL3 field laboratory, making the entire module ready for a future use.

Macrolides and associated antibiotics based on similar mechanism of action like lincosamides in malaria.

Malaria, a parasite vector-borne disease, is one of the biggest health threats in tropical regions, despite the availability of malaria chemoprophylaxis. The emergence and rapid extension of Plasmodium falciparum resistance to various anti-malarial drugs has gradually limited the potential malaria therapeutics available to clinicians. In this context, macrolides and associated antibiotics based on similar mechanism of action like lincosamides constitute an interesting alternative in the treatment of malaria. These molecules, whose action spectrum is similar to that of tetracyclines, are typically administered to children and pregnant women. Recent studies have examined the effects of azithromycin and the lincosamide clindamycin, on isolates from different continents. Azithromycin and clindamycin are effective and well tolerated in the treatment of uncomplicated malaria in combination with quinine. This literature review assesses the roles of macrolides and lincosamides in the prophylaxis and treatment of malaria.

Tetracyclines in malaria.

Malaria, a parasite vector-borne disease, is one of the greatest health threats in tropical regions, despite the availability of malaria chemoprophylaxis. The emergence and rapid extension of Plasmodium falciparum resistance to various anti-malarial drugs has gradually limited the number of potential malaria therapeutics available to clinicians. In this context, doxycycline, a synthetically derived tetracycline, constitutes an interesting alternative for malaria treatment and prophylaxis. Doxycycline is a slow-acting blood schizontocidal agent that is highly effective at preventing malaria. In areas with chloroquine and multidrug-resistant P. falciparum parasites, doxycycline has already been successfully used in combination with quinine to treat malaria, and it has been proven to be effective and well-tolerated. Although not recommended for pregnant women and children younger than 8 years of age, severe adverse effects are rarely reported. In addition, resistance to doxycycline is rarely described. Prophylactic and clinical failures of doxycycline have been associated with both inadequate doses and poor patient compliance. The effects of tetracyclines on parasites are not completely understood. A better comprehension of the mechanisms underlying drug resistance would facilitate the identification of molecular markers of resistance to predict and survey the emergence of resistance.

Absence of association between Plasmodium falciparum small sub-unit ribosomal RNA gene mutations and in vitro decreased susceptibility to doxycycline.

BACKGROUND: Doxycycline is an antibiotic used in combination with quinine or artesunate for malaria treatment or alone for malaria chemoprophylaxis. Recently, one prophylactic failure has been reported, and several studies have highlighted in vitro doxycycline decreased susceptibility in Plasmodium falciparum isolates from different areas. The genetic markers that contribute to detecting and monitoring the susceptibility of P. falciparum to doxycycline, the pfmdt and pftetQ genes, have recently been identified. However, these markers are not sufficient to explain in vitro decreased susceptibility of P. falciparum to doxycycline. In this paper, the association between polymorphism of the small sub-unit ribosomal RNA apicoplastic gene pfssrRNA (PFC10_API0057) and in vitro susceptibilities of P. falciparum isolates to doxycycline were investigated. METHODS: Doxycycline IC50 determinations using the hypoxanthine uptake inhibition assay were performed on 178 African and Thai P. falciparum isolates. The polymorphism of pfssrRNA was investigated in these samples by standard PCR followed by sequencing. RESULTS: No point mutations were found in pfssrRNA in the Thai or African isolates, regardless of the determined IC50 values. CONCLUSIONS: The pfssrRNA gene is not associated with in vitro decreased susceptibility of P. falciparum to doxycycline. Identifying new in vitro molecular markers associated with reduced susceptibility is needed, to survey the emergence of doxycycline resistance.

Molecular Markers and In Vitro Susceptibility to Doxycycline in Plasmodium falciparum Isolates from Thailand.

Determinations of doxycycline 50% inhibitory concentrations (IC50) for 620 isolates from northwest Thailand were performed via the isotopic method, and the data were analyzed by the Bayesian method and distributed into two populations (mean IC50s of 13.15 µM and 31.60 µM). There was no significant difference between the group with low IC50s versus the group with high IC50s with regard to copy numbers of the Plasmodium falciparum tetQ (pftetQ) gene (P = 0.11) or pfmdt gene (P = 0.87) or the number of PfTetQ KYNNNN repeats (P = 0.72).

Multinormal in vitro distribution of Plasmodium falciparum susceptibility to piperaquine and pyronaridine.

BACKGROUND: In 2002, the World Health Organization recommended that artemisinin-based combination therapy (ACT) be used to treat uncomplicated malaria. Dihydroartemisinin-piperaquine and artesunate-pyronaridine are two of these new combinations. The aim of the present work was to assess the distribution of the in vitro values of pyronaridine (PND) and piperaquine (PPQ) and to define a cut-off for reduced susceptibility for the two anti-malarial drugs. METHODS: The distribution and range of the 50% inhibitory concentration values (IC50) of PND and PPQ were determined for 313 isolates obtained between 2008 and 2012 from patients hospitalized in France for imported malaria. The statistical Bayesian analysis was designed to answer the specific question of whether Plasmodium falciparum has different phenotypes of susceptibility to PND and PPQ. RESULTS: The PND IC50 values ranged from 0.6 to 84.6 nM, with a geometric mean of 21.1 ± 16.0 nM (standard deviation). These values were classified into three components. The PPQ IC50 values ranged from 9.8 to 217.3 nM, and the geometric mean was 58.0 ± 34.5 nM. All 313 PPQ values were classified into four components. Isolates with IC50 values greater than 60 nM or four-fold greater than 3D7 IC50 are considered isolates that have reduced susceptibility to PND and those with IC50 values greater than 135 nM or 2.3-fold greater than 3D7 IC50 are considered isolates that have reduced susceptibility to PPQ. CONCLUSION: The existence of at least three phenotypes for PND and four phenotypes for PPQ was demonstrated. Based on the cut-off values, 18 isolates (5.8%) and 13 isolates (4.2%) demonstrated reduced susceptibility to PND and PPQ, respectively.

Nocardia veterana: disseminated infection with urinary tract infection

Nocardia spp. are a group of aerobic actinomycetes widely distributed in soil, and associated with severe opportunistic infections, essentially pulmonary infections. We report the first case of disseminated infection associated with urinary tract infection caused by Nocardia veterana. The diagnosis was difficult; despite the presence of pulmonary nodules, the lung biopsies remained negative while only one aerobic blood culture and the urine culture were positive for N. veterana, identified after a 16S rDNA gene sequence analysis. Few cases of clinical importance due to N. veterana have been published since its characterization. The bacteriological diagnosis of nocardiosis can be difficult to establish because of the delayed growth and the specific techniques that are required. This case illustrates the necessity of performing specific investigations in immunocompromised patients who present with infectious disease because the severity of this infection requires early diagnosis and quick initiation of appropriate antibiotic therapy.