A Multi-Laboratory Evaluation of Commercial Monkeypox Virus Molecular Tests.

In this report, we describe the first national scale multi-laboratory evaluation of monkeypox virus (MPXV) DNA commercial PCR kits. The objective of this study was to evaluate 2 kits by different diagnostic laboratories across Israel. Ten standardized samples were tested simultaneously using the Novaplex (15 laboratories) and Bio-Speedy (seven laboratories) kits. An in-house assay based on previously published reactions was used as reference. Comparison of the results showed high intra-assay agreement between laboratories, with small variations for most samples. The in-house assay had an analytical detection limit of less than 10 copies per reaction. While the 2 commercial kits were able to detect specimens with low viral loads similarly to the in-house assay, significant differences were observed, in the Cq values and relative fluorescence (RF), between the assays. The RF signal of the in-house and Bio-Speedy assays ranged between 5,000 and 10,000 RFU, while the signal in the Novaplex assay was less than 600 RFU. Due to the kit measurement protocol, the Cq values of the Bio-Speedy kit were 5 to 7.5 cycles lower than those of the in-house assay. On the contrary, the Cq values of the Novaplex kit were significantly higher than those of the in-house assay, with differences of 3 to 5 cycles per sample. Our results suggest that while all assays were similar in their overall sensitivity, direct comparison of Cq values between them may be misleading. To our knowledge, this is the first methodical evaluation of commercial MPX test kits. We therefore anticipate that this study would help diagnostic laboratories in choosing a specific MPX detection assay. IMPORTANCE To the best of our knowledge, this study is the first methodical evaluation of commercial kits designed for Monkeypox virus detection. This was done by performing the same tests using the same sample set in multiple laboratories, simultaneously, on a national scale. It therefore provides important and unique information on the performance of such kits and provides a guideline for choosing the assay of choice for monkeypox virus diagnosis in a standard diagnostic laboratory. It also demonstrates potential complications when trying to compare the results of different assays, even when testing exactly the same samples, under identical conditions.

Delftibactin-A, a Non-ribosomal Peptide With Broad Antimicrobial Activity.

The rapid emergence of drug resistant bacteria is occurring worldwide, outpacing the development of new antibiotics. It is known that some of the main sources of antibiotics are the bacteria themselves, many of which are secondary metabolites of Gram positive bacteria. Siderophores, which are secondary metabolites, function as natural chelators (e.g., iron). They are produced and secreted by many bacteria and have been experimented on as "carriers" of several types of antibiotics that pass the cell membrane of challenging Gram negative bacteria. Delftibactin A is a non-ribosomal peptide (NRP), which is known to detoxify gold in Delftia spp. and form gold nuggets, and is considered to be a siderophore. In this study we demonstrate that the supernatant from novel environmental isolates of Delftia spp. have antimicrobial activity. We characterized the active fraction and identified delftibactin A as a compound with antimicrobial activity. Delftibactin A exhibits potent antimicrobial activity against Gram positive multi drug resistant (MDR) bacteria like Methicillin-resistant Staphylococcus aureus (MRSA), and Vancomycin resistant Enterococcus (VRE), and also against the Gram negative pathogens Acinetobacter baumannii and Klebsiella pneumoniae. We discovered that the production of delftibactin A is greatly influenced by temperature. Furthermore, we have demonstrated the possibility of utilizing delftibactin A as a siderophore carrier of toxic metals such as gallium into Gram negative bacteria. These findings expose new opportunities of yet unexploited natural products such as delftibactin A, which have been known for other bacterial uses, as potent factors in the battle against MDR bacteria.

A reprofiled drug, auranofin, is effective against metronidazole-resistant Giardia lamblia.

Giardiasis is one of the most common causes of diarrheal disease worldwide. Treatment is primarily with 5-nitro antimicrobials, particularly metronidazole. Resistance to metronidazole has been described, and treatment failures can occur in up to 20% of cases, making development of alternative antigiardials an important goal. To this end, we have screened a chemical library of 746 approved human drugs and 164 additional bioactive compounds for activity against Giardia lamblia. We identified 56 compounds that caused significant inhibition of G. lamblia growth and attachment. Of these, 15 were previously reported to have antigiardial activity, 20 were bioactive but not approved for human use, and 21 were drugs approved for human use for other indications. One notable compound of the last group was the antirheumatic drug auranofin. Further testing revealed that auranofin was active in the low (4 to 6)-micromolar range against a range of divergent G. lamblia isolates representing both human-pathogenic assemblages A and B. Most importantly, auranofin was active against multiple metronidazole-resistant strains. Mechanistically, auranofin blocked the activity of giardial thioredoxin oxidoreductase, a critical enzyme involved in maintaining normal protein function and combating oxidative damage, suggesting that this inhibition contributes to the antigiardial activity. Furthermore, auranofin was efficacious in vivo, as it eradicated infection with different G. lamblia isolates in different rodent models. These results indicate that the approved human drug auranofin could be developed as a novel agent in the armamentarium of antigiardial drugs, particularly against metronidazole-resistant strains.

Α1-giardin based live heterologous vaccine protects against Giardia lamblia infection in a murine model.

Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide, yet preventive medical strategies are not available. A crude veterinary vaccine has been licensed for cats and dogs, but no defined human vaccine is available. We tested the vaccine potential of three conserved antigens previously identified in human and murine giardiasis, α1-giardin, α-enolase, and ornithine carbamoyl transferase, in a murine model of G. lamblia infection. Live recombinant attenuated Salmonella enterica Serovar Typhimurium vaccine strains were constructed that stably expressed each antigen, maintained colonization capacity, and sustained total attenuation in the host. Oral administration of the vaccine strains induced antigen-specific serum IgG, particularly IgG(2A), and mucosal IgA for α1-giardin and α-enolase, but not for ornithine carbamoyl transferase. Immunization with the α1-giardin vaccine induced significant protection against subsequent G. lamblia challenge, which was further enhanced by boosting with cholera toxin or sublingual α1-giardin administration. The α-enolase vaccine afforded no protection. Analysis of α1-giardin from divergent assemblage A and B isolates of G. lamblia revealed >97% amino acid sequence conservation and immunological cross-reactivity, further supporting the potential utility of this antigen in vaccine development. Together. These results indicate that α1-giardin is a suitable candidate antigen for a vaccine against giardiasis.

Impaired parasite attachment as fitness cost of metronidazole resistance in Giardia lamblia.

Infections with the diarrheagenic protozoan pathogen Giardia lamblia are most commonly treated with metronidazole (Mz). Treatment failures with Mz occur in 10 to 20% of cases and Mz resistance develops in the laboratory, yet clinically, Mz-resistant (Mz(r)) G. lamblia has rarely been isolated from patients. To understand why clinical Mz(r) isolates are rare, we questioned whether Mz resistance entails fitness costs to the parasite. Our studies employed several newly generated and established isogenic Mz(r) cell lines with stable, high-level resistance to Mz and significant cross-resistance to tinidazole, nitazoxanide, and furazolidone. Oral infection of suckling mice revealed that three of five Mz(r) cell lines could not establish infection, while two Mz(r) cell lines infected pups, albeit with reduced efficiencies. Failure to colonize resulted from a diminished capacity of the parasite to attach to the intestinal mucosa in vivo and to epithelial cells and plastic surfaces in vitro. The attachment defect was related to impaired glucose metabolism, since the noninfectious Mz(r) lines consumed less glucose, and glucose promoted ATP-independent parasite attachment in the parental lines. Thus, resistance of Giardia to Mz is accompanied by a glucose metabolism-related attachment defect that can interfere with colonization of the host. Because glucose-metabolizing pathways are important for activation of the prodrug Mz, it follows that a fitness trade-off exists between diminished Mz activation and reduced infectivity, which may explain the observed paucity of clinical Mz(r) isolates of Giardia. However, the data also caution that some forms of Mz resistance do not markedly interfere with in vivo infectivity.

New approaches to the treatment of giardiasis.

PURPOSE OF REVIEW: Giardiasis is one of the most common causes of diarrheal disease worldwide, yet existing antimicrobial therapies are not always effective and drug resistance occurs in vivo and in vitro. The review focuses on recent advances in the development of new antigiardial drug candidates. RECENT FINDINGS: Modification of existing drug leads is a major strategy to develop new high-potency drugs. Complex derivatives of 5-nitroimidazole, the core structure of the most commonly used antigiardial drug, metronidazole, have shown significantly improved activities against Giardia and the ability to overcome metronidazole resistance. Derivatives of benzimidazole, the structural core of the effective antigiardial albendazole, are also exhibiting promising new activities. Beyond lead modifications, several new classes of antigiardial drug candidates have recently been identified by high-throughput screening of large compound libraries, and first efforts have been reported on the development of drugs tailored to known molecular targets in Giardia. SUMMARY: The pipeline of new antigiardial drug candidates has significantly expanded over the last few years, but this expansion has so far not been accompanied by demonstration of efficacy in animal models or by a clear understanding of the action mechanisms, particularly in regard to new nitro antimicrobials. Many challenges are still to be expected before clinical utility of new antigiardial drugs can be established.

Association between renal injury and reduced interleukin-15 and interleukin-15 receptor levels in acute kidney injury.

Interleukin (IL)-15 serves as a survival factor for a broad array of cells. Renal cells express both IL-15 and its receptor (IL-15R); however, the role of IL-15 in the kidney is yet to be determined. We examined IL-15 and IL-15R levels in sepsis-related renal injury, ischemia-reperfusion injury (IRI), and cisplatin-induced nephrotoxicity. To test the anti-apoptotic effect of IL-15, Bcl-2/Bax mRNA levels were assessed in kidneys of IL-15Ralpha(-/-) mice and in IL-15-stimulated renal epithelial cells (RECs). In addition, RECs were exposed to cisplatin and apoptosis was evaluated by TUNEL staining, caspase-3 activity, and cell cycle analysis. Intrarenal IL-15 levels decreased 24 h after initiation of all three examined pathologies by 5.8-fold (sepsis), 11-fold (IRI), and 23-fold (cisplatin-induced nephrotoxicity). Further experiments revealed that while addition of rIL-15 (1 ng/mL) to wild-type (WT) RECs increased Bcl-2/Bax ratio by 2-fold, kidneys of IL-15Ralpha(-/-) mice exhibited 4-fold lower Bcl-2/Bax ratio compared to WT mice. Accordingly, IL-15 lowered the apoptotic rate in cisplatin-treated cultured REC, and IL-15Ralpha(-/-) renal cells exhibited a higher rate of cisplatin-induced apoptosis. Furthermore, IL-15 levels negatively correlated with BUN of cisplatin-treated mice (R = -0.69, P = 0.003), suggesting that a decline in renal-derived IL-15 is detrimental to renal cell survival and kidney function during pathological stress.

Involvement of graft-derived interleukin-15 in islet allograft rejection in mice.

The possibility that islets play a role in graft rejection during islet transplantation for type-1 diabetes patients holds promise for ex vivo islet manipulation and for specific anti-rejection therapy. Interleukin (IL)-15 is a T cell growth factor and chemoattractant that is expressed by non-T cells. Intragraft expression of IL-15 is elevated during acute rejection in patients and in mice, and systemic blockade of IL-15 in mice prolongs allograft survival. However, the source of IL-15 in these conditions is undetermined. Since epithelial cell-derived IL-15 promotes lymphocyte proliferation in culture, we sought to determine whether islet-derived IL-15 promotes rejection in mice. We designed antisense oligodeoxyribonucleotide molecules that target mouse IL-15. Uptake of FITC-labeled antisense molecules and efficacy of IL-15 inhibition in IFNgamma-stimulated islets were evaluated. Islets exhibited typical cytoplasmatic distribution of antisense molecules and produced IL-15 levels that were comparable to non-stimulated cells. Antisense-treated islet allografts, that were transplanted across multiple minor-histocompatibility-antigen mismatched strains of mice, were accepted at a higher rate than control-antisense treated islets or untreated islets (88.9% vs. 37.5% and 20%, respectively). Our results suggest that islet-derived IL-15 may be involved in acute islet allograft rejection.

Renal cells express a functional interleukin-15 receptor.

BACKGROUND: Interleukin (IL)-15 is a pleiotropic cytokine known to be involved in graft rejection and to serve as a survival factor for leukocytes and epithelial cells, including renal cells. It utilizes a heterotrimeric receptor complex that consists of the IL-2 receptor betagammac subunits (IL-2/15Rbetagammac) and a unique high-affinity alpha-chain responsible for IL-15 specificity. METHODS: The cDNA of IL-15Ralpha main mRNA product was isolated from primary human tubular epithelial cells (TEC) and sequenced. IL-15R expression in TEC and in murine renal tissue was demonstrated using western blotting, ligand binding and flow cytometry. TEC were activated with combinations of IL-15, IL-2 and interferon-gamma (IFN-gamma), and mRNA and protein levels of IL-15R were determined. Jak-STAT tyrosine phosphorylation was assayed following IL-15 exposure. RESULTS: The full-length alpha-chain mRNA bearing exons 1-7 is expressed in TEC. IL-15Ralpha protein was detected on intact cells by flow cytometry and in extracts of human and mice renal cells using a specific anti-IL-15Ralpha antibody and by ligand-binding assay. The three subunits of the IL-15R were similarly expressed in cortex and medulla of mice kidney. Stimulation of TEC with IFN-gamma upregulated the alpha-chain while IL-2 and IL-15 had no effect on its expression. A short IL-15 stimulation of TEC induced tyrosine phosphorylation of the main IL-15 signalling molecules (Jak-1, Jak-3, STAT-3 and STAT-5). CONCLUSIONS: Our data demonstrate the presence of a functional IL-15 receptor in the kidney. Since renal cells produce IL-15, this cytokine may have an autocrine/paracrine regulatory role in the kidney.