Comparison of disc diffusion, Etest and broth microdilution for testing susceptibility of carbapenem-resistant P. aeruginosa to polymyxins

Background

New prophylactic and therapeutic tools are needed for the treatment of herpes simplex virus infections. Several essential oils have shown to possess antiviral activity in vitro against a wide spectrum of viruses.

Aim

The present study was assess to investigate the activities of the essential oil obtained from leaves of Artemisia arborescens against HSV-1 and HSV-2

Methods

The cytotoxicity in Vero cells was evaluated by the MTT reduction method. The IC₅₀ values were determined by plaque reduction assay. In order to characterize the mechanism of action, yield reduction assay, inhibition of plaque development assay, attachment assay, penetration assay and post-attachment virus neutralization assay were also performed.

Results

The IC₅₀ values, determined by plaque reduction assay, were 2.4 and 4.1 μg/ml for HSV-1 and HSV-2, respectively, while the cytotoxicity assay against Vero cells, as determined by the MTT reduction method, showed a CC₅₀ value of 132 μg/ml, indicating a CC₅₀/IC₅₀ ratio of 55 for HSV-1 and 32.2 for HSV-2. The antiviral activity of A. arborescens essential oil is principally due to direct virucidal effects. A poor activity determined by yield reduction assay was observed against HSV-1 at higher concentrations when added to cultures of infected cells. No inhibition was observed by attachment assay, penetration assay and post-attachment virus neutralization assay. Furthermore, inhibition of plaque development assay showed that A. arborescens essential oil inhibits the lateral diffusion of both HSV-1 and HSV-2.

Conclusion

This study demonstrates the antiviral activity of the essential oil in toto obtained from A. arborescens against HSV-1 and HSV-2. The mode of action of the essential oil as antiherpesvirus agent seems to be particularly interesting in consideration of its ability to inactivate the virus and to inhibit the cell-to-cell virus diffusion.     

Effects of non toxic doses of acyclovir on nitric oxide and cellular death responses in herpesvirus types 1 and 2 infected hep-2 cells

Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are among the most "successful" pathogens and code for a variety of proteins to direct the apoptosis/necrosis responses of the cells they infect. Nitric oxide (NO) is an important intracellular signaling molecule in pathological processes. Acyclovir (ACV) is a chain terminator that targets the viral DNA polymerase as an antiviral agent. In this study, NO signals, and apoptosis/necrosis responses of HEp-2 cells were compared when infected by HSV-1 and -2 for 24 hours against non toxic doses (starting from 48.8, 24.4, 12.2, 6.1, 3 to 1.5 microg/mL) of ACV. In 48.8, 24.4 and 12.2 microg/mL of ACV, HSV-1 had an "upregulating effect" whereas HSV-2 had a "downregulating effect" on NO production, and in 6.1, 3 and 1.5 microg/mL of ACV HSV-1 had a "down-regulating effect" whereas HSV-2 had an "upregulating effect" on NO responses (HSV-1 had a "downregulating effect" on NO production whereas HSV-2 had an "upregulating effect" on NO production without any ACV). In 48.8, 24.4 and 12.2 microg/mL of ACV, HSV-1 had an "anti-apoptotic effect" whereas HSV-2 had a stimulation on "apoptotic effect", and in 6.1, 3 and 1.5 microg/mL of ACV HSV-1 had an "apoptotic effect" and HSV-2 turned to "its natural viral apoptotic effect level" (HSV-1 had an "natural viral apoptotic effect" whereas HSV-2 had a "natural viral apoptotic effect" on apoptosis response without any ACV). In 48.8, and 24.4 microg/mL of ACV, HSV-1 had significant "necrotic effect" on necrotic cellular death, "necrosis" increased in 12.2, 6.1, 3 and 1.5 microg/mL of ACV (HSV-1 had a negligible "necrotic effect" on HEp-2 cells alone), and HSV-2 had a "natural viral necrotic effect" alone; and also in all non toxic ACV concentrations. These results showed that HSV-1 and -2 had different "strategies" on apoptosis/necrosis and NO with and without non toxic ACV. These differences deserve further studies in order to explain the interactions between apoptotic/anti apoptotic, necrotic genes and NO, and ACV in HSV-1 and HSV-2 infections respectively.     

Virus Type-Specific Thymidine Kinase in Cells Biochemically Transformed by Herpes Simplex Virus Types 1 and 2

Transformation of mouse cells (Ltk(-)) and human cells (HeLa Bu) from a thymidine kinase (TK)-minus to a TK(+) phenotype (herpes simplex virus [HSV]-transformed cells) has been induced by infection with ultraviolet-irradiated HSV type 2 (HSV-2), as well as by HSV type 1 (HSV-1). Medium containing methotrexate, thymidine, adenine, guanosine, and glycine was used to select for cells able to utilize exogenous thymidine. We have determined the kinetics of thermal inactivation of TK from cells lytically infected with HSV-1 or HSV-2 and from HSV-1- and HSV-2-transformed cells. Three hours of incubation at 41 C produces a 20-fold decrease in the TK activity of cell extracts from HSV-2-transformed cells and Ltk(-) cells lytically infected with HSV-2. The same conditions produce only a twofold decrease in the TK activities from HSV-1-transformed cells and cells lytically infected with HSV-1. This finding supports the hypothesis that an HSV structural gene coding for TK has been incorporated in the HSV-transformed cells.     

Establishment of mutant murine mammary carcinoma FM3A cell strains transformed with the herpes simplex virus type 2 thymidine kinase gene

To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.     

SOS-red fluorescent protein (RFP) bioassay system for monitoring of antigenotoxic activity in plant extracts

An optical antigenotoxicity assay using genetically engineered red fluorescent bacteria is presented. Exposure of Escherichia coli RS4U to genotoxicants [mitomycin C (MMC), nalidixic acid (NA) and hydrogen peroxide (HP)] resulted in phenotypic red fluorescence proportional to the concentration of the inducer. Except for tannic acid (TA), co-treatment of the genotoxicant-activated bacteria with ascorbic acid (AA) and aqueous plant extracts (Mangifera indica, Psidium guajava and Syzygium cumini) afforded protection against all three genotoxicants. TA was effective in suppressing the genotoxic effect of MMC and HP. The antigenotoxic effect is seen as inhibition of the genotoxicant-triggered red fluorescence. The IC50 of the plant extracts and AA varied with the genotoxicant used. Rec assay verified the antigenotoxic activity of the plant extracts. Folin-Ciocalteu test, FeCl3 test and DPPH assay confirmed the presence of polyphenolic compounds and hydrolyzable tannins in the plant extracts and the antioxidant capacity of the plant samples.     

Anti-herpes simplex virus efficacies of 2-aminobenzamide derivatives as novel HSP90 inhibitors

After the widespread use of the acyclic purine nucleoside analogues for therapy of herpes simplex virus (HSV) infection since the 1980s, new antiviral strategies are urgently needed to counter the emergence of drug-resistant clinical isolates. In this report, we define the anti-HSV efficacies of three optimized 2-aminobenzamide derivatives in vitro and in vivo. The synthetic analogues SNX-25a, SNX-2112 and SNX-7081, which selectively bind to the N-terminal ATP pocket of heat shock protein 90 (HSP90), exhibited significant anti-HSV-1 and HSV-2 activities at non-cytotoxic concentrations in Vero cells, with EC(50) values close to that of acyclovir (ACV). The in vivo antiviral potentials were then confirmed using a herpes simplex keratitis (HSK) rabbit model, where eye gels containing 0.1% or 0.025% SNX-25a displayed the highest efficacies against HSV-1 infection, which were better than that obtained with 0.1% ACV. SNX-2112 and SNX-7081 gels were also effective against HSV-1 with different magnitude of activities. Our results for the first time confirmed the anti-HSV efficacies of these 2-aminobenzamide derivatives and suggest that with alternative mechanisms of action these novel HSP90 inhibitors, especially SNX-25a, could be potent as new anti-HSV clinical trial candidates.     

Efficacy of topical acyclovir monophosphate, acyclovir, or penciclovir in orofacial HSV-1 infections of mice and genital HSV-2 infections of guinea pigs

The purpose of these studies was to compare the efficacy of acyclovir monophosphate (ACVMP), acyclovir (ACV), or penciclovir (PCV) against HSV-1 in an orofacial infection of mice and against ACV sensitive and resistant genital HSV-2 infections of guinea pigs. Treatment was initiated 24, 48, or 72 hours post inoculation with 5% ACVMP, 5% ACV (Zovirax) or 1% PCV (Denavir). In all experiments, similar efficacy was obtained for ACVMP and ACV, whereas PCV was considerably less effective.     

靶向UL27shRNA抑制Ⅱ型单纯疱疹病毒在HEK293细胞中的复制

按照shRNA(small hairpin RNA)设计原则,针对Ⅱ型单纯疱疹病毒(herpes simplex virus type 2, HSV-2)的UL27基因序列保守区域筛选设计、合成4条干扰靶序列并构建表达UL27序列特异性siRNA(short interfering RNA)的质粒载体pGPU6/GFP/Neo．通过脂质体介导重组表达载体转染HEK293细胞 (human embryonic kidney 293 cell)再接种HSV-2．采用实时荧光定量PCR(real-time fluorescent quantitative PCR)技术检测UL27各组的mRNA转录水平,终点滴定法检测细胞上清液中的病毒滴度,四甲基偶氮唑盐(four methyl thiazolyl tetrazolium, MTT)法测定细胞存活率,Western印迹法检测蛋白表达效果．结果显示,UL27shRNA75组对UL27基因mRNA表达抑制效果最佳,同时能显著抑制感染细胞的CPE(cytopathic effect, CPE),降低上清液中的病毒感染滴度,提高细胞的生存率,抑制UL27基因的蛋白表达．提示本研究构建的pGPU6/GFP/Neo-UL27表达载体能在细胞水平上不同程度地干扰HSV-2 UL27基因表达,抑制HSV-2在HEK293细胞中复制．     

Herpes Simplex Virus Type 1 Renders Infected Cells Resistant to Cytotoxic T-Lymphocyte-Induced Apoptosis

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the “lethal hit,” inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4⁺ CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.     

敲减单纯疱疹病毒Ⅱ型（HSV-2）UL30蛋白表达可抑制HSV-2复制

按照shRNA（small hairpin RNA）设计要求,选择编码单纯疱疹病毒Ⅱ型DNA多聚酶催化亚单位的U_L30（unique long 30,U_L30）基因序列保守区域,设计、合成并构建表达U_L30序列特异性siRNA（short interfering RNA）的质粒载体pUL30．通过磷酸钙转染法将其转染入HEK（human embryonic kidney）293细胞中,用蛋白印迹法检测对HSV-2 UL30蛋白表达的影响,观察受染细胞病变效应（cytopathic effect,CPE）,终点滴定法测定细胞上清液中病毒感染滴度（50% tissue culture infective dose,TCID₅₀）．结果表明,针对U_L30基因的siRNA能有效抑制UL30蛋白表达,同时显著抑制受染细胞的CPE,降低上清液中病毒感染滴度．提示本研究建立的针对U_L30基因特异性siRNA能有效阻断HSV-2在HEK293细胞内的复制,U_L30基因是一个潜在的抗HSV-2复制的药物靶标．     

Combination of Azidothymidine (AZT) and (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU) Inhibits the Replication of Herpes Simplex Virus Type 1 (HSV-1) and Type 2 (HSV-2) and Varicella Zoster Virus (VZV) Strains That Are Deficient in the Expression of the Viral Thymidine Kinase (tk)

Abstract

Combinations of high concentrations of AZT with BVDU, acyclovir (ACV) or ganciclovir (GCV) show antagonism against TK⁺ HSV-1, but not TK⁺ VZV strains, in cell cultures. When BVDU and AZT were used in combination against TK^? HSV-1, TK^? HSV-2 and TK^? VZV strains, a pronounced inhibition of viral replication was observed. This potentiating effect was not seen if AZT was combined with ACV or GCV.     

Fluorosubstitution and 7-alkylation as prospective modifications of biologically active 6-aryl derivatives of tricyclic acyclovir and ganciclovir analogues

A series of fluorine containing tricyclic analogues of acyclovir (ACV, 1) and ganciclovir (GCV, 2) were synthesized and evaluated for their activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and cytostatic activity against HSV-1 thymidine kinase (TK) gene-transduced human osteosarcoma tumour cells. It was found that fluorine substitution reduced the antiviral activity, but most of the new compounds were pronounced cytostatic agents with potency and selectivity similar to those of parental ACV and GCV. Compounds 12, 13 and 16 seem to be promising as labeled substrates for (19)F NMR studies of the HSV TK-ligand interaction and/or monitoring of their metabolites in cells expressing HSV TK.     

Antiviral efficacy of Limonium densiflorum against HSV-1 and influenza viruses

Viral infections remain a major threat to humans and animals and there is a crucial need for new antiviral agents especially with the development of resistant viruses. Several Limonium genus members (Plumbaginacea) have been widely used in traditional medicine for the treatment of infections. In this study, we investigated the antiviral activities of different fractions after successive extraction (hexane, dichloromethane, ethanol and methanol) of the halophyte Limonium densiflorum against H1N1 influenza and HSV-1 herpes viruses. In addition, TLC phytochemicals of the shoot extracts were analyzed. All extracts were tested for their cytotoxicity using a fluorometric resazurin assay. The antiviral activity of extracts was tested using four modes of action: virucidal test, pretreatment of cells with samples before infection, attachment assay and plaque reduction test. A good antiviral activity was found with ethanol and methanol extracts. They were most potent in HSV-1 inhibition than H1N1 influenza virus. The most potent inhibition was observed with ethanol extract, and it exhibited high levels of virucidal activity against HSV-1 (IC₅₀ = 6 μg/mL). It inhibits the replication of the virus by 75% when added after penetration of the virus, and by 100% when added during the viral attachment. It protects MDCK cells against influenza virus by abolishing virus to entry into the host cell (IC₅₀ = 55 μg/mL). After attachment of influenza virus, the ethanol extract displayed an appreciable inhibition of virus replication (IC₅₀ = 193 μg/mL). Methanol extract showed a moderate antiviral capacity against both viruses. While dichloromethane has excellent antiherpes potential, results were inappropriate because it was toxic to Vero cells, hexane extract has no effect. TLC analysis of these extracts showed that flavonoids and saponins were the major classes of natural products found in the shoot extracts that may be responsible for these antiviral activities.     

58种植物提取液对葡萄霜霉病菌的抑菌活性筛选研究

通过 5 8种植物提取液对葡萄霜霉病菌 (Plasmoparaviticola)的游动孢子囊萌发抑制率及接种叶圆盘的发病控制效果的研究 ,表明 :虎耳草 (ChlorisvirgataSwartz) ,黄檀 (DalbergiahuoeanaHance) ,马尾松 (PinusmassonianaLarmb .) ,牡丹 (Paeoniasuffruticosa) ,刺槐 (RobiniapseudoacciaL .) 5种植物提取液对葡萄霜霉病有抑菌效果 ,对游动孢子囊萌发抑制率分别为 :84 70 %、87 5 2 %、88 4 6 %、83 78%、83 15 % ,而叶圆盘生测结果 ,其发病率均为 0级 ,该效果和目前常用药剂波尔多液的效果等同 ,有的还优于对照。该研究为开发植物源控制葡萄霜霉病杀菌剂奠定了基础。     

Similarities and differences in the Fc-binding glycoprotein (gE) of herpes simplex virus types 1 and 2 and tentative mapping of the viral gene for this glycoprotein.

We performed affinity chromatography and immunoprecipitation experiments to determine whether cells infected with herpes simplex virus type 2 (HSV-2) expressed a glycoprotein that was functionally and antigenically related to the HSV-1 Fc-binding glycoprotein designated gE. We found that a protein from extracts of HSV-2-infected HEp-2 cells bound specifically to an Fc affinity column and that the electrophoretic mobility of this protein in sodium dodecyl sulfate-acrylamide gels was slightly less than the mobility of HSV-1 gE. Immunoprecipitation experiments performed with an antiserum prepared against HSV-1 gE revealed that (i) extracts from HSV-2-infected cells contained a glycoprotein that was antigenically related to HSV-1 gE; (ii) the electrophoretic mobility of the HSV-2 gE was indistinguishable from the mobility of the HSV-2 Fc-binding protein; (iii) the antiserum reacted with both newly synthesized transient forms and stable fully processed forms of both HSV-1 gE and HSV-2 gE; and (iv) the transient and stable forms of HSV-2 gE all had lower electrophoretic mobilities than their HSV-1 counterparts. Electrophoretic analyses of gE precipitated from extracts of HEp-2 cells infected with two sets of HSV-1 x HSV-2 intertypic recombinant viruses suggested that the gene for gE is located at the right end of the HSV genome (0.85 to 0.97 map units) in the unique portion of the S component.     

Herpes simplex virus type 2 glycoprotein gF and type 1 glycoprotein gC have related antigenic determinants.

The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.     

Efficacy of foliar extracts against pre- and post-harvest diseases of sponge-gourd fruits

S.K. AHMAD AND J.S. PRASAD. 1995. The inhibitory potential of aqueous foliar extracts of Adhatoda vasica, Azadirachta indica, Catharanthes roseus, Datura fistulosa, Lantana camara, Muraya exotica, Ocimum sanctum, Ricinus communis and Strychnos nux-vomica were evaluated against soft rot diseases of sponge-gourd fruits caused by Helminthosporium spiciferum and Fusarium scirpi. Conidial germination of F. scirpi and H. spiciferum were reduced to about 75% when their spores were treated with A. indica, C. roseus, D. fistulosa, L. camara, M. exotica and O. sanctum. Considerable reductions (50%) in mycelial dry weights and colony diameters were recorded in liquid media containing (20%) aqueous extracts of C. roseus, D. fistulosa and M. exotica . Other extracts reduced the mycelial growth but to a lesser extent. Post-infection treatment of sponge-gourd fruits with the extracts of A. indica, L. camara, M. exotica, O. sanctum, D. fistulosa and C. roseus almost fully inhibited the spread of disease. Treatment of fruits with A. indica and C. roseus before fungal infection reduced the spread of disease caused by both the pathogens, whereas M. exotica, O. sanctum and D. fistulosa reduced the spread of soft-rot caused by F. scirpi but not by H. spiciferum .     

Flow cytometric evaluation of antiviral agents against human herpesvirus 6

Antiviral activities of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV), penciclovir (9-[4-hydroxy-3-(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9-(1,3-dihydroxy-2-propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV-6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV-6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV-6 followed by PFA, PCV and ACV. For HHV-6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 microM and 34.7 microM respectively, whereas the IC50 of ACV and PCV were found to be 53.7 microM and 37.9 microM respectively. For HHV-6B, the IC50 of GCV and PFA were found to be 5.7 microM and 71.4 microM respectively, whereas the IC50 of ACV and PCV were found to be 119.0 microM and 77.8 microM respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV-6.     

Drug resistance patterns of recombinant herpes simplex virus DNA polymerase mutants generated with a set of overlapping cosmids and plasmids

Herpes simplex virus (HSV) DNA polymerase (Pol) mutations can confer resistance to all currently available antiherpetic drugs. However, discrimination between mutations responsible for drug resistance and those that are part of viral polymorphism can be difficult with current methodologies. A new system is reported for rapid generation of recombinant HSV type 1 (HSV-1) DNA Pol mutants based on transfection of a set of overlapping viral cosmids and plasmids. With this approach, twenty HSV-1 recombinants with single or dual mutations within the DNA pol gene were successfully generated and subsequently evaluated for their susceptibilities to acyclovir (ACV), foscarnet (FOS), cidofovir (CDV), and adefovir (ADV). Mutations within DNA Pol conserved regions II (A719T and S724N), VI (L778M, D780N, and L782I), and I (F891C) were shown to induce cross-resistance to ACV, FOS, and ADV, with two of these mutations (S724N and L778M) also conferring significant reduction in CDV susceptibility. Mutant F891C was associated with the highest levels of resistance towards ACV and FOS and was strongly impaired in its replication capacity. One mutation (D907V) lying outside of the conserved regions was also associated with this ACV-, FOS-, and ADV-resistant phenotype. Some mutations (K522E and Y577H) within the delta-region C were lethal, whereas others (P561S and V573M) induced no resistance to any of the drugs tested. Recombinants harboring mutations within conserved regions V (N961K) and VII (Y941H) were resistant to ACV but susceptible to FOS. Finally, mutations within conserved region III were associated with various susceptibility profiles. This new system allows a rapid and accurate evaluation of the functional role of various DNA Pol mutations, which should translate into improved management of drug-resistant HSV infections.