Real-world evidence of a novel tetravalent immunoglobulin Y effectiveness and safety in patients with the refractory Helicobacter pylori infection.

BACKGROUND: Refractory Helicobacter pylori (H. pylori) infection inevitably increase the difficulty of drug selection. Here, we described our experience with the use of a novel tetravalent IgY against H. pylori for the treatment of patients with refractory H. pylori infection. METHODS: Patients were randomly assigned to receive the standard quadruple therapy (amoxicillin, clarithromycin, omeprazole and bismuth potassium citrate ) for 2 weeks or 250 mg of avian polyclonal IgY orally twice a day for 4 weeks. The binding efficacy of IgY to H. pylori antigens was detected by western blotting13. C-urea breath test was performed to evaluate the eradication therap's efficacy. The side effects of IgY were evaluated via various routine tests. The questionnaire was used to gather clinical symptoms and adverse reactions. RESULTS: Western blot analysis showed that tetravalent IgY simultaneously bind to VacA, HpaA, CagA and UreB of H. pylori. Tetravalent IgY had an eradication rate of 50.74% in patients with refractory H. pylori and an inhibition rate of 50.04% against DOB (delta over baseline) of 13C-urea. The symptom relief rate was 61.76% in thirty-four patients with clinical symptoms, and no adverse reactions were observed during tetravalent IgY treatment period. CONCLUSIONS: Polyclonal avian tetravalent IgY reduced H. pylori infection, and showed good efficacy and safety in the treatment of refractory H. pylori infection patients, which represented an effective therapeutic option of choice for patients with refractory H. pylori infection.

Effect of Maternal Immunization With 10-Valent Pneumococcus Conjugate Vaccine (PCV-10), 23-Valent Pneumococcus Polysaccharide Vaccine, or Placebo on the Immunogenicity of PCV-10 in Human Immunodeficiency Virus-Exposed Uninfected Infants: A Randomized Clinical Trial.

BACKGROUND: The effect of pneumococcal vaccination of mothers with human immunodeficiency virus (HIV) on infant responses to childhood vaccination has not been studied. We compared the immunogenicity of 10-valent pneumococcus conjugate vaccine (PCV-10) in HIV-exposed uninfected infants born to mothers who received PCV-10, 23-valent pneumococcus polysaccharide vaccine (PPV-23), or placebo during pregnancy. METHODS: Antibody levels against 7 serotypes were measured at birth, before the first and second doses of PCV-10m and after completion of the 2-dose regimen in 347 infants, including 112 born to mothers who received PPV-23, 112 who received PCV-10, and 119 who received placebo during pregnancy. Seroprotection was defined by antibody levels ≥0.35 µg/mL. RESULTS: At birth and at 8 weeks of life, antibody levels were similar in infants born to PCV-10 or PPV-23 recipients and higher than in those born to placebo recipient. After the last dose of PCV-10, infants in the maternal PCV-10 group had significantly lower antibody levels against 5 serotypes than those in the maternal PPV-23 group and against 3 serotypes than those in the maternal placebo group, and they did not have higher antibody levels against any serotype. The seroprotection rate against 7 serotypes was 50% in infants in the maternal PCV-10 group, compared with 71% in both of the maternal PPV-23 and placebo groups (P < .001). CONCLUSIONS: Administration of PCV-10 during pregnancy was associated with decreased antibody responses to PCV-10 and seroprotection rates in infants. Considering that PCV-10 and PPV-23 had similar immunogenicity in pregnant women with HIV and that administration of PPV-23 did not affect the immunogenicity of PCV-10 in infants, PPV-23 in pregnancy may be preferred over PCV-10.

Antibody activities in hyperimmune plasma against the Rhodococcus equi virulence -associated protein A or poly-N-acetyl glucosamine are associated with protection of foals against rhodococcal pneumonia.

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide ß-1→6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C՛1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n = 119) or subclinical pneumonia at Farm B (n = 114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity ≤ the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities ≤ the population median for PNAG or C՛1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C՛1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.

Investigation of a monoclonal antibody against enterotoxigenic Escherichia coli, expressed as secretory IgA1 and IgA2 in plants.

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68-81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68-61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

Pre-treatment with Bifidobacterium infantis and its specific antibodies enhance targeted radiosensitization in a murine model for lung cancer.

PURPOSE: The hypoxic microenvironments of solid tumours are complex and reduce the susceptibility of cancer cells to chemo- and radiotherapy. Conventional radiosensitisers have poor specificity, unsatisfactory therapeutic effects, and significant side effects. Anaerobic bacteria colonise and destroy hypoxic areas of the tumour and consequently enhance the effects of radiation. METHODS: In this study, we treated a Lewis lung carcinoma transplant mouse model with Bifidobacterium infantis (Bi) combined with its specific monoclonal antibody (mAb) and radiotherapy (RT) to investigate its ability to radiosensitise the tumour. The tumour metabolism and hypoxia in the tumour tissue were monitored by micro-18F-FDG and 18F-FMISO PET/CT imaging. Immunohistochemistry was used to detect phosphorylated histone (γ-H2AX), proliferation (Ki-67), platelet endothelial cell adhesion molecules (CD31), tumour necrosis factor-α (TNF-α), hypoxia-inducible factor-1α (HIF-1α), and glucose transporter 1 (Glut-1) levels. RESULTS: Tumour growth was slowed and survival time was markedly prolonged in mice subjected to the combination of B. infantis, specific antibody, and radiotherapy. Levels of HIF-1α, Glut-1, Ki-67, and CD31 expression, as well as uptake of FDG and FMISO, were the lowest in the combination-treated mice. In contrast, γ-H2AX and TNF-α expression levels were elevated and hypoxia in tumour tissue was reduced compared with controls. CONCLUSION: In conclusion, our data indicated that the curative effect of radiotherapy for lung cancer was enhanced by pre-treating mice with a combination of B. infantis and its specific monoclonal antibody.

A new passive immune strategy based on IgY antibodies as a key element to control neonatal calf diarrhea in dairy farms.

BACKGROUND: Neonatal diarrhea remains one of the main causes of morbi-mortality in dairy calves under artificial rearing. It is often caused by infectious agents of viral, bacterial, or parasitic origin. Cows vaccination and colostrum intake by calves during the first 6 h of life are critical strategies to prevent severe diarrhea but these are still insufficient. Here we report the field evaluation of a product based on IgY antibodies against group A rotavirus (RVA), coronavirus (CoV), enterotoxigenic Escherichia coli, and Salmonella sp. This product, named IgY DNT, has been designed as a complementary passive immunization strategy to prevent neonatal calf diarrhea. The quality of the product depends on the titers of specific IgY antibodies to each antigen evaluated by ELISA. In the case of the viral antigens, ELISA antibody (Ab) titers are correlated with protection against infection in calves experimentally challenged with RVA and CoV (Bok M, et al., Passive immunity to control bovine coronavirus diarrhea in a dairy herd in Argentina, 2017), (Vega C, et al., Vet Immunol Immunopathol, 142:156-69, 2011), (Vega C, et al., Res Vet Sci, 103:1-10, 2015). To evaluate the efficiency in dairy farms, thirty newborn Holstein calves were randomly assigned to IgY DNT or control groups and treatment initiated after colostrum intake and gut closure. Calves in the IgY DNT group received 20 g of the oral passive treatment in 2 L of milk twice a day during the first 2 weeks of life. Animals were followed until 3 weeks of age and diarrhea due to natural exposure to infectious agents was recorded during all the experimental time. RESULTS: Results demonstrate that the oral administration of IgY DNT during the first 2 weeks of life to newborn calves caused a delay in diarrhea onset and significantly reduced its severity and duration compared with untreated calves. Animals treated with IgY DNT showed a trend towards a delay in RVA infection with significantly shorter duration and virus shedding compared to control calves. CONCLUSIONS: This indicates that IgY DNT is an effective product to complement current preventive strategies against neonatal calf diarrhea in dairy farms. Furthermore, to our knowledge, this is the only biological product available for the prevention of virus-associated neonatal calf diarrhea.

Contemporary approaches and future perspectives of antibacterial photodynamic therapy (aPDT) against methicillin-resistant Staphylococcus aureus (MRSA): A systematic review.

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) causing skin and soft tissue infections in both the community and healthcare settings challenges the limited options of effective antibiotics and motivates the search for alternative therapeutic solutions, such as antibacterial photodynamic therapy (aPDT). While many publications have described the promising anti-bacterial activities of PDT in vitro, its applications in vivo and in the clinic have been very limited. This limited availability may in part be due to variabilities in the selected photosensitizing agents (PS), the variable testing conditions used to examine anti-bacterial activities and their effectiveness in treating MRSA infections. We thus sought to systematically review and examine the evidence from existing studies on aPDT associated with MRSA and to critically appraise its current state of development and areas to be addressed in future studies. In 2018, we developed and registered a review protocol in the International Prospective Register of Systematic Reviews (PROSPERO) with registration No: CRD42018086736. Three bibliographical databases were consulted (PUBMED, MEDLINE, and EMBASE), and a total of 113 studies were included in this systematic review based on our eligibility criteria. Many variables, such as the use of a wide range of solvents, pre-irradiation times, irradiation times, light sources and light doses, have been used in the methods reported by researchers, which significantly affect the inter-study comparability and results. On another note, new approaches of linking immunoglobulin G (IgG), antibodies, efflux pump inhibitors, and bacteriophages with photosensitizers (PSs) and the incorporation of PSs into nano-scale delivery systems exert a direct effect on improving aPDT. Enhanced activities have also been achieved by optimizing the physicochemical properties of the PSs, such as the introduction of highly lipophilic, poly-cationic and site-specific modifications of the compounds. However, few in vivo studies (n = 17) have been conducted to translate aPDT into preclinical studies. We anticipate that further standardization of the experimental conditions and assessing the efficacy in vivo would allow this technology to be further applied in preclinical trials, so that aPDT would develop to become a sustainable, alternative therapeutic option against MRSA infection in the future.

Bypassing Phase Variation of Lipooligosaccharide (LOS): Using Heptose 1 Glycan Mutants To Establish Widespread Efficacy of Gonococcal Anti-LOS Monoclonal Antibody 2C7.

The sialylatable lacto-N-neotetraose (LNnT; Gal-GlcNAc-Gal-Glc) moiety from heptose I (HepI) of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae undergoes positive selection during human infection. Lactose (Gal-Glc) from HepII, although phase variable, is commonly expressed in humans; loss of HepII lactose compromises gonococcal fitness in mice. Anti-LOS monoclonal antibody (MAb) 2C7, a promising antigonococcal immunotherapeutic that elicits complement-dependent bactericidal activity and attenuates gonococcal colonization in mice, recognizes an epitope comprised of lactoses expressed simultaneously from HepI and HepII. Glycan extensions beyond lactose on HepI modulate binding and function of MAb 2C7 in vitro Here, four gonococcal LOS mutants, each with lactose from HepII but fixed (unable to phase-vary) LOS HepI glycans extended beyond the lactose substitution of HepI (lactose alone, Gal-lactose, LNnT, or GalNAc-LNnT), were used to define how HepI glycan extensions affect (i) mouse vaginal colonization and (ii) efficacy in vitro and in vivo of a human IgG1 chimeric derivative of MAb 2C7 (2C7-Ximab) with a complement-enhancing E-to-G Fc mutation at position 430 (2C7-Ximab-E430G). About 10-fold lower 2C7-Ximab-E430G concentrations achieved similar complement-dependent killing of three gonococcal mutants with glycan extensions beyond lactose-substituted HepI (lactose alone, LNnT, or GalNAc-LNnT) as 2C7-Ximab (unmodified Fc). The fourth mutant (Gal-lactose) resisted direct complement-dependent killing but was killed approximately 70% by 2C7-Ximab-E430G in the presence of polymorphonuclear leukocytes and complement. Only mutants with (sialylatable) LNnT from HepI colonized mice for >3 days, reiterating the importance of LNnT sialylation for infection. 2C7-Ximab-E430G significantly attenuated colonization caused by the virulent mutants.

Inhibition of Francisella tularensis phagocytosis using a novel anti-LPS scFv antibody fragment.

Francisella tularensis (Ft), the causative agent of lethal tularemia, is classified as a category A biological warfare threat agent. While Ft infection is treatable by antibiotics, many failed antibiotic treatments were reported, highlighting the need for effective new treatments. It has been demonstrated that binding of antibody-coated bacteria to the Fc receptor located on phagocytic cells is a key process needed for efficient protection against Ft. Yet, Ft utilizes the same receptor to enter the phagocytic cells in order to escape the immune system. To address the question whether an anti-Ft LPS antibody lacking the ability to bind the Fc receptor may inhibit the entry of Ft into host cells, a soluble scFv (TL1-scFv) was constructed from an anti Ft-LPS antibody (TL1) that was isolated from an immune single-chain (scFv) phage-display library. Bacterial uptake was assessed upon infection of macrophages with Ft live attenuated strain (LVS) in the presence of either TL1 or TL1-scFv. While incubation of LVS in the presence of TL1 greatly enhanced bacterial uptake, LVS uptake was significantly inhibited in the presence of TL1-scFv. These results prompt further experiments probing the therapeutic efficacy of TL1-scFv, alone or in combination with antibiotic treatment.

Questionable Efficacy of Therapeutic Antibodies in the Treatment of Anthrax.

Inhalational anthrax caused by Bacillus anthracis, a spore-forming Gram-positive bacterium, is a highly lethal infection. Antibodies targeting the protective antigen (PA) binding component of the toxins have recently been authorized as an adjunct to antibiotics, although no conclusive evidence demonstrates that anthrax antitoxin therapy has any significant benefit. We discuss here the rational basis of anti-PA development regarding the pathogenesis of the disease. We argue that inductive reasoning may induce therapeutic bias. We identified anthrax animal model analysis as another bias. Further studies are needed to assess the benefit of anti-PA antibodies in the treatment of inhalational anthrax, while a clearer consensus should be established around what evidence should be proven in an anthrax model.

Prophylactic and therapeutic protection of human IgG purified from sera containing anti-exotoxin A titers against pneumonia caused by Pseudomonas aeruginosa.

Antibodies are effective alternative tools to combat infections caused by Pseudomonas aeruginosa (PA), especially multi-drug-resistant PA. Thus, to solve the urgent need for an anti-PA antibody drug, we hypothesized that anti-PA intravenous immunoglobulins could be a practical attempt. Exotoxin A (ETA) is one of the most important factors for PA infection and is also a critical target for the development of immune interventions. In this study, a total of 320 sera were collected from healthy volunteers. The concentration of ETA-specific antibodies was determined by a Luminex-based assay and then purified by affinity chromatography. The purified IgGs were able to neutralize the cytotoxicity of ETA in vitro. We showed they had a prophylactic and therapeutic protective effect in PA pneumonia and ETA toxemia models. In addition, administration of nonspecific IgGs also provided partial protection. Collectively, our results provide additional evidence for IVIG-based treatment of infections caused by multi-drug-resistant PA and suggest that patients at high risk of PA pneumonia could be prophylactically treated with anti-ETA IgGs or even with nonspecific IgGs.

Monoclonal antibody-based therapies for bacterial infections.

PURPOSE OF REVIEW: This review highlights recent developments in the development of monoclonal antibodies to treat bacterial disease, including preclinical advances and the status of current clinical trials. RECENT FINDINGS: Monoclonal antibody (mAb) therapy is becoming increasingly promising in the infectious disease field. Though bacterial exotoxins continue to be a mainstay of mAb targets, searches for protein targets on the surface of bacteria have uncovered new mechanisms of antibody-mediated action against bacteria. Additionally, surveys of the polysaccharide serotype prevalence among antibiotic-resistant bacterial populations have yielded opportunities to leverage human selective pressures to our clinical advantage. Several mAb candidates are progressing through clinical development with great promise, especially those with structures altered to provide maximum benefit. Although other clinical trials have recently proved unsuccessful, these failures and lessons from immune profiling provide opportunities to understand how vulnerabilities of certain targets may change in different disease states. SUMMARY: Despite the hurdles of identifying effective targets and understanding how mAbs provide protection within different infections, we show that the progress made in these fields is a positive indication of mAbs becoming more widely accepted as the future for treating bacterial infections.

Synthetic and immunological studies of Salmonella Enteritidis O-antigen tetrasaccharides as potential anti-Salmonella vaccines.

The first synthetic carbohydrate based potential anti-Salmonella Enteritidis vaccine has been developed by conjugating a synthetic tetrasaccharide antigen with bacteriophage Qß. High levels of specific and long lasting anti-glycan IgG antibodies were induced by the conjugate, which completely protected mice from lethal bacterial challenges in a passive transfer model.

Cellulitis in human brucellosis: An atypical presentation.

INTRODUCTION: Brucellosis is a zoonotic disease with variable clinical manifestations and atypical presentation in humans. Human brucellosis cases are not seen often in Malaysia. CASE REPORT: This is a case report of 19 years old gentleman who presented with fever, lower limb redness, pain and swelling. He was initially treated as cellulitis. However, based on the recovery of Brucella melitensis from his blood culture, he was later diagnosed to have brucellosis. He had a history of consumption of fresh goat's milk and uncooked meat which could have been the possible modes of transmission. Brucella serology IgM and IgG were both positive, and anti-Brucella immunocapture agglutination test (BrucellaCapt) was also positive with a titer of 1:2560. He was treated with six weeks of oral doxycycline 100 mg twice daily and oral rifampin 450 mg twice daily. DISCUSSION: This is a case of human brucellosis with atypical cutaneous involvement.

Holistic Approach in Patients With Presumed Lyme Borreliosis Leads to Less Than 10% of Confirmation and More Than 80% of Antibiotic Failures.

BACKGROUND: There is no precise idea whether patients with chronic symptoms attributed to Lyme borreliosis (LB) have LB or another disease. METHODS: We evaluated patients consulting for a presumed LB with a holistic approach including presumptive treatment. We included symptomatic patients consulting for presumed LB. They were classified as confirmed LB when they met four criteria, and possible LB if three with a positive clinical response to presumptive treatment. RESULTS: Amongst the 301 patients, 275 (91%) were exposed to tick bites, and 165 (54%) were bitten by a tick. At presentation, 151 patients (50.1%) had already been treated with a median of one (1-22) course of antimicrobials, during 34 (28-730) days. Median number of symptoms was three (1-12) with a median duration of 16 (1-68) months. Median number of signs was zero (0-2). ELISA was positive in 84/295 (28.4%) for IgM and 86/295 (29.1%) for IgG, and immunoblot was positive in 21/191 (10.9%) for IgM and 50/191 (26.1 %) for IgG. Presumptive treatment after presentation failed in 46/88 patients (52%). Diagnosis of LB was confirmed in 29 patients (9.6%), and possible in 9 (2.9%). Of the 243 patients with non-LB diagnosis, diseases were psychological, musculoskeletal, neurological or other origin in 76 (31.2%), 48 (19.7%), 37 (15.2%) and 82 (33.7%) patients respectively. Patients with other diseases were significantly younger, having more symptoms, longest duration of symptoms, less clinical signs and less frequent LB positive serologies. CONCLUSIONS: Overdiagnosis and overtreatment of LB is worsening. Health authorities should investigate this phenomenon.

Human monoclonal anti-protective antigen antibody for the low-dose post-exposure prophylaxis and treatment of Anthrax.

BACKGROUND: Disease caused by Bacillus anthracis is often accompanied by high mortality primarily due to toxin-mediated injury. In the early disease course, anthrax toxins are secreted; thus, antibiotic use is limited to the early stage. In this regard, antibodies against the toxin component, protective antigen (PA), play an important role in protecting against anthrax. Therefore, we developed PA21, a fully human anti-PA immunoglobulin G monoclonal antibody. METHODS: Combining human Fab was screened from a phage library with human heavy constant regions. Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test evaluated the binding ability of PA21. Moreover, the affinity and neutralizing activity of the antibody was detected in vitro while the protective effectiveness in 60 rats was also examined in vivo. RESULTS: The Fischer 344 rats challenged with the lethal toxin can be protected by PA21 at a concentration of 0.067 mg/kg. All six rats remained alive although PA21 was injected 24 h before the toxin challenge. PA21 did not influence the binding of PA to cell receptors and that of a lethal factor to PA. CONCLUSION: The PA21 monoclonal antibody against PA can be used for emergency prophylaxis and anthrax treatment.

Combination of Antibodies and Antibiotics as a Promising Strategy Against Multidrug-Resistant Pathogens of the Respiratory Tract.

The emergence of clinical isolates associated to multidrug resistance is a serious threat worldwide in terms of public health since complicates the success of the antibiotic treatment and the resolution of the infectious process. This is of great concern in pathogens affecting the lower respiratory tract as these infections are one of the major causes of mortality in children and adults. In most cases where the respiratory pathogen is associated to multidrug-resistance, antimicrobial concentrations both in serum and at the site of infection may be insufficient and the resolution of the infection depends on the interaction of the invading pathogen with the host immune response. The outcome of these infections largely depends on the susceptibility of the pathogen to the antibiotic treatment, although the humoral and cellular immune responses also play an important role in this process. Hence, prophylactic measures or even immunotherapy are alternatives against these multi-resistant pathogens. In this sense, specific antibodies and antibiotics may act concomitantly against the respiratory pathogen. Alteration of cell surface structures by antimicrobial drugs even at sub-inhibitory concentrations might result in greater exposure of microbial ligands that are normally hidden or hardly exposed. This alteration of the bacterial envelope may stimulate opsonization by natural and/or specific antibodies or even by host defense components, increasing the recognition of the microbial pathogen by circulating phagocytes. In this review we will explain the most relevant studies, where vaccination or the use of monoclonal antibodies in combination with antimicrobial treatment has demonstrated to be an alternative strategy to overcome the impact of multidrug resistance in respiratory pathogens.