Exploration of reducing and stabilizing phytoconstituents in Arisaema dracontium extract for the effective synthesis of Silver nanoparticles and evaluation of their antibacterial and toxicological proprties.

Medicinal plants have been widely used for their antimicrobial properties against various microorganisms. Arisaema dracontium a familiar medicinal plant, was analyzed and silver nanoparticles (AgNPs) were synthesized using extracts of different parts of its shoot including leaves and stem. Further, the antimicrobial activity of different solvent extracts such as ethyl acetate, n-hexane, ethanol, methanol, and chloroform extracts were analyzed. AgNPs were prepared using aqueous silver nitrate solution and assessed their antibacterial activity against multidrug-resistant (MDR) and Non-multidrug-resistant bacteria. The characterization of AgNPs was done by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), UV-visible spectroscopy, Fourier Transform Infrared (FTI), and X-ray Diffraction approaches. The leaf extract contained Tannins, Flavonoids, Terpenoids, and Steroids while Alkaloids, Saponins, and Glycosides were undetected. The stem extract contained Alkaloids, Tannins, Flavonoids, Saponins, Steroids, and Glycosides while Terpenoids were not observed. The AgNPs synthesized from stem and leaf extracts in the current study had spherical shapes and ranged in size from 1 to 50 nm and 20-500 nm respectively as were visible in TEM. The leaf extract-prepared AgNPs showed significantly higher activities i.e., 27.75 mm ± 0.86 against the MDR strains as compared to the stem-derived nanoparticles i.e., 24.33 ± 0.33 by comparing the zones of inhibitions which can be attributed to the differences in their phytochemical constituents. The acute toxicity assay confirmed that no mortality was noticed when the dosage was 100 mg per kg which confirms that the confirms that the AgNPs are not toxic when used in low quantities. It is concluded that leaf extract from A. dracontium could be used against pathogenic bacteria offering economic and health benefits compared to the chemical substances.

Antimicrobial Resistance of Shigella flexneri in Pakistani Pediatric Population Reveals an Increased Trend of Third-Generation Cephalosporin Resistance.

The rapid emergence of resistance to third-generation cephalosporins in Shigella flexneri is crucial in pediatric shigellosis management. Limited studies have been conducted on molecular pattern of antibiotic resistance of S. flexneri in diarrhea endemic areas of Pakistan. The aim of the study was to analyze the antimicrobial resistance of S. flexneri isolated from pediatric diarrheal patients in Peshawar, Pakistan. A total of 199 S. flexneri isolates (clinical, n = 1 55 and non-clinical, n = 44) were investigated for drug resistance and mutational analysis of selected drug resistance genes. All isolates were found to be highly resistant to amoxicillin/clavulanic acid (88%), followed by trimethoprim-sulfamethoxazole (77%), chloramphenicol (43%), and quinolones (41.6%). About 34.5% S. flexneri isolates were found to be resistant to third-generation cephalosporin. None of the isolates was resistant to imipenem, piperacillin-tazobactam, and amikacin. Interestingly high frequency of third-generation cephalosporin resistance was observed in S. flexneri isolated from non-clinical samples (49%) when compared to clinical samples (30.5%). Furthermore, the most prevalent phenotypic-resistant patterns among third-generation cephalosporin-resistant isolates were AMC,CAZ,CPD,CFM,CRO,SXT (13%) followed by OFX,AMC,CAZ,CPD,CFM,CRO,SXT,NA,CIP (10%). The most frequently detected resistance genes were trimethoprim-sulfamethoxazole (sul2 = 84%), beta-lactamase genes (blaOXA = 87%), quinolones (qnrS = 77%), and chloramphenicol (cat = 64%). No mutation was detected in any drug-resistant genes. We are reporting for the first time the sequence of the blaTEM gene in S. flexneri. Furthermore, high third-generation cephalosporin resistance was observed in the patients who practiced self-medication as compared to those who took medication according to physician prescription. This study shows the high emergence of third-generation cephalosporin-resistant S. flexneri isolates, which is a potential threat to the community in the country. This finding will be helpful to develop a suitable antibiotic prescription regime to treat shigellosis.

Epidemiology of molecular probes in Xpert MTB/RIF assay in Khyber Pakhtunkhwa, Pakistan.

Regardless of a plethora of advanced diagnostics, TB and drug resistance remains a principal killer. We proposed gold nanoparticles (AuNPs) attached with probes to enhance the efficiency of GeneXpert MTB/RIF assay instead of conventional dye probes for molecular detection. A total of 15,000 samples were collected from TB suspects and subjected to Xpert MTB/RIF assay, where 6800 (45.3%) were detected as MTB positive, 280 (4.3%) were detected to harbor mutations in the RRDR, while invalid /errors were found in 690 (4.6%) cases. The mutations were detected by probe E, 199 (71.1%), while probes B and D, 30 and 26 (10% and 9%), respectively. In the Xpert MTB/RIF Assay were found mutations picked by probes E and B codons 529-533 (71%) and 512-518 (10%), respectively. The fast-rising works of TB nano-diagnostics, of Xpert probes, may improve by the applications of gold nanoparticle probes.

Cystic echinococcosis: an emerging zoonosis in southern regions of Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Cystic echinococcosis (CE) is one of the principal causes of economic loss to the livestock industry because of its morbidity and mortality of food-producing animals and condemnation of important visceral organs. Pakistan being an agricultural country having an extensive livestock sector, is mostly practiced by poor people, which has a fundamental role in the economy. The present study was aimed to conduct a cross-sectional survey and PCR based confirmation of Echinococcus granulosus in sheep, goats, cows, and buffaloes from southern regions (three districts: Lakki Marwat, Bannu, and Karak) of Khyber Pakhtunkhwa, Pakistan. During the study, a total of 2833 animals were examined randomly including; sheep (n = 529), goats (n = 428), cows (n = 1693), and buffaloes (n = 183). Hydatid cysts were collected and examined for the presence of protoscoleces using microscopy. Detection of DNA was performed by using PCR and two mitochondrial genetic markers namely; NAD-1 and COX-1 were amplified. RESULTS: The overall prevalence of CE was found to be (9%) among the examined animals. The hydatid cyst infection was highly prevalent in buffaloes (12%), followed by sheep (10%), cows (9%), and goats (5.1%). Cystic echinococcosis was more prevalent (10%; 96/992) in district Lakki Marwat followed by district Bannu (9%; 112/1246) and Karak (7%; 39/595). Female animals were more likely to be infected with CE (11.6%) than male animals (5.3%) (p = 0.001). Similarly, the infection was higher in the older group of animals as compared to younger (p = 0.001). Mostly (52.2%; n = 129) of hydatid cysts were found in the liver, while (64.4%; n = 159) cysts of the infected animals were infertile. PCR based identification confirmed the presence of E. granulosus sensu stricto (s.s) in the study area. CONCLUSION: Cystic echinococcosis was found to be highly prevalent in southern regions of Khyber Pakhtunkhwa and could be a potential threat to human health. Moreover, molecular sequencing and phylogenetic analyses should be carried out in future to identify the prevailing genotype (s) of E. granulosus s.s.

Burden of Drug resistant Tuberculosis in newly diagnosed Tuberculosis patients of Khyber Pakhtunkhwa, Pakistan.

OBJECTIVE: To explore the burden of drug-resistant tuberculosis in Khyber Pakhtunkhwa, Pakistan. METHODS: The cross-sectional study was conducted from March 1, 2016, to February 28, 2017, at the Khyber Pakhtunkhwa Tuberculosis Reference Laboratory, Hayatabad Medical Complex, Peshawar, Pakistan, and comprised referred suspected tuberculosis patient samples. Drug Susceptibility testing on all Mycobacterium tuberculosis complex strains was performed and data was subjected to statistical analysis. RESULTS: Of the 8220 samples, 4230 (51.5%) were related to females and 3990 (48.5%) to males. Also, 1978 (24%) were related to patients aged 15-24 years. Of the total, 1351 (16.5%) samples were positive on culture. Drug susceptibility testing showed 525 (39%) samples to be resistant to at least one of the first- and second-line drugs. Among the culture-positive cases, 5 (0.4%) were extensively drug-resistant, 62 (4.6%) multi-drug resistant, 243 (18%) polyresistant, 215 (16%) monoresistant and 826 (61%) were pan-sensitive. CONCLUSIONS: Drug-resistant tuberculosis in newly-diagnosed tuberculosis patients was alarmingly high in Khyber Pakhtunkhwa region.

Surveillance of genetic markers associated with Plasmodium falciparum resistance to artemisinin-based combination therapy in Pakistan, 2018-2019.

BACKGROUND: The spread of artemisinin resistance in the Greater Mekong Subregion of Southeast Asia poses a significant threat for current anti-malarial treatment guidelines globally. The aim of this study was to assess the current prevalence of molecular markers of drug resistance in Plasmodium falciparum in the four provinces with the highest malaria burden in Pakistan, after introducing artemether-lumefantrine as first-line treatment in 2017. METHODS: Samples were collected during routine malaria surveillance in Punjab, Sindh, Baluchistan, and Khyber Pakhtunkhwa provinces of Pakistan between January 2018 and February 2019. Plasmodium falciparum infections were confirmed by rapid diagnostic test or microscopy. Plasmodium falciparum positive isolates (n = 179) were screened by Sanger sequencing for single nucleotide polymorphisms (SNPs) in the P. falciparum kelch 13 (pfk13) propeller domain and in P. falciparum coronin (pfcoronin). SNPs in P. falciparum multidrug resistance 1 (pfmdr1) N86Y, Y184F, D1246Y and P. falciparum chloroquine resistance transporter (pfcrt) K76T were genotyped by PCR-restriction fragment length polymorphism. RESULTS: No artemisinin resistance associated SNPs were identified in the pfk13 propeller domain or in pfcoronin. The pfmdr1 N86, 184F, D1246 and pfcrt K76 alleles associated with reduced lumefantrine sensitivity were present in 83.8% (150/179), 16.9% (29/172), 100.0% (173/173), and 8.4% (15/179) of all infections, respectively. The chloroquine resistance associated pfcrt 76T allele was present in 98.3% (176/179) of infections. CONCLUSION: This study provides an update on the current prevalence of molecular markers associated with reduced P. falciparum sensitivity to artemether and/or lumefantrine in Pakistan, including a first baseline assessment of polymorphisms in pfcoronin. No mutations associated with artemisinin resistance were observed in pfk13 or pfcoronin. However, the prevalence of the pfmdr1 N86 and D1246 alleles, that have been associated with decreased susceptibility to lumefantrine, remain high. Although clinical and molecular data suggest that the current malaria treatment guidelines for P. falciparum are presently effective in Pakistan, close monitoring for artemisinin and lumefantrine resistance will be critical to ensure early detection and enhanced containment of emerging ACT resistance spreading across from Southeast Asia.

Molecular epidemiology of Shigella flexneri isolated from pediatrics in a diarrhea-endemic area of Khyber Pakhtunkhwa, Pakistan.

Shigella flexneri is considered as an important causative agent of Shigellosis causing diarrhea in the countries with a low socioeconomic status. No study has been carried out on the molecular prevalence of S. flexneri in Khyber Pakhtunkhwa, Pakistan. So this study was designed to evaluate the molecular prevalence of S. flexneri and their associated risk factors. A total of 2014 diarrheal stool samples were collected from January 2016 to May 2017 from pediatrics patients of Khyber Pakhtunkhwa followed by identification of S. flexneri through biochemical, serological, and molecular methods. The overall prevalence of Shigella species was found to be 7.9% (n = 160). The predominant Shigella specie was S. flexneri (n = 155, 96.8%) followed by S. boydii (n = 5, 3.1%). Interestingly, no sample was found positive for S. sonnei and S. dysenteriae. The majority of Shigellosis cases occurred from June to September. Potential risk factors related with Shigellosis were unhygienic latrine usage, bad hand washing, and consumption of unhygienic food and water, and pipe leakage in the sewage system. In this study, we have observed a high number of Shigellosis cases especially those caused by S. flexneri. It is suggested that effective health awareness programs should be organized by the regional health authorities to minimize the magnitude of pediatrics Shigellosis.

CD40 ligand deficiency causes functional defects of peripheral neutrophils that are improved by exogenous IFN-γ.

BACKGROUND: Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches. OBJECTIVES: We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function. METHODS: We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function. RESULTS: Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients. CONCLUSION: Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

Epidemiology of Cutaneous Leishmaniasis Outbreak, Waziristan, Pakistan.

During 2013-2015, prevalence of cutaneous leishmaniasis in war-affected Waziristan areas was 3.61% by PCR. Youths (1-15 years of age) were more susceptible. Internal transcribed spacer 1 PCR followed by restriction fragment length polymorphism analysis identified Leishmania tropica in 215 samples and Leishmania major in 6 samples.

Baseline characteristics of infected foot ulcers in patients with diabetes at a tertiary care hospital in Pakistan.

OBJECTIVE:: Studies on diabetic foot ulcers (DFU) involving a representative sample of patients in Pakistan are scarce. This study aimed to determine baseline characteristics of infected DFUs in patients hospitalised at the Pakistan Institute of Medical Sciences Islamabad. METHOD:: In this cross-sectional study, carried out during May 2015 and June 2016, foot ulcer characteristics of patients with DFUs were investigated and documented. From infected DFUs, aerobic bacterial pathogens were isolated, identified and evaluated for antimicrobial susceptibility. RESULTS:: A total of 214 patients were recruited to the study, 62.6% of which were male, 90.2% were aged ≥40 years, 76.2% had type 1 diabetes and 78.5% had poor glycaemic control at time of presentation to hospital. Most patients had grade 3/moderate ulceration (based on the Wagner and International Working Group on the Diabetic Foot/Infectious Diseases Society of America criteria). Over half of the DFUs (57.9%) were of ≤3 months' duration and 70.1% were ≥3 cm2. Of the patients with deep infection grade ulcers, 26.6% underwent amputation, accounting for their prolonged hospital stay (≥20 days). Significant differences were observed between patients with type 1 and type 2 diabetes with DFUs in relation to gender (p≤0.0001), ulcer size (p=0.0421) and duration of hospital stay (p=0.0253). The most significant predictors for lower extremity amputation were osteomyelitis (p=0.0114), retinopathy (p=0.0001) and neuropathy (p=0.0001. Piperacillin/tazobactam was found to be an effective antibiotic against the most commonly isolated Staphylococcus non-aureus (35.48%), Pseudomonas aeruginosa (22.26%), and Staphylococcus aureus (20.96%) species indentified in the DFU infections. CONCLUSION:: The findings of this study may be helpful in the optimal management and appropriate treatment of patients with infected DFUs.

Human CD40 ligand deficiency dysregulates the macrophage transcriptome causing functional defects that are improved by exogenous IFN-γ.

BACKGROUND: CD40 ligand (CD40L) deficiency predisposes to opportunistic infections, including those caused by fungi and intracellular bacteria. Studies of CD40L-deficient patients reveal the critical role of CD40L-CD40 interaction for the function of T, B, and dendritic cells. However, the consequences of CD40L deficiency on macrophage function remain to be investigated. OBJECTIVES: We sought to determine the effect of CD40L absence on monocyte-derived macrophage responses. METHODS: After observing the improvement of refractory disseminated mycobacterial infection in a CD40L-deficient patient by recombinant human IFN-γ (rhIFN-γ) adjuvant therapy, we investigated macrophage functions from CD40L-deficient patients. We analyzed the killing activity, oxidative burst, cytokine production, and in vitro effects of rhIFN-γ and soluble CD40 ligand (sCD40L) treatment on macrophages. In addition, the effect of CD40L absence on the macrophage transcriptome before and after rhIFN-γ treatment was studied. RESULTS: Macrophages from CD40L-deficient patients exhibited defective fungicidal activity and reduced oxidative burst, both of which improved in the presence of rhIFN-γ but not sCD40L. In contrast, rhIFN-γ and sCD40L ameliorate impaired production of inflammatory cytokines. Furthermore, rhIFN-γ reversed defective control of Mycobacterium tuberculosis proliferation by patients' macrophages. The absence of CD40L dysregulated the macrophage transcriptome, which was improved by rhIFN-γ. Additionally, rhIFN-γ increased expression levels of pattern recognition receptors, such as Toll-like receptors 1 and 2, dectin 1, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin in macrophages from both control subjects and patients. CONCLUSION: Absence of CD40L impairs macrophage development and function. In addition, the improvement of macrophage immune responses by IFN-γ suggests this cytokine as a potential therapeutic option for patients with CD40L deficiency.

Paracoccidioidomycosis Associated With a Heterozygous STAT4 Mutation and Impaired IFN-γ Immunity.

Background: Mutations in genes affecting interferon-γ (IFN-γ) immunity have contributed to understand the role of IFN-γ in protection against intracellular pathogens. However, inborn errors in STAT4, which controls interleukin-12 (IL-12) responses, have not yet been reported. Our objective was to determine the genetic defect in a family with a history of paracoccidioidomycosis. Methods: Genetic analysis was performed by whole-exome sequencing and Sanger sequencing. STAT4 phosphorylation (pSTAT4) and translocation to the nucleus, IFN-γ release by patient lymphocytes, and microbicidal activity of patient monocytes/macrophages were assessed. The effect on STAT4 function was evaluated by site-directed mutagenesis using a lymphoblastoid B cell line (B-LCL) and U3A cells. Results: A heterozygous missense mutation, c.1952 A>T (p.E651V) in STAT4 was identified in the index patient and her father. Patient's and father's lymphocytes showed reduced pSTAT4, nuclear translocation, and impaired IFN-γ production. Mutant B-LCL and U3A cells also displayed reduced pSTAT4. Patient's and father's peripheral blood mononuclear cells and macrophages demonstrated impaired fungicidal activity compared with those from healthy controls that improved in the presence of recombinant human IFN-γ, but not rhIL-12. Conclusion: Our data suggest autosomal dominant STAT4 deficiency as a novel inborn error of IL-12-dependent IFN-γ immunity associated with susceptibility to paracoccidioidomycosis.

Novel RAG1 mutation and the occurrence of mycobacterial and Chromobacterium violaceum infections in a case of leaky SCID.

Severe combined immunodeficiency (SCID) is a potentially fatal primary immunodeficiency (PID) that is caused by mutations in genes such as IL2RG, JAK3, IL7RA, RAG1, RAG2, and ADA. The products of these genes are involved in the development of several immune cells such as T, B and natural killer (NK) cells. Most of the SCID forms are autosomal recessive with the exception of IL2RG defects that cause an X-linked SCID. Among the different SCID types, there is a rare SCID form called leaky SCID, which is less severe when compared to the other classical SCID phenotypes. Leaky SCID can be caused by hypomorphic mutations in RAG1 and RAG2 that result in only partial loss of enzymatic function of the proteins respectively encoded by these genes. Here we report a novel missense mutation (c. 307C > T/p.H103Y) in the RAG1 gene in a patient with leaky SCID. In addition, we characterize the clinical and immunological features of this patient that developed along with other severe and recurrent infections such as mycobacterial diseases (BCGitis and pulmonary tuberculosis), the first occurrence of Chromobacterium violaceum in a patient with SCID. Understanding the increased susceptibility to mycobacteria presented by the patient, in which a functional investigation of IL-12/IFN-γ axis was performed, which demonstrated reduced production of IFN-γ in the supernatans of peripheral blood mononuclear cell cultures from the patient compared with those from healthy subjects. In conclusion, our data expands the molecular and clinical spectrum associated with the leaky SCID phenotype.

Expanding the clinical and genetic spectrum of G6PD deficiency: The occurrence of BCGitis and novel missense mutation.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway that ensures sufficient production of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) by catalyzing the reduction of NADP+ to NADPH. Noteworthy, the latter mediates the production of reactive oxygen species (ROS) by phagocytic cells such as neutrophils and monocytes. Therefore, patients with severe forms of G6PD deficiency may present impaired NADPH oxidase activity and become susceptible to recurrent infections. This fact, highlights the importance to characterize the immunopathologic mechanisms underlying the susceptibility to infections in patients with G6PD deficiency. Here we report the first two cases of G6PD deficiency with Bacille Calmette-Guérin (BCG) adverse effect, besides jaundice, hemolytic anemia and recurrent infections caused by Staphylococcus aureus. The qualitative G6PD screening was performed and followed by oxidative burst analysis using flow cytometry. Genetic and in silico analyses were carried out by Sanger sequencing and mutation pathogenicity predicted using bioinformatics tools, respectively. Activated neutrophils and monocytes from patients displayed impaired oxidative burst. The genetic analysis revealed the novel missense mutation c.1157T>A/p.L386Q in G6PD. In addition, in silico analysis indicated that this mutation is pathogenic, thereby hampering the oxidative burst of neutrophils and monocytes from patients. Our data expand the clinical and genetic spectrum of G6PD deficiency, and suggest that impaired oxidative burst in this severe primary immune deficiency is an underlying immunopathologic mechanism that predisposes to mycobacterial infections.

Punica granatum peel extracts: HPLC fractionation and LC MS analysis to quest compounds having activity against multidrug resistant bacteria.

BACKGROUND: Medicinal plants are rich source of traditional herbal medicine around the globe. Most of the plant's therapeutic properties are due to the presence of secondary bioactive compounds. METHODS: The present study analyzed the High Pressure Liquid Chromatography (HPLC) fractions of Puncia granatum (peel) extracts (aqueous, chloroform, ethanol and hexane) against multidrug resistant bacterial pathogens (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus). All the fractions having antibacterial activity was processed for bioactive compounds identification using LC MS/MS analysis. RESULTS: Among total HPLC fractions (n = 30), 4 HPLC fractions of P. granatum (peel) showed potential activity against MDR pathogens. Fraction 1 (F1) and fraction 4 (F4) collected from aqueous extract showed maximum activity against P. aeruginosa. Fraction 2 (F2) of hexane showed antibacterial activity against three pathogens, while ethanol F4 exhibited antibacterial activity against A. baumannii. The active fractions were processed for LC MS/MS analysis to identify bioactive compounds. Valoneic acid dilactone (aqueous F1 and F4), Hexoside (ethanol F4) and Coumaric acid (hexane F2) were identified as bioactive compounds in HPLC fractions. CONCLUSION: Puncia granatum peel extracts HPLC fractions exhibited potential inhibitory activity against MDR bacterial human pathogens. Several bioactive compounds were identified from the HPLC fractions. Further characterization of these compounds may be helpful to conclude it as therapeutic lead molecules against MDR pathogens.

A novel missense mutation in the NADPH binding domain of CYBB abolishes the NADPH oxidase activity in a male patient with increased susceptibility to infections.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the five structural genes (CYBB, CYBA, NCF1, NCF2, and NCF4) that typically results in a decrease in function or inability to generate a respiratory burst, leading to defective killing of pathogens, including fungi and intracellular bacteria. Mutations in CYBB, encoding the gp91phox (also known as NOX2) result in X-linked CGD account for approximately 65% of CGD cases. Here, we aimed the characterization of a novel missense mutation c.1226C > A/p.A409E in the CYBB gene in a patient with X-linked CGD. Relevant clinical data of a male patient whose family was positive for XCGD was reviewed. Oxidative burst and NADPH protein expression was evaluated by flow cytometry, while Genetic analysis was performed by Sanger sequencing. Monocyte-derived macrophages (MDMs) were evaluated for their capacity for phagocytosis and growth suppression of the intracellular Mycobacterium tuberculosis (M. tuberculosis). We thus report the absence of an oxidative burst in the phagocytes of the patient. Flow cytometry evaluation revealed a normal expression of NADPH oxidase components in neutrophils and genetic analysis proved the existence of a novel missense c.1226C > A mutation in the CYBB gene resulting in p.A409E. Further, we have showed that the patient's MDMs were unhindered in their ability to take up mycobacteria normally. Instead, the MDMs failed to control the intracellular proliferation of M. tuberculosis, a phenotype that improved in the presence of recombinant human interferon-gamma (rhIFN-γ). This work expands the genetic spectrum of X-linked CGD and demonstrates improvement in macrophage function in X91+CGD patient by rhIFN-γ.

Interferon-gamma reduces the proliferation of M. tuberculosis within macrophages from a patient with a novel hypomorphic NEMO mutation.

X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by mutations in the nuclear factor-kappa B essential modulator (NEMO) gene. Here, we report the clinical and genetic features of a XL-EDA-ID patient who developed bacillus Calmette-Guérin infection. Patient lymphocytes failed to degrade IκB-α, and sequencing of NEMO identified the novel mutation c.1238A>C/p.H413P. Furthermore, patient monocyte-derived macrophages ingested Mycobacterium tuberculosis normally, but failed to control the intracellular proliferation of bacilli, a defect which was improved in the presence of interferon-gamma (IFN-γ). This work expands the genetic spectrum of XL-EDA-ID and demonstrates improvement in macrophage function in a NEMO-deficient patient by IFN-γ.

Effects of Luteolin and Quercetin in Combination with Some Conventional Antibiotics against Methicillin-Resistant Staphylococcus aureus.

The present study was designed to evaluate the effects of flavonoids luteolin (L) and quercetin + luteolin (Q + L) in combination with commonly used antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates and S. aureus (ATCC 43300). Minimum inhibitory concentrations (MICs) of L and Q + L, as well as the MICs of flavonoids in combination with antibiotics were determined and results showed an increased activity of flavonoids with antibiotics. The synergistic, additive, or antagonistic relationships between flavonoids (L and Q + L) and antibiotics were also evaluated, and additive and synergistic effects were observed for some antibiotic + flavonoid combinations. In addition, some combinations were also found to damage the bacterial cytoplasmic membrane, as assessed through potassium leakage assay. The effects of flavonoids and flavonoids + antibiotics on mecA gene mutations were also tested, and no functional variation was detected in the coding region.

First report of the Hyper-IgM syndrome Registry of the Latin American Society for Immunodeficiencies: novel mutations, unique infections, and outcomes.

Hyper-IgM (HIGM) syndrome is a heterogeneous group of disorders characterized by normal or elevated serum IgM levels associated with absent or decreased IgG, IgA and IgE. Here we summarize data from the HIGM syndrome Registry of the Latin American Society for Immunodeficiencies (LASID). Of the 58 patients from 51 families reported to the registry with the clinical phenotype of HIGM syndrome, molecular defects were identified in 37 patients thus far. We retrospectively analyzed the clinical, immunological and molecular data from these 37 patients. CD40 ligand (CD40L) deficiency was found in 35 patients from 25 families and activation-induced cytidine deaminase (AID) deficiency in 2 unrelated patients. Five previously unreported mutations were identified in the CD40L gene (CD40LG). Respiratory tract infections, mainly pneumonia, were the most frequent clinical manifestation. Previously undescribed fungal and opportunistic infections were observed in CD40L-deficient patients but not in the two patients with AID deficiency. These include the first cases of pneumonia caused by Mycoplasma pneumoniae, Serratia marcescens or Aspergillus sp. and diarrhea caused by Microsporidium sp. or Isospora belli. Except for four CD40L-deficient patients who died from complications of presumptive central nervous system infections or sepsis, all patients reported in this study are alive. Four CD40L-deficient patients underwent successful bone marrow transplantation. This report characterizes the clinical and genetic spectrum of HIGM syndrome in Latin America and expands the understanding of the genotype and phenotype of this syndrome in tropical areas.

Etiology, pathology, and host-impaired immunity in medical implant-associated infections.

Host impaired immunity and pathogens adhesion factors are the key elements in analyzing medical implant-associated infections (MIAI). The infection chances are further influenced by surface properties of implants. This review addresses the medical implant-associated pathogens and summarizes the etiology, pathology, and host-impaired immunity in MIAI. Several bacterial and fungal pathogens have been isolated from MIAI; together, they form cross-kingdom species biofilms and support each other in different ways. The adhesion factors initiate the pathogen's adherence on the implant's surface; however, implant-induced impaired immunity promotes the pathogen's colonization and biofilm formation. Depending on the implant's surface properties, immune cell functions get slow or get exaggerated and cause immunity-induced secondary complications resulting in resistant depression and immuno-incompetent fibro-inflammatory zone that compromise implant's performance. Such consequences lead to the unavoidable and straightforward conclusion for the downstream transformation of new ideas, such as the development of multifunctional implant coatings.