Transmission patterns of C1-INH deficiency hereditary angioedema favors a wild-type male offspring: Our experience at Chandigarh, India.

BACKGROUND: Deficiency of C1-inhibitor (C1-INH) protein, caused by pathogenic variants in the Serpin family G member 1 (SERPING1) gene, is the commonest pathophysiological abnormality (in ∼95 % cases) in patients with hereditary angioedema (HAE). C1-INH protein provides negative control over kallikrein-kinin system (KKS). Although the inheritance of the HAE-C1-INH is autosomal dominant, female predominance has often been observed in patients with HAE. OBJECTIVE: To analyze the risk of transmission of SERPING1 gene variant from father or mother to their offspring. METHODS: Pedigree charts of 42 families with a confirmed diagnosis of HAE-C1-INH and a pathogenic variant in the SERPING1 gene were analysed. Patients with HAE who had had at least one child were included for analyses to assess the risk of transmission from the father or mother to their offspring. RESULTS: Overall, 49 % (189/385) of all offspring inherited the genetic defect. In the subgroup analyses, 54.8 % (90/164) female offspring and 44.8 % (99/221; p < 0.02) male offspring inherited the genetic defect. Inheritance of the genetic defect was significantly lower in male offspring. Fathers with SERPING1 gene variant had a statistically significant skewed transmission of the wild type to the male offspring as compared to the variant (57.8 % wild type vs. 42.1 % variant; p < 0.02), whereas no statistically significant difference was found when a father transmitted the variant to a female offspring. Mothers with SERPING1 gene variant had no statistically significant difference in variant transmission to male or female offsprings. CONCLUSION: Results of the study suggest that the transmission pattern of SERPING1 gene variant favours the transmission of wild-type alleles in males, especially when the father is the carrier; hence, overall, fewer males and more female offspring inherited the variant. This could be because of a selection of wild-type male sperms during spermatogenesis, as the KLK system has been reported to play a crucial role in the regulation of spermatogenesis. Although, a similar pattern was observed in the maternal transmission of the SERPING1 gene variant; the difference was not statistically significant, likely because of a small sample size.

X-Linked Lymphoproliferative Syndrome: A Spectrum of Clinical and Immunological Profile and Novel Pathogenic Variants from Chandigarh, India.

INTRODUCTION: X-linked lymphoproliferative syndrome (XLP) is a rare primary immune deficiency. Two types of XLP have been described: XLP-1 and XLP-2. METHODS: We found 7 patients with XLP (3 had XLP-1 and 4 had XLP-2) after reviewing the data from Pediatric Immunodeficiency Clinic from 1997 to 2021. RESULTS: Mean age at diagnosis was 3.8 years, and mean delay in diagnosis was 2.6 years. Five patients had recurrent episodes of infections. Four patients developed at least one episode of hemophagocytic lymphohistiocytosis (HLH) (2 with XLP-1 and 2 with XLP-2). Of these, 2 had recurrent HLH (both with XLP-2). Epstein-Barr virus (EBV) infection was detected in 2 (1 with XLP-1 and 1 with XLP-2). Both these patients had HLH. One child with XLP-2 had inflammatory bowel disease. Hypogammaglobulinemia was seen in 3 (2 with XLP-1 and 1 with XLP-2). Genetic analysis showed previously reported variants in 5, while 2 had novel variants (one in exon 7 of XIAP gene [c.1370dup p.Asn457Lysfs Ter16] and other had splice site variant in intron 1 of SH2D1A gene [c.138-2_138-1insG]). Episodes of HLH were managed with intravenous immunoglobulin (IVIg), methylprednisolone, oral prednisolone, cyclosporine, and rituximab. Inflammatory bowel disease was managed using oral prednisolone and azathioprine. One patient underwent haploidentical hematopoietic stem cell transplantation. One child with XLP-2 and WAS died because of fulminant pneumonia. DISCUSSION/CONCLUSIONS: XLP should be considered as a strong possibility in any patient with features of HLH, repeated infections with hypogammaglobulinemia, persistent EBV infection, and early-onset IBD.

Immune and metabolic perturbations in COVID-19-associated pulmonary mucormycosis: A transcriptome analysis of innate immune cells.

BACKGROUND AND OBJECTIVES: The mechanisms underlying COVID-19-associated pulmonary mucormycosis (CAPM) remain unclear. We use a transcriptomic analysis of the innate immune cells to investigate the host immune and metabolic response pathways in patients with CAPM. PATIENTS AND METHODS: We enrolled subjects with CAPM (n = 5), pulmonary mucormycosis (PM) without COVID-19 (n = 5), COVID-19 (without mucormycosis, n = 5), healthy controls (n = 5) without comorbid illness and negative for SARS-CoV-2. Peripheral blood samples from cases were collected before initiating antifungal therapy, and neutrophils and monocytes were isolated. RNA sequencing was performed using Illumina HiSeqX from monocytes and neutrophils. Raw reads were aligned with HISAT-2 pipeline and DESeq2 was used for differential gene expression. Gene ontology (GO) and metabolic pathway analysis were performed using Shiny GO application and R packages (ggplot2, Pathview). RESULTS: The derangement of core immune and metabolic responses in CAPM patients was noted. Pattern recognition receptors, dectin-2, MCL, FcRγ receptors and CLEC-2, were upregulated, but signalling pathways such as JAK-STAT, IL-17 and CARD-9 were downregulated; mTOR and MAP-kinase signalling were elevated in monocytes from CAPM patients. The complement receptors, NETosis, and pro-inflammatory responses, such as S100A8/A9, lipocalin and MMP9, were elevated. The major metabolic pathways of glucose metabolism-glycolysis/gluconeogenesis, pentose phosphate pathway, HIF signalling and iron metabolism-ferroptosis were also upregulated in CAPM. CONCLUSIONS: We identified significant alterations in the metabolic pathways possibly leading to cellular iron overload and a hyperglycaemic state. Immune responses revealed altered recognition, signalling, effector functions and a pro-inflammatory state in monocytes and neutrophils from CAPM patients.

Clinical, immunological and molecular profiles of DOCK8 deficiency in six patients from a tertiary care centre in North India.

BACKGROUND: Dedicator of cytokinesis protein 8 (DOCK8) deficiency is an autosomal recessive form of combined immunodeficiency. This rare disorder is characterized by an increased predisposition to allergy, autoimmunity and malignancies. OBJECTIVES: To analyse clinical, immunological and molecular profiles of patients with DOCK8 deficiency. METHODS: Clinic records of all patients attending the primary immunodeficiency clinic from 2018 to 2021 were reviewed. Six patients from five families were found to have DOCK8 deficiency. RESULTS: Median age at diagnosis was 7.5 years (range 2-13), with a male/female ratio of 5 : 1. Among the six patients, recurrent eczematous skin lesions were the predominant cutaneous manifestation, present in five patients (83%). Warts and molluscum contagiosum were evident in two patients (33%) and one patient (16%), respectively. Two patients had recalcitrant prurigo nodularis lesions and two had epidermodysplasia verruciformis-like lesions. Food allergies and asthma were reported by one patient each. Of the six patients, recurrent sinopulmonary infections were detected in five (83%). Epstein-Barr virus-driven non-Hodgkin lymphoma with liver metastases was the only case of malignancy, in a 4-year-old boy. IgE was elevated in all patients. Lymphopenia and eosinophilia were observed in three patients (50%) and five patients (83.3%), respectively. Genetic analysis showed DOCK8 pathogenic variants in all patients: homozygous deletion mutations in two patients, compound heterozygous deletion mutations in one, and homozygous nonsense mutations in two. A novel pathogenic homozygous missense variant in the DOCK8 gene was identified in one patient. CONCLUSIONS: DOCK8 deficiency should be considered as a possibility in any patient with early onset eczema, cutaneous viral infections and increased predisposition to allergy, autoimmunity and malignancy.

Delays in diagnosis is the commonest proximate reason for mortality in hereditary angioedema: our experience at Chandigarh, India.

BACKGROUND: Hereditary angioedema (HAE) is a rare autosomal dominant disorder characterized clinically by recurrent episodes of non-pruritic subcutaneous and/or submucosal edema. Laryngeal edema is the commonest cause of mortality in patients with HAE. Prior to the availability of first-line treatment options for management of HAE, the mortality used to be as high as 30%. The mortality has significantly reduced in countries where first-line treatment options are available and patients can access these therapies. There is paucity of literature on the outcomes of patients with HAE in developing countries where availability and access to first-line treatment options is still a challenge. OBJECTIVE: To report our experience with mortality in patients with HAE and to report factors associated with death of these patients. METHODS: We carried out a record review of all patients diagnosed to have HAE between January 1996 and August 2022. Families with HAE who had reported death of at least one family member/relative because of laryngeal edema were studied in detail. RESULTS: Of the sixty-five families (one hundred and seventy patients) registered in the clinic, sixteen families reported death of at least one family member/relative because of laryngeal edema (total thirty-six deaths). Of these sixteen families, fourteen families reported that one or more family members had experienced at least one attack of laryngeal edema. One patient died during follow-up when she was taking long-term prophylaxis with stanozolol and tranexamic acid while remaining thirty-five patients were not diagnosed to have HAE at the time of their death. At the time of death of all thirty-six patients, at least one other family member had symptoms suggestive of HAE but the diagnosis was not established for the family. CONCLUSIONS: This is the largest single centre cohort of patients with HAE in India reporting mortality data and factors associated with death in these families. Delay in diagnosis is the commonest reason for mortality.

Tuberculosis and Bacillus Calmette-Guérin Disease in Patients with Chronic Granulomatous Disease: an Experience from a Tertiary Care Center in North India.

Chronic granulomatous disease (CGD) is a phagocytic defect characterized by recurrent bacterial and fungal infections. We report clinical profile of patients with CGD and mycobacterial infections in a cohort from North India. A review of clinical and laboratory records was carried out for patients with CGD registered at our center between 1990 and 2021. Of the 99 patients with CGD, 22 had mycobacterial infections-Mycobacterium tuberculosis and M. bovis-BCG in 11 each. Among the children with M. bovis-BCG infection, 6 had localized and 5 had disseminated BCG disease. Median age at onset of symptoms and diagnosis of BCG disease was 5 months and 15 months, respectively. While disseminated forms of BCG were noted only in CYBB defect, none of the patients with NCF1 defect developed complications due to BCG vaccine. A recurring radiological feature was left axillary lymph node calcification, which was present in around 50% of CGD patients with BCG infections. Of 11 patients with tuberculosis, pulmonary, pleuro-pulmonary, abdominal, and disseminated forms were present in 6, 1, 2, and 2, respectively. Median age at onset of symptoms and diagnosis of tuberculosis was 129 months and 130 months, respectively. Molecular defects were identified in CYBB (5), NCF1 (4), and CYBA (1). Incidence of tuberculosis and BCG-related complications in patients with CGD is higher than the normal population. Screening for CGD is warranted in any patient with adverse reactions to BCG vaccination, calcification of left axillary lymph node, and persistent, recurrent or disseminated forms of tuberculosis.

Kawasaki Disease and Inborn Errors of Immunity: Exploring the Link and Implications.

The exact etiopathogenesis of Kawasaki disease (KD), the most common childhood vasculitis, remains unknown; however, an aberrant immune response, possibly triggered by an infectious or environmental agent in genetically predisposed children, is believed to be the underlying pathogenetic mechanism. Patients with inborn errors of immunity (IEI) are predisposed to infections that trigger immune dysregulation due to an imbalance in various arms of the immune system. KD may develop as a complication in both primary and secondary immunodeficiencies. KD may occur either at disease presentation or have a later onset in IEIs. These include X-linked agammaglobulinemia (XLA), selective IgA deficiency, transient hypogammaglobulinemia of infancy; Wiskott-Aldrich syndrome (WAS), hyper IgE syndrome (HIES); chronic granulomatous disease (CGD), innate and intrinsic immunity defects, and autoinflammatory diseases, including PFAPA. Hitherto, the association between KD and IEI is confined to specific case reports and case series and, thus, requires extensive research for a comprehensive understanding of the underlying pathophysiological mechanisms. IEIs may serve as excellent disease models that would open new insights into the disease pathogenesis of children affected with KD. The current review highlights this critical association between KD and IEI supported by published literature.

Microbiome and Its Dysbiosis in Inborn Errors of Immunity.

Inborn errors of immunity (IEI) can present with infections, autoimmunity, lymphoproliferation, granulomas, and malignancy. IEIs are due to genetic abnormalities that disrupt normal host-immune response or immune regulation. The microbiome appears essential for maintaining host immunity, especially in patients with a defective immune system. Altered gut microbiota in patients with IEI can lead to clinical symptoms. Microbial dysbiosis is the consequence of an increase in pro-inflammatory bacteria or a reduction in anti-inflammatory bacteria. However, functional and compositional differences in microbiota are also involved. Dysbiosis and a reduced alpha-diversity are well documented, particularly in conditions like common variable immunodeficiency. Deranged microbiota is also seen in Wiskott-Aldrich syndrome, severe combined immunodeficiency, chronic granulomatous disease, selective immunoglobulin-A deficiency, Hyper IgE syndrome (HIGES), X-linked lymphoproliferative disease-2, immunodysregulation, polyendocrinopathy, enteropathy, x-linked syndrome, and defects of IL10 signalling. Distinct gastrointestinal, respiratory, and cutaneous symptoms linked to dysbiosis are seen in several IEIs, emphasizing the importance of microbiome identification. In this study, we discuss the processes that maintain immunological homeostasis between commensals and the host and the disruptions thereof in patients with IEIs. As the connection between microbiota, host immunity, and infectious illnesses is better understood, microbiota manipulation as a treatment strategy or infection prevention method would be more readily employed. Therefore, optimal prebiotics, probiotics, postbiotics, and fecal microbial transplantation can be promising strategies to restore the microbiota and decrease disease pathology in patients with IEIs.

T Cell Abnormalities in X-Linked Agammaglobulinaemia: an Updated Review.

X-linked agammaglobulinaemia (XLA) is a primary immunodeficiency (PID) resulting from a defect in the B cell development. It has conventionally been thought that T cells play a major role in the development and function of the B cell compartment. However, it has also been shown that B cells and T cells undergo bidirectional interactions and B cells also influence the structure and function of the T cell compartment. Patients with XLA offer a unique opportunity to understand the effect of absent B cells on the T cell compartment. In this review, we provide an update on abnormalities in the T cell compartment in patients with XLA. Studies have shown impaired memory T cells, follicular helper T cells, T regulatory cells and T helper 17 in patients with XLA. In addition, these patients have also been reported to have abnormal delayed cell-mediated immune responses and vaccine-specific T cell-mediated immune responses; defective T helper cell polarization and impaired T cell receptor diversity. At present, the clinical significance of these T cell abnormalities has not been studied in detail. However, these abnormalities may result in an increased risk of viral infections, autoimmunity, autoinflammation and possibly chronic lung disease. Abnormal response to SARS-Cov2 vaccine in patients with XLA and prolonged persistence of SARS-Cov2 virus in the respiratory tract of these patients may be related to abnormalities in the T cell compartment.

Kawasaki disease and the environment: an enigmatic interplay.

Kawasaki disease (KD) is a common systemic vasculitis of childhood. Although it has been almost 6 decades since Dr. Tomisaku Kawasaki reported the first case series of KD, the underlying cause remains a mystery. KD is a self-limiting disease. However, a dreaded complication is development of coronary artery abnormalities (CAAs). KD is the most common cause of acquired heart disease in children in the developed world and is being increasingly reported from developing countries too. Over the years, significant observations have been made about epidemiology of KD. It usually affects children below 5, has male preponderance and has significantly higher incidence in North East Asian countries. While several hypotheses have been proffered for etiology of KD, none have been conclusive. These include associations of KD epidemics in Japan and the United Stated with changes in tropospheric wind patterns suggesting wind-borne agents, global studies showing peaks of incidence related to season, and increased rates in populations with a higher socioeconomic profile related to hygiene hypothesis and vaccination. Furthermore, the self-limiting, febrile nature of KD suggests an infectious etiology, more so with sudden decline noted in cases in Japan with onset of COVID-19 mitigation measures. Finally, single nucleotide polymorphisms have been identified as possible risk alleles in patients with KD and their significance in the pathogenesis of this disease are also being defined. The purpose of this review is to elucidate the puzzling associations of KD with different environmental factors. Looking at patterns associated with KD may help us better predict and understand this disease.

Challenges in the diagnosis of periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome in developing countries-A decade of experience from North India.

Background: Reports of periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome from developing countries are sparse. Recognizing PFAPA is often challenging in these regions due to a higher incidence of infectious illnesses and significant resource constraints. Herein, we present our experience from North India regarding the diagnosis and management of PFAPA syndrome. Methods: We reviewed cases of non-monogenic periodic fever syndrome diagnosed at our center from January 2011 to December 2021. A total of 17 children who fulfilled the Marshall criteria for PFAPA syndrome were included. Data regarding basic clinical features, treatment/outcome, and performance of the recently proposed Eurofever/PRINTO and Takeuchi criteria were analyzed. Results: Besides recurrent fever, the triad of oral aphthae, pharyngitis, and adenitis was noted in only 18% of patients. Episodes of exudative pharyngitis/tonsillitis were documented in 24%. These figures were lower than the values reported from developed countries. The Takeuchi and Eurofever/PRINTO criteria were fulfilled in 76% and 71% cases, respectively. In addition to antipyretics and supportive care, intermittent steroid therapy was the main treatment modality used. Additional treatment with colchicine (n = 3) and thalidomide (n = 1) was used successfully in a few patients. Before the diagnosis of PFAPA, all patients had received multiple courses of antimicrobials (without microbiological confirmation). These included multiple courses of antibacterials for fever, pharyngotonsillitis, and/or cervical adenitis in all patients and antivirals for fever and aphthous stomatitis in a patient. Empiric antitubercular therapy had also been administered in two patients. Conclusions: A significant proportion of patients with PFAPA seem to remain undiagnosed in the Indian subcontinent. Increased awareness and improvement in basic healthcare facilities are crucial in enhancing the recognition of PFAPA, which would eliminate the unprecedented scale of undesirable antimicrobial use in such children.

Serum glucose-regulated protein 78 (GRP78) levels in COVID-19-associated mucormycosis: results of a case-control study.

BACKGROUND: In experimental models, the expression of glucose-regulated protein 78 (GRP78) in endothelial cells played a role in the pathogenesis of mucormycosis. However, the role of GRP78 in COVID-19-associated mucormycosis (CAM) has not been studied. We hypothesized that serum GRP78 levels are elevated in subjects with CAM. OBJECTIVE: To compare the serum GRP78 levels in subjects with CAM and COVID-19 controls without mucormycosis. DESIGN AND SETTING: We performed a hospital-based, case-control study between 1 April 2021 and 31 May 2021. PARTICIPANTS: We enrolled 24 subjects each of CAM and COVID-19 subjects without mucormycosis. We also measured serum GRP78 levels in ten healthy controls. EXPOSURE: The primary exposure studied was serum GRP78 concentration, estimated using a commercially available ELISA kit in stored serum samples. RESULTS: We found the mean ± standard deviation (SD) serum GRP78 levels significantly higher (p = 0.0001) among the CAM (374.3 ± 127.3 pg/mL) than the COVID-19 (246.4 ± 67.0 pg/mL) controls. The proportion of subjects with an abnormal GRP78 level (> mean [184.8 pg/mL] plus two SD [23.2 pg/mL] of GRP78 from healthy participants) was 87.5% and 45.8% in the CAM group and COVID-19 controls, respectively. Serum GRP78 level was independently associated with CAM (odds ratio 1.011; 95% confidence interval [1.002-1.019]) after adjusting for diabetes mellitus and hypoxemia during acute COVID-19. CONCLUSION: Serum GRP78 levels were significantly higher in CAM than in COVID-19 controls. Further studies are required to the role of GRP78 in the pathogenesis of CAM.

Deficiency of Human Adenosine Deaminase Type 2 - A Diagnostic Conundrum for the Hematologist.

Deficiency of adenosine deaminase type 2 (DADA2) was first described in 2014 as a monogenic cause of polyartertitis nodosa (PAN), early onset lacunar stroke and livedo reticularis. The clinical phenotype of DADA2 is, however, very broad and may involve several organ systems. Apart from vasculitis, children may present with i) Hematological manifestations (ii) Lymphoproliferation and iii) Immunodeficiencies. Patients with DADA2 can have variable patterns of cytopenias and bone marrow failure syndromes. Patients with DADA2 who have predominant haematological manifestations are associated with ADA2 gene variants that result in minimal or no residual ADA2 activity. Lymphoproliferation in patients with DADA2 may range from benign lymphoid hyperplasia to lymphoreticular malignancies. Patients may present with generalized lymphadenopathy, splenomegaly, autoimmune lymphoproliferative syndrome (ALPS) like phenotype, Hodgkin lymphoma, T-cell large granular lymphocytic infiltration of bone marrow and multicentric Castleman disease. Immunodeficiencies associated with DADA are usually mild. Affected patients have variable hypogammaglobulinemia, decrease in B cells, low natural killer cells, common variable immunodeficiency and rarely T cell immunodeficiency. To conclude, DADA2 has an extremely variable phenotype and needs to be considered as a differential diagnosis in diverse clinical conditions. In this review, we describe the evolving clinical phenotypes of DADA2 with a special focus on haematological and immunological manifestations.

Mechanisms of Immune Dysregulation in COVID-19 Are Different From SARS and MERS: A Perspective in Context of Kawasaki Disease and MIS-C.

Coronaviruses have led to three major outbreaks to date-Severe Acute Respiratory Syndrome (SARS; 2002), Middle East Respiratory Syndrome (MERS; 2012) and the ongoing pandemic, Coronavirus Disease (COVID-19; 2019). Coronavirus infections are usually mild in children. However, a few children with MERS had presented with a severe phenotype in the acute phase resulting in progressive pneumonic changes with increasing oxygen dependency and acute respiratory distress requiring ventilatory support. A subset of children with a history of SARS-CoV-2 infection develops a multisystem hyper-inflammatory phenotype known as Multisystem Inflammatory Syndrome in Children (MIS-C). This syndrome occurs 4-6 weeks after infection with SARS-CoV-2 and has been reported more often from areas with high community transmission. Children with MIS-C present with high fever and often have involvement of cardiovascular, gastrointestinal and hematologic systems leading to multiorgan failure. This is accompanied by elevation of pro-inflammatory cytokines such as IL-6 and IL-10. MIS-C has several similarities with Kawasaki disease (KD) considering children with both conditions present with fever, rash, conjunctival injection, mucosal symptoms and swelling of hands and feet. For reasons that are still not clear, both KD and MIS-C were not reported during the SARS-CoV and MERS-CoV outbreaks. As SARS-CoV-2 differs from SARS-CoV by 19.5% and MERS by 50% in terms of sequence identity, differences in genomic and proteomic profiles may explain the varied disease immunopathology and host responses. Left untreated, MIS-C may lead to severe abdominal pain, ventricular dysfunction and shock. Immunological investigations reveal reduced numbers of follicular B cells, increased numbers of terminally differentiated CD4+T lymphocytes, and decreased IL-17A. There is still ambiguity about the clinical and immunologic risk factors that predispose some children to development of MIS-C while sparing others. Host-pathogen interactions in SARS, MERS and COVID-19 are likely to play a crucial role in the clinical phenotypes that manifest. This narrative review focuses on the immunological basis for development of MIS-C syndrome in the ongoing SARS-CoV-2 pandemic. To the best of our knowledge, these aspects have not been reviewed before.

Lymphoproliferation in Inborn Errors of Immunity: The Eye Does Not See What the Mind Does Not Know.

Inborn errors of immunity (IEIs) are a group of heterogeneous disorders characterized by a broad clinical spectrum of recurrent infections and immune dysregulation including autoimmunity and lymphoproliferation (LP). LP in the context of IEI may be the presenting feature of underlying immune disorder or may develop during the disease course. However, the correct diagnosis of LP in IEI as benign or malignant often poses a diagnostic dilemma due to the non-specific clinical features and overlapping morphological and immunophenotypic features which make it difficult to treat. There are morphological clues to LP associated with certain IEIs. A combination of ancillary techniques including EBV-associated markers, flow cytometry, and molecular assays may prove useful in establishing a correct diagnosis in an appropriate clinical setting. The present review attempts to provide comprehensive insight into benign and malignant LP, especially the pathogenesis, histological clues, diagnostic strategies, and treatment options in patients with IEIs.

Sociodemographic characteristics and spectrum of Malassezia species in individuals with and without seborrhoeic dermatitis/dandruff: A comparison of residents of the urban and rural populations.

Seborrhoeic dermatitis/dandruff (SD/D) is a common, persistent, relapsing inflammatory condition affecting the areas rich in sebaceous glands. SD/D is widely prevalent in India but Malassezia species implicated are not well studied. To estimate the prevalence and spectrum of Malassezia species causing SD/D and understand the sociodemographic characteristics of SD/D in rural and urban populations, a total of 200 SD/D patients and 100 healthy controls (HC) from both rural and urban backgrounds were enrolled in this study. SD/D severity was clinically graded as mild, moderate, severe, and very severe. The isolates were identified by phenotypic characters and confirmed by ITS2 PCR-RFLP and sequencing of the ITS region of rDNA. Severe (59%) and very severe (71%) form of SD/D was higher in the rural population compared to the urban population (P = .004). The isolation rate of Malassezia was significantly higher in overall SD/D patients scalp (82%) compared to HC (67%) (P = .005). From the scalp of SD/D patients, M. globosa (36.2%) was predominantly isolated followed by M. restricta (31.3%), M. furfur (15.7%), a mixture of M. globosa and M. restricta (12%) or M. arunalokei (4.8%). Similarly, M. globosa (49.3%) was predominately isolated from the scalp of HC followed by M. restricta (22.4%). M. restricta was significantly higher in the scalp of SD/D patients compared to HC and/or nasolabial fold of both SD/D patients and HC (P = .0001). Our findings indicate that M. restricta has a high association with SD/D. More severe disease frequency was observed in the rural population. PRECIS: Dandruff is associated with Malassezia restricta and very severe cases are higher in rural population, probably due the poor hygiene. Moderate to severe hair loss and itching were strongly associated with dandruff. Use of soaps to cleanse scalp appears to be better than shampoo in preventing dandruff.

Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues.

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species levelGap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation.Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues.Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae.Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues.Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

In vitro antifungal activity of a novel topical triazole PC945 against emerging yeast Candida auris.

OBJECTIVES: Management of Candida auris infection is difficult as this yeast exhibits resistance to different classes of antifungals, necessitating the development of new antifungals. The aim of this study was to investigate the susceptibility of C. auris to a novel antifungal triazole, PC945, optimized for topical delivery. METHODS: A collection of 50 clinical isolates was obtained from a tertiary care hospital in North India. Nine isolates from the UK, 10 from a CDC panel (USA) and 3 from the CBS-KNAW culture collection (Japanese and South Korean isolates) were also obtained. MICs (azole endpoint) of PC945 and other triazoles were determined in accordance with CLSI M27 (third edition). Quality control strains were included [Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258)]. RESULTS: Seventy-four percent of isolates tested showed reduced susceptibility to fluconazole (≥64 mg/L). PC945 (geometric mean MIC = 0.058 mg/L) was 7.4-fold and 1.5-fold more potent than voriconazole and posaconazole, respectively (both P < 0.01). PC945 MIC values correlated with those of voriconazole or posaconazole, and only three isolates were found to be cross-resistant between PC945 and other azoles. ERG11 sequence analysis revealed several mutations, but no correlation could be established with the MIC of PC945. Tentative epidemiological cut-off values (ECOFFs) evaluated by CLSI's ECOFF Finder (at 99%) with 24 h reading of MICs were 1, 4 and 1 mg/L for PC945, voriconazole and posaconazole, respectively. MIC values for quality control strains of all triazoles were in the normal ranges. CONCLUSIONS: PC945 was found to be a more potent inhibitor than posaconazole, voriconazole and fluconazole of C. auris isolates collected globally, warranting further laboratory and clinical evaluations.

Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps.

The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 µg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

Pharmacodynamics of Voriconazole against Wild-Type and Azole-Resistant Aspergillus flavus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis.

Invasive aspergillosis (IA) due to Aspergillus flavus is associated with high mortality. Although voriconazole (VRC) is widely recommended as the first-line treatment for IA, emergence of azole resistance in Aspergillus spp. is translating to treatment failure. We evaluated the efficacy of voriconazole in a nonneutropenic murine model of disseminated A. flavus infection using two voriconazole-resistant isolates (one harboring the Y319H substitution in the cyp51C gene) and two wild-type isolates without mutations. All isolates exhibited a dose-response relationship, and voriconazole treatment improved mouse survival in a dose-dependent manner. At 40 mg/kg of body weight, 100% efficacy was observed for 1 susceptible isolate and 1 resistant isolate (with mutation), whereas for another susceptible isolate and resistant isolate (without mutation), survival rates were 81% and 72%, respectively. The Hill equation with a variable slope fitted the relationship between the area under the concentration-time curve (AUC)/MIC ratio and 14-day survival well for each strain. An F test showed the 50% effective doses to be significantly different from each other (P = 0.0023). However, contrary to expectation, there was a significant difference in exposure-response relationships between strains, and it appeared that the susceptible strains required a relatively higher exposure than the resistant ones to result in the same treatment effect, the 50% effective pharmacokinetic/pharmacodynamic (PK/PD) index (EI50) required being negatively and log-linearly related to the MIC (P = 0.04). We conclude that the efficacy of voriconazole depended on drug exposure and the voriconazole MIC of the isolates, but lower exposures are required for strains with higher MICs. These findings may have profound significance in clinical practice with respect to dosing and drug choice.