Fungal microbiota in newborn infants with and without respiratory distress syndrome.

BACKGROUND: Pneumocytis jirovecii infection in preterm newborns has recently been associated with neonatal respiratory distress syndrome and bronchopulmonary dysplasia. Changes in the bacterial microbiota of the airways have also been described in infants with bronchopulmonary dysplasia. However, until now there has been no information on the airway mycobiota in newborns. The purpose of this study was to describe the airway mycobiota in term and preterm newborns and its possible association with respiratory distress syndrome. METHODS: Twenty-six matched preterm newborns with and without respiratory distress syndrome were studied, as well as 13 term babies. The identification of the fungal microbiota was carried out using molecular procedures in aspirated nasal samples at birth. RESULTS: The ascomycota phylum was identified in 89.7% of newborns, while the basidiomycota phylum was found in 33.3%. Cladosporium was the predominant genus in both term and preterm infants 38.4% vs. 73% without statistical differences. Candida sake and Pneumocystis jirovecii were only found in preterm infants, suggesting a potential relationship with the risk of prematurity. CONCLUSIONS: This is the first report to describe the fungal microbiota of the airways in term and preterm infants with and without respiratory distress syndrome. Although no differences have been observed, the number of cases analyzed could be small to obtain conclusive results, and more studies are needed to understand the role of the fungal microbiota of the airways in neonatal respiratory pathology.

Biophysical evaluation of antiparallel triplexes for biosensing and biomedical applications.

Polypyrimidine sequences can be targeted by antiparallel clamps forming triplex structures either for biosensing or therapeutic purposes. Despite its successful implementation, their biophysical properties remain to be elusive. In this work, PAGE, circular dichroism and multivariate analysis were used to evaluate the properties of PPRHs directed to SARS-CoV-2 genome. Several PPRHs designed to target various polypyrimidine sites within the viral genome were synthesized. These PPRHs displayed varying binding affinities, influenced by factors such as the length of the PPRH and its GC content. The number and position of pyrimidine interruptions relative to the 4 T loop of the PPRH was found a critical factor, affecting the binding affinity with the corresponding target. Moreover, these factors also showed to affect in the intramolecular and intermolecular equilibria of PPRHs alone and when hybridized to their corresponding targets, highlighting the polymorphic nature of these systems. Finally, the functionality of the PPRHs was evaluated in a thermal lateral flow sensing device showing a good correspondence between their biophysical properties and detection limits. These comprehensive studies contribute to the understanding of the critical factors involved in the design of PPRHs for effective targeting of biologically relevant genomes through the formation of triplex structures under neutral conditions.

Fungal microbiota dynamics and its geographic, age and gender variability in patients with cystic fibrosis.

OBJECTIVES: In cystic fibrosis (CF), there is a predisposition to bronchial colonization by potentially pathogenic microorganisms, such as fungi. Our aims were to describe the dynamics of respiratory mycobiota in patients with CF and to evaluate the geographic, age and gender variability in its distribution. METHODS: Cohort study in which 45 patients with CF from four hospitals in three Spanish cities were followed up during a 1-year period, obtaining spontaneous sputum samples every 3 to 6 months. Fungal microbiota were characterized by Internal Transcribed Spacer sequencing and Pneumocystis jirovecii was identified by nested PCR in a total of 180 samples. RESULTS: The presence of fungi were detected in 119 (66.11%) of the 180 samples and in 44 (97.8%) of the 45 patients: 19 were positive and 1 negative throughout all follow-ups and the remaining 25 presented alternation between positive and negative results. A total of 16 different genera were identified, with Candida spp. (50/180, 27.78%) and Pneumocystis spp. (44/180, 24.44%) being the most prevalent ones. The distribution of fungal genera was different among the evaluated centres (p < 0.05), by age (non-adults aged 6-17 years vs. adults aged ≥18 years) (p < 0.05) and by gender (p < 0.05). DISCUSSION: A high prevalence of fungal respiratory microbiota in patients with CF was observed, whose dynamics are characterized by the existence of multiple cycles of clearance and colonization, reporting the existence of geographic, age and gender variability in the distribution of fungal genera in this disease.

Pneumocystis jirovecii among patients with cystic fibrosis and their household members.

We conducted a pilot study of patients with cystic fibrosis (CF) to assess intra-family transmission of P. jirovecii and compare it with data on other prevalent pathogens such as P. aeruginosa and S. pneumoniae, in which respiratory transmission has already been documented. Oral swab samples from 10 patients with CF and 15 household members were collected at baseline and 2 weeks later. P. aeruginosa and S. pneumoniae were assessed using standardized culture methods and PCR, and P. jirovecii was assessed using real and nested PCR, genotyping the positive samples by direct sequencing. P. aeruginosa cultures were positive for 7/10 (70%) of patients with CF at baseline and was identified by PCR in 8/10 (80%) of cases at baseline and 2 weeks later. S. pneumoniae cultures were negative for all patients, but the microorganism was identified by PCR in two cases. P. jirovecii was detected by real time and nested PCR in 5/10 (50%) of the patients at the two time points. In the household members, P. aeruginosa and P. jirovecii were identified in 7/15 (46.7%), and S. pneumoniae was identified in 8/15 (53,3%). The concordance of positive or negative pairs of patients with CF and their household members was 33.3% (5/15) for P. aeruginosa, 46.7% (7/15) for S. pneumonia and 93.3% (14/15) for P. jirovecii. The concordance for P. jirovecii genotypes among five pairs with available genotype was 100%. This study suggests for the first time the possible transmission of Pneumocystis in the home of patients with CF, indicating that patients and their household members are reservoirs and possible sources of infection. LAY SUMMARY: This study suggests for the first time the possible transmission of Pneumocystis in the family environment of patients with cystic fibrosis, indicating that patients and their household members are reservoirs and possible sources of this infection.

Changing Trends in the Epidemiology and Risk Factors of Pneumocystis Pneumonia in Spain.

Objective: The information about the epidemiology of Pneumocystis pneumonia (PcP) in Europe is scarce, and in Spain there are only data nationwide on patients with HIV infection. This study has been carried out with the aim of knowing in our country the current epidemiological spectrum and the risk factors of PcP. Methods: Observational, descriptive transversal study that included all patients admitted in Spain with diagnosis upon discharge of PcP registered in the National Health System's Hospital Discharge Records Database of Spain, between 2008 and 2012. Results: Four thousand five hundred and fifty four cases of PcP were reported, 1,204 (26.4%) in HIV-negative patients. During the study period, mean annual incidence (cases per million) was 19.4, remaining globally stable, increasing from 4.4 to 6.3 in HIV-negative patients and decreasing from 15.5 to 13.4 among HIV-infected patients. Risk factors were identified in 85.5% of HIV-negative cases, the most frequent being hematological neoplams (29%), chronic lung diseases (15.9%), and non-hematological cancers (14.9%). Mean mortality and hospitalization cost were high (25.5% and 12,000 euros, respectively). Conclusions: The results of this first nationwide study in Spain allow a change in the misconception that, after the AIDS epidemic, PcP is an infrequent disease, showing that today it is an emerging problem in patients without HIV infection. These findings underlines the need for increased efforts toward a better characterization of risk groups to improve prophylactic strategies and reduce the burden of disease.

Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth: A cross-sectional survey.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection is the most prevalent congenital infection acquired worldwide, with higher incidence in developing countries and among HIV-exposed children. Less is known regarding vertical transmission of parvovirus B19 (B19V) and enterovirus (EV). We aimed to assess the prevalence of CMV, B19V and EV vertical transmission and compare results of screening of congenital CMV obtained from two different specimens in a semirural Mozambican maternity. METHODS: A cross sectional study was conducted among pregnant mothers attending Manhiça District Hospital upon delivery. Information on maternal risk factors was ascertained. Dried umbilical cord (DUC) samples were collected in filter paper for CMV, B19V and EV detection by real-time polymerase chain reaction (RT-PCR), and nasopharyngeal aspirates (NPA) to test for CMV by RT-PCR. Maternal blood samples and placental biopsy samples were also obtained to investigate CMV maternal serology, HIV status and immunopathology. RESULTS: From September 2014 to January 2015, 118 mothers/newborn pairs were recruited. Prevalence of maternal HIV infection was 31.4% (37/118). CMV RT-PCR was positive in 3/115 (2.6%) of DUC samples and in 3/96 (6.3%) of NPA samples obtained from neonates. The concordance of the RT-PCR assay through DUC with their correspondent NPA sample was moderate (Kappa = 0.42 and p<0.001. No differences on cCMV prevalence were found among HIV-exposed and unexposed. All (100%) mothers were seropositive for CMV IgG. RT-PCR of EV and B19V in DUC were both negative in all screened cases. No histological specific findings were found in placental tissues. No risk factors associated to vertical transmission of these viral infections were found. CONCLUSIONS: This study indicates the significant occurrence of vertical transmission of CMV in southern Mozambique. Larger studies are needed to evaluate the true burden, clinical relevance and consequences of congenital infections with such pathogens in resource-constrained settings.

Prevalence and genotype distribution of Pneumocystis jirovecii in Cuban infants and toddlers with whooping cough.

This study describes the prevalence and genotype distribution of Pneumocystis jirovecii obtained from nasopharyngeal (NP) swabs from immunocompetent Cuban infants and toddlers with whooping cough (WC). A total of 163 NP swabs from 163 young Cuban children with WC who were admitted to the respiratory care units at two pediatric centers were studied. The prevalence of the organism was determined by a quantitative PCR (qPCR) assay targeting the P. jirovecii mitochondrial large subunit (mtLSU) rRNA gene. Genotypes were identified by direct sequencing of mtLSU ribosomal DNA (rDNA) and restriction fragment length polymorphism (RFLP) analysis of the dihydropteroate synthase (DHPS) gene amplicons. qPCR detected P. jirovecii DNA in 48/163 (29.4%) samples. mtLSU rDNA sequence analysis revealed the presence of three different genotypes in the population. Genotype 2 was most common (48%), followed in prevalence by genotypes 1 (23%) and 3 (19%); mixed-genotype infections were seen in 10% of the cases. RFLP analysis of DHPS PCR products revealed four genotypes, 18% of which were associated with resistance to sulfa drugs. Only contact with coughers (prevalence ratio [PR], 3.51 [95% confidence interval {CI}, 1.79 to 6.87]; P = 0.000) and exposure to tobacco smoke (PR, 1.82 [95% CI, 1.14 to 2.92]; P = 0.009) were statistically associated with being colonized by P. jirovecii. The prevalence of P. jirovecii in infants and toddlers with WC and the genotyping results provide evidence that this population represents a potential reservoir and transmission source of P. jirovecii.

Pneumocystis jirovecii pneumonia in Latin America. A public health problem?

Pneumocystis jirovecii pneumonia (PcP) is a well-recognized major opportunistic infection in HIV-infected patients. During the 1980s, the HIV pandemic turned PcP into a major worldwide medical and public health problem. With the introduction of Pneumocystis chemoprophylaxis and the development of highly active antiretroviral therapy (ART) for the treatment of HIV infection, there has been a decrease in PcP incidence in developed countries. However, the prevalence of AIDS-related PcP in developing countries remains high because a lot of people do not have access to ART or ignore their HIV infection status. This article discusses the information available about PcP among Latin American countries where there is a great regional heterogeneity in the prevalence of HIV infection and in ART coverage, as well as in the observed frequencies of PcP that range from 5.9 to 55% in this area.

High prevalence of Pneumocystis jirovecii colonization in Brazilian cystic fibrosis patients.

A high rate of Pneumocystis jirovecii colonization was observed in Brazilian cystic fibrosis (CF) patients (13 out of 34; 38.2%) who underwent bronchoscopy between March 2006 and August 2009 at the Hospital de Clinicas de Porto Alegre, Brazil. Bronchoalveolar lavage samples were collected from these patients and studied by nested PCR amplification of the mitochondrial gene coding for the large subunit ribosomal RNA (mtLSUrDNA). The observed rate of colonization was higher than that reported in European populations. Genotypic characterization of the mtLSUrDNA locus revealed a predominance of the polymorphisms 85C/248C (genotype 1) and 85T/248C (genotype 3), with all samples possessing the wild-type genotype of dihydropteroate synthase. These findings suggest that cystic fibrosis patients could be an important reservoir and source of P. jirovecii infection. Further studies are required to elucidate the role of this common fungal colonization in the evolution of CF patients.

Pneumocystis jirovecii colonization in patients treated with infliximab.

BACKGROUND: Infliximab, a chimeric antitumour necrosis factor (TNF) monoclonal antibody, has become an established effective therapy for inflammatory rheumatic disease. However, TNF is a critical factor in host defence, and the suppression of its biological activity may be associated with the increased risk of opportunistic infections. The frequent use of infliximab in clinical practice has identified Pneumocystis jirovecii pneumonia (PcP) as a serious complication. Individuals colonized with Pneumocystis may be at high risk of development of PcP when they have undergone immunosuppression. Hence, we addressed the question of the frequency of Pneumocystis colonization among patients treated with infliximab. DESIGN: We examined 125 oropharyngeal washes collected from 78 individuals with rheumatoid arthritis, 30 with ankylosing spondylitis and 17 with psoriatic arthritis, half of them underwent infliximab therapy, using a real-time polymerase chain reaction assay that employs specific primers from a portion of the mitochondrial large-subunit rRNA gene of P. jirovecii. RESULTS: Pneumocystis jirovecii colonization was detected in 32 (25·6%) patients. In a multivariate regression model, only duration of infliximab treatment for more than 3 years and use of corticosteroid were significantly and independently associated with risk of Pneumocystis colonization. However, the effect of corticosteroid on P. jirovecii colonization rate was not linearly dose dependent as showed other logistic regression analysis. CONCLUSIONS: There is a high rate of P. jirovecii colonization among patients with rheumatologic diseases treated with infliximab. The identification of patients colonized by P. jirovecii before starting the treatment with infliximab could be a strategy for PcP prevention.

Pneumocystis jirovecii and cystic fibrosis.

Pneumocystis jirovecii is an atypical opportunistic fungus with lung tropism and worldwide distribution that causes pneumonia in immunosuppressed individuals. The development of sensitive molecular techniques has led to the recognition of a colonization or carrier state of P. jirovecii, in which low levels of the organism are detected in persons who do not have pneumonia. Pneumocystis colonization has been described in individuals with various lung diseases, and accumulating evidence suggests that it may be a relevant issue with potential clinical impact. Only a few published studies carried out in Europe have evaluated the prevalence of Pneumocystis colonization in patients with cystic fibrosis, reporting ranges from 1.3-21.6%. The evolution of P. jirovecii colonization in cystic fibrosis patients is largely unknown. In a longitudinal study, none of the colonized patients developed pneumonia during a 1-year follow-up. Since patients with cystic fibrosis could act as major reservoirs and sources of infection for susceptible individuals further research is thus warranted to assess the true scope of the problem and to design rational preventive strategies if necessary. Moreover, it's necessary to elucidate the role of P. jirovecii infection in the natural history of cystic fibrosis in order to improve the clinical management of this disease.

Pneumocystis jiroveci dihydropteroate synthase gene mutations among colonized individuals and Pneumocystis pneumonia patients from Spain.

Cotrimoxazole, an association of trimethoprim and sulfamethoxazole, and dapsone, are mainstays for the prophylaxis and treatment of Pneumocystis pneumonia (PcP). The inability to culture Pneumocystis prevents routine susceptibility testing and detection of drug resistance. Instead, molecular techniques have been used to detect Pneumocystis jiroveci dihydropteroate synthase (DHPS) mutations that cause sulfa resistance in other microorganisms. The most frequent DHPS mutations occur at nucleotide positions 165 and 171, which lead to an amino acid change at positions 55 and 57. Several studies suggest that these mutations are associated with the failure of chemoprophylaxis for PcP. The aim was to establish the frequency and characteristics of P jiroveci DHPS mutations among colonized individuals and PcP patients from Spain. A total of 50 colonized individuals and 25 PcP patients were studied. DHPS polymorphisms were identified by restriction fragment length polymorphism assay. The analysis provided a rate of 28% of DHPS gene mutations in our population, with the presence of all possible polymorphisms described. The presence of mutations was higher in PcP patients than in colonized subjects (40% vs 22%), probably because of the chemoprophylaxis used in PcP patients. The comparison between patients with and without DHPS mutations did not show statistical differences due to age, sex, steroid use, sulfa drug exposure, or smoking. A high rate of DHPS mutations in our area of Spain, not only confined to patients previously exposed to sulfa drugs, is shown in this study. As well as PcP patients, colonized individuals who harbor P jiroveci strains with DHPS mutations could play a major role in the transmission cycle of these mutations, representing a reservoir and source of infection for susceptible individuals. Further research is thus warranted to assess the true scope of the problem and to design rational preventive strategies.

Pneumocystis infection in humans: diagnosis and treatment.

Pneumocystis jirovecii is an atypical fungus exhibiting pulmonary tropism and a highly defined host specificity. It is generally regarded as an opportunistic microorganism causing serious pneumonia in AIDS patients. However, with the currently rising number of patients receiving immunosuppressive therapies for malignancies, allogeneic organ transplantations and autoimmune diseases, Pneumocystis pneumonia is becoming more and more recognized in non-HIV immunosuppressed individuals. The clinical presentation in HIV-infected patients may differ from that in other immunocompromised patients and its diagnosis continues to be challenging as there are no specific symptoms or signs. Cotrimoxazole is the drug of choice for prophylaxis and therapy of any form or severity of Pneumocystis pneumonia, but there are only a few options for other alternative treatments. The management of this pneumonia remains a major challenge for all physicians caring for immunosuppressed patients.

Primary lymphoma of the central nervous system and HTLV-I infection.

Only a few cases of AIDS-related primary lymphomas of the central nervous system (CNS) show a T-cell phenotype. We have recently studied two intravenous drug users with HIV infection who had primary CNS T-cell lymphomas. In both cases, the enzyme immunoassay (EIA) for HTLV gave a positive result. In the first case, study by western-blot (WB) and specific PCR confirmed the human T-cell lymphotropic virus type I (HTLV-I) infection and serological study by EIA for HTLV of his mother was negative. In the second case, analysis of ante-mortem serum samples by two different WBs showed an indeterminate pattern suggestive of HTLV-I infection, but adequate samples for PCR were not available. We speculate about the possibility that the horizontal transmission of HTLV-I infection could have facilitated the devepolment of a primary CNS T-cell lymphoma in these HIV patients, although they cannot be strictly considered as ATLL cases.