Genetic diversity of Leptospira in northwestern Colombia: first report of Leptospira santarosai as a recognised leptospirosis agent.

The region of Antioquia in northeastern Colombia has the highest number of reported leptospirosis cases in the country. It also shows high seroprevalence indexes in the general population and socio-environmental conditions favourable for the transmission of the disease between humans and animals. In this study, 25 Leptospira isolates from Colombia's Antioquia department were identified to the species level as L. santarosai (12), L. interrogans (9) and L. meyeri (4) using phylogenetic analysis of the Amidohydrolase gene. Typing at the serovar level was performed using multilocus sequence typing (MLST) and monoclonal antibodies. The serovars Canalzonae, Babudieri, Alice, Beye, and Copenhageni have been identified as causing human or animal infections in Antioquia, Colombia. The four environmental isolates were not identified to the serovar level. L. santarosai serovar Canalzonae and Alice were identified as new etiologic agents of human leptospirosis in Antioquia, Colombia. This paper reports species and serovars that were previously unknown in the region.

Highly Multiplexed Serology for Nonhuman Mammals.

Emerging infectious diseases represent a serious and ongoing threat to humans. Most emerging viruses are maintained in stable relationships with other species of animals, and their emergence within the human population results from cross-species transmission. Therefore, if we want to be prepared for the next emerging virus, we need to broadly characterize the diversity and ecology of viruses currently infecting other animals (i.e., the animal virosphere). High-throughput metagenomic sequencing has accelerated the pace of virus discovery. However, molecular assays can detect only active infections and only if virus is present within the sampled fluid or tissue at the time of collection. In contrast, serological assays measure long-lived antibody responses to infections, which can be detected within the blood, regardless of the infected tissues. Therefore, serological assays can provide a complementary approach for understanding the circulation of viruses, and while serological assays have historically been limited in scope, recent advancements allow thousands to hundreds of thousands of antigens to be assessed simultaneously using <1 µL of blood (i.e., highly multiplexed serology). The application of highly multiplexed serology for the characterization of the animal virosphere is dependent on the availability of reagents that can be used to capture or label antibodies of interest. Here, we evaluate the utility of commercial immunoglobulin-binding proteins (protein A and protein G) to enable highly multiplexed serology in 25 species of nonhuman mammals, and we describe a competitive fluorescence-linked immunosorbent assay (FLISA) that can be used as an initial screen for choosing the most appropriate capture protein for a given host species. IMPORTANCE Antibodies are generated in response to infections with viruses and other pathogens, and they help protect against future exposures. Mature antibodies are long lived, are highly specific, and can bind to their protein targets with high affinity. Thus, antibodies can also provide information about an individual's history of viral exposures, which has important applications for understanding the epidemiology and etiology of disease. In recent years, there have been large advances in the available methods for broadly characterizing antibody-binding profiles, but thus far, these have been utilized primarily with human samples only. Here, we demonstrate that commercial antibody-binding reagents can facilitate modern antibody assays for a wide variety of mammalian species, and we describe an inexpensive and fast approach for choosing the best reagent for each animal species. By studying antibody-binding profiles in captive and wild animals, we can better understand the distribution and prevalence of viruses that could spill over into humans.

High Diversity of Leptospira Species Infecting Bats Captured in the Urabá Region (Antioquia-Colombia).

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the genus Leptospira. This zoonotic disease affects humans, domestic animals and wild animals. Colombia is considered an endemic country for leptospirosis; Antioquia is the second department in Colombia, with the highest number of reported leptospirosis cases. Currently, many studies report bats as reservoirs of Leptospira spp. but the prevalence in these mammals is unknown. The goal of this study was to better understand the role of bats as reservoir hosts of Leptospira species and to evaluate the genetic diversity of circulating Leptospira species in Antioquia-Colombia. We captured 206 bats in the municipalities of Chigorodó (43 bats), Carepa (43 bats), Apartadó (39 bats), Turbo (40 bats), and Necoclí (41 bats) in the Urabá region (Antioquia-Colombia). Twenty bats tested positive for Leptospira spp. infection (20/206-9.70%) and the species of infected bats were Carollia perspicillata, Dermanura rava, Glossophaga soricina, Molossus molossus, Artibeus planirostris, and Uroderma convexum. These species have different feeding strategies such as frugivorous, insectivores, and nectarivores. The infecting Leptospira species identified were Leptospira borgpetersenii (3/20-15%), Leptospira alexanderi (2/20-10%), Leptospira noguchii (6/20-30%), Leptospira interrogans (3/20-15%), and Leptospira kirschneri (6/20-30%). Our results showed the importance of bats in the epidemiology, ecology, and evolution of Leptospira in this host-pathogen association. This is the first step in deciphering the role played by bats in the epidemiology of human leptospirosis in the endemic region of Urabá (Antioquia-Colombia).

Hematologically important mutations: X-linked chronic granulomatous disease (third update).

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations.

New molecular target for the phylogenetic identification of Leptospira species directly from clinical samples: an alternative gene to 16S rRNA.

INTRODUCTION: Phylogenetic analysis of the 16S ribosomal gene initial region is used to identify Leptospira isolates at the species level from clinical samples. Unfortunately, this method cannot differentiate between some intermediates and saprophytic species. METHODS: We used comparative genomic analysis between 35 Leptospira species to find new molecular targets for Leptospira species identification. RESULTS: We proposed the use of the rpoC gene, encoding the DNA-directed RNA polymerase ß-subunit, for identifying 35 Leptospira species. CONCLUSIONS: The rpoC gene can be a molecular target to identify the main species of the Leptospira genus directly from clinical samples.

[Molecular and clinical characterization of Colombian patients suffering from type III glycogen storage disease].

INTRODUCTION: Type III glycogen storage disease (GSD III) is an autosomal recessive disorder in which a mutation in the AGL gene causes deficiency of the glycogen debranching enzyme. The disease is characterized by fasting hypoglycemia, hepatomegaly and progressive myopathy. Molecular analyses of AGL have indicated heterogeneity depending on ethnic groups. The full spectrum of AGL mutations in Colombia remains unclear. OBJECTIVE: To describe the clinical and molecular characteristics of ten Colombian patients diagnosed with GSD III. MATERIALS AND METHODS: We recruited ten Colombian children with a clinical and biochemical diagnosis of GSD III to undergo genetic testing. The full coding exons and the relevant exon-intron boundaries of the AGL underwent Sanger sequencing to identify mutation. RESULTS: All patients had the classic phenotype of the GSD III. Genetic analysis revealed a mutation p.Arg910X in two patients. One patient had the mutation p.Glu1072AspfsX36, and one case showed a compound heterozygosity with p.Arg910X and p.Glu1072AspfsX36 mutations. We also detected the deletion of AGL gene 3, 4, 5, and 6 exons in three patients. The in silico studies predicted that these defects are pathogenic. No mutations were detected in the amplified regions in three patients. CONCLUSION: We found mutations and deletions that explain the clinical phenotype of GSD III patients. This is the first report with a description of the clinical phenotype and the spectrum of AGL mutations in Colombian patients. This is important to provide appropriate prognosis and genetic counseling to the patient and their relatives.

Variants in LTA, TNF, IL1B and IL10 genes associated with the clinical course of sepsis.

The aim of this study was to explore the association between some SNPs of the TNF, LTA, IL1B and IL10 genes with cytokine concentrations and clinical course in Colombian septic patients. We conducted a cross-sectional study to genotype 415 septic patients and 205 patients without sepsis for the SNPs -308(G/A) rs1800629 of TNF; +252 (G/A) rs909253 of LTA; -511(A/G) rs16944 and +3953(C/T) rs1143634 of IL1B; and -1082(A/G) rs1800896, -819(C/T) rs1800871 and -592(C/A) rs1800872 of IL10. The association of theses SNPs with the following parameters was evaluated: (1) the presence of sepsis; (2) severity and clinical outcomes; (3) APACHE II and SOFA scores; and (4) procalcitonin, C-reactive protein, tumor necrosis factor, lymphotoxin alpha, interleukin 1 beta and interleukin 10 plasma concentrations. We found an association between the SNP LTA +252 with the development of sepsis [OR 1.29 (1.00-1.68)]; the SNP IL10 -1082 with sepsis severity [OR 0.53 (0.29-0.97)]; the TNF -308 with mortality [OR 0.33 (0.12-0.95)]; and the IL10 -592 and IL10 -1082 with admission to the intensive care unit (ICU) [OR 3.36 (1.57-7.18)] and [OR 0.18 (0.04-0.86)], respectively. None of the SNPs were associated with cytokine levels, procalcitonin and C-reactive protein serum concentrations, nor with APACHE II and SOFA scores. Our results suggest that these genetic variants play an important role in the development of sepsis and its clinical course.

Successful immune reconstitution by means of hematopoietic stem cell transplantation in a Colombian patient with chronic granulomatous disease.

INTRODUCTION: Chronic granulomatous disease is a primary immunodeficiency that results from mutations in proteins of the NADPH oxidase system that affect the microbicidal activity of phagocytes. Immune reconstitution by hematopoietic stem cell transplantation is currently the only curative therapy for this disease.  OBJECTIVE: To describe the clinical and molecular characterization of a patient with X-linked chronic granulomatous disease and the successful immune reconstitution by means of a hematopoietic stem cell transplantation.  METHODS: The respiratory burst was measured by flow cytometry using the dihydrorodamine 123 (DHR) oxidation test in neutrophils of peripheral blood. Mutational analysis of CYBB was performed by PCR amplification in complementary DNA, as well as sequencing and comparative genomic hybridization in genomic DNA. HLA-identical stem cells from the patient's younger brother were used for the transplantation and reduced intensity pre-transplantation conditioning was administered. Post-transplantation immune reconstitution was evaluated periodically by serial complete blood counts and DHR 123 in peripheral blood neutrophils.  RESULTS: The diagnosis of X-linked chronic granulomatous disease resulted from a hemizygous deletion affecting Xp21.1 that included the entire CYBB. Post-transplantation engraftment was documented in platelets and peripheral blood neutrophils at days 10 and 11, respectively. Total hematological reconstitution was achieved by day 30 post-transplantation and no complications or infections have been observed in the three years since the transplantation.  CONCLUSION: Hemopoietic stem cell transplantation allows for total reconstitution of the immune function related to microbicidal activity of phagocytic cells from patients with X-linked chronic granulomatous disease.

New molecular target for the phylogenetic identification of Leptospira species directly from clinical samples: an alternative gene to 16S rRNA

Abstract INTRODUCTION: Phylogenetic analysis of the 16S ribosomal gene initial region is used to identify Leptospira isolates at the species level from clinical samples. Unfortunately, this method cannot differentiate between some intermediates and saprophytic species. METHODS: We used comparative genomic analysis between 35 Leptospira species to find new molecular targets for Leptospira species identification. RESULTS: We proposed the use of the rpoC gene, encoding the DNA-directed RNA polymerase β-subunit, for identifying 35 Leptospira species. CONCLUSIONS: The rpoC gene can be a molecular target to identify the main species of the Leptospira genus directly from clinical samples.

[Evaluation as a function of granulocytes in hyperimmunoglobulinemia E syndrome with recurrent infections]. / Evaluación de la función de los granulocitos en el síndrome de hiperinmunoglobulinemia E con infecciones recurrentes.

The hyper-IgE syndrome with recurrent infections (HIESRI) is characterized by skin and respiratory infections due to Staphylococcus aureus and several fungi infections which are frequently associated with tissue damage. A deficiency in the chemotaxis of phagocytic cells has been documented to explain these findings; however, the expression of adhesion molecules, the secretion of cytokines that activate granulocytes and the production of oxygen reactive molecules have not been evaluated in HIESRI. Six HIESRI patients were evaluated for the following parameters: (1) secretion of GM-CSF and IL-5 by mitogen and antigen-activated mononuclear cells, (2) the chemotactic response of FMLP-activated granulocytes, (3) the respiratory burst of PMA-activated granulocytes, and (4) the expression of L-selectin and CD11b in PMA-activated granulocytes. Human recombinant GM-CSF and culture supernatants were evaluated for capacity to modulate granulocytic function. Compared to controls, HIESRI patients showed a normal production of GM-CSF and an increase in the basal secretion of IL-5. No significant differences were observed for chemotaxis, respiratory burst or L-selectin and CD11b expression. The GM-CSF did not modulate these functions in granulocytes from HIESRI patients, but culture supernatants applied to granulocytes inhibited chemotaxis, increased respiratory burst and caused the shedding of L-selectin from the granulocyte surface. The 6 HIESRI patients were nonsymptomatic during the time of this research due to a program of continued treatment; findings suggest that granulocytes are activated more easily in response to proinflammatory factors and that production of these factors is higher in HIESRI.

Brotes emergentes de leptospirosis del Amazonas colombiano / Emerging leptospirosis outbreaks in the Colombian Department of Amazonas

Se describe por primera vez una serie de nueve casos con clínica indicativa de leptospirosis en el municipio Puerto Nariño en el departamento Amazonas, Colombia. Se muestran evidencias serológicas de exposición con Rickettsia del grupo de las fiebres manchadas. Los casos fueron clínicamente considerados como síndrome febril de origen desconocido. Se descartó infección por dengue y malaria. El diagnóstico de Leptospira se realizó mediante el método de reacción en cadena de la polimerasa en tiempo real. Igualmente, se detectó la presencia de anticuerpos contra rickettsias del grupo de las fiebres manchadas por inmunofluorescencia Indirecta. Finalmente, se realiza revisión del tema(AU)

A description is provided for the first time of a series of nine cases with a clinical examination suggestive of leptospirosis in the municipality of Puerto Nariño, Department of Amazonas, Colombia. Serological evidence is presented of exposure to Rickettsia, spotted fever group. The cases were clinically considered as febrile syndrome of unknown origin. Infection with dengue or malaria was ruled out. Diagnosis of leptospirosis was achieved by real-time polymerase chain reaction. Additionally, indirect immunofluorescence detected the presence of antibodies against rickettsia, spotted fever group. Finally, a review was conducted about the topic(AU)

Enfermedad por almacenamiento de glucógeno de tipo III en pacientes colombianos: caracterización clínica y molecular / Molecular and clinical characterization of Colombian patients suffering from type III glycogen storage disease

Resumen Introducción. La enfermedad por almacenamiento de glucógeno de tipo III es una alteración autosómica recesiva, en la cual las mutaciones del gen AGL causan una deficiencia en la enzima desramificadora de glucógeno. Se caracteriza por hipoglucemia, hepatomegalia y miopatías progresivas. El análisis molecular del gen AGL ha evidenciado mutaciones que difieren según la población estudiada. En la actualidad, no existen reportes que describan mutaciones en el AGL de pacientes colombianos con esta condición. Objetivo. Describir las características clínicas y moleculares de diez pacientes colombianos con enfermedad por almacenamiento del glucógeno de tipo III. Materiales y métodos. Se analizaron diez pacientes pediátricos colombianos con la enfermedad y se hizo su estudio genético mediante la secuenciación de las regiones que codifican y las intrónicas circundantes del gen AGL con el método de Sanger. Resultados. Todos los pacientes tenían el fenotipo clásico de la enfermedad. El estudio genético reveló la mutación p.Arg910X en dos pacientes. Uno presentó la mutación p.Glu1072AspfsX36 y otro resultó heterocigoto compuesto con las mutaciones p.Arg910X y p.Glu1072AspfsX36. Asimismo, en tres pacientes se detectó la deleción de los exones 4, 5 y 6 del gen AGL. Los estudios de simulación computacional predijeron que estos defectos eran patogénicos. En tres pacientes no se encontraron mutaciones en las regiones amplificadas. Conclusión. Se encontraron mutaciones y deleciones que explican el fenotipo clínico de los pacientes. Este es el primer reporte en el que se describe el fenotipo clínico y el espectro de mutaciones en el gen AGL de pacientes colombianos, lo cual es importante para ofrecer un apropiado pronóstico, y asesoría genética al paciente y a su familia.

Abstract Introduction: Type III glycogen storage disease (GSD III) is an autosomal recessive disorder in which a mutation in the AGL gene causes deficiency of the glycogen debranching enzyme. The disease is characterized by fasting hypoglycemia, hepatomegaly and progressive myopathy. Molecular analyses of AGL have indicated heterogeneity depending on ethnic groups. The full spectrum of AGL mutations in Colombia remains unclear. Objective: To describe the clinical and molecular characteristics of ten Colombian patients diagnosed with GSD III. Materials and methods: We recruited ten Colombian children with a clinical and biochemical diagnosis of GSD III to undergo genetic testing. The full coding exons and the relevant exon-intron boundaries of the AGL underwent Sanger sequencing to identify mutation. Results: All patients had the classic phenotype of the GSD III. Genetic analysis revealed a mutation p.Arg910X in two patients. One patient had the mutation p.Glu1072AspfsX36, and one case showed a compound heterozygosity with p.Arg910X and p.Glu1072AspfsX36 mutations. We also detected the deletion of AGL gene 3, 4, 5, and 6 exons in three patients. The in silico studies predicted that these defects are pathogenic. No mutations were detected in the amplified regions in three patients. Conclusion: We found mutations and deletions that explain the clinical phenotype of GSDIII patients. This is the first report with a description of the clinical phenotype and the spectrum of AGLmutations in Colombian patients. This is importantto provide appropriate prognosis and genetic counseling to the patient and their relatives.

Genetic diversity of Leptospira in northwestern Colombia: first report of Leptospira santarosai as a recognised leptospirosis agent

The region of Antioquia in northeastern Colombia has the highest number of reported leptospirosis cases in the country. It also shows high seroprevalence indexes in the general population and socio-environmental conditions favourable for the transmission of the disease between humans and animals. In this study, 25 Leptospira isolates from Colombia’s Antioquia department were identified to the species level as L. santarosai (12), L. interrogans (9) and L. meyeri (4) using phylogenetic analysis of the Amidohydrolase gene. Typing at the serovar level was performed using multilocus sequence typing (MLST) and monoclonal antibodies. The serovars Canalzonae, Babudieri, Alice, Beye, and Copenhageni have been identified as causing human or animal infections in Antioquia, Colombia. The four environmental isolates were not identified to the serovar level. L. santarosai serovar Canalzonae and Alice were identified as new etiologic agents of human leptospirosis in Antioquia, Colombia. This paper reports species and serovars that were previously unknown in the region.

Reconstitución inmune exitosa mediante trasplante de células madre hematopoyéticas en un paciente colombiano afectado con enfermedad granulomatosa crónica / Successful immune reconstitution by means of hematopoietic stem cell transplantation in a Colombian patient with chronic granulomatous disease

Introducción. La enfermedad granulomatosa crónica es una inmunodeficiencia primaria causada por mutaciones en los genes que codifican para las proteínas del sistema de la oxidasa de NADPH ( Nicotinamide Adenine Dinucleotide Phosphate ) de las células fagocíticas, las cuales afectan la producción de especies reactivas del oxígeno y la actividad microbicida. Actualmente, la única terapia curativa para esta enfermedad es la reconstitución inmune mediante el trasplante de células madre hematopoyéticas. Objetivo. Reportar la caracterización clínica y molecular de un paciente con enfermedad granulomatosa crónica ligada al cromosoma X y su reconstitución inmunitaria exitosa mediante el trasplante de células madre hematopoyéticas. Materiales y métodos. El estallido respiratorio en neutrófilos de sangre periférica se midió por citometría de flujo mediante la prueba de oxidación de la dihidrorrodamina 123 (DHR 123). El análisis de las mutaciones del gen CYBB se hizo mediante reacción en cadena de la polimerasa (PCR) en el ADN complementario y la secuenciación e hibridación genómica comparativa en el ADN genómico. En el trasplante se emplearon células madre del hermano menor con HLA idéntico, y previamente se hizo un acondicionamiento de intensidad reducida. La reconstitución inmunitaria después del trasplante se evaluó periódicamente con hemoleucogramas y la prueba DHR 123 en neutrófilos de sangre periférica. Resultados. El diagnóstico de la enfermedad granulomatosa crónica ligada al cromosoma X se estableció como resultado de una deleción hemicigota en la banda Xp21.1 que implicó la deleción completa del CYBB . La toma de injerto postrasplante para plaquetas y neutrófilos fue en los días 10 y 11, respectivamente. En el día 30 después del trasplante se logró la reconstitución hematológica completa y en los tres años siguientes no se observaron complicaciones ni infecciones. Conclusión. El trasplante de células madre hematopoyéticas permite la reconstitución completa de la función inmunitaria relacionada con la actividad microbicida de las células fagocíticas de pacientes con enfermedad granulomatosa crónica ligada al cromosoma X.

Introduction: Chronic granulomatous disease is a primary immunodeficiency that results from mutations in proteins of the NADPH oxidase system that affect the microbicidal activity of phagocytes. Immune reconstitution by hematopoietic stem cell transplantation is currently the only curative therapy for this disease. Objective: To describe the clinical and molecular characterization of a patient with X-linked chronic granulomatous disease and the successful immune reconstitution by means of a hematopoietic stem cell transplantation. Materials and methods: The respiratory burst was measured by flow cytometry using the dihydrorodamine 123 (DHR) oxidation test in neutrophils of peripheral blood. Mutational analysis of CYBB was performed by PCR amplification in complementary DNA, as well as sequencing and comparative genomic hybridization in genomic DNA. HLA-identical stem cells from the patient´s younger brother were used for the transplantation and reduced intensity pre-transplantation conditioning was administered. Post-transplantation immune reconstitution was evaluated periodically by serial complete blood counts and DHR 123 in peripheral blood neutrophils. Results: The diagnosis of X-linked chronic granulomatous disease resulted from a hemizygous deletion affecting Xp21.1 that included the entire CYBB . Post-transplantation engraftment was documented in platelets and peripheral blood neutrophils at days 10 and 11, respectively. Total hematological reconstitution was achieved by day 30 post-transplantation and no complications or infections have been observed in the three years since the transplantation. Conclusion: Hemopoietic stem cell transplantation allows for total reconstitution of the immune function related to microbicidal activity of phagocytic cells from patients with X-linked chronic granulomatous disease.

[Report of a new mutation in CYBB gene in two patients with X linked chronic granulomatous disease]. / Identificación de mutaciones en el gen CYBB que llevan al fenotipo de la enfermedad granulomatosa crónica ligada al cromosoma X: reporte de una nueva mutación.

BACKGROUND: The X-linked form of chronic granulomatous disease (CGD) is a primary immunodeficiency that affects phagocytes of the innate immune system and is characterized by an increased susceptibility to severe bacterial and fungal infections. It is caused by mutations in the CYBB gene, which encodes the 91-kD subunit of phagocyte NADPH oxidase. AIM: To identify the mutation in the CYBB gene in two unrelated patients from Chile with the diagnosis of X-linked CGD and their families. PATIENTS AND METHODS: The molecular genetic defects of two unrelated patients from Chile with X-linked CGD caused by defects in the CYBB gene were investigated. The underlying mutation was investigated by single strand conformation polymorphism (SSCP) analysis of PCR-amplified genomic DNA and by sequencing of the affected gene region. RESULTS: We found an insertion c.1267_1268insA in exon 10 leading to a frameshift mutation. This mutation is a novel report. We also identified a splice site mutation in the other patient, that presented a c.1326 +1 G>A substitution in intron 10. The mutation was also detectable in his heterozygous mother. CONCLUSIONS: This is the first report of the clinical and molecular characterization of Chilean patients with mutations in CYBB gene.

Chronic granulomatous disease in Latin American patients: clinical spectrum and molecular genetics.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by early onset of recurrent and severe infections. The molecular defects causing CGD are heterogeneous and lead to absence, low expression, or malfunctioning of one of the phagocyte NADPH oxidase components. The aim of this study was to analyze the clinical features and to investigate the molecular genetic defects of Latin American patients with CGD. PROCEDURES: The study included 14 patients. The diagnosis was based on a history of recurrent severe infections, impaired respiratory burst, and the demonstration of an underlying mutation by single strand conformation polymorphism (SSCP) or RT-PCR analysis, followed by genomic DNA or cDNA sequencing. RESULTS: Seven unrelated patients were found to have the X-linked form of CGD (X-CGD). Heterogeneous mutations affected the CYBB gene: two insertions, one substitution, and four splice site defects; two of them are novel. Seven patients presented with one of the autosomal recessive forms of CGD (A47-CGD); all had the most common mutation, a DeltaGT deletion in exon 2 of the NCF1 gene. Pneumonia was the most frequent clinical feature, followed by pyoderma, sinusitis, otitis, and liver abscess. Patients with X-CGD were more likely to have initial infections before age 2 years and to have inflammatory obstructive granulomas later. None of the patients had severe adverse reactions to BCG immunization. CONCLUSIONS: X-CGD patients from Latin America showed a high degree of molecular heterogeneity, including two novel mutations. Their clinical characteristics included early onset of infections and eventual obstructive granulomas. A47-CGD represented 50% of the reported cases, a higher prevalence than reported in other series.

Association of glucose-6-phosphate dehydrogenase deficiency and X-linked chronic granulomatous disease in a child with anemia and recurrent infections.

Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 +/- 2.1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus-transformed B-lymphocytes from the proband was similar to healthy controls. Molecular studies showed that the G6PD deficiency was due a 202 G-->A mutation, the A- variant common in African ethnic groups. The proband also exhibited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-->A substitution at the 3' splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of X-linked CGD. We propose that clinicians treating a patient with G6PD deficiency during a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes' microbicidal activity and the eventual association of G6PD deficiency and chronic granulomatous disease.

Identificación de mutaciones en el gen CYBB que llevan al fenotipo de la enfermedad granulomatosa crónica ligada al cromosoma X: Reporte de una nueva mutación / Report of a new mutation in CYBB gene in two patients with X linked chronic granulomatous disease

Background: The X-linked form of chronic granulomatous disease (CGD) is a primary immunodeficiency that affects phagocytes of the innate immune system and is characterized by an increased susceptibility to severe bacterial and fungal infections. It is caused by mutations in the CYBB gene, which encodes the 91-kD subunit of phagocyte NADPH oxidase. Aim: To identify the mutation in the CYBB gene in two unrelated patients from Chile with the diagnosis of X-linked CGD and their families. Patients and methods: The molecular genetic defects of two unrelated patients from Chile with X-linked CGD caused by defects in the CYBB gene were investigated. The underlying mutation was investigated by single strand conformation polymorphism (SSCP) analysis of PCR-amplified genomic DNA and by sequencing of the affected gene region. Results: We found an insertion c.1267_1268insA in exon 10 leading to a frameshift mutation. This mutation is a novel report. We also identified a splice site mutation in the other patient, that presented a c.1326 +1 G>A substitution in intron 10. The mutation was also detectable in his heterozygous mother. Conclusions: This is the first report of the clinical and molecular characterization of Chilean patients with mutations in CYBB gene.