¿La detección de toxinas de Clostridioides difficile es necesaria cuando se detecta la enzima glutamato deshidrogenasa? / Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

Resumen Introducción: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. Objetivo: Definir si la determinación de GDH es redundante a la de las toxinas. Métodos: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. Resultados: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. Conclusiones: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.

Abstract Introduction: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. Objective: To define if GDH determination is redundant to that of toxins. Methods: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. Results: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. Conclusions: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

Estudo epidemiológico e caracterização de coorte de pacientes com infecção por Clostridioides difficile internados no Hospital das Clínicas da UFMG / Epidemiological study and characterization of a cohort of patients with Clostridioides difficile infection hospitalized at the Hospital das Clínicas of UFMG

Introdução: a infecção por Clostridioides difficile (ICD) é a principal causa bacteriana de diarreia infecciosa associada aos cuidados de saúde e é responsável por significativa morbimortalidade, assim como por custos elevados relacionados ao tratamento em todo o mundo. Na Europa e nos Estados Unidos a densidade de incidência varia entre 2,9 e 8,3 casos/10.000 pacientes-dia. Não existem dados precisos sobre a taxa de incidência na América Latina. Objetivos: obter a medida da densidade de incidência da ICD associada aos cuidados de saúde em hospital de alta complexidade e descrever o perfil desta coorte de pacientes. Métodos: foi realizada busca ativa diária de casos de diarreia durante o período de 3 meses, entre abril e julho de 2021. Os casos suspeitos foram submetidos ao teste rápido para pesquisa da glutamato desidrogenase (GDH) e das toxinas A e B de C. difficile. Nas amostras positivas apenas para o GDH, a confirmação diagnóstica foi feita por meio da cultura toxigênica. Resultados: foram identificados 104 pacientes com diarreia e o C. difficile toxigênico foi responsável por 21 casos. A densidade de incidência foi de 9,2 casos para cada 10.000 pacientes-dia. A mediana de idade dos pacientes com ICD foi de 63 (19-80) anos, 57,1% eram do sexo masculino e a média do Índice de Comorbidades de Charlson foi de 4,10 (±2,49). Dezessete pacientes (81%) fizeram uso de antibiótico (ATB) nos 3 meses que precederam a infecção e a média do número de ATB foi de 3,29 (±2,72). A ICD foi considerada grave em 11 pacientes (52,4%). Vancomicina foi opção inicial de tratamento em 14 pacientes (66,7%), e 11 pacientes (52,4%) apresentaram resposta até o quinto dia. Dois pacientes estavam no segundo episódio de ICD e um paciente apresentou recorrência após período de recrutamento. Ocorreram três óbitos, provavelmente não relacionados à ICD. Conclusão: a medida da densidade de incidência foi alta e aponta para a necessidade de medidas que visem melhor controle da infecção. A amostra de pacientes foi caracterizada como complexa, com múltiplas comorbidades, uso recente de vários ATB e mortalidade alta.

Introduction: Clostridioides difficile (ICD) infection is the leading bacterial cause of healthcare-associated infectious diarrhea and it is responsible for significant morbidity and mortality rates, as well as treatment-related costs worldwide. In Europe and the United States, the incidence density varies between 2.9 and 8.3 cases/10,000 patient-days. There is no precise data about the incidence rate in Latin America. Objectives: get the measure of the incidence density of healthcare-related ICD in a high-complexity hospital and to define the profile of this cohort of patients. Methods: daily active search for diarrhea cases was carried out during a 3-month period, between April and July 2021. Suspected cases were submitted to a rapid test for glutamate dehydrogenase (GDH) and C. difficile toxins A and B. In samples positive only for GDH, diagnostic confirmation was made through toxigenic culture. Results: 104 patients with diarrhea were identified and toxigenic C. difficile was responsible for 21 cases. The incidence density was 9.2 cases for every 10,000 patient-days. The median age of patients with ICD was 63 (19-80) years, 57.1% were male and the mean Charlson Comorbidity Index was 4.10 (±2.49). Seventeen patients (81%) used antibiotics (ATB) in the 3 months preceding the infection and the mean number of ATB was 3.29 (±2.72). ICD was considered severe in 11 patients (52.4%). Vancomycin was the initial treatment option in 14 patients (66.7%) and 11 patients (52,4%) responded by the fifth day. Two patients were in the second episode of ICD and one patient had recurrence after the recruitment period. There were three deaths, probably unrelated to CDI. Conclusion: The measure of incidence density was high and points to the need for measures aimed at better infection control. The sample of patients was characterized as complex, with multiple comorbidities, recent use of multiple ATB and high mortality.

Laboratory Diagnosis of Clostridium difficile Infection in Korea: The First National Survey

In May 2015, we conducted a voluntary online survey on laboratory diagnostic assays for Clostridium difficile infection (CDI) across clinical microbiology laboratories in Korea. Responses were obtained from 66 laboratories, including 61 hospitals and five commercial laboratories. Among them, nine laboratories reported having not conducted CDI assays. The toxin AB enzyme immunoassay (toxin AB EIA), nucleic acid amplification test (NAAT), and C. difficile culture, alone or in combination with other assays, were used in 51 (89.5%), 37 (64.9%), and 37 (64.9%) of the remaining 57 laboratories, respectively, and 23 (40.4%) of the laboratories performed all three assays. Only one laboratory used the glutamate dehydrogenase assay. Nine laboratories used the toxin AB EIA as a stand-alone assay. The median (range) of examined specimens in one month for the toxin AB EIA, NAAT, and C. difficile culture was 160 (50–2,060), 70 (7–720), and 130 (9–750), respectively. These findings serve as valuable basic data regarding the current status of laboratory diagnosis of CDI in Korea, offering guidance for improved implementation.

Simultaneous Detection of Clostridioides difficile Glutamate Dehydrogenase and Toxin A/B: Comparison of the C. DIFF QUIK CHEK COMPLETE and RIDASCREEN Assays

Various commercial assays have recently been developed for detecting glutamate dehydrogenase (GDH) and/or toxin A/B to diagnose Clostridioides difficile infection (CDI). We compared the performance of two assays for the simultaneous detection of C. difficile GDH and toxin A/B, using 150 stool samples: C. DIFF QUIK CHEK COMPLETE (QCC; TechLab, Blacksburg, VA, USA) and RIDASCREEN Clostridium difficile GDH (RC-GDH) and Toxin A/B (RC-Toxin A/B; R-Biopharm, Darmstadt, Germany). For GDH detection, QCC and RC-GDH showed satisfactory sensitivity (95.7% and 94.3%, respectively) and specificity (92.5% and 93.8%, respectively) compared with C. difficile culture. For toxin A/B detection, QCC showed higher sensitivity than RC-Toxin A/B (60.0% vs 33.3%, P < 0.001) compared with toxigenic C. difficile culture. When the results of QCC or RC-GDH+RC-Toxin A/B were used as the first step of a two-step algorithm for diagnosing CDI, QCC permitted more accurate discrimination than RC of positive or negative results for CDI (77.3% and 65.3%, respectively). QCC is useful for the simultaneous detection of C. difficile GDH and toxin A/B as a part of the two-step algorithm for diagnosing CDI.

Correlation of glutamate dehydrogenase with several tumors / 生物工程学报

Most organisms contain glutamate dehydrogenase (E.C. 1.4.1.2-1.4.1.4). In eukaryotes, the enzyme is mainly present in mitochondria. This enzyme plays a vital role in the metabolism of nitrogen and carbon and the signaling pathway. Studies have found that glutamate dehydrogenase has a certain relationship with the occurrence and development of tumors, which is significant for tumor research, but reviews on its relationship with human tumors are rare. This review summarized the relationship between glutamate dehydrogenase and breast cancer, glioma, colorectal cancer and ovarian cancer, etc, thus providing assistance for related research.

Glutamato deshidrogenasa: Su valor diagnóstico en la diarrea por Clostridioides difficile / Glutamate dehydrogenase: Its diagnostic value in Clostridioides difficile diarrhea

Clostridioides difficile es el principal agente causal de diarreas asociadas al cuidado de la salud. Esta bacteria produce toxinas y una enzima que se encuentra muy conservada en la especie: la glutamato deshidrogenasa (GDH). El diagnóstico rápido y el tratamiento efectivo permiten la pronta mejoría del paciente, con el consecuente control de la diseminación del microorganismo. Sin embargo, aún no se cuenta con un método diagnóstico óptimo y se propone la realización de diversas pruebas, cuyos resultados se interpretan en el contexto de ciertos algoritmos. En este trabajo se evaluó el desempeño de la GDH como prueba de tamizaje en el diagnóstico de la diarrea por c. difficile. Se estudiaron 615 muestras de materia fecal. Se determinó la presencia de GDH y de toxinas mediante el equipo diagnóstico de enzimoinmunoensayo de membrana C. DIFF QUIK-CHEK COMPLETE® (TECHLAB) y se realizaron cultivos para la búsqueda de C. difficile. Se calcularon los valores de sensibilidad, especificidad, VPP y VPN con un nivel de significación p < 0,05. Se detectó GDH en 266 muestras (43,25%), con una sensibilidad del 100% y una especificidad del 87,10%, IC95: 84,58-91,42. Se hallaron toxinas en 218 muestras (35,45%) y C. difficile desarrolló en 235 cultivos (38,21%). De 48 muestras GDH positivas y sin producción de toxina/s, 17 fueron positivas al cultivo de C. difficile, con 15 aislamientos toxigénicos y 2 no toxigénicos. No hubo desarrollo de C. difficile en las 31 muestras restantes. Ninguna muestra GDH negativa dio resultado positivo de toxina/s ni desarrollo en el cultivo, por lo cual el VPN de la GDH fue del 100%, mientras que el VPP fue del 81,9%. Concluimos que la determinación de GDH representa un screening adecuado para descartar casos de diarrea por C. difficile, por lo tanto de valor en los algoritmos diagnósticos de las diarreas infecciosas.

Clostridioides difficile is the main etiological agent of diarrhea associated with health care, it produces toxins and glutamate dehydrogenase (GDH), an enzyme that is highly conserved in this species. Rapid diagnosis and effective treatment produce prompt improvement of the patient and subsequent control of the microorganism spread. There are several techniques whose results are interpreted in the context of algorithms. However, the optimal diagnostic method is yet unknown. The performance of GDH as a screening test for the diagnosis of C. difficile diarrhea was assessed. Six hundred and fifteen stool samples were studied. The presence of GDH and toxins presence was determined by TECHLAB® C. DIFF QUIK-CHEK COMPLETE and the samples were cultured for the search of C. difficile. The values of sensitivity, specificity, PPV and NPV were calculated with a p value of 0.05 or less. GDH was detected in 266 samples (43.25%), with a sensitivity of 100% and specificity of 87.10%, IC95: 84.58-91.42; toxin/s were detected in 218 (35.45%) and C. difficile developed in 235 cultures (38.21%). From 48 samples with positive GDH and negative toxin/s, 15 toxigenic and 2 non-toxigenic isolates were obtained, the remaining 31 samples were negative for C. difficile. All GDH-negative samples were negative for toxins or culture, therefore, GDH NPV was 100%, while PPV was 81.9%. We conclude that GDH is a suitable screening test for the diagnostic algorithm of C. difficile diarrhea.

Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

A thermophilic microorganism from Deception Island, Antarctica with a thermostable glutamate dehydrogenase activity

BACKGROUND: The Antarctic continent is a source of extreme microorganisms. Millions of years of isolation have produced unique biodiversity with adaptive responses to its extreme environment. Although the Antarctic climate is mainly cold, the presence of several geothermal sites, including thermal springs, fumaroles, hot soils and hydrothermal vents, provides ideal environments for the development of thermophilic and hyperthermophilic microorganisms. Their enzymes, called thermoenzymes, are the focus of interest in both academic and industrial research, mainly due to their high thermal activity and stability. Glutamate dehydrogenase, is an enzyme that plays a key role in the metabolism of carbon and nitrogen catalyzing reversibly the oxidative deamination of glutamate to alpha-ketoglutarate and ammonium. It belongs to the family of oxidoreductases, is widely distributed and it has been highly regarded for use as biosensors, particularly for their specificity and ability to operate in photochemical and electrochemical systems. However, the use of enzymes as biosensors is relatively problematic due to their instability to high temperatures, organic solvents and denaturing agents. The purpose of this study is to present the partial characterization of a thermophilic microorganism isolated from Deception Island, Antarctica, that displays glutamate dehydrogenase activity. RESULTS: In this work, we report the isolation of a thermophilic microorganism called PID15 from samples of Deception Island collected during the Antarctic Scientific Expedition ECA 46. This microorganism is a thermophile that grows optimally at 50 °C and pH 8.0. Scanning electron microscopy shows rod cells of 2.0 to 8.0 µm of length. Phylogenetic analysis of 16S rRNA gene revealed that this microorganism is closely related to Bacillus gelatini. This microorganism contains a thermostable glutamate dehydrogenase with optimal activity at pH 8.0 and temperatures for its activity from 37 to 50 °C, range of temperature of interest for biotechnological applications. This glutamate dehydrogenase is a highly thermostable enzyme. CONCLUSION: This is the first report of a microorganism from Antarctica containing a thermostable glutamate dehydrogenase that maintains its activity in a broad range of temperatures making it of potential interest for biotechnological applications.

Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci

ABSTRACT Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

Genotipos de Giardia duodenalis en muestras de niños de las guarderías del Instituto Colombiano de Bienestar Familiar y de perros en Ibagué, Colombia / Giardia duodenalis genotypes found in the Instituto Colombiano de Bienestar Familiar day care centers and dogs in Ibagué, Colombia

Introducción. Se han descrito ocho genotipos de Giardia duodenalis, del A al H. Los genotipos A y B se han aislado de humanos y de una gran variedad de mamíferos; sin embargo, los genotipos del C al H han mostrado mayor especificidad de huésped. Objetivo. Identificar los genotipos de G. duodenalis a partir de quistes obtenidos en heces de niños de las guarderías del Instituto Colombiano de Bienestar Familiar (ICBF) y de perros en Ibagué, mediante PCR-RFLP de los genes de la beta giardina y la glutamato deshidrogenasa. Materiales y métodos. Los quistes de las muestras positivas para G. duodenalis fueron sometidos a concentración; se extrajo su ADN y se efectuó el análisis de PCR-RFLP de los genes de la beta giardina y de la glutamato deshidrogenasa. Como control positivo se utilizó la cepa MHOM/CO/04/G40 procedente del Grupo de Parasitología del Instituto Nacional de Salud. Resultados. De las muestras tomadas de niños, 11/23 (48 %) correspondieron al genotipo A y, 12/23 (52 %), al genotipo B. Cuatro muestras de perros presentaron los genotipos C y D, específicos de este huésped. Conclusiones. En los niños solamente se encontraron los genotipos asociados a infecciones humanas (AII, BIII y BIV) y en los perros, los genotipos específicos para este huésped (C y D). Debido al reducido tamaño de las muestras analizadas provenientes de perros, y dado que estos no estuvieron en contacto con los niños de las guarderías del ICBF, no fue posible determinar una interacción entre el ciclo de transmisión de los humanos y el de los animales.

Introduction: Eight Giardia duodenalis genotypes (A-H) have been described to date. Genotypes A and B have been isolated from humans and a wide range of mammals; however, genotypes C-H have shown greater host specificity. Objective: Identifying G. duodenalis genotypes from cysts in faeces obtained from children attending the Instituto Colombiano de Bienestar Familiar (ICBF) day care centres and from dogs in Ibagué by PCR-RFLP targeting both the b -giardin and glutamate dehydrogenase genes. Materials and methods: Cysts from G. duodenalis positive samples were concentrated, DNA was extracted and the b -giardin and glutamate dehydrogenase genes were analysed by PCR-RFLP. The MHOM/CO/04/G40 strain was used as positive control (this was obtained from the Grupo de Parasitología at the Instituto Nacional de Salud ). Results: Of the total human samples, 11/23 (48%) were genotyped as A and 12/23 (52%) as B; PCR-RFLP revealed that four canine samples were genotypes C and D, these being host-specific. Conclusions: Only genotypes associated with human infection (AII, BIII and BIV) were found in the children and host-specific genotypes were observed in canines (C and D). No interaction could be established between animal and human transmission cycles due to the small canine sample size and as the former did not come into contact with children attending ICBF day-care centres.

Incidence of giardia lamblia subspecies by PCR-RFLP in stool specimens of hospitalized children at Urmia Mutahhari hospital, West Azerbaijan province, Iran

Giardia lamblia is one of the most prevalent intestinal flagellate protozoa that infects a wide range of vertebrate hosts causing severe intestinal disorder in children. This study was performed to determine subspecies of G.lamblia by the PCRRFLP method, targeting the glutamate dehydrogenase[gdh]locus, in hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province,Iran and determining the infection transformational storages in this area. Overall,720 stool specimens were collected from the hospitalized children, 34 samples were positive and Giardia cysts were detected under the microscope. Cysts were partially purified by the sucrose density gradient method and then washed with sterile distilled water to remove effectively the PCR inhibitors. Genomic DNA of G. lamblia isolates was extracted by freeze-thaw cycles followed by phenol/ chloroform/ isoamyl alcohol method. The single step PCR-RFLP assay was used to differentiate the assemblages between A and B, which were found in humans. In this method, 432 bp expected size was amplified, and then for detection of subspecies, specific restriction RsaI and BspLI enzymes were used. Totally 34 samples were positive in terms of Giardia cyst out of 720 examined samples microscopically, so the parasite spread rate is reported 4.72%. Analysis PCR-RFLP on these samples revealed that 28 samples [93.3%] have the genotype BIII and 2 samples [6.7%] belong to the subgroup BIV. PCR-RFLP is a proper analytical method for determining the genotype among parasite types, using the glutamate dehydrogenizes zone's genes. Based on the results, an animal origin of infection cycle is suggested

Genotyping of Giardia duodenalis Isolates from Dogs in Guangdong, China Based on Multi-Locus Sequence

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.

Evaluation of a Rapid Membrane Enzyme Immunoassay for the Simultaneous Detection of Glutamate Dehydrogenase and Toxin for the Diagnosis of Clostridium difficile Infection

We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMerieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome due to a mutation in the glutamate dehydrogenase gene / Síndrome de hiperinsulinemia/hiperamonemia devido a uma mutação no gene da glutamato desidrogenase

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a rare autosomal dominant disease manifested by hypoglycemic symptoms triggered by fasting or high-protein meals, and by elevated serum ammonia. HI/HA is the second most common cause of hyperinsulinemic hypoglycemia of infancy, and it is caused by activating mutations in GLUD1, the gene that encodes mitochondrial enzyme glutamate dehydrogenase (GDH). Biochemical evaluation, as well as direct sequencing of exons and exon-intron boundary regions of the GLUD1 gene, were performed in a 6-year old female patient presenting fasting hypoglycemia and hyperammonemia. The patient was found to be heterozygous for one de novo missense mutation (c.1491A>G; p.Il497Met) previously reported in a Japanese patient. Treatment with diazoxide 100 mg/day promoted complete resolution of the hypoglycemic episodes. Arq Bras Endocrinol Metab. 2012;56(8):485-9.

A síndrome de hiperinsulinemia/hiperamonemia (HI/HA) é uma condição rara, de herança autossômica dominante, que se manifesta por sintomas de hipoglicemia desencadeada por jejum ou refeições de alto conteúdo proteico, juntamente com elevação da concentração de amônia sérica. HI/HA é a segunda causa de hipoglicemia hiperinsulinêmica da infância e é causada por mutações ativadoras no GLUD1, o gene que codifica a enzima mitocondrial glutamato desidrogenase (GDH). A avaliação bioquímica, bem como o sequenciamento direto dos éxons e junções éxon-íntron do gene GLUD1, foi realizada em uma paciente de 6 anos de idade com hipoglicemia de jejum e hiperamonemia. A paciente apresentava uma mutação de novo missense (c.1491A>G; p.Il497Met) em heterozigose, que havia sido previamente relatada em um paciente japonês. O tratamento com diazóxido 100 mg/dia promoveu resolução completa dos episódios hipoglicêmicos. Arq Bras Endocrinol Metab. 2012;56(8):485-9.

A Case of Hyperinsulinism/Hyperammonemia (HI/HA) Syndrome due to a Mutation in the Glutamate Dehydrogenase Gene (GLUD1) / 대한소아내분비학회지

Hyperinsulinism/Hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism (CHI) caused by a mutation in the GLUD1 gene. It is characterized by hyperinsulinemic hypoglycemia accompanying hyperammonemia. A 6-month-old male infant presented with seizure caused by fasting-induced hypoglycemia. At the time of seizure, the serum glucose and ammonia levels were 17 mg/dL and 203 micron mol/L, respectively. Even though he was fed as usual, his blood glucose level reduced to below 50 mg/dL with an increased plasma insulin level. He was thought to have hyperinsulinemic hypoglycemia associated with hyperammonemia. Analysis of the GLUD1 gene revealed a heterozygous c.1337G>A (p.Gly446Asp) mutation. He was administered diazoxide, following which his blood glucose levels were maintained within the normal range. Because HI/HA syndrome is a diazoxide-responsive form of CHI, early detection and appropriate management are important to prevent brain injury. Since patients with HI/HA syndrome may have neurological complications such as developmental delay, and cognitive impairment, careful and repeated neurologic evaluation is needed.

Comparison of Simultaneous Use of C. DIFF QUIK CHEK and VIDAS C. difficile Toxin A&B to detect C. difficile in Fecal Specimen / 임상검사와정도관리

BACKGROUND: Clostridium difficile is the most common pathogen of antibiotic-associated diarrhea. Toxigenic strains produce toxin A and toxin B. The pathogenicity of C. difficile is due to the production of these two exotoxins. This study aimed to evaluate diagnostic value of two enzyme immunoassay by comparison of concordance rate to diagnose C. difficile-associated infection. METHODS: C. DIFF QUIK CHEK (TECHLAB, USA) that detect glutamate dehydrogenase antigen and VIDAS C. difficile Toxin A&B (BioMerieux, France) that detect toxin A and toxin B were done in 122 fecal specimens to detect C. difficile. RESULTS: In the total 122 stool specimens, 17 cases showed positive results in both tests. One specimen showed discrepancy that positive result in VIDAS C. difficile Toxin A&B (relative fluorescence value, RFV=2.93) but negative result in C. DIFF QUIK CHEK. Therefore, the concordance rate between two tests was 95.1% (116/122). Both anaerobic culture and in-house PCR for toxin B were negative in the discrepant fecal specimen and there was no clinical evidence that support C. difficile-associated diarrhea, so we concluded result in VIDAS C. difficile Toxin A&B as false positive. CONCLUSIONS: Although these two enzyme immunoassays targeted different antigen, they showed high concordance rate. The discrepant case was concluded to false positive in VIDAS C. difficile Toxin A&B test because it showed negative results in culture and PCR for toxin B and there were no clinical evidences of C. difficile-associated infection. It could be needed for analysis about conditions that cause false positive result in enzyme immunoassays to detect C. difficile toxin.

The Changes in the Expression of gamma-Aminobutyric Acid Related Enzymes in the Mouse Hippocampus Following Ketogenic Diet / 대한해부학회지

The ketogenic diet (KD) has been used to treat intractable childhood epilepsy. However, its mechanism of action remains unknown. In the present study, we examined the effects of KD on the expression of multiple constituents of the GABAergic system in the hippocampus through immunohistochemistry and northern blot analysis. From the results, we have shown that KD increased expression of GABA and decreased GABA transporter1 (GABATp) and GABA transaminase (GABA-T) mRNA levels in the hippocampus. These results suggest that the neuroinhibitory effect of KD may be mediated, at least in part, by the increment of GABAergic activity in the hippocampus. KD may increase the GABA levels in the synaptic space by limiting GABA reuptake and in the presynaptic nerve terminal by inhibiting GABA degradation.