La derivación protocolizada a endoscopía asociada a la detección de Helicobacter pylori mediante antígeno en deposiciones disminuye lista de espera para endoscopía y optimiza la detección de lesiones pre-malignas y cáncer gástrico incipiente / Protocolized referral to endoscopy and Helicobacter pylori detected in stools aimed to decrease endoscopy waiting lists

Background Chile has one of the highest mortality rates by gastric cancer (GC) worldwide. Primary prevention of GC and detection of pre-neoplastic and early neoplastic lesions should be a national priority. Aim To assess the impact of the protocolization of endoscopy referral and the use of H. pylori stool antigen test (HPSA) in the management of dyspepsia to decrease the waiting list for endoscopy and increase the detection of gastric pre-neoplastic and early neoplastic lesions. Material and Methods We included all patients referred to the Endoscopy Unit of a regional hospital, from January 2015 to December 2017. We also included patients with known pre-neoplastic lesions and all those with first degree relatives with GC. We implemented protocols for referral of patients with dyspepsia considering the use of HPSA test, prioritizing to endoscopy those with a higher risk of GC. Results A total of 4,641 endoscopies and 2,631 HPSA tests were carried out. After the adoption of these protocols, we observed a 52% decrease in the waiting time for endoscopy. The GC detection rate in this period was 1.8 to 3.1 cases per 100 endoscopies. After the adoption of the protocols, we observed a significant increase in early GC detection rate (from none in 2015 to 13% in 2017, p = 0.03). Conclusions The protocolization of the referral for endoscopy associated with widespread use of HPSA test in the management of patients with dyspepsia, are successful strategies to decrease waiting lists for endoscopy and optimize the detection rate of pre-neoplastic lesions and early GC.

Detección y expresión de superantígenos y de resistencia antimicrobiana en aislamientos obtenidos de mujeres portadoras de Staphylococcus aureus que cuidan y alimentan niños / Detection and expression of superantigens and resistance in isolates from women carriers of Staphylococcus aureus who take care of and feed children

Resumen Introducción. Staphylococcus aureus coloniza mucosas y piel, y causa graves infecciones en el hombre y los animales. Es importante establecer el estatus de portadoras de cepas enterotoxigénicas de este microorganismo en manipuladoras de alimentos, con el fin de prevenir intoxicaciones alimentarias. Objetivo. Establecer las correlaciones entre los genes de enterotoxinas clásicas, el gen tsst-1, la producción de toxinas en cultivo y la resistencia antimicrobiana en aislamientos de S. aureus provenientes de manipuladoras de alimentos que cuidan niños en sus comunidades. Materiales y métodos. Se cultivaron muestras de las fosas nasales y las yemas de los dedos de las manos, y se identificó S. aureus empleando las pruebas de rutina y métodos automatizados. La extracción de ADN se hizo mediante el método de bromuro de cetil-trimetil-amonio (Cetyl-Trimethyl- Ammonium Bromide, CTAB) modificado. Para la detección de superantígenos se emplearon pruebas de reacción en cadena de la polimerasa (PCR) simple y múltiple, y para la de toxinas, estuches comerciales. Resultados. Se encontró que el 22,0 % de los aislamientos correspondía a portadoras de S. aureus: 17,0 % en los aislamientos de fosas nasales; 5,0 % en los de las manos y 6,7 % simultáneamente en los dos sitios. La prevalencia de superantígenos fue de 73,7 %. El genotipo más frecuente fue el sea-tsst-1, con 10,0 %. La resistencia a un solo antibiótico fue de 74,7 % y, a cuatro antibióticos, de 3,2 %; de los aislamientos, el 93,7 % correspondía a cepas productoras de betalactamasas. La detección de genes clásicos y de tsst-1 mediante PCR fue de 48,4 % y la de toxinas en el sobrenadante, de 42,1 %, con una correlación de 95,7 %. Las mayores correlaciones se establecieron entre las toxinas TSST-1 (22/22) y SEA (17/18). La correlación del gen tsst-1 con la proteína y la resistencia fue de 100 %. Todos los aislamientos con el genotipo sea-tsst-1 t fueron resistentes y productores de las toxinas. Conclusión. La tasa de aislamientos de S. aureus toxigénicos y resistentes obtenidos de mujeres que cuidan y preparan alimentos para niños fue de más de 70 %, lo que demostró su gran virulencia y la consecuente necesidad de aplicar estrictamente las normas higiénicas y sanitarias vigentes para evitar el riesgo de intoxicación alimentaria.

Abstract Introduction: Staphylococcus aureus colonizes mucous membranes and skin causing severe infections in humans and animals. It is important to determine carrier status of enterotoxigenic strains of this microorganism in food handlers to prevent food poisoning. Objective: To establish the correlations among classic enterotoxigenic genes, tsst-1 gene, the production of toxins in cultures and antimicrobial resistance in S. aureus isolates from women who handle the food, feed and take care of children in their communities. Materials and methods: Nasal swab and finger samples were cultured and S. aureus was identified using routine methods and automated systems. DNA extraction was done by the CTAB modified method, and superantigen detection by simple and multiplex PCR, while toxins were detected using commercial kits. Results: We found that 22.0% of subjects were S. aureus carriers: 17.0% corresponded to nose samples, 5.0% to hands and 6.7% to both nose and hands. The prevalence of superantigens was 73.7%. The most frequent genotype was sea-tsst-1 with 10%. Resistance to one antibiotic was 74.7%, and to four antibiotics, 3.2%; 93.7% of the isolates were betalactamase-positive. Classical genes and tsst-1 gene were detected by PCR in 48.4% of samples and toxins in supernatant were detected in 42.1% of them with 95.7% of correlation.The highest correlations were established for TSST-1 and SEA with 100% and 94.4%, respectively. The correlation of tsst-1 gene with toxin production and resistance was 100%. All isolates with genotype sea-tsst-1 were toxin-positive and resistant. Conclusion: The rate of toxigenic and resistant S. aureus isolates from women in charge of feeding and taking care of children was higher than 70%, which demonstrates its high virulence. This requires the strict application of hygienic and sanitary regulations in order to avoid the risk of food poisoning.

Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

Salivary anti-PGL-1 IgM may indicate active transmission of Mycobacterium leprae among young people under 16 years of age

Abstract Considering that the main route of Mycobacterium leprae transmission is the upper respiratory tract, detection of salivary antibodies can be a useful tool for diagnosing early infection. The study aimed to analyze salivary anti-PGL-1 IgA and IgM antibodies in 169 children aged 4-16 years old, who lived nearby or inside the house of multibacillary or paucibacillary leprosy patients in two endemic cities in Alagoas State - Brazil. Salivary anti-PGL-1 antibodies were quantified by modified ELISA method. The frequency of contact and clinical form of the index case were significantly associated with salivary antibody levels. High frequency of IgM positivity strongly suggests active transmission of M. leprae in these communities. We suggest in the present work that salivary anti-PGL IgA and IgM are important biomarkers to be used for identifying communities with probable active transmission of M. leprae.

Comparisons of IgA response in saliva and colostrum against oral streptococci species

Abstract The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies’ samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.

Is the immunocromatographic fecal antigen test effective for primary diagnosis of helicobacter pylori infection in dyspeptic patients? / Teste imunocromatográfico do antígeno fecal: efetivo no diagnóstico primário da infecção por Helicobacter pylori em pacientes dispépticos?

ABSTRACT Background The diagnosis of H. pylori infection can be performed by non-invasive and invasive methods.The identification through a fecal antigen test is a non-invasive, simple, and relatively inexpensive test. Objective To determine the diagnostic performance of fecal antigen test in the identification of H. pylori infection. Methods H. pylori antigens were identified in the stools of dyspeptic patients undergoing upper gastrointestinal endoscopy. For the identification of H. pylori antigen, we use ImmunoCard STAT! HpSA with immunochromatography technique. Histopathology plus urease test were the gold standard. Results We studied 163 patients, 51% male, mean age of 56.7± 8.5years. H. pylori infection was present in 49%. Fecal test presented: sensitivity 67.5% (CI95% 60.6-72.9); specificity 85.5% (CI95% 78.9-90.7); positive predictive value 81.8% (CI95% 73.4-88.4) and negative predictive value 73,2% (CI95% 67.5-77.6); Positive likelihood ratio was 4.7 (CI95% 2.9-7.9) and Negative Likelihood Ratio 0.4 (CI95% 0.3-0.5). The prevalence odds ratio for a positive test was 12.3 (CI95% 5.7-26.3).The index kappa between FAT and histology/urease test was 0.53 (CI95% 0.39-0.64). Conclusion Immunochromatographic FAT is less expensive than the other methods and readily accepted by the patients but its diagnostic performance does not recommend its use in the primary diagnosis, when the patient may have an active infection.

RESUMO Contexto O diagnóstico da infecção por Helicobacter pylori (H. pylori) pode ser realizado por métodos invasivos e não invasivos. A identificação através do teste do antígeno fecal é um método não invasivo, simples, fácil e relativamente barato. Objetivo Determinar o desempenho diagnóstico do teste fecal imunocromatográfico na identificação da infecção pelo H. pylori. Métodos A pesquisa de antígenos fecais do H. pylori foi realizada através do ImmunoCard STAT! HpSA em pacientes dispépticos submetidos à endoscopia digestiva alta com coleta de biópsias para histopatologia e teste da urease, utilizados como padrão ouro. Resultados Foram estudados 163 pacientes, 51% do sexo masculino, com idade média de 56,7± 8,5 anos. A infecção por H. pylori esteve presente em 49%. O teste fecal apresentou o seguinte desempenho diagnóstico: sensibilidade 67,5% (IC95% 60,6-72,9), especificidade 85,5% (IC95% 78,9-90,7), valor preditivo positivo 81,8% (IC95% 73,4-88,4) e valor preditivo negativo 73,2% (IC95% 67,5-77,6). A razão de probabilidade positiva foi 4,7 (IC95% 2,9-7,9) e a razão de probabilidade negativa foi 0,4 (IC95% 0,3-0,5). A razão de chances de prevalência para teste fecal positivo foi 12,3 (IC95% 5,7-26,3). O índice kappa para a concordância do teste fecal com histologia/teste da urease foi 0,53 (IC95% 0,39-0,64) Conclusão O teste fecal imunocromatográfico apresenta baixo custo e é facilmente aceito pelos pacientes, no entanto seu desempenho diagnóstico não o recomenda para diagnóstico primário.

Investigation of Helicobacter pylori antigen in stool samples of patients with upper gastrointestinal complaints

Abstract Helicobacter pylori infection is usually acquired in early childhood and it can persist throughout life without antibiotic treatment. This study aimed to compare the accuracy of the noninvasive H. pylori Stool Antigen Test-applied on the stool samples with the invasive gold standart Rapid Urease Test-applied on the gastric biopy samples of patients with upper gastrointestinal complaints. After endoscopy, biopsy and stool specimens were taken in 122 patients. The infection was detected with rapid urease test which is accepted as gold standart test. Rapid, one-step H. pylori card test was applied to all patients stool specimens. In this study 106 of the 122 patients (86.8%) were positive for H. pylori infection, while 16 of the 122 patients (13.2%) were negative. H. pylori card test was negative in 13 of the 16 patients and was positive in 98 of the 106. The sensitivity, specifity, positive and negative predictive values were 92.45%, 81.25%, 97.02%, and 61.90%, respectively. H. pylori card test is rapid, easy, noninvasive and inexpensive methods for detection H. pylori infection. This test showed high sensitivity and specificity. Additionally, it may be a good alternative to invasive tests for the detection of H. pylori infections especially in children.

Correlations between the CagA Antigen and Serum Levels of Anti-Helicobacter pylori IgG and IgA in Children

We tested correlations between anti-Helicobacter pylori IgG and IgA levels and the urease test, anti-CagA protein antibody, degree of gastritis, and age. In total, 509 children (0-15 years) were enrolled. Subjects were stratified as 0-4 years (n = 132), 5-9 years (n = 274), and 10-15 years (n = 103) and subjected to the urease test, histopathology, ELISA, and western blot using whole-cell lysates of H. pylori strain 51. The positivity rate in the urease test (P = 0.003), the degree of chronic gastritis (P = 0.021), and H. pylori infiltration (P < 0.001) increased with age. The median titer for anti-H. pylori IgG was 732.5 IU/mL at 0-4 years, 689.0 IU/mL at 5-9 years, and 966.0 IU/mL at 10-15 years (P < 0.001); the median titer for anti-H. pylori IgA was 61.0 IU/mL at 0-4 years, 63.5 IU/mL at 5-9 years, and 75.0 IU/mL at 10-15 years (P < 0.001). The CagA-positivity rate was 26.5% at 0-4 years, 36.5% at 5-9 years, and 46.6% at 10-15 years for IgG (P = 0.036), and 11.3% at 0-4 years, 18.6% at 5-9 years, and 23.3% at 10-15 years for IgA (P < 0.001). Anti-H. pylori IgG and IgA titers increased with the urease test grade, chronic gastritis degree, active gastritis, and H. pylori infiltration. Presence of CagA-positivity is well correlated with a high urease test grade and high anti-H. pylori IgG/IgA levels.

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

PURPOSE: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7x10(4) CFU/mL and 2.0x10(6) CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Diagnosis of Helicobacter pylori infection by invasive and noninvasive tests

Although several invasive and noninvasive tests have been developed for the diagnosis of Helicobacter pylori infection, all of the tests have their limitations. We conducted a study to investigate and compare the suitability of rapid urease test (RUT), serology, histopathology and stool antigen tests with polymerase chain reaction (PCR) for detection of H. pylori, and correlate the diagnostic methods with PCR. Eighty nine patients (61 adults, 28 children) referred to the Firoozgar Hospital and Children Medical Center Hospital for diagnostic upper gastrointestinal endoscopy entered to the study and noninvasive tests such as immunoassay for serological antibodies against H. pylori and detection of its antigen in feces were measured. The biopsies were utilized for histological examination, RUT and PCR. The H. pylori statuses were evaluated by the positivity of ureC PCR in biopsy specimens and 53 subjects had H. pylori positive result. Histopathology showed high overall performance in adults and children with sensitivity and specificity 100% and 90%, respectively. Sensitivity, specificity, and accuracy for stool antigen test were 87.8%, 75% and 82%, respectively. Correlation of RUT, serology (IgG), histopathology and stool antigen tests with PCR were 0.82, 0.32, 0.91 and 0.63, respectively. In conclusion, the RUT and histopathology are as accurate as the PCR of biopsy and stool antigen test can consider as appropriate noninvasive test for detection of H. pylori infection.

Impacto do uso da prova rápida para estreptococo beta-hemolítico do grupo A no diagnóstico e tratamento da faringotonsilite aguda em pronto-socorro de Pediatria / Impact of the rapid antigen detection test in diagnosis and treatment of acute pharyngotonsillitis in a Pediatric emergency room / Impacto del uso de la prueba rápida para estreptococos beta-hemolíticos del grupo A en el diagnóstico y tratamiento de la faringotonsilitis en emergencia de Pediatría

OBJETIVO: Avaliar o impacto da realização rotineira da prova rápida para pesquisa de estreptococo do grupo A no diagnóstico e tratamento da faringotonsilite aguda em crianças. MÉTODOS: Estudo prospectivo e observacional que contou com a utilização de protocolo de pesquisa estabelecido na Unidade de Emergência do Hospital Universitário da Universidade de São Paulo para o atendimento de crianças e adolescentes com faringotonsilite aguda. RESULTADOS: Com base na avaliação clínica, dos 650 pacientes estudados, antimicrobianos seriam prescritos para 389 indivíduos (59,8%) e, com o uso da pesquisa de estreptococo do grupo A, foram prescritos em 286 pacientes (44,0%). Das 261 crianças que não receberiam antibiótico pelo quadro clínico, 111 (42,5%) tiveram pesquisa de estreptococo do grupo A positiva. O diagnóstico baseado no quadro clínico apresentou sensibilidade de 61,1%, especificidade de 47,7%, valor preditivo positivo de 44,9% e valor preditivo negativo de 57,5%. CONCLUSÕES: O diagnóstico clínico da faringotonsilite estreptocócica mostrou baixa sensibilidade e especificidade. O uso rotineiro da prova rápida para pesquisa de estreptococo permitiu a redução do uso de antibióticos e a identificação de um grupo de risco para as complicações da infecção estreptocócica, pois 42,5% dos pacientes com prova rápida positiva não receberiam antibióticos, se levado em consideração apenas o diagnóstico clínico.

OBJECTIVE: To evaluate the impact of the routine use of rapid antigen detection test in the diagnosis and treatment of acute pharyngotonsillitis in children. METHODS: This is a prospective and observational study, with a protocol compliance design established at the Emergency Unit of the University Hospital of Universidade de São Paulo for the care of children and adolescents diagnosed with acute pharyngitis. RESULTS: 650 children and adolescents were enrolled. Based on clinical findings, antibiotics would be prescribed for 389 patients (59.8%); using the rapid antigen detection test, they were prescribed for 286 patients (44.0%). Among the 261 children who would not have received antibiotics based on the clinical evaluation, 111 (42.5%) had positive rapid antigen detection test. The diagnosis based only on clinical evaluation showed 61.1% sensitivity, 47.7% specificity, 44.9% positive predictive value, and 57.5% negative predictive value. CONCLUSIONS: The clinical diagnosis of streptococcal pharyngotonsillitis had low sensitivity and specificity. The routine use of rapid antigen detection test led to the reduction of antibiotic use and the identification of a risk group for complications of streptococcal infection, since 42.5% positive rapid antigen detection test patients would not have received antibiotics based only on clinical diagnosis.

OBJETIVO: Evaluar el impacto de la realización de rutina de la prueba rápida para investigación de estreptococos del grupo A en el diagnóstico y tratamiento de la faringotonsilitis aguda en niños. MÉTODOS: Estudio prospectivo y observacional que contó con el uso de protocolo de investigación establecido en la Unidad de Emergencia del Hospital Universitario de la USP para la atención a niños y adolescentes con faringotonsilitis aguda. RESULTADOS: Con base en la evaluación crítica, de los 650 pacientes estudiados, antimicrobianos serían prescritos a 389 individuos (59,8%) y, con el uso de la investigación de estreptococos del grupo A se los prescribieron a 286 pacientes (44,0%). De los 261 niños que no recibirían antibióticos por el cuadro clínico, 111 (42,5%) tuvieron investigación de estreptococos del grupo A positiva. El diagnóstico basado en el cuadro clínico presentó sensibilidad del 61,1%, especificidad del 47,7%, valor predictivo positivo del 44,9% y valor predictivo negativo del 57,5%, CONCLUSIONES: En este estudio, el diagnóstico clínico de la faringotonsilitis estreptocócica mostró baja sensibilidad y especificidad. El uso de rutina de la prueba rápida para investigación de estreptococos permitió la reducción del uso de antibióticos y la identificación de un grupo de riesgo para las complicaciones de la infección estreptocócica, pues el 42,5% de los pacientes con prueba rápida positiva no recibirían antibióticos si se llevara en consideración solamente el diagnóstico clínico.

Evaluation of an immunochromatographic test for discrimination between Mycobacterium tuberculosis complex & non tuberculous mycobacteria in clinical isolates from extra-pulmonary tuberculosis.

Background & objectives: Accurate diagnosis of tuberculosis (TB) is crucial to facilitate early treatment of the patients, and to reduce its spread. Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non tuberculous mycobacteria (NTM) may or may not be the same, but the treatment regimen is always different for both the infections. Differentiation between MTBC and NTM by routine laboratory methods is time consuming and cumbersome. This study was aimed to evaluate an immunochromatographic test (ICT), based on mouse monoclonal anti-MPT64, for simple and rapid discrimination between MTBC and NTM in clinical isolates from extra-pulmonary tuberculosis cases. Methods: A total of 800 clinical samples were collected from patients suspected to have extra-pulmonary tuberculosis. Preliminary diagnosis has been done by direct Ziehl–Neelsen (ZN) staining followed by culture in BACTEC system. A total of 150 clinical isolates, which were found positive in BD 460 TB system during September 2009 to September 2010 were selected for the screening by ICT test. p-nitro-α-acetylamino- β-hydroxy propiophenone (NAP) test was performed for differentiation of MTBC and NTM. M. tuberculosis complex was further confirmed by IS6110 PCR of BACTEC culture positive isolates, this served as the reference method for MTBC identification and comparative evaluation of the ICT kit. Results: Of the 150 BACTEC culture positive isolates tested by ICT kit, 101 (67.3%) were found positive for MTBC and remaining 49 (32.7%) were considered as NTM. These results were further confirmed by IS6110 PCR that served as the reference method for detection of MTBC. H37Rv reference strain was taken as a control for ICT test and IS6110 PCR. The reference strain showed the presence of MPT64 antigen band in the ICT test. Similar bands were formed in 101 of 102 MTBC isolates tested, proving 99.1 per cent sensitivity and no bands were detected in 48 (100%) NTM isolates tested, proving 100 per cent specificity of the ICT kit. Interpretation & conclusions: Our findings show that ICT test can be used on direct culture positive specimens. It does not require any special equipment, is simple and less time consuming. It can easily discriminate between MTBC and NTM and thus can help in appropriate management of tuberculosis.

Immunoasssay chromatographic antigen test for rapid diagnosis of Group A beta hemolytic Streptococcus pharyngitis in children: a cross/ sectional study

Group A beta-hemolytic streptococcus [GABHS] is an important pharyngotonsillitis etiologic agent in children. The objective of this study was diagnosis of streptococcal pharyngitis based on rapid antigen detection test and conventional pharyngeal culture. The rapid GABHS antigen detection test was compared to culture on blood agar, the gold standard for the diagnosis of this etiologic agent. Streptococcal antigen was detected in pharyngeal specimens of 34.5% of cases by rapid strip test. We detected group A Streptococcus in 17.2% of pharyngeal culture. There was no agreement between two methods [PV < 0.1]. The negative pharyngeal culture results are probably due to antibiotic usage in 43.2% of patients. Positive rapid test results in pharyngeal swab was age dependent [P < 0.05]. There was good correlation between observing the [petechia in pharynx of patients] and positive rapid test in pharyngeal swab [P < 0.004]. Throat culture results were relatated to previous antibiotic usage [P < 0.03]. The rapid test in pharyngeal swab is helpful for rapid diagnosis and treatment of GABHS pharyngitis. Diagnosis of GABHS pharyngitis based on soley clinical findings is misleading in the majority of cases. Petechia observed in pharynx of the cases was highly predictive of streptococcal pharyngitis

Estudo da mucosa nasal de contatos de hanseníase, com positividade para o antígeno glicolipídio fenólico 1 / Nasal mucosa study of leprosy contacts with positive serology for the phenolic glycolipid 1 antigen

A hanseníase é uma doença infecciosa de evolução crônica causada pelo Mycobacterium leprae que acomete com maior frequência a mucosa nasal. Esse acometimento independe da forma clínica da doença e pode ocorrer mesmo antes do aparecimento de lesões na pele ou em outras partes do corpo. Faz-se necessário a vigilância epidemiológica dos contatos de casos novos de hanseníase para o diagnóstico precoce da doença. OBJETIVOS: Identificar lesões específicas e precoces de hanseníase por meio de exame endoscópico, baciloscópico, histopatológico e da reação em cadeia da polimerase em Tempo Real da mucosa das cavidades nasais dos contatos domiciliares e peridomiciliares com sorologia positiva para o antígeno glicolipídio fenólico. MATERIAL E MÉTODOS: Estudo prospectivo transversal em 31 contatos de pacientes de hanseníase com sorologia positiva (PGL-1), 05 controles negativos e 01 positivo no período de 2003 a 2006. RESULTADOS: Entre os contatos soropositivos a PCR-RT foi positiva para a presença de DNA de M. leprae em 06 (19,35 por cento) destes e o maior número de cópias do genoma do bacilo foi encontrado no contato que adoeceu. CONCLUSÃO: Isoladamente os exames da mucosa nasal não permitiram o diagnóstico precoce da hanseníase, mas com a combinação de vários métodos, o exame dos contatos pôde ajudar na identificação da infecção subclínica e monitoramento daqueles que poderiam ter papel importante na transmissão da doença.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. The disease more frequently affects the nasal mucosa and can occur independently of its clinical form or even before lesions on the skin or on other parts of the body. It is necessary to employ epidemiological surveillance of household contacts with new leprosy cases for early disease diagnosis. AIM: identify specific and early leprosy lesions through endoscopic, baciloscopy, histopathology exams, and real time polymerase chain reaction of the nasal cavity mucosa on household and peridomiciliary contacts with positive serology for the phenolic glycolipid 1 antigen. METHODOLOGY: Between 2003 at 2006 there was a prospective cross-sectional clinical study with 31 contacts with patients with leprosy with positive serology against PGL-1, 05 negative controls and 01 positive control. RESULTS: Between seropositive contacts, real-time PCR was positive for M. leprae DNA in 06 (19.35 percent) of them and the higher number of genome copies were found in contacts who became sick. CONCLUSION: Nasal mucosa tests alone did not enable the early diagnosis of Leprosy. However, through the combination of various methods, tests on the contacts can help identify subclinical infection and monitor the contacts that could be responsible for spreading the disease.

Validation of a rapid stool antigen test for diagnosis of Helicobacter pylori infection / Validação de teste rápido de antígeno fecal para diagnóstico de infecção por Helicobacter pylori

The aim of this study was to validate the rapid lateral flow Helicobacter pylori stool antigen test (One step H. pylori antigen test, ACON laboratories, San Diego, USA; Prime diagnostics, São Paulo), using 13C-Urea Breath Test as the gold standard for H. pylori infection diagnosis. A total of 98 consecutive patients, asymptomatic or dyspeptic, entered the study. Sixty-nine were women, with a mean age of 45.76 ± 14.59 years (14 to 79 years). In the H. pylori-positive group, the rapid stool antigen test detected H. pylori antigen in 44 of the 50 positive patients (sensitivity 88 percent; 95 percent CI: 75.7-95.5 percent), and six false-negative; and in the H. pylori-negative group 42 presented negative results (specificity 87.5 percent; 95 percent CI: 74.7-95.3 percent), and six false-positive, showing a substantial agreement (Kappa Index = 0.75; p < 0.0001; 95 percent CI: 0.6-0.9). Forty four of fifty patients that had positive stool antigen were H. pylori-positive, the PPV of the stool antigen test was 88 percent (95 percent CI: 75.7-95.5 percent), and 42 patients with negative stool antigen test were H. pylori-negative, the NPV of the stool antigen test was 87.5 percent (95 percent CI: 74.7-95.3 percent). We conclude that the lateral flow stool antigen test can be used as an alternative to breath test for H. pylori infection diagnosis especially in developing countries.

O objetivo desse trabalho foi avaliar o teste rápido de antígeno de H. pylori nas fezes (One step H. pylori antigen test, ACON laboratories, San Diego, USA; Prime diagnostics, São Paulo), usando teste respiratório com uréia marcada com 13C (TRU-13C), como padrão ouro. Noventa e oito pacientes assintomáticos ou com dispepsia participaram do estudo. Sessenta e nove eram mulheres; a média de idade dos pacientes foi de 45.76 ± 14.59 (14 a 79 anos). No grupo H. pylori positivo, o teste rápido detectou antígenos de H. pylori nas fezes em 44 dos 50 pacientes positivos (sensibilidade de 88 por cento; 95 por cento IC: 75.7-95.5 por cento), com seis falso-negativos; e no grupo H. pylori negativo, 42 apresentaram resultados negativos (especificidade de 87,5 por cento; 95 por cento IC: 74.7-95.3 por cento), com seis falso-positivos, mostrando concordância substancial (índice Kappa = 0.75; p < 0.0001; 95 por cento IC: 0.6-0.9). Quarenta e quatro dos 50 que tiveram teste de antígeno fecal positivo eram H. pylori positivos, sendo o VPP do teste 88 por cento (95 por cento IC: 75.7-95.5 por cento), e 42 pacientes com teste de antígeno fecal negativo eram H. pylori negativos, com VPN de 87,5 por cento (95 por cento IC: 74.7-95.3 por cento). Concluímos que o teste de antígeno fecal imunocromatográfico pode ser usado como alternativa ao teste respiratório para diagnóstico de infecção pelo H. pylori, principalmente em países em desenvolvimento.

Neumonía grave de la comunidad (NGC), 145 casos internados en terapia intensiva / Severe community acquired pneumonia (SCAP), 145 cases admitted to the intensive care unit

Objetivos: analizar la epidemiología, mortalidad y factores asociados a la presencia de neumonía grave de la comunidad (NGC). Métodos: análisis de datos de pacientes internados por neumonía durante 6 años. Resultados: 145/687 (21.1%) pacientes internados por Neumonìa Aguda de la Comunidad (NAC) tenían NGC, 71 eran varones; la edad media fue 71 ± 16; 85 recibieron asistencia respiratoria mecánica (ARM); en 67 se determinó la etiología (S. pneumoniae 46.3%, P. aeruginosa 12.8%; S. aureus 11.5%, polimicrobiana 26.9%); la mortalidad global fue 45.5%. Se relacionaron significativamente con la mortalidad: la ARM, score de Glasgow £ 14, PaO2/FIO2 < 250, patógeno gram-negativo (no Haemophilus), S. aureus, infección polimicrobiana, tensión arterial sistólica < 90 mmHg, derrame pleural, y frecuencia respiratoria > 30/min y se relacionó negativamente un patógeno atípico (excluyendo Legionella). En el análisis multivariado solo permanecieron relacionados con la mortalidad los primeros 4 factores arriba mencionados; los odds ratio y los intervalos de confianza (IC 5-95%) fueron respectivamente: 6.04 (5.16 - 6.91); 2.30 (1.49 - 3.11); 2.64 (1.73 - 3.55); 4.49 (3.08 - 5.89). Pacientes con bajos scores del índice de gravedad de neumonía (PSI) y CURB-65 fueron internados en la UTI y mostraron una mortalidad mayor a la observada en los que se internaron en una sala general. La internación en la UTI luego de las primeras 24 horas (tardíamente) mostró una tendencia a mayor mortalidad. La mayoría de los pacientes internados en forma temprana y todos los internados tardíamente cumplían los criterios de las normas de la ATS sobre NGC. Discusión: La NGC tiene alta mortalidad y epidemiología diferente. Es conocido que el tratamiento debe ser efectivo y precoz teniendo en cuenta los patógenos probables. El examen clínico, los gases en sangre y la radiografía permiten identificar al ingreso unmayor riesgo de muerte. Muchos de los hallazgos habitualmente considerados ...

Objectives: to analyze the epidemiology, mortality rate and associated risk factors in SCAP. Methods: secondary analysis from the data of the patients admitted for Community-Acquired Pneumonia (CAP) during the last 6 years. Results: 145/687 (21.1%) patients admitted for CAP had SCAP; there were 71 males; mean age was 71 ± 16; 85 patients received mechanical ventilation (MV); in 67 the etiology was identified (S. pneumoniae 46.3%, P. aeruginosa 12.8%; S. aureus 11.5%,polimicrobial 26.9%); mortality rate was 45.5%. Variables significantly associated with mortality were: MV, Glasgow coma score £ 14, PaO2/FIO2 < 250, a gram-negative pathogen(excluding Haemophilus), S. aureus, polimicrobial etiology, systolic arterial pressure < 90 mmHg, pleural effusion; while it was negatively associated with the presence of an atypical pathogen (excluding Legionella). In the multivariate analysis only the 4 firstabove mentioned factors remained related with mortality; the odds ratios and confidence intervals (CI 5-95%) were respectively: 6.04 (5.16 - 6.91); 2.30 (1.49 - 3.11); 2.64 (1.73 - 3.55); 4.49 (3.08 - 5.89). Some patients with low class pneumonia severity indexand CURB-65 scores who were admitted into the ICU, presented a higher mortality rate than that observed in those admitted into a general ward. ICU admission after the first 24 hours was associated with a trend towards higher mortality rate. All those patients admitted late met the severity criteria recommended by the ATS guidelines. Discussion: SCAP has high mortality rate and a different epidemiology. It is well known that therapy should be administered early and addressed to be effective against the probable pathogens. Clinical exam, blood gases and chest X-ray help to recognize agreater risk of death. A number of facts commonly considered to predict mortality were not confirmed in this study.

Do Archaea and bacteria co-infection have a role in the pathogenesis of chronic chagasic cardiopathy?

Chronic cardiopathy (CC) in Chagas disease is a fibrotic myocarditis with C5b-9 complement deposition. Mycoplasma and Chlamydia may interfere with the complement response. Proteolytic enzymes and archaeal genes that have been described in Trypanosoma cruzi may increase its virulence. Here we tested the hypothesis that different ratios of Mycoplasma, Chlamydia and archaeal organisms, which are frequent symbionts, may be associated with chagasic clinical forms. MATERIALS AND METHODS: eight indeterminate form (IF) and 20 CC chagasic endomyocardial biopsies were submitted to in situ hybridization, electron and immunoelectron microscopy and PCR techniques for detection of Mycoplasma pneumoniae (MP), Chlamydia pneumoniae(CP), C5b-9 and archaeal-like bodies. RESULTS: MP and CP-DNA were always present at lower levels in CC than in IF (p < 0.001) and were correlated with each other only in CC. Electron microscopy revealed Mycoplasma, Chlamydia and two types of archaeal-like bodies. One had electron dense lipid content (EDL) and was mainly present in IF. The other had electron lucent content (ELC) and was mainly present in CC. In this group, ELC correlated negatively with the other microbes and EDL and positively with C5b-9. The CC group was positive for Archaea and T. cruzi DNA. In conclusion, different amounts of Mycoplasma, Chlamydia and archaeal organisms may be implicated in complement activation and may have a role in Chagas disease outcome.

Bacterial meningitis: rapid diagnosis and microbial profile: a multicentered study.

Meningitis continues to be a formidable illness with high morbidity and mortality among children in India. The present study was undertaken to prospectively look for the prevalence of pyogenic meningitis at Gulbarga and to find out the utility of gram stain, Latex Agglutination Test and (LTA) and C-reactive protein in the rapid diagnosis of pyogenic meningitis from children. Over a 48-months period, 535 children with a presumptive clinical diagnosis of acute bacterial meningitis were investigated by direct microscopy, CRP, bacterial culture, latex agglutination test (L TA), cell count and cell type and biochemical tests. Latex Agglutination Test (LA T) was done for detection of the antigens of Streptococcus pneumoniae, Group B Streptococci, E. coli, Neisseria meningitidis and Haemophilus influenzae type b. Among 535 suspected meningitis cases, 291 cases were diagnosed as pyogenic meningitis cases based on biochemical tests, cell count and cell type. Out of 291 cases, 55 cases have already received antibiotic treatment. Among 236 cases of untreated pyogenic meningitis cases, 199 CSF samples were culture positive. Streptococcus pneumoniae (44.7%) was the predominant organism identified, followed by H influenzae (25.6%) and Gp. B. Streptococci (9.5%). 208 of 236 cases were gram-stain positive, 129 cases had elevated CSF-CRP and 214 cases were diagnosed as pyogenic meningitis by the detection of bacterial antigens by latex agglutination test. Among 55 pretreated cases, only 05 (9.1%) CSF samples were culture positive, bacteria was observed in 36 gram stain smear, CRP was elevated in 16 CSF samples and 52 pretreated cases of suspected meningitis were diagnosed as pyogenic meningitis by latex agglutination test for detection of bacterial antigens. Many of the bacterial isolates were sensitive to gentamicin, cefotaxime and ceftriaxone and least sensitive to tetracycline and gentamicin. 13.1% of gram-negative bacilli were ESBL producers. To conclude, inclusion of latex agglutination test for detection of bacterial antigen in the routine diagnosis adds a valuable adjunct in the rapid and accurate diagnosis of pyogenic meningitis.