[Peripheral facial palsy in a paediatric patient with no risk factors within the context of infection by SARS-CoV-2]. / Parálisis facial periférica en un paciente pediátrico sin factores de riesgo en el contexto de infección por SARS-CoV-2.

TITLE: Parálisis facial periférica en un paciente pediátrico sin factores de riesgo en el contexto de infección por SARS-CoV-2.

Th22 response induced by Mycobacterium tuberculosis strains is closely related to severity of pulmonary lesions and bacillary load in patients with multi-drug-resistant tuberculosis.

The role of interleukin-22 (IL-22) in the pathogenesis or tissue repair in human tuberculosis (TB) remains to be established. Here, we aimed to explore the ex-vivo and in-vitro T helper 22 (Th22) response in TB patients and healthy donors (HD) induced by different local multi-drug-resistant (MDR) Mvcobacterium tuberculosis (Mtb) strains. For this purpose, peripheral blood mononuclear cells from drug-susceptible (S-TB) MDR-TB patients and HD were stimulated with local MDR strains and the laboratory strain H37Rv. IL-22 and IL-17 expression and senescent status were assessed in CD4+ and CD8+ cells by flow cytometry, while IL-22 amount was measured in plasma and culture supernatants by enzyme-linked immunosorbent assay (ELISA). We found lower IL-22 amounts in plasma from TB patients than HD, together with a decrease in the number of circulating T cells expressing IL-22. In a similar manner, all Mtb strains enhanced IL-22 secretion and expanded IL-22+ cells within CD4+ and CD8+ subsets, being the highest levels detected in S-TB patients. In MDR-TB, low systemic and Mtb-induced Th22 responses associated with high sputum bacillary load and bilateralism of lung lesions, suggesting that Th22 response could be influencing the ability of MDR-TB patients to control bacillary growth and tissue damage. In addition, in MDR-TB patients we observed that the higher the percentage of IL-22+ cells, the lower the proportion of programmed cell death 1 (PD-1)+ or CD57+ T cells. Furthermore, the highest proportion of senescent T cells was associated with severe lung lesions and bacillary load. Thus, T cell senescence would markedly influence Th22 response mounted by MDR-TB patients.

Eosinofilia grave en lactante con proctocolitis alérgica / Severe eosinophilia in breast feeding infant with allergic proctocolitis

La eosinofilia en el recién nacido puede ser secundaria a múltiples etiologías. Presentamos el caso de un recién nacido que presentó eosinofilia severa, rectorragia y desmedro como manifestación de proctocolitis alérgica a proteínas de leche de vaca. Hacemos el diagnóstico diferencial de la eosinofilia en el período neonatal

Eosinophilia in newborn is caused for several causes. We presented a newborn who showed severe eosinofilia, rectorrhagia and lose weight due to allergic proctocolitis. We revised the differencial diagnosis of eosinophilia in newborn

Detection of multidrug-resistant tuberculosis from stored DNA Samples: A multicenter study.

Background: In low-income countries, rapid detection of tuberculosis (TB) drug resistance is often restricted by the difficulties of transporting and storing sputum samples from remote health centers to the reference laboratories where molecular tests are available. The aim of this study was to evaluate the performance of four transport and storage systems for molecular detection of rifampicin (RIF) and isoniazid (INH) resistance. Methods: This was a multicenter study. Molecular detection of RIF and INH resistance was performed directly from smear-positive TB sputa spotted on a slide, FTA card, GenoCard, and ethanol using the Genotype MTBDRplus assay. The performance of the DNA extraction method from each storage support to detect drug resistance was assessed by calculating their sensitivity and specificity compared to the phenotypic method. Results: From all sites, the overall sensitivity and specificity for RIF-resistance detection was 88% and 85%, respectively, for slides, 86% and 92%, respectively, for GenoCard, 87% and 89%, respectively, for FTA card, and 88% and 92%, respectively, for ethanol. For INH-resistance detection, the overall sensitivity and specificity was 82% and 90%, respectively, for slides, 85% and 96%, respectively, for GenoCard, 86% and 92%, respectively, for FTA card, and 86% and 94%, respectively, for ethanol. Conclusion: Smear slides and filter cards showed to be very useful tools to facilitate DNA extraction from sputum samples with the potential to accelerate the detection of drug resistance in remote areas.

[Presumed perinatal ischemic stroke: risk factors and clinical and radiological findings]. / Ictus isquemico presumiblemente perinatal: factores de riesgo, hallazgos clinicos y radiologicos.

INTRODUCTION: Presumed perinatal ischemic stroke is a frequent cause of neurological sequelae. We aimed to describe the different clinical findings and risk factors and to analyse the differences according the vascular origin. PATIENTS AND METHODS: Retrospective, descriptive study of patients diagnosed with presumed perinatal ischemic stroke attended at a tertiary pediatric hospital from 1990 to 2015. RESULTS: 44 patients were included. A total of 24 patients (55%) had arterial ischemic stroke and 20 (45%) had periventricular venous infarction. Delay in diagnosis was significantly higher in patients with periventricular venous infarction compared to those with arterial ischemic stroke (14 and 8 months respectively; p = 0.025). Most patients presented with asymmetrical motor development (90%), only < 5% with seizures or non motor delays. Subsequent epilepsy at follow-up was significantly more prevalent in arterial ischemic stroke group (p = 0.020). We determined risk factors theoretically involved in the pathogenesis of presumed perinatal ischemic stroke: prenatal, obstetrical, perinatal, prothrombotic and cardiac. No significant differences between risk factors and vascular origin were found. Prothrombotic abnormalities were common (48.3%). CONCLUSIONS: Investigation in risk factors implicated in presumed perinatal ischemic stroke is required to develop prevention strategies. Delay in diagnosis is higher in periventricular venous infarction group.

TITLE: Ictus isquemico presumiblemente perinatal: factores de riesgo, hallazgos clinicos y radiologicos.Introduccion. El ictus isquemico presumiblemente perinatal es una causa frecuente de secuelas neurologicas importantes. Los objetivos del estudio son describir las caracteristicas clinicas y los factores de riesgo implicados, y analizar las diferencias segun su origen vascular. Pacientes y metodos. Estudio descriptivo retrospectivo que incluye pacientes con diagnostico de ictus isquemico presumiblemente perinatal atendidos en un hospital terciario entre 1990-2015. Resultados. Se incluyeron 44 pacientes: 24 (55%) fueron de origen arterial, frente a 20 (45%) de origen venoso. El diagnostico fue significativamente mas tardio en los de origen venoso que en los de origen arterial (14 y 8 meses respectivamente; p = 0,025). La mayoria comenzo con un deficit motor (90%), y las crisis epilepticas y el retraso psicomotor global fueron menos frecuentes en ambos grupos (< 5%). La prevalencia de epilepsia posterior fue significativamente mas frecuente entre los de origen arterial (p = 0,020). Se analizaron los factores de riesgo teoricamente implicados en su patogenia: prenatales, obstetricos, perinatales, protromboticos y cardiacos, sin hallarse diferencias significativas en la presencia de estos entre los infartos arteriales y los venosos. Encontramos la presencia de al menos una alteracion en el estudio de hipercoagulabilidad en el 48,3% de los pacientes. Conclusion. Es preciso investigar el papel que desempeñan los factores de riesgo implicados en el ictus isquemico presumiblemente perinatal para establecer medidas preventivas. Su diagnostico es mas tardio si el origen es venoso.

Genetic diversity of Mycobacterium avium complex strains isolated in Argentina by MIRU-VNTR.

Mycobacterium avium sp. avium (MAA), M. avium sp. hominissuis (MAH), and M. avium sp. paratuberculosis (MAP) are the main members of the M. avium complex (MAC) causing diseases in several hosts. The aim of this study was to describe the genetic diversity of MAC isolated from different hosts. Twenty-six MAH and 61 MAP isolates were recovered from humans and cattle, respectively. GenoType CM® and IS1311-PCR were used to identify Mycobacterium species. The IS901-PCR was used to differentiate between MAH and MAA, while IS900-PCR was used to identify MAP. Genotyping was performed using a mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) scheme (loci: 292, X3, 25, 47, 3, 7, 10, 32) and patterns (INMV) were assigned according to the MAC-INMV database (http://mac-inmv.tours.inra.fr/). Twenty-two (22/26, 84·6%) MAH isolates were genotyped and 16 were grouped into the following, INMV 92, INMV 121, INMV 97, INMV 103, INMV 50, and INMV 40. The loci X3 and 25 showed the largest diversity (D: 0·5844), and the global discriminatory index (Hunter and Gaston discriminatory index, HGDI) was 0·9300. MAP (100%) isolates were grouped into INMV 1, INMV 2, INMV 11, INMV 8, and INMV 5. The HGDI was 0·6984 and loci 292 and 7 had the largest D (0·6980 and 0·5050). MAH presented a higher D when compared with MAP. The MIRU-VNTR was a useful tool to describe the genetic diversity of both MAH and MAP as well as to identify six new MAH patterns that were conveniently reported to the MAC-INMV database. It was also demonstrated that, in the geographical region studied, human MAC cases were produced by MAH as there was no MAA found among the human clinical samples.

[Language disorders in acute cerebellitis: beyond dysarthria]. / Alteraciones del lenguaje en la cerebelitis aguda: mas alla de la disartria.

INTRODUCTION: Acute cerebellitis is one of the main causes of cerebellar syndrome in infancy. Among the wide range of manifestations, headache and ataxia being the most predominant, we can find other less frequent, although nonetheless interesting, ones, such as language disorders, which go beyond the well-known cerebellar dysarthria. The different combinations in which the symptoms can appear, especially when not accompanied by ataxia, make the condition a real challenge for the clinician. CASE REPORTS: Two patients, aged 2 and 4 years, with clinical features, lab tests and neuroimaging results consistent with parainfectious acute cerebellitis. Both of them also presented a striking language disorder, one in the form of cerebellar mutism and the other in the form of hypofluency and agrammatism, the latter also developing in the absence of ataxia. Both cases progressed favourably, and mild speech alterations persisted in the follow-up visits. CONCLUSIONS: Cases such as these expand the range of clinical manifestations of acute cerebellitis. The involvement of the cerebellum in neurocognitive processes like language is becoming increasingly more important and, although many aspects are still only speculations, managing to define its true role will have important repercussions on the diagnosis, treatment and long-term prognosis of these patients.

TITLE: Alteraciones del lenguaje en la cerebelitis aguda: mas alla de la disartria.Introduccion. La cerebelitis aguda es una de las principales causas de sindrome cerebeloso en la infancia. Entre un amplio elenco de manifestaciones, en el que predominan la cefalea y la ataxia, podemos encontrar otras menos habituales, aunque interesantes, como las alteraciones del lenguaje, mas alla de la bien conocida disartria cerebelosa. Las diferentes combinaciones en que pueden aparecer los sintomas, especialmente cuando no se acompañan de ataxia, hacen de este cuadro un verdadero reto para el clinico. Casos clinicos. Se presentan dos pacientes, de 2 y 4 años, con clinica, pruebas de laboratorio y neuroimagen compatibles con cerebelitis aguda parainfecciosa, que asociaron una llamativa alteracion del lenguaje, uno en forma de mutismo cerebeloso y otro en forma de hipofluencia y agramatismo, y este ultimo cursaba ademas en ausencia de ataxia. La evolucion de ambos casos fue buena, y persistieron leves alteraciones del habla en el seguimiento posterior. Conclusiones. Casos como estos amplian el espectro de manifestaciones clinicas de la cerebelitis aguda. Cada vez cobra mayor importancia la participacion del cerebelo en procesos neurocognitivos como el lenguaje y, aunque muchos aspectos son aun especulativos, alcanzar a definir su verdadero papel tendra una repercusion en el diagnostico, el tratamiento y el pronostico a largo plazo de estos pacientes.

Molecular and phenotypic characterisation of Mycobacterium tuberculosis resistant to anti-tuberculosis drugs.

SETTING: Dr Cetrángolo Hospital, Buenos Aires, Argentina. OBJECTIVES: To characterise drug-resistant (DR), multidrug-resistant (MDR-) and extensively drug-resistant (XDR-) Mycobacterium tuberculosis isolates, and identify their genetic profiles, drug resistance levels and resistance-conferring mutations. DESIGN: Phenotypic drug susceptibility testing methods were used to determine drug resistance profiles. Minimal inhibitory concentrations (MICs) of isoniazid (INH), rifampicin (RMP) and levofloxacin (LVX) from 169 DR tuberculosis (TB) isolates, 78 of them monoresistant to INH, 13 to RMP, 7 to LVX, and 71 MDR-TB, were determined. Multiplex allele-specific polymerase chain reaction and DNA sequencing were used to detect mutations in katG, rpoB and gyrA/B genes. Genotyping was performed using spoligotyping and insertion sequence 6110 restriction fragment length polymorphism. RESULTS: In total, 38.9% of the INH-resistant (INH(R)) isolates had an MIC ≥ 32 g/ml; 61.3% of RMP-resistant (RMP(R)) isolates had an MIC ≥ 64 g/ml and 55.6% of the LVX-resistant (LVX(R)) isolates had an MIC 4 ≥ 16 g/ml. The main mutations found in INH(R) isolates were katG315 (53.7%) and inhAP-15 (25.5%), whereas in RMP(R) isolates the main mutations were rpoB531 (61.9%), followed by rpoB526 (16.7%). LVX(R) isolates showed mutations in gyrA94/90. Haarlem, LAM and T were the main spoligotyping families found. katG315 was mainly associated with Haarlem and LAM, whereas inhAP-15 was associated with T. CONCLUSIONS: Several isolates showed an association between high INH(R) levels and katG mutation; others from the Haarlem family were prone to becoming MDR-TB and continue to circulate in the community.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis by multiplex allele-specific polymerase chain reaction.

SETTING: Dr Cetrangolo Hospital, Buenos Aires Province, Argentina. OBJECTIVE: To evaluate a multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis (MDR-TB) clinical isolates and to describe the main mutations conferring resistance to isoniazid (INH) and rifampicin (RMP). DESIGN: Drug-resistant Mycobacterium tuberculosis clinical isolates were tested to detect mutations using MAS-PCR. The genes involved were katG, inhA promoter and rpoB. RESULTS: Among 193 clinical isolates included in the study, 52.6% of the INH-resistant isolates presented a mutation in the katG (315) gene, 28.1% in the inhAP (-15) and 3.0% in both. For the rpoB gene, 60% of the RMP-resistant isolates showed a mutation in codon 531, 17.5% in 526 and 2.5% in 516. Results were compared with those obtained by sequencing, and 100% concordance was obtained for the detection of the mutation in katG (315), 94.1% for inhAP (-15), and 97.8% for rpoB. The global concordance between both methods was 98%. CONCLUSIONS: The MAS-PCR system allowed the simultaneous and rapid detection of approximately 80.0% of the drug-resistant clinical isolates. This method could be used as a rapid and simple screening tool to detect drug-resistant TB in clinical practice.

Evaluation of MGIT 960 and the colorimetric-based method for tuberculosis drug susceptibility testing.

SETTING: Dr Cetrángolo Hospital, Buenos Aires Province, Argentina. OBJECTIVE: Evaluation of the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system and the colorimetric-based method (CMM) for first- and second-line drug susceptibility testing (FL-DST, SL-DST) against Mycobacterium tuberculosis. DESIGN: FL-DST was studied using SIRE MGIT 960. Minimal inhibitory concentrations (MICs) for isoniazid (INH), streptomycin, rifampicin (RMP), ethambutol (EMB) and levofloxacin (LVX) were also determined by CMM using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). MICs for amikacin (AMK), kanamycin (KM), capreomycin (CPM), ethionamide (ETH), cycloserine, ofloxacin (OFX), linezolide (LZ) and moxifloxacin (MFX) were determined on 94 multidrug-resistant M. tuberculosis isolates by MGIT 960 and CMM. Statistical methods were applied to define drug-susceptible and drug-resistant isolates on the basis of the comparison between results obtained by gold standards. RESULTS: A total of 1626 clinical isolates were studied. Critical drug concentrations could be defined in less than 10 days for both CMM and MGIT 960. CMM was cheaper but more laborious than MGIT 960. The highest performances of both methods were achieved for AMK, RMP, OFX, LZ and MFX, followed by INH, ETH, KM, CPM and LVX (tested only by CMM). CONCLUSIONS: Both methods could be implemented as rapid diagnostic tools to detect drug-resistant isolates in clinical practice.

Molecular typing of Mycobacterium bovis isolates in Argentina: first description of a person-to-person transmission case.

Bovine tuberculosis is caused by Mycobacterium bovis, a mycobacterium highly similar to M. tuberculosis that belongs to the M. tuberculosis complex. The main host of M. bovis is cattle but it also affects many other mammalians including humans. Tuberculosis in humans caused by either M. bovis or M. tuberculosis is clinically hard to distinguish. During 2004-2005, samples from 448 patients with diagnosis of TB were collected from different regions of Argentina. The PRA technique identified 400 isolates with representative patterns of mycobacterium. The predominant ones were the M. tuberculosis complex, the M. avium-M. intracellulare complex and M. gordonae. Samples with M. tuberculosis complex PRA restriction profiles were analyzed with a multiplex PCR to differentiate between M. tuberculosis and M. bovis. Multiplex PCR identified nine M. bovis. The results allowed the possibility to establish that 2% of pulmonary tuberculosis was due to M. bovis. Isolates of M. bovis from humans were examined using spoligotyping. These isolates presented five different spoligotypes. The main spoligotype was also the most frequently one found in cattle. The remaining human spoligotypes (grouped in clusters) are occasionally found in cattle. Variable number tandem repeat (VNTR) analysis identified five different patterns. By combining the results of spoligotyping and VNTR analysis, we were able to differentiate seven M. bovis isolates. The remaining two M. bovis samples showed the same spoligotype and VNTR profile and belonged to household contacts. An MDR-M. bovis was isolated from the samples of these household contacts. The identification of two epidemiologically linked cases of human M. bovis infection suggests person-to-person transmission of an MDR-M. bovis.

Tuberculosis transmission of predominant genotypes of Mycobacterium tuberculosis in northern suburbs of Buenos Aires city region.

In 2003, the incidence of tuberculosis in Argentina showed an increase compared to 2002. The severe national crisis at the end of the 90s has probably strongly contributed to this situation. The goal of this work was to estimate the extent of the spread of the most predominant Mycobacterium tuberculosis strains and to assess the spread of predominant M. tuberculosis clusters as determined by spoligotyping and IS6110 RFLP. The study involved 590 pulmonary, smear-positive TB cases receiving medical attention at health centers and hospitals in Northern Buenos Aires (NBA) suburbs, from October 2001 to December 2002. From a total of 208 clinical isolates belonging to 6 major clusters, 63 (30.2%) isolates had identical spoligotyping and IS6110 RFLP pattern. Only 22.2% were shown to have epidemiological connections with another member of their respective cluster. In these major clusters, 30.2% of the 208 TB cases studied by both molecular techniques and contact tracing could be convincingly attributable to a recently acquired infection. This knowledge may be useful to assess the clonal distribution of predominant M. tuberculosis clusters in Argentina, which may make an impact on TB control strategies.

Tuberculosis transmission of predominant genotypes of Mycobacterium tuberculosis in Northern suburbs of Buenos Aires city region / Transmisión de la tuberculosis por genotipos predominantes de Mycobacterium tuberculosis en la región Gran Buenos Aires Norte

In 2003, the incidence of tuberculosis in Argentina showed an increase compared to 2002. The severe national crisis at the end of the 90s has probably strongly contributed to this situation. The goal of this work was to estimate the extent of the spread of the most predominant Mycobacterium tuberculosis strains and to assess the spread of predominant M. tuberculosis clusters as determined by spoligotyping and IS6110 RFLP. The study involved 590 pulmonary, smear-positive TB cases receiving medical attention at health centers and hospitals in Northern Buenos Aires (NBA) suburbs, from October 2001 to December 2002. From a total of 208 clinical isolates belonging to 6 major clusters, 63 (30.2%) isolates had identical spoligotyping and IS6110 RFLP pattern. Only 22.2% were shown to have epidemiological connections with another member of their respective cluster. In these major clusters, 30.2% of the 208 TB cases studied by both molecular techniques and contact tracing could be convincingly attributable to a recently acquired infection. This knowledge may be useful to assess the clonal distribution of predominant M. tuberculosis clusters in Argentina, which may make an impact on TB control strategies.

La incidencia de la tuberculosis en Argentina mostró en 2003 un incremento en comparación con 2002. La grave crisis nacional a fines de los 90 ha probablemente contribuido en gran medida a esta situación. El objetivo del presente trabajo fue determinar la diversidad genética de aislamientos de Mycobacterium tuberculosis y el grado de dispersión de algunas cepas mayoritarias genéticamente relacionadas. El estudio involucró 590 aislamientos clínicos provenientes de muestras respiratorias con examen directo positivo, de pacientes atendidos en los hospitales y centros de salud que conforman la región Gran Buenos Aires Norte (NBA), de octubre de 2001 a diciembre de 2002. De 208 aislamientos que se encontraron en los 6 mayores clusters, 63 (30,2%) tenían patrones idénticos de spoligotyping y de IS6110 RFLP. En el 22,2% de los casos fue posible verificar la conexión epidemiológica con otro miembro del respectivo cluster. Concluimos que el 30,2% de estos agrupamientos principales pueden ser atribuidos a una infección reciente. Estos resultados pueden ser útiles para determinar la distribución clonal de los grupos predominantes de M. tuberculosis en Argentina, lo que puede impactar en las estrategias de control de la tuberculosis.

[Evaluation of a colorimetric micromethod for determining the minimal inhibitory concentration of antibiotics against Mycobacterium tuberculosis]. / Evaluación de un micrométodo colorimétrico para determinar la concentración inhibitoria mínima de drogas antituberculosas frente a Mycobacterium tuberculosis.

Multidrug-resistant tuberculosis (MDR) caused by strains resistant to both isoniazid and rifampin is now considered a serious sanitary problem worldwide. New technical tools for the early detection of these strains are urgently needed to avoid their spread within the community. We have evaluated a microplate colorimetric-based method to determine the minimal inhibitory concentration (MIC) of first-line antituberculosis drugs by using 3-(4, 5 dimethylthiazolyl 1-2 yl)-2,5 diphenyl tetrazolium bromide as a bacterial growth indicator (MTT) (M-MTT). A total of 603 clinical isolates, 507 from respiratory cases (84.1%) and 96 from non-respiratory cases (15.9%) were processed. The proportion method on a Löwenstein-Jensen medium (PM) with isoniazid (INH), 0.20 microg/ml; streptomycin (SM), 4.00 microg/ml; ethambutol (EMB), 2.00 microg/ml and rifampin (RMP), 40.00 microg/ml, was used as the gold standard. The drugs and the concentration range tested were: INH, 1.00-0.03 microg/ml; SM, 8.00-0.25 microg/ml; EMB, 32.00-1.00 microg/ml and RMP, 2.00-0.06 microg/ml. MIC results were obtained on an average of 8 days (range: 7-12). The cut-off values for each drug, calculated by the ROC curve method, were: INH, 0.25 microg/ml, RMP, 0.50 microg/ml, SM, 4.00 microg/ml and EMB, 4.00 microg/ml. Sensitivity and specificity for RMP were 100 %, while for INH, they were 97.8% and 99.5% respectively. The results obtained suggested that M-MTT is a low cost and easy to set up method that could be applied to MDR clinical diagnosis in developing countries.

Evaluación de un micrométodo colorimétrico para determinar la concentración inhibitoria mínima de drogas antituberculosas frente a Mycobacterium tuberculosis / Evaluation of a colorimetric micromethod for determining the minimal inhibitory concentration of antibiotics against Mycobacterium tuberculosis

La tuberculosis multidrogorresistente (MDR), originada por aislamientos de cepas simultáneamente resistentes a isoniacida y rifampicina, es reconocida en la actualidad como un problema sanitario mundial. Nuevas técnicas que permitan detectar en forma temprana cepas MDR son necesarias para evitar su dispersión en la comunidad. En este trabajo hemos evaluado el empleo de un micrométodo colorimétrico para determinar las concentraciones inhibitorias mínimas (CIM) de las drogas de primera línea frente a dichas cepas, usando el indicador: bromuro de 3-(4,5 dimetiltiazol-2-yl)-2,5 difeniltetrazolio (M-MTT). Junto a la cepa de referencia H37Rv se procesaron 603 aislamientos clínicos, 507 provenientes de casos pulmonares (84,1%) y 96 de extrapulmonares (15,9%). Como estándar de referencia se utilizó el método de proporciones en medio de Löwenstein-Jensen (MP), con isoniacida (INH), 0,20 µg/ml; estreptomicina (SM), 4,00 µg/ml; etambutol (EMB), 2,00 µg/ml y rifampicina (RMP), 40,00 µg/ml. Los intervalos de concentraciones de las drogas empleadas en el M-MTT fueron: INH, 1,00-0,03 µg/ml; SM, 8,00-0,25 µg/ml; EMB, 32,00-1,00 µg/ml y RMP: 2,00-0,06 µg/ml. El resultado de la CIM por el M-MTT fue obtenido en un tiempo promedio de 8 días (rango total: 7 a 12 días). Los puntos de corte para cada una de las drogas, calculados mediante el análisis de la curva ROC, fueron: INH, 0,25 µg/ml; RMP, 0,50 µg/ml; SM, 4,00 µg/ml y EMB, 4,00 µg/ml. Los valores de sensibilidad y especificidad fueron 100% en el caso de RMP; y 97,8% y 99,5%, respectivamente, para INH. El análisis estadístico de los resultados permitió concluir que el M-MTT es un método seguro para la rápida detección de MDR. Por su sencillez y bajo costo, podría ser aplicado en los países en vías de desarrollo.

Multidrug-resistant tuberculosis (MDR) caused by strains resistant to both isoniazid and rifampin is now considered a serious sanitary problem worldwide. New technical tools for the early detection of these strains are urgently needed to avoid their spread within the community. We have evaluated a microplate colorimetric-based method to determine the minimal inhibitory concentration (MIC) of first-line antituberculosis drugs by using 3-(4, 5 dimethylthiazolyl 1-2 yl)-2,5 diphenyl tetrazolium bromide as a bacterial growth indicator (MTT) (M-MTT). A total of 603 clinical isolates, 507 from respiratory cases (84.1%) and 96 from non-respiratory cases (15.9%) were processed. The proportion method on a Löwenstein-Jensen medium (PM) with isoniazid (INH), 0.20 µg/ml; streptomycin (SM), 4.00 µg/ml; ethambutol (EMB), 2.00 µg/ml and rifampin (RMP), 40.00 µg/ml, was used as the gold standard. The drugs and the concentration range tested were: INH, 1.00-0.03 µg/ml; SM, 8.00-0.25 µg/ml; EMB, 32.00-1.00 µg/ml and RMP, 2.00-0.06 µg/ml. MIC results were obtained on an average of 8 days (range: 7-12). The cut-off values for each drug, calculated by the ROC curve method, were: INH, 0.25 µg/ml, RMP, 0.50µg/ml, SM, 4.00 µg/ml and EMB, 4.00 µg/ml. Sensitivity and specificity for RMP were 100 %, while for INH, they were 97.8% and 99.5% respectively. The results obtained suggested that M-MTT is a low cost and easy to set up method that could be applied to MDR clinical diagnosis in developing countries.

Evaluation of a rapid culture method for tuberculosis diagnosis: a Latin American multi-center study.

SETTINGS: Tuberculosis (TB) diagnostic laboratories in Latin America. OBJECTIVES: Evaluation of thin-layer agar (TLA) compared to Löwenstein-Jensen (LJ) culture for the diagnosis of TB. DESIGN: Phase II prospective study in six laboratories. Samples included sputum and extra-pulmonary specimens from patients with a clinical diagnosis of TB. Respiratory samples were decontaminated using NaOH/ NALC; all samples were centrifuged, stained with Ziehl-Neelsen for acid-fast bacilli (AFB), cultured on LJ and TLA and identified according to recommended procedures. Sensitivity and likelihood ratios (LR), growth detection time and contamination rate were calculated for both media. RESULTS: A total of 1118 clinical specimens were studied. Cultures detected Mycobacterium tuberculosis in all AFB-positive samples, whereas for AFB-negative specimens LJ detected 3.2% and TLA 4.4%. Sensitivity was 92.6% (95%CI 87.9-95.9) and 84.7% (95%CI 78.8-89.0) for TLA and LJ, respectively. Positive and negative LRs were similar. Contamination was 5.1% for TLA and 3.0% for LJ. Median time to detection of a positive culture was 11.5 days (95%CI 9.3-15.0) for TLA and 30.5 days (95%CI 26.9-39.0) for LJ (P < 0.0001). CONCLUSION: Difference in the characteristics of the participating laboratories, the disease prevalence and the number and type of specimens processed did not affect the overall performance of TLA as compared to LJ, supporting the robustness of the method and its feasibility in different laboratory settings.

First description of Mycobacterium tuberculosis Beijing genotype in Argentina.

During a population-based study to genotype isolates of Mycobacterium tuberculosis from Buenos Aires Northern suburbs, we found isolates with molecular patterns related to those of the Beijing genotype. Five out of 590 (0.85%) patients had isolates with spoligopattern identical to that of the Beijing family. Since two of these isolates showed identical IS6110RFLP pattern, we found only four different patterns containing 11 to 19 bands. The isolates were obtained from young people (including a 7 years-old child) who were born in Argentina, and were living in a small area of our region. However, conventional contact tracing did not prove epidemiological linkage among them. These isolates were fully drug-susceptible to the first-line drugs. The comparison of the IS6110RFLP patterns from our isolates against a set of 19 reference Beijing patterns from the RIVM (The Netherlands) confirmed that the strains belonged to the Beijing lineage. These findings might be partially explained by the important migration phenomena occurred during the last decade. Further surveillance studies would help in the following of Beijing family strain dissemination in our community.

Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs.

OBJECTIVE: A multicentre evaluation was performed to assess two rapid low-cost methods, MTT (3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) and resazurin assays, for testing the susceptibility of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and streptomycin (SM). METHODS: Thirty coded M. tuberculosis strains were sent to seven laboratories located in Latin America, representing six countries. Each site performed the colorimetric assays, MTT and resazurin, blind for the first-line drugs RMP, INH, EMB and SM. The minimum inhibitory concentration results obtained were compared to the conventional proportion method on Lowenstein-Jensen medium. RESULTS: After establishing the breakpoint concentrations, excellent results were obtained for RMP, INH and EMB, with levels of specificity and sensitivity of between 96% and 99%. CONCLUSION: MTT and resazurin assays are promising, accessible new alternative methods for middle- and low-resource countries that need low-cost methods to perform rapid susceptibility testing of M. tuberculosis to key anti-tuberculosis drugs.

First description of Mycobacterium tuberculosis Beijing genotype in Argentina

During a population-based study to genotype isolates of Mycobacterium tuberculosis from Buenos Aires Northern suburbs, we found isolates with molecular patterns related to those of the Beijing genotype. Five out of 590 (0.85

) patients had isolates with spoligopattern identical to that of the Beijing family. Since two of these isolates showed identical IS6110RFLP pattern, we found only four different patterns containing 11 to 19 bands. The isolates were obtained from young people (including a 7 years-old child) who were born in Argentina, and were living in a small area of our region. However, conventional contact tracing did not prove epidemiological linkage among them. These isolates were fully drug-susceptible to the first-line drugs. The comparison of the IS6110RFLP patterns from our isolates against a set of 19 reference Beijing patterns from the RIVM (The Netherlands) confirmed that the strains belonged to the Beijing lineage. These findings might be partially explained by the important migration phenomena occurred during the last decade. Further surveillance studies would help in the following of Beijing family strain dissemination in our community.