Programme for Harmonization to the International Scale in Latin America for BCR-ABL1 quantification in CML patients: findings and recommendations.

Objectives The quantitation of BCR-ABL1 mRNA is mandatory for chronic myeloid leukemia (CML) patients, and RT-qPCR is the most extensively used method in testing laboratories worldwide. Nevertheless, substantial variation in RT-qPCR results makes inter-laboratory comparability hard. To facilitate inter-laboratory comparative assessment, an international scale (IS) for BCR-ABL1 was proposed. Methods The laboratory-specific conversion factor (CF) to the IS can be derived from the World Health Organization (WHO) genetic reference panel; however, this material is limited to the manufacturers to produce and calibrate secondary reference reagents. Therefore, we developed secondary reference calibrators, as lyophilized cellular material, aligned to the IS. Our purpose was both to re-evaluate the CF in 18 previously harmonized laboratories and to propagate the IS to new laboratories. Results Our field trial including 30 laboratories across Latin America showed that, after correction of raw BCR-ABL1/ABL1 ratios using CF, the relative mean bias was significantly reduced. We also performed a follow-up of participating laboratories by annually revalidating the process; our results support the need for continuous revalidation of CFs. All participating laboratories also received a calibrator to determine the limit of quantification (LOQ); 90% of them could reproducibly detect BCR-ABL1, indicating that these laboratories can report a consistent deep molecular response. In addition, aiming to investigate the variability of BCR-ABL1 measurements across different RNA inputs, we calculated PCR efficiency for each individual assay by using different amounts of RNA. Conclusions In conclusion, for the first time in Latin America, we have successfully organized a harmonization platform for BCR-ABL1 measurement that could be of immediate clinical benefit for monitoring the molecular response of patients in low-resource regions.

Comparative genomic analysis of Mycobacterium tuberculosis Beijing-like strains revealed specific genetic variations associated with virulence and drug resistance.

Isolates of the Mycobacterium tuberculosis lineage 2/East-Asian are considered one of the most successful strains due to their increased pathogenicity, hyper-virulence associated with drug resistance, and high transmission. Recent studies in Colombia have shown that the Beijing-like genotype is associated with multidrug-resistance and high prevalence in the southwest of the country, but the genetic basis of its success in dissemination is unknown. In contribution to this matter, we obtained the whole sequences of six genomes of clinical isolates assigned to the Beijing-like genotype. The genomes were compared with the reference genome of M. tuberculosis H37Rv and 53 previously published M. tuberculosis genomes. We found that the six Beijing-like isolates belong to a modern Beijing sub-lineage and share specific genomic variants: i.e. deletion in the PPE8 gene, in Rv3806c (ubiA) responsible of high ethambutol resistance and in Rv3862c (whiB6) which is involved in granuloma formation and virulence, are some of them. Moreover, each isolated has exclusively single nucleotide polymorphisms (SNPs) in genes related with cell wall processes and cell metabolism. We identified polymorphisms in genes related to drug resistance that could explain the drug-resistant phenotypes found in the six isolates from Colombia. We hypothesize that changes due to these genetic variations contribute to the success of these strains. Finally, we analyzed the IS6110 insertion sequences finding very low variance between them, suggesting that SNPs is the major cause of variability found in Beijing-like strains circulating in Colombia.

Genotypic Analysis of Genes Associated with Independent Resistance and Cross-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Clinical Isolates.

Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n = 57 ETH-resistant MDR-TB isolates; n = 3 ETH-susceptible MDR-TB isolates; and n = 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB.

Population structure among mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Colombia.

BACKGROUND: Phylogeographic composition of M. tuberculosis populations reveals associations between lineages and human populations that might have implications for the development of strategies to control the disease. In Latin America, lineage 4 or the Euro-American, is predominant with considerable variations among and within countries. In Colombia, although few studies from specific localities have revealed differences in M. tuberculosis populations, there are still areas of the country where this information is lacking, as is a comparison of Colombian isolates with those from the rest of the world. PRINCIPAL FINDINGS: A total of 414 M. tuberculosis isolates from adult pulmonary tuberculosis cases from three Colombian states were studied. Isolates were genotyped using IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and 24-locus Mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTRs). SIT42 (LAM9) and SIT62 (H1) represented 53.3% of isolates, followed by 8.21% SIT50 (H3), 5.07% SIT53 (T1), and 3.14% SIT727 (H1). Composite spoligotyping and 24-locus MIRU- VNTR minimum spanning tree analysis suggest a recent expansion of SIT42 and SIT62 evolved originally from SIT53 (T1). The proportion of Haarlem sublineage (44.3%) was significantly higher than that in neighboring countries. Associations were found between M. tuberculosis MDR and SIT45 (H1), as well as HIV-positive serology with SIT727 (H1) and SIT53 (T1). CONCLUSIONS: This study showed the population structure of M. tuberculosis in several regions from Colombia with a dominance of the LAM and Haarlem sublineages, particularly in two major urban settings (Medellín and Cali). Dominant spoligotypes were LAM9 (SIT 42) and Haarlem (SIT62). The proportion of the Haarlem sublineage was higher in Colombia compared to that in neighboring countries, suggesting particular conditions of co-evolution with the corresponding human population that favor the success of this sublineage.

Biosafety evaluation of the DNA extraction protocol for Mycobacterium tuberculosis complex species, as implemented at the Instituto Nacional de Salud, Colombia

Introducción. El trabajo con Mycobacterium tuberculosis se considera un factor de riesgo para el personal de laboratorio que manipula especímenes clínicos y cultivos. Uno de los procesos que requiere de una alta concentración de microorganismos es la extracción de ADN para realizar metodologías moleculares. Se han reportado casos de tuberculosis pulmonar en profesionales que realizan procedimientos moleculares en los que se requiere previa manipulación del microorganismo en masa, lo cual ha motivado la investigación sobre la bioseguridad del protocolo de extracción, sin que a la fecha haya consenso sobre los riesgos del proceso. Objetivo. Evaluar la bioseguridad del protocolo de extracción de ADN reportado por van Soolingen et al., 2002, mediante la determinación de la viabilidad de M. tuberculosis en cada etapa del proceso. Materiales y métodos. Se realizaron 880 cultivos a partir de 220 aislamientos clínicos de M. tuberculosis que se procesaron para las tres primeras fases de extracción de ADN. A los cultivos positivos se les realizó identificación molecular por PRA hsp65 y caracterización por spoligotyping. Resultados. Se obtuvo crecimiento en uno de los procedimientos realizados. Por caracterización molecular, se determinó que no correspondió al aislamiento analizado originalmente, sino que fue producto de contaminación cruzada. Conclusión. Se determinó que el protocolo de extracción de ADN descrito por van Soolingen et al. (2002) e implementado en el Instituto Nacional de Salud de Colombia, es seguro para el personal de laboratorio y el medio ambiente.

Introduction. Manipulating Mycobacterium tuberculosis clinical specimens and cultures represents a risk factor for laboratory personnel. One of the processes that requires high concentrations of microorganisms is DNA extraction for molecular procedures. Pulmonary tuberculosis cases have occurred among professionals in charge of molecular procedures that require manipulation of massive quantities of microorganisms. This has prompted research studies on biosafety aspects of extraction protocols; however, as yet, no consensus has been reached regarding risks associated with the process. Objective. The biosafety was evaluated for the DNA extraction protocol of van Soolingen, et al. 2002 by determining M. tuberculosis viability at each process stage. Materials and methods. Eight hundred eighty cultures were grown from 220 M. tuberculosis clinical isolates that had been processed through the first three DNA extraction stages. Molecular identifications of positive cultures used a PCR isolation of a fragment of the heat shock protein PRA-hsp65 and examination of its restriction enzyme profile (spoligotyping). Results. Growth was seen in one culture with one of the procedures used. The molecular characterization did not correspond to the initially analyzed isolate, and therefore was deduced to be the product of a cross-contamination. Conclusion. The DNA extraction protocol, as described by van Soolingen, et al. 2002 and as implemented at the Instituto Nacional de Salud, was established to be safe for laboratory personnel as well as for the environment.

Biosafety evaluation of the DNA extraction protocol for Mycobacterium tuberculosis complex species, as implemented at the Instituto Nacional de Salud, Colombia.

INTRODUCTION: Manipulating Mycobacterium tuberculosis clinical specimens and cultures represents a risk factor for laboratory personnel. One of the processes that requires high concentrations of microorganisms is DNA extraction for molecular procedures. Pulmonary tuberculosis cases have occurred among professionals in charge of molecular procedures that require manipulation of massive quantities of microorganisms. This has prompted research studies on biosafety aspects of extraction protocols; however, as yet, no consensus has been reached regarding risks associated with the process. OBJECTIVE: The biosafety was evaluated for the DNA extraction protocol of van Soolingen, et al. 2002 by determining M. tuberculosis viability at each process stage. MATERIALS AND METHODS: Eight hundred eighty cultures were grown from 220 M. tuberculosis clinical isolates that had been processed through the first three DNA extraction stages. Molecular identifications of positive cultures used a PCR isolation of a fragment of the heat shock protein PRA-hsp65 and examination of its restriction enzyme profile (spoligotyping). RESULTS: Growth was seen in one culture with one of the procedures used. The molecular characterization did not correspond to the initially analyzed isolate, and therefore was deduced to be the product of a cross-contamination. CONCLUSION: The DNA extraction protocol, as described by van Soolingen, et al. 2002 and as implemented at the Instituto Nacional de Salud, was established to be safe for laboratory personnel as well as for the environment.

Accessory mitral valve without left ventricular outflow tract obstruction in an adult.

Accessory mitral valve is a rare congenital abnormality and an unusual cause of subvalvular obstruction of the left ventricular outflow tract. Accessory mitral valves are usually detected in children due to symptomatic obstruction; isolated nonobstructive accessory mitral valve is rarely seen in adults. We describe the echocardiographic diagnosis of accessory mitral valve as an isolated congenital anomaly not associated with a substantial degree of obstruction of the left ventricular outflow tract in an asymptomatic adult patient. This case highlights the importance of transthoracic and transesophageal echocardiography in the diagnosis and follow-up of this uncommon congenital anomaly.

Intrapericardial primary thymic carcinoma in a 73-year-old man.

Thymic carcinoma is a rare, highly aggressive type of tumor that typically occurs in the anterior mediastinum. We describe the case of a 73-year-old man who presented with weakness, cough, dyspnea, anorexia, and weight loss. An echocardiogram showed an intrapericardial mass that occupied the space around the lateral walls of the left ventricle and distally compressed the right ventricle. Magnetic resonance imaging and a biopsy confirmed the presence of intrapericardial primary thymic carcinoma. The patient underwent surgical excision of the tumor and died of right ventricular rupture during the procedure. This case highlights the importance of considering thymic carcinoma whenever an otherwise unexplained intrapericardial mass is encountered.

Kawasaki disease in the adult: a case report and review of the literature.

Kawasaki disease, predominantly a disease of childhood, includes such symptoms as acute vasculitis, mucosal inflammation, rash, cervical adenopathy, and edema. Its most severe forms are associated with coronary artery aneurysms. We report a rare case of this disease in an asymptomatic adult and review its epidemiology, etiology, diagnosis, treatment, and prognosis.

Rare chronic gastric volvulus associated with left atrial and mediastinal compression.

We report a case of chronic gastric volvulus associated with left atrial compression in a 75-year-old woman who presented with chest pain, shortness of breath, and hypotension after elective hemiarthroplasty of the left hip. The patient's medical history included a paraesophageal hernia and gastric volvulus diagnosed in 1997 but left untreated. The present diagnosis of gastric volvulus was made on the basis of a chest radiograph and subsequent computed tomography. Echocardiography showed the volvulus compressing the left atrium. Surgery to repair the defect was successful, and there were no operative or postoperative complications. A review of the world medical literature revealed that gastric volvulus is rarely reported to cause hemodynamic compromise or compression of the heart and mediastinal structures.