Publications by authors named "Nora Morcillo"

28 Publications

  • Page 1 of 1

Effect of the deletion of lprG and p55 genes in the K10 strain of Mycobacterium avium subspecies paratuberculosis.

Res Vet Sci 2021 Sep 28;138:1-10. Epub 2021 May 28.

Instituto de Agrobiotecnología y Biología Molecular (IABIMO), INTA-CONICET, Hurlingham, Buenos Aires 1686, Argentina.

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.
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http://dx.doi.org/10.1016/j.rvsc.2021.05.019DOI Listing
September 2021

Surveillance and characterization of drug-resistant isolated in a reference hospital from Argentina during 8 years' period.

Int J Mycobacteriol 2019 Jul-Sep;8(3):223-228

Tuberculosis and Mycobacterioses Laboratory, Dr. Cetrángolo Hospital, Vicente López, Argentina.

Background: Argentina is considered a country with a middle tuberculosis (TB) incidence. However, according to the last national epidemiological report released in 2018, since 2013, the trends are steadily increasing. The aims of this study were to determine the drug-resistance (DR), multi-DR and extensively DR (MDR/XDR-TB), and rifampicin resistance (RIF-R) burden as a part of the local TB diagnosis (June 2010-August 2018); to detect the mutations associated to isoniazid (INH) and RIF-R and their geographical distribution; and to analyze the lineage relationship among the genetic patterns of the isolates circulating in the community.

Methods: Respiratory and extrapulmonary specimens were processed by Ziehl-Neelsen stain and cultured on specific media. Drug-susceptibility testing of isolates was performed by the MGIT 960 and a colorimetric micro-method. Mutations conferring DR were detected by Genotype and DNA sequencing.

Results: The study showed a DR-TB prevalence of approximately 20% of the isolated strains, while M/XDR-TB-and particularly RIF-R-affected more than 5.0% of the total amount of cases. DR geographical distribution revealed isolates carrying mutations in the inhA gene promoter region only constrained to three districts where it was also registered two same family relatives' cases with the infrequent rpoB S522 L/Q mutation. The fact that most DR/MDR-TB isolates were not grouped in genetic clusters suggested that these cases may mostly have occurred due to endogenous reactivation rather than recently transmission.

Conclusion: According to the obtained results, it would be convenient, in highly MDR-TB suspected individuals, to confirm phenotypically, the INH and RIF susceptibility detected by molecular tests.
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http://dx.doi.org/10.4103/ijmy.ijmy_94_19DOI Listing
January 2020

Exploring the "Latin American Mediterranean" family and the RD lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela.

BMC Microbiol 2019 06 13;19(1):131. Epub 2019 Jun 13.

Laboratório de Biologia Molecular aplicada às Micobactérias, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, 21045-900, Brazil.

Background: The Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RD) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RD genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C SNP and the regions of difference RD and RD174. IS6110-RFLP results were also used when available.

Results: Genotyping of 413 M. tuberculosis isolates from three Latin-American countries detected LAM (46%) and the ill-defined T clade (16%) as the most frequent families. The highest clustering rate was detected in the sample population from the city of Caracas in Venezuela. We observed considerable differences in the presence of the RD lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C, MIRU02-MIRU40 signature) of the RD lineage were essentially confirmed. For the LAM family, the most polymorphic loci were MIRU40, MIRU31, MIRU10, MIRU26, MIRU16 and the least polymorphic MIRU24, MIRU20, MIRU04, MIRU23.

Conclusions: Our results suggest a differential adaptation of LAM-sublineages in neighboring populations and that RD strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RD) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.
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http://dx.doi.org/10.1186/s12866-019-1479-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567603PMC
June 2019

Crystal violet decolorization assay for rapid detection of multidrug-resistant isolates: A multicenter study.

Int J Mycobacteriol 2018 Oct-Dec;7(4):310-314

Mycobacteria Service, INEI ANLIS "Dr. Carlos G. Malbran," Buenos Aires, Argentina.

Background: Effective control of tuberculosis is achieved by early diagnosis and drug susceptibility testing for initiation of appropriate treatment. The performance of crystal violet decolorization assay (CVDA) for susceptibility testing of Mycobacterium tuberculosis to isoniazid (INH) and rifampicin (RIF) was compared in a multicenter study.

Methods: Seventy-two M. tuberculosis isolates were tested in two phases by CVDA.

Results: In Phase I, the specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and agreement for INH were 100%, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 98.2%, 100%, 94.1%, 100%, and 98.6%, respectively. In Phase II, specificity, sensitivity, PPV, NPV, and agreement were 98%, 100%, 95.4%, 100%, and 98.6% for INH, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 96.3%, 88.2%, 88.2%, 96.3%, and 94.4%, respectively. Results in the study were obtained on average 10.9 ± 3.1 days in Phase I and 9.8 ± 2.2 days in Phase II.

Conclusion: CVDA can be performed for drug susceptibility testing in developed and developing countries. In addition, further studies with larger sample size are needed for evaluation of this method.
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http://dx.doi.org/10.4103/ijmy.ijmy_141_18DOI Listing
June 2019

String test: A new tool for tuberculosis diagnosis and drug-resistance detection in children.

Int J Mycobacteriol 2018 Apr-Jun;7(2):162-166

Université catholique de Louvain (UCL), Institute of Experimental and Clinical Research, Laboratory of Medical Microbiology, Brussels, Belgium.

Background: There is a critical need to improve the diagnostic accuracy of tuberculosis (TB) in children. Several techniques have been developed to improve the quality of sputum samples; however, these procedures are very unpleasant and invasive and require hospitalization and trained personnel. This study aims to explore the potential use of a new and noninvasive tool, "string test," for TB diagnosis in children and in adults not able to render sputum samples and at risk of developing multidrug-resistant TB (MDR-TB).

Methods: Children with clinical suspicion of TB attending the pediatric consultation at the Cetrangolo or Cordero Hospitals and adults suspected of MDR-TB and unable to produce sputum attending the Infectious Disease Unit of Cetrangolo Hospital were included in this study.

Subjects And Methods: The "string test" is a string that is swallowed by the patients and exposed to gastrointestinal secretions that were late analyzed for TB diagnosis and drug-resistance detection by GenoType MTBDRplus. MedCalc software was used to perform statistical analysis.

Results: This technique could be applied on 62.1% of selected children. About 11 (30.6%) children were diagnosed as TB cases, 8 (22.2%) from gastric aspirate and using the "string test." Six out of 19 adults were also diagnosed. Genotype directly on the string specimen detected two MDR-TB in adults and two isoniazid-resistant cases before obtaining the isolate.

Conclusion: This test was safe, cheap, and easily implemented without requiring hospitalization. This research could represent a significant step forward to diagnose and rapidly detect drug-resistant TB in children.
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http://dx.doi.org/10.4103/ijmy.ijmy_54_18DOI Listing
March 2019

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates.

Rev Argent Microbiol 2017 Oct - Dec;49(4):332-338. Epub 2017 Sep 15.

Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr. Cetrangolo Hospital, Italia 1750, Florida 1602, Buenos Aires Province, Argentina.

Background: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments.

Methods: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method.

Results: MZ and IPZ showed bacteriostatic activity against M. tuberculosis strains. MIC and MIC to ECO was 4.0μg/ml, while MIC to CLO was 4.0μg/ml and MIC was 8.0μg/ml respectively.

Conclusion: All azole compounds tested in the study showed inhibitory activity against MDR M. tuberculosis clinical isolates.
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http://dx.doi.org/10.1016/j.ram.2017.02.008DOI Listing
February 2019

Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis.

PLoS One 2015 2;10(10):e0139382. Epub 2015 Oct 2.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.

Background: Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus.

Methods: Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing.

Results: For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%.

Conclusion: The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139382PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591989PMC
June 2016

Fitness of drug resistant Mycobacterium tuberculosis and the impact on the transmission among household contacts.

Tuberculosis (Edinb) 2014 Dec 15;94(6):672-7. Epub 2014 Aug 15.

Biotechnology Institute, National Institute of Agricultural Technology (INTA), Hurlingham, Buenos Aires Province, Argentina. Electronic address:

There has been an on-going debate on whether the development of drug resistance in Mycobacterium tuberculosis reduces its relative fitness and its ability to cause disease. The aim of this study was to explore this relationship. For this purpose, we evaluated the in vitro growth of clinical isolates and the transmission of the strains within the patients' households. Clinical and epidemiological data from patients in households, drug-susceptibility and genetic patterns of the isolates were collected. BACTEC MGIT 960™ system with the Epicenter™ software was used to perform fitness experiments and calculate the relative fitness (RF) comparing with the H73Rv reference strain. From 39 households, 124 patients and 388 contacts were included. Concerning transmission, 20 Multi drug-resistant (MDR) and 16 drug sensitive (DS) index cases generated 23 and 28 secondary cases, respectively. An average RF drop of 16.7% was found for MDR strains, but only mutations in rpoB codons 531 were associated with reduced fitness. When the strains were transmitted, their RF tended to decrease, and strains with low RF were less frequently transmitted. Within the limitations of this study, the results showed that the decrease in RF was associated to a limited transmission among the households' contacts.
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http://dx.doi.org/10.1016/j.tube.2014.08.003DOI Listing
December 2014

Characterization of a Mycobacterium avium subsp. avium operon associated with virulence and drug detoxification.

Biomed Res Int 2014 20;2014:809585. Epub 2014 May 20.

Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria, Hurlingham, Buenos Aires 1686, Argentina.

The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo.
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http://dx.doi.org/10.1155/2014/809585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055363PMC
February 2015

Evaluation of Mycobacterium tuberculosis cross-resistance to isoniazid, rifampicin and levofloxacin with their respective structural analogs.

J Antibiot (Tokyo) 2014 Nov 4;67(11):749-54. Epub 2014 Jun 4.

Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr Cetrángolo Hospital, Florida, Buenos Aires Province, Argentina.

The emergence of drug-resistant, multidrug-resistant and extensively drug-resistant tuberculosis (TB) is of major public health concern in several countries. In this study, the pharmacodynamic relationships among the structural analogs of antibiotics belonging to the same family were taken into consideration. The aim of this study was to compare the susceptibility of Mycobacterium tuberculosis to isoniazid (INH), rifampicin and levofloxacin (LX) to their respective structural analogs, which are frequently used as second-line agents. The microplate colorimetric method was used to determine the MIC to INH, ethionamide (ETH), rifampicin, rifabutin, LX and moxifloxacin (MOX) in clinical isolates previously shown to be drug resistant. Mutations conferring drug resistance were detected by GenoType MTBDR plus and DNA sequencing. INH and ETH cross-resistance was found in 95.12% (39/41) of the INH-resistant isolates harboring a mutation in inhAP or inhA open reading frame, but rifabutin cross-resistance was observed in 90.0% (63/70) of the clinical isolates originally shown to be resistant to rifampicin. Isolates with high LX-resistance levels also showed high MIC to MOX. Fluoroquinolone cross-resistance was verified in isolates containing the gyrA94 and the gyrA90 mutation. In general, isolates with high INH, rifampicin and LX-resistance levels also displayed high MIC values for their structural analogs. These findings suggest the need to test in vitro the second-line drugs before their incorporation in the therapeutic schemes.
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http://dx.doi.org/10.1038/ja.2014.61DOI Listing
November 2014

Predictive value of direct nitrate reductase assay and its clinical performance in the detection of multi- and extensively drug-resistant tuberculosis.

J Med Microbiol 2014 Apr 20;63(Pt 4):522-527. Epub 2014 Jan 20.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, K.L Ledeganckstraat 35, B-9000 Gent, Belgium.

Conventional culture and drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and time consuming. For this reason alternative rapid culture and DST techniques are urgently needed to shorten the time for drug-resistance detection. A total of 222 smear-positive sputum samples were evaluated by the direct nitrate reductase assay (D-NRA) on Lowenstein-Jensen medium, for the rapid and simultaneous detection of resistance to isoniazid, rifampicin, kanamycin and ofloxacin. p-Nitrobenzoic acid was also included for identification of the M. tuberculosis complex. Results were compared with the BACTEC MGIT 960 as gold standard. The general performance of the D-NRA was very good, reaching a global value of 97 %. D-NRA had a turn-around time of 16.9 days to obtain results while that of the indirect MGIT 960 system was 29 days. D-NRA is a low-cost technology, easy to set up in clinical laboratories and suitable to be used for DST of M. tuberculosis in all smear-positive samples.
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http://dx.doi.org/10.1099/jmm.0.070219-0DOI Listing
April 2014

Prospective multicentre evaluation of the direct nitrate reductase assay for the rapid detection of extensively drug-resistant tuberculosis.

J Antimicrob Chemother 2014 Feb 5;69(2):441-4. Epub 2013 Sep 5.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium.

Objectives: To perform a multicentre study evaluating the performance of the direct nitrate reductase assay (NRA) for the detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in sputum samples.

Methods: The study was conducted in six laboratories performing tuberculosis diagnosis that were located in six different countries. The NRA was performed directly on sputum samples in parallel with the reference method used at each site. Detection of resistance was performed for rifampicin, isoniazid, ofloxacin and kanamycin.

Results: Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 93.7%-100% for rifampicin, 88.2%-100% for isoniazid, 94.6%-100% for ofloxacin and 100% for kanamycin. The majority of NRA results were available at day 21 for sites 1, 2 and 5. Site 3 had a turnaround time of 13.9 days, at site 4 it was 18.4 days and at site 6 it was 16.2 days. The contamination rate ranged between 2.5% and 12%.

Conclusions: Rapid detection of drug resistance by the direct NRA on sputum smear-positive samples was accurate and easy to implement in clinical diagnostic laboratories, making it a good alternative for rapid screening for MDR and XDR tuberculosis.
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http://dx.doi.org/10.1093/jac/dkt353DOI Listing
February 2014

The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study.

Eur Respir J 2013 Dec 18;42(6):1604-13. Epub 2013 Apr 18.

A full list of the authors' affiliations can be found in the Acknowledgements.

A significant knowledge gap exists concerning the geographical distribution of nontuberculous mycobacteria (NTM) isolation worldwide. To provide a snapshot of NTM species distribution, global partners in the NTM-Network European Trials Group (NET) framework (www.ntm-net.org), a branch of the Tuberculosis Network European Trials Group (TB-NET), provided identification results of the total number of patients in 2008 in whom NTM were isolated from pulmonary samples. From these data, we visualised the relative distribution of the different NTM found per continent and per country. We received species identification data for 20 182 patients, from 62 laboratories in 30 countries across six continents. 91 different NTM species were isolated. Mycobacterium avium complex (MAC) bacteria predominated in most countries, followed by M. gordonae and M. xenopi. Important differences in geographical distribution of MAC species as well as M. xenopi, M. kansasii and rapid-growing mycobacteria were observed. This snapshot demonstrates that the species distribution among NTM isolates from pulmonary specimens in the year 2008 differed by continent and differed by country within these continents. These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location.
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http://dx.doi.org/10.1183/09031936.00149212DOI Listing
December 2013

First evaluation in Argentina of the GenoType® MTBDRplus assay for multidrug-resistant Mycobacterium tuberculosis detection from clinical isolates and specimens.

Rev Argent Microbiol 2012 Oct-Dec;44(4):283-9

Laboratorio de Referencia del Programa de Control de la Tuberculosis de la Provincia de Buenos Aires, Hospital Dr. Antonio Cetrángolo, Italia 1750, Florida (1602) Buenos Aires.

Tuberculosis (tB) and multidrug and extensively drug-resistant (dR) tB are important public health problems that are spreading worldwide. The aims of this study were to determine the sensitivity and specificity of the genotype® mtBdRplus assay from smear-positive clinical specimens and isolates and to explore its possible application in routine work. Clinical samples were previously decontaminated using naoH-n-acetyl-l-cystein or naoH-Clna hypertonic solution for Ziehl-neelsen staining and cultures. The leftover sediments of smear-positive samples were stored at -20 °C, 70 of which were selected to be included in this study according to their dR profile. thirty dR Mycobacterium tuberculosis isolates were also assessed. Sequencing was used as gold standard to detect mutations conferring isoniazid (InH) and rifampicin (RIF) resistance. Valid results were obtained in 94.0 % of the samples and 85.5 % (53/62) of the InH-R samples were properly identified. mutations in the katGS315t gene and inhA C-15t gene promoter region were present in 59.7 % (37/62) and 25.8 % (16/62) of the InH-R samples, respectively. the system could also identify 97.7 % (41/42) of the RIF-R samples; the mutations found were rpoBS531l (66.7 %, 28/42), d516V (19.0 %, 8/42), H526Y and S531p/W (4.8 %, 2/42 each one), and S522l/Q (2.4 %, 1/42). a 98.8 % concordance between the genotype assay and sequencing was obtained. genotype® mtBdRplus has demonstrated to be easy to implement and to perform in clinical laboratories and useful for a rapid detection of dR M. tuberculosis from decontaminated sputa and clinical isolates. Therefore, this assay could be applied as a rapid tool to predict InH-R and/or RIF-R in dR risk cases.
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April 2013

[Zoonotic tuberculosis in Argentina].

Medicina (B Aires) 2012 ;72(6):514-20

Programa de Control de Tuberculosis, Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), Buenos Aires, Argentina.

There are significant achievements in the control of animal tuberculosis (tB) in Argentina. the percentage of bovines with apparent tB lesions at the slaughterhouse inspection decreased from 6.7% to 0.6% between 1969 and 2011. on the other hand, the mean percentage of human tB cases due to M. bovis among all those bacteriologically confirmed, was in Santa Fe, an agro-industrial province, 2.3% in the period 1977-2001. It fell to 1.6% by 2011. In the Cetrángolo Hospital (Buenos Aires), it was 0.34% in the period 2001-2005, and 0.36% in 2006-2011. At the Muñiz Hospital, these percentages decreased from 1.75% in 1971 to 0.22% in 2006. Frequency of HIV infection among M. bovis cases varied from 5.9% in santa Fe to 11.1% and 20.5% respectively, in Cetrángolo and Muñiz Hospitals (a reference institution for aids) in Buenos Aires. According to these data M. bovis infection predominates in agro-industrial/ rural areas, showing a slow decrease there as well as in Buenos Aires. Co-infection with HIV is more frequent among patients with M. bovis that in all cases of tB. The situation of M. bovis in Argentina is here compared with that in USA and in several European and Latin American countries. Trends followed by tB in cattle, in humans, and the percentages of M. bovis among them, are not always closely related to move towards the common goal of eradicating tB; the employment of appropriate strategies and the strengthening of control measures are critical in both programs.
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May 2014

Disease caused by non-tuberculous mycobacteria: diagnostic procedures and treatment evaluation in the North of Buenos Aires Province.

Rev Argent Microbiol 2012 Jan-Mar;44(1):3-9

Laboratorio de Referencia del Programa de Control de la Tuberculosis de la Buenos Aires, Hospital Dr. Antonio Cetrángolo, Italia 1750, Florida, Buenos Aires, Argentina.

Non-tuberculous mycobacteria (NTM) have emerged as pathogens frequently associated to HIV co-infection. The aims of this study were to describe the clinical importance of NTM in patients from the North of Buenos Aires Province and the drug-susceptibility patterns in relation with the therapy used. A total of 23,624 clinical specimens were investigated during the period 2004-2010. Ziehl-Neelsen stain and cultures were used for diagnosis. Molecular and biochemical tests were performed to identify the mycobacteria. TB and mycobacterioses cases were 2 118 and 108 respectively. Sixteen NTM species were found: Mycobacterium avium and Mycobacterium intracellulare as the main causative agents. Infections produced by more than one species at the same time were confirmed (4 cases). Macrolides and fluoroquinolones were the most active in vitro drugs. Treatment evaluation showed that 68.0 % of the cases completed the therapy, 20 % died; and 12 % were relapses. The cases in which the treatment outcome was evaluated received an individual tailor-made therapeutic scheme including those drugs showing in vitro activity and presumed in vivo usefulness. More than a quarter of the patients had HIV co-infection and the majority of the deaths were associated with this co-infection.
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http://dx.doi.org/10.1590/S0325-75412012000100002DOI Listing
July 2012

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing.

J Clin Microbiol 2012 Jun 29;50(6):2026-33. Epub 2012 Mar 29.

Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.
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http://dx.doi.org/10.1128/JCM.06664-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372151PMC
June 2012

Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds.

BMC Infect Dis 2011 Jul 16;11:195. Epub 2011 Jul 16.

Instituto de Biotecnología, CICVyA-INTA, N, Repetto and De los Reseros, Buenos Aires, Argentina.

Background: The P27-P55 (lprG-Rv1410c) operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis.

Method: In order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions.

Results: The mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant.

Conclusions: The results clearly indicate that P27 and P55 are functionally connected in processes that involve the preservation of the cell wall and the transport of toxic compounds away from the cells.
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http://dx.doi.org/10.1186/1471-2334-11-195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146831PMC
July 2011

Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain.

Infect Genet Evol 2012 Jun 21;12(4):711-7. Epub 2011 Jun 21.

Instituto Nacional de Enfermedades Infecciosas ANLIS Carlos Malbrán, Vélez Sarsfield 563, 1281 Buenos Aires, Argentina.

Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD(Rio) LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders.
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http://dx.doi.org/10.1016/j.meegid.2011.06.006DOI Listing
June 2012

Multicentre laboratory validation of the colorimetric redox indicator (CRI) assay for the rapid detection of extensively drug-resistant (XDR) Mycobacterium tuberculosis.

J Antimicrob Chemother 2011 Apr 19;66(4):827-33. Epub 2011 Jan 19.

Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium.

Objectives: To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin.

Methods: The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method.

Results: Phase I: a strain was considered resistant by the CRI assay if the MIC was ≥0.5 mg/L for rifampicin, ≥0.25 mg/L for isoniazid, ≥4.0 mg/L for ofloxacin and ≥5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively.

Conclusions: Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.
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http://dx.doi.org/10.1093/jac/dkq527DOI Listing
April 2011

Rifampin-isoniazid oligonucleotide typing: an alternative format for rapid detection of multidrug-resistant Mycobacterium tuberculosis.

J Clin Microbiol 2010 Dec 29;48(12):4386-91. Epub 2010 Sep 29.

Corporacion CorpoGen, Carrera 5 No. 66A-34, Bogotá, D. C., Colombia.

A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources.
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http://dx.doi.org/10.1128/JCM.00448-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008445PMC
December 2010

Cost-effectiveness analysis of introduction of rapid, alternative methods to identify multidrug-resistant tuberculosis in middle-income countries.

Clin Infect Dis 2008 Aug;47(4):487-95

Instituto de Medicina Tropical Alexander Von Humboldt, Lima, Peru.

Background: Resistance to commonly used antituberculosis drugs is emerging worldwide. Conventional drug-susceptibility testing (DST) methods are slow and demanding. Alternative, rapid DST methods would permit the early detection of drug resistance and, in turn, arrest tuberculosis transmission.

Methods: A cost-effectiveness analysis of 5 DST methods was performed in the context of a clinical trial that compared rapid with conventional DST methods. The methods under investigation were direct phage-replication assay (FASTPlaque-Response; Biotech), direct amplification and reverse hybridization of the rpoB gene (INNO-LiPA; Innogenetics), indirect colorimetric minimum inhibitory concentration assay (MTT; ICN Biomedicals), and direct proportion method on Löwenstein-Jensen medium. These were compared with the widely used indirect proportion method on Löwenstein-Jensen medium.

Results: All alternative DST methods were found to be cost-effective, compared with other health care interventions. DST methods also generate substantial cost savings in settings of high prevalence of multidrug-resistant tuberculosis. Excluding the effects of transmission, the direct proportion method on Löwenstein-Jensen medium was the most cost-effective alternative DST method for patient groups with prevalences of multidrug-resistant tuberculosis of 2%, 5%, 20%, and 50% (cost in US$2004, $94, $36, $8, and $2 per disability-adjusted life year, respectively).

Conclusion: Alternative, rapid methods for DST are cost-effective and should be considered for use by national tuberculosis programs in middle-income countries.
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http://dx.doi.org/10.1086/590010DOI Listing
August 2008

Human Mycobacterium bovis infection in ten Latin American countries.

Tuberculosis (Edinb) 2008 Jul 8;88(4):358-65. Epub 2008 Jan 8.

Tuberculosis Consultants Group, World Health Organization, Av. Libertador 7504, 16 A, 1429 Buenos Aires, Argentina.

The aim of this work was to obtain the best possible estimate of the relevance of bovine tuberculosis (BTB) in humans in Argentina, Brazil, Chile, Colombia, Costa Rica, Dominican Republic, Ecuador, Peru, Uruguay and Venezuela. Sources of information were a questionnaire filled by the participant laboratories, and a search of published literature (1970-2007). Only four of these countries reported bacteriologically confirmed cases of BTB in humans. Most of these were diagnosed in Argentina, where the mean percentage of Mycobacterium bovis cases in relation to those due to Mycobacterium tuberculosis (2000-2006) ranged from 0.34% to 1.0%, according to the region. A slowly decreasing trend was observed in non HIV as well as in HIV/AIDS patients in Buenos Aires. In most of these countries, the low coverage of culture methods, especially of those including pyruvate-containing media, appropriate to isolate M. bovis, contributes to an underestimate of the problem. It was confirmed that BTB in humans exists, even though its relevance seems to be low. Milk pasteurization, sanitary controls to dairy products, and meat inspection at slaughterhouses contribute to the protection of human health. However, occupational aerogenous exposure to TB cattle and their carcasses remains a source of infection in the region.
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http://dx.doi.org/10.1016/j.tube.2007.11.007DOI Listing
July 2008

New simple decontamination method improves microscopic detection and culture of mycobacteria in clinical practice.

Infect Drug Resist 2008 20;1:21-6. Epub 2008 Aug 20.

Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr. Cetrángolo Hospital, Buenos Aires, Argentina;

This study was carried out at Dr. Cetrángolo Hospital, Buenos Aires, Argentina. The objective was to compare two digestion-decontamination procedures: the N-acetyl-L-cysteine-sodium citrate-NaOH (NALC-NaOH) and a combination of 7% NaCl plus NaOH, the hypertonic saline-sodium hydroxide (HS-SH) method, in detection and recovery of mycobacteria. Microscopy detection rates before and after concentration of specimens by both methods, were also compared. The study had two phases. Phase I: comparison of the gold standard NALC-NaOH and HS-SH on paired samples involving respiratory clinical specimens by means of receiver operating characteristic curve analysis. Phase II: blinded, randomized trial to assess the performance of HS-SH versus NALC-NaOH in clinical practice. Phase I: Positive microscopy rate was significantly increased in around 2.2% after concentration in comparison to that of specimens without concentration. The calculated sensitivity values for microscopy detection increased between 15.2% (HS-SH: 73.5%) to 16.7% (NALC-NaOH: 75.0%) over those without concentration (58.3%). Phase II: similar diagnostic rates by microscopy and cultures were obtained by either HS-SH or NALC-NaOH. The clinical performances were also very similar. These results and the low cost of the HS-SH procedure indicate the possibility of its implementation in clinical laboratories with high burden of tuberculosis cases and low resources.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108717PMC
http://dx.doi.org/10.2147/idr.s3838DOI Listing
July 2011

[Anti- tuberculosis treatment evaluation in northern districts of Buenos Aires suburbs].

Medicina (B Aires) 2007 ;67(2):131-5

Programa de Control de la Tuberculosis de la Región Sanitaria V, Hospital Dr. A. A. Cetrángolo, Vicente López, Provincia de Buenos Aires.

The aim of this study was to analyze the epidemiologic impact produced by Direct Observed Treatment Strategy (DOTS) application regarding to its success in the 51h Sanitary Region during the year 2 003. The cure was evaluated by a cohort study comparing two groups. Group 1: Districts in which DOTS were applied in 65% or more TB patients; group 2: Districts applying DOTS in 64% or less, or without its implementation. The global mortality was analyzed on HIV (-) as well as on HIV (+) patients in both groups. In Municipalities where DOTS strategy was applied in >65% of notified cases, the treatment success was 85.7%; the cure rate of bacteriological confirmed pulmonary disease was 86.2% and non-adherence was proved in 8.8% of cases. When DOTS was applied in equal or less than 64% of the cases, the global cure reached 67.6%; in confirmed pulmonary disease it was 68.1%, and non-adherence was proved in 21.8% of cases. Global mortality due to TB was 5.2%; 22.3% in HIV (+) and 4.1% in HIV (-). While In group 1, the cure rate of co-infected TB/HIV patients was 55.2%, non-adherence was 6.9%, in group 2 these figures were 46.4% and 19.7% respectively. Mortality rate in HIV (+) patients was 31.0% in group 1, and 16.1% in group 2. These results show that DOTS application proved to be an efficient tool to cure most of the patients, reaching the proposed goal of 85%, therefore reducing the risk of illness in the community.
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July 2008

Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis.

J Microbiol Methods 2005 Nov;63(2):145-50

Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat, 155, Antwerp, B-2000, Belgium.

The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on Löwenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities.
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http://dx.doi.org/10.1016/j.mimet.2005.03.004DOI Listing
November 2005

In vitro susceptibility testing of Mycobacterium tuberculosis complex strains isolated from seals to antituberculosis drugs.

Biomedica 2004 Jun;24 Supp 1:85-91

Reference Laboratory of Tuberculosis for MERCOSUR-DILACOT-SENASA, National Service of Sanity and Agricultural Quality (SENASA), Buenos Aires, Argentina.

Mycobacteria strains belonging to the Mycobacterium tuberculosis complex were isolated from seals found in the South Atlantic. The animals were received in Mundo Marino installations and treated for Mycobacterium tuberculosis complex by conventional therapy of intensive care and enriched food supply; however, in all cases treatment failed. Necropsies of all animals revealed extensive lesions compatible with tuberculosis involving lungs, liver, spleen and lymphatic nodes. Classical biochemical methods as well as molecular techniques using the IS6110 probes were performed for mycobacterial identification. Furthermore, the LCx M. tuberculosis assay (Abbott Laboratories) identified all strains as Mycobacterium tuberculosis complex members. The in vitro susceptibility pattern was examined in mycobacterial strains isolated from seven seals and in 3 reference strains--BCG, H37Rv (M. tuberculosis) and AN5 (Mycobacterium bovis)--to 4 medications--isoniazid, rifampin, streptomycin and ethambutol. Minimal inhibitory drug concentrations were determined by the Mycobacterial Growth Indicator Tube (BD Argentina) method and a microdilution and colorimetric assay using 3-(4-5 dimethyltiazol-2)-2,5 diphenyltetrazolium bromide. All the isolates and the reference strains BCG and AN5 were inhibited by MIC values similar to those of H37Rv with good agreement obtained by both techniques. These findings suggest that a therapeutic regimen aimed to seals diagnosed with tuberculosis play an important role in the prevention of tuberculosis transmission from infected animals to humans that are in routine contact with them.
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June 2004
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