Lucilaine Ferrazoli | Instituto Adolfo Lutz (original) (raw)

Papers by Lucilaine Ferrazoli

Genome Medicine

Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a seri... more Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB (further resistance against bedaquiline and/or linezolid) rates. Still, factors driving the MDR/pre-XDR epidemic in India are not well defined. Methods In a retrospective study, we analyzed 1852 consecutive MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai by whole genome sequencing (WGS). Univariate and multivariate statistics was used to investigate factors associated with pre-XDR. Core genome multi locus sequence typing, time scaled haplotypic density (THD) method and homoplasy analysis were used to analyze epidemiological success, and positive selection in different strain groups, respectively. Result...

Clinical Microbiology and Infection, Aug 1, 2018

Objectives: To describe the prevalence, associated factors, treatment outcomes and transmission o... more Objectives: To describe the prevalence, associated factors, treatment outcomes and transmission of extensively drug-resistant (XDR) tuberculosis (TB) in the state of São Paulo, Brazil, for 2011 to 2013. Methods: Drug susceptibility testing to firstand second-line drugs was performed by BACTEC MGIT 960 and molecular typing, by IS6110 restriction fragment length polymorphism. Clinical, epidemiologic and demographic data were obtained from surveillance information systems for TB. Patients were divided into three groups: multidrug resistant (MDR) TB (resistance to at least isoniazid and rifampicin), pre eXDR-TB (MDR-TB resistant to a fluoroquinolone or to at least one of the second-line injectable drugs) and XDR-TB (MDR-TB resistant to a fluoroquinolone and to at least one of the second-line injectables). Results: Among the 313 MDR-TB patients identified, the prevalence of XDR-TB and preeXDR-TB was 10.2% (n ¼ 32) and 19.2% (n ¼ 60), respectively. Compared to MDR-TB patients, XDR-TB patients were more likely to be female (odds ratio (OR) ¼ 2.74, 95% confidence interval (CI), 1.29e5.83), have a history of TB (OR ¼ 5.16; 95% CI, 1.52e17.51) and present higher death rates (OR¼ 3.74; 95% CI 1.70e8.25). XDR-TB transmission was observed in households, between neighbours and between a patient and a healthcare worker in a hospital. Conclusions: The prevalence of XDR-TB in the state of São Paulo is close to that estimated globally. Most of the XDR-TB patients were treated previously for TB and presented the lowest successful outcome rates. Because transmission of XDR-TB occurred, it is important that timely diagnosis of drug resistance is performed.

Memorias Do Instituto Oswaldo Cruz, 2020

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuber... more BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drugresistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

European Journal of Clinical Microbiology & Infectious Diseases, Jul 23, 2021

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in... more We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.

M. tuberculosis-positive cultures were obtained from 228 patients seen in our service and drug se... more M. tuberculosis-positive cultures were obtained from 228 patients seen in our service and drug sensitivity assays were carried out from January 1992 to December 1994. A survey of the medical records of these patients showed resistance to one or more drugs in 47 (20.6%), 25 of whom (10.9%), who reported previous treatment, were considered to have acquired resistance. Among the antecedents investigated, only previous treatment and alcoholism were the factors independently associated with the occurrence of resistance. The survival of patients with resistant strains was lower than that of patients attacked by non-resistant M. tuberculosis. We conclude that in the present series M. tuberculosis resistance to tuberculostatic agents was predominantly of the acquired type.No período de janeiro de 1992 a dezembro de 1994, foram obtidas culturas positivas para M. tuberculosis e foram realizados testes de sensibilidade a drogas em 228 pacientes atendidos no Centro de Referência DST/AIDS-SP. At...

European Journal of Clinical Microbiology & Infectious Diseases, 2021

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in... more We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.

Memorias Do Instituto Oswaldo Cruz, Nov 1, 2004

Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberc... more Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species-M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti"-were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, São Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.

International Journal of Tuberculosis and Lung Disease, Aug 1, 2017

To measure the association between diabetes mellitus (DM) among household contacts and recent-tra... more To measure the association between diabetes mellitus (DM) among household contacts and recent-transmission TB (RT TB).

Revista Da Sociedade Brasileira De Medicina Tropical, 2023

Background: We aimed to evaluate the costs of GenoType ® MTBDRplus and MTBDRsl incurred during th... more Background: We aimed to evaluate the costs of GenoType ® MTBDRplus and MTBDRsl incurred during the diagnosis of first-and secondline drug-resistant tuberculosis (TB) in São Paulo, Brazil. Methods: Mean and activity-based costs of GenoType ® were calculated in a referral laboratory for TB in Brazil. Results: The mean cost value and activity-based cost of GenoType ® MTBDRplus were USD 19.78 and USD 35.80 and those of MTBDRsl were USD 54.25 and USD 41.85, respectively. Conclusions: The cost of GenoType ® MTBDRplus was reduced owing to the high number of examinations performed and work optimization.

Revista do Instituto Adolfo Lutz (Impresso), 2012

Caracterização dos surtos causados pelo grupo Mycobacterium abscessus Characterization of the out... more Caracterização dos surtos causados pelo grupo Mycobacterium abscessus Characterization of the outbreaks caused by the Mycobacterium abscessus group RESUMO O gênero Mycobacterium contempla espécies do complexo M. tuberculosis e as denominadas micobactérias não tuberculosas (MNT). As micobactérias, quando em contato com o homem e alguns animais, podem causar doenças por meio de quebra da barreira do hospedeiro. Em virtude de sua natureza ambiental e muitas vezes oportunista, as micobactérias de crescimento rápido podem causar infecções nosocomiais, e com maior frequência pela espécie Mycobacterium abscessus. O M. abscessus causa diversos tipos de infecções teciduais e é altamente resistente à maioria dos quimioterápicos. Foi realizada uma revisão da literatura sobre os surtos de ocorrência nacional e internacional, com o objetivo de averiguar as principais causas que facilitaram a sua proliferação. Em 28 publicações, foram descritas as características das MNT e 15 trabalhos foram referentes ao relato de surtos, dos quais três nacionais associados aos procedimentos clínicos invasivos e 12 internacionais, correlacionados aos procedimentos médicos não invasivos. Todos os artigos relataram a frequente ocorrência de práticas inadequadas de limpeza, de procedimentos e de desinfecção. Estes fatos mostram a necessidade de sistema de qualidade mais eficiente e de estudos adicionais sobre a natureza do agente patogênico para tomada de medidas profiláticas mais efetivas. Palavras-chave. micobactéria de crescimento rápido, Mycobacterium abscessus, surtos.

Journal of Hospital Infection, Nov 1, 2018

Adolfo Lutz Institute in Sao Paolo state, performs identification of mycobacteria for many health... more Adolfo Lutz Institute in Sao Paolo state, performs identification of mycobacteria for many health care units and in identified a possible outbreak involving patients submitted for bronchoscopy at the same hospital. This study aimed to analyze the clonality of isolates. M. abscessus subsp. massiliense isolated from 28 patients and water from one bronchoscope and four automated endoscope reprocessing machines, which presented high similarity by PFGE. This strain was not found in the water supply, and it was hypothesized that an infected patient contaminated the bronchoscope, with further false positive cultures from subsequent patients.

Revista do Instituto Adolfo Lutz (Impresso), 2012

Mycobacterium genus comprises the species of the M. tuberculosis complex and those called as nont... more Mycobacterium genus comprises the species of the M. tuberculosis complex and those called as nontuberculous mycobacteria (NTM). When humans and some animals come into contact with mycobacteria, these microorganisms might cause severe pathogenic infections by breaking the host barrier. Due to their environmental nature and frequently as opportunistic infection, the rapid growing mycobacteria have been related to nosocomial infections, and M. abscessus has been one of the mostly frequent species. This microorganism may cause many types of tissue infections and it has been highly resistant to the majority of chemotherapeutic agents. A literature review on international and national outbreaks caused by this pathogen was performed to search the foremost causes which facilitate its proliferation. Twenty-eight publications described the NTM characteristics. The NTM outbreaks were reported in 15 articles, being three of them national studies which were related to invasive medical procedures, and 12 were international investigations linked to noninvasive procedures. All of the papers reported the occurrence of inadequate cleaning practices and disinfection procedures, indicating that a highly efficient quality system are needed, and also the further studies on the pathogen nature for carrying on the more effective preventive measures.

Journal of Clinical Laboratory Analysis, May 12, 2016

Background: Mycobacterium abscessus group has heterogeneous susceptibility pattern among species.... more Background: Mycobacterium abscessus group has heterogeneous susceptibility pattern among species. The species is most common cause of nosocomial infections. Macrolides minimum Inhibitory concentration (MIC) determination is essential for the treatment. Methods: Thirty-six strains were randomly selected for performing Resazurin Microtiter Assay (REMA) for clarithromycin testing in comparison to MIC test according to Clinical and Laboratory Standards Institute (2011) recommendation. REMA has been used for detection of drug resistance in M. tuberculosis. Extended incubation was performed to detect induced resistance. Results: Thirty microliters of resazurin (0.01%) was added after visually taking MIC reading. Resistance was observed in 11.1% of M. bolletti and 4.8% of M. abscessus strains; and induced resistance was detected in 77.8% and 95.2% of M. bolletti and M. abscessus strains, respectively. All strains of M. massiliense were susceptible. The samples presented same MIC value both by visual reading and through resazurin. Conclusion: The present study showed 100% concordance between both readings, with REMA providing easier to read and report results benefit. This change in reading can also reflect on the MIC determination and report, improving the test. J. Clin. Lab. Anal.

Memorias Do Instituto Oswaldo Cruz, Nov 1, 2004

Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infectio... more Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infection and classical identification of this pathogen needs a time consuming phenotypic tests. Polymerase chain reactionrestriction fragment lenght polymorphism analysis (PRA) of the gene enconding for the 65kDa heat shock (hsp65) protein offers an easy, rapid, and inexpensive procedure to identify and subtype M. kansasii isolates. In the present study, we performed a retrospective analysis of patients who had mycobacteria identified on the basis of phenotypic tests by means of a review of database at Mycobacteria Laboratory of the Instituto Adolfo Lutz in the period 1995-1998. A total of 9381 clinical isolates were analyzed of which 7777 (82.9%) were identified as M. tuberculosis complex and 1604 (17.1%) as nontuberculous mycobacteria. Of the 296 M. kansasii isolates, 189 (63.8%) isolates obtained from 119 patients were viable and were analyzed by PRA-hsp65. Hundred eight two (98.9%) were classified as M. kansasii type I. Two isolates were classified as type II and III and five isolates were characterized as other Mycobacterium species. Clinical isolates of M. kansasii in the state of São Paulo was almost exclusively subtype I regardless of HIV status.

Revista Do Instituto De Medicina Tropical De Sao Paulo, Sep 1, 2014

Mendes MURICY(1), Romilda Aparecida LEMES(2), Sidney BOMBARDA(3), Lucilaine FERRAZOLI(2) & Erica ... more Mendes MURICY(1), Romilda Aparecida LEMES(2), Sidney BOMBARDA(3), Lucilaine FERRAZOLI(2) & Erica CHIMARA(2) SUMMARY New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT TM) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients.

Journal of Medical Microbiology, Oct 1, 2005

Mycobacterium kansasii is the second most common cause of non-tuberculosis mycobacterial diseases... more Mycobacterium kansasii is the second most common cause of non-tuberculosis mycobacterial diseases in Sao Paulo, Brazil. An important component of the management of infections caused by this organism is antibiotic susceptibility testing. The objective of this study was to determine the drug susceptibility profiles and genotypes of clinical isolates of M. kansasii obtained from patients with or without an infection that met the American Thoracic Society's case definition criteria of M. kansasii disease. One hundred and sixty-nine clinical isolates of M. kansasii collected between 1993 and 1998 in Sao Paulo, Brazil, were tested consecutively. The isolates were genotyped by PCR restriction-enzyme pattern analysis (PRA). Most of the M. kansasii strains were susceptible to isoniazid, streptomycin, rifabutin, rifampicin, clarithromycin, ethionamide, amikacin, clofazimine and cycloserine, and resistant to ethambutol, ciprofloxacin and doxycycline. Of 169 isolates, 167 belonged to the type I PRA genotype and one each belonged to type II and III genotypes. There was no correlation between PRA subtype and M. kansasii disease according to the American Thoracic Society case definition. Clinical trials may be needed to better correlate MIC values with treatment outcomes to identify appropriate parameters for drug-resistance testing of M. kansasii.

Tubercle and Lung Disease, Oct 1, 1995

Genome Medicine

Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a seri... more Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB (further resistance against bedaquiline and/or linezolid) rates. Still, factors driving the MDR/pre-XDR epidemic in India are not well defined. Methods In a retrospective study, we analyzed 1852 consecutive MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai by whole genome sequencing (WGS). Univariate and multivariate statistics was used to investigate factors associated with pre-XDR. Core genome multi locus sequence typing, time scaled haplotypic density (THD) method and homoplasy analysis were used to analyze epidemiological success, and positive selection in different strain groups, respectively. Result...

Clinical Microbiology and Infection, Aug 1, 2018

Objectives: To describe the prevalence, associated factors, treatment outcomes and transmission o... more Objectives: To describe the prevalence, associated factors, treatment outcomes and transmission of extensively drug-resistant (XDR) tuberculosis (TB) in the state of São Paulo, Brazil, for 2011 to 2013. Methods: Drug susceptibility testing to firstand second-line drugs was performed by BACTEC MGIT 960 and molecular typing, by IS6110 restriction fragment length polymorphism. Clinical, epidemiologic and demographic data were obtained from surveillance information systems for TB. Patients were divided into three groups: multidrug resistant (MDR) TB (resistance to at least isoniazid and rifampicin), pre eXDR-TB (MDR-TB resistant to a fluoroquinolone or to at least one of the second-line injectable drugs) and XDR-TB (MDR-TB resistant to a fluoroquinolone and to at least one of the second-line injectables). Results: Among the 313 MDR-TB patients identified, the prevalence of XDR-TB and preeXDR-TB was 10.2% (n ¼ 32) and 19.2% (n ¼ 60), respectively. Compared to MDR-TB patients, XDR-TB patients were more likely to be female (odds ratio (OR) ¼ 2.74, 95% confidence interval (CI), 1.29e5.83), have a history of TB (OR ¼ 5.16; 95% CI, 1.52e17.51) and present higher death rates (OR¼ 3.74; 95% CI 1.70e8.25). XDR-TB transmission was observed in households, between neighbours and between a patient and a healthcare worker in a hospital. Conclusions: The prevalence of XDR-TB in the state of São Paulo is close to that estimated globally. Most of the XDR-TB patients were treated previously for TB and presented the lowest successful outcome rates. Because transmission of XDR-TB occurred, it is important that timely diagnosis of drug resistance is performed.

Memorias Do Instituto Oswaldo Cruz, 2020

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuber... more BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drugresistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

European Journal of Clinical Microbiology & Infectious Diseases, Jul 23, 2021

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in... more We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.

M. tuberculosis-positive cultures were obtained from 228 patients seen in our service and drug se... more M. tuberculosis-positive cultures were obtained from 228 patients seen in our service and drug sensitivity assays were carried out from January 1992 to December 1994. A survey of the medical records of these patients showed resistance to one or more drugs in 47 (20.6%), 25 of whom (10.9%), who reported previous treatment, were considered to have acquired resistance. Among the antecedents investigated, only previous treatment and alcoholism were the factors independently associated with the occurrence of resistance. The survival of patients with resistant strains was lower than that of patients attacked by non-resistant M. tuberculosis. We conclude that in the present series M. tuberculosis resistance to tuberculostatic agents was predominantly of the acquired type.No período de janeiro de 1992 a dezembro de 1994, foram obtidas culturas positivas para M. tuberculosis e foram realizados testes de sensibilidade a drogas em 228 pacientes atendidos no Centro de Referência DST/AIDS-SP. At...

European Journal of Clinical Microbiology & Infectious Diseases, 2021

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in... more We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.

Memorias Do Instituto Oswaldo Cruz, Nov 1, 2004

Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberc... more Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species-M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti"-were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, São Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.

International Journal of Tuberculosis and Lung Disease, Aug 1, 2017

To measure the association between diabetes mellitus (DM) among household contacts and recent-tra... more To measure the association between diabetes mellitus (DM) among household contacts and recent-transmission TB (RT TB).

Revista Da Sociedade Brasileira De Medicina Tropical, 2023

Background: We aimed to evaluate the costs of GenoType ® MTBDRplus and MTBDRsl incurred during th... more Background: We aimed to evaluate the costs of GenoType ® MTBDRplus and MTBDRsl incurred during the diagnosis of first-and secondline drug-resistant tuberculosis (TB) in São Paulo, Brazil. Methods: Mean and activity-based costs of GenoType ® were calculated in a referral laboratory for TB in Brazil. Results: The mean cost value and activity-based cost of GenoType ® MTBDRplus were USD 19.78 and USD 35.80 and those of MTBDRsl were USD 54.25 and USD 41.85, respectively. Conclusions: The cost of GenoType ® MTBDRplus was reduced owing to the high number of examinations performed and work optimization.

Revista do Instituto Adolfo Lutz (Impresso), 2012

Caracterização dos surtos causados pelo grupo Mycobacterium abscessus Characterization of the out... more Caracterização dos surtos causados pelo grupo Mycobacterium abscessus Characterization of the outbreaks caused by the Mycobacterium abscessus group RESUMO O gênero Mycobacterium contempla espécies do complexo M. tuberculosis e as denominadas micobactérias não tuberculosas (MNT). As micobactérias, quando em contato com o homem e alguns animais, podem causar doenças por meio de quebra da barreira do hospedeiro. Em virtude de sua natureza ambiental e muitas vezes oportunista, as micobactérias de crescimento rápido podem causar infecções nosocomiais, e com maior frequência pela espécie Mycobacterium abscessus. O M. abscessus causa diversos tipos de infecções teciduais e é altamente resistente à maioria dos quimioterápicos. Foi realizada uma revisão da literatura sobre os surtos de ocorrência nacional e internacional, com o objetivo de averiguar as principais causas que facilitaram a sua proliferação. Em 28 publicações, foram descritas as características das MNT e 15 trabalhos foram referentes ao relato de surtos, dos quais três nacionais associados aos procedimentos clínicos invasivos e 12 internacionais, correlacionados aos procedimentos médicos não invasivos. Todos os artigos relataram a frequente ocorrência de práticas inadequadas de limpeza, de procedimentos e de desinfecção. Estes fatos mostram a necessidade de sistema de qualidade mais eficiente e de estudos adicionais sobre a natureza do agente patogênico para tomada de medidas profiláticas mais efetivas. Palavras-chave. micobactéria de crescimento rápido, Mycobacterium abscessus, surtos.

Journal of Hospital Infection, Nov 1, 2018

Adolfo Lutz Institute in Sao Paolo state, performs identification of mycobacteria for many health... more Adolfo Lutz Institute in Sao Paolo state, performs identification of mycobacteria for many health care units and in identified a possible outbreak involving patients submitted for bronchoscopy at the same hospital. This study aimed to analyze the clonality of isolates. M. abscessus subsp. massiliense isolated from 28 patients and water from one bronchoscope and four automated endoscope reprocessing machines, which presented high similarity by PFGE. This strain was not found in the water supply, and it was hypothesized that an infected patient contaminated the bronchoscope, with further false positive cultures from subsequent patients.

Revista do Instituto Adolfo Lutz (Impresso), 2012

Mycobacterium genus comprises the species of the M. tuberculosis complex and those called as nont... more Mycobacterium genus comprises the species of the M. tuberculosis complex and those called as nontuberculous mycobacteria (NTM). When humans and some animals come into contact with mycobacteria, these microorganisms might cause severe pathogenic infections by breaking the host barrier. Due to their environmental nature and frequently as opportunistic infection, the rapid growing mycobacteria have been related to nosocomial infections, and M. abscessus has been one of the mostly frequent species. This microorganism may cause many types of tissue infections and it has been highly resistant to the majority of chemotherapeutic agents. A literature review on international and national outbreaks caused by this pathogen was performed to search the foremost causes which facilitate its proliferation. Twenty-eight publications described the NTM characteristics. The NTM outbreaks were reported in 15 articles, being three of them national studies which were related to invasive medical procedures, and 12 were international investigations linked to noninvasive procedures. All of the papers reported the occurrence of inadequate cleaning practices and disinfection procedures, indicating that a highly efficient quality system are needed, and also the further studies on the pathogen nature for carrying on the more effective preventive measures.

Journal of Clinical Laboratory Analysis, May 12, 2016

Background: Mycobacterium abscessus group has heterogeneous susceptibility pattern among species.... more Background: Mycobacterium abscessus group has heterogeneous susceptibility pattern among species. The species is most common cause of nosocomial infections. Macrolides minimum Inhibitory concentration (MIC) determination is essential for the treatment. Methods: Thirty-six strains were randomly selected for performing Resazurin Microtiter Assay (REMA) for clarithromycin testing in comparison to MIC test according to Clinical and Laboratory Standards Institute (2011) recommendation. REMA has been used for detection of drug resistance in M. tuberculosis. Extended incubation was performed to detect induced resistance. Results: Thirty microliters of resazurin (0.01%) was added after visually taking MIC reading. Resistance was observed in 11.1% of M. bolletti and 4.8% of M. abscessus strains; and induced resistance was detected in 77.8% and 95.2% of M. bolletti and M. abscessus strains, respectively. All strains of M. massiliense were susceptible. The samples presented same MIC value both by visual reading and through resazurin. Conclusion: The present study showed 100% concordance between both readings, with REMA providing easier to read and report results benefit. This change in reading can also reflect on the MIC determination and report, improving the test. J. Clin. Lab. Anal.

Memorias Do Instituto Oswaldo Cruz, Nov 1, 2004

Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infectio... more Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infection and classical identification of this pathogen needs a time consuming phenotypic tests. Polymerase chain reactionrestriction fragment lenght polymorphism analysis (PRA) of the gene enconding for the 65kDa heat shock (hsp65) protein offers an easy, rapid, and inexpensive procedure to identify and subtype M. kansasii isolates. In the present study, we performed a retrospective analysis of patients who had mycobacteria identified on the basis of phenotypic tests by means of a review of database at Mycobacteria Laboratory of the Instituto Adolfo Lutz in the period 1995-1998. A total of 9381 clinical isolates were analyzed of which 7777 (82.9%) were identified as M. tuberculosis complex and 1604 (17.1%) as nontuberculous mycobacteria. Of the 296 M. kansasii isolates, 189 (63.8%) isolates obtained from 119 patients were viable and were analyzed by PRA-hsp65. Hundred eight two (98.9%) were classified as M. kansasii type I. Two isolates were classified as type II and III and five isolates were characterized as other Mycobacterium species. Clinical isolates of M. kansasii in the state of São Paulo was almost exclusively subtype I regardless of HIV status.

Revista Do Instituto De Medicina Tropical De Sao Paulo, Sep 1, 2014

Mendes MURICY(1), Romilda Aparecida LEMES(2), Sidney BOMBARDA(3), Lucilaine FERRAZOLI(2) & Erica ... more Mendes MURICY(1), Romilda Aparecida LEMES(2), Sidney BOMBARDA(3), Lucilaine FERRAZOLI(2) & Erica CHIMARA(2) SUMMARY New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT TM) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients.

Journal of Medical Microbiology, Oct 1, 2005

Mycobacterium kansasii is the second most common cause of non-tuberculosis mycobacterial diseases... more Mycobacterium kansasii is the second most common cause of non-tuberculosis mycobacterial diseases in Sao Paulo, Brazil. An important component of the management of infections caused by this organism is antibiotic susceptibility testing. The objective of this study was to determine the drug susceptibility profiles and genotypes of clinical isolates of M. kansasii obtained from patients with or without an infection that met the American Thoracic Society's case definition criteria of M. kansasii disease. One hundred and sixty-nine clinical isolates of M. kansasii collected between 1993 and 1998 in Sao Paulo, Brazil, were tested consecutively. The isolates were genotyped by PCR restriction-enzyme pattern analysis (PRA). Most of the M. kansasii strains were susceptible to isoniazid, streptomycin, rifabutin, rifampicin, clarithromycin, ethionamide, amikacin, clofazimine and cycloserine, and resistant to ethambutol, ciprofloxacin and doxycycline. Of 169 isolates, 167 belonged to the type I PRA genotype and one each belonged to type II and III genotypes. There was no correlation between PRA subtype and M. kansasii disease according to the American Thoracic Society case definition. Clinical trials may be needed to better correlate MIC values with treatment outcomes to identify appropriate parameters for drug-resistance testing of M. kansasii.

Tubercle and Lung Disease, Oct 1, 1995