Molecular analysis of Mycobacterium isolates from extrapulmonary specimens obtained from patients in Mexico

  • Cosme Alvarado-Esquivel1Email author,

    Affiliated with

    • Nora García-Corral2,

      Affiliated with

      • David Carrero-Dominguez3,

        Affiliated with

        • José Antonio Enciso-Moreno4,

          Affiliated with

          • Teodoro Gurrola-Morales5,

            Affiliated with

            • Leopoldo Portillo-Gómez6,

              Affiliated with

              • Rudi Rossau7 and

                Affiliated with

                • Wouter Mijs7

                  Affiliated with

                  BMC Clinical Pathology20099:1

                  DOI: 10.1186/1472-6890-9-1

                  Received: 13 July 2008

                  Accepted: 09 March 2009

                  Published: 09 March 2009

                  Abstract

                  Background

                  Little information is available on the molecular epidemiology in Mexico of Mycobacterium species infecting extrapulmonary sites in humans. This study used molecular methods to determine the Mycobacterium species present in tissues and body fluids in specimens obtained from patients in Mexico with extrapulmonary disease.

                  Methods

                  Bacterial or tissue specimens from patients with clinical or histological diagnosis of extrapulmonary tuberculosis were studied. DNA extracts from 30 bacterial cultures grown in Löwenstein Jensen medium and 42 paraffin-embedded tissues were prepared. Bacteria were cultured from urine, cerebrospinal fluid, pericardial fluid, gastric aspirate, or synovial fluid samples. Tissues samples were from lymph nodes, skin, brain, vagina, and peritoneum. The DNA extracts were analyzed by PCR and by line probe assay (INNO-LiPA MYCOBACTERIA v2. Innogenetics NV, Gent, Belgium) in order to identify the Mycobacterium species present. DNA samples positive for M. tuberculosis complex were further analyzed by PCR and line probe assay (INNO-LiPA Rif.TB, Innogenetics NV, Gent, Belgium) to detect mutations in the rpoB gene associated with rifampicin resistance.

                  Results

                  Of the 72 DNA extracts, 26 (36.1%) and 23 (31.9%) tested positive for Mycobacterium species by PCR or line probe assay, respectively. In tissues, M. tuberculosis complex and M. genus were found in lymph nodes, and M. genus was found in brain and vagina specimens. In body fluids, M. tuberculosis complex was found in synovial fluid. M. gordonae, M. smegmatis, M. kansasii, M. genus, M. fortuitum/M. peregrinum complex and M. tuberculosis complex were found in urine. M. chelonae/M. abscessus was found in pericardial fluid and M. kansasii was found in gastric aspirate. Two of M. tuberculosis complex isolates were also PCR and LiPA positive for the rpoB gene. These two isolates were from lymph nodes and were sensitive to rifampicin.

                  Conclusion

                  1) We describe the Mycobacterium species diversity in specimens derived from extrapulmonary sites in symptomatic patients in Mexico; 2) Nontuberculous mycobacteria were found in a considerable number of patients; 3) Genotypic rifampicin resistance in M. tuberculosis complex infections in lymph nodes was not found.

                  Background

                  The genus Mycobacterium has been classified into many species [1]. A group of Mycobacterium species called M. tuberculosis complex that comprise M. tuberculosis, M. bovis, M. microti, and M. africanum is of utmost clinical importance since it causes tuberculosis in humans worldwide [24]. Mycobacterium species other than those of the tuberculosis complex, also called nontuberculous mycobacteria, are widely distributed in the environment and may colonize and occasionally cause infections in humans [57]. Mycobacteria of the M. tuberculosis complex and nontuberculous mycobacteria have been found to cause infections in immunocompetent and immunocompromised subjects and cause pathology in pulmonary and extrapulmonary sites [811]. Most epidemiological studies on mycobacteria have been focused on pulmonary infections, while extrapulmonary infections have been poorly explored. Extrapulmonary tuberculosis accounts for about 10% to 20% of tuberculosis cases in immunocompetent subjects but this frequency increases markedly in immunocompromised subjects [12]. Extrapulmonary sites are affected in up to 60% of patients suffering from acquired immunodeficiency syndrome and tuberculosis [12]. Molecular diagnosis of mycobacterial infections has enabled rapid detection of species in clinical specimens, detection of drug resistance, and typing for epidemiological studies [4, 6, 9, 13]. Little is known on the worldwide molecular epidemiology of Mycobacterium species infecting extrapulmonary sites. In addition, genotypic resistance to drugs in M. tuberculosis isolates from extrapulmonary sites has been poorly studied. There is scarce information on the molecular epidemiology in Mexico of Mycobacterium species infecting extrapulmonary sites. Therefore, in this study on samples obtained from such patients, we used molecular methods to identify the Mycobacterium species in tissue samples and body fluids. In addition, we analyzed the gene encoding for the β-subunit of the RNA polymerase (rpoB) for identification of mutations associated with rifampicin resistance in DNA extracts positive for M. tuberculosis complex.

                  Methods

                  Patients and specimens

                  Seventy-two patients with clinical or histological findings compatible with extrapulmonary tuberculosis were studied. Patients attended four public hospitals in the Mexican cities of Durango, Zacatecas and Guadalajara. Durango City is located in north central Mexico, while Zacatecas and Guadalajara Cities are located in central Mexico. Specimens from the patients were prepared as either paraffin-embedded tissue or cultures of body fluids. For paraffin-embedded tissues, we studied recent and stored specimens obtained from the pathology departments of two hospitals from 2002 to 2008. In total, tissues of 42 patients were studied: twelve of them were obtained in Durango City and 30 in Zacatecas City. For body fluids, we studied recent and stored cultures in Löwenstein Jensen medium from urine, pericardial fluid, gastric aspirate, synovial fluid and cerebrospinal fluid obtained from 30 patients. These specimens were obtained in two hospitals in Guadalajara City from 2007 to 2008. The studied specimens were obtained only from the participating hospitals and are not representative of specimens from all hospitals in these cities. DNA was extracted from all specimens using a commercially available kit (QIAamp DNA Mini Kit, QIAGEN. Germany) following the instructions of the manufacturer. Prior to extraction, paraffin-embedded tissues were treated with xylene and alcohol to remove the paraffin, and bacterial cultures were heated at 95°C for 10 minutes.

                  Molecular analysis of Mycobacterium species

                  DNA extracted from the 72 specimens was amplified by PCR using the INNO-LiPA MYCOBACTERIA v2 Amp. (Innogenetics NV, Gent, Belgium) kit according to the manufacturers' instructions. PCR parameters were: denaturation at 95°C for 1 min followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 62°C for 30 sec, and extension at 72°C for 30 sec. Electrophoresis of amplified products was performed in a 2% agarose gel. The gel was then stained with ethidium bromide and visualized by ultraviolet transillumination. In addition, amplified products were analyzed by line probe assay (INNO-LiPA MYCOBACTERIA v2. Innogenetics NV, Gent, Belgium) following the manufacturers' instructions. This DNA probe test targets the 16S–23S ribosomal RNA spacer region and detects and identifies the genus Mycobacterium and 16 different mycobacterial species. As a quality control for amplifications, we included water samples as negative controls, and M. tuberculosis complex DNA as positive controls in each run.

                  Analysis of rifampicin resistance of M. tuberculosis

                  Amplification of the rifampicin resistance region of the gene encoding for the β-subunit of the RNA polymerase (rpoB) in DNA samples positive for M. tuberculosis complex by INNO-LiPA MYCOBACTERIA v2 was performed using the INNO-LiPA Rif.TB Amplification kit (Innogenetics NV, Gent, Belgium). The PCR parameters were denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and an elongation at 72°C for 10 min. After electrophoresis of the amplified products in 2% agarose gel, the gel was stained with ethidium bromide and visualized by ultraviolet transillumination. In addition, amplification products were analyzed by line probe assay (INNO-LiPA Rif.TB. Innogenetics NV, Gent, Belgium) for detection of rifampicin resistance. This assay was performed following the manufacturers' instructions. As a quality control for amplifications, we included water samples as negative controls, and M. tuberculosis complex DNA as positive controls in each run.

                  Results and discussion

                  Rate of positive PCR and LiPA samples

                  As shown in Tables 1 and 2, only 26 (36.1%) and 23 (31.9%) of the 72 DNA extracts were PCR positive for the 16S–23S ribosomal RNA spacer region of Mycobacterium species and LiPA hybridization, respectively. The low rate of positive samples could be explained by: 1) No Mycobacterium species were present in the samples: pathology diagnosis is not conclusive of tuberculosis; 2) DNA of Mycobacterium was present in the tissues but at levels that were undetectable by a single-round PCR assay; 3) DNA preservation in some samples was sub-optimal; and 4) the presence of PCR inhibitors. Most samples tested were archival and a comparable rate of positive PCR results in archival tissue samples was reported in a previous study [14]. Moreover, our study was based in a single determination and it is possible that repeating assays could increase the rate of positive samples.
                  Table 1

                  Clinical and histological data of paraffin embedded tissues from the study population.

                  Case No.

                  Age

                  Gender

                  Place of origin

                  Clinical diagnosis

                  Specimen

                  Pathology diagnosis

                  ZN staining

                  PCR

                  Mycobacteria

                  T1

                  39

                  M

                  Dgo

                  TB

                  Brain

                  TB

                  +

                  +

                  M. genus

                  T2

                  34

                  M

                  Dgo

                  Tumour

                  Lymph node

                  TB

                  +

                  +

                  M. tuberculosis complex

                  T3

                  42

                  M

                  Dgo

                  Lymphadenitis

                  Lymph node

                  TB

                  -

                  -

                   

                  T4

                  45

                  F

                  Dgo

                  Tumour

                  Vagina

                  TB

                  +

                  +

                  M. genus

                  T5

                  45

                  M

                  Dgo

                  Lymphadenitis

                  Lymph node

                  TB

                  +

                  +

                  M. genus

                  T6

                  18

                  M

                  Dgo

                  Tumour

                  Lymph node

                  TB

                  +

                  -

                   

                  T7

                  40

                  M

                  Dgo

                  TB

                  Lymph node

                  TB

                  -

                  -

                   

                  T8

                  13

                  M

                  Dgo

                  Acute abdomen

                  Epiplon

                  TB

                  +

                  -

                   

                  T10

                  21

                  F

                  Dgo

                  Lymphadenitis

                  Lymph node

                  TB

                  -

                  -

                   

                  T11

                  29

                  M

                  Dgo

                  Lymphadenitis

                  Lymph node

                  TB

                  ND

                  -

                   

                  T12

                  16

                  M

                  Dgo

                  Acute abdomen

                  Peritoneum

                  TB

                  -

                  -

                   

                  T14

                  20

                  M

                  Dgo

                  Epidermic cyst

                  Skin

                  TB

                  +

                  -

                   

                  T41

                  9

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  +

                  M. tuberculosis complex

                  T42

                  57

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T43

                  31

                  M

                  Zac

                  TB

                  Lymph node

                  TB

                  +

                  -

                  M. tuberculosis complex

                  T44

                  56

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                  M. genus

                  T45

                  1

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                  M. genus

                  T46

                  39

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T47

                  39

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T48

                  1

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T49

                  53

                  M

                  Zac

                  Lymphoma

                  Lymph node

                  Lymphoma

                  ND

                  -

                   

                  T50

                  1

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T51

                  1

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T52

                  62

                  F

                  Zac

                  Hyperplasia

                  Lymph node

                  Hyperplasia

                  ND

                  -

                   

                  T53

                  19

                  M

                  Zac

                  Lymphoma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T54

                  67

                  F

                  Zac

                  Lymphoma

                  Lymph node

                  Lymphoma

                  ND

                  -

                  M. tuberculosis complex

                  T55

                  37

                  F

                  Zac

                  Hyperplasia

                  Lymph node

                  Hyperplasia

                  ND

                  -

                  M. tuberculosis complex

                  T56

                  4

                  M

                  Zac

                  Granuloma

                  Lymph node

                  Lymphoma

                  ND

                  -

                   

                  T57

                  44

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T58

                  32

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T59

                  32

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T60

                  74

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T61

                  38

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T62

                  9

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T63

                  0.4

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  +

                   

                  T64

                  53

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T65

                  45

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T66

                  1

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  +

                  -

                   

                  T67

                  3

                  M

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T68

                  54

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  T69

                  72

                  F

                  Zac

                  Lymphoma

                  Lymph node

                  Lymphoma

                  ND

                  -

                   

                  T70

                  28

                  F

                  Zac

                  Granuloma

                  Lymph node

                  TB

                  ND

                  -

                   

                  ND: Not done. Dgo: Durango. Zac: Zacatecas.

                  Table 2

                  Clinical data of patients with Löwenstein Jensen cultures strains.

                  Case No.

                  Age

                  Gender

                  Clinical diagnosis

                  Specimen

                  ZN staining

                  PCR

                  Mycobacteria

                  C1

                  65

                  M

                  RTB

                  Urine

                  -

                  -

                   

                  C2

                  10

                  M

                  RTB

                  Urine

                  +

                  +

                   

                  C3

                  39

                  F

                  RTB

                  Urine

                  +

                  +

                  M. gordonae

                  C4

                  29

                  M

                  RTB

                  Urine

                  ND

                  +

                   

                  C5

                  48

                  F

                  RTB

                  Urine

                  +

                  -

                   

                  C6

                  29

                  F

                  RTB

                  Urine

                  +

                  -

                   

                  C7

                  12

                  M

                  RTB

                  Urine

                  +

                  -

                   

                  C8

                  23

                  F

                  RTB

                  Urine

                  -

                  -

                   

                  C9

                  79

                  F

                  RTB

                  Urine

                  +

                  -

                   

                  C10

                  23

                  F

                  RTB

                  Urine

                  ND

                  +

                   

                  C11

                  20

                  M

                  TB

                  Pericardial fluid

                  ND

                  +

                  M. chelonae complex group III. M. abscessus

                  C12

                  29

                  F

                  RTB

                  Urine

                  ND

                  +

                  M. tuberculosis complex

                  C13

                  38

                  F

                  RTB

                  Urine

                  -

                  +

                  M. smegmatis

                  C14

                  53

                  F

                  RTB

                  Urine

                  -

                  +

                  M. smegmatis

                  C15

                  63

                  F

                  RTB

                  Urine

                  +

                  -

                  M. smegmatis

                  C16

                  13

                  F

                  RTB

                  Urine

                  ND

                  +

                  M. kansasii group II

                  C17

                  3

                  F

                  RTB

                  Urine

                  ND

                  +

                  M. genus

                  C18

                  35

                  F

                  TB

                  Gastric aspirate

                  ND

                  +

                  M. kansasii group I

                  C19

                  9

                  F

                  RTB

                  Urine

                  ND

                  +

                   

                  C20

                  13

                  F

                  RTB

                  Urine

                  ND

                  +

                  M. fortuitum-M. peregrinum complex

                  C21

                  48

                  F

                  RTB

                  Urine

                  +

                  +

                  M. smegmatis

                  C22

                  34

                  F

                  RTB

                  Urine

                  ND

                  +

                  M. genus

                  C23

                  23

                  F

                  RTB

                  Urine

                  ND

                  +

                   

                  C24

                  29

                  M

                  RTB

                  Urine

                  ND

                  +

                   

                  C25

                  35

                  M

                  RTB

                  Urine

                  ND

                  +

                   

                  C26

                  10

                  M

                  RTB

                  Urine

                  +

                  -

                   

                  C27

                  1

                  F

                  TB

                  CSF

                  +

                  -

                   

                  C28

                  30

                  F

                  TB

                  Synovial fluid

                  +

                  +

                  M. tuberculosis complex

                  C29

                  45

                  M

                  TB

                  CSF

                  -

                  -

                   

                  C30

                  56

                  F

                  RTB

                  Urine

                  +

                  +

                   

                  CSF: Cerebrospinal fluid

                  RTB: Renal tuberculosis

                  Mycobacterium species

                  Table 1 shows the Mycobacterium species identified in paraffin-embedded tissues, and Table 2 shows results obtained for body fluids. In paraffin-embedded tissues, M. tuberculosis complex and M. genus were found in 5 and 3 lymph nodes, respectively. The predominant M. tuberculosis complex infection in lymph nodes found in our patients is consistent with results in patients with lymphadenitis reported by other researchers [4, 15, 16]. With respect to other tissues, M. genus was found in specimens of brain and vagina. Hybridization with M. genus probes indicates that amplified DNA belongs to mycobacteria but it is not one of the 16 Mycobacterium species included in the kit. In body fluids, M. tuberculosis complex and nontuberculous mycobacteria were found in 1 (10%) and 9 (90%) of urine samples with positive hybridization, respectively. The frequency of M. tuberculosis complex infection in these patients is lower than that reported in a study performed in Turkey, where 12 (70.5%) of 17 strains isolated from urine cultures of patients with suspected urinary tuberculosis were identified as M. tuberculosis complex [17]. The finding of a high frequency of nontuberculous mycobacteria in urine may indicate contamination by mycobacteria in the environment. Nevertheless, all urine samples were obtained from symptomatic subjects with clinical diagnosis of renal tuberculosis and had obtained clinical improvement after anti-tuberculosis therapy. Many nontuberculous mycobacteria have recently been recognized as pathogenic [18]. The species of nontuberculous mycobacteria found in the urine of our patients have been found in symptomatic patients in several studies: 1) M. fortuitum was found in 2 of 5 renal transplant recipients with urinary symptoms [19]; 2) a case report of a woman suffering from urinary complaints describes that M. tuberculosis and other pathogens were not detected, but that M. gordonae was repeatedly isolated from urine. The patient responded to a standard anti-tuberculosis regimen [20]; and 3) positive urine culture for M. kansasii in patients with persistent fever who were suffering from hairy cell leukemia and disseminated atypical mycobacterial infection [21]. On the other hand, M. smegmatis has been isolated from skin and soft tissue infections [22], but to the best of our knowledge there is not any report of its isolation in urine from patients with urinary disease. The finding of nontuberculous mycobacteria in any specimen should always be correlated with clinical data of the patients to make appropriated therapeutic decisions. Since most Mycobacterium species found in extrapulmonary specimens in our symptomatic patients did not belong to the M. tuberculosis complex, our results emphasize the importance of performing identification of mycobacterial species. Discrimination of infecting Mycobacterium species is important since treatment differs [23]. PCR and LiPA technology has been successfully used in identifying both Mycobacterium species and rifampicin resistance in M. tuberculosis isolates in either Mycobacterium grown in culture or directly in some clinical samples [2427]. However, this study is the first to identify and classify Mycobacterium species and to detect genotypic rifampicin resistance from paraffin-embedded tissues by using LiPA technology. In addition, this is the first report of Mycobacterium species diversity detected by molecular methods in symptomatic patients in Mexico suffering from extrapulmonary disease. Our results indicate that LiPA can be used successfully in analyzing DNA extracts of paraffin-embedded tissues.

                  Rifampicin resistance

                  Two of the seven M. tuberculosis complex samples were positive for amplification of the rpoB gene and showed hybridization patterns compatible with wild type M. tuberculosis. Samples negative for LiPA were not further analyzed. The two positive samples were obtained from lymph nodes and were considered sensitive to rifampicin. These results further provide molecular characterization of the M. tuberculosis complex isolates. Patients suffering from extrapulmonary tuberculosis have a delay in diagnosis [28]. Therefore, fast molecular analysis of Mycobacterium using LiPA technology provides an aid for rapid diagnosis and the opportunity to take optimal preventive and treatment measures.

                  Conclusion

                  1) We describe the Mycobacterium species diversity in specimens derived from extrapulmonary sites in symptomatic patients in Mexico; 2) Nontuberculous mycobacteria were found in a considerable number of patients; 3) genotypic rifampicin resistance in M. tuberculosis complex infections in lymph nodes was not found.

                  Declarations

                  Acknowledgements

                  The authors would like to thank Anne Farmer for English reviewing of the manuscript.

                  Authors’ Affiliations

                  (1)
                  Department of Microbiology, Faculty of Medicine, Juárez University of Durango State
                  (2)
                  Department of Pharmacology, Faculty of Medicine, Juárez University of Durango State
                  (3)
                  Regional Laboratory of Epidemiological Reference, Mexican Social Security Institute
                  (4)
                  Medical Research Unit, Mexican Social Security Institute
                  (5)
                  Department of Pathology, Faculty of Medicine, Juárez University of Durango State
                  (6)
                  Department of Microbiology and Parasitology, University Center of Health Sciences, University of Guadalajara
                  (7)
                  R&D Diagnostics, Innogenetics NV

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                  29. Pre-publication history

                    1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1472-6890/​9/​1/​prepub

                  Copyright

                  © Alvarado-Esquivel et al. 2009

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.