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Journal of Medical Microbiology (2006), 55, 127–131 Detection of mixed infections with Mycobacteriumlentiflavum and Mycobacterium avium by moleculargenotyping methods Philip Suffys,1 Adalgiza da Silva Rocha,1 Adeilton Branda˜o,2Bart Vanderborght,3 Wouter Mijs,4 Geert Jannes,4 Fernanda C. Q. Mello,3Heloisa da Silveira Paro Pedro,5 Leila de Souza Fonseca,6Rosaˆngela Siqueira de Oliveira,7 Sylvia Cardoso Lea˜o7and Maria Helena Fe´res Saad1 Department of Mycobacterioses1 and Department of Tropical Medicine2, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil 3,6University Hospital Clementino Fraga Filho3 and Microbiology Institute6, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 4Innogenetics N. V., Technologiepark 6, B-9052 Ghent, Belgium 5Adolfo Lutz Institute, Sa˜o Paulo, Brazil 7Department of Microbiology, Immunology and Parasitology, Paulista School of Medicine, Three mycobacterial isolates, one from the blood of an HIV-infected patient and two consecutiveisolates from a woman with unknown HIV status, had been identified as belonging to theMycobacterium avium complex by conventional procedures. In both patients, using genetic analysisprocedures such as PCR–restriction enzyme analysis (PRA) of the hsp65 gene, a commerciallyavailable reverse hybridization-based assay (INNO-LiPA MYCOBACTERIA) and/or sequencinganalysis of the 16S–23S internal transcribed spacer (ITS), the presence of Mycobacteriumlentiflavum was also demonstrated. At the time of detection, both cases were also infected with M.
avium, suggesting an underestimation of infection with M. lentiflavum and co-infection with different 2002). The main reservoir in the environment has not been Mycobacterium lentiflavum is a slowly growing acid-fast firmly established, but organisms with M. lentiflavum-like bacillus (AFB) that has biochemical characteristics identical 16S rRNA gene sequences were detected in soil samples from to those of organisms belonging to the Mycobacterium the UK and from France (Mendum et al., 2000) and the avium complex (MAC) and mycolic acid and fatty acid species seems to be frequently present in drinking water chromatography patterns very similar to those of Myco- distribution systems in Finland (Torvinen et al., 2004). We bacterium simiae, so genetic analysis is necessary for con- here report the detection of a co-infection with M. aviumand M. lentiflavum in the blood or lung of two different clusive identification (Springer et al., 1996). This organism has been isolated from sterile clinical samples in Italy,Switzerland, Germany, France and Spain (Springer et al.,1996; Tortoli et al., 1997; Niobe et al., 2001; Ibanez et al.,2002) and from sputum samples in Brazil (da Silva Rocha et al., 1999) and Italy (Molteni et al., 2005) and, recently, cases of human disease have been reported, includingchronic pulmonary disease (Molteni et al., 2005), cervical Case 1. In December 1994, a 27-year-old Caucasian male was sub- lymphadenitis (Cabria et al., 2002), liver abscess (Tortoli mitted to a renal biopsy at the Clementino Fraga Filho University et al., 2002) and fatal disseminated infection (Ibanez et al., Hospital in Rio de Janeiro and membranoproliferative glomerulo-nephritis type I was diagnosed. The individual was submitted tointravenous corticoid treatment and discharged from the hospital Abbreviations: AFB, acid-fast bacillus; ITS, internal transcribed spacer; while continuing oral methylprednisone treatment that was gradu- MAC, Mycobacterium avium complex; PRA, PCR–restriction enzyme ally reduced until interruption in December 1995. By that time, the patient had developed lymphopenia (as shown by CD4 lymphocyte counts of 500 cells mm23) that was held under control, but, in M. intracellulare and M. avium, respectively, according to Thierry September 1996, he returned with weight loss, cough, fever and oral et al. (1993), while the presence of the insertion sequence IS1245 is candidiasis. A chest X-ray revealed a diffuse reticulonodular infiltrate indicative of M. avium (Guerrero et al., 1995). Because the two cases suggestive of active pulmonary disease. Sputum bacteriology showed were investigated independently by different research groups, the no evidence of AFB, but pneumocystosis, a definitive marker of methodologies performed on the isolates from the two patients were AIDS, was diagnosed and infection with HIV was confirmed. Treat- ment of pneumocystosis resulted in clinical improvement but,3 weeks later, the patient returned with high fever and aqueous diar-rhoea. A blood culture was performed and was positive for AFB.
Upon treatment with streptomycin, sulphamethoxazole/trimethro-prim and corticoid, the patient improved and was discharged from the hospital in October 1996. Two weeks later he returned to thehospital and received zidovidine and didanosine and was discharged Blood culture yielded small, pale-yellow, smooth-domed with the recommendation to return for regular re-evaluation. The colonies of AFB after 28 days of incubation at 37 uC in patient died a few months later in another hospital and no data are LJ medium and the culture was identified as MAC by available on the exact cause of death.
conventional biochemical methods (negative for Tweenhydrolysis, nitrate reduction and urease and positive for Case 2. In 1996, a 64-year-old woman was diagnosed with tubercu- catalase and tellurite reduction). Part of the sample was losis in the State of Mato Grosso. Despite treatment for tuberculosis treated by heat shock and submitted to genetic characteri- since February 1997, she presented to Public Health Care in Votu- zation by PRA of part of the hsp65 gene (Telenti et al., 1993; poranga, Sa˜o Paulo, with cough, fever, coryza, weakness, lack ofappetite, severe weight loss and dyspnoea. The X-ray showed bilat- da Silva Rocha et al., 2002): no digestion with BstEII and eral thin-walled cavitations and sputum collection revealed AFB. She 145/125 bp fragments with HaeIII were obtained (Fig. 1a).
was therefore enrolled in 1998 at the ‘Nu´cleo de Gesta˜o Assistencial’, According to the literature, this pattern is identical to M.
a health care service. During the following 4 years, she was sub- lentiflavum I (Springer et al., 1996), but this pattern has also mitted to several treatment schemes for tuberculosis, including anti- been described for M. simiae (da Silva Rocha et al., 2002).
biotics such as clarithromycin, ethambutol, clofazimine, rifampicin The isolate was submitted to INNO-LiPA MYCOBACTERIA and doxycycline. During this period, the patient did not showimprovement but rather a gradual worsening of the clinical symp- and hybridized with the Mycobacterium species probe but toms was observed with frequent positive bacilloscopy and mycobac- not with the probes specific for MAC, confirming that the terial cultures. In August 2002 she died of uterine cancer at 26 kg in isolate did not belong to this complex. Since the INNO-LiPA assay does not contain probes specific for M. lentiflavum orM. simiae, sequencing of the 16S–23S ITS was performed Culture, conventional identification and drug susceptibility and the isolate was confirmed to be M. lentiflavum.
testing. AFB staining was performed by the Ziehl–Neelsen method.
For case 1, a blood culture was performed using the lysis-centrifugation A subculture obtained from a small amount of the original method (Fandinho et al., 1997) and colonies of AFB were grown byincubation on Lo¨wenstein–Jensen (LJ) medium. For case 2, sputum bacterial mass in the tube containing LJ medium before culture on LJ medium was performed after decontamination using submitting it to heat shock was grown in Middlebrook 7H9 the Petroff method and centrifugation (Kent & Kubica, 1985). Sub- and reanalysed by PRA. On this occasion, the M. avium II culture was carried out on LJ medium and colonies were submitted restriction digest was observed and no (not even weak) to conventional identification (Kent & Kubica, 1985).
bands corresponding to the M. lentiflavum pattern werefound.
Genetic characterization. For genetic analysis, part of the samplewas submitted to three consecutive cycles of snap freezing andboiling in 10 mM Tris/HCl, 1 mM EDTA and 1 % Triton X-100.
The INNO-LiPA MYCOBACTERIA assay is commercially available(Innogenetics) for detection of Mycobacterium and identification of Among the 15 Mycobacterium cultures obtained from 1997 members of the Mycobacterium tuberculosis (MTB) complex, Myco- to 2001 on LJ medium, 12 were identified as MAC and 3 as bacterium kansasii, M. xenopi, M. gordonae, M. avium, M. intracellu- M. gordonae using conventional biochemical identification lare, M. scrofulaceum and M. chelonae, targeting the 16S–23S rRNA procedures (data not shown). Among these, two cultures, internal transcribed spacer (ITS) region (Miller et al., 2000); this one obtained in June 1998 and the other in September 2000 assay was evaluated in Brazil with excellent results (Suffys et al., and both identified as MAC, were submitted to molecular 2001). The PCR–restriction enzyme analysis (PRA) assay was origin-ally described by Telenti et al. (1993) and is based on amplification identification procedures. Both cultures were identified as of part of hsp65 and restriction analysis with HaeIII and BstEII; M. intracellulare I by PRA; however, amplification results evaluation of the assay in Brazil also demonstrated the straightfor- using primers from the M. intracellulare-specific DT1 wardness of the method (da Silva Rocha et al., 2002). Sequencing fragment and the M. avium-specific DT6 and IS1245 was performed on the amplified ITS that was used for INNO-LiPA fragments were contradictory. The first isolate was negative using a QIAquick PCR purification kit (Qiagen) and sequenced in the three PCR systems, while the more recent isolate was using an ABI PRISM BigDye Terminator cycle sequencing ready positive upon amplification of DT6 and IS1245; both results reaction kit on an ABI PRISM 377 sequencer (Applied Biosystems).
Sequence analysis was performed by comparison against a database were therefore inconsistent with the identification as M.
present at Innogenetics and against GenBank using SEQED and FASTA of the Wisconsin Package (version 9.1; Genetics Computer Group).
Some PCR systems for differentiation of M. intracellulare and M.
For that reason, subcultures were prepared on 7H10-OADC avium were used: amplification of DT1 or DT6 is characteristic for plates and the molecular tests were repeated on isolated Mycobacterium lentiflavum and M. avium co-infection contaminated bronchoscope, gastric juice and urinesamples (Springer et al., 1996), lymph nodes (Tortoli et al.,1997; Haase et al., 1997), bronchoalveolar lavage fluid and,very recently, sputum (Molteni et al., 2005); the presentreport is to our knowledge the second to demonstratedetection of M. lentiflavum in Brazil. Infection with MACis frequent in Brazilian AIDS patients (Barreto et al., 1993)and because routine identification procedures do not differ-entiate some Mycobacterium species (including M. lenti-flavum) from MAC, use of genetic methods for identifi-cation on a more routine basis would give a better idea of theprevalence of such species in this country.
Niobe et al. (2001) published the ITS sequences of a clinicalisolate of M. lentiflavum and of the type strain ATCC 51985and, although the 16S rRNA gene sequence of the clinicalisolate had 100 % identity to the sequence of the type strain,the ITS sequence of the clinical isolate had only 92?6 %identity to that of strain ATCC 51985, suggesting the need tostudy ITS variability in this species. The ITS sequence of oneM. lentiflavum isolate described here was identical to thatpublished by Niobe et al. (2001), as was the case for anotherisolate obtained from a sputum sample of an HIV-positiveindividual from Rio de Janeiro (data not shown). Thissuggests that the ITS sequence variability, at least in Brazil, islimited or non-existent while, interestingly, the isolatespresented different PRA patterns (Springer et al., 1996).
Bearing in mind that the whole ITS sequence was evaluatedwhile restriction enzyme analysis only demonstrates crea-tion or destruction of restriction sites, this finding suggests Fig. 1. Ethidium-bromide-stained agarose gels showing PRA of that mutation in the two genes seems to occur at different the hsp65 gene. (a) PRA patterns of isolates from Case 1.
HaeIII (lane 1) and BstEII (2) digestion products of M. lentifla-vum I and HaeIII (3) and BstEII (4) digestion products of M.
In both patients, mixed mycobacterial populations were avium II; lanes M contain a 50 bp ladder. (b) PRA patterns of present upon genetic analysis. Although an M. lentiflavum isolated colonies of the second isolate of Case 2, including pattern was observed in the original sample from the first BstEII of M. avium II (lanes 1 and 3) and of M. lentiflavum III patient, M. avium was observed exclusively in the sample (2) and HaeIII of M. avium II (4 and 6) and of M. lentiflavum III that was obtained after culturing of a fraction of that sample, (5); lane M contains a 50 bp ladder and lane m contains a suggesting the selection of M. avium either by fractionating the sample during collection or because of the faster growthof M. avium; M. lentiflavum has been reported to be lessvirulent than M. intracellulare in mice (Saito et al., 2000), colonies. In the second isolate, a mixed population contain- and this needs to be further investigated. This suggests the ing colonies with two different morphologies was observed; importance of submitting identical fractions to different PRA analysis resulted in the M. lentiflavum III pattern for identification procedures and considering analysis of the small yellow colonies, while the pale colonies were isolated colonies, as confirmed by the results of character- identified as M. avium II. This confirms the presence of a ization of separate colonies of the second isolate of case 2.
mixed infection with two different species and explains the Besides observing different colony morphology using this earlier results obtained using amplification systems for DT1, strategy, two genotypes belonging to different species were DT6 and IS1245. On the other hand, analysis of the earlier isolated culture yielded small yellow colonies only and PRApatterns were all M. lentiflavum III.
In the initial identification, both isolates of the secondpatient were identified as M. intracellulare by PRA. Com-paring PRA patterns of M. intracellulare and M. lentiflavum III, the BstEII fragments are indistinguishable and there is a In contrast to M. avium, a species isolated frequently from one-band difference upon HaeIII digestion, a 60 bp band in the blood of HIV-infected individuals, isolation of M.
M. intracellulare I that is absent from M. lentiflavum III.
lentiflavum from blood was described only recently (Niobe Failure to observe the 60 bp band or the appearance of et al., 2001). Previous reports describe isolates from the 60 bp band from M. avium could have led to this misinterpretation (Fig. 1b); the influence of such bands on correct pattern interpretation has been observed before (da This study was supported by CNPq (PRONEX 661028/1998-4), an Silva Rocha et al., 1999; Lea˜o et al., 2005).
International Collaborations in Disease Research (ICIDR) grant andFAPESP 98/11746-1. R. S. de O. was supported by CAPES and Sequencing also confirmed that the isolates were actually A. da S. R. by Faperj. We thank Lucilaine Ferrazoli for patient record M. lentiflavum rather than M. intracellulare. In our earlier information and Dr Lee Riley for his thoughts on existing but not yet study, the PRA pattern M. lentiflavum III was also observed in about half of the M. intracellulare isolates, adding onemore identifying pattern for the latter species (da SilvaRocha et al., 1999). The fact that these two species that are hard to separate by conventional identification and have thesame PRA type co-exist in Brazil demonstrates the need to Barreto, J. A., Palaci, M., Ferrazoli, L. & 7 other authors (1993).
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