Actinomadura is a filamentous bacterium found in soil. Although it was once believed to be a fungus, the information later attained about its ultrastructural cellular properties showed that Actinomadura is in fact an aerobic actinomycetes. However, for the reason that most of the diagnostic procedures related to Actinomadura are still held in mycology laboratories in many centers, the discussion on Actinomadura is included in this website. Actinomadura madurae , Actinomadura pelletieri , and Actinomadura dassonvillei are the species included in the genus Actinomadura. Actinomadura madurae is distinguished from A. It is one of the common causes of actinomycotic mycetoma maduramycosis or madura foot , characterized by formation of granules containing branched filaments.
|Published (Last):||11 May 2008|
|PDF File Size:||3.64 Mb|
|ePub File Size:||9.43 Mb|
|Price:||Free* [*Free Regsitration Required]|
Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms. A year-old woman from northern Mexico with a diagnosis of diabetes presented with a year history of a gradually enlarging right foot demonstrating multiple draining lesions.
She reported a long course of originally pruritic lesions on the plantar instep of her right foot which over many years developed into multiple bulbous nodules with central pinpoint ulcerations expressing purulent and granular material. Although the patient remained ambulatory throughout the course of her disease, she gradually had to modify her footwear to accommodate the increasing size of her foot, which at the time of presentation was causing her great pain.
Approximately 4 years prior to the current presentation, the diagnosis of actinomycotic mycetoma was reportedly established by histology, and she was initiated on antibiotic therapy with penicillin mg orally [p.
Despite treatment, the lesions continued to steadily grow, and 2 years prior to presentation she received a 6-week course of intravenous penicillin therapy with continuous daily infusions 20 million units intravenously [i.
The patient noted some retreat of the lesions and improvement in her pain and pruritis while on this treatment, and she was transitioned to oral penicillin mg p.
QID , ciprofloxacin mg p. BID , and doxycycline mg p. BID for suppression. No new lesions evolved while she was on this therapy, but approximately 1 year prior to presentation the patient self-discontinued ciprofloxacin and shortly thereafter noted increasing drainage from her foot. Intravenous penicillin 20 million units i. QD was resumed for an 8-week course, but no response in the amount of discharge or reduction in the size of the nodules was achieved and the antibiotic was consequently discontinued.
Culture of discharged material reportedly returned with overgrowth of skin flora and provided no evidence of infection with Actinomyces species. The patient repeatedly declined the recommendation of surgical debridement. The patient was referred to the Infectious Disease clinic at Harborview Medical Center for further evaluation. A punch biopsy specimen of the lesion was submitted to pathology, revealing marked acute and chronic inflammation and an inclusion of abundant filamentous structures consistent with aerobic actinomycetes Fig.
Grocott's methenamine silver GMS staining for fungal organisms was negative. Based on these findings, the diagnosis of actinomycotic mycetoma was suggested. A portion of the specimen was concurrently submitted for culture in order to establish the identity of the aerobic actinomycetes-like organism and to evaluate its antibiotic sensitivities; however, the culture quickly became overgrown by Staphylococcus aureus , preventing culture for slower-growing aerobic actinomycetes species.
A second biopsy specimen was submitted for culture and was again positive for S. Histological section of actinomycotic mycetoma. A Punch biopsy material stained using hematoxylin and eosin. An inclusion of filamentous, basophilic organisms consistent with Actinomyces species is seen. Formalin-fixed paraffin-embedded FFPE material from the biopsy specimen was concurrently submitted for molecular characterization of the pathogen to the University of Washington Molecular Diagnosis Microbiology Section.
DNA was amplified from the specimen in two separate reactions, in which sequencing adapters were incorporated on opposite ends of the amplicon, enabling bidirectional sequencing across the full length of 16S rRNA variable regions 1 and 2 3. PCR products were purified using 0. A template procedure for Ion Torrent sequencing was performed using a OneTouch 2 system Life Technologies , and sequencing was performed on an Ion PGM sequencer Life Technologies using a bp sequencing kit and a v2 chip according to the manufacturer's instructions.
Base calling was performed using TorrentServer software, version 3. Cumulatively, those low-abundance classifications accounted for 2, reads 7. Table 1 details the classification of deep-sequencing reads from the specimen submitted for analysis. The tallies and percent abundance of reads corresponding to each organism are indicated. For some sequences, multiple organisms met the identity threshold for species-level classification; in such cases, species names separated by a slash and genus names separated by a semicolon list the possible taxonomic assignments.
Consistent with culture results, reads classified as S. A number of other organisms were also detected, reflecting the polymicrobial nature of such specimens 5. Nevertheless, deep sequencing recovered reads 2. This pathogen is one of the classical agents of actinomycotic mycetoma and is consistent with the organism visualized histologically.
Based on this information, a course of sulfamethoxazole-trimethoprim therapy was considered. However, on imaging, the patient's osteomyelitis was found to involve all bones of the foot, including the calcaneus. Given the extent of infection, neither additional antibiotic therapy nor debridement with subsequent reconstruction was felt to be a viable option, and a recommendation was made for below-the-knee amputation.
The patient agreed to this procedure, and was discharged on a course of oral sulfamethoxazole-trimethoprim. Many sites of the human body are colonized by complex communities of microbes in both health and various disease states 6. Chronic infections 7 and diabetic foot ulcers 5 , 8 , 9 , in particular, can contain highly diverse bacterial populations.
Polymicrobial specimens may be difficult or even impossible to fully characterize by techniques in common clinical use: culture introduces bias against fastidious or slow-growing organisms 10 and can be practically employed to classify only a limited number of species, while molecular methods such as 16S rRNA gene sequencing 11 may detect only the predominant organism in a sample or may generate a mixed and uninterpretable signal Both of these diagnostic limitations complicated analysis of the patient specimen in this report, a biopsy specimen from a diabetic foot ulcer.
Consequently, the likely causative organism of the patient's foot infection could not be identified by existing clinical diagnostic approaches. Although the application of deep sequencing to microbial communities traces its origins to metagenomics research 13 , we recently demonstrated the feasibility of using deep sequencing to interrogate the composition of polymicrobial specimens in a clinical context by sequencing bacterial 16S rRNA amplified directly from patient material 3. In this case report, we have utilized the approach to investigate the causative agent of biopsy-proven actinomycotic mycetoma.
Deep sequencing successfully detected the presence of A. Although the disease can be caused by a number of fungal or bacterial agents that are typically introduced through traumatic inoculation from contaminated soil 15 , A. Several treatment regimens for actinomycotic mycetoma have been published 18 — 20 , and in general, the condition shows response to a wide range of antibiotics, including aminoglycosides, rifampin, amoxicillin-clavulanic acid, doxycycline, and sulfamethoxazole-trimethoprim, although combination antibiotic therapy is recommended In one small, prospective study, initial treatment with intravenous gentamicin, intravenous penicillin, and oral sulfamethoxazole-trimethoprim followed by oral sulfamethoxazole-trimethoprim and oral amoxicillin maintenance was found effective in empirically treating disease minimally involving the bones, while regimens incorporating intravenous amikacin and oral sulfamethoxazole-trimethoprim for initial treatment and oral sulfamethoxazole-trimethoprim for maintenance therapy were useful in cases of more extensive bony involvement The diagnosis of A.
Sequence reads originating from A. This finding is consistent with the results of both culture and Sanger sequencing for the specimen, which were dominated by S. The portion of the biopsy material sequenced may have contained little of the aerobic actinomycetes inclusion, which represented a focal area within the larger sample Fig. Further, it should be noted that read counts in 16S rRNA amplicon deep-sequencing studies are semiquantitative and correlate only roughly with the relative abundances of organisms due to bias introduced through PCR 23 , 24 , differing DNA extraction efficiencies for particular organisms 25 , and organism-specific differences in 16S rRNA operon counts Thus, the relative abundance of A.
Deep sequencing has previously been used to characterize cultured bacterial isolates 27 and to explore the composition of microbial populations in metagenomic research 28 , Here, we have extended the application of next-generation DNA sequencing technologies to perform molecular diagnosis in a clinical context. The case exemplifies several capabilities of deep sequencing as a clinical diagnostic tool, specifically, deconvoluting the identity of individual organisms within polymicrobial samples, classifying organisms directly from patient specimens without the need for culture, and characterizing nonviable or unculturable organisms in this case, organisms killed by FFPE processing prior to pathology examination.
The consensus sequence determined in this work has been submitted to GenBank under accession no. Published ahead of print 9 October National Center for Biotechnology Information , U. Journal List J Clin Microbiol v. J Clin Microbiol. Stephen J. Salipante , a Dhruba J.
SenGupta , a Daniel R. Hoogestraat , a Lisa A. Cummings , a Bronwyn H. Monsaas , a Mimosa Chau , a Lindley A. Barbee , c, d Christopher Rosenthal , a Brad T. Cookson , a, b and Noah G. Hoffman a. Dhruba J. Daniel R. Lisa A. Bronwyn H. Peter W. Lindley A. Brad T. Noah G. Author information Article notes Copyright and License information Disclaimer. Departments of Laboratory Medicine a. Corresponding author. Address correspondence to Stephen J. Salipante, ude.
All Rights Reserved. This article has been cited by other articles in PMC. Abstract Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Open in a separate window. Fig 1. Table 1 Deep-sequencing results. Nucleotide sequence accession number. Footnotes Published ahead of print 9 October Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia.
Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms. A year-old woman from northern Mexico with a diagnosis of diabetes presented with a year history of a gradually enlarging right foot demonstrating multiple draining lesions. She reported a long course of originally pruritic lesions on the plantar instep of her right foot which over many years developed into multiple bulbous nodules with central pinpoint ulcerations expressing purulent and granular material. Although the patient remained ambulatory throughout the course of her disease, she gradually had to modify her footwear to accommodate the increasing size of her foot, which at the time of presentation was causing her great pain. Approximately 4 years prior to the current presentation, the diagnosis of actinomycotic mycetoma was reportedly established by histology, and she was initiated on antibiotic therapy with penicillin mg orally [p. Despite treatment, the lesions continued to steadily grow, and 2 years prior to presentation she received a 6-week course of intravenous penicillin therapy with continuous daily infusions 20 million units intravenously [i.
Nonmycetomic Actinomadura madurae infection in a patient with AIDS.
The genus Actinomadura is one of four genera of actinobacteria that belong to the family Thermomonosporaceae. It contains aerobic, Gram-positive, non-acid-fast, non-motile, chemo-organotrophic actinomycetes that produce well-developed, non-fragmenting vegetative mycelia and aerial hyphae that differentiate into surface-ornamented spore chains. These chains are of various lengths and can be straight, hooked or spiral. The phospholipid pattern is PI diphosphatidylglycerol and phosphatidylinositol are present as major phospholipids and the fatty acid pattern is type 3a branched saturated and unsaturated fatty acids plus tuberculostearic acid.