Non-tuberculous mycobacterial infections of the hand.

UNLABELLED: Non-tuberculous mycobacterial infections of the hand are difficult to treat and require a long time before remission. But how long should we wait to see an improvement? To answer this question, the published scientific literature was reviewed in English, French and German. Tuberculosis, arthritis and osteomyelitis cases were excluded. A total of 241 non-tuberculous mycobacterial hand infections in 38 scientific publications were retrieved. Most were case reports or series. The median age of the patients was 58years and one third was female. Patients were immunocompromised in 17 episodes. The most common species were Mycobacterium marinum in 198 episodes (82%), followed by M. chelonae in 13 cases (5%). There were no cases of mixed infection. Most infections were aquatic in origin and community-acquired, and were treated with a combination of surgical debridement and long-duration systemic combination antibiotic therapy (14 different regimens; no local antibiotics) for a median duration of 6months. The median number of surgical procedures was 2.5 (range 1-5). Clinical success was not immediate: a median period of 3months (range 2-6) was necessary before the first signs of improvement were observed. The majority (173 cases; 76%) remained entirely cured after a median follow-up time of 1.7years (range, 1-6). Only two microbiological recurrences occurred (1%). However, 49 patients (21%) had long-term sequelae such as pain, stiffness and swelling. The approach of long-duration antibiotic treatment in combination with repeated surgery for mycobacterial soft tissue infections of the hand leads to few recurrences. However, clinical success is not immediate and may take up to 3months. TYPE OF STUDY: Therapeutic study: systematic review of level III studies. LEVEL OF EVIDENCE: III.

Characterization of human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi.

In this study, 16 human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi were evaluated using phenotypic methods, including traditional and commercial (API Coryne) biochemical tests, antimicrobial susceptibility testing, and 16S rRNA gene and gyrB gene sequencing. Positive results for both the hydrolysis of adenine and Christie-Atkins-Munch-Petersen (CAMP) reaction allowed for differentiation between the Dietzia isolates and the type strain of Rhodococcus equi; however, traditional and commercial phenotypic profiles could not be used to reliably identify Dietzia species. The analysis of 16S rRNA gene and gyrB gene sequences could discriminate all Dietzia strains from the type strain of R. equi. Most Dietzia species had distinct 16S rRNA gene and gyrB gene sequences; however, the 16S rRNA gene sequences of the type strains of D. schimae and D. cercidiphylli were identical to D. maris and D. natronolimnaea, respectively. Based on comparative sequence analysis, five clinical isolates clustered with D. maris/D. schimae and nine with D. natronolimnaea/D. cercidiphylli. The two remaining isolates were found to be most closely related to the D. cinnamea/D. papillomatosis clade. Even though molecular analyses were not sufficiently discriminative to accurately identify all Dietzia species, the method was able to reliably identify isolates that were previously misidentified by phenotypic methods to the genus level.

Molecular methods for the identification of Aspergillus species.

Invasive aspergillosis (IA) is a leading cause of morbidity and mortality in immunocompromised hosts. In some institutions, species of Aspergillus less susceptible to amphotericin B than Aspergillus fumigatus are becoming more common, making an accurate identification of species important. However, species identification has traditionally relied on macroscopic colony characteristics and microscopic morphology, which may require several days of culture. Additional sub-culturing on specialized media may be required to induce conidia formation; in some cases conidia may never form, confounding identification. Therefore, rapid, nucleic acid-based methods that identify species of Aspergillus independent of morphology are now being developed to augment or replace phenotypic identification methods. The most successful methods to date have employed polymerase chain reaction (PCR) amplification of target sequences within the ribosomal RNA gene complex, including the 28S ribosomal subunit (D1-D2 region) and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). We therefore developed a PCR-based assay to differentiate medically important species of Aspergillus from one another, and from other opportunistic moulds and yeasts, by employing universal, pan-fungal primers directed to conserved ribosomal genes and species-specific DNA probes directed to the highly variable ITS2 region. Amplicons were then detected in a simple, colorimetric enzyme immunoassay format (PCR-EIA). DNA sequencing of the ITS1 and ITS2 regions and of the D1-D2 region was also conducted for the differentiation of species by comparative GenBank sequence analysis. The PCR-EIA method was found to be rapid, sensitive, and specific for the identification and differentiation of the most medically important species of Aspergillus. In addition, methods to identify species of Aspergillus by comparative GenBank sequence analysis were found to be more reliable using the ITS1 and ITS2 regions than the D1-D2 region.

Tropheryma whippelii DNA in saliva of patients without Whipple's disease.

Tropheryma whippelii is the causative agent of Whipple's disease, a difficult to diagnose systemic illness. Amplification of part of its 16S ribosomal RNA gene(s) has become a standard diagnostic method because of increased sensitivity as compared to classical histopathological analysis. Recently, we demonstrated the presence of T. whippelii DNA by PCR in duodenal biopsies and/or gastric juice of a considerable fraction of individuals without clinical signs of Whipple's disease. In this follow-up study, saliva and dental plaques of the same patients were screened for the presence of T. whippelii DNA. Six out of the 14 previously PCR-positive persons but none of the 17 controls had T. whippelii DNA in their saliva. Our results suggest that Whipple bacteria are ubiquitous environmental or commensal organisms causing Whipple's disease only in a particular subset of individuals, possibly those with an as yet uncharacterized immunological defect.

Analysis of the actinobacterial insertion in domain III of the 23S rRNA gene of uncultured variants of the bacterium associated with Whipple's disease using broad-range and 'Tropheryma whippelii'-specific PCR.

Heterogeneity in the 16S-23S rDNA spacer of uncultured 'Tropheryma whippelii' has previously been reported. In this study, the hypervariable insertion in the 23S rDNA domain III characteristic for actinobacteria was analysed. The finding of a unique sequence virtually identical among the subtypes supports the classification of 'T. whippelii' among the actinobacteria and the concept of three subtypes rather than three subspecies, and provides an alternative target for diagnostic assays.

Evaluation of a specific nested PCR targeting domain III of the 23S rRNA gene of "Tropheryma whippelii" and proposal of a classification system for its molecular variants.

"Tropheryma whippelii"-associated infections are usually confirmed histopathologically by using light microscopy. PCR assays targeting the 16S rRNA gene (16S rDNA) of "T. whippelii" are increasingly being applied for this purpose. Compared to microscopic analysis, PCR seems to be more sensitive, as indicated by the fact that several cases of Whipple's disease with negative histopathological findings but positive PCR results have been reported. Considering the lack of pathognomonic clinical features for this disease and the fact that "T. whippelii" DNA has repeatedly been found in patients without clinical Whipple's disease, such PCR results should be confirmed by additional tests. We have, therefore, evaluated a "T. whippelii"-specific nested PCR targeting domain III of the 23S rDNA with 41 clinical specimens known to contain "T. whippelii" 16S rDNA. All of these specimens were also positive for "T. whippelii" 23S rDNA. The specificity of the test was shown by sequencing of the amplicons and by the absence of amplicons in 38 negative controls. We consider this PCR test to be a suitable tool for confirming the presence of "T. whippelii" DNA in specimens with inconclusive histopathological findings. The information derived from sequencing of the partial "T. whippelii" 23S rDNA was then combined with our recent data of the 16S-23S rDNA spacer region of this organism. Overall, four different rDNA types are recognized in our proposed classification system for molecular variants of "T. whippelii." This preliminary scheme may provide a basis for further epidemiological and clinical studies with "T. whippelii" and associated diseases.

Detection of three different types of 'Tropheryma whippelii' directly from clinical specimens by sequencing, single-strand conformation polymorphism (SSCP) analysis and type-specific PCR of their 16S-23S ribosomal intergenic spacer region.

The 16S-23S rDNA intergenic spacer region of organisms identical with or closely related to 'Tropheryma whippelii', the uncultivated causative agent of Whipple's disease, was analysed directly from 38 clinical specimens of 28 patients using a specific nested PCR followed by direct sequencing. As compared to the reference sequence in public databases, two novel 'T. whippelii' spacer types were recognized. In the absence of DNA-DNA hybridization data it is uncertain whether the three types found represent subtypes of a single species or three different but closely related species. Methods were developed to detect all three variants by single-strand conformation polymorphism analysis and by type-specific PCR assays, thus allowing the screening of large numbers of specimens. Further studies may provide a clue to the possible associations between the type of infecting strain and the various clinical presentations of Whipple's disease.

Homogeneity of 16S-23S ribosomal intergenic spacer regions of Tropheryma whippelii in Swiss patients with Whipple's disease.

The current genetic strategies used to identify Tropheryma whippelii, the putative agent of Whipple's disease, are based on PCR-mediated amplification of a part of its 16S rRNA gene (16S rDNA). Because there is very little intraspecies variation in these molecules, they are not suitable as targets for epidemiologic investigations. However, the intergenic spacer region between the 16S and 23S rDNAs is usually much more variable and has repeatedly been used for epidemiologic purposes. We have therefore amplified the spacer region of T. whippelii directly from clinical specimens from nine independent Swiss patients with Whipple's disease by PCR with primers complementary to the 3' and 5' ends of the 16S and 23S rDNAs, respectively. The amplicons were directly sequenced and the sequences were compared to the T. whippelii reference sequence in GenBank/EMBL (accession no. X99636). Complete sequence homogeneity was found between the samples from our nine patients; the spacer sequence was also identical to the reference sequence. However, the sequences corresponding to the 3' and 5' ends of the 16S and the 23S rDNAs of T. whippelii, respectively, differed from the respective sequences in GenBank/EMBL. The same sequence found in our patients was then found in a sample from the German patient from which the published sequence had been derived. We conclude that the 16S-23S rDNA spacer region seems to be very conserved in T. whippelii and that the respective reference entry in public databases should be revised.