Precision diagnostics in paediatric dermatology: Advancing management of tinea capitis through dermatophyte PCR.

BACKGROUND: Tinea capitis (TC) is the most frequent dermatophyte infection in children requiring systemic and topical treatment for several weeks. Traditionally, diagnosis and treatment monitoring were based on microscopic examination and fungal culture of scales and plucked hairs, which both have significant limitations. OBJECTIVES: To investigate the role of dermatophyte polymerase chain reaction (PCR) in the treatment of TC. METHODS: Scales and plucked hairs of children with TC were investigated by dermatophyte PCR, microscopic examination and fungal culture at baseline and during antifungal treatment. RESULTS: Seventeen children with TC were included. At baseline, sensitivity of PCR was 100% as compared to 60% and 87% for direct microscopy and fungal culture, respectively. Species identification by PCR and fungal culture was consistent in all cases. During follow-up, analysis of 38 samples under treatment showed a sensitivity of PCR, direct microscopy and fungal culture of 68%, 26% and 89% while specificity was 84%, 100% and 100%, respectively. PCR during therapy proved to be false-negative in six and false-positive in three instances. The latter turned negative after 4 weeks without further systemic treatment. CONCLUSIONS: Dermatophyte PCR is an excellent tool for baseline diagnostics of TC providing rapid and accurate results. Our findings suggest that due to the fast and reliable results, it may replace direct microscopy and fungal culture to confirm or exclude TC in children. In the treatment course, diagnostic accuracy and performance of PCR seem reduced as compared to fungal culture, limiting its value for treatment monitoring. Mycological cure ascertained by fungal culture should currently remain the therapeutic goal.

Dysbiosis of skin microbiota with increased fungal diversity is associated with severity of disease in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a multifactorial inflammatory skin disease and an altered skin microbiota with an increase of Staphylococcus aureus has been reported. However, the role of fungi remains poorly investigated. OBJECTIVES: We aimed to improve the understanding of the fungal skin microbiota, the mycobiota, in AD in relation to the bacterial colonization. METHODS: Skin swabs of 16 AD patients and 16 healthy controls (HC) from four different skin sites, that is antecubital crease, dorsal neck, glabella and vertex from multiple time points were analysed by DNA sequencing of the internal transcribed spacer region 1 (ITS1) and 16S rRNA gene for fungi and bacteria, respectively. RESULTS: Malassezia spp. were the predominant fungi in all subjects but with a decreased dominance in severe AD patients in favour of non-Malassezia fungi, for example Candida spp. For bacteria, a decrease of Cutibacterium spp. in AD patients in favour of Staphylococcus spp., particularly S. aureus, was observed. Further, both bacterial and fungal community compositions of severe AD patients significantly differed from mild-to-moderate AD patients and HC with the latter two having overall similar microbiota showing some distinctions in bacterial communities. CONCLUSIONS: We conclude that severe AD is associated with a pronounced dysbiosis of the microbiota with increased fungal diversity. Potentially infectious agents, for example Staphylococcus and Candida, were increased in severe AD.

Epidemiological and clinical aspects of Trichophyton mentagrophytes/Trichophyton interdigitale infections in the Zurich area: a retrospective study using genotyping.

BACKGROUND: Trichophyton mentagrophytes (formerly Arthroderma vanbreuseghemii) and its clonal offshoot Trichophyton interdigitale, which are leading causes of dermatophytoses, have recently been recognized as two separate species. Over the last 20 years, several internal transcribed spacer (ITS) genotypes of Trichophyton mentagrophytes and Trichophyton interdigitale have been identified, some of which have specific characteristics and lead to typical clinical manifestations. OBJECTIVES: The aim of this study was to determine the current epidemiology of Trichophyton mentagrophytes and Trichophyton interdigitale genotypes in Switzerland, particularly in the Zurich area. METHODS: Consecutive cases diagnosed by ITS sequencing between 2009 and 2019 were retrospectively analysed. RESULTS: A total of 81 Trichophyton mentagrophytes and 81 Trichophyton interdigitale cases were investigated. T. mentagrophytes infections clearly differed from T. interdigitale infections by affecting younger and more frequently female patients, targeting almost exclusively head and body rather than feet and toenails, leading to inflammatory dermatophytosis and often requiring a combination of systemic and topical treatment. Seven different T. mentagrophytes genotypes (II*, III, III*, IV, VII, VIII and XXVI) were observed, with genotype XXVI being discovered in this study. Genotype III occurred most frequently (56% of all T. mentagrophytes cases) and affected predominantly children. Genotypes III* and VII led to inflammatory tinea in most cases. Four strains that proved to be terbinafine resistant belonged to the 'Indian genotype' VIII, which mostly caused tinea glutealis and inguinalis. CONCLUSION: Being able to distinguish between Trichophyton mentagrophytes and Trichophyton interdigitale is of paramount importance as the two species cause different clinical presentations. In addition, ITS genotyping allows recognizing sources of infection and potential terbinafine resistance. The latter needs to be confirmed by resistance testing or by sequencing part of the squalene epoxidase (SQLE) gene.

Emerging antifungal treatment failure of dermatophytosis in Europe: take care or it may become endemic.

BACKGROUND: Dermatophytosis is a world-wide distributed common infection. Antifungal drug resistance in dermatophytosis used to be rare, but unfortunately the current Indian epidemic of atypical widespread recalcitrant and terbinafine-resistant dermatophytosis is spreading and has sporadically been reported in Europe. OBJECTIVES: To explore the occurrence of clinical and mycological proven antifungal drug resistance in dermatophytes in Europe. METHODS: A standardized questionnaire was distributed through the EADV Task Force of Mycology network to dermatologists in Europe. RESULTS: Representatives from 20 countries completed the questionnaires of which 17 (85 %) had observed clinical and/or mycological confirmed antifungal resistance, two countries published cases of antifungal resistance and one country had no known cases. CONCLUSIONS: This pilot study confirms that both clinical and mycological antifungal resistance exist in Europe.

Development and evaluation of a pan-dermatophyte polymerase chain reaction with species-level identification using sloppy molecular beacon probes.

BACKGROUND: Conventional laboratory diagnosis of dermatophyte infection is cumbersome and time-consuming. OBJECTIVES: We aimed to establish a simple, robust and rapid molecular diagnostic assay for the detection of dermatophytes and optionally nondermatophytes in clinical specimens. MATERIALS AND METHODS: We developed a two-tube pan-dermatophyte polymerase chain reaction (PCR) assay using six sloppy molecular beacon (SMB) probes. The first PCR uses dermatophyte-specific primers and enables detection and identification of most dermatophyte species. The second PCR with pan-fungal primers allows further differentiation of Trichophyton interdigitale and T. mentagrophytes/T. quinckeanum, T. violaceum and T. soudanense, and T. tonsurans and T. equinum, and detection of nondermatophytes. The test was evaluated with 306 clinical specimens by comparing it with the results of microscopy and culture. RESULTS: In melting-curve analyses, species-specific melting temperature signatures of the SMBs were defined, and thus, our new PCR enabled detection and species-level identification of at least 19 dermatophyte species. Sensitivity and specificity of PCR for detection of dermatophytes in clinical samples were estimated to be 96·9% and 90·4%, for culture 46·7% and 98·7%, and for microscopy 91·4% and 84·0%, respectively. The detection of nondermatophytes by PCR and culture did not correlate. CONCLUSIONS: The new assay showed excellent performance characteristics for the detection of dermatophytes and is significantly faster than culturing techniques, which makes it very promising for routine diagnostics of dermatophytosis. We noted that the detection of nondermatophytes in our assay currently has no benefit.

In vitro efficacy of antifungal agents alone and in shampoo formulation against dandruff-associated Malassezia spp. and Staphylococcus spp.

OBJECTIVE: Dandruff is a complex skin condition characterized by unpleasant itching and flaking of the scalp. It is primarily attributed to the over colonization of Malassezia yeasts such as Malassezia globosa and Malassezia restricta. Some studies also suggest the involvement of staphylococci bacteria in dandruff disease pathogenesis. We aimed to access the effectiveness of anti-dandruff treatments by determining the efficacy of the active antifungal agents alone or in commercial shampoo formulations against Malassezia and Staphylococcus. METHODS: The minimum inhibitory concentrations of three anti-dandruff shampoo antifungals (zinc pyrithione, ketoconazole and ciclopirox) and the witch hazel extract, hamamelitannin were tested against commensal Malassezia and Staphylococcus species using broth microdilution methods. In experiments simulating shampoo exposure and washing conditions on the scalp, we also tested the ability of the above agents in shampoo formulation (Head and Shoulders® (H&S), Ketomed® , Sebiprox® , Erol Healthcare Hair Shampoo® respectively) along with a generic over-the-shelf shampoo to inhibit microbial growth. RESULTS: Ketomed® and H&S shampoo were the most effective treatments against Malassezia in in vitro assays and washing simulation experiments. Erol Healthcare Hair Shampoo® was less effective against Malassezia as it required a longer contact time to achieve growth inhibition for some species. Sebiprox® showed variable efficacy in washing and contact time experiments whereas the generic over-the-shelf shampoo was the least effective in inhibiting Malassezia and Staphylococcus growth. CONCLUSION: From these findings, it is reasonable that patients with dandruff may benefit from applying specific antifungal shampoo although results may vary with microbial species, time of contact and shampoo formulation components.

OBJECTIFS: Les pellicules sont une affection cutanée complexe caractérisée par des démangeaisons et une desquamation du cuir chevelu. Elles sont principalement attribuées à une colonisation excessive par des levures du genre Malassezia telles que Malassezia globosa et Malassezia restricta. Certaines études suggèrent également que des bactéries comme les staphylocoques sont impliquées dans la pathogenèse des pellicules. Nous désirions évaluer l'efficacité des traitements antipelliculaires en déterminant l'efficacité des antifongiques actifs seuls ou dans des formulations commerciales de shampooing contre Malassezia et les bactéries du genre Staphylococcus. MÉTHODES: Les concentrations minimales inhibitrices de trois antifongiques présents dans des shampooings antipelliculaires (pyrithione de zinc, kétoconazole et ciclopirox) ainsi que l'hamamélan, extrait d'hamamélis, ont été évaluées contre des espèces commensales de Malassezia et Staphylococcus en utilisant des méthodes de microdilution en culture. Dans des expériences simulant l'exposition au shampooing et les conditions de lavage sur le cuir chevelu, nous avons également testé la capacité à inhiber la croissance microbienne des agents décrits ci-dessus dans la formulation de shampooings (Head and Shoulders (H&S), Ketomed, Sebiprox, Erol Healthcare Hair Shampoo, respectivement) avec un produit générique trouvé dans le commerce. RÉSULTATS: Les shampooings Ketomed et H&S ont été les traitements les plus efficaces contre Malassezia dans des essais in vitro et dans des expériences de simulation de lavage. Le shampooing Erol Healthcare était moins efficace contre Malassezia in vitro car nécessitant un temps de contact plus long pour obtenir une inhibition de la croissance de certaines espèces. Sebiprox a montré une efficacité variable dans les expériences de lavage et de temps de contact alors que le shampooing générique était le moins efficace pour inhiber la croissance de Malassezia et Staphylococcus. CONCLUSION: Ces résultats suggèrent que les patients avec des pellicules peuvent raisonnablement retirer un bénéfice de l'utilisation d'un shampooing antifongique spécifique bien que les résultats puissent varier selon les espèces microbiennes, la durée du contact et des composants entrant dans la formulation du shampooing.

EAACI Molecular Allergology User's Guide.

The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

Tinea capitis and tinea faciei in the Zurich area - an 8-year survey of trends in the epidemiology and treatment patterns.

BACKGROUND: Tinea capitis and tinea faciei are dermatophyte infections of the scalp and glabrous skin of the face affecting mainly prepubertal children. During the past 30 years, a significant increase and a change in the pattern of infectious agents has been noted for tinea capitis. OBJECTIVES: The aim of this study was to determine trends in the current epidemiological situation of tinea capitis and tinea faciei in the Zurich area, Switzerland and adjacent Central and Eastern Switzerland. METHODS: Consecutive cases diagnosed between 2006 and 2013 were studied retrospectively. RESULTS: A total of 90 tinea capitis and 40 tinea faciei cases were observed. Anthropophilic isolates (primarily Trichophyton violaceum and Microsporum audouinii) accounted for 76% of tinea capitis cases. In contrast, zoophilic isolates (primarily T. interdigitale) were responsible for 73% of tinea faciei cases. The peak incidence in both conditions was in 4-8 year-old children. While the annual number of tinea faciei cases remained stable over the past 8 years, a trend for an increase in T. violaceum-positive tinea capitis has been observed. This was mainly due to patients of African ethnicity. CONCLUSIONS: Anthropophilic isolates accounted for three quarters of tinea capitis and one quarter of tinea faciei cases. T. violaceum-positive tinea capitis was primarily linked to patients of African ethnicity. Tinea capitis caused by Microsporum spp. was more refractory to therapy and needed longer treatment than Trichophyton spp.-induced infection.

Cryptococcus gattii meningoencephalitis in an immunocompetent person 13 months after exposure.

A 53-year old immunocompetent Swiss female is described who developed severe meningoencephalitis due to infection with Cryptococcus gattii 13 months following exposure on Vancouver Island, Canada. Diagnosis was based on cerebrospinal fluid (CSF) examination, i.e., positive India-ink staining, positive latex particle agglutination, and positive culture. Species identification was performed by growth on L-canavanine-glycine-bromthymol blue medium and by sequencing of the intergenic and internal transcribed spacer regions of the rRNA genes. After initial therapy with fluconazole by which the patient did not improve, therapy was changed to amphotericin B and flucytosine and later to high-dose fluconazole and amphotericin B. Despite long-term treatment and external drainage of the CSF, the patient's condition improved only slowly. The patient was discharged after 132 days of hospitalization.

A multicenter prospective trial to asses a new real-time polymerase chain reaction for detection of Treponema pallidum, herpes simplex-1/2 and Haemophilus ducreyi in genital, anal and oropharyngeal ulcers.

Treponema pallidum, herpes simplex virus types 1 or 2 (HSV-1/2) and Haemophilus ducreyi are sexually transmitted pathogens that can cause genital, anal and oropharyngeal ulcers. Laboratory evaluation of these pathogens in ulcers requires different types of specimens and tests, increasing the risk of improper specimen handling and time lapse until analysis. We sought to develop a new real-time PCR (TP-HD-HSV1/2 PCR) to facilitate the detection of T. pallidum, HSV-1/2 and H. ducreyi in ulcers. The TP-HD-HSV1/2 PCR was tested (i) in a retrospective study on 193 specimens of various clinical origin and (ii) in a prospective study on 36 patients with genital, anal or oropharyngeal ulcers (ClinicalTrials.gov # NCT01688258). The results of the TP-HD-HSV1/2 PCR were compared with standard diagnostic methods (T. pallidum: serology, dark field microscopy; HSV-1/2: PCR; H. ducreyi: cultivation). Sensitivity and specificity of the TP-HD-HSV1/2 PCR for T. pallidum were both 100%, for HSV-1 100% and 98%, and for HSV-2 100% and 98%, respectively. T. pallidum and HSV-1/2 were detected in 53% and 22% of patients in the prospective study; H. ducreyi was not detected. In the prospective study, 5/19 (26%) specimens were true positive for T. pallidum in the TP-HD-HSV1/2 PCR but non-reactive in the VDRL. The TP-HD-HSV1/2 PCR is sensitive and specific for the detection of T. pallidum and HSV-1/2 in routine clinical practice and it appears superior to serology in early T. pallidum infections.

Nodular secondary syphilis in a woman.

We report the case of a 21-year-old woman with symmetrically distributed, ulcerated nodules and plaques on the face, neck and arms. Initial differential diagnoses included pyoderma or sarcoidosis based on the clinical presentation and histopathology with non-caseating granulomas. After inefficient treatment with topical and systemic fusidic acid and steroids, we diagnosed nodular secondary syphilis owing to positive serology and immunohistochemical staining of Treponema pallidum in lesional skin. After treatment with benzathine penicillin, skin lesions improved and antibody titres declined significantly within 3 months. Nodular skin lesions in secondary syphilis are rare with 15 reported cases within the last 20 years. Furthermore, the granulomatous histology is often misleading. Our patient's case suggests that the physicians should be aware of syphilis as a possible differential diagnosis also in patients outside a high-risk population for sexually transmitted diseases and with an unusual clinical presentation.

Evaluation of the colorimetric VITEK 2 card for identification of gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing.

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.

Internal transcribed spacer sequencing versus biochemical profiling for identification of medically important yeasts.

In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as Candida rugosa or Candida utilis. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region.

16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory.

Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non-Pseudomonas aeruginosa) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.

Seasonal and spatial community dynamics in the meromictic Lake Cadagno.

The seasonal and spatial variations in the community structure of bacterioplankton in the meromictic alpine Lake Cadagno were examined by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Two different amplifications were performed, one specific for the domain Bacteria (Escherichia coli positions 8-536) and another specific for the family Chromatiaceae (E. coli positions 8-1005). The latter was followed by semi-nested reamplification with the bacterial primer set, allowing comparison of the two PCR approaches by TTGE. The TTGE patterns of samples from the chemocline and the anoxic monimolimnion were essentially identical, whereas the oxic mixolimnion displayed distinctively different banding patterns. For samples from the chemocline and the monimolimnion, dominant bands in the Bacteria-specific TTGE profiles comigrated with bands obtained by the semi-nested PCR approach specific for Chromatiaceae. This observation suggested that Chromatiaceae are in high abundance in the anoxic water layer. All dominant bands were excised and sequenced. Changes in the community structure, as indicated by changes in the TTGE profiles, were observed in samples taken at different times of the year. In the chemocline, Chomatium okenii was dominant in the summer months, whereas Amoebobacter purpureus populations dominated in autumn and winter. This change was confirmed by fluorescent in situ hybridization.

Comparison of conventional and molecular methods for identification of aerobic catalase-negative gram-positive cocci in the clinical laboratory.

Over a period of 18 months we have evaluated the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic catalase-negative gram-positive cocci in the clinical laboratory. A total of 171 clinically relevant strains were studied. The results of molecular analyses were compared with those obtained with a commercially available phenotypic identification system (API 20 Strep system; bioMérieux sa, Marcy l'Etoile, France). Phenotypic characterization identified 67 (39%) isolates to the species level and 32 (19%) to the genus level. Seventy-two (42%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 138 (81%) isolates to the species level and 33 (19%) to the genus level. For 42 of 67 isolates assigned to a species with the API 20 Strep system, molecular analyses yielded discrepant results. Upon further analysis it was concluded that among the 42 isolates with discrepant results, 16S rDNA sequencing was correct for 32 isolates, the phenotypic identification was correct for 2 isolates, and the results for 8 isolates remained unresolved. We conclude that 16S rDNA sequencing is an effective means for the identification of aerobic catalase-negative gram-positive cocci. With the exception of Streptococcus pneumoniae and beta-hemolytic streptococci, we propose the use of 16S rDNA sequence analysis if adequate species identification is of concern.

Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation).

We have evaluated over a period of 18 months the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: (i) 37 clinical strains of gram-positive rods well identified by phenotypic tests, and (ii) 136 clinical isolates difficult to identify by standard microbiological investigations, i.e., identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2%) isolates at the genus level; 65 of 136 (47.8%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4%) isolates at the species level, 43 of 136 (31.6%) isolates at the genus level, and 4 of 136 (2.9%) isolates at the family level. We conclude that (i) rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques, and (ii) molecular identification procedures are not required for isolates well identified by phenotypic investigations.

Quantitative detection of Streptococcus pneumoniae in nasopharyngeal secretions by real-time PCR.

Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 10(6) cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient of S. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae, Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were > or =10(6) cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections.