Treatment of community acquired pneumonia (CAP) patients with antibiotics before laboratory-confirmed diagnosis leads to loss of knowledge on the causative bacterial pathogen. Therefore, an increasing number of pneumococcal infections is identified using non-culture based techniques. However, methods for serotyping directly on the clinical specimen remain scarce. Here we present three approaches for detection and serotyping of pneumococci using samples from patients with CAP.
The first approach is quantitative PCR (qPCR) analysis on blood samples (n = 211) followed by capsular sequence typing (CST) to identify the serotype. The second approach, a urinary antigen assay (n = 223), designated as inhibition multiplex immunoassay (IMIA), is based on Luminex technology targeting 14 serotypes. The third approach is a multiplex immunoassay (MIA) (n = 171) also based on Luminex technology which detects serologic antibody responses against 14 serotypes. The three alternative assays were performed on samples obtained from 309 adult hospitalized CAP patients in 2007–2010 and the results were compared with those obtained from conventional laboratory methods to detect pneumococcal CAP, i.e. blood cultures, sputum cultures and BinaxNOW® urinary antigen tests.
Using qPCR, MIA and IMIA, we were able to detect the pneumococcus in samples of 56% more patients compared to conventional methods. Furthermore, we were able to assign a serotype to the infecting pneumococcus from samples of 25% of all CAP patients, using any of the three serotyping methods (CST, IMIA and MIA).
This study indicates the usefulness of additional molecular methods to conventional laboratory methods for the detection of pneumococcal pneumonia. Direct detection and subsequent serotyping on clinical samples will improve the accuracy of pneumococcal surveillance to monitor vaccine effectiveness.
Streptococcus pneumoniae; Pneumococcus; Community acquired pneumonia; Detection; Serotype; Blood; Urine
Since the separation of Pseudallescheria boydii and P. apiosperma in 2010, limited data on species-specific susceptibility patterns of these and other species of Pseudallescheria and its anamorph Scedosporium have been reported. This study presents the antifungal susceptibility patterns of members affiliated with both entities. Clinical and environmental isolates (n = 332) from a wide range of sources and origins were identified down to species level and tested according to CLSI M38-A2 against eight antifungal compounds. Whereas P. apiosperma (geometric mean MIC/minimal effective concentration [MEC] values of 0.9, 2.4, 7.4, 16.2, 0.2, 0.8, 1.5, and 6.8 μg/ml for voriconazole, posaconazole, isavuconazole, itraconazole, micafungin, anidulafungin, caspofungin, and amphotericin B, respectively) and P. boydii (geometric mean MIC/MEC values of 0.7, 1.3, 5.7, 13.8, 0.5, 1.4, 2.3, and 11.8 μg/ml for voriconazole, posaconazole, isavuconazole, itraconazole, micafungin, anidulafungin, caspofungin, and amphotericin B, respectively) had similar susceptibility patterns, those for S. aurantiacum, S. prolificans, and S. dehoogii were different from each other. Voriconazole was the only drug with significant activity against S. aurantiacum isolates. The MIC distributions of all drugs except voriconazole did not show a normal distribution and often showed two subpopulations, making a species-based prediction of antifungal susceptibility difficult. Therefore, antifungal susceptibility testing of all clinical isolates remains essential for targeted antifungal therapy. Voriconazole was the only compound with low MIC values (MIC90 of ≤2 μg/ml) for P. apiosperma and P. boydii. Micafungin and posaconazole showed moderate activity against the majority of Scedosporium strains.
Schizophyllum commune (n = 30) showed lowest geometric mean MICs of isavuconazole (0.19 μg/ml), itraconazole (0.2 μg/ml), voriconazole (0.24 μg/ml), and amphotericin B (0.29 μg/ml) and high geometric mean MICs of fluconazole (19.39 μg/ml) and flucytosine (17.28 μg/ml). Five cases (of 8) of allergic bronchopulmonary mycosis that were treated with itraconazole had no recrudescence after 6 to 24 months of follow-up. One case each of invasive pulmonary mycosis and fungal ball were treated successfully with voriconazole and itraconazole.
Simple, low-cost PCR/PCR-restriction fragment length polymorphism (RFLP) assays targeting cyp51A promoter and codon 98 regions were developed for the detection of triazole-resistant Aspergillus fumigatus strains carrying TR34/L98H mutations. The assays were evaluated using 40 itraconazole-susceptible isolates and 35 itraconazole-resistant isolates. The prevalence of TR34/L98H mutations in clinical/environmental A. fumigatus isolates may now be determined easily from resource-poor settings.
The incidence of invasive pneumococcal disease (IPD) rises with age. Among adult IPD patients, the avidity of antipneumococcal polysaccharide antibodies against the infecting serotype increased with age and severity of disease, indicating that susceptibility to IPD in the elderly may rather be due to flaws in other aspects of opsonophagocytosis.
Fusarium species are among the most common fungi present in the environment and some species have emerged as major opportunistic fungal infection in human. However, in immunocompromised hosts they can be virulent pathogens and can cause death. The pathogenesis of this infection relies on three factors: colonization, tissue damage, and immunosuppression. A novel Fusarium species is reported for the first time from keratitis in an agriculture worker who acquired the infection from plant material of maize. Maize plants are the natural host of this fungus where it causes stalk rot and seeding malformation under temperate and humid climatic conditions. The clinical manifestation, microbiological morphology, physiological features and molecular data are described.
Diagnosis was established by using polymerase chain reaction of fungal DNA followed by sequencing portions of translation elongation factor 1 alpha (TEF1 α) and beta-tubulin (BT2) genes. Susceptibility profiles of this fungus were evaluated using CLSI broth microdilution method.
The analyses of these two genes sequences support a novel opportunist with the designation Fusarium temperatum. Phylogenetic analyses showed that the reported clinical isolate was nested within the Fusarium fujikuroi species complex. Antifungal susceptibility testing demonstrated that the fungus had low MICs of micafungin (0.031 μg/ml), posaconazole (0.25 μg/ml) and amphotericin B (0.5 μg/ml).
The present case extends the significance of the genus Fusarium as agents of keratitis and underscores the utility of molecular verification of these emerging fungi in the human host.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-014-0588-y) contains supplementary material, which is available to authorized users.
Keratitis; Fusarium temperatum; Maize; Molecular phylogenetics; Infection
Cyphellophora guyanensis (n = 15), other Cyphellophora species (n = 11), Phialophora europaea (n = 43), and other Phialophora species (n = 12) were tested in vitro against nine antifungal drugs. The MIC90s across all of the strains (n = 81) were, in increasing order, as follows: posaconazole, 0.063 μg/ml; itraconazole, 0.5 μg/ml; voriconazole, 1 μg/ml; micafungin, 1 μg/ml; terbinafine, 2 μg/ml; isavuconazole, 4 μg/ml; caspofungin, 4 μg/ml; fluconazole, 8 μg/ml; amphotericin B, 16 μg/ml.
A. fumigatus cyp51A–mediated resistance to azole drugs is rare in the United States.
Emergence of Aspergillus fumigatus strains containing mutations that lead to azole resistance has become a serious public health threat in many countries. Nucleotide polymorphisms leading to amino acid substitutions in the lanosterol demethylase gene (cyp51A) are associated with reduced susceptibility to azole drugs. The most widely recognized mutation is a lysine to histidine substitution at aa 98 (L98H) and a duplication of the untranscribed promoter region, together known as TR34/L98H. This mechanism of resistance has been reported in Europe, Asia, and the Middle East, and is associated with resistance to all azole drugs and subsequent treatment failures. To determine whether isolates with this mutation are spreading into the United States, we conducted a passive surveillance–based study of 1,026 clinical isolates of A. fumigatus from 22 US states during 2011–2013. No isolates harboring the TR34/L98H mutation were detected, and MICs of itraconazole were generally low.
antifungal resistance; azole; azole resistance; fungi; Aspergillus fumigatus; CYP51A; TR34/L98H; susceptibility testing; passive surveillance; United States
Cryptococcus gattii; the Netherlands; Europe; fungi; climate
Nocardia species are rare, opportunistic organisms that cause disease in both immunocompetent and immunocompromised individuals.
To investigate the clinical presentations of various Nocardia infections based on the 16S ribosomal RNA gene of the isolate, as well as related risk factors and susceptibility patterns to antimicrobial agents
Thirteen patients with a diagnosis of nocardiosis were included in the present study. Seven Nocardia species were identified by 16S ribosomal RNA. Susceptibility testing was performed using six antimicrobial agents.
Five patients were immunocompromised, and eight were immunocompetent with predisposing factors including cystic fibrosis, tuberculosis and ophthalmic infections. Nocardia caused pulmonary infections in eight patients (61.5%), invasive systemic infections in three patients (23%) and local (ophthalmic) infections in two patients (15.4%). In the patients with pulmonary disease, nocardiosis was caused by six species (Nocardia cyriacigeorgica, Nocardia otitidiscaviarum, Nocardia farcinica, Nocardia carnea, Nocardia testacea and Nocardia asiatica). The seventh species identified in the present study was Nocardia crassostreae.
N crassostreae is a multidrug-resistant organism that was reported to be an emerging human pathogen causing invasive nocardiosis in a patient with non-Hodgkin’s lymphoma. N farcinica was isolated from blood in a patient with breast cancer. None of the Nocardia isolates were resistant to linezolid. One N otitidiscaviarum isolate was a multidrug-resistant organism. All patients in the present study were treated with the appropriate antibiotics and their condition resolved without further sequelae.
The present study is the first report on N crassostreae as a human pathogen. The detection of multidrug-resistant species necessitate molecular identification and susceptibility testing, and should be performed for all Nocardia infections. Nocardiosis manifests various clinical features depending on the Nocardia species and underlying conditions.
Antibiotic susceptibility; Clinical cases; Molecular identification; Nocardia; Nocardia crassostreae
We describe a calorimetric assay for detection of voriconazole-resistant Aspergillus fumigatus within 8 h. Among 27 genetically distinct strains, all 21 resistant and all 6 susceptible strains were correctly identified by measurement of fungal heat production in the presence of voriconazole. This proof-of-concept study demonstrates the potential of microcalorimetry for rapid detection of azole resistance in A. fumigatus.
Nonsporulating molds (NSMs), especially basidiomycetes, have predominantly been reported as human pathogens responsible for allergic and invasive disease. Their conventional identification is problematic, as many isolates remain sterile in culture. Thus, inconclusive culture reports might adversely affect treatment decisions. The clinical significance of NSMs in pulmonary mycoses is poorly understood. We sequenced the internal transcribed spacer (ITS) region and D1/D2 domain of the larger subunit (LSU) of 52 NSMs isolated from respiratory specimens. The basidiomycetes were the predominant NSMs, of which Schizophyllum commune was the most common agent in allergic bronchopulmonary mycosis (ABPM), followed by Ceriporia lacerata in invasive fungal disease. Porostereum spadiceum, Phanaerochaete stereoides, Neosartorya fischeri, and Marasmiellus palmivorus were the other molds observed. Application of ITS and LSU region sequencing identified 92% of the isolates. The antifungal susceptibility data revealed that all basidiomycetes tested were susceptible to amphotericin B and resistant to caspofungin, fluconazole, and flucytosine. Except for 3 isolates of S. commune and a solitary isolate of M. palmivorus, all basidiomycetes had low MICs for itraconazole, posaconazole, and voriconazole. Basidiomycetes were isolated from patients with ABPM, invasive pulmonary mycosis/pneumonia, or fungal balls. In addition, the majority of the basidiomycetes were isolated from patients with chronic respiratory disorders who were sensitized to one of the basidiomycetous fungi and demonstrated precipitating antibodies against the incriminating fungi, indicating an indolent tissue reaction. Thus, isolation of basidiomycetes from the lower respiratory tract could be significant, and it is important to monitor these patients in order to prevent subsequent lung damage.
We report the first case of community-acquired bacteremia with Burkholderia fungorum, a newly described member of the Burkholderia cepacia complex. A 9-year-old girl sought treatment with septic arthritis in her right knee and ankle with soft tissue involvement. Commercial identification systems did not identify the causative microorganism.
Burkholderia fungorum; Burkholderia infections; Burkholderia cepacia complex; bacteremia; infection
To develop new approaches for the treatment of invasive infections caused by Scedosporium prolificans, the in vitro interaction between amphotericin B and pentamidine against 30 clinical isolates was evaluated using a checkerboard microdilution method based on the National Committee for Clinical Laboratory Standards M38-P guidelines. The interaction between the drugs was analyzed using fractional inhibitory concentration index (FICI) analysis and response surface modeling. Amphotericin B alone was inactive against all the isolates. The geometric mean MIC for pentamidine was 57 μg/ml (range, 8 to 256 μg/ml; MIC at which 50% of the isolates tested were inhibited [MIC50], 64 μg/ml; MIC90, 128 μg/ml). The combination was synergistic against 28 of 30 isolates (93.3%) by FICI analysis and 30 of 30 (100%) by response surface modeling analysis. Antagonism was not observed.
Three isolates of zygomycetes belonging to three different genera (Rhizopus microsporus, Absidia corymbifera, and Apophysomyces elegans) were used to produce a disseminated infection in nonimmunocompromised mice. The therapeutic efficacy of amphotericin B, given intraperitoneally at doses ranging from 0.5 to 4.5 mg/kg of body weight/day, oral itraconazole at 100 mg/kg/day, and oral terbinafine at 150 mg/kg/day was evaluated in this model. The markers of antifungal efficacy were the median survival time, the mortality rate, and the percentage of infected organs. Organ culture was performed along with microscopic direct examinations of tissues to assess the presence of an active infection. An acute and lethal infection was obtained in untreated mice challenged with each of the three strains. The data obtained for direct examinations and qualitative cultures indicate that, due to the nonseptate nature of the hyphae, each technique gives different information and should be used together with the others. Against all three strains, amphotericin B yielded a 90 to 100% survival rate. Itraconazole was inactive against R. microsporus but significantly reduced mortality in mice infected with A. corymbifera or A. elegans. Terbinafine had no beneficial effects against R. microsporus and A. corymbifera despite documented absorption of the drug. Overall, only limited correlations were observed between MICs determined in vitro and in vivo efficacy of the drugs. The efficacy of itraconazole in these models of zygomycosis suggests that this drug, as well as the new azole compounds presently under development, warrants close evaluation.
The susceptibilities of 25 clinical isolates of Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, and A. ustus to itraconazole and amphotericin B were determined by an agar diffusion-dilution method (the Etest method) and a colorimetric broth microdilution method (the Sensititre method); and the results were compared with those obtained by the NCCLS proposed standard M-38P method for antifungal susceptibility testing of filamentous fungi. Various MIC endpoints for the three methods were determined visually by four different observers in three blinded experiments, and the reproducibilities among the observers (interobserver agreement) and among the replicates (interexperimental agreement) as well as the levels of agreement between the NCCLS, the Etest, and the Sensititre methods were calculated. High levels of reproducibility (within 1 twofold dilution) were found for the NCCLS method (>95%) with the MIC-0 endpoint (complete inhibition of growth) for both drugs and with the MIC-1 endpoint (slight growth) for itraconazole and for the Sensititre method (>90%) with all MIC endpoints, although for the latter the interexperimental agreement for itraconazole was comparatively lower (83 to 93%). The Etest method was less reproducible (67 to 87%) for both drugs. Using the recommended MIC endpoints, high levels of agreement (within one twofold dilution) between the NCCLS and the Sensititre methods for all species were found for amphotericin B (>77%) but not for itraconazole (<66%), for which the MICs by the Sensititre method were up to 3 twofold dilutions lower than the corresponding MICs by the NCCLS method. The use of the first blue well as an endpoint for the Sensititre method and 48 h of incubation improved the levels of agreement with the NCCLS method. Low levels of agreement between the NCCLS and the Etest methods using the recommended MIC endpoints were found for most species, especially after 48 h of incubation (<50%), when the MICs obtained by the Etest method were up to 9 twofold dilutions higher than the corresponding MICs obtained by the NCCLS method. Relatively better agreement was found after 24 h, although it was species dependent, with the highest levels of agreement (>82%) found for A. terreus and A. ustus for amphotericin B and A. fumigatus for both drugs. Overall, better agreement was found when MIC-0 was used as the MIC endpoint for the NCCLS method for both drugs and when the MICs by the Etest method were determined after 48 h of incubation for itraconazole and after 24 h of incubation for amphotericin B.
Combinations of antimicrobial agents were tested against 35 strains of zygomycetes. The interaction between amphotericin B and rifampin was synergistic or additive. Flucytosine alone was inactive and, upon combination with amphotericin B, synergy was not achieved. The combination of amphotericin B with terbinafine was synergistic for 20% of strains, and the interaction between terbinafine and voriconazole was synergistic for 44% of strains. Antagonism was not observed.
The susceptibilities of 13 clinical isolates of Scedosporium apiospermum and 55 clinical isolates of S. prolificans to new and conventional drugs belonging to three different classes of antifungal agents, the azoles (miconazole, itraconazole, voriconazole, UR-9825, posaconazole), the polyenes (amphotericin B, nystatin and liposomal nystatin), and allylamines (terbinafine), were studied by use of proposed standard M38-P of NCCLS. Low growth-inhibitory antifungal activities were found in vitro for most of the drugs tested against S. prolificans isolates, with the MICs at which 90% of isolates are inhibited (MIC90s) being >8 μg/ml; the MIC90s of voriconazole and UR-9825, however, were 4 μg/ml. S. apiospermum isolates were more susceptible in vitro, with the highest activity exhibited by voriconazole (MIC90s, 0.5 μg/ml), followed by miconazole (MIC90s, 1 μg/ml), UR-9825 and posaconazole (MIC90s, 2 μg/ml), and itraconazole (MIC90s, 4 μg/ml). The MICs of terbinafine, amphotericin B, and the two formulations of nystatin (for which no statistically significant differences in antifungal activities were found for the two species) for S. apiospermum isolates were high. Cross-resistance was observed among all the azoles except posaconazole and among all the polyenes except the lipid formulation. A distribution analysis was performed with the MICs of each drug and for each species. Bimodal and skewed MIC distributions were obtained, and cutoffs indicating the borders of different MIC subpopulations of the distributions were determined on the basis of the normal plot technique. These cutoffs were in many cases reproducible between 48 and 72 h.
A colorimetric assay for antifungal susceptibility testing of Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, and Aspergillus ustus) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 μg of XTT/ml with 25 μM menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 102 CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.
A new clonal strain of Candida auris is an emerging etiologic agent of fungemia in Delhi, India. In 12 patients in 2 hospitals, it was resistant to fluconazole and genotypically distinct from isolates from South Korea and Japan, as revealed by M13 and amplified fragment length polymorphism typing.
Candida auris; fungemia; M13 fingerprinting; AFLP; antifungal susceptibility; India; fungi; parasitic diseases
Cryptococcosis is an important fungal disease in Asia with an estimated 140,000 new infections annually the majority of which occurs in patients suffering from HIV/AIDS. Cryptococcus neoformans variety grubii (serotype A) is the major causative agent of this disease. In the present study, multilocus sequence typing (MLST) using the ISHAM MLST consensus scheme for the C. neoformans/C. gattii species complex was used to analyse nucleotide polymorphisms among 476 isolates of this pathogen obtained from 8 Asian countries. Population genetic analysis showed that the Asian C. neoformans var. grubii population shows limited genetic diversity and demonstrates a largely clonal mode of reproduction when compared with the global MLST dataset. HIV-status, sequence types and geography were found to be confounded. However, a correlation between sequence types and isolates from HIV-negative patients was observed among the Asian isolates. Observations of high gene flow between the Middle Eastern and the Southeastern Asian populations suggest that immigrant workers in the Middle East were originally infected in Southeastern Asia.
The species diversity and identification of black fungi belonging to Cyphellophora and Phialophora, which colonize and infect human skin and nails, were studied using amplified fragment length polymorphism (AFLP). A total of 76 Cyphellophora and Phialophora isolates were evaluated, and their delimitation was compared to earlier studies using multilocus sequencing. The results of the AFLP analysis and sequencing were in complete agreement with each other. Seven species-specific padlock probes for the most prevalent species were designed on the basis of the ribosomal DNA internal transcribed spacer region, and identification of the respective species could easily be achieved with the aid of rolling circle amplification.