Evaluation of a membrane hybridization array for detection of Mycobacterium tuberculosis complex and resistance to isoniazid and rifampin in sputum specimens, mycobacterial liquid cultures, and clinical isolates.

The gold standard of antituberculosis susceptibility testing is based on culture method which takes weeks. Rapid detection of resistance to isoniazid (INH) and rifampin (RIF) to avoid inappropriate regimens and to prevent transmission of resistant strains are important. A membrane array (BluePoint MTBDR) was developed to identify Mycobacterium tuberculosis complex (MTBC) and the genetic mutations responsible for resistance to RIF and INH. We aimed to evaluate the performance of this array for diagnosing drug-resistant MTBC. A total of 261 acid-fast bacilli positive sputum specimens, 1025 positive mycobacteria growth indicator tube (MGIT) cultures and 544 clinical isolates were analyzed. Antituberculosis susceptibility testing was the gold standard and was performed on MTBC isolated from positive MGIT cultures and on 544 clinical isolates. The sensitivity and specificity of the array to detect MTBC were 62.2% and 88.1% for sputum specimens, 100% and 97.9% for MGIT cultures. For detection of drug-resistant MTBC in positive MGIT tubes, the sensitivities of the array were 100% for RIF and 97.1% for INH, while the specificities were 99.7% and 100%, respectively. Interestingly, we noticed four genotypically RIF-resistant but phenotypically RIF-susceptible isolates and eight genotypically INH resistant but phenotypically INH-susceptible isolates. Comparing with conventional culture methods for species identification and drug susceptibility testing, the BluePoint MTBDR assay demonstrated to be a rapid test with high sensitivity and specificity to identify MTBC and to detect isoniazid and rifampin resistance when it is applied to broth culture specimens and clinical isolates.

Tsukamurella tyrosinosolvens bacteremia with coinfection of Mycobacterium bovis pneumonia: case report and literature review.

INTRODUCTION: We describe an immunocompromised patient with Tsukamurella tyrosinosolvens bacteremia and coinfection of Mycobacterium bovis pneumonia. CASE DESCRIPTION: A 75-year-old male was admitted to our hospital complaining of persistent fever with general malaise. His medical history showed that he had diabetes mellitus (HbA1C 9.2%). A chest computed tomography (CT) showed left upper lung consolidation . Two sets of blood culture at admission finally showed Tsukamurella tyrosinosolvens. Moreover, three transbronchoscopy washing specimen cultures revealed Mycobacterium bovis. DISCUSSION AND EVALUATION: The organism Tsukamurella tyrosinosolvens was identified using conventional biochemical identification methods, PCR-restriction DNA fragment analysis, and 16S rRNA gene sequencing. The clinical mycobacterial isolates were identified to the species level by combining Polymerase Chain Reaction (PCR) with an oligonucleotide microarray to detect the M. bovis amplicons. CONCLUSION: According to our literature review, our patient's case was the first of a coinfection with Tsukamurella tyrosinosolvens and Mycobacterium bovis. Prolonged antibiotic treatment and underlying disease control are necessary for this type of patient.

Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array.

Phenotypic identification of coagulase-negative staphylococci (CoNS) is difficult and many staphylococcal species carry mecA. This study developed an array that was able to detect mecA and identify 30 staphylococcal species by targeting the internal transcribed spacer regions. A total of 129 target reference strains (30 species) and 434 clinical isolates of staphylococci were analyzed. Gene sequencing of 16S rRNA, gap or tuf genes was the reference method for species identification. All reference strains (100%) were correctly identified, while the identification rates of clinical isolates of S. aureus and CoNS were 98.9% and 98%, respectively. The sensitivity and specificity for mecA detection were 99% and 100%, respectively, in S. aureus isolates, and both values were 100% in isolates of CoNS. The assay takes 6 h from a purified culture isolate, and so far it has not been performed directly on patient samples.

Multiple Brain Abscesses Due to Aspergillus Fumigatus in a Patient With Liver Cirrhosis: A Case Report.

Invasive cerebral aspergillosis always developed in immunocompromised host. Early diagnosis may save life in this critical condition; however, it is difficult to reach. Herein, we presented an unusual case of invasive cerebral aspergillosis in a cirrhotic patient. A 47-year-old man presented with progressive deterioration of consciousness for three days. The patient had a history of alcoholic liver cirrhosis, Child-Pugh class C. Magnetic resonance imaging (MRI) of brain showed multi-focal parenchymal lesions, which was consistent with multiple brain abscesses. The diagnosis of invasive cerebral aspergillosis was made by molecular based laboratory methods including Aspergillus galactomannan antigen assay and oligonucleotide array. Despite treatment with the antifungal agent, Amphotericin B, the patient died at the ninth day of hospitalization. Our findings suggest that liver cirrhosis can be one of risk factors of invasive cerebral aspergillosis, and support the diagnosing usefulness of MRI, Aspergillus galactomannan antigen assay, and oligonucleotide array.

A cluster of endophthalmitis caused by Mycobacterium abscessus after cataract surgery.

We report two cases of postoperative endophthalmitis after cataract surgery caused by the same strain of Mycobacterium abscessus confirmed by arbitrarily primed polymerase chain reaction, sequencing of the erythromycin ribosome methyltransferase gene and pulsed-field gel electrophoresis. The outcomes were poor despite aggressive treatments. This is the first report of nontuberculous mycobacteria as a causative pathogen for a cluster of endophthalmitis.

Optimization of the score cutoff value for routine identification of Staphylococcus species by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Staphylococcus species are important pathogens. We evaluated 2 score cutoffs (2.0 and 1.7) and the replicate number (a single or a duplicate test) on the identification of staphylococci using the Bruker matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A collection of 440 clinical isolates (11 species) and 144 reference strains (36 species) was evaluated. For clinical isolates using a cutoff of 2.0 and duplicate tests, the rates of species, genus, and unreliable identifications were 93.4%, 5.7%, and 0.9% respectively, while the respective values were 99.3%, 0.2%, and 0.5% when the cutoff was 1.7. The species identification rates were significantly higher (P<0.01) when a cutoff of 1.7 or a duplicate test was used. Similar results were obtained for reference strains. In conclusion, a cutoff of 1.7 and duplicate tests are recommended for identification of staphylococci using MALDI-TOF MS.

Performance Assessment of the BluePoint MycoID Plus Kit for Identification of Mycobacterium tuberculosis, Including Rifampin- and Isoniazid-resistant Isolates, and Nontuberculous Mycobacteria.

The performance of the BluePoint MycoID plus kit (Bio Concept Corporation, Taichung, Taiwan), which was designed to simultaneously detect Mycobacterium tuberculosis (MTB), rifampin- and isoniazid-resistant MTB, and nontuberculous mycobacteria (NTM) was first evaluated with 950 consecutive positive cultures in Mycobacterium Growth Indicator Tube (MGIT) system (BACTEC, MGIT 960 system, Becton-Dickinson, Sparks) from clinical respiratory specimens. The discrepant results between kit and culture-based identification were finally assessed by 16S rRNA gene sequencing and clinical diagnosis. The accuracy rate of this kit for identification of all Mycobacterium species was 96.3% (905/940). For MTB identification, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the kit were 99.7%, 99.3%, 99.0% and 99.8%, respectively. For rifampicin-resistant MTB identification, the sensitivity, specificity, PPV, and NPV of the kit were 100.0%, 99.4%, 91.3%, and 100.0%, respectively, while the corresponding values of isoniazid-resistant MTB identification were 82.6%, 99.4%, 95.0%, and 97.6%, respectively. In identifying specific NTM species, the kit correctly identified 99.3% of M. abscessus (147/148) complex, 100% of M. fortuitum (32/32), M. gordonae (38/38), M. avium (39/39), M. intracellulare (90/90), M. kansasii (36/36), and M. avium complex species other than M. avium and M. intracellulare (94/94). In conclusions, the diagnostic value of the BluePoint MycoID plus kit was superior to culture method for recoveries and identification of NTM to species level. In addition, the diagnostic accuracy of BluePoint MycoID plus kit in MTB identification was similar to conventional culture method with high accuracy rate of rifampicin-resistant M. tuberculosis identification.

Early diagnosis and successful treatment of Cryptococcus albidus keratitis: a case report and literature review.

Cryptococcus albidus keratitis is a rare and difficult diagnosed disease. Here we report a case of C albidus keratitis early diagnosed by dot hybridization assay and successfully treated with intrastromal injection of Amphotericin B (AB).A 45-year-old man presented with left red eye for 2 days. The slit lamp examination exhibited deep corneal infiltrations. Smears and cultures were performed but revealed negative findings. Molecular detection of pathogens was performed by dot hybridization assay, and C albidus keratitis was diagnosed. Despite the identification of C albidus, the clinical condition still worsened due to deep corneal infiltration. After performing intrastromal injection of AB, the corneal infiltration gradually improved.C albidus is a rare cause of diseases in humans and should be considered as a potential pathogen of corneal ulcer. The prognosis of C albidus keratitis will improve if the condition is recognized early and treated properly.

Increased resistin may suppress reactive oxygen species production and inflammasome activation in type 2 diabetic patients with pulmonary tuberculosis infection.

Although it has been known for decades that patients with type 2 diabetes mellitus (DM) are more susceptible to severe tuberculosis (TB) infection, the underlying immunological mechanisms remain unclear. Resistin, a protein produced by immune cells in humans, causes insulin resistance and has been implicated in inhibiting reactive oxygen species (ROS) production in leukocytes. Recent studies suggested that IL-1ß production in patients with Mycobacteria tuberculosis infection correlates with inflammasome activation which may be regulated by ROS production in the immune cells. By investigating the level of resistin in different patient groups, we found that serum resistin levels were significantly higher in severe TB and DM-only groups when compared with mild TB cases and healthy controls. Moreover, elevation of serum resistin correlated with impairment of ROS production of neutrophils in patients with both DM and TB. In human macrophages, exogenous resistin inhibits the production of ROS which are important in the mycobacterium-induced inflammasome activation. Moreover, macrophages with defective ROS production had poor IL-1ß production and ineffective control of mycobacteria growth. Our results suggest that increased resistin in severe TB and DM patients may suppress the mycobacterium-induced inflammasome activation through inhibiting ROS production by leukocytes.

Rapid detection of dermatophytes and Candida albicans in onychomycosis specimens by an oligonucleotide array.

BACKGROUND: Onychomycosis is a fungal infection of nails, leading to the gradual destruction of the nail plate. Treatment of onychomycosis may need long-time oral antifungal therapy that can have potential side effects, thus accurate diagnosis of the disease before treatment is important. Culture for diagnosis of onychomycosis is time-consuming and has high false-negative rates. To expedite the diagnosis, an oligonucleotide array, based on hybridization between immobilized oligonucleotide probes and PCR products, for direct detection of dermatophytes and Candida albicans in clinical specimens was evaluated. METHODS: Species-specific oligonucleotide probes designed from the internal transcribed spacer (ITS) regions of the rRNA gene were immobilized on a nylon membrane. The assay procedures consisted of PCR amplification of the ITS using universal primers, followed by hybridization of the digoxigenin-labeled amplicons to probes on the array. Thirty two nail samples (29 patients) were analyzed by the array, and the results were compared with those obtained by culture. Array-positive but culture-negative samples were confirmed by cloning and re-sequencing of the amplified ITS and by reviewing patient's clinical data. The total recovery of culture and confirmed array-positive but culture-negative results was considered 100% and was used for performance evaluation of both methods. RESULTS: Concordant results were obtained in 21 samples (10 positives and 11 negatives) by both methods. Eleven samples were array-positive but culture-negative; among them, 9 samples were considered true positives after discrepant analysis. Comparing with culture, the array had significantly higher sensitivity [100% (95% CI 82.2% -100%) vs 52.6% (28.9% -75.5%), p <0.001] and negative predictive value [100% (71.3% -100%) vs 59.1% (36.4% -79.3%), p <0.05), while no significant differences were observed in specificity (84.6% vs 100%, p =0.48) and positive predictive value (90.5% vs 100%, p =1.0). The whole procedures of the array were about 24 h, whilst results from culture take 1 to 3 weeks. CONCLUSIONS: The array offers an accurate and rapid alternative to culture. Rapid diagnosis can expedite appropriate antifungal treatment of onychomycosis. However, the single site nature of this study conducted at a referral hospital invites caution.

Evaluation of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of blood isolates of Acinetobacter species.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Biotyper) was able to accurately identify 98.6% (142/144) of Acinetobacter baumannii isolates, 72.4% (63/87) of A. nosocomialis isolates, and 97.6% (41/42) of A. pittii isolates. All Acinetobacter junii, A. ursingii, A. johnsonii, and A. radioresistens isolates (n = 28) could also be identified correctly by Bruker Biotyper.

Acute meningitis caused by Cladosporium sphaerospermum.

Phaeohyphomycosis of the central nervous system is rare but typically associated with high mortality. Treatment has not been standardized, but the combination of antifungal chemotherapy with surgical debridement is recommended. We report a 73-year-old, retired, male timber merchant with acute meningitis caused by Cladosporium sphaerospermum. The patient, who had well-controlled type 2 diabetes mellitus, presented with fever and weakness of the lower limbs. No brain abscess was apparent by cranial computed tomography. C. sphaerospermum was isolated from the cerebral spinal fluid and identified based on both morphology and DNA sequencing. He was treated with combination antifungal chemotherapy with amphotericin B and voriconazole for 28 days, followed by voriconazole monotherapy for 46 days. To date, the patient has recovered without significant sequelae. This patient represents the first reported case of cerebral phaeohyphomycosis caused by C. sphaerospermum. Moreover, the therapy was successful for totally less than 3 months of treatment duration.

Acrophialophora fusispora brain abscess in a patient with acquired immunodeficiency syndrome: a case report and review of the literature.

We reported a fatal case of brain abscess caused by Acrophialophora fusispora in a patient with acquired immunodeficiency syndrome. Identification of the fungus was based on microscopic morphology and sequence analyses of the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of ribosomal RNA gene from the isolate recovered from brain abscess. Four published cases were reviewed as well.

Evaluation of a membrane array for detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria in positive liquid cultures.

Molecular identification of mycobacteria in positive Mycobacteria Growth Indicator Tube (MGIT) cultures can accelerate mycobacterial diagnosis. A membrane hybridization array (Blue Point) was evaluated for this purpose in 284 positive MGIT cultures. Discrepant results were resolved by testing with the GenoType Mycobacterium kit, TBc ID test, sequencing of the 16S rRNA gene and internal transcribed spacer. Total recovery from culture and the array (if confirmed) was considered 100%. The sensitivity, specificity, positive, and negative predictive values of the array for detection of Mycobacterium tuberculosis complex were 99.4%, 100%, 100%, and 99.2%, respectively, while the corresponding values of culture were 95.1%, 100%, 100%, and 93.8%, respectively, with significant differences in sensitivity and negative predictive value being found between the 2 methods. The recoveries of nontuberculous mycobacteria and mixed cultures of the array were also significantly higher than those of culture. The array can be adopted in routine mycobacteriology laboratory.

Catheter-related fungemia caused by Candida dubliniensis.

Infections caused by Candida dubliniensis in humans are rare and have never been reported in Taiwan. We report two cancer patients with catheter-related fungemia due to C. dubliniensis infection in Taiwan. The two isolates were confirmed to the species level using an oligonucleotide array system and sequence analysis, and both showed high in vitro susceptibilities to nine antifungal agents. The catheters were removed, and both patients responded well to antifungal treatment. Although this type of infection is rare, physicians should consider C. dubliniensis as one of the possible pathogens causing catheter-related infections in Taiwan.

Impact of molecular diagnosis on treating Mendelian susceptibility to mycobacterial diseases.

BACKGROUND/OBJECTIVE: The IL-12-IFN-γ axis is critical for immune defense against mycobacterial infections. Inherited mutations that affect normal activation of this self-amplifying cytokine reaction lead to increased chances of mycobacterial infections, known as Mendelian susceptibility to mycobacterial diseases (MSMD). Delayed diagnosis and difficulty in identifying pathogenic mycobacteria hinder proper treatment of patients, so the aim of this study was to facilitate the diagnosis of mycobacterial infections in MSMD patients using an oligonucleotide array method. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from three MSMD patients in the same family. A series of immunologic studies, including testing for cytokine secretion after leukocyte stimulation, cell-surface marker analysis, and cDNA sequencing, were then performed. An oligonucleotide array was used to rapidly identify pathogens. RESULTS: Cytokine secretion testing showed normal IFN-γ secretion after IL-12 stimulation but low IL-12 secretion after IFN-γ stimulation, which indicates a defect in the IFN-γ receptor or its intracellular signaling. Cell-surface receptor analysis showed IFN-γ receptor 1 overexpression, suggesting an autosomal dominant IFN-γ receptor 1 deficiency. cDNA sequencing identified the IFNGR1 818del4 mutation in three members of the family with known MSMD, and an oligonucleotide array identified Mycobacterium tuberculosis complex and Mycobacterium abscessus as pathogens. CONCLUSIONS: Patients with suspected MSMD should undergo molecular diagnosis of the primary immunodeficiency. Oligonucleotide array methods may be a tool for rapid identification of pathogens and for guiding antimicrobial treatment in immunodeficient patients.

Identification of lethal Aspergillus at early growth stages based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Delayed and incorrect diagnoses are potential risk factors leading to high mortality of invasive aspergillosis (IA). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to acquire a wide mass spectral range and characterize the early process of asexual sporulation of lethal IA pathogens recovered on agar plates. Proteins were extracted using trifluoroacetic acid and soft ionized using an ultraviolet laser with the assistance of ferulic acid. At the second stage of sporulation with various differentiated structures, there are more specific peaks that can be used to discriminate different Aspergillus species than at the first stage, which features vegetative hyphae. Certain specific peaks are found in different strains of the same species, Aspergillus fumigatus. In addition, the relative standard deviations of the m/z ratios are much smaller than those of the relative intensities in these peaks. Therefore, common lethal Aspergillus species can be identified after short-term cultivation by matching species-specific m/z values.