Tsukamurella Conjunctivitis: a Novel Clinical Syndrome

In this report, we describe the first three cases of Tsukamurella conjunctivitis in the literature. All three patients presented with congestion of one eye with small amounts of serous discharge for 1 to 2 days. All three recovered after 10 days of treatment with polymyxin B-neomycin or chloramphenicol eyedrops. Sequencing of the 16S rRNA genes of the three isolates recovered from the serous discharge of the three patients showed that they were all Tsukamurella species. The phenotypic characteristics of the isolate obtained from one patient best fit the phenotypic profile of Tsukamurella pulmonis, whereas those of the other two best fit that of Tsukamurella tyrosinosolvens

Agar Block Smear Preparation: a Novel Method of Slide Preparation for Preservation of Native Fungal Structures for Microscopic Examination and Long-Term Storage▿

We describe a novel method of fungal slide preparation named “agar block smear preparation.” A total of 510 agar block smears of 25 fungal strains obtained from culture collections, 90 QC fungal strains, and 82 clinical fungal strains from our clinical microbiology laboratory, which included a total of 137 species of yeasts, molds, and thermal dimorphic fungi, were prepared and examined. In contrast to adhesive tape preparation, agar block smears preserved the native fungal structures, such as intact conidiophores of Aspergillus species and arrangements of conidia in Scopulariopsis brevicaulis. Furthermore, agar block smears allowed examination of fungal structures embedded in the agar, such as the ascomata with ascomal hairs in Chaetomium funicola; pycnidium of Phoma glomerata; the intercalary ovoidal chlamydospores arranged in chains of Fusarium dimerum; and the lateral, spherical chlamydospores arranged in pairs of Fusarium solani. After 1 year of storage, morphological integrity was found to have been maintained in 459 (90%) of the 510 agar block smears. After 3 years of storage, morphological integrity was found to have been maintained in 72 (71%) of the 102 smears prepared in 2006. Agar block smear preparation preserves the native fungal structures and allows long-term storage and examination of fungal structures embedded in the agar, hence overcoming the major drawbacks of adhesive tape preparation. The major roles of agar block smear should be diagnosis for difficult cases, accurate identification of fungal species for clinical management of patients and epidemiological studies, and long-term storage for transportation of slides and education purposes

Complication of Corticosteroid Treatment by Acute Plasmodium malariae Infection Confirmed by Small-Subunit rRNA Sequencing▿

We report a case of acute Plasmodium malariae infection complicating corticosteroid treatment for membranoproliferative glomerulonephritis in a patient from an area where P. malariae infection is not endemic. A peripheral blood smear showed typical band-form trophozoites compatible with P. malariae or Plasmodium knowlesi. SSU rRNA sequencing confirmed the identity to be P. malariae

Lasiodiplodia theobromae Pneumonia in a Liver Transplant Recipient▿

We report a case of Lasiodiplodia theobromae pneumonia in a patient who died 14 days after cadaveric-liver transplantation. His condition was complicated by Enterococcus faecium peritonitis. Direct microscopy analysis of the bronchoalveolar lavage specimens showed septate hyphae. A dematiaceous mold was recovered and identified as L. theobromae by microscopic morphology and EF1α gene sequencing

Seronegative Bacteremic Melioidosis Caused by Burkholderia pseudomallei with Ambiguous Biochemical Profile: Clinical Importance of Accurate Identification by 16S rRNA Gene and groEL Gene Sequencing

An aerobic gram-negative bacterium was isolated from the blood and sputum of an 84-year-old, chair-bound nursing home resident with acute bacteremic pneumonia. Although the phenotypic characteristics suggested that the bacterium could be Burkholderia pseudomallei, the Vitek 1 system (GNI+), which can successfully identify 99% of B. pseudomallei strains, showed that the bacterium was “unidentified.” Immunoglobulin G against the lipopolysaccharide (LPS) of B. pseudomallei, as detected by an LPS-based enzyme-linked immunosorbent assay with 95% sensitivity, was negative in both the acute-phase and convalescent-phase sera. Sequencing of the groEL gene showed that the isolate was B. pseudomallei. Proper identification of the bacterium in this study is crucial, since there would be a radical difference in the duration of antimicrobial therapy

Internal Transcribed Spacer Region Sequence Heterogeneity in Rhizopus microsporus: Implications for Molecular Diagnosis in Clinical Microbiology Laboratories ▿

Although internal transcribed spacer region (ITS) sequence heterogeneity has been reported in a few fungal species, it has very rarely been reported in pathogenic fungi and has never been described in Mucorales, causes of the highly fatal mucormycosis. In a recent outbreak investigation of intestinal mucormycosis due to Rhizopus microsporus infection in patients with hematological malignancies, PCR of the ITS of four of the 28 R. microsporus strains, P11, P12, D3-1, and D4-1, showed thick bands at about 700 bp. Direct sequencing of the purified bands showed frequent double peaks along all of the sequence traces and occasional triple peaks for P12, D3-1, and D4-1. The thick bands of the four R. microsporus strains were purified and cloned. Sequencing of 10 clones for each strain revealed two different ITS sequences for P11 and three different ITS sequences for P12, D3-1, and D4-1. Variations in ITS sequence among the different ribosomal DNA (rDNA) operons in the same strain were observed in only ITS1 and ITS2 and not the 5.8S rDNA region. One copy of P11, P12, and D4-1, respectively, and one copy of P11, P12, D3-1, and D4-1, respectively, showed identical sequences. This represents the first evidence of ITS sequence heterogeneity in Mucorales. ITS sequence heterogeneity is an obstacle to molecular identification and genotyping of fungi in clinical microbiology laboratories. When thick bands and double peaks are observed during PCR sequencing of a gene target, such a strain should be sent to reference laboratories proficient in molecular technologies for further identification and/or genotyping

Diversity of phenotypically non-dermatophyte, non- Aspergillus

ABSTRACTOnychomycosis is most commonly caused by dermatophytes. In this study, we examined the spectrum of phenotypically non-dermatophyte and non-Aspergillus fungal isolates recovered over a 10-year period from nails of patients with onychomycosis in Hong Kong. A total of 24 non-duplicated isolates recovered from 24 patients were included. The median age of the patients was 51 years, and two-thirds of them were males. One-third and two-thirds had finger and toe nail infections respectively. Among these 24 nail isolates, 17 were confidently identified as 13 different known fungal species, using a polyphasic approach. These 13 species belonged to 11 genera and ≥9 families. For the remaining seven isolates, multilocus sequencing did not reveal their definite species identities. These seven potentially novel species belonged to four different known and three potentially novel genera of seven families. 33.3%, 41.7% and 95.8% of the 24 fungal isolates possessed minimum inhibitory concentrations of >1 µg/mL to terbinafine, itraconazole and fluconazole, respectively, the first line treatment of onychomycosis. A high diversity of moulds was associated with onychomycosis. A significant proportion of the isolates were potentially novel fungal species. To guide proper treatment, molecular identification and antifungal susceptibility testing should be performed for these uncommonly isolated fungal species

Outbreak of Intestinal Infection Due to Rhizopus microsporus▿

Sinopulmonary and rhinocerebral zygomycosis has been increasingly found in patients with hematological malignancies and bone marrow transplantation, but intestinal zygomycosis remains very rare in the literature. We investigated an outbreak of intestinal infection due to Rhizopus microsporus in 12 patients on treatment for hematological malignancies over a period of 6 months in a teaching hospital. The intake of allopurinol during hospitalization (P < 0.001) and that of commercially packaged ready-to-eat food items in the preceding 2 weeks (P < 0.001) were found to be independently significant risk factors for the development of intestinal zygomycosis. A total of 709 specimens, including 378 environmental and air samples, 181 food samples, and 150 drug samples, were taken for fungal culture. Among them, 16 samples of allopurinol tablets, 3 prepackaged ready-to-eat food items, and 1 pair of wooden chopsticks were positive for Rhizopus microsporus, which was confirmed by ITS1-5.8S-ITS2 rRNA gene cluster (internal transcribed spacer [ITS]) sequencing. The mean viable fungal counts of allopurinol obtained from wards and pharmacy were 4.22 × 103 CFU/g of tablet (range, 3.07 × 103 to 5.48 × 103) and 3.24 × 103 CFU/g of tablet (range, 2.68 × 103 to 3.72 × 103), respectively, which were much higher than the mean count of 2 × 102 CFU/g of food. Phylogenetic analysis by ITS sequencing showed multiple clones from isolates of contaminated allopurinol tablets and ready-to-eat food, of which some were identical to patients' isolates, and with one isolate in the cornstarch used as an excipient for manufacture of this drug. We attempted to type the isolates by random amplification of polymorphic DNA analysis, with limited evidence of clonal distribution. The primary source of the contaminating fungus was likely to be the cornstarch used in the manufacturing of allopurinol tablets or ready-to-eat food. Rhizopus microsporus is thermotolerant and can multiply even at 50°C. The long holding time of the intermediates during the manufacturing process of allopurinol amplified the fungal load. Microbiological monitoring of drugs manufactured for highly immunosuppressed patients should be considered

A novel dirofilaria species causing human and canine infections in Hong Kong

Dirofilariasis is globally the commonest manifestation of zoonotic filariasis. We report the detection of a novel canine species causing human and canine dirofilariasis in Hong Kong. Three human cases occurring over 10 months were identified, one presenting with cervical lymphadenopathy, one with an abdominal subcutaneous mass, and one with a subconjunctival nodule. Transected worms recovered from the resected abdominal subcutaneous mass were morphologically compatible with Dirofilaria. The cox1 gene sequences of the three human isolates were identical; however, they were only 96.2% and 89.3% identical to the cox1 gene of Dirofilaria repens and Dirofilaria immitis, respectively. Sequencing of the 18S-ITS1-5.8S gene cluster was successful in the intact worm, and the nucleotide sequences were 94.0% and 94.9% identical to those of D. repens and D. immitis, respectively. Screening of the blood samples from 200 dogs and 100 cats showed the presence of the novel Dirofilaria species in 3% (6/200) of the dogs' but none of the cats' blood samples. Nucleotide sequences of the cox1 gene and 18S-ITS1-5.8S gene clusters of the dogs' samples were identical to those in the human samples. The sera of canines infected by this novel Dirofilaria species were negative when tested with the SNAP 4Dx D. immitis detection kit, except in the case of dogs with a mixed infection with D. immitis as detected by PCR. The results from this study suggest that this novel Dirofilaria species is a cause of filarial infection in humans and dogs in Hong Kong. We propose to name this Dirofilaria species "Candidatus Dirofilaria hongkongensis." Copyright © 2012, American Society for Microbiology. All Rights Reserved.link_to_subscribed_fulltex