Optimization of the detection of microbes in blood from immunocompromised patients with haematological malignancies

Skovbjerg, Susann; Welinder‐Olsson, Christina; Kondori, Nahid; Kjellin, Eva; Nowrouzian, Forough L.; Wold, Agnes E.; Stockelberg, Dick; Larsson, Peter; Wennerås, Christine

doi:10.1111/j.1469-0691.2009.02796.x

Cited by 16 publications

(11 citation statements)

References 9 publications

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“…The resulting PCR products were loaded on a 2% agarose gel, stained with ethidium bromide, and visualized using UV fluorescence. The DNA bands of the expected size were cut out from the gel, purified with QIAquick gel extraction kit (Qiagen, Hilden, Germany) and subjected to cycle sequencing in both directions as previously described (28). The DNA sequences of the PCR products were purified by ethanol precipitation and then analyzed using the BigDye Terminator v. 3.1 kit and an ABI Prism 3130 genetic analyzer (Applied Biosystems, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

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Multilocus Sequence Analysis of Clinical “Candidatus Neoehrlichia mikurensis” Strains from Europe

et al. 2015

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h "Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ, clpB, gatB, lipA, groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed "Ca. Neoehrlichia" infection from Sweden (n ‫؍‬ 9), the Czech Republic (n ‫؍‬ 2), and Germany (n ‫؍‬ 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ, gatB, and groEL. According to the MLSA, among the Anaplasmataceae, "Ca. Neoehrlichia" is most closely related to Ehrlichia ruminantium, less so to Anaplasma phagocytophilum, and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious "Ca. Neoehrlichia" were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

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Section: Methodsmentioning

confidence: 99%

“…PCR amplification of the 16S rRNA gene of the Swedish "Ca. Neoehrlichia mikurensis" strains was performed as previously described (28). The resulting PCR products were loaded on a 2% agarose gel, stained with ethidium bromide, and visualized using UV fluorescence.…”

Section: Methodsmentioning

confidence: 99%

Multilocus Sequence Analysis of Clinical “Candidatus Neoehrlichia mikurensis” Strains from Europe

et al. 2015

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“…For PCR and DNA sequence analysis, the conserved primers UNI1 (forward primer; 5Ј-GTC AAA CTT GGT CAT TTA-3Ј) and UNI2 (reverse primer; 5Ј-TTC TTT TCC TCC GCT TAT TGA-3Ј) were used to amplify the region encompassing the rRNA internal transcribed spacer regions 1 (ITS1) and 2 (ITS2), including the 5.8S rRNA (3,26) moiety. Each PCR was performed in a total volume of 50 l, as described previously in detail (23). The PCR amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, Düsseldorf, Germany), following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

High Rate of Exophiala dermatitidis Recovery in the Airways of Patients with Cystic Fibrosis Is Associated with Pancreatic Insufficiency

et al. 2011

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The black-pigmented fungus Exophiala dermatitidis is considered to be a harmless colonizer of the airways of cystic fibrosis (CF) patients. The aim of this study was to establish the recovery rate of E. dermatitidis in respiratory specimens from CF patients, transplant recipients, and subjects with other respiratory disorders in Sweden. Second, we wished to determine if particular clinical traits were associated with E. dermatitidis colonization of the airways and the antifungal susceptibility profiles of Exophiala strains. Sputum and bronchoalveolar lavage samples (n ‫؍‬ 492) derived from 275 patients were investigated. E. dermatitidis was isolated in respiratory specimens from 19% (18/97) of the CF patients but in none of the other patient categories. All isolates were recovered after 6 to 25 days of incubation on erythritol-chloramphenicol agar (ECA) medium. Morphological and genetic analyses confirmed species identity. Pancreatic insufficiency was positively associated with the presence of E. dermatitidis in sputum samples (P ‫؍‬ 0.0198). Antifungal susceptibility tests demonstrated that voriconazole and posaconazole had the lowest MICs against E. dermatitidis. In conclusion, E. dermatitidis is a frequent colonizer of the respiratory tract in CF patients in Sweden and appears to be associated with more advanced disease. Whether E. dermatitidis is pathogenic remains to be elucidated.

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“…As suspicion of sepsis remained, blood culture samples in eight bottles from the BacT/Alert system (bioMérieux), including four aerobic and four anaerobic flasks, were analyzed by a PCR assay targeting the panbacterial 16S rRNA gene as described previously (12). These blood samples had been collected on two separate occasions on 23 September and had not revealed any signs of bacterial growth after incubation in the automated thermostat for 6 days.…”

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confidence: 99%

First Case of Human “ Candidatus Neoehrlichia mikurensis” Infection in a Febrile Patient with Chronic Lymphocytic Leukemia

Welinder‐Olsson¹,

Kjellin²,

Vaht³

et al. 2010

J Clin Microbiol

148

115

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An immunocompromised patient presented with febrile episodes, an erysipelas-like rash, and thromboembolic complications. Amplification of 16S rRNA gene sequences from blood and sequence analysis revealed "Candidatus Neoehrlichia mikurensis." We report the first case of human disease caused by "Ca. Neoehrlichia mikurensis." CASE REPORTA 77-year-old man with B-cell chronic lymphocytic leukemia developed autoimmune anemia in 2007 and started long-term treatment with corticosteroids. In September of the same year, he had a transitory ischemic attack. Since his hemolytic anemia worsened despite treatment with corticosteroids, he was given courses of cyclophosphamide during the second half of 2008. The patient developed autoimmune thrombocytopenia (platelet count, 38 ϫ 10 9 /liter; reference range for healthy adults, 145 ϫ 10 9 to 355 ϫ 10 9 /liter) and was splenectomized laparoscopically on 4 June 2009, with subsequent normalization of platelet counts.While kayaking on 3 July 2009, the patient developed acute diarrhea, which was followed by fever and chills and a short episode of loss of consciousness the same night. When admitted to Kungälv Hospital, Kungälv, Sweden, the next day under suspicion of sepsis, he was hypotensive (blood pressure [BP], 85/60 mm Hg) and febrile (temperature, 38.5°C; reference, Ͻ38.0°C). Deep vein thrombosis in the left lower extremity encompassing the groin and pulmonary embolism were also discovered. The patient was treated intravenously (i.v.) with ceftazidime for 1 week, but no microbe was identified. The patient's systemic inflammatory reaction (C-reactive protein level of 92 mg/liter [reference, Ͻ5 mg/liter] and fever) was judged to result from widespread thromboembolism, and the patient was discharged on 10 July with low-molecular-weight heparin medication.A month later, the patient was readmitted to Sahlgrenska University hospital with a fever of 39.5°C, BP of 105/55 mm Hg, and an erysipelas-like rash on the inside of the left leg. The patient was anemic (hemoglobin [Hb], 85 g/liter; reference range, 134 to 170 g/liter) and had leukocytosis (white blood cell [WBC] count, 11 ϫ 10 9 /liter; reference range, 3.5 ϫ 10 9 to 8.8 ϫ 10 9 /liter) with a pronounced left shift, a normal platelet count, and a C-reactive protein level of 54 mg/liter (reference, Ͻ5 mg/liter). Hyponatremia was present (sodium level, 134 mmol/liter; reference range, 137 to 145 mmol/liter). The patient was taking warfarin, oral prednisolone, omeprazole, and vitamin B tablets. He was treated with i.v. cloxacillin for 2 days, followed by oral floxacillin (flucloxacillin) for 2 days and, finally, i.v. meropenem for 7 days (Fig. 1). Fever, elevated levels of C-reactive protein, and hyponatremia resolved within 1 week, apparently after the institution of meropenem (Fig. 1). All cultures (three blood cultures, two urinary cultures, and one oral swab culture) were negative. A chest X ray revealed scant infiltrates around the hili and in the basal part of the right lung, but computed tomography (CT) scans of the thorax and ...

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Optimization of the detection of microbes in blood from immunocompromised patients with haematological malignancies

Cited by 16 publications

References 9 publications

Multilocus Sequence Analysis of Clinical “Candidatus Neoehrlichia mikurensis” Strains from Europe

Multilocus Sequence Analysis of Clinical “Candidatus Neoehrlichia mikurensis” Strains from Europe

High Rate of Exophiala dermatitidis Recovery in the Airways of Patients with Cystic Fibrosis Is Associated with Pancreatic Insufficiency

First Case of Human “ Candidatus Neoehrlichia mikurensis” Infection in a Febrile Patient with Chronic Lymphocytic Leukemia

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