Detecting the Diversity of Mycoplasma and Ureaplasma Endosymbionts Hosted by Trichomonas vaginalis Isolates.

Objectives: The symbiosis of Trichomonas vaginalis and Mycoplasma hominis is the first described association between two obligate human parasites. Trichomonas is the niche and the vector for the transmission of M. hominis infection. This clinically significant symbiosis may affect T. vaginalis virulence and susceptibility to treatment. The aims of this study were to investigate the intracellularly present Mycoplasma and Ureaplasma species in T. vaginalis strains isolated from the vaginal discharge of infected women as well as to trace the diversity pattern among the species detected in the isolated strains. Methods: Hundred pure T. vaginalis cultures were isolated from ~7,500 patient specimens presented with clinical purulent vaginitis. PCR and sequencing for Mycoplasma/Ureaplasma spp. were performed in DNA extracted from the pure cultures. In addition, vaginal discharge samples were cultured for the presence of M. hominis and U. urealyticum. Phylogenetic analysis assisted the identification of interspecies relationships between the Mycoplasma and Ureaplasma isolates. Results: Fifty four percentage of T. vaginalis isolates were harboring Mycoplasma spp. Phylogenetic analysis revealed three distinct clusters, two with already characterized M. hominis and Ureaplasma spp. (37% of total Mycoplasma spp.), whereas one group formed a distinct cluster matched with the newly identified species Candidatus Mycoplasma girerdii (59.3%) and one or more unknown Mycoplasma spp. (3.7%). Conclusions:T. vaginalis strains associated with vaginal infection might host intracellular mycoplasmas or ureaplasmas. Intracellular Mollicutes that remain undetected in the extracellular environment when conventional diagnostic methods are implemented may comprise either novel species, such as Candidatus M. giredii, or unknown species with yet unexplored clinical significance.

Nationwide surveillance of resistance rates of Staphylococcus aureus clinical isolates from Greek hospitals, 2012-2013.

Purpose To evaluate the in vitro efficacy of several anti-staphylococcal agents against a nationwide collection of contemporary Staphylococcus aureus clinical isolates from several healthcare centres in Greece. Methods Thirty hospitals throughout Greece (18 in Attica) provided all clinical isolates of S.aureus from April 2012 to May 2013 to a central lab to be re-submitted to susceptibility testing. The MICs were evaluated by Vitek® 2 with the exception of ceftaroline (OXOID M.I.C. Evaluator™). Vancomycin and daptomycin MICs were also evaluated by Etest®. Heterogeneously vancomycin-intermediate strains (hVISA) were detected by the Etest® GRD. VISA phenotype was confirmed by PAP-AUC. Results A total of 1005 isolates (39% MRSA) were studied. Susceptibility rates were: erythromycin 66.5%, clindamycin 79.2%, SXT 98.9%, rifampicin 97.3%, fusidic acid 67%, moxifloxacin 78.8%, vancomycin 99.9%, ceftaroline 92.9% and linezolid, tigecycline and daptomycin 100%. For mupirocin, high level resistance could be excluded for 98.9% of isolates. Vancomycin Etest® MIC50/90 were 1.5/1.5 mg/L, 58.5% of isolates exhibited a MIC > 1 and 8.7% a MIC of 2 mg/L, while Vitek® MIC50/90 were 1/1 and 3.1% showed MIC > 1 mg/L. One VISA strain was detected. Among the selected 175 isolates that were screened for hVISA phenotype, six (3.4%) were positive. In 315 bloodstream isolates, 64.1% had a vancomycin Etest® MIC > 1 mg/L. Conclusions This multi-centre surveillance study revealed that a significant percentage of contemporary S.aureus isolates from Greek patients have a vancomycin MIC (> 1 mg/L) that may compromise the clinical efficacy of the drug for the treatment of serious infections. The in vitro activity of SXT, rifampicin, mupirocin, linezolid, tigecycline, daptomycin and ceftaroline remains excellent.

Genetic basis of aminoglycoside resistance following changes in aminoglycoside prescription patterns.

Aminoglycosides (AG) offer an important therapeutic option for the treatment of infections caused by multiresistant Enterobacteriaceae. We observed a change in AG usage patterns in our institution between 1997 and 2006, namely a reduction in use of all AG except amikacin. We studied the changes in AG susceptibility rates in these time periods and correlated with prevalence of different molecular resistance mechanisms. Enterobacteriaceae isolated from blood cultures from 1997 and 2006 were studied. Susceptibilities to AG were determined with the disk diffusion method. PCR was used to detect genes encoding AG-modifying enzymes and methylases. Gentamicin resistance rates dropped from 14·5 to 8·8%, whereas resistance rates to other AG remained unchanged. The AAC(6')-I+AAC(3)-I combination was more common in 1997, whereas AAC(6')-I was the most common mechanism in 2006. Reduction in gentamicin use may preserve the usefulness of this agent against severe infections by multiresistant bacteria such as carbapenemase-producing Enterobacteriaceae.

Autochthonous Plasmodium vivax malaria in a Greek schoolgirl of the Attica region.

In August 2009, one case of autochthonous malaria due to Plasmodium vivax was diagnosed in Greece in a young woman residing in the Eastern Attica region. The source of infection could not be identified. No other autochthonous malaria cases have been described in the Attica region since 1974. This was a sporadic case with no evidence of further local transmission, and no more cases have been reported in Attica up to now, two years later.

Consecutive Serratia marcescens multiclone outbreaks in a neonatal intensive care unit.

BACKGROUND: This report describes 3 consecutive outbreaks caused by genetically unrelated Serratia marcescens clones that occurred in a neonatal intensive care unit (NICU) over a 35-month period. METHODS: Carriage testing in neonates and health care workers and environmental investigation were performed. An unmatched case-control study was conducted to identify risk factors for S marcescens isolation. RESULTS: During the 35-month period, there were 57 neonates with S marcescens isolation in the NICU, including 37 carriers and 20 infected neonates. The prevalence rate of S marcescens isolation was 12.3% in outbreak 1, 47.4% in outbreak 2, and 42% in outbreak 3. Nine of the 20 infected neonates died (45% case fatality rate). A total of 10 pulsed field gel electrophoresis types were introduced in the NICU in various times; 4 of these types accounted for the 9 fatal cases. During outbreak 3, a type VIII S marcescens strain, the prevalent clinical clone during this period, was detected in the milk kitchen sink drain. Multiple logistic regression revealed that the only statistically significant factor for S marcencens isolation was the administration of total parenteral nutrition. CONCLUSIONS: Total parenteral nutrition solution might constitute a possible route for the introduction of microorganisms in the NICU. Gaps in infection control should be identified and strict measures implemented to ensure patient safety.

Flow cytometry as a diagnostic tool in the early diagnosis of aggressive lymphomas mimicking life-threatening infection.

Aggressive lymphomas can present with symptoms mimicking life-threatening infection. Flow cytometry (FC) is usually recommended for the classification and staging of lymphomas in patients with organomegaly and atypical cells in effusions and blood, after the exclusion of other possible diagnoses. FC may also have a place in the initial diagnostic investigation of aggressive lymphoma. Three cases are presented here of highly aggressive lymphomas in young adults, which presented with the clinical picture of fever of unknown origin (FUO) in patients severely ill. All followed a life-threatening clinical course, and two developed the hemophagocytic syndrome (HPS), but microbiological, immunological, and morphological evaluation and immunohistochemistry (IHC) failed to substantiate an early diagnosis. FC was the technique that provided conclusive diagnostic evidence of lymphoma, subsequently verified by IHC. Our experience with these three cases highlights the potential role of FC as an adjunct methodology in the initial assessment of possible highly aggressive lymphoma presenting with the signs and symptoms of life-threatening infection, although the definitive diagnosis should be established by biopsy. In such cases, FC can contribute to the diagnosis of lymphoma, independently of the presence of HPS.

An improved flow cytometric assay for detection and discrimination between malignant cells and atypical mesothelial cells, in serous cavity effusions.

BACKGROUND: The aim of this study was to evaluate a flow cytometric assay for the detection of malignant effusions. METHODS: During the last 4-year period, 125 effusions suspicious for malignancy were prospectively analyzed by flow cytometry and conventional cytology. A three-step flow cytometric assay was performed, beginning with an initial informative panel of two protocols, containing SYTO-16, 7-AAD, CD71-PE, CD45-ECD, and CD66abce-FITC, CD64-PE, CD45-ECD, CD16-PECy5, CD14-PECy7, respectively. This was followed by a basic immunophenotypic panel of seven three-color combinations, containing in the first position, EMA, Ber-EP4, CD66abce, CD56, and intracellular desmin-33, combined with CD71-PE and CD45-PeCy5 in each tube. Finally, a cytokeratin-FITC/propidium iodide DNA panel was conducted, for the detection of aneuploidy in cytokeratin positive cells. RESULTS: The sensitivity and specificity of flow cytometry were 85.1 and 97.8%, and of cytology 93.2 and 95.6%, respectively. A significant association was observed between the results of the two techniques (P < 0.001). Among eight atypical cases detected by cytology, five had been precisely characterized as malignant by flow cytometry. EMA and Ber-EP4 proved the most sensitive markers for malignancy diagnosis, while the detection of desmin-33 negative/cytokeratin positive cells had the simultaneous highest positive and negative predictive values. CD66abce was very specific, although nonsensitive, while DNA ploidy analysis was nonspecific, as hyperploidy was observed in reactive mesothelial cells. CONCLUSIONS: A flow cytometric assay of high sensitivity and specificity is proposed for the routine identification of carcinoma cells in effusions and their distinction from atypical mesothelial cells, as an ancillary to conventional cytology.

Intestinal schistosomiasis caused by both Schistosoma intercalatum and Schistosoma mansoni.

A case is presented of intestinal schistosomiasis due to both Schistosoma intercalatum and Schistosoma mansoni in a 30-year-old man from Senegal with discussion of diagnostic approach, species identification and determination of the effect of treatment. The patient was admitted to hospital for investigation of renal failure, arterial hypertension and hypereosinophilia. Repeated stool examinations for ova and parasites were negative. Ultrasonography (US) and computed tomography (CT) of the abdomen showed no abnormalities. US of the urinary tract showed kidneys of borderline size with increased echogenicity. Cystoscopy and histopathological examination of bladder biopsy specimens were normal. Flexible colonoscopy revealed numerous nodular lesions in the rectosigmoid region and a few similar lesions in the transverse colon, the histopathological examination of which showed deposition of Schistosoma ova with granuloma formation. Examination of multiple crush biopsy specimens from the rectosigmoid region revealed numerous granulomas formed around Schistosoma eggs which had a terminal spine and were identified as S. intercalatum (longer than Schistosoma haematobium and with a slightly curved terminal spine) and a very few S. mansoni eggs. Crush biopsies from the lesions in the transverse colon showed only S. mansoni eggs. In conclusion, the examination of multiple crush biopsy specimens is a very sensitive and specific technique for species identification of Schistosoma, especially in mixed infections, and for defining the location and extent of the granulomas evoked by each species.

Fibrous subcutaneous nodule caused by Dirofilaria repens.

A case is described of subcutaneous dirofilariasis in a Greek woman who had visited many countries around the world, including areas of sub-Saharan Africa. The patient presented with a single hard subcutaneous nodule on the right cheek, with no cutaneous manifestations of early or long-standing onchocercal dermatitis or eye lesions. The nodule was removed surgically and the filarial adult worm Onchocerca volvulus was initially diagnosed, based on the history, the hardness and large size of the fibrous nodule and the absence of cuticular longitudinal ridges of the parasite in the initial histological sections. Bloodless skin snips taken from the regions of the scapula, iliac crest and lateral calf were negative for O. volvulus microfilariae. Serial cross-sections of the fibrous nodule and gross examination of a portion of the adult worm removed from the nodule revealed the characteristic longitudinal ridges which allowed the identification of the worm as Dirofilaria repens. The aim of this report on tropical and non-tropical filarial worms affecting the skin and eyes is to point out the key features for precise identification of the parasites and differential diagnosis of the infections caused by the filiform nematode worms of O. volvulus, and Dirofilaria species.

Subcutaneous dirofilariasis caused by Dirofilaria repens in Greece: a case report.

Dirofilaria repens (formerly Dirofilaria conjunctiva) is a natural parasite of the subcutaneous tissues of dogs, cats and wild carnivores in Europe, Africa and Asia. Microfilariae are transmitted to humans by various species of mosquito. An autochthonous case of subcutaneous dirofilariasis is reported in a Greek patient from the island of Corfu. The clinical manifestation of the infection was a palpable, painless, subcutaneous nodule in the region of the groin, which 2 days before the patient consulted the doctor developed symptoms and signs of inflammation (pain, edema and redness). The entire lesion was surgically removed, and the nematode worm D. repens was identified on histological sections of biopsy material. The aim of this report was (a) to describe the microscopic morphological features of D. repens that enable identification of the parasite on histological examination and (b) to emphasize the importance of consideration of subcutaneous dirofilariasis in the differential diagnosis of subcutaneous nodules with inflammatory eosinophilic infiltration in countries where the infection is endemic.

Report of an HIV and HHV-8 negative case of primary effusion lymphoma with idiopathic T4 lymphocytopenia.

Although primary effusion lymphoma (PEL) is usually associated with human herpes virus-8/Kaposi sarcoma herpes virus (HHV-8/KSHV) and human immunodeficiency virus (HIV), there are several reports of HHV-8/KSHV and HIV negative cases, mainly in the setting of immunodeficiency. Here, we report the second case of PEL associated with idiopathic T4 lymphocytopenia (ICL), which was HHV-8/KSHV negative, HIV negative and Epstein-Barr virus positive, while no other causative agents for immunodeficiency were documented. Flow cytometry revealed a hyperdiploid and highly mitotic large B-cell population, CD30, EMA, CD66, CD38 and CD71 positive. The malignant lymphoma cells showed atypia with prominent nuclei and basophilic vacuolated cytoplasm, while cytogenetic analysis with fluorescent in situ hybridization showed trisomy 18. The patient was administered R-COP chemotherapy, but no remission was achieved, up to 3 months from diagnosis.

Evaluation of imipenem/imipenem+EDTA disk method for detection of metallo-beta-lactamase-producing Klebsiella pneumoniae isolated from blood cultures.

The objective of this study was to evaluate the imipenem (IMP) and IMP+EDTA (IMP/IMP+EDTA) disk method for the detection of metallo-beta-lactamases (MBLs) in clinical isolates of Klebsiella pneumoniae with various MIC levels to IMP. Forty-one blood isolates of K. pneumoniae with MIC to IMP ranging from < or =0.5 to > or =16 microg/ml were examined. The MICs were determined by VITEK-2 (bioMerieux Vitek two, France). Disks of 10 microg IMP with and without the addition of 0.5 M EDTA were used for the IMP/IMP+EDTA disk method. The E-test (AB Biodisk, Solna, Sweden) for MBL detection was also used. All isolates were examined for the bla (VIM-1) gene by PCR and for clonality of VIM-1-producing isolates by pulsed-field gel electrophoresis (PFGE). All isolates with MIC values of IMP < or =0.5 microg/ml exhibited no differences in inhibition zone diameters (IZD) produced by IMP and IMP+EDTA disks, whereas the isolates with MICs > or =1 microg/ml showed an increase in IZD, ranging from 8 to 26 mm. All isolates with MIC values of > or =1 microg/ml were found positive for the bla (VIM-1) gene by PCR and for MBL production by the E-test, whereas none of isolates with MICs <0.5 microg/ml was found positive by any of the tests. DNA restriction fragments generated by PFGE of VIM-1-producing isolates were classified in four main types. The IMP/IMP+EDTA disk method is simple to perform, sensitive, and specific for detection of MBL-producing K. pneumoniae clinical isolates. K. pneumoniae isolates with MICs of IMP > or =1 microg/ml and/or IZD produced by IMP disk <19 mm should be tested for MBL production.

Detection of Mycobacterium tuberculosis DNA in respiratory and nonrespiratory specimens by the Amplicor MTB PCR.

To evaluate the diagnostic performance of a commercially available Mycobacterium tuberculosis PCR assay (Amplicor MTB-ROCHE), 2296 respiratory and nonrespiratory specimens from 2296 patients with suspected tuberculosis (TB) were collected prospectively in an 8-year period. Clinical data for each patient were abstracted, and all samples were examined blindly by direct microscopy, culture, and PCR. M. tuberculosis DNA was detected in 93 of 113 culture-positive samples and in 29 of 38 samples from patients with probable TB. The lowest sensitivity was observed in pleural fluid and abscess aspirates. The sensitivity, specificity, and positive predictive value of the assay were 97.2%, 100%, and 100% for smear-positive specimens and 75.3%, 97.0%, and 47.5% for smear-negative specimens, respectively. The PCR cost per additional correct clinical decision was Euro 2826 but would have declined to Euro 308 if the test was applied only to smear-positive specimens. The overall performance of Amplicor MTB test was excellent for smear-positive, but suboptimal for smear-negative specimens.