Combination of commercially available molecular assays and culture based methods in diagnosis of tuberculosis and drug resistant tuberculosis

ABSTRACT Early diagnosis of tuberculosis is of major clinical importance. Among 4733 clinical specimens collected from 3363 patients and subjected to Ziehl-Neelsen microscopy, 4109 were inoculated onto Löwenstein-Jensen slants and 3139 in Bactec/9000MB. Polymerase Chain Reaction (PCR) was performed in 3139 specimens, whereas, a genotypic assay was directly applied in 93 Mycobacterium tuberculosis complex PCR-positive for isoniazid and rifampicin resistance detection specimens (GenoType MTBDRplus). Recovered M. tuberculosis isolates (64) as well as, 21 more sent from Regional Hospitals were tested for antimycobacterial resistance with a phenotypic (manual MGIT-SIRE) and a genotypic assay (GenoType MTBDRplus). PCR in the clinical specimens showed excellent specificity (97.4%) and accuracy (96.8%), good sensitivity (70.4%), but low positive predictive value (40.3%). MGIT-SIRE performed to M. tuberculosis did not confer a reliable result in 16 isolates. Of the remaining 69 isolates, 15 were resistant to streptomycin, seven to isoniazid, seven to ethambutol and five to rifampicin. GenoType MTBDRplus correctly detected isoniazid (seven) and rifampicin-resistant M. tuberculosis strains (five), showing an excellent performance overall (100%). Susceptibility results by the molecular assay applied directly to clinical specimens were identical to those obtained from recovered isolates of the corresponding patients. Combining molecular and conventional methods greatly contribute to early diagnosis and accurate susceptibility testing of tuberculosis.

Combination of commercially available molecular assays and culture based methods in diagnosis of tuberculosis and drug resistant tuberculosis.

Early diagnosis of tuberculosis is of major clinical importance. Among 4733 clinical specimens collected from 3363 patients and subjected to Ziehl-Neelsen microscopy, 4109 were inoculated onto Löwenstein-Jensen slants and 3139 in Bactec/9000MB. Polymerase Chain Reaction (PCR) was performed in 3139 specimens, whereas, a genotypic assay was directly applied in 93 Mycobacterium tuberculosis complex PCR-positive for isoniazid and rifampicin resistance detection specimens (GenoType MTBDRplus). Recovered M. tuberculosis isolates (64) as well as, 21 more sent from Regional Hospitals were tested for antimycobacterial resistance with a phenotypic (manual MGIT-SIRE) and a genotypic assay (GenoType MTBDRplus). PCR in the clinical specimens showed excellent specificity (97.4%) and accuracy (96.8%), good sensitivity (70.4%), but low positive predictive value (40.3%). MGIT-SIRE performed to M. tuberculosis did not confer a reliable result in 16 isolates. Of the remaining 69 isolates, 15 were resistant to streptomycin, seven to isoniazid, seven to ethambutol and five to rifampicin. GenoType MTBDRplus correctly detected isoniazid (seven) and rifampicin-resistant M. tuberculosis strains (five), showing an excellent performance overall (100%). Susceptibility results by the molecular assay applied directly to clinical specimens were identical to those obtained from recovered isolates of the corresponding patients. Combining molecular and conventional methods greatly contribute to early diagnosis and accurate susceptibility testing of tuberculosis.

Interspecies spread of Staphylococcus aureus clones among companion animals and human close contacts in a veterinary teaching hospital. A cross-sectional study in Greece.

Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) prevalence among companion animals and veterinary personnel (VP) was investigated. Strains' molecular characteristics were evaluated in order to assess S. aureus transmission. Specimens (224) from colonized and infected sites of 102 animals (92 dogs, 10 cats) and 18 VP were collected during 2012 and 2013. Antibiotic susceptibility was performed by the disk diffusion method and Etest. mecA, mecC, tst (toxic shock syndrome toxin) and lukF/lukS-PV (Panton-Valentine leukocidin, PVL) genes were investigated by PCR. Genotypes were identified by Multi Locus Sequence Typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), accessory gene regulator group (agr), spa and Pulsed Field Gel Electrophoresis (PFGE). S. aureus prevalence among pets and VP was 36.3% (37/102) and 38.9% (7/18), respectively. Younger companion animals, those living in rural areas, having a disease upon admission or Coagulase-negative staphylococci co-carriage showed significantly higher prevalence of S. aureus isolation (p<0.05). Twenty-six pets and five VP carried PVL-positive S. aureus. In total, 60 S. aureus strains were recovered (53 from pets, seven from VP) of which 16 were MRSA (26.7%), 12 mecA- and four mecC-positive. MRSA showed higher resistance rates against other antimicrobials as compared to methicillin-susceptible ones. Strains were classified by MLST in 13 STs, with the predominance of ST80 and ST15. In MRSA, SCCmec types II, IV and XI were identified. The most frequent spa types were t5559 and t7558. Fifty-six strains were classified into 15 PFGE types. Comparison of genetic markers shows that identical or very similar strains disseminate among animals and VP. Companion animals harbor PVL-positive clones constituting a possible source for transmission to humans.

Inhibition of Bacterial Attachment on Surfaces by Immobilization of Tobramycin-Loaded Liposomes.

Stainless steel surfaces were processed by gold deposition in order to immobilize tobramycin-loaded liposomes which were functionalized on their surface with thiol-groups (through maleimide (MAL) derivatization with thiols). After optimizing the tobramycin loading in liposomes (LIPs), and the immobilization of THIOL-MAL-functionalized LIPs on gold-sputtered surfaces, the coated surfaces were challenged with two reference Staphylococcus epidermidis strains: ATCC 35984 (slime-positive) and ATCC 12228 (slime-negative), in order to measure the degree of surface protection from biofilm formation. Moreover, the effect of the reference and two well characterized clinical S. epidermidis strains on the integrity of LIPs (composed of PC or DSPC) was evaluated, in order to investigate whether specific interactions between LIPs and bacteria occur, and if they are affected by LIP membrane composition and/or bacterial strain type. Bacteria growth on surfaces is substantially inhibited by TOBR-loaded-LIP immobilization, especially in the case of the non-biofilm forming bacterial strain. Gold sputtered surfaces were moderately (albeit significantly) protected, from both reference strains tested (compared to bare surfaces). Interestingly, LIP integrity is significantly decreased in the presence of bacteria (at specific lipid/bacteria ratios); the biofilm-forming bacteria being most potent for LIP disruption, whereas, less rigid liposomal membranes (PC) are affected more compared to rigid (DSPC) ones. The clinical strains are also reactive against LIP. This interaction indicates a potential for triggered release of LIP-encapsulated drugs in presence of biofilm-forming bacteria, therefore LIP encapsulation/immobilization may be envisioned as a potential platform technology for triggered antimicrobial therapy.

Dissemination of Methicillin-Susceptible CC398 Staphylococcus aureus strains in a rural Greek area.

A large collection of Staphylococcus aureus including a. 745 clinically significant isolates that were consecutively recovered from human infections during 2012-2013, b. 19 methicillin-susceptible (MSSA), randomly selected between 2006-2011 from our Staphylococcal Collection, c. 16 human colonizing isolates, and d. 10 strains from colonized animals was investigated for the presence and the molecular characteristics of CC398. The study was conducted in Thessaly, a rural region in Greece. The differentiation of livestock-associated clade from the human clade was based on canSNPs combined with the presence of the φ3 bacteriophage and the tetM, scn, sak, and chp genes. Among the 745 isolates, two MRSA (0.8% of total MRSA) and thirteen MSSA (2.65% of total MSSA) were found to belong to CC398, while, between MSSA of our Staphylococcal Collection, one CC398, isolated in 2010, was detected. One human individual, without prior contact with animals, was found to be colonized by a MSSA CC398. No CC398 was identified among the 10 S. aureus isolated from animals. Based on the molecular markers, the 17 CC398 strains were equally placed in the livestock-associated and in the human clades. This is the first report for the dissemination of S. aureus CC398 among humans in Greece.

Pulmonary infection by Rhodococcus equi presenting with positive Ziehl-Neelsen stain in a patient with human immunodeficiency virus: a case report.

INTRODUCTION: Patients with human immunodeficiency virus carry a significant risk of contracting opportunistic infections. The worldwide increased incidence of tuberculosis has instituted pulmonary tuberculosis as an important diagnostic consideration in patients with human immunodeficiency virus presenting with lower respiratory tract infection. A positive result on the readily-available Ziehl-Neelsen stain usually leads to the initiation of antituberculous treatment, since tuberculosis may exert a rapid and even fatal clinical progress in human immunodeficiency virus coinfection. However, a number of other acid-fast bacteria might be implicated as offending pathogens. This case highlights the importance of broadening the list of pathogens that can account for a positive Ziehl-Neelsen stain in this select group of patients. CASE PRESENTATION: We describe the case of a 34-year-old, Albanian man with untreated human immunodeficiency virus, presenting with clinical and radiologic signs of pulmonary tuberculosis and a positive Ziehl-Neelsen sputum specimen, who was finally diagnosed with pulmonary infection by Rhodococcus equi. CONCLUSIONS: Rhodococcus equi is a rare cause of pulmonary disease, even in patients with human immunodeficiency virus, and a positive Ziehl-Neelsen sputum specimen often misleads clinicians to more common organisms such as mycobacteria. A high index of suspicion, broadening the spectrum of optional pathogens, and effective communication between clinicians and microbiologists can ensure an efficient diagnostic and therapeutic approach.

Coagulase-negative staphylococcal bloodstream and prosthetic-device-associated infections: the role of biofilm formation and distribution of adhesin and toxin genes.

Coagulase-negative staphylococci (CNS), especially Staphylococcus epidermidis and Staphylococcus haemolyticus, have emerged as opportunistic pathogens in immunocompromised patients and those with indwelling medical devices. In this study, CNS recovered from patients with bloodstream infections (BSIs) or prosthetic-device-associated infections (PDAIs) were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution, and carriage of adhesin and toxin genes. A total of 226 CNS isolates (168 S. epidermidis and 58 S. haemolyticus) recovered from hospital inpatients with BSIs (100 isolates) or PDAIs (126 isolates) were tested for biofilm formation, antimicrobial susceptibility, and mecA, ica operon, adhesin (aap, bap, fnbA, atlE, fbe) and toxin (tst, sea, sec) genes. The selected CNS were classified into pulsotypes by PFGE and assigned to sequence types by multilocus sequence typing. In total, 106/226 isolates (46.9%) produced biofilm, whereas 150 (66.4%) carried the ica operon. Most isolates carried mecA and were multidrug resistant (90.7%). CNS recovered from BSIs were significantly more likely to produce biofilm (P=0.003), be resistant to antimicrobials and carry mecA (P<0.001), as compared with isolates derived from PDAIs. CNS from PDAIs were more likely to carry the aap and bap genes (P=0.006 and P=0.045, respectively). No significant differences in the carriage of toxin genes were identified (P>0.05). Although PFGE revealed genetic diversity, especially among S. epidermidis, analysis of representative strains from the main PFGE types by multilocus sequence typing revealed three major clones (ST2, ST5 and ST16). A clonal relationship was found with respect to antimicrobial susceptibility and ica and aap gene carriage, reinforcing the premise of clonal expansion in hospital settings. The results of this study suggest that the pathogenesis of BSIs is associated with biofilm formation and high-level antimicrobial resistance, whereas PDAIs are related to the adhesion capabilities of S. epidermidis and S. haemolyticus strains.

Relapsing Bacillus cereus peritonitis in a patient treated with continuous ambulatory peritoneal dialysis.

INTRODUCTION: Peritonitis is a severe complication of peritoneal dialysis (PD) due to associated morbidity and mortality. Although Bacillus cereus is mostly considered as a contaminant, its role as a causative agent in a few cases of PD peritonitis has been documented. Peritonitis due to B. cereus has been associated with high rates of catheter removal and resistance to beta-lactam antibiotics. CASE PRESENTATION: A case of relapsing peritonitis caused by B. cereus in a 69-year-old man with end-stage renal disease on continuous ambulatory PD for 3 years is described. B. cereus was recovered from the patient's peritoneal fluid and was identified by phenotypic and molecular methods. The patient was treated, according to the susceptibility test, with tobramycin for 14 days. Cultures became sterile and the patient was discharged from hospital. Three days after discharge, the patient reported recurrence of abdominal pain and a new antibiotic regimen based on the previous culture results was initiated consisting of vancomycin and ciprofloxacin. The presence of B. cereus in the peritoneal fluid was confirmed, whereas repeated cultures for the next 15 days were positive. All B. cereus isolates produced biofilm. On day 16, the PD catheter was removed and the patient was transferred to haemodialysis. A review of previously reported cases is also presented. CONCLUSION: Since peritonitis is the most common cause of transition to haemodialysis, isolation of B. cereus from PD patients, even though rare, should not be considered as a contaminant. An appropriate antibiotic regimen and, whenever necessary, catheter removal should be applied.

Biofilm development by clinical isolates of Staphylococcus spp. from retrieved orthopedic prostheses.

BACKGROUND: Biofilms are considered the key factor in the development of implant-related infections. However, only a few reports have dealt with the ability of organisms isolated from such infections to develop biofilms in vitro. METHODS: We evaluated different phenotypic techniques (2 microtiter plate assays and confocal laser scanning microscopy (CLSM) and genotypic techniques (detection of the ica operon) related to biofilm development by clinical isolates of Staphylococcus spp. RESULTS: All 26 strains tested (from 23 specimens) were biofilm producers. Stepanovic test detected biofilm formation in 85% of the strains, microtiter plate assay in 65%, and CLSM in 39%. The ica operon was detected in 73% of all strains (all 13 S. aureus strains and 6 of the 13 coagulase-negative Staphylococcus strains). 7 ica-negative strains were biofilm-positive by phenotypic methods. INTERPRETATION: The detection of ica genes could not be related to the phenotypic ability of the strains to develop a biofilm in vitro, so both studies (genetic and phenotypic) are required for a better evaluation of the biofilm-producing ability of clinical strains of Staphylococcus isolated from orthopedic infections.

Emergence of a new clone carrying Panton-Valentine leukocidin genes and staphylococcal cassette chromosome mec type V among methicillin-resistant Staphylococcus aureus in Greece.

Clonal analysis and PCR screening for the presence of Panton-Valentine leukocidin (PVL) genes was performed among 694 methicillin-resistant Staphylococcus aureus (MRSA) cases collected during a 2-y period in Greece. The detection rate of PVL-positive MRSA is high, both in the community and in hospital. Clonal analysis revealed the predominance among the PVL-positive strains of the clonal complex CC80 (ST80-IV) and the emergence of ST377 clone carrying agr1 allele and SCCmec type V.