Characterisation of clinical Staphylococcus aureus isolates harbouring mecA or Panton-Valentine leukocidin genes from four tertiary care hospitals in Indonesia.

OBJECTIVES: To determine the prevalence, antimicrobial susceptibility profiles and clonal distribution of either methicillin-resistant Staphylococcus aureus (MRSA) or Panton-Valentine leukocidin (PVL)-positive S. aureus obtained from clinical cultures in Indonesian hospitals. METHODS: S. aureus isolates from clinical cultures of patients in four tertiary care hospitals in Denpasar, Malang, Padang and Semarang were included. We assessed the antimicrobial susceptibility profiles using the Vitek2(®) system, determined the presence of the mecA gene and genes encoding PVL using PCR and analysed the clonal relatedness with Raman spectroscopy. SCCmec typing was performed for all MRSA isolates. Multilocus sequence typing (MLST) was performed for a subset of isolates. RESULTS: In total, 259 S. aureus strains were collected. Of these, 17/259 (6.6%) and 48/259 (18.5%) were MRSA and PVL-positive methicillin-susceptible S. aureus (MSSA), respectively. The prevalence of MRSA and PVL-positive MSSA ranged between 2.5-8.9% and 9.5-29.1%, respectively and depended on geographic origin. PVL-positive MRSA were not detected. Raman spectroscopy of the strains revealed multiple Raman types with two predominant clusters. We also showed possible transmission of a ST239-MRSA-SCCmec type III strain and a ST121 PVL-positive MSSA in one of the hospitals. CONCLUSIONS: We showed that MRSA and PVL-positive MSSA are of clinical importance in Indonesian hospitals. A national surveillance system should be set-up to further monitor this. To reduce the prevalence of MRSA in Indonesian hospitals, a bundle of intervention measures is highly recommended.

Reprint of "GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC)".

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.

[Laboratory-based evaluation of PVL-RPLA "Seiken" to detect Panton-Valentine leukocidin produced by Staphylococcus aureus].

It is well known that some isolates of Staphylococcus aureus produce pathogenic toxin, Panton-Valentine leukocidin (PVL), and that the toxin has been reported to be highly associated with community acquired methicillin resistant S. aureus (CA-MRSA). Currently, the PCR method using specific primers for the PVL gene (LukS-PV-lukF-PV) have been widely used to detect PVL. In this study, we evaluated the PVL-RPLA "Seiken", diagnostic reagent based on a reserved passive latex agglutination reaction with a specific monoclonal antibody for detecting PVL. A total of 630 clinical isolates were used. PCR method detected 34 PVL-positive (28 MRSA and 6 MSSA), and, of these, PVL-RPLA "Seiken" read positive for 32 isolates (27 MRSA and 5 MSSA), the result indicating two false negative occurrences. The concordance rate was 99.7%. In addition the recommended BHI broth, CCY medium, Dolman broth and Todd-Hewitt broth were applied for toxin preparation media. Toxin concentration produced in CCY medium was significantly higher than those in the remaining culture medium (p < 0.05). PVL-RPLA "Seiken" is a method for detecting the PVL in the culture broth by antigen antibody reaction after an overnight shaking culture. This method does not require any expensive equipments or facilities. Thus this reagent provides us with rapid, easy-to-perform, less expensive test method to detect PVL in clinical microbiology laboratories.

[Frequency of diseases caused by group A streptococci among invasive infections of soft tissues and characteristics of the causative agent].

AIM: Determine frequency of diseases caused by group A streptococci (GAS) among invasive infections of soft tissues; identify emm-types of the isolated streptococci, determine the presence of bacteriophage integrases and toxin genes in their genomes. MATERIALS AND METHODS: 4750 case histories of patients with soft tissue infections of the purulent-surgical department of the 23rd City Clinical Hospital.of Moscow "Medsantrud" in 2008 - 2011 were analyzed. 46 strains of GAS isolated from patients with invasive streptococcus infection (ISI) were studied. GAS identification was carried out by latex-agglutination method. GAS emm-type was determined by molecular-genetic methods, as well as the presence of bacteriophage integrases int2, int3, int4, int5, int6, int7, int49, bacteriophage toxins speA, speI, sla, speC/J, speL, speH, speC, ssa and speB gene present on the chromosomal DNA. RESULTS: 132 cases (2.8%) were attributed to invasive infections. In 46 cases of invasive infections (35%) GAS were isolated. 22 different emm-types of invasive GAS strains were detected. Only speB gene among all the toxin genes (as well as the expression of the gene--SpeB toxin) was detected in all the strains, whereas sla and speI genes were not detected in any of the strains. Genes of the other toxins (ssa, speL, speC, speA, speH, speC/J) occurred in a number of strains. Genes of phage integrases were detected among all the strains however in varying combinations (from 1 to 4 genes). CONCLUSION: Invasive infections caused by GAS are more frequently spread than had been previously assumed and a high degree of genetic heterogeneity of invasive GAS strains was detected.

Activation of neutrophils by the two-component leukotoxin LukE/D from Staphylococcus aureus: proteomic analysis of the secretions.

Staphylococcus aureus is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions.

Isolation of methicillin-resistant Staphylococcus aureus (MRSA) from retail meats in Hong Kong.

OBJECTIVES: The presence of methicillin-resistant Staphylococcus aureus (MRSA) on meat purchased from retail outlets may allow its spread to households and represents a risk for colonization and possibly infection of consumers. Improved isolation methods have indicated that more than 10% of samples are positive. We aimed to determine rates of MRSA contamination of meat samples, including comparison of fresh and frozen samples. We characterized isolates and determined their antibiotic susceptibility. METHODS: Samples of raw meats commonly consumed in Hong Kong were investigated for MRSA contamination using a double-enrichment isolation method. Isolates were characterized by antibiotic susceptibility testing, presence of mecA, SCCmec type, staphylococcal enterotoxins, Panton-Valentin leukocidin (PVL), and spa type. Differences in rates of MRSA contamination between meat types, rearing method, locations, sources, and fresh or frozen storage were compared. RESULTS: MRSA was recovered from 21.9% of pork samples (78/355), 6.8% chicken (31/455), and 4.4% of beef (17/380). Isolation was considerably higher from fresh pork (47%) than frozen (0.6%), whereas contamination rates in fresh (6%) and frozen (7%) chicken were similar. All strains were multidrug resistant. All contaminated fresh pork and most frozen chicken originated from China. Most isolates belonged to CC9, being SCCmec IVb and spa type t899 or closely related spa types, but one chicken sample yielded ST398. Five strains carried spa types associated with human isolates. The egc enterotoxin group was present in the majority of isolates, but PVL in only three from chicken. CONCLUSIONS: The predominance of t899 in isolates indicates that the primary source of contamination may be pig carcasses, previously demonstrated to frequently harbor CC9-positive MRSA in Hong Kong and China. The high rates of meat contamination suggest that improvements in food safety and personal hygiene guidelines may be advisable to reduce risk of spread of these MRSA strains in the community.

Panton-Valentine leukocidin positive sequence type 80 methicillin-resistant Staphylococcus aureus carrying a staphylococcal cassette chromosome mec type IVc is dominant in neonates and children in an Algiers hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major antimicrobial drug-resistant pathogen causing serious infections. It was first detected in healthcare settings, but in recent years it has also become disseminated in the community. Children and young adults are most susceptible to infection by community-acquired (CA) MRSA strains. In this study 25 MRSA isolates implicated in infections of neonates and children admitted to an Algiers hospital during an 18 month period were characterized by molecular methods including staphylococcal cassette chromosome (SCC) mec typing, PCR amplification of pvl genes, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Fifteen out of 25 isolates were from hospital-acquired infections. Twenty-four isolates carried SCCmec type IVc and belonged to the sequence type (ST) 80, one isolate carried SCCmec type II and was ST 39. Twenty-two out of 24 ST80-MRSA-IVc isolates carried pvl genes. Our results suggest that the Panton-Valentine leukocidin positive ST80- MRSA-IVc is the dominant MRSA clone causing disease in neonates and children in Algiers.

Frequency and clinical association of Panton-Valentine leukocidin-positive Staphylococcus aureus isolates: a study from Kuwait.

OBJECTIVE: This study was undertaken to determine the frequency of Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus among strains isolated in our laboratory and to study the association of PVL-positive strains with clinical disease. MATERIALS AND METHODS: A total of 291 S. aureus isolates obtained from different clinical specimens from June 1, 2009, to March 31, 2010, at the Farwania Hospital Laboratory were investigated for antimicrobial susceptibility, carriage of genes for PVL, and SCCmec elements. Antimicrobial susceptibility testing was performed by standard methods. The presence of mecA genes for PVL SCCmec typing was determined by PCR. RESULTS: Of the 291 S. aureus isolates, 89 (30.6%) were methicillin-resistant S. aureus (MRSA), whereas 202 (69.4%) were methicillin susceptible (MSSA). Genes for PVL were detected in 13 (14.6%) and 24 (12.0%) of the MRSA and MSSA isolates, respectively. The majority of the PVL-producing MRSA and MSSA were isolated from 12 (30.7%) and 19 (21.8%) cases of skin and soft tissue infections (SSTI), respectively. Although both MSSA and MRSA strains were uniformly susceptible to rifampicin, teicoplanin, and vancomycin, multidrug resistance was observed among PVL-producing and nonproducing MRSA isolates. Both MRSA types carried SCCmec type III, IV, IVc, and V genetic elements. CONCLUSION: This study revealed the presence of genes for PVL in both MSSA and MRSA, associated mostly with SSTI and respiratory tract infections, supporting previous observations that PVL production is widespread among S. aureus strains obtained from different clinical sources.

Time course of virulence factors produced by group A streptococcus during a food-borne epidemic.

We studied the protein amount and activity of the major virulence factors hemolysin, cysteine protease streptococcal pyrogenic exotoxin B (SpeB), and NAD glycohydrolase (NADase), which are produced by Streptococcus pyogenes type T-25, with a food poisoning outbreak. The three virulence factors were analyzed by activity and amount of protein using supernatants at 2-30 h of culture. All these virulence factors were confirmed by their activity. Streptolysin O (SLO), SpeB, and NADase were immunochemically confirmed at protein level by Western blot analysis. Two hemolytic forms (70 and 60 kDa) of SLO were identified. SpeB was detected as a 44-kDa precursor form and a 30-kDa mature form. NADase was 50 kDa. SLO protein peaked at 8 h of culture, which corresponded with the hemolytic activity peak. Conversion from precursor to SpeB protein peaked at 14 h of culture. The conversion peak corresponded to the activity expression time. Also, mature SpeB protein peaked at 24 h of culture and corresponded to SpeB activity peak. Electrophoretic analysis clarified the relationship between SLO protein and SpeB protein, although amounts of SLO and SpeB have been reported to be inversely proportional to activity. NADase protein peaked at 12 h of culture, but protein level did not correspond to the peak. Because the NADase protein peak was closer to SpeB activity than SLO protein, our results suggested NADase protein was degraded at 12 h of culture. The time course production of these virulence factors is discussed.

Drug-resistant human Staphylococcus aureus in sanctuary apes pose a threat to endangered wild ape populations.

Reintroduction of sanctuary apes to natural habitat is considered an important tool for conservation; however, reintroduction has the potential to endanger resident wild apes through the introduction of human pathogens. We found a high prevalence of drug-resistant, human-associated lineages of Staphylococcus aureus in sanctuary chimpanzees (Pan troglodytes) from Zambia and Uganda. This pathogen is associated with skin and soft tissue diseases and severe invasive infections (i.e. pneumonia and septicemia). Colonization by this bacterium is difficult to clear due to frequent recolonization. In addition to its pathogenic potential, human-related S. aureus can serve as an indicator organism for the transmission of other potential pathogens like pneumococci or mycobacteria. Plans to reintroduce sanctuary apes should be reevaluated in light of the high risk of introducing human-adapted S. aureus into wild ape populations where treatment is impossible.

Epidemiology of Staphylococcus aureus in pigs and farmers in the largest farm in Dakar, Senegal.

Between December 2009 and November 2011, we collected 57 (12.3%) Staphylococcus aureus isolates from 464 pigs and 16 (30.8%) isolates from 52 farmers in the largest farm in Dakar. Fifty-one isolates (70%) belonged to four major multilocus sequence typing clonal complexes (CCs): CC152 (26.0%), CC15 (19.2%), CC5 (13.7%), and CC97 (10.9%). The CC variability among the pigs was similar to that observed among the farmers. Six isolates that were recovered only among pigs were resistant to methicillin (10.5%). They were assigned to the ST5-staphylococcal cassette chromosome mec type (SCCmec) IV (n = 5) and ST88-SCCmec IV (n = 1) clones. The luk-PV genes encoding Panton-Valentine leukocidin (PVL), present in 43 (58.9%) isolates overall, including all major CCs and the MRSA ST5-SCCmec IV clone, were highly prevalent compared to data from industrialized countries. This finding is of major concern with regard to the potential virulence of these strains.

Characterization of antigenic determinants in ApxIIA exotoxin capable of inducing protective immunity to Actinobacillus pleuropneumoniae challenge.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors of the pathogen, ApxIIA, a bacterial exotoxin, is expressed by many serotypes and presents a plausible target for vaccine development. We characterized the region within ApxIIA that induces a protective immune response against bacterial infection using mouse challenge model. Recombinant proteins spanning the length of ApxIIA were produced and antiserum to the full-length ApxIIA was induced in mice. This antiserum recognized fragments #2, #3 and #5 with high binding specificity, but showed poor recognition for fragments #1 and #4. Of the antisera induced in mice by injection of each fragments, only the antiserum to fragment #4 failed to efficiently recognize the full-length antigen, although the individual antisera recognized their cognate antigens with almost equal efficiency. The protective potency of the immunogenic proteins against a challenge injection of bacteria in vivo correlated well with the antibody titer. Fragment #5 induced the highest level of protective activity, comparable to that by the full-length protein. These results support the use of fragment #5 to produce a vaccine against A. pleuropneumoniae challenge, since the small antigen peptide is easier to handle than is the full-length protein and can be expressed efficiently in heterologous expression systems.

Cases of community-acquired meticillin-resistant Staphylococcus aureus in an asylum seekers centre in Germany, November 2010.

In an asylum seeker centre in Schleswig-Holstein, a resident was diagnosed with furuncle caused by a Panton-Valentine leukocidine (PVL)-positive community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA). As a result of active case finding, 232 of 427 persons (54% of all residents) were screened for MRSA and two further PVL-positive CA-MRSA cases were identified

Characterization and biological properties of a new staphylococcal exotoxin.

Staphylococcus aureus strain D4508 is a toxic shock syndrome toxin 1-negative clinical isolate from a nonmenstrual case of toxic shock syndrome (TSS). In the present study, we have purified and characterized a new exotoxin from the extracellular products of this strain. This toxin was found to have a molecular mass of 25.14 kD by mass spectrometry and an isoelectric point of 5.65 by isoelectric focusing. We have also cloned and sequenced its corresponding genomic determinant. The DNA sequence encoding the mature protein was found to be 654 base pairs and is predicted to encode a polypeptide of 218 amino acids. The deduced protein contains an NH2-terminal sequence identical to that of the native protein. The calculated molecular weight (25.21 kD) of the recombinant mature protein is also consistent with that of the native molecules. When injected intravenously into rabbits, both the native and recombinant toxins induce an acute TSS-like illness characterized by high fever, hypotension, diarrhea, shock, and in some cases death, with classical histological findings of TSS. Furthermore, the activity of the toxin is specifically enhanced by low quantities of endotoxins. The toxicity can be blocked by rabbit immunoglobulin G antibody specific for the toxin. Western blotting and DNA sequencing data confirm that the protein is a unique staphylococcal exotoxin, yet shares significant sequence homology with known staphylococcal enterotoxins, especially the SEA, SED, and SEE toxins. We conclude therefore that this 25-kD protein belongs to the staphylococcal enterotoxin gene family that is capable of inducing a TSS-like illness in rabbits.

Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli.

Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application.

Skin and soft tissue infections caused by community-associated methicillin-resistant staphylococcus aureus among children in China.

AIM: To investigate the characteristic of community-associated methicillin-resistant staphylococcus aureus (CA-MRSA) skin and soft tissue infections (SSTIs) among children in China. METHODS: Forty-seven children with CA-MRSA SSTIs were enrolled in this study. Clinical information was collected and analysed. The strains from the children were analysed by multilocus sequence typing (MLST), staphylococcus cassette chromosome mec (SCCmec) typing and spa typing. The Panton-Valentine leukocidin (PVL) gene was also detected. RESULTS: The majority of the 47 cases were impetigo (20; 42.6%) and abscesses (14; 29.8%). The rest was cellulites, infected wounds, omphalitis, paronychia and conjunctivitis combined folliculitis. Thirty-two of the isolates (68.1%) were PVL-positive, and the abscesses infected with PVL-positive strains usually required incision and drainage (87.5% vs. 16.7%, p = 0.026). Most of the isolates belonged to ST type 59, which accounted for 46.8%, followed by ST1 (7/47, 14.9%) and ST910 (5/47, 10.6%). The clone of ST59-MRSA-IV with t437 was the most prevalent one. The multiresistant rate of these strains was 93.6%. CONCLUSION: The most common disease of CA-MRSA SSTIs was impetigo, and PVL-positive abscess was associated with incision and drainage. ST59-MRSA-IV with t437 was the most prevalent clone, and the multiresistant rate was high in Chinese children.

Staphylococcus aureus Exotoxins and Their Detection in the Dairy Industry and Mastitis.

Staphylococcus aureus constitutes a major food-borne pathogen, as well as one of the main causative agents of mastitis in dairy ruminants. This pathogen can produce a variety of extracellular toxins; these include the shock syndrome toxin 1 (TSST-1), exfoliative toxins, staphylococcal enterotoxins (SE), hemolysins, and leukocidins. S. aureus expresses many virulence proteins, involved in evading the host defenses, hence facilitating microbial colonization of the mammary glands of the animals. In addition, S. aureus exotoxins play a role in the development of both skin infections and mastitis. Indeed, if these toxins remain in dairy products for human consumption, they can cause staphylococcal food poisoning (SFP) outbreaks. As a result, there is a need for procedures to identify the presence of exotoxins in human food, and the methods used must be fast, sensitive, reliable, and accurate. It is also essential to determine the best medical therapy for human patients suffering from S. aureus infections, as well as establishing the relevant veterinary treatment for infected ruminants, to avoid economic losses in the dairy industry. This review summarizes the role of S. aureus toxins in the development of mastitis in ruminants, their negative effects in the food and dairy industries, and the different methods used for the identification of these toxins in food destined for human consumption.

Characterization of different virulent factors in methicillin-resistant Staphylococcus aureus isolates recovered from Iraqis and Syrian refugees in Duhok city, Iraq.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious public health problem. There is limited information regarding the genetics of MRSA strains among the native Iraqi and incoming Syrian refugee communities. We aimed to characterize the genotypes and different virulence factors of MRSA in strains isolated from these two communities. Frozen MRSA strains (125) isolated from the native Iraqi and Syrian refugee communities were used in this study. PCR (singleplex and multiplex) and agr typing was used for the genotypic analysis of different virulence genes. We tested for the presence of virulence genes including pvl, arcA, tst, lukE/lukD, hla, hlb, eta, etb and agr. Prevalence of arcA MRSA in the Iraqi community (56.58%) was significantly higher (p = 0.008) than that in the Syrian refugee community (32.66%). Prevalence of lukE-lukD was also significantly higher (p = 0.001) in the Iraqi (82.89%) compared to that in the Syrian refugee community (57.14%). Further, prevalence of hla MRSA in the Iraqi community was (93.4%) and in the Syrian refugee community was (71.4%); (p = 0.0008). No significant differences were observed in the prevalence of pvl, tst, eta, etb and hlb. The most dominant agr types in both Iraqi (76.1% and 10.5%) and Syrian refugee (44.9% and 18.37%) communities were I and III. To sum up, no significant differences were observed between the groups for a majority of virulence factors. This is the first investigation of MRSA genotypes and virulence in both these communities. These results could be useful for further studies that assess the genetic relatedness of strains in the region for epidemiological and monitoring purposes, which would be crucial to limiting the spread of MRSA.

A pentaplex PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus and Panton-Valentine Leucocidin.

BACKGROUND: Staphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. RESULTS: The present study focused on the development of a pentaplex PCR assay for the rapid detection of MRSA. The assay simultaneously detected five genes, namely 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, lukS that encodes production of Panton-Valentine leukocidin (PVL), a necrotizing cytotoxin, and one internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. The analytical sensitivity and specificity of the pentaplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the pentaplex PCR at the DNA level was found to be 10 ng DNA. The analytical specificity was evaluated with 34 reference staphylococci and non-staphylococcal strains and was found to be 100%. The diagnostic evaluation of MRSA carried out using 230 clinical isolates, showed 97.6% of sensitivity, 99.3% of specificity, 98.8% of positive predictive value and 98.6% of negative predictive value compared to the conventional method. The presence of an internal control in the pentaplex PCR assay is important to exclude false-negative cases. CONCLUSION: The pentaplex PCR assay developed was rapid and gave results within 4 h, which is essential for the identification of Staphylococcus spp., virulence and their resistance to methicillin. Our PCR assay may be used as an effective surveillance tool to survey the prevalence of MRSA and PVL-producing strains in hospitals and the community.

Pleuropulmonary complications of PVL-positive Staphylococcus aureus infection in children.

UNLABELLED: It is increasingly recognized world-wide that Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus (PVL-SA) is associated with a highly aggressive and often fatal form of community-acquired pneumonia. We report four children who presented with severe pleuropulmonary complications due to infection by community-acquired methicillin-sensitive S. aureus (CA-MSSA), producing PVL toxin. The complications included bilateral multilobular infiltrates, pneumatocoeles, recurrent pneumothoraces, pleural effusion, empyema, lung abscess and diaphragmatic paralysis. This case series highlights the diverse pleuropulmonary manifestations of this potentially lethal infection and the importance of heightened awareness, early recognition and aggressive therapy. CONCLUSION: Complicated pneumonia in a previously fit young patient with a history of preceding 'flu-like' illness or skin/soft tissue infection should raise the suspicion of infection by PVL-positive Staphylococcus aureus (PVL-SA). Severe pleuropulmonary complications are a feature of this disease.