R. Cisterna

Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae

Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type beta-lactamases in Enterobacteriaceae. TEM-type and OXA-1 enzymes are the major plasmid-borne beta-lactamases implicated in amoxicillin-clavulanic acid resistance in Escherichia coli isolates. We have developed a rapid and simple multiplex polymerase chain reaction (PCR) which discriminates bla(TEM), bla(SHV) and bla(OXA-1) genes by generating fragments of 516, 392 and 619 bp respectively. Multiplex PCR analysis of 51 amoxicillin-clavulanate resistant E. coli isolates detected bla(TEM) and bla(SHV) genes in 45 and two strains, respectively, and only one strain harboured a bla(OXA-1) gene. Twenty-three of the 40 cefotaxime-resistant Enterobacteriaceae isolates produced amplicons with a size compatible with the presence of bla(TEM) (13 strains), bla(SHV) (six strains) genes or the association of both genes (four strains). These results were verified by colony hybridisation. Therefore, multiplex PCR is a suitable tool for initial rapid screening of bla genes in Enterobacteriaceae.

Value of detection of antibodies toCandida albicans germ tube in the diagnosis of systemic candidosis

To test the value of detection of anti-Candida albicans germ tube antibodies by indirect immunofluorescence assay in the diagnosis of systemic candidosis, a retrospective study was done using 126 sera from 27 patients with presumptive systemic candidosis (13 immunocompromised), 165 sera from 45 patients with aspergillosis (29 immunocompromised), 35 sera from eight patients with cryptococcosis (6 immunocompromised), and 101 sera from 101 blood donors. While 21 of 27 patients with systemic candidosis (77.8%) had anti-germ tube antibodies, these antibodies were absent in all patients with cryptococcosis and in all blood donors. They were however detected in 5 of 45 patients with aspergillosis (11.1%). Ten of 13 (76.9%) immunocompromised patients with candidosis had anti-germ tube antibodies; similar results were obtained in immunocompetent patients with candidosis (78.6%). The specificity was 96.8%, indicating a high degree of discrimination was possible between systemic candidosis and other invasive mycoses in the patients studied. Anti-germ tube responses did not appear to be significantly reduced in immunocompromised patients.

Molecular Epidemiology of Panton-Valentine Leukocidin-Positive Staphylococcus aureus in Spain: Emergence of the USA300 Clone in an Autochthonous Population

ABSTRACT We characterized all of the Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus isolates collected between 2005 and 2008 in the Bilbao, Spain, area. For the first time, the USA300 clone is reported as predominant among PVL-positive clones in a European autochthonous population, requiring active monitoring of the incidence of USA300 in Spain and throughout Europe.

Detection of antibodies toCandida albicans germ tube in the diagnosis of systemic candidiasis

Sera from 109 subjects were tested for the presence oftantiCandida albicansantibodies by an indirect immunofluorescence assay. Aliquots of the sera were adsorbed with heat-killed blastospores to remove the antibodies against the surface of the yeast-phase cell wall and tested for anti-germ tube cell wall antibodies. Unadsorbed sera stained the entire cell wall of yeast and germ-tubes. Immunoglobulin G (IgG) antibodies were found in all patients with systemic candidiasis and in 81.2% of patients withCandida albicansisolated from skin and mucous membranes. IgA and IgG were found in 67.4 and 57.1 %, respectively, of controls without evidence of candidiasis. After the adsorption only sera from patients with systemic candidiasis showed antibodies, predominantly IgA, against germ tube cell wall. Adsorption of the sera thus increased the specificity, efficiency, and positive and negative predictive values of the test. The test achieved the highest sensitivity in adsorbed sera for the combination of IgA and IgG.

Nosocomial outbreak of linezolid-resistant Enterococcus faecalis infection in a tertiary care hospital

We describe 12 cases of linezolid-resistant Enterococcus faecalis. The present study was done in 2 wards of Hospital Universitario La Paz in Madrid, Spain. The 2 wards involved were the intensive care unit (ICU) and reanimation unit. Twelve clinical strains of E. faecalis reported by the clinical laboratory as linezolid resistant based on MICs determined by E-test (AB Biodisk, Solna, Sweden) were collected between September 2005 and October 2006. The MIC of linezolid for all the resistant isolates was >128 microg/mL. The isolates were analyzed for the presence of the G2576T mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and pyrosequencing. Pyrosequencing showed that the first isolate had G and T at position 2576 in a 1:1 ratio, whereas the remaining ones had a wild type to mutant ratio of 1:3. PCR-RFLP showed that the mutations were in alleles 1, 3, and 4. The 12 isolates under investigation came from different patients but were indistinguishable by pulsed-field gel electrophoresis (n = 7) and repetitive extragenic palindromic sequence (REP)-PCR (n = 12). This is the first report of a clonal outbreak of linezolid-resistant E. faecalis in Spain. To prevent or minimize the emergence of resistance, we should use linezolid strictly after the therapeutic indications, courses of treatment should be kept as short as possible, and risk factors for resistance development should be considered before starting. In addition, we suggest that susceptibility testing of clinically significant Gram-positive pathogens should be done in all cases of treatment failure, and, depending on the local epidemiology of each ICU, it might be advisable to do it before starting treatment with linezolid.

In vitro susceptibility ofAeromonas caviae, Aeromonas hydrophila andAeromonas sobria to fifteen antibacterial agents

In vitro testing of the activity of 15 antibacterial agents against 522 clinical isolates ofAeromonas species demonstrated some species-associated trends. Amoxicillin plus clavulanic acid was effective against approximately 45% ofAeromonas caviae andAeromonas hydrophila, but allAeromonas sobria isolates were resistant. Aztreonam, piperacillin and mezlocillin were highly active against all the strains ofAeromonas tested. Ticarcillin was equally effective againstAeromonas caviae andAeromonas hydrophila, but more than 50% ofAeromonas sobria isolates were resistant. The latter species was more susceptible to cephalosporins thanAeromonas hydrophila andAeromonas caviae. Chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole were extremely active against all threeAeromonas species, likewise ofloxacin and ciprofloxacin. Aztreonam, third-generation cephalosporins, chloramphenicol and the quinolones can thus be considered for therapy of infections whenAeromonas is implicated.

Seroprevalence of hepatitis B and C, and human immunodeficiency type 1 viruses in a rural population from the Republic of Equatorial Guinea

The seroprevalence of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) markers was evaluated in a group of 2042 subjects from a rural area in the Republic of Equatorial Guinea, to obtain a better understanding of the transmission patterns of these viruses. Antibodies to HIV-1 were detected in 12 subjects (0.6%); the seroprevalence did not differ significantly by age or gender. Overall seroprevalence for HCV was 1.7% (in patients aged > 40 years, 5.6%). Hepatitis B surface antigen was detected in 8.8% of subjects, with the higher seroprevalence in children aged < or = 18 years of 13.4% contrasting with the higher seroprevalence of HCV in older subjects of the Equatoguinean studied population. These results indicate differences in the distribution of the viruses and, probably, different routes of transmission. The study demonstrates the existence of a high HBV carrier rate in children, concluding that hepatitis B vaccine should be incorporated into the Expanded Programme on Vaccination in Equatorial Guinea.

Two Lymphogranuloma Venereum Cases in a Heterosexual Couple in Bilbao (spain)

LYMPHOGRANULOMA VENEREUM (LGV) WAS AN unusual disease in industrialized countries until 4 years ago; before this when it did appear, it had generally been acquired in some endemic area (Africa, Asia, South America, or the Caribbean Islands). In December 2003, an outbreak of LGV was detected in Rotterdam, Netherlands.1 Since then, more than 1000 cases have been reported in the Netherlands, France, the United Kingdom, Germany, Belgium, Switzerland, Sweden, Portugal, Denmark, and Spain.2–10 In the United States and Canada, the first cases were recorded in 2004 and between then and November 2006, 85 cases were reported in Canada.1,11 All these cases had remarkably uniform characteristics: men who have sex with men (MSM), a high rate of HIV seropositivity and frequent concurrent infection with other STIs. Most of them had rectal symptoms and only a few inguinal lymphadenopathy or classic genital ulcers.12 All confirmed cases were of the genotype L2. There was no evidence that this infection had extended beyond these nuclear groups, until 3 LGV cases were reported among Portuguese women during 2007.9 The aim of this article is to describe the first LGV cases diagnosed in a heterosexual couple in Bilbao (Spain). We directly documented sexual transmission by identifying genital Chlamydia trachomatis type L2 infection in both patients. The results also suggest that primary classic LGV can take the form of urethritis and cervicitis, without genital ulceration. A further 2 cases of LGV had previously been recorded in Spain, but both of them were in MSM.10 In June 2006, a 33-year-old Spanish man came to see us complaining of 2 months of gradually progressive and painful right inguinal lymphadenopathy (swelling). The onset of swelling was accompanied by radiating low back pain, myalgia in the lower extremities, and dysuria. He denied any history of previous genital ulceration, inguinal buboes, fever, malaise, proctitis, or urethral discharge. His general practitioner had treated him with an antibiotic and antiinflammatory drug whose name he did not know. Dysuria remitted with treatment but the swelling did not. The patient reported multiple unprotected heterosexual contacts during the previous year but he had not traveled abroad. He also denied having had sexual intercourse with men or immigrants. Physical examination revealed bilateral inguinal lymph nodes of firm consistency, those on the right being bigger. Urethral, rectal, and pharyngeal samples were taken for microbiologic diagnosis. Samples for direct fluorescent antibody and culture for C. trachomatis were negative. So were culture for Neisseria gonorrhoeae and herpes simplex virus. There was no serological evidence of syphilis or HIV infection. The laboratory extracted DNA from all samples using the Amplicor CT/NG extraction kit and tested them for C. trachomatis using an in-house TaqMan real-time PCR assay, which targeted the cryptic plasmid. C. trachomatis PCR was positive in the urethral sample and negative in other sites. Ultrasound-guided fine needle aspiration of the right lymph node was performed during a second visit in July 2006. The material obtained was processed in the same way as described above and C. trachomatis PCR was positive. Urethral and lymph node DNA samples were tested for LGV using a TaqMan based real-time PCR that used the polymorphic membrane protein H gene as a PCR target.13 The results of this assay were positive. Both samples were identified as LGV serovar 2 and not L2b genovar, by sequencing an ompA segment spanning variable segments 1 and 2 using an ABI Genetic Analyzer 3130 and Big Dye Terminator kit version 3.1 according to the manufacturer’s protocol. The man’s partner, a 29 year-old Spanish woman, described a history of painful bilateral inguinal swellings. She was 8-weeks pregnant and denied any sexual contact other than with her partner over the previous 2 years. Examination found painless bilateral inguinal nodes of firm consistency. Vaginal, endocervical, and Correspondence: Dr. Josefina López de Munain, Hospital de Basurto, Servicio de Enfermedades Infecciosas, Avenida de Montevideo 18, 48013 Bilbao, Spain. E-mail: josefina.lopezdemunain@osakidetza.net. Received for publication October 22, 2007, and accepted May 2, 2008. From the *Infectious Diseases Service, and the Clinical Microbiology Service, Basurto Hospital, Basque Health Service, Bilbao, Spain Sexually Transmitted Diseases, November 2008, Vol. 35, No. 11, p.918–919 DOI: 10.1097/OLQ.0b013e31817e9228 Copyright

Sentinel surveillance of invasive candidiasis in Spain: epidemiology and antifungal susceptibility

In order to know the epidemiology and the changes of antifungal resistance in invasive candidiasis (IC) we carried out this prospective study of Candida strains belonging to patients admitted to 26 Spanish hospitals from June 2011 to June 2012 diagnosed with IC. Clinical information and the identity of the Candida species were collected and antifungal susceptibility was tested using broth microdilution in five agents: amphotericin B, fluconazole, voriconazole, caspofungin and anidulafungin. A total of 705 cases-isolates were documented. Most of the patients suffered from candidemia and several underlying diseases and more than half of them were neutropenic or under immunosuppressive therapy, factors associated with higher mortality. Thirty percent of global mortality was documented. C. albicans was the most frequently isolated species, although an increase of non-C. albicans species was observed. Resistance to fluconazole was of 3.4%, lower than in previous years (6.3%). C. parapsilosis presented a higher MIC90 of echinocandins compared to other species.

Detection of hepatitis C virus RNA in serum and peripheral blood mononuclear cells in patients with chronic hepatitis C treated with interferon alpha

PCR was used to detect hepatitis C virus (HCV) RNA in serum and peripheral blood mononuclear cells (PBMCs) for evaluation of a six-month course of Interferon therapy in 18 patients with histologically confirmed chronic hepatitis C. At follow-up six months after the end of therapy positive-stranded (genomic) and negative-stranded (anti-genomic, presumptive replicative intermediate) HCV RNA could be detected in PBMCs of all ten patients who either did not respond to therapy or suffered a relapse; genomic strand RNA was detected in five patients who responded but then relapsed. The study confirms that interferon therapy leads to inhibition of HCV replication but not eradication of the virus. Persistence of the virus at extrahepatic sites may explain its reactivation after cessation of interferon therapy.