Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1.

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.

Trachea, lung, and tracheobronchial lymph nodes are the major sites where antigen-presenting cells are detected after nasal vaccination of mice with human papillomavirus type 16 virus-like particles.

Vaccination by the nasal route has been successfully used for the induction of immune responses. Either the nasal-associated lymphoid tissue (NALT), the bronchus-associated lymphoid tissue, or lung dendritic cells have been mainly involved. Following nasal vaccination of mice with human papillomavirus type 16 (HPV16) virus-like-particles (VLPs), we have previously shown that interaction of the antigen with the lower respiratory tract was necessary to induce high titers of neutralizing antibodies in genital secretions. However, following a parenteral priming, nasal vaccination with HPV16 VLPs did not require interaction with the lung to induce a mucosal immune response. To evaluate the contribution of the upper and lower respiratory tissues and associated lymph nodes (LN) in the induction of humoral responses against HPV16 VLPs after nasal vaccination, we localized the immune inductive sites and identified the antigen-presenting cells involved using a specific CD4(+) T-cell hybridoma. Our results show that the trachea, the lung, and the tracheobronchial LN were the major sites responsible for the induction of the immune response against HPV16 VLP, while the NALT only played a minor role. Altogether, our data suggest that vaccination strategies aiming to induce efficient immune responses against HPV16 VLP in the female genital tract should target the lower respiratory tract.

European guidelines for empirical antibacterial therapy for febrile neutropenic patients in the era of growing resistance: summary of the 2011 4th European Conference on Infections in Leukemia.

Owing to increasing resistance and the limited arsenal of new antibiotics, especially against Gram-negative pathogens, carefully designed antibiotic regimens are obligatory for febrile neutropenic patients, along with effective infection control. The Expert Group of the 4(th) European Conference on Infections in Leukemia has developed guidelines for initial empirical therapy in febrile neutropenic patients, based on: i) the local resistance epidemiology; and ii) the patient's risk factors for resistant bacteria and for a complicated clinical course. An 'escalation' approach, avoiding empirical carbapenems and combinations, should be employed in patients without particular risk factors. A 'de-escalation' approach, with initial broad-spectrum antibiotics or combinations, should be used only in those patients with: i) known prior colonization or infection with resistant pathogens; or ii) complicated presentation; or iii) in centers where resistant pathogens are prevalent at the onset of febrile neutropenia. In the latter case, infection control and antibiotic stewardship also need urgent review. Modification of the initial regimen at 72-96 h should be based on the patient's clinical course and the microbiological results. Discontinuation of antibiotics after 72 h or later should be considered in neutropenic patients with fever of unknown origin who are hemodynamically stable since presentation and afebrile for at least 48 h, irrespective of neutrophil count and expected duration of neutropenia. This strategy aims to minimize the collateral damage associated with antibiotic overuse, and the further selection of resistance.

Molecular characterization of G and P-types bovine rotavirus strains from Goiás, Brazil: high frequency of mixed P-type infections

In this study, 331 samples from calves less than one month old from a dairy herd in the district of Piracanjuba, state of Goiás, Brazil were tested for rotavirus. Thirty-three samples (9.9%) tested positive for rotavirus. Out of those, 31 were submitted to G and P characterization by reverse transcription followed by semi-nested polymerase chain reaction. Two samples were characterized as G6P[1], three as G10P[11] and five as G6P[11]. The majority of the samples (51.6%) displayed multiple P genotypes (P-genotype mixtures), including typical human genotypes P[4] and P[6M], suggesting the occurrence of co-infections and genetic reassortment. Also, the detection of human genotypes in bovine samples may be considered evidence of the zoonotic potential of rotaviruses. To our knowledge, this is the first report of such a high frequency of P genotype mixtures in bovine rotavirus samples. It also increases data on G and P rotavirus genotypes circulating in dairy herds in Brazil and can help in the development of more efficient immunization approaches, thereby controlling infection and reducing economical losses.

Correlation between koilocytes and human papillomavirus detection by PCR in oral and oropharynx squamous cell carcinoma biopsies

The purpose of this study was to compare the histopathological analysis with polymerase chain reaction (PCR) methods to predict the presence of human papillomavirus (HPV) infection in oral squamous cell carcinoma biopsies. Eighty-three paraffin-embedded tissue specimens from patients with oropharynx and mouth floor squamous cell carcinoma were submitted to histopathological analysis under light microscopy, specifically for the determination of the presence of koilocytes. Subsequently, DNA was purified from the same paraffin-embedded specimens and submitted to PCR. Fisher's exact test showed no statistically significant correlation between the two methods. The results suggest that the presence of koilocytes is unreliable for the detection of HPV presence in oral and oropharynx squamous cell carcinoma.

Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent

Human rhinoviruses (HRV) are usually associated with mild respiratory symptoms in children. However, some studies have found that HRV can cause severe disease, especially when the patient is co-infected with a second virus. In this study, 532 nasopharyngeal aspirates (NPAs) were collected over a nine-year period from children at the Clinics Hospital of Uberlândia. The collected NPAs were then tested for HRV RNA using the reverse transcription-polymerase chain reaction. Eighty-three specimens from children diagnosed with lower respiratory tract illness (LRTI) were positive for HRV RNA and were then tested for the presence of eight other respiratory viruses. A second virus was detected in 37.3% (31/83) of the samples. The most frequent clinical diagnosis was bronchiolitis, followed by other LRTI and then pneumonia. The frequency of severe disease in children infected with more than one virus was not significantly different from the frequency of severe disease in children infected with HRV alone. Children infected with both HRV and parainfluenza virus (1.5 m.o.) were significantly younger than those infected by HRV alone (5.0 m.o.) (p = 0.0454). Overall, these results suggest that infection with a second virus does not lead to a higher frequency of severe syndromes in children presenting with LRTI.

Epidemiology of fungal infections in liver transplant recipients: a six-year study of a large Brazilian liver transplantation centre

Liver transplant seems to be an effective option to prolong survival in patients with end-stage liver disease, although it still can be followed by serious complications. Invasive fungal infections (ifi) are related to high rates of morbidity and mortality. The epidemiology of fungal infections in Brazilian liver transplant recipients is unknown. The aim of this observational and retrospective study was to determine the incidence and epidemiology of fungal infections in all patients who underwent liver transplantation at Albert Einstein Israeli Hospital between 2002-2007. A total of 596 liver transplants were performed in 540 patients. Overall, 77 fungal infections occurred in 68 (13%) patients. Among the 77 fungal infections, there were 40 IFI that occurred in 37 patients (7%). Candida and Aspergillus species were the most common etiologic agents. Candida species accounted for 82% of all fungal infections and for 67% of all IFI, while Aspergillus species accounted for 9% of all fungal infections and for 17% of all IFI. Non-albicans Candida species were the predominant Candida isolates. Invasive aspergillosis tended to occur earlier in the post-transplant period. These findings can contribute to improve antifungal prophylaxis and therapy practices in Brazilian centres.

Complicated intra-abdominal infections in a worldwide context: an observational prospective study (CIAOW Study).

Despite advances in diagnosis, surgery, and antimicrobial therapy, mortality rates associated with complicated intra-abdominal infections remain exceedingly high. The World Society of Emergency Surgery (WSES) has designed the CIAOW study in order to describe the clinical, microbiological, and management-related profiles of both community- and healthcare-acquired complicated intra-abdominal infections in a worldwide context. The CIAOW study (Complicated Intra-Abdominal infection Observational Worldwide Study) is a multicenter observational study currently underway in 57 medical institutions worldwide. The study includes patients undergoing surgery or interventional drainage to address complicated intra-abdominal infections. This preliminary report includes all data from almost the first two months of the six-month study period. Patients who met inclusion criteria with either community-acquired or healthcare-associated complicated intra-abdominal infections (IAIs) were included in the study. 702 patients with a mean age of 49.2 years (range 18-98) were enrolled in the study. 272 patients (38.7%) were women and 430 (62.3%) were men. Among these patients, 615 (87.6%) were affected by community-acquired IAIs while the remaining 87 (12.4%) suffered from healthcare-associated infections. Generalized peritonitis was observed in 304 patients (43.3%), whereas localized peritonitis or abscesses was registered in 398 (57.7%) patients.The overall mortality rate was 10.1% (71/702). The final results of the CIAOW Study will be published following the conclusion of the study period in March 2013.

Complicated intra-abdominal infections in Europe: a comprehensive review of the CIAO study.

The CIAO Study ("Complicated Intra-Abdominal infection Observational" Study) is a multicenter investigation performed in 68 medical institutions throughout Europe over the course of a 6-month observational period (January-June 2012).Patients with either community-acquired or healthcare-associated complicated intra-abdominal infections (IAIs) were included in the study.2,152 patients with a mean age of 53.8 years (range: 4-98 years) were enrolled in the study. 46.3% of the patients were women and 53.7% were men. Intraperitoneal specimens were collected from 62.2% of the enrolled patients, and from these samples, a variety of microorganisms were collectively identified.The overall mortality rate was 7.5% (163/2.152).According to multivariate analysis of the compiled data, several criteria were found to be independent variables predictive of patient mortality, including patient age, the presence of an intestinal non-appendicular source of infection (colonic non-diverticular perforation, complicated diverticulitis, small bowel perforation), a delayed initial intervention (a delay exceeding 24 hours), sepsis and septic shock in the immediate post-operative period, and ICU admission.Given the sweeping geographical distribution of the participating medical centers, the CIAO Study gives an accurate description of the epidemiological, clinical, microbiological, and treatment profiles of complicated intra-abdominal infections (IAIs) throughout Europe.

Infections with human coronaviruses NL63 and OC43 among hospitalised and outpatient individuals in São Paulo, Brazil

The incidence and clinical features of human coronaviruses (HCoVs) among Brazilian patients with respiratory illness are not well known. We investigated the prevalence of HCoVs among Brazilian outpatients and hospitalised patients with respiratory illnesses during 2009 and 2010. To identify the HCoVs, pancoronavirus and species-specific reverse-transcriptase polymerase chain reaction assays were performed. Five of 394 samples were positive for HCoVs (1.2%): 1/182 (0.5%) outpatients and 4/212 (1.8%) hospitalised patients. The OC43 and NL63 HCoVs were identified. Two patients were admitted to the intensive care unit. Underlying chronic disease was reported in cases and one diabetic adult died. HCoVs can cause lower respiratory infections and hospitalisation. Patients with pre-existing conditions and respiratory infections should be evaluated for HCoV infections.

Differential outcome of concurrent radiotherapy plus epidermal growth factor receptor inhibitors versus radiotherapy plus cisplatin in patients with human papillomavirus-related head and neck cancer.

BACKGROUND Human papillomavirus (HPV)-related head and neck cancer has been associated with an improved prognosis in patients treated with radiotherapy (RT) +/- chemotherapy (CT); however, RT combined with epidermal growth factor receptor (EGFR) inhibitors has not been fully studied in this group of patients. METHODS Immunohistochemical expression of p16 and PCR of HPV16 DNA were retrospectively analyzed in tumor blocks from 108 stage III/IV head and neck cancer patients treated with RT+CT (56) or RT+EGFR inhibitors (52). Disease-free survival (DFS) and overall survival (OS) were analyzed by the Kaplan-Meier method. RESULTS DNA of HPV16 was found in 12 of 108 tumors (11%) and p16 positivity in 18 tumors (17%), with similar rates in both arms of treatment. After a median follow-up time of 35 months (range 6-135), p16-positive patients treated with RT+EGFR inhibitors showed improved survival compared with those treated with RT+CT (2-year OS 88% vs. 60%, HR 0.18; 95% CI 0.04 to 0.88; p = 0.01; and 2-year DFS 75% vs. 47%, HR 0.17; 95% CI 0.03 to 0.8; p = 0.01). However, no differences were observed in p16-negative patients (2-year OS 56% vs. 53%, HR 0.97; 95% CI 0.55 to 1.7; p = 0.9; and 2-year DFS 43% vs. 45%, HR 0.99; 95% CI 0.57 to 1.7; p = 0.9). CONCLUSIONS This is the first study to show that p16-positive patients may benefit more from RT+EGFR inhibitors than conventional RT+CT. These results are hypothesis-generating and should be confirmed in prospective trials.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Unveiling healthy carriers and subclinical infections among household contacts of leprosy patients who play potential roles in the disease chain of transmission

Leprosy transmission still occurs despite the availability of highly effective treatment. The next step towards successfully eliminating leprosy is interrupting the chain of transmission of the aetiological agent, Mycobacterium leprae. In this investigation, we provide evidence that household contacts (HHCs) of leprosy patients might not only have subclinical infections, but may also be actively involved in bacilli transmission. We studied 444 patients and 1,352 contacts using anti-phenolic glycolipid-I (PGL-I) serology and quantitative polymerase chain reaction (qPCR) to test for M. leprae DNA in nasal swabs. We classified the patients according to the clinical form of their disease and the contacts according to the characteristics of their index case. Overall, 63.3% and 34.2% of patients tested positive by ELISA and PCR, respectively. For HHCs, 13.3% had a positive ELISA test result and 4.7% had a positive PCR test result. The presence of circulating anti-PGL-I among healthy contacts (with or without a positive PCR test result from nasal swabs) was considered to indicate a subclinical infection. DNA detected in nasal swabs also indicates the presence of bacilli at the site of transmission and bacterial entrance. We suggest that the concomitant use of both assays may allow us to detect subclinical infection in HHCs and to identify possible bacilli carriers who may transmit and disseminate disease in endemic regions. Chemoprophylaxis of these contacts is suggested.

Trends in yearly prevalence of third-generation cephalosporin and fluoroquinolone resistant Enterobacteriaceae infections and antimicrobial use in Spanish hospitals, Spain, 1999 to 2010.

Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp. are a major cause of infections in hospitalised patients. The aim of our study was to evaluate rates and trends of resistance to third-generation cephalosporins and fluoroquinolones in infected patients, the trends in use for these antimicrobials, and to assess the potential correlation between both trends. The database of national point prevalence study series of infections and antimicrobial use among patients hospitalised in Spain over the period from 1999 to 2010 was analysed. On average 265 hospitals and 60,000 patients were surveyed per year yielding a total of 19,801 E. coli, 3,004 K. pneumoniae and 3,205 Enterobacter isolates. During the twelve years period, we observed significant increases for the use of fluoroquinolones (5.8%-10.2%, p<0.001), but not for third-generation cephalosporins (6.4%-5.9%, p=NS). Resistance to third-generation cephalosporins increased significantly for E. coli (5%-15%, p<0.01) and for K. pneumoniae infections (4%-21%, p<0.01) but not for Enterobacter spp. (24%). Resistance to fluoroquinolones increased significantly for E. coli (16%30%, p<0.01), for K. pneumoniae (5%-22%, p<0.01), and for Enterobacter spp. (6%-15%, p<0.01). We found strong correlations between the rate of fluoroquinolone use and the resistance to fluoroquinolones, third-generation cephalosporins, or co-resistance to both, for E. coli (R=0.97, p<0.01, R=0.94, p<0.01, and R=0.96, p<0.01, respectively), and for K. pneumoniae (R=0.92, p<0.01, R=0.91, p<0.01, and R=0.92, p<0.01, respectively). No correlation could be found between the use of third-generation cephalosporins and resistance to any of the latter antimicrobials. No significant correlations could be found for Enterobacter spp.. Knowledge of the trends in antimicrobial resistance and use of antimicrobials in the hospitalised population at the national level can help to develop prevention strategies.

Time-based distribution of Staphylococcus saprophyticus pulsed field gel-electrophoresis clusters in community-acquired urinary tract infections

The epidemiology of urinary tract infections (UTI) by Staphylococcus saprophyticus has not been fully characterised and strain typing methods have not been validated for this agent. To evaluate whether epidemiological relationships exist between clusters of pulsed field gel-electrophoresis (PFGE) genotypes of S. saprophyticus from community-acquired UTI, a cross-sectional surveillance study was conducted in the city of Rio de Janeiro, Brazil. In total, 32 (16%) female patients attending two walk-in clinics were culture-positive for S. saprophyticus. Five PFGE clusters were defined and evaluated against epidemiological data. The PFGE clusters were grouped in time, suggesting the existence of community point sources of S. saprophyticus. From these point sources, S. saprophyticus strains may spread among individuals.