Semen searching when sperm is absent.

Sexual assault cases have varying factors that may mask semen findings when analysing evidence at the forensic laboratory. Semenogelin (Sg) is a potential marker for the identification of semen even at azoospermy or when few sperm cells are found. The current study examined Sg in normospermic and azoospermic donors as an internal evaluation of sensitivity, specificity and interference. The impact of a historical review of 53 judicial sexual assault cases over a five-year period was also analysed. The use of varying tests was of importance to prioritize certain samples within cases. Semen findings by Sg were then compared to prostate-specific antigen (PSA), phosphatase enzyme (AP) and Y-chromosome presence, the latter being used in an attempt to link semen fluid identification with obtaining a male DNA profile. Test findings were the highest ever registered for Sg (1:400,000), PSA (1:800,000), AP (1:25,000) and sperm cytology (SC) (1:50,000). Our results demonstrated the usefulness of using the Sg marker to avoid a false semen-negative result (6% cases), particularly in cases where sperm was absent or scarce (11% spermatozoa positive cases). Results were expressed in categories according to the set: Sg-PSA-AP. Thus, categories I (full positive, 46%), VI (full negative, 27%) and III (Sg/PSA positive; 11%) were the most frequent and Y-chromosome was obtained in 59%, 12% and 12% ratios, respectively. In conclusion, Sg was recommended for the workflow procedure of semen investigation when sperm absence is expected either from azoospermic/oligospermic or normospermic semen, especially before/after ejaculation.

Emergence of high level azithromycin-resistant Neisseria gonorrhoeae strain isolated in Argentina.

One Neisseria gonorrhoeae strains highly resistant to azithromycin AzHLR (MIC >2048 mg/L) was isolated in Argentina in 2001 and it has been characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) as ST696, suggesting a different event to other isolates in Europe. Neither, mtrR mutations or presence of mef gene were detected.

Real-time polymerase chain reaction detection of Neisseria meningitidis in formalin-fixed tissues from sudden deaths.

Accurate identification of meningococcal sudden deaths is needed to avoid underestimation of the true incidence of the disease. This study analyzed the usefulness of a real-time polymerase chain reaction (PCR) protocol using MGB (3'-minor groove binder) probes to detect Neisseria meningitidis in formalin-fixed paraffin-embedded tissues from sudden deaths where a meningococcal fulminating infection was suspected. The protocol included detection of meningococcal DNA (ctrA gene), multiplex B/C PCR serogrouping (siaD gene), and rapid confirmation of PCR products by microcapillary electrophoresis. Sixty-nine tissues from 15 culture-confirmed meningococcal sudden deaths were analyzed (positive cases). Validation studies were performed. In each positive case, both the ctrA and the B/C siaD genes were detected. The ctrA was detected in 81.2% of the samples, whereas the serogroup (B or C) was identified in 44.9% of them. Therefore, this protocol may improve nonculture diagnosis and case ascertainment in meningococcal disease deaths, particularly when formalin-fixed tissues are the only available specimen.

Antibiotic resistant meningococci in Europe: any need to act?

Meningococcal disease is a serious and rapidly progressing illness. It is therefore very important to monitor changes in the level of antibiotic susceptibility among clinical isolates. Different aspects such as interpretation of laboratory results, determination of breakpoints predicting treatment failure as well as definition of susceptibility levels in clinical samples using molecular methods are critical points for the surveillance of antibiotic resistance in Neisseria meningitidis. Within the strategic framework outlined by the EU.MenNet project, several objectives were identified to analyze 'The spread of antibiotic resistant meningococci in Europe', including the extent of antimicrobial resistance, its association with particular meningococcal lineages and geographical areas, as well as molecular characterization of the mechanisms involved, particularly in penicillin resistance. A heterogeneous figure for the frequency of intermediate resistance to penicillin appears across Europe. This heterogeneity may reflect different clonal lineages and/or uneven access to antibiotics in each country. In addition, the use of different criteria for the methodology used for definition cannot be avoided. The description of five specific changes associated with intermediate resistance to penicillin also allows the design of PCR-based tools to objectify results and for application in clinical samples.

[Evolution of Neisseria meningitidis sensitivity to various antimicrobial drugs over the course of chemoprophylaxis during an epidemic outbreak]. / Evolución de la sensibilidad de Neisseria meningitidis a diversos antimicrobianos en el curso de intervenciones con quimioprofilaxis durante un brote epidémico.

INTRODUCTION: The aim of this study was to assess the evolution of the population MICs for various antimicrobial drugs against Neisseria meningitidis isolates obtained from asymptomatic carriers during a chemoprophylaxis campaign carried out for an epidemic outbreak of meningococcal disease in Nerva, a small town in Huelva province (Spain). MATERIAL AND METHODS: A nasopharyngeal carrier study including 427 people was carried out to determine the incidence rate of the epidemic strain among the general population. On the basis of the results, chemoprophylaxis with rifampicin was administered to the population aged 15 to 29 years (age group showing the highest incidence of the epidemic strain among carriers) living in Nerva. Three months later a new carrier study was performed (507 people) to evaluate the effects of chemoprophylaxis. Given the evolution of the outbreak, seven months later a new intervention was required with ciprofloxacin chemoprophylaxis; a second carrier study (399 people) was performed two months later to evaluate its effect. RESULTS: The number of strains isolated during the three carrier studies was 59 (13.8%), 33 (6.5%), and 22 (5.5%), respectively. Analysis of the changes in the MIC50 and MIC90 for the various antibiotics from the first to the second carrier study (rifampicin chemoprophylaxis) showed statistical differences only in the distribution of rifampicin MICs. Similarly, when changes from the second to the third study were analyzed (ciprofloxacin chemoprophylaxis), significant variations were detected for the cefotaxime MICs. Nevertheless, although there were changes in the MICs, the percentages of susceptibility from the beginning to the end of the study did not vary. CONCLUSIONS: Massive chemoprophylaxis in the age group with the highest incidence of the epidemic strain among carriers did not clearly modify the antibiotic susceptibility of the isolates. However, a slight increase in the MIC50 and MIC90 was observed for rifampicin after the first chemoprophylactic intervention and for cefotaxime at the end of the study. Consecutive chemoprophylactic interventions with rifampicin and ciprofloxacin had an impact on the percentage of meningococcal carriers in the overall population, with a clearly decreasing trend.

Evolución de la sensibilidad de Neisseria meningitidis a diversos antimicrobianos en el curso de intervenciones con quimioprofilaxis durante un brote epidémico / Evolution of Neisseria meningitidis sensitivity to various antimicrobial drugs over the course of chemoprophylaxis during an epidemic outbreak

Introducción. El objetivo del estudio fue evaluar la evolución de las concentraciones inhibitorias mínimas (CIM) poblacionales frente a diversos antimicrobianos en aislamientos de Neisseria meningitidis obtenidos de portadores asintomáticos en el contexto de un brote epidémico de enfermedad meningocócica localizado en la provincia de Huelva en el que se realizó una intervención de quimioprofilaxis en población general. Material y métodos. El primer estudio de portadores se realizó en 427 individuos con objeto de conocer la presencia de la cepa causante del brote en población general. Como resultado se procedió a utilizar quimioproflaxis con rifampicina en la población entre 15 y 29 años (grupo etario con mayor presencia de la cepa epidémica), residentes en la población de Nerva. A los 3 meses se realizó un nuevo estudio de portadores (507 personas) para evaluar el efecto de dicha quimioprofilaxis. Dada la evolución del brote epidémico fue necesario realizar a los 7 meses una nueva intervención con ciprofloxacino y transcurridos 2 meses un nuevo estudio de portadores (399 personas) para evaluar su efecto. Resultados. El número de cepas aisladas en los 3 estudios de portadores realizados fue de 59 (13,8%), 33 (6,5%) y 22 (5,5%), respectivamente. El análisis de la evolución de la CIM50 y CIM90 de los distintos antibióticos del primer al segundo estudio de portadores (quimioprofilaxis con rifampicina) únicamente detectó cambios con significación estadística en las CIM de rifampicina. Así mismo, cuando se analizó la variación en la distribución de las CIM del segundo al tercer estudio (quimioprofilaxis con ciprofloxacino) fueron detectados cambios significativos particularmente en las CIM de cefotaxima. Aunque existieron variaciones de CIM, los porcentajes de sensibilidad al principio y final del estudio no variaron. Conclusiones. El empleo de quimioprofilaxis masiva en el grupo de edad con mayor porcentaje de portadores de la cepa de N. meningitidis responsable del brote no modificó sensiblemente la sensibilidad antibiótica de los aislados, si bien puede observarse un aumento de los valores de la CIM50 y CIM90 en el caso de la rifampicina tras la intervención con este antimicrobiano, y en el caso de la cefotaxima tras las 2 intervenciones realizadas. Las sucesivas intervenciones de quimioprofilaxis con rifampicina y posteriormente con ciprofloxacino tuvieron un claro reflejo en el porcentaje portadores en la población general, con una tendencia claramente decreciente (AU)

Introduction. The aim of this study was to assess the evolution of the population MICs for various antimicrobial drugs against Neisseria meningitidis isolates obtained from asymptomatic carriers during a chemoprophylaxis campaign carried out for an epidemic outbreak of meningococcal disease in Nerva, a small town in Huelva province (Spain). Material and methods. A nasopharyngeal carrier study including 427 people was carried out to determine the incidence rate of the epidemic strain among the general population. On the basis of the results, chemoprophylaxis with rifampicin was administered to the population aged 15 to 29 years (age group showing the highest incidence of the epidemic strain among carriers) living in Nerva. Three months later a new carrier study was performed (507 people) to evaluate the effects of chemoprophylaxis. Given the evolution of the outbreak, seven months later a new intervention was required with ciprofloxacin chemoprophylaxis; a second carrier study (399 people) was performed two months later to evaluate its effect. Results. The number of strains isolated during the three carrier studies was 59 (13.8%), 33 (6.5%), and 22 (5.5%), respectively. Analysis of the changes in the MIC50 and MIC90 for the various antibiotics from the first to the second carrier study (rifampicin chemoprophylaxis) showed statistical differences only in the distribution of rifampicin MICs. Similarly, when changes from the second to the third study were analyzed (ciprofloxacin chemoprophylaxis), significant variations were detected for the cefotaxime MICs. Nevertheless, although there were changes in the MICs, the percentages of susceptibility from the beginning to the end of the study did not vary. Conclusions. Massive chemoprophylaxis in the age group with the highest incidence of the epidemic strain among carriers did not clearly modify the antibiotic susceptibility of the isolates. However, a slight increase in the MIC50 and MIC90 was observed for rifampicin after the first chemoprophylactic intervention and for cefotaxime at the end of the study. Consecutive chemoprophylactic interventions with rifampicin and ciprofloxacin had an impact on the percentage of meningococcal carriers in the overall population, with a clearly decreasing trend (AU)

Locus NMB0035 codes for a 47-kDa surface-accessible conserved antigen in Neisseria / El locus NMB0035 codifica un antígeno de superficie conservado de 47 kDa en Neisseria

A47 kDa neisserial outer-membrane antigenic protein (P47) was purified to homogeneity and used to prepare polyclonal anti-P47 antisera. Protein P47 was identified by MALDI-TOF fingerprinting analysis as the hypothetical lipoprotein NMB0035. Two-dimensional diagonal SDS-PAGE results suggested that, contrary to previous findings, P47 is not strongly associated with other proteins in membrane complexes. Western blotting with the polyclonal monospecific serum showed that linear P47 epitopes were expressed in similar amounts in the 27 Neisseria meningitidis strains tested and, to a lesser extent, in commensal Neisseria, particularly N. lactamica. However, dot-blotting assays with the same serum demonstrated binding variability between meningococcal strains, indicating differences in surface accessibility or steric hindrance by other surface structures. Specific anti-P47 antibodies were bactericidal against the homologous strain but had variable activity against heterologous strains, consistent with the results from dot-blotting experiments. An in-depth study of P47 is necessary to evaluate its potential as a candidate for new vaccine designs (AU)

El tratamiento con Triton X-114, seguido de la separación por SDS-PAGE permitió la purificación de una proteína antigénica de 47 kDa (P47) de la membrana externa de Neisseria de forma homogénea, lo que permitió la preparación de sueros policlonales anti-P47. La P47 se identificó como la lipoproteína (hipotética) NMB0035 mediante el análisis de la huella molecular realizado por MALDI-TOF. Los resultados obtenidos por SDS-PAGE diagonal bidimensional sugieren que, a diferencia de otros resultados previos, la P47 no parece estar fuertemente asociada a otras proteínas en complejos de membrana. Análisis de inmunoelectrotransferencia (Western blot) empleando el suero policlonal monoespecífico demostró que los epítopos lineales de la P47 se expresaron de forma similar en las 27 cepas de Neisseria meningitidis ensayadas y, aunque con menor intensidad, también lo hicieron en algunas Neisseria comensales, especialmente en N. lactamica. Sin embargo, resultados obtenidos por transferencia de mancha con el mismo suero demostraron que entre las cepas meningocócicas existe variabilidad en la capacidad de unión, lo que sugiere que o bien hay diferencias en la accesibilidad en superficie o bien una ocultación estérica producida por otras estructuras de superficie. Los anticuerpos específicos anti-P47 tenían efecto bactericida contra la cepa homóloga pero mostraron una actividad variable contra cepas heterólogas, lo cual concuerda con los resultados de la transferencia de mancha, y hace pensar que es necesario estudiar esta proteína más profundamente para evaluar su potencial como candidata para nuevos diseños de vacunas (AU)

Locus NMB0035 codes for a 47-kDa surface-accessible conserved antigen in Neisseria.

A47 kDa neisserial outer-membrane antigenic protein (P47) was purified to homogeneity and used to prepare polyclonal anti-P47 antisera. Protein P47 was identified by MALDI-TOF fingerprinting analysis as the hypothetical lipoprotein NMB0035. Two-dimensional diagonal SDS-PAGE results suggested that, contrary to previous findings, P47 is not strongly associated with other proteins in membrane complexes. Western blotting with the polyclonal monospecific serum showed that linear P47 epitopes were expressed in similar amounts in the 27 Neisseria meningitidis strains tested and, to a lesser extent, in commensal Neisseria, particularly N. lactamica. However, dot-blotting assays with the same serum demonstrated binding variability between meningococcal strains, indicating differences in surface accessibility or steric hindrance by other surface structures. Specific anti-P47 antibodies were bactericidal against the homologous strain but had variable activity against heterologous strains, consistent with the results from dot-blotting experiments. An in-depth study of P47 is necessary to evaluate its potential as a candidate for new vaccine designs.

Antigenic and/or phase variation of PorA protein in non-subtypable Neisseria meningitidis strains isolated in Spain.

The PorA protein is a potential candidate as a vaccine component against meningococcal disease. However, this protein experiences antigenic variation and is subject to phase variations to evade immune selective pressure. In this study, the mechanisms responsible for altered expression of the PorA protein were analysed in 50 non-subtypable strains isolated from patients with meningococcal disease in Spain. The porA gene was amplified from 47 of the 50 strains. The majority of isolates were not recognized by the subtyping panel, as a result of non-synonymous base changes in the variable regions of the porA gene. Two of these strains revealed a premature stop codon before the variable region VR1 of PorA due to a single base-pair substitution at position 109 of the porA coding region. Another two presented a homopolymeric tract of eight adenine residues in the coding region, producing a DNA strand-slippage mechanism and PorA phase variation.

Sequencing of Neisseria meningitidis penA gene: the key to success in defining penicillin G breakpoints.

Testing of susceptibility to penicillin G by E-test and sequencing of an internal fragment of the penA gene were done for 43 meningococcal strains. Those strains for which the MIC was >/=0.094 micro g/ml showed mosaic alleles, so 0.094 micro g/ml is suggested as the penicillin G intermediate breakpoint when E-test is used.

Dynamics of the penA gene in serogroup C meningococcal strains.

The transpeptidase encoding region of the penA gene was sequenced in 44 meningococcal strains (41 serogroup C [23 characterized as serotype 2b and 18 as serotype 2a] and 3 serogroup B [B:2b:P1.2,5]). All strains were characterized by multilocus sequence typing and were determined to be susceptible or intermediate resistant to penicillin (Pen(s) or Pen(i), respectively). A high degree of homology was found among the penA alleles identified in the Pen(s) strains. All the Pen(i) C:2b strains, which belonged to 2 different clonal complexes, showed the same penA gene allele. This fact suggests that 1 of the clonal complexes acquired that allele, spreading it to the other by horizontal transfer. The same allele also was found in the B:2b strains studied, indicating that 1 of the Pen(i) C:2b strains underwent a capsular switching event. A different mosaic penA allele was identified in the Pen(i) C:2a strains, which belonged to the ET37 cluster.

Capsule switching among C:2b:P1.2,5 meningococcal epidemic strains after mass immunization campaign, Spain.

A mass immunization campaign for 18-month to 19-year-olds was undertaken in Spain in 1996-1997 because of an epidemic of serogroup C meningococcal disease associated with a C:2b:P1.2,5 strain belonging to the A4 lineage. Surveillance for the "capsule-switching" phenomenon producing B:2b:P1.2,5 isolates was undertaken. Of 2,975 meningococci characterized, B:2b:P1.2,5 and B:2b:P1.2 antigenic combinations were found in 18 isolates; 15 meningococci were defined as serogroup B belonging to the A4 lineage.