Tularaemia of middle ear with suppurative lymphadenopathy and retropharyngeal abscess.

OBJECTIVE: We report an extremely rare case of otitis media due to Francisella tularensis, complicated by multiple suppurative cervical lesions and a lasting conductive hearing loss. CASE REPORT: A young woman presented with otitis media, several neck swellings and a retropharyngeal swelling. Polymerase chain reaction testing of aspirated fluid and serology confirmed the diagnosis of tularaemia. Specific antibiotic therapy initiated six weeks after the onset of initial symptoms did not resolve the disease, and open surgical drainage was necessary. CONCLUSIONS: Otitis media unresponsive to conventional therapy and accompanied by unusually pronounced lymphadenopathy should prompt the clinician to consider tularaemia as a differential diagnosis, in order to initiate timely, specific therapy.

An outbreak of oropharyngeal tularaemia linked to natural spring water.

A tularaemia outbreak was investigated involving 188 suspected cases in the Kocaeli region of Turkey between December 2004 and April 2005. A case-control study comprising 135 laboratory-confirmed cases and 55 controls was undertaken to identify risk factors for the development of the outbreak and to evaluate laboratory diagnostic methods. Tularaemia was confirmed by a microagglutination test (MAT) titre of >or=1 : 160 in 90 of the patients. In MAT-negative sera, 23/44 (52 %) were positive by ELISA with Francisella tularensis LPS and 1/9 (11 %) by Western blotting with this antigen. A species-specific PCR was positive in 16/25 (64 %) throat swabs and 8/13 (62 %) lymph node aspirates. Multivariate analysis showed that drinking natural spring water was the leading risk factor for the development of tularaemia (P=0.0001, odds ratio 0.165, 95 % CI 0.790-0.346). The outbreak ceased after abandonment of the suspected natural water springs.

A novel screening ELISA and a confirmatory Western blot useful for diagnosis and epidemiological studies of tularemia.

A novel enzyme-linked immunosorbent assay (ELISA) and a confirmatory Western blot (WB) to detect human antibodies against Francisella tularensis were evaluated. The ELISA was based on partially purified lipopolysaccharide (LPS), the WB on whole antigen of F. tularensis. Positive WB showed a typical LPS ladder. Sensitivity and specificity of the ELISA, as assessed in 104 positive sera and 1149 'normal' sera from healthy young adults, were 99.0% and 97.1% respectively. Sensitivity of the WB was close to 100%, whereas specificity was 99.6%. Antibodies against the LPS of F. tularensis were detected in four of the 'normal' sera in both ELISA and WB. The assays were further evaluated using sera of individuals from Norway, Sweden and Kosovo suspected to be infected in tularemia outbreaks. Results revealed that the combination of ELISA and WB is suitable for laboratory confirmation of tularemia as well as for large-scale epidemiological studies.

A combination of different polymerase chain reaction (PCR) assays for the presumptive identification of Yersinia pestis.

A combination of four polymerase chain reaction (PCR) assays targeting the Yersinia pestis-specific plasmoidal genes of the fraction 1 capsular antigen and plasminogen activator/coagulase, the gene of the V antigen of the Yersinia virulence plasmid, and the chromosomal 16S rRNA gene was evaluated for the identification of Y. pestis isolates. All four assays were subjected to the same sample preparation technique, reagents and cycling conditions. Eighteen Y. pestis, 66 Y. pseudotuberculosis, 40 Y. enterocolitica strains, the type strains of the other Yersinia species, and 20 other pathogenic bacterial strains were investigated. By using the proposed combination of PCR assays all Y. pestis strains were identified correctly. The applicability of this combination of PCR assays was demonstrated by the detection of Y. pestis DNA in spiked tissues from Rattus norwegicus and fleas (Xenopsylla cheopis and Ctenocephalides spp.). As little as 60 genome equivalents were detected. This system is applicable for monitoring Y. pestis and its vectors in enzootic natural foci and in the diagnosis of plague in humans and animals.

A review on melioidosis with special respect on molecular and immunological diagnostic techniques.

Melioidosis is an 'emerging' tropical disease which causes diagnostic problems in endemic and especially in nonendemic areas e.g. Germany when imported. In the last decade, many efforts have been made to develop new molecular and immunological techniques for diagnostic use. However, an actual comprehensive review does not exist. Apart from classical microbiological procedures (microscopy, culture and biochemical identification) efforts have been made to identify Burkholderia pseudomallei using specific antibodies and PCR. The direct antigen detection can be done within a few hours leading to diagnosis days before cultural proof. ELISA and immunoblot techniques were examined for their ability to replace indirect hemagglutination and immunofluorescence test in serology. The diagnostic value of serological procedures for early detection of melioidosis is limited, however. The rapid and reliable diagnosis of melioidosis is required for an adequate onset of therapy. But no evaluated test kit based on the detection of specific antibodies, specific antigens, or on the amplification of species-specific DNA sequences is commercially available up to now. Even PCR testing--primers can easily be ordered by many gene technology companies--can not be recommended as B. pseudomallei DNA for positive controls is not available. Therefore, only microscopy and biochemical identification systems like the API 20NE can be used in the routine laboratory up to now. At this point, it has to be stressed that B. pseudomallei is a level 3 agent in many countries e.g. Germany and that only laboratories with high containment are allowed to handle it. In all cases the help of an experienced reference laboratory is adviced.

Serodiagnosis of human plague by an anti-F1 capsular antigen specific IgG/IgM ELISA and immunoblot.

Plague is a re-emerging disease endemic in at least 24 countries. Non-endemic countries should be able to confirm plague to prevent outbreaks due to imported cases. We established a combination of a IgG/IgM screening ELISA and a confirmation immunoblot employing F1 capsular antigen (CA) for the serodiagnosis of plague in countries where yersiniosis is present. The ELISA and the immunoblot assay showed a specificity of 96.1% and 100% among sera from healthy German blood donors. This group had a seroprevalence of 39% of anti-yersinia outer protein (YOP) antibodies obviously caused by previous Y. enterocolitica infection. The ELISA detected anti-F1 CA antibodies in 22 and the immunoblot in 20 out of 26 sera of plague vaccinees. Five control sera from bacteriologically confirmed plague cases from Madagascar reacted positively. It can be concluded that anti-YOP antibodies do not affect assays based on purified F1 CA.

Serotonin acts as a radical scavenger and is oxidized to a dimer during the respiratory burst of human mononuclear and polymorphonuclear phagocytes.

Isolated human mononuclear and polymorphonuclear phagocytes were stimulated in the presence and absence of serotonin (5-HT), the major secretory product of activated platelets, and the release of reactive oxygen metabolites during the respiratory burst was assessed by luminol-enhanced chemiluminescence. In the presence of 5-HT, a dose-dependent suppression of the chemiluminescence signal occurred, irrespective of the stimulus used to elicit the respiratory burst. A similar suppression of luminol-enhanced chemiluminescence was also seen in a radical generating cell free system. 5-HT was found to be oxidized by the reactive oxygen species released by stimulated phagocytes. This oxidation is prevented in the presence of other antioxidants. The major 5-HT oxidation product was isolated by gel chromatography and identified by mass-spectrometry as a 5-HT dimer, probably 5,5'-dihydroxy-4,4'-bitryptamine. It is concluded that the 5-HT released from activated thrombocytes at sites of inflammation and endothelial cell damage acts as a true scavenger of reactive oxygen species generated during the respiratory burst of stimulated phagocytes and may thus modulate various aspects of cell-mediated defence reactions.