Effects of 6-week azithromycin treatment on the <Emphasis Type="Italic">Wolbachia</Emphasis> endobacteria of <Emphasis Type="Italic">Onchocerca volvulus</Emphasis>

The effects of azithromycin treatment on the presence of Wolbachia endobacteria and on the embryogenesis and microfilariae production of Onchocerca volvulus were studied. In 2002, in an endemic area in Ghana, 37 onchocerciasis patients were treated for 6 weeks with azithromycin: 23 patients received 250 mg every day, and 14 took 1,200 mg once a week. After 6 and/or 12 months, all palpable worm nodules were extirpated from 31 treated and nine additional untreated patients, and the presence of Wolbachia and embryogenesis were assessed by immunohistology. In nodules taken 6 months after treatment with either dose and 12 months after 1,200 mg/week, the Wolbachia loads of the worms were not different from those of untreated worms. However, 12 months after the 250-mg/day azithromycin regimen, significantly less female worms (65% compared to 92% untreated ones) presented many Wolbachia, although the reduction was less pronounced than observed in other studies after treatment with doxycycline. Embryogenesis and microfilariae production were not reduced. It is concluded that azithromycin administered alone for 6 weeks at 250 mg/day or 1,200 mg/week is not suitable for treatment of human onchocerciasis. But daily azithromycin should be studied in combination with other drugs and with other doses.     

Efficacy of 2- and 4-week rifampicin treatment on the <Emphasis Type="Italic">Wolbachia</Emphasis> of <Emphasis Type="Italic">Onchocerca volvulus</Emphasis>

The microfilaricidal and temporarily sterilizing drug ivermectin is used for mass treatment of filarial infections. Filariae containing Wolbachia endobacteria can also be treated by the antibiotic doxycycline. The loss of Wolbachia results in sterilization of Onchocerca volvulus and macrofilaricidal effects. Besides doxycycline, other antibiotics may be effective in depleting Wolbachia. A preliminary study on the effects of rifampicin on the endobacteria, embryogenesis and microfilariae production of O. volvulus was carried out in the year 2000 in Ghana. Twenty-six onchocerciasis patients were treated for 2 or 4 weeks with 10 mg/kg/day rifampicin. From 17 treated and nine untreated patients, all palpable nodules were extirpated 1 or 18 months after the start of the study and examined for Wolbachia and embryogenesis using immunohistology. One and 18 months after rifampicin treatment, the proportion of Wolbachia-positive worms was significantly reduced compared to the untreated group. In patients treated 4 weeks with rifampicin, only 21% and 18% of living female filariae contained Wolbachia after 1 and 18 months, respectively, compared to 92% in the untreated patients. The reduction of Wolbachia after 2 weeks rifampicin was less but also significant. Embryogenesis and microfilariae production were reduced after 4 weeks rifampicin treatment, rendering rifampicin an antibiotic with anti-wolbachial efficacy in human onchocerciasis. This treatment is less efficient than treatment with 6 weeks doxycycline, but might be an alternative for cases that cannot be treated with doxycycline, e.g. children, or might be further developed for combination therapy.     

Immunopathogenesis of <Emphasis Type="Italic">Onchocerca volvulus</Emphasis> keratitis (river blindness): a novel role for endosymbiotic <Emphasis Type="Italic">Wolbachia</Emphasis> bacteria

River blindness is thought to occur as a result of the host response to degenerating microfilariae in the eye. Utilizing a murine model of corneal inflammation (keratitis) to investigate the immune and inflammatory responses associated with river blindness, we recently demonstrated an important role for endotoxin-like products from endosymbiotic bacteria and for activation of Toll-like receptor 4 (TLR4). These observations have led to a new understanding of the pathogenesis of this disease     

No evidence of<Emphasis Type="Italic"> Wolbachia</Emphasis> endosymbiosis with<Emphasis Type="Italic"> Loa loa</Emphasis> and<Emphasis Type="Italic"> Mansonella perstans</Emphasis>

Endosymbiotic Wolbachia bacteria from different filarial species, including major pathogens of humans such as Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus, seem to play an important role in the development, viability and fertility of these worms. Wolbachia trigger inflammatory host responses as well as adverse reactions against standard treatment regimens and are therefore under investigation as novel treatment targets. We investigated whether Wolbachia are also endosymbiotic in Loa loa and Mansonella perstans. In both male and female adult L. loa, we found no evidence of bacteria by light or transmission electron microscopy. Furthermore, Wolbachia-specific PCR was negative in both L. loa and M. perstans microfilariae. The absence of Wolbachia in both filarial species therefore discourages the use of antibiotics as an adjunct or alternative approach to current treatment concepts for both loiasis and mansonelliasis perstans.     

Study of specific IgG subclass antibodies for diagnosis of<Emphasis Type="Italic"> Gnathostoma spinigerum</Emphasis>

Gnathostoma spinigerum infection is endemic in Thailand and many Asian countries. Current diagnosis is the skin test and enzyme-linked immunosorbent assay (ELISA) for IgG antibody against the G. spinigerum third-stage larvae (L3), but cross-reactivity is common. We evaluated the sensitivity and specificity of anti-G. spinigerum L3 IgG subclass antibodies for diagnosis of 43 patients with gnathostomiasis. The majority of patients with gnathostomiasis (91%) had eosinophilia. While the anti-G. spinigerum L3 IgG1 antibody provided the highest sensitivity (98%), the anti-G. spinigerum L3 IgG2 antibody had the highest specificity (88%). The ELISA that detected anti-G. spinigerum L3 IgG1 antibody could be a reliable laboratory screening test, while anti-G. spinigerum L3 IgG2 antibody could be used to confirm the diagnosis.     

Expression of<Emphasis Type="Italic"> Tri15</Emphasis> in<Emphasis Type="Italic"> Fusarium sporotrichioides</Emphasis>

In the fungus Fusarium sporotrichioides, biosynthesis of trichothecene mycotoxins requires at least three genetic loci: a core 12-gene cluster, a smaller two-gene cluster, and a single-gene locus. Here, we describe the Tri15 gene, which represents a fourth locus involved in trichothecene biosynthesis. Tri15 is predicted to encode a Cys₂-His₂ zinc finger protein and is expressed in a manner similar to genes in the core trichothecene gene cluster. However, disruption of F. sporotrichioides Tri15 does not affect production of T-2 toxin, the major trichothecene produced by this fungus. This result suggests that Tri15 is not necessary for the production of toxin. Cultures with exogenously added T-2 toxin have high levels of Tri15 expression and no detectable expression of the trichothecene biosynthetic genes Tri5 and Tri6. The expression analysis is consistent with Tri15 being a negative regulator of at least some of the trichothecene biosynthetic genes. In F. graminearum, Tri15 has been mapped to linkage group 2 and is therefore unlinked to the main trichothecene biosynthetic gene cluster.Communicated by U. Kück     

Phylogenetic analysis of<Emphasis Type="Italic"> Theileria</Emphasis> species transmitted by<Emphasis Type="Italic"> Haemaphysalis qinghaiensis</Emphasis>

The phylogenetic relationships between six isolates of Theileria spp. infective to small ruminants, and two isolates of Theileria spp. infective to yak, all transmitted by Haemaphysalis qinghaiensis, together with the Theileria orientalis/sergenti/buffeli group and T. sinensis, were analyzed using the 18S ssrRNA gene sequence. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was used either for direct sequencing or was ligated to the PCR II vector for sequencing. The length of the 18S ssrRNA gene of all Theileria spp. involved in this study was around 1,740 bp. Two phylogenetic trees were inferred based on the 18S ssrRNA gene sequence of the Chinese isolates only, and Chinese isolates and other species of Theileria available in GenBank. In the first tree, the Theileria sp. infective to yaks was found to be T. sinensis. The Theileria sp. infective to small ruminants was found to be composed of two separate species of Theileria. Theileria sp. from Qinghai, Madang, Ningxian and Lintan, which was identical to the unidentified Theileria sp. described previously, is designated Theileria sp (China 1). The Theileria sp. from Longde, Zhangjiachuan and Lintan, which has not been described previously, is designated Theileria sp. (China 2) in order to avoid confusion. In the second tree, Theileria sp. (China 1) was closely related to benign Theileria, such as T. buffeli and T. sergenti, while Theileria sp. (China 2) was separated from other Theileria spp. The results indicate that H. qinghaiensis transmit at least three species of Theileria, two which are infective to sheep and goats, but not yak and one which is infective to yaks and cattle, but not to sheep and goats.     

Selective activity of extracts of <Emphasis Type="Italic">Margaritaria discoidea</Emphasis> and <Emphasis Type="Italic">Homalium africanum</Emphasis> on <Emphasis Type="Italic">Onchocerca ochengi</Emphasis>

Background  

The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon.     

Import and assembly of<Emphasis Type="Italic"> Neurospora crassa</Emphasis> Tom40 into mitochondria of<Emphasis Type="Italic"> Trypanosoma brucei</Emphasis> in vivo

The TOM complex (translocase of the mitochondrial outer membrane) is a dynamic, multisubunit protein complex. Tom40 is the major component of the complex and forms the preprotein conducting pore. To determine if a heterologous Tom40 could be properly targeted and assembled into the Trypanosoma brucei mitochondrial outer membrane, an ectopic copy of a gene encoding Neurospora crassa Tom40 (NcTom40) was expressed in procyclic trypanosomes from a tetracycline regulated procyclic acidic repetitive protein promoter. The level of NcTom40 expression was found to be maximal within 20–26 h of induction with tetracycline. Immunoblot analysis of subcellular fractions showed that NcTom40 was enriched in the mitochondrial fraction. Alkali extraction of isolated mitochondria revealed that NcTom40 was assembled as an integral membrane protein and limited proteolysis demonstrated that it was present in the outer membrane of the mitochondria. These data demonstrate that a heterologous mitochondrial protein containing internal targeting information can be correctly targeted to T. brucei mitochondria. Following blue native gel electrophoresis, the NcTom40 protein was found in a 370 kDa complex which may contain T. brucei Tom components. A 16 kDa protein was coimmunoprecipitated from T. brucei mitochondria containing NcTom40 using antisera developed against the N. crassa protein. The 16 kDa protein may represent a component of the T. brucei TOM complex that associates with NcTom40.Communicated by M. BrunnerThis revised version was published in October 2003. Throughout the text, owing to a technical problem, degree signs were erroneously inserted before µg/ml and µl.     

Chromosome rearrangements in isolates that escape from<Emphasis Type="Italic"> het-c</Emphasis> heterokaryon incompatibility in<Emphasis Type="Italic"> Neurospora crassa</Emphasis>

Chromosomal rearrangement is implicated in human cancers and hereditary diseases. Mechanisms generating chromosomal rearrangements may be shared by a variety of organisms. Spontaneous chromosomal rearrangements, especially large deletions, take place at high frequency in isolates that escape from heterokaryon incompatibility in Neurospora crassa. In this study, chromosomal rearrangements were detected in strains that had escaped from het-c heterokaryon incompatibility in N. crassa. A vc1 mutant carried a 20-kbp deletion covering five ORFs. A vc2 mutant carried a complex chromosome rearrangement with an 8-kbp deletion covering three ORFs, a 34-bp deletion and an 80-kbp inversion. The break-points of chromosome rearrangements in the vc1 and vc2 mutants all have direct repeats of 2 bp, similar to the break-points of some chromosome rearrangements associated with human cancer and genetic diseases. An ahc mutant carried a 31-kbp deletion covering at least 11 ORFs and a het-c deletion mutant carried a 7-kbp deletion covering two ORFs. Additional chromosomal rearrangements occurred in these two strains. These results indicate that escape from heterokaryon incompatibility can be used as a model system for chromosome rearrangement and DNA-repair studies. The impact of the chromosomal rearrangements is discussed, especially the deletion of the predicted ORFs on the phenotype of mutants.Communicated by U. Kück     

Development of<Emphasis Type="Italic"> Trichostrongylus colubriformis</Emphasis> and<Emphasis Type="Italic"> Trichostrongylus vitrinus</Emphasis>, parasites of ruminants in the rabbit and comparison with<Emphasis Type="Italic"> Trichostrongylus retortaeformis</Emphasis>

The parasitic phase of development of both Trichostrongylus colubriformis and Trichostrongylus vitrinus, parasites of ruminants, was studied in detail in the rabbit. In T. colubriformis, the third moult appeared by 4 days after infection (DAI) and the last moult occurred between 10 and 11 DAI. In T. vitrinus, the third moult occurred between 8 and 11 DAI and the last one between 12 and 15 DAI. The prepatent period lasted 16-17 days for T. colubriformis and 20 days for T. vitrinus. The chronology of the life cycles and the distribution of the parasites along the small intestine for various Trichostrongylus spp. from lagomorphs and ruminants in the natural host or in the experimental host were compared. All of these biological parameters indicated a lower level of adaptation of T. vitrinus compared to the other species of Trichostrongylus. The results are fully compatible with the evolutionary scheme based on morphological analyses.     

Economic Impact of<Emphasis Type="Italic"> Candida</Emphasis> Colonization and<Emphasis Type="Italic"> Candida</Emphasis> Infection in the Critically Ill Patient

The objective of the study presented here was to assess the economic impact of Candida colonization and Candida infection in critically ill patients admitted to intensive care units (ICUs). For this purpose, a prospective, cohort, observational, and multicenter study was designed. A total of 1,765 patients over the age of 18 years who were admitted for at least 7 days to 73 medical-surgical ICUs in 70 Spanish hospitals between May 1998 and January 1999 were studied. From day 7 of ICU admission to ICU discharge, samples of tracheal aspirates, pharyngeal exudates, gastric aspirates and urine were collected every week for culture. Prolonged length of stay was associated with severity of illness, Candida colonization or infection, infection by other fungi, antifungal therapy, treatment with more than one antifungal agent, and toxicity associated with this therapy. Compared to non-colonized, non-infected patients (n=720), patients with Candida colonization (n=880) had an extended ICU stay of 6.2 days (OR, 1.69; 95%CI, 1.53–1.87; P<0.001) and an extended hospital stay of 8.6 days (OR, 1.27; 95%CI, 1.16–1.40; P<0.001). The corresponding figures for patients with Candida infection (n=105) were 12.7 days for ICU stay (OR, 2.13; 95%CI, 1.72–2.64; P<0.001) and 15.5 days for hospital stay (OR, 1.23; 95%CI, 0.99–1.52; P=0.060). Candida colonization resulted in an additional 8,000 EUR in direct costs and Candida infection almost 16,000 EUR. Both Candida colonization and Candida infection had an important economic impact in terms of cost increases due to longer stays in both the ICU and in the hospital.     

Dissemination of<Emphasis Type="Italic"> emm</Emphasis>28 Erythromycin-, Clindamycin- and Bacitracin-Resistant<Emphasis Type="Italic"> Streptococcus pyogenes</Emphasis> in Spain

Reported here is an unusual cluster of non-invasive infections caused by an emm28 Streptococcus pyogenes strain resistant to bacitracin, erythromycin and clindamycin detected in Santander, Spain. Since one of the characteristics of group A streptococci is their almost uniform susceptibility to bacitracin, this finding was unusual, and a search for bacitracin-resistant Streptococcus pyogenes strains was conducted in two other distant cities of Spain (Madrid and San Sebastián) where their presence was confirmed. These strains were frequently associated with erythromycin- and clindamycin-resistance, and most of them belonged to a unique emm28 T28, ST52 clone.     

Isolation and characterisation of the mating-type (<Emphasis Type="Italic">MAT</Emphasis>) locus from<Emphasis Type="Italic"> Rhynchosporium secalis</Emphasis>

The mating-type (MAT) genes from Rhynchosporium secalis were isolated using PCR-based methods. Characterisation of the MAT idiomorphs suggests that R. secalis is closely related to the discomycetes Pyrenopeziza brassicae and Tapesia yallundae in terms of sequence and MAT locus gene composition. The MAT1-2 idiomorph contains a single gene encoding a protein with a high-mobility group (HMG) DNA-binding domain. The MAT1-1 idiomorph contains two genes, one encoding a protein with a HMG domain and the other encoding an alpha box domain. A second, previously undescribed, intron was identified within the P. brassicae MAT1-2-1 gene. Two introns were also present in the corresponding gene in R. secalis and this showed the similarity between these genes at the discomycete MAT1-2 locus. Using PCR, we identified isolates of both mating types from barley crops in different parts of the UK and showed that the composition of the MAT idiomorphs is conserved in these isolates. These findings support the hypothesis that R. secalis is a heterothallic discomycete which has an as yet unidentified teleomorph.Communicated by J. Heitman     

Rapid diagnosis of<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> bacteremia using <Emphasis Type="Italic">S. aureus</Emphasis> PNA FISH

In the study presented here, the performance of the S. aureus PNA FISH assay was evaluated using 285 blood cultures (from 104 patients) that had gram-positive cocci resembling staphylococci on Gram stain. The new molecular test is based on a fluorescence in situ hybridization assay using peptide nucleic acid probes targeting Staphylococcus aureus 16S rRNA and is designed for the rapid identification of Staphylococcus aureus directly from positive blood cultures. The sensitivity, specificity, and positive and negative predictive values of the S. aureus PNA FISH for the rapid identification of Staphylococcus aureus directly from positive blood culture bottles were 100, 99.4, 99.2 and 100%, respectively.