Human-infecting Species Research Articles

Pulmonary mycetoma caused by an atypical isolate of Paecilomyces species in an immunocompetent individual: case report and literature review of Paecilomyces lung infections

The first case of pulmonary fungus ball caused by Paecilomyces species is reported. The diagnosis was established by isolation of the fungus in culture from specimens obtained by percutaneous transthoracic needle aspiration in an immunocompetent individual. The isolate was atypical, as it failed to produce fruiting structures on routine mycological media. Identification was achieved by sequencing polymorphisms of the internal transcriber spacer regions of the ribosomal DNA. The antifungal susceptibility profile was also determined. This report underscores the increasing importance of Paecilomyces species in human infections. Paecilomyces should be included among the etiological agents of pulmonary mycetoma.

Detection of Cryptosporidium and Identification to the Species Level by Nested PCR and Restriction Fragment Length Polymorphism

Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools.

An Analysis of the Microsporidian Genus Brachiola, with Comparisons of Human and Insect Isolates of Brachiola algerae

The genus Brachiola is the newest microsporidian genus established for a human infection with the type species being B. vesicularum in skeletal muscle. Subsequently, the microsporidium, Nosema algerae, identified from mosquitoes, was added to this genus because of morphological and physiological similarities. The present report illustrates a confirmed case of Brachiola algerae infecting skeletal muscle in a 56-year-old woman who was being treated for rheumatoid arthritis with immunosuppressive drugs. In the following study, these two human-infecting microsporidian species are ultrastructurally compared from human biopsy tissue. Additionally, Brachiola algerae from mosquitoes as reference B. algerae, was grown in athymic mice and compared to the human isolate in vivo, and in culture. B. algerae is morphologically identical in the host situations presented and different from B. vesicularum in human skeletal muscle. B. algerae has a consistently, slightly longer spore that typically contains one row of polar filament coils, while B. vesicularum typically contains two rows of polar filament coils and occasionally, one or three rows. In proliferative development, B. vesicularum forms protoplasmic extensions which do not occur on B. algerae, nor have they been reported on any other microsporidium. This report demonstrates that B. vesicularum and B. algerae are two different species of Brachiola that infect human skeletal muscle.

Detection and differentiation of Cryptosporidium oocysts in water by PCR-RFLP.

Consumption of contaminated water has been implicated as a major source of Cryptosporidium infection in various outbreak investigations and case control studies. Surveys conducted in various regions of the United States demonstrated the presence of Cryptosporidium oocysts in 67-100% of wastewaters, 24-100% of surface waters, and 17-26.8% of drinking waters. The identity and human infective potential of these waterborne oocysts are not known, although it is likely that not all oocysts are from human-infecting Cryptosporidium species. Likewise, the source of the oocyst contamination is also not fully clear. Farm animals and human sewage discharge are generally considered to be the major sources of surface water contamination with C. parvum. Because Cryptosporidium infection is common in wildlife, it is conceivable that wildlife can also be a source for Cryptosporidium oocysts in waters. The presence of host-adapted Cryptosporidium spp. and genotypes makes it possible to develop molecular tools to assess the human infection potential and source of Cryptosporidium oocysts in water.Currently, the identification of Cryptosporidium oocysts in environmental samples is largely made by the use of immunofluorescent assay (IFA) after concentration processes (Environmental Protection Agency [EPA] recommended information collection rule [ICR] method or method 1622/1623 or similar techniques). Because IFA detects oocysts from all Cryptosporidium parasites, the species distribution of Cryptosporidium parasites in environmental samples cannot be assessed. Although many surface water samples contain Cryptosporidium oocysts, it is unlikely that all these oocysts are from human-pathogenic species or genotypes, because only five genotypes of Cryptosporidium parasites (the C. parvum human and bovine genotypes, C. meleagridis, C. canis, and C. felis) are responsible for most human infections. Information on the source of C. parvum contamination is necessary for effective evaluation and selection of management practices for reducing C. parvum contamination of surface water and the risk of cryptosporidiosis. Thus, identification of oocysts to species and genotype levels is of public health importance.

A newly-identified lineage of Schistosoma

Because of their role in causing schistosomiasis, flukes of the genus Schistosoma are the best known of all digeneans. The genus has traditionally been divided into four familiar species groups. Here we report on three poorly known species of Schistosoma, one of which, Schistosoma hippopotami, is known from the hippopotamus, one of which is provisionally identified as Schistosoma edwardiense, another hippo parasite, and a third that has not previously been described. All were collected from freshwater snails obtained from Lake Edward, western Uganda, the type locality for both known hippo schistosomes. The three different kinds of schistosome cercariae differ from one another in size, and all are readily differentiated by their long tail stems from the cercariae of human-infecting species. Furthermore, each was recovered from a different genus of snail host, Biomphalaria sudanica, Bulinus truncatus or Ceratophallus natalensis. Molecular analysis, based on 8350 bases of combined nuclear and mitochondrial DNA, groups these three long tail-stem cercariae into a well supported clade that does not associate with any of the recognised species groups. The placement of this clade, basal to all African species plus several Asian species, suggests that there has been an ancient association between Schistosoma and hippos. This new African Schistosoma clade advocates the need for further modification of the traditional species group-based classification. Two of the four species groups are paraphyletic. It also suggests that Schistosoma has been remarkably plastic with respect to adapting to snail hosts—three distantly related genera of planorbid snails have been exploited by worms within a single clade. Finally, it adds a new layer of complexity to deciphering the origins of Schistosoma, often considered to be African but recently challenged as being Asian. In the late Cenozoic the distribution of hippo species straddled both Africa and Asia and they may have provided a means for the introduction of blood flukes to Africa.

DETECTION OF CRYPTOSPORIDIUM PARVUM AND GIARDIA LAMBLIA CARRIED BY SYNANTHROPIC FLIES BY COMBINED FLUORESCENT IN SITU HYBRIDIZATION AND A MONOCLONAL ANTIBODY

Wild-caught synanthropic flies were tested for the presence of Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their digestive tracks by fluorescent in situ hybridization and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (MAb) against Cryptosporidium and Giardia cell wall epitopes. The levels of C. parvum were positively correlated with the levels of G. lamblia, indicating a common source of contamination. The majority of oocysts and cysts were potentially viable (C. parvum = 80% and G. lamblia = 69%). More G. lamblia cysts occurred on the exoskeleton of the flies than within the digestive tracts; the opposite relationship was observed for C. parvum. No genotype other than C. parvum G2 was found to be associated with flies. Because filth flies carry viable C. parvum oocysts and G. lamblia cysts acquired naturally from unhygienic sources, they can be involved in the epidemiology of cryptosporidiosis and giardiasis. Fluorescent oligonucleotide probes used together with FITC-conjugated MAb represent a convenient and cost-effective technique for rapid and specific identification of human-infectious species of Cryptosporidium and Giardia mechanically transported by flies, and for the assessment of the viability of these pathogens.

Development of PCR assays for detection of Streptococcus canis

Streptococcus canis isolates, also including S. canis of artificially contaminated milk, could be identified by polymerase chain reaction (PCR) amplification using oligonucleotide primers designed according to species-specific parts of the 16S rRNA gene and, after sequencing, according to S. canis-specific parts of the 16S–23S rDNA intergenic spacer region and with oligonucleotide primers detecting an internal fragment of the group G streptococcal CAMP factor gene cfg. The 16S rRNA gene- and CAMP factor gene cfg-specific oligonucleotide primers could be used together in a multiplex PCR. No cross-reactivities could be observed with other group G streptococcal isolates or with any of the other control strains of various streptococcal species and serogroups. The PCR methods presented in this study allowed a rapid and reliable identification of S. canis and might help to improve the diagnosis of this bacterial species in animal and human infections.

Plasmodium chabaudi: rosetting in a rodent malaria model

Rosetting is a property of many malaria parasite species that has been linked to virulence in the major species infecting humans, Plasmodium falciparum. Here, the basic properties of rosettes in the rodent malaria laboratory model, P. chabaudi, were studied with a view to future studies on the role of rosetting in malaria parasite virulence and transmission. Rosetting occurred in 14 out of the 15 P. chabaudi clones studied, varied consistently between clones, and ranged between 9 and 37% at full parasite maturity. Rosetting frequency markedly declined after the mouse reached peak parasitemia, possibly due to host immunity. Consistent with P. falciparum and P. vivax, rosettes in P. chabaudi were disrupted by treatment with trypsin and EDTA. However, P. chabaudi rosettes were insensitive to sulfated glycoconjugates (heparin, heparan sulfate and fucoidan). The molecular basis of rosetting in P. chabaudi is unknown at present, but the results suggest that the molecules involved may differ from those in human-infecting species. Index Descriptors and Abbreviations: Plasmodium chabaudi chabaudi, P. c. adami; rodent malaria; rosette; CI, confidence interval; EDTA, ethylene-diamine-tetraacetic acid; SEM, standard error of mean; BSA, bovine serum albumin.

Rapid diagnosis of lyme disease: Flagellin gene-based nested polymerase chain reaction for identification of causative Borrelia species

Objective: Each of Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii has characteristic restriction sites in its flagellin gene. The authors focused on this gene and developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for rapid diagnosis of Lyme disease. Methods: External and internal primer sets were designed for nested PCR to amplify an approximately 580 by fragment of the flagellin gene that includes species-specific restriction sites. DNA extracted from tissue samples of mice and humans were used as templates for PCR. The amplicons obtained were digested with HapII, HhaI, CelII HincII, or Ddel endonuclease. Results: In mice experimentally infected with each of B. burgdorferi sensu stricto, B. garinii, and B. afzelii, borrelial DNA was detected irrespective of differences in the causative species. However, RFLP of the amplicons was able to identify the species. Skin biopsy samples from 11 Japanese patients with erythema migrans were subjected to both PCR and culture tests. Borrelial infections were detected in seven cases (64%) by PCR and eight cases (73%) by culture. All PCR-positive samples were also positive by culture. The causative species in human infections was easily identified as B. garinii by RFLP analysis of the amplicons. Conclusion: The nested PCR-RFLP system appears to be an easy and reliable diagnostic tool for the detection and species identification of borreliae in human cutaneous biopsies.