Differentiating Dracunculus medinensis from D. insignis, by the sequence analysis of the 18S rRNA gene.

This study, undertaken as a component of the global Dracunculiasis Eradication Program (DEP), was designed to provide molecular tools to distinguish Dracunculus medinensis, the nematode causing human dracunculiasis, from other tissue-dwelling nematodes, including other Dracunculus species that infect humans and other animals. DNA was extracted from D. medinensis and from a closely related species that infects North American carnivores, D. insignis, so that the genes coding for the small-subunit ribosomal RNA (18S rRNA) of the parasites could be amplified, sequenced and compared. Sequences were obtained for 20 specimens of D. medinensis (from humans in Pakistan, Yemen and six African countries endemic for dracunculiasis) and three of D. insignis (from raccoons trapped in the state of Georgia in the southern U.S.A.). All of the D. medinensis 18S-rRNA sequences were found to be 1819 bases long and identical. The three D. insignis 18S-rRNA sequences were also found to be identical to each other but were 1821 bases long and differed from the D. medinensis 18S- rRNA sequence at eight positions (representing a difference of 0.44%). The 18S-rRNA coding region of a Guinea worm extracted from a dog in Ghana was indistinguishable from that of the D. medinensis isolates from human cases. These results provide the basis for the molecular differentiation of D. medinensis that will permit the DEP to determine, rapidly and accurately, whether a worm recovered from an area considered dracunculiasis-free is a specimen of D. medinensis or not.

Preliminary molecular characterization of Cryptosporidium parvum isolates of wildlife rodents from Poland.

Isolates of Cryptosporidium were collected from 3 species of woodland and field rodents (Clethrionomys glareolus, Microtus arvalis, and Apodemus flavicollis) and were characterized by polymerase chain reaction amplification and sequencing of fragments of the oocyst wall protein (COWP) gene and of the 18S ribosomal RNA gene. Sequence analysis of these markers revealed that the animals were infected with C. parvum, and that the genotype involved was almost identical to the mouse genotype previously described from Mus musculus. Thus, small rodents should be considered as an important reservoir of C. parvum genotypes closely related to the zoonotic genotype 2 and potentially hazardous to humans.

Usefulness of PCR for diagnosis of imported malaria in Poland.

The aim of the present study was to use the polymerase chain reaction (PCR) to detect and identify Plasmodium spp. in diagnostic specimens, especially in those from patients diagnosed by microscopy as having possible mixed infections, and in those demonstrating low parasitemia or those that were parasite-negative. For most of the specimens, the PCR results were in accordance with microscopic findings, and in 16.2% of the cases with low parasitemia PCR enhanced the results by identifying the parasite species. This method detected one additional case of Plasmodium falciparum malaria among the patients with fever of unknown origin. The sensitivity of PCR for detecting Plasmodium DNA was found to correspond to 1.35-0.38 and 0.12 for Plasmodium falciparum and Plasmodium vivax parasites per microliter of blood, respectively. Follow-up examinations demonstrated that most of the patients became negative for Plasmodium DNA from 1 to 4 days after the disappearance of parasitemia, as determined by examination of blood films. In conclusion, PCR performed by the reference laboratory significantly enhanced the microscopic diagnosis of malaria and proved very helpful in cases of low parasitemia and in cases of mixed infection.

Sequence variation in the cytochrome oxidase I, internal transcribed spacer 1, and Ts14 diagnostic antigen sequences of Taenia solium isolates from South and Central America, India, and Asia.

We examined the genetic variability in the pig-human tapeworm, Taenia solium, by sequencing the genes for cytochrome oxidase I, internal transcribed spacer 1, and a diagnostic antigen, Ts14, from individual cysts isolated from Peru, Colombia, Mexico, India, China, and the Philippines. For these genes, the rate of nucleotide variation was minimal. Isolates from these countries can be distinguished based on one to eight nucleotide differences in the 396 nucleotide cytochrome oxidase I (COI) sequence. However, all of the 15 isolates from within Peru had identical COI sequences. The Ts14 sequences from India and China were identical and differed from the Peru sequence by three nucleotides in 333. These data indicate that there is minimal genetic variability within the species T. solium. Minimal variability was also seen in the ITS1 sequence, but this variation was observed within the individual. Twenty-two cloned sequences from six isolates sorted into 13 unique sequences. The variability observed within the sequences from individual cysts was as great as the variability between the isolates.

Effect of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of P carinii pneumonia in patients with HIV-1: a prospective study.

BACKGROUND: Investigators have reported that patients infected with Pneumocystis carinii containing mutations in the DHPS (dihydropteroate synthase) gene have a worse outcome than those infected with P carinii containing wild-type DHPS. We investigated patients with HIV-1 infection and P carinii pneumonia to determine if DHPS mutations were associated with poor outcomes in these patients. METHODS: We compared presence of mutations at the DHPS locus with survival and response of patients to co-trimoxazole or other drugs. FINDINGS: For patients initially given co-trimoxazole, nine (14%) of 66 with DHPS mutant died, compared with nine (25%) of 36 with wild type (risk ratio50.55 [95% CI=0.24-1.25]; p=0.15). Ten (15%) of 66 patients with a DHPS mutant did not respond to treatment, compared with 13 (36%) of 36 patients with the wild type (0.42 [0.20-0.86]; p=0.02). For patients aged 40 years or older, four (14%) of 29 with the mutant and nine (56%) of 16 with the wild type died (0.25 [0.09-0.67]; p=0.005). INTERPRETATION: These results, by contrast with those of previous studies, suggest that patients with wild-type P carinii do not have a better outcome than patients with the mutant when given co-trimoxazole. Our results suggest that presence of a DHPS mutation should be only one of several criteria guiding the choice of initial drug treatment of P carinii pneumonia in patients with HIV-1 infection.

Cryptosporidium parvum genotype 2 infections in free-ranging mountain gorillas (Gorilla gorilla beringei) of the Bwindi Impenetrable National Park, Uganda.

For behavioral research and due to growing ecotourism, some populations of free-ranging mountain gorillas (Gorilla gorilla beringei) have become habituated to humans. Molecular analysis of two Cryptosporidium sp. oocyst isolates originating from two human-habituated gorilla groups and two oocyst isolates from non-habituated gorillas yielded positive identification of C. parvum Genotype 2 (G2; i.e., "cattle", "animal-adapted", or "zoonotic"). As G2 is cross-transmissible between humans and animals, C. parvum infections can be propagated in the habitats of human-habituated, free-ranging gorillas through both zoonotic and anthroponotic transmission cycles.

Cryptosporidium parvum oocysts in zebra mussels (Dreissena polymorpha): evidence from the St Lawrence River.

Molluscan shellfish can recover and concentrate environmentally derived waterborne pathogens and can be used for the sanitary assessment of water quality. Oocysts of Cryptosporidium parvum (genotype 1) were identified in zebra mussels (Dreissena polymorpha) from the St. Lawrence River, Quebec. Approximately 67 oocysts/ml of hemolymph and 129 oocysts/g of soft tissue were recovered. The adjusted concentration of oocysts per gram of tissue was 2.2 x 10(2), and approximately 4.4 x 10(2) oocysts were recovered from a single mussel. Zebra mussels can serve as biological indicators of waterborne contamination with Cryptosporidium.

In vitro culture, ultrastructure, antigenic, and molecular characterization of Encephalitozoon cuniculi isolated from urine and sputum samples from a Spanish patient with AIDS.

In this report we describe the cultivation of two isolates of microsporidia, one from urine and the other from sputum samples from a Spanish AIDS patient. We identified them as Encephalitozoon cuniculi, type strain III (the dog genotype), based on ultrastructure, antigenic characteristics, PCR, and the sequence of the ribosomal DNA internal transcribed spacer region.

Acute and long-term humoral immunity following active immunization of rabbits with inactivated spores of various Encephalitozoon species.

Microsporidia of the genus Encephalitozoon are increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. helleri, or E. intestinalis is capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoon antigens was determined by IIF and immunogold electron microscopy; however, only the E. hellem immune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinalis were fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections.

Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission.

To study transmission patterns of Pneumocystis carinii pneumonia (PCP) in persons with AIDS, we evaluated P. carinii isolates from patients in five U.S. cities for variation at two independent genetic loci, the mitochondrial large subunit rRNA and dihydropteroate synthase. Fourteen unique multilocus genotypes were observed in 191 isolates that were examined at both loci. Mixed infections, accounting for 17.8% of cases, were associated with primary PCP. Genotype frequency distribution patterns varied by patients' place of diagnosis but not by place of birth. Genetic variation at the two loci suggests three probable characteristics of transmission: that most cases of PCP do not result from infections acquired early in life, that infections are actively acquired from a relatively common source (humans or the environment), and that humans, while not necessarily involved in direct infection of other humans, are nevertheless important in the transmission cycle of P. carinii f. sp. hominis.

Morphologic, host specificity, and molecular characterization of a Hungarian Cryptosporidium meleagridis isolate.

This study was undertaken in order to characterize Cryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis and Cryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location of C. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridis isolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridis sequence recently described for a North Carolina isolate. Thus, C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) and Cryptosporidium felis. Because birds are susceptible to C. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.

Babesia microti and Borrelia bissettii transmission by Ixodes spinipalpis ticks among prairie voles, Microtus ochrogaster, in Colorado.

An endemic transmission cycle of Babesia microti was discovered in Colorado in the foothills of the Rocky Mountains. B. microti were found by PCR in 4 of 25 Ixodes spinipalpis tick pools tested (a 3.2 % minimum infection rate) and in 87% (13 of 15) of Microtus ochrogaster (the prairie vole) spleen and blood samples. Using naturally infected I. spinipalpis collected from wild-caught M. ochrogaster as vectors, B. microti and Borrelia bissettii were successfully transmitted to laboratory-born M. ochrogaster. Neither I. spinipalpis, nor M. ochrogaster (the prairie vole) have been previously reported as a vector or a reservoir host of B. microti. Unlike the east coast of the United States where Peromyscus leucopus is an important reservoir for B. microti, evidence for Peromyscus spp. (neither P. maniculatus nor P. difficilis) as B. microti reservoirs was not found in this study.

Mechanical transport and transmission of Cryptosporidium parvum oocysts by wild filth flies.

Over the course of six months wild filth flies were collected from traps left for 7-10 days in a barn with or without a calf shedding Cryptosporidium parvum Genotype 2 oocysts in diarrheic feces. The oocysts of C. parvum transported on the flies' exoskeletons and eluted from their droplets left on visited surfaces were infectious for mice. The mean number of oocysts carried by a fly varied from 4 to 131, and the total oocyst number per collection varied from 56 to approximately 4.56 x 10(3). Fly abundance and intensity of mechanical transmission of infectious C. parvum oocysts were positively correlated, and both increased significantly when an infected calf was in the barn. Molecular data showed that the oocysts shed by infected calves were carried by flies for at least 3 weeks. Filth flies can acquire infectious C. parvum oocysts from unsanitary sites, deposit them on visited surfaces, and therefore may be involved in human or animal cryptosporidiosis.

Morphologic and molecular characterization of new Cyclospora species from Ethiopian monkeys: C. cercopitheci sp.n., C. colobi sp.n., and C. papionis sp.n.

In recent years, human cyclosporiasis has emerged as an important infection, with large outbreaks in the United States and Canada. Understanding the biology and epidemiology of Cyclospora has been difficult and slow and has been complicated by not knowing the pathogen s origins, animal reservoirs (if any), and relationship to other coccidian parasites. This report provides morphologic and molecular characterization of three parasites isolated from primates and names each isolate: Cyclospora cercopitheci sp.n. for a species recovered from green monkeys, C. colobi sp.n. for a parasite from colobus monkeys, and C. papionis sp.n. for a species infecting baboons. These species, plus C. cayetanensis, which infects humans, increase to four the recognized species of Cyclospora infecting primates. These four species group homogeneously as a single branch intermediate between avian and mammalian Eimeria. Results of our analysis contribute toward clarification of the taxonomic position of Cyclospora and its relationship to other coccidian parasites.

Encephalitozoon cuniculi: light and electron microscopic evidence for di-, tetra-, and octosporous sporogony and a note on the molecular phylogeny of encephalitozoonidae.

We demonstrate, based on the light, electron microscopic, and immunofluorescence studies carried out on two isolates of Encephalitozoon cuniculi established in culture, that E. cuniculi exhibits di-, tri-, tetra- and octosporous sporogony. We therefore propose that the generic characters of Encephalitozoon should be amended to include tetra-sporous sporogony as generic features. Additionally, the molecular phylogenetic analysis indicates that E. cuniculi, E. hellem, and E. (Septata) intestinalis form a cohesive group.

New cryptosporidium genotypes in HIV-infected persons.

Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes.

Fast and reliable extraction of protozoan parasite DNA from fecal specimens.

BACKGROUND: Polymerase chain reaction (PCR) detection of intestinal protozoa in fecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this study we describe a novel method for DNA extraction from such specimens containing spores and oocysts of Enterocytozoon bieneusi and Cryptosporidium parvum, respectively. METHODS AND RESULTS: Extraction was done using commercial kits modified to maximize the recovery and purity of extracted DNA. In comparison with a procedure we previously reported, we estimate that this method may increase the sensitivity of parasite DNA detection in fecal specimens up to tenfold. An additional advantage of this method is that up to 12 samples may be processed simultaneously within 2 hours. CONCLUSIONS: By using this method, we were able to increase reproducibility of PCR amplification on fecal specimens and significantly reduce the hands-on time required to process the samples.