A longitudinal study of Babesia microti infection in seropositive blood donors.

BACKGROUND: Babesia infection is caused by intraerythrocytic tick-borne parasites. Cases of transfusion-transmitted babesiosis have been increasingly recognized. To date, no Babesia test has been licensed for screening US blood donors. We conducted a longitudinal study to assess the course and markers of Babesia infection among seropositive donors identified in a seroprevalence study. STUDY DESIGN AND METHODS: Eligible donors had B. microti indirect fluorescent antibody (IFA) titers of 64 or greater. Enrollees were monitored up to 3 years, by IFA and three methods for evidence of parasitemia: B. microti nested polymerase chain reaction (PCR) analysis (at two laboratories), hamster inoculation, and blood-smear examination. RESULTS: Among 115 eligible donors, 84 (73%) enrolled. Eighteen enrollees (21%) had evidence of parasitemia for 30 total specimens (17% of 181), which were collected in 9 different months and tested positive by various approaches: PCR (25 specimens/16 persons), hamster inoculation (13 specimens/8 persons), and blood smear (one specimen positive by all three approaches). Overall, 14 persons had one or more specimen with positive PCR results at both laboratories (12 persons) and/or had parasitologically confirmed infection (eight persons). Three of nine persons who had more than one specimen with evidence of parasitemia had nonconsecutive positives. Several enrollees likely had been infected at least 1 year when their last positive specimen was collected. The final three specimens for seven persons tested negative by all study methods, including IFA. CONCLUSION: Seropositive blood donors can have protracted low-level parasitemia that is variably and intermittently detected by parasitologic and molecular methods. Donor-screening algorithms should include serologic testing and not solely rely on molecular testing.

Caracterización molecular de aislados humanos de Cryptosporidium spp. procedentes de 2 diferentes localizaciones de España / Molecular characterization of Cryptosporidium spp. isolated in humans in two different locations in Spain

Las técnicas de diagnóstico molecular por PCR permiten distinguir entre las diferentes especies de Cryptosporidium morfológicamente idénticas capaces de infectar a humanos. De las 23 especies actualmente reconocidas en el género, al menos 9 son capaces de infectar a humanos. Por ello, y debido a que la intensidad de las manifestaciones clínicas, la patogenicidad, la excreción de ooquistes y la incidencia varían entre ellas, la realización de estudios moleculares es crucial para una mejor comprensión de la epidemiología de la criptosporidiosis humana. En el presente trabajo se analizan muestras procedentes de 2 estudios independientes: uno formado por 23 muestras procedentes de Madrid y otro compuesto por 72 muestras procedentes de La Coruña, todas ellas positivas para Cryptosporidium spp. por métodos microscópicos y pertenecientes a casos aislados de criptosporidiosis. Para la identificación a nivel de especie se utilizaron las regiones de diagnóstico descritas para el ADNr 18S y las regiones de diagnóstico del gen de la COWP. De las 95 muestras analizadas, se consiguió extraer y amplificar ADN en 77 casos, en los que las especies causantes de la infección fueron: C. parvum (40 casos: 2 Madrid y 38 La Coruña), C. hominis (30 casos: 10 Madrid y 20 La Coruña) y C. meleagridis (2 casos: uno Madrid y uno La Coruña). En otros 5 casos fue imposible detectar la especie responsable de la infección, aunque se confirmara su positividad por PCR (4 Madrid y uno La Coruña). Los genotipos aislados en estos pacientes se correlacionaron con los hallados en animales de las mismas regiones (AU)

Molecular PCR based diagnostic techniques have enabled us to distinguish between the different, morphologically identical, Cryptosporidium species that can infect humans. Of the 23 recognized species in the genus, at least 9 are able to infect humans. As the intensity of the clinical manifestations, pathogenicity, excretion of oocysts, and incidence, are different between this species, molecular studies are crucial for a better understanding of the epidemiology of human cryptosporidiosis. Samples form two independent studies are analyzed in this publication. One included 23 samples from Madrid, and the other, 72 samples from La Coruña. All of them positive for Cryptosporidium spp. by microscopic methods and belonging to isolated cases of human cryptosporidiosis. For the identification of the species responsible for the infection, the 18S rDNA diagnostic region and the COWP gene diagnostic regions were used. Out of the 95 samples tested, in 77 cases we were able to extract and amplify DNA. In those cases the species responsible for the infection were: C. parvum (40 cases, 2 Madrid and 38 La Coruña), C. hominis (30 cases, 10 Madrid and 20 La Coruña) and C. meleagridis (2 cases, 1 Madrid and 1 La Coruña). In 5 samples it was impossible to detect the species responsible for the infection, but their positivity was confirmed by PCR (4 Madrid and 1 La Coruña). The genotypes of the isolates from patients correlated well with animals from the same regions

[Molecular characterization of Cryptosporidium spp. isolated in humans in two different locations in Spain]. / Caracterización molecular de aislados humanos de Cryptosporidium spp. procedentes de 2 diferentes localizaciones de España.

Molecular PCR based diagnostic techniques have enabled us to distinguish between the different, morphologically identical, Cryptosporidium species that can infect humans. Of the 23 recognized species in the genus, at least 9 are able to infect humans. As the intensity of the clinical manifestations, pathogenicity, excretion of oocysts, and incidence, are different between this species, molecular studies are crucial for a better understanding of the epidemiology of human cryptosporidiosis. Samples form two independent studies are analyzed in this publication. One included 23 samples from Madrid, and the other, 72 samples from La Coruña. All of them positive for Cryptosporidium spp. by microscopic methods and belonging to isolated cases of human cryptosporidiosis. For the identification of the species responsible for the infection, the 18S rDNA diagnostic region and the COWP gene diagnostic regions were used. Out of the 95 samples tested, in 77 cases we were able to extract and amplify DNA. In those cases the species responsible for the infection were: C. parvum (40 cases, 2 Madrid and 38 La Coruña), C. hominis (30 cases, 10 Madrid and 20 La Coruña) and C. meleagridis (2 cases, 1 Madrid and 1 La Coruña). In 5 samples it was impossible to detect the species responsible for the infection, but their positivity was confirmed by PCR (4 Madrid and 1 La Coruña). The genotypes of the isolates from patients correlated well with animals from the same regions.

The third described case of transfusion-transmitted Babesia duncani.

BACKGROUND: Almost all of the reported US tick-borne and transfusion-associated Babesia cases have been caused by Babesia microti, which is endemic in the Northeast and upper Midwest. We investigated a case caused by B. duncani (formerly, the WA1-type parasite), in a 59-year-old California resident with sickle cell disease (HbSS) whose only risk factor for infection was receipt of red blood cell transfusions. CASE REPORT: The patient's case was diagnosed in September 2008: intraerythrocytic parasites were noted on a blood smear, after a several-month history of increasing transfusion requirements. Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024). The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991. The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA. The implicated donor-a 67-year-old California resident-had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008. CONCLUSIONS: This patient had the third documented transfusion case caused by B. duncani. His case underscores the fact that babesiosis can be caused by agents not detected by molecular or serologic analyses for B. microti.

Tubulinosema sp. microsporidian myositis in immunosuppressed patient.

The Phylum Microsporidia comprises >1,200 species, only 15 of which are known to infect humans, including the genera Trachipleistophora, Pleistophora, and Brachiola. We report an infection by Tubulinosema sp. in an immunosuppressed patient.

Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2.

Isoenzyme analysis of cultured parasites is the conventional approach for Leishmania species identification. Molecular approaches have the potential to be more sensitive and rapid. We designed PCR generic primers to amplify a segment of the rRNA internal transcribed spacer 2 (ITS2) from multiple Leishmania species. To validate the selected ITS2 fragment, we tested clinical specimens and compared the species results obtained by the molecular approach (PCR followed by DNA sequencing analysis) with those from the parasitologic approach (in vitro culture followed by isoenzyme analysis). Among the 159 patients with clinical specimens positive by both approaches, a total of eight Leishmania species were identified. The species results were concordant for all but two patients: for one patient, the results were Leishmania (Viannia) guyanensis by the molecular approach versus L. (V.) braziliensis by the parasitologic approach; for the other patient, the results were L. (Leishmania) tropica versus L. (L.) major, respectively. ITS2 PCR, followed by sequencing analysis, can be used to detect and discriminate among Leishmania species. The results confirmed our hypothesis that a region of the ITS2 gene can complement the characterization of Leishmania parasites at the species level. The approach we developed can be used as a diagnostic tool in reference laboratories with adequate infrastructure to perform molecular characterization of pathogens.

Paravahlkampfia francinae n. sp. masquerading as an agent of primary amoebic meningoencephalitis.

Paravahlkampfia francinae n. sp., a new species of the free-living amoeba genus Paravahlkampfia, designated as CDC:V595, was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, typical symptoms of primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri. The isolate grew at 33 degrees C, 37 degrees C, 40 degrees C, and 42 degrees C and destroyed mammalian cell cultures. However, it did not kill young mice upon intranasal inoculation. P. francinae does not produce flagellates and does not grow on agar plates coated with Gram-negative bacteria such as Escherichia coli, the usual food source of Paravahlkampfia ustiana, the type species of the genus. The trophozoite at light microscopy exhibited eruptive locomotion and possessed a single vesicular nucleus. Ultrastructurally, the trophozoites had numerous mitochondria with discoidal cristae but did not have a Golgi apparatus. The trophozoites differentiated into cysts after consuming most of the monolayer. The cyst had an inner well-differentiated endocyst and an outer thin, wrinkled, and wavy ectocyst with no pores. During excystation trophozoites ruptured the cyst wall and emerged from the cysts. A unique feature seen in the cysts was the presence of bacterial endosymbionts, both in the endoplasm and within the cyst wall. Full-length sequencing analysis of the 18S and 5.8S RNA genes of P. francinae showed that they were distinct from those of other Paravahlkampfia species. The patient recovered within a few days indicating that some of the previously reported cases of PAM that survived may have been due to P. francinae.

Prevalence of intestinal microsporidiosis in human immunodeficiency virus-infected patients with diarrhea in major United States cities.

To determine the prevalence of intestinal microsporidiosis in HIV-infected patients, we performed a prospective study of HIV-infected patients with diarrheal illnesses in three US hospitals and examined an observational database of HIV-infected patients in 10 US cities. Among 737 specimens from the three hospitals, results were positive for 11 (prevalence 1.5%); seven (64%) acquired HIV through male-to-male sexual contact, two (18%) through male-to-male sexual contact and injection drug use, and one (9%) through heterosexual contact; one (9%) had an undetermined mode of transmission. Median CD4 count within six months of diagnosis of microsporidiosis was 33 cells/microL (range 3 to 319 cells/microL). For the national observational database (n = 24,098), the overall prevalence of microsporidiosis was 0.16%. Prevalence of microsporidiosis among HIV-infected patients with diarrheal disease is low, and microsporidiosis is most often diagnosed in patients with very low CD4+ cell counts. Testing for microsporidia appears to be indicated, especially for patients with very low CD4+ cell counts.

Prevalence of intestinal microsporidiosis in Human Immunodeficiency Virus-infected patients with diarrhea in major United States cities

To determine the prevalence of intestinal microsporidiosis in HIV-infected patients, we performed a prospective study of HIV-infected patients with diarrheal illnesses in three US hospitals and examined an observational database of HIV-infected patients in 10 US cities. Among 737 specimens from the three hospitals, results were positive for 11 (prevalence 1.5 percent); seven (64 percent) acquired HIV through male-to-male sexual contact, two (18 percent) through male-to-male sexual contact and injection drug use, and one (9 percent) through heterosexual contact; one (9 percent) had an undetermined mode of transmission. Median CD4 count within six months of diagnosis of microsporidiosis was 33 cells/µL (range 3 to 319 cells/µL). For the national observational database (n = 24,098), the overall prevalence of microsporidiosis was 0.16 percent. Prevalence of microsporidiosis among HIV-infected patients with diarrheal disease is low, and microsporidiosis is most often diagnosed in patients with very low CD4+ cell counts. Testing for microsporidia appears to be indicated, especially for patients with very low CD4+ cell counts.

Para determinar a prevalência de microsporidiose intestinal em pacientes infectados pelo HIV foi realizado um estudo prospectivo em três hospitais dos Estados Unidos da América do Norte (EUA) e analizada uma base de dados nacional composta de dados coletados de pacientes infectados pelo HIV em 10 cidades dos EUA. De um total de 737 amostras de fezes de pacientes infectados pelo HIV que apresentavam diarréia, amostras de 11 pacientes (prevalência de 1,5 por cento) foram positivas para microsporídios. Todos os positivos eram do sexo masculino e, entre eles, sete (64 por cento) pacientes adquiriram a infecção pelo HIV através de relação homossexual, dois (18 por cento) através de relação sexual e drogas injetáveis e um (9 por cento) através de contato heterosexual, enquanto que em um paciente o modo de transmissão do HIV não foi determinado. A contagem média de linfócitos CD4 realizada até seis meses do diagnóstico de microsporidiose foi de 33 células/microlitro (3 a 319 células/microlitro). A análise da base de dados nacional (n = 24.098) mostrou uma prevalência de microsporidiose de 0,16 por cento. A prevalência de microsporidiose em pacientes HIV-positivos com diarréia é baixa. Entretando, como a microsporidiose é mais frequentemente diagnosticada em pacientes com contagens de CD4 muito baixas, a indicação de pesquisa de microsporídios é justificada, especialmente para estes pacientes.

Detection and molecular characterization of Cryptosporidium bovis-like isolate from a newborn lamb in Spain.

Species of Cryptosporidium infect a broad variety of animals. Because morphological features of the secreted oocysts are not useful in identifying the parasite at the species level, molecular tools were used to accomplish this task, leading to discovery of new Cryptosporidium species. With the use of this approach, Cryptosporidium bovis has recently been described as a new species infecting bovines and several other hosts, but clearly distinct from C. parvum. In this report, we present a description of a Cryptosporidium sp. isolate from a newborn lamb from a farm in Spain. The isolate seemed to be very similar to C. bovis based on the analysis of the gene that codes for the 18S rRNA.

PCR as a confirmatory technique for laboratory diagnosis of malaria.

We compared a nested PCR assay and microscopic examination of Giemsa-stained blood films for detection and identification of Plasmodium spp. in blood specimens. PCR was more sensitive than microscopy and capable of identifying malaria parasites at the species level when microscopy was equivocal.

Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa.

The development in Plasmodium falciparum of the resistance to chloroquine (CQ) constitutes a public health priority, due to its direct influence in childhood mortality. The molecular basis for CQ resistance (CQR) is still unclear but, recently, a new relevant gene, named pfcrt, with several point mutations was identified in P. falciparum. Two mutations, K76T and A220S, have been considered crucial for CQR in further studies, making the pfcrt a good candidate as determinant for CQR in P. falciparum. To contribute to this topic, we have undertaken a molecular screening on 164 P. falciparum isolates from Africa: 120 isolates were Italian imported malaria cases, 27 and 17 isolates were from a school-children survey from Congo and Tanzania, respectively. In vitro tests (pLDH and WHO-Mark III tests) for CQ sensitivity have been also carried out on 28 plasmodial isolates and results compared to those obtained by molecular analysis in the same isolates. The SVIET pfcrt haplotype has been identified in the samples from Congo, and this is the first time that this haplotype is detected in Africa. Our results give further evidence to the reliability of the 76T (and the linked 74I-75E) pfcrt point mutation as molecular marker for CQR.

Myosporidium merluccius n. g., n. sp. infecting muscle of commercial hake (Merluccius sp.) from fisheries near Namibia.

A new species of Microsporidia classified to a new genus was observed in the trunk muscle of commercial hake (Merluccius capensis/paradoxus complex) from Namibian fisheries. Macroscopic examination revealed thin and dark filaments inserted among muscle fibers. Inside the filaments were many sporophorous vesicles with about 30-50 spores per vesicle. The shape of the spore was pyriform and the extruded polar filament was of moderate length (up to 4.29 microm, n=12). This new species of Microsporidia is described using macrophotography, microphotography, staining, and transmission electron microscopy (TEM), as well as molecular methods. Its 16S rRNA was found to be similar to that of Microsporidium prosopium Kent et al., 1999, while both sequences were quite different from 16S rRNA sequences known for other Microsporidia. Nevertheless, this new species is separated morphologically from M. prosopium by the presence of 11-12 anisofilar coils and the formation of the xenoma at the site of infection. Type species.

Babesia divergens-like infection, Washington State.

Most reported U.S. zoonotic cases of babesiosis have occurred in the Northeast and been caused by Babesia microti. In Washington State, three cases of babesiosis have been reported previously, which were caused by WA1 (for "Washington 1")-type parasites. We investigated a case of babesiosis in Washington in an 82-year-old man whose spleen had been removed and whose parasitemia level was 41.4%. The complete 18S ribosomal RNA gene of the parasite was amplified from specimens of his whole blood by polymerase chain reaction. Phylogenetic analysis showed the parasite is most closely related, but not identical, to B. divergens (similarity score, 99.5%), a bovine parasite in Europe. By indirect fluorescent-antibody testing, his serum reacted to B. divergens but not to B. microti or WA1 antigens. This case demonstrates that babesiosis can be caused by novel parasites detectable by manual examination of blood smears but not by serologic or molecular testing for B. microti or WA1-type parasites.

Molecular characterization of a non-Babesia divergens organism causing zoonotic babesiosis in Europe.

In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.

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.

Molecular and morphologic characterization of a Cryptosporidium genotype identified in lemurs.

This study reports the molecular and morphologic characterization of a Cryptosporidium sp., identified in stools of captive lemurs Propithecus verreauxi coquereli. Stool samples were collected from seven animals (n=7) presenting episodes of diarrhea. Bright-field light microscopy of stool smears stained with modified acid-fast technique revealed the presence of Cryptosporidium sp. oocysts in four of the stool samples analyzed. All microscopically positive samples were confirmed by PCR using primers designed to amplify DNA fragments from two independent loci, i.e. the Cryptosporidium oocyst wall protein (COWP) gene and the small subunit ribosomal RNA (ssrRNA) gene. Phylogenetic analysis based on the full-length ssrRNA gene placed this isolate within a clade that contains all currently known C. parvum species/genotypes, closely related to the C. parvum pig genotype. Comparison with partial ssrRNA sequences available in the GenBank revealed 100% sequence identity with the genotype previously identified in Canadian patients. This finding was confirmed further by comparison of the COWP gene partial sequences.

Transmission of Babesia microti in Minnesota through four blood donations from the same donor over a 6-month period.

BACKGROUND: Babesiosis is a tick-borne zoonosis caused by intraerythrocytic protozoa. More than 40 US cases of Babesia microti infection acquired by blood transfusion have been reported. This report describes the identification of a transfusion-associated case of babesiosis and the subsequent identification of the infected blood donor and three other infected recipients of cellular blood components from three other donations by this donor. STUDY DESIGN AND METHODS: Serum specimens from the donors of blood that had been made into cellular components received by the index recipient and from other recipients of such components from the implicated donor were tested by the indirect fluorescent antibody (IFA) assay for antibodies to B. microti. Whole blood from IFA-positive persons was tested by PCR for B. microti DNA. RESULTS: IFA testing of serum from 31 of 36 donors implicated a 45-year-old man (titer, 1 in 256), whose donation had been used for RBCs. He likely became infected when bitten by ticks while camping in Minnesota in June 1999 and had donated blood four times thereafter. As demonstrated by PCR, he remained parasitemic for at least 10 months. Of the five other surviving recipients of cellular blood components from the implicated donor, three recipients (one for each of the three other donations) had become infected through either RBC or platelet transfusions. CONCLUSIONS: Babesiosis should be included in the differential diagnosis of posttransfusion febrile illness, and effective means for preventing transmission by blood transfusion are needed.

A single genotype of Encephalitozoon intestinalis infects free-ranging gorillas and people sharing their habitats in Uganda.

Microsporidian spores have been detected by Chromotrope 2R and calcofluor stains in fecal samples of three free-ranging human-habituated mountain gorillas in Uganda and in two people who share gorilla habitats. All spore isolates have been identified by PCR with species-specific primers and fluorescent in situ hybridization with a species-specific oligonucleotide probe to be Encephalitozoon intestinalis. Sequencing analyses of the full length SSUrRNA amplified from all spore isolates were identical with Enc. intestinalis SSUrRNA GenBank SIU09929. Sequences generated from a fragment containing the internal transcribed spacer of these isolates were identical to GenBank sequence Y11611, i.e., Enc. intestinalis of anthroponotic origin. A single pathogen genotype in two genetically distant but geographically united host groups indicates anthropozoonotic transmission of Enc. intestinalis. It is highly unlikely that these two identical Enc. intestinalis genotypes were acquired independently by gorillas and people; it is much more probable that one group initiated infection of the other.