A method using a single-round PCR coupled to pyrosequencing was developed for the detection and d... more A method using a single-round PCR coupled to pyrosequencing was developed for the detection and differentiation of members of the Entamoeba complex. The technique was evaluated using DNA isolated directly from faecal specimens and compared with a duplex real-time PCR targeting Entamoeba histolytica and Entamoeba dispar, and a conventional single-round PCR for the detection of Entamoeba moshkovskii. Tetranucleate cysts from 102 faecal specimens from Swedish, Danish and Dutch patients test-positive for the ...
The spot hybridization assay for the detection of Plasmodium falciparum reported here uses as pro... more The spot hybridization assay for the detection of Plasmodium falciparum reported here uses as probe a repetitive DNA sequence from this species and exhibits a high degree of species specificity. Isolates from African, Asian, and South American patients were positive in the assay and gametocytes could be detected at the same level of parasitaemia as asexual parasites. An RNA probe containing the same repetitive sequence as the DNA probe has a detection limit of 1 parasite per 10(6) red blood cells. Comparison of the results of the assay with those obtained by microscopic examination of blood films indicated that the assay was more sensitive than microscopy if the blood films were examined for only 10 minutes; however, 40 minutes' examination by microscopy was slightly more sensitive than the assay.
SUMMARYIn March 2013, a veterinary student tested positive forCryptosporidium; four classmates re... more SUMMARYIn March 2013, a veterinary student tested positive forCryptosporidium; four classmates reported similar gastrointestinal symptoms. We aimed to identify source(s) and risk factors forCryptosporidiuminfection in university persons symptomatic between 21 January and 14 April 2013. Sixty-four (79%) students from a cohort of 81 fourth-year veterinary students completed questionnaires, identifying 13 cases; four wereCryptosporidium parvumGP60 subtype IIaA16G1R1b, two were IIdA24G1, seven did not submit stool samples. Thirteen cases attended the university's field clinic before symptom onset (13/37 attendees, 35%); 11 visited at least one of four farms where students recalled seeing calves with diarrhoea.C. parvumsubtype IIaA16G1R1b was identified in calves at one of the farms. Entering pens of calves with diarrhoea [relative risk (RR) 7·6, 95% confidence interval (CI) 1·7–33·5] and eating in clinic cars (RR 9·1, 95% CI 1·3–65·8) were associated with being a case. Washing hands...
Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype... more Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype is responsible for many waterborne outbreaks as well as sporadic cases and is considered virulent and highly important in the epidemiology of cryptosporidiosis. Due to low heterogeneity within the genome of C. hominis it has been difficult to identify epidemiological markers with higher resolution than gp60. However, new markers are required in order to improve outbreak investigations and studies of the transmission dynamics of this clinically important subtype. Based on the whole genome sequences of 17 C. hominis isolates, we have identified several differential loci and developed a new sequence based typing panel with higher resolution than gp60. An amplicon sequencing method was also developed which is based on a one-step PCR which can be sequenced using a Next Generation Sequencing (NGS) platform. Such a system provides a rapid and high-throughput workflow. A panel of nine loci with 10 single nucleotide variants (SNV) was selected and evaluated using clinical IbA10G2 isolates from sporadic, cluster and outbreak associated cases. The specimens were separated into 10 different genetic profiles named sequence types (STs). All isolates within an outbreak or cluster belonged to the same ST, including several samples from the two large waterborne outbreaks which occurred in Sweden between 2010 and 2011 indicating that these outbreaks might be linked. The results demonstrate the methods suitability for improved genotyping of C. hominis IbA10G2.
The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worl... more The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. The aim of this study was to expand the knowledge on the molecular epidemiology of human cryptosporidiosis in Sweden to better understand transmission patterns and potential zoonotic sources. Cryptosporidium-positive fecal samples were collected between January 2013 and December 2014 from 12 regional clinical microbiology laboratories in Sweden. Species and subtype determination was achieved using small subunit ribosomal RNA and 60 kDa glycoprotein gene analysis. Samples were available for 398 patients, of whom 250 (63%) and 138 (35%) had acquired the infection in Sweden and abroad, respectively. Species identification was successful for 95% (379/398) of the samples, revealing 12 species/genotypes: Cryptosporidium parvum (n = 299), C. hominis (n = 49), C. meleagridis (n = 8), C. cuniculus (n = 5), Cryptosporidium chipmunk genotype I (n = 5), C. felis (n = 4), C. erinacei (n = 2), ...
Tropical medicine and parasitology: official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft für Technische Zusammenarbeit (GTZ)
Pf155 (RESA), a malaria antigen in the membrane of infected red cells, was recently identified as... more Pf155 (RESA), a malaria antigen in the membrane of infected red cells, was recently identified as a possible future malaria vaccine candidate. In this study the seroreactivities against this antigen were compared with those against crude parasitic antigens in 195 subjects from a Somali village with mesoendemic malaria. The seroreactivities were determined with immunofluorescence. With age, there was an increased seroreactivity to both Pf155 and the crude parasitic antigens. However, the acquisition of seroreactivity was much slower against Pf155. Hence in the age group 15-24 years, only half of the subjects had detectable antibodies against Pf155.
A method using a single-round PCR coupled to pyrosequencing was developed for the detection and d... more A method using a single-round PCR coupled to pyrosequencing was developed for the detection and differentiation of members of the Entamoeba complex. The technique was evaluated using DNA isolated directly from faecal specimens and compared with a duplex real-time PCR targeting Entamoeba histolytica and Entamoeba dispar, and a conventional single-round PCR for the detection of Entamoeba moshkovskii. Tetranucleate cysts from 102 faecal specimens from Swedish, Danish and Dutch patients test-positive for the ...
The spot hybridization assay for the detection of Plasmodium falciparum reported here uses as pro... more The spot hybridization assay for the detection of Plasmodium falciparum reported here uses as probe a repetitive DNA sequence from this species and exhibits a high degree of species specificity. Isolates from African, Asian, and South American patients were positive in the assay and gametocytes could be detected at the same level of parasitaemia as asexual parasites. An RNA probe containing the same repetitive sequence as the DNA probe has a detection limit of 1 parasite per 10(6) red blood cells. Comparison of the results of the assay with those obtained by microscopic examination of blood films indicated that the assay was more sensitive than microscopy if the blood films were examined for only 10 minutes; however, 40 minutes' examination by microscopy was slightly more sensitive than the assay.
SUMMARYIn March 2013, a veterinary student tested positive forCryptosporidium; four classmates re... more SUMMARYIn March 2013, a veterinary student tested positive forCryptosporidium; four classmates reported similar gastrointestinal symptoms. We aimed to identify source(s) and risk factors forCryptosporidiuminfection in university persons symptomatic between 21 January and 14 April 2013. Sixty-four (79%) students from a cohort of 81 fourth-year veterinary students completed questionnaires, identifying 13 cases; four wereCryptosporidium parvumGP60 subtype IIaA16G1R1b, two were IIdA24G1, seven did not submit stool samples. Thirteen cases attended the university's field clinic before symptom onset (13/37 attendees, 35%); 11 visited at least one of four farms where students recalled seeing calves with diarrhoea.C. parvumsubtype IIaA16G1R1b was identified in calves at one of the farms. Entering pens of calves with diarrhoea [relative risk (RR) 7·6, 95% confidence interval (CI) 1·7–33·5] and eating in clinic cars (RR 9·1, 95% CI 1·3–65·8) were associated with being a case. Washing hands...
Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype... more Cryptosporidium hominis gp60 subtype IbA10G2 is a common cause of cryptosporidiosis. This subtype is responsible for many waterborne outbreaks as well as sporadic cases and is considered virulent and highly important in the epidemiology of cryptosporidiosis. Due to low heterogeneity within the genome of C. hominis it has been difficult to identify epidemiological markers with higher resolution than gp60. However, new markers are required in order to improve outbreak investigations and studies of the transmission dynamics of this clinically important subtype. Based on the whole genome sequences of 17 C. hominis isolates, we have identified several differential loci and developed a new sequence based typing panel with higher resolution than gp60. An amplicon sequencing method was also developed which is based on a one-step PCR which can be sequenced using a Next Generation Sequencing (NGS) platform. Such a system provides a rapid and high-throughput workflow. A panel of nine loci with 10 single nucleotide variants (SNV) was selected and evaluated using clinical IbA10G2 isolates from sporadic, cluster and outbreak associated cases. The specimens were separated into 10 different genetic profiles named sequence types (STs). All isolates within an outbreak or cluster belonged to the same ST, including several samples from the two large waterborne outbreaks which occurred in Sweden between 2010 and 2011 indicating that these outbreaks might be linked. The results demonstrate the methods suitability for improved genotyping of C. hominis IbA10G2.
The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worl... more The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. The aim of this study was to expand the knowledge on the molecular epidemiology of human cryptosporidiosis in Sweden to better understand transmission patterns and potential zoonotic sources. Cryptosporidium-positive fecal samples were collected between January 2013 and December 2014 from 12 regional clinical microbiology laboratories in Sweden. Species and subtype determination was achieved using small subunit ribosomal RNA and 60 kDa glycoprotein gene analysis. Samples were available for 398 patients, of whom 250 (63%) and 138 (35%) had acquired the infection in Sweden and abroad, respectively. Species identification was successful for 95% (379/398) of the samples, revealing 12 species/genotypes: Cryptosporidium parvum (n = 299), C. hominis (n = 49), C. meleagridis (n = 8), C. cuniculus (n = 5), Cryptosporidium chipmunk genotype I (n = 5), C. felis (n = 4), C. erinacei (n = 2), ...
Tropical medicine and parasitology: official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft für Technische Zusammenarbeit (GTZ)
Pf155 (RESA), a malaria antigen in the membrane of infected red cells, was recently identified as... more Pf155 (RESA), a malaria antigen in the membrane of infected red cells, was recently identified as a possible future malaria vaccine candidate. In this study the seroreactivities against this antigen were compared with those against crude parasitic antigens in 195 subjects from a Somali village with mesoendemic malaria. The seroreactivities were determined with immunofluorescence. With age, there was an increased seroreactivity to both Pf155 and the crude parasitic antigens. However, the acquisition of seroreactivity was much slower against Pf155. Hence in the age group 15-24 years, only half of the subjects had detectable antibodies against Pf155.
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