Jessica Beser

Frequent In Vitro Recombination in Internal Transcribed Spacers 1 and 2 during Genotyping of Pneumocystis jirovecii

ABSTRACT Pneumocystis jirovecii is the causative agent of Pneumocystis pneumonia (PCP) in immunocompromised persons. Knowledge of the transmission and epidemiology of PCP is still incipient, and investigations on these subjects are based exclusively on applications of molecular typing techniques. The polymorphic internal transcribed spacers ITS1 and ITS2 in the ribosomal DNA operon, which in the P. jirovecii genome exist as single-copy DNA, are commonly used as target loci for isolate typing. In the course of genotyping P. jirovecii in respiratory specimens from PCP patients by amplification and cloning of a large number of ITS sequences, we found mixed infections (two or more types) in 50% of the samples. In a majority of the specimens with mixed infections, we detected many ITS haplotypes (combinations of ITS1 and ITS2 types) that appeared to be products of recombination between globally common ITS haplotypes present in the same sample. Here we present results of a series of experiments showing that essentially all ITS recombinants are chimeras formed during the genotyping process. Under standard conditions, as many as 37% of the amplified sequences could be hybrid DNA artifacts. We show that by modifying PCR amplification conditions, ITS chimera formation could be largely abolished and the erroneous establishment of artifactual haplotypes avoided. The accurate assessment of genetic diversity is fundamental for a better understanding of the epidemiology and biology of P. jirovecii infections.

High Applicability of a Novel Method for gp60-Based Subtyping of Cryptosporidium meleagridis

ABSTRACT Cryptosporidium meleagridis is a common cause of cryptosporidiosis in avian hosts and the third most common species involved in human cryptosporidiosis. Sequencing of the highly polymorphic 60-kDa glycoprotein (gp60) gene is a frequently used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium. However, few studies have included gp60 sequencing of C. meleagridis. One explanation may be that the gp60 primers currently in use are based on Cryptosporidium hominis and Cryptosporidium parvum sequence data, potentially limiting successful amplification of the C. meleagridis gp60 gene. We therefore aimed to design primers for better gp60 subtyping of C. meleagridis. Initially, ∼1,440 bp of the gp60 locus of seven C. meleagridis isolates were amplified using primers flanking the open reading frame. The obtained sequence data (∼1,250 bp) were used to design primers for a nested PCR targeting C. meleagridis. Twenty isolates (16 from human and 4 from poultry) previously identified as C. meleagridis by analysis of small subunit (SSU) rRNA genes were investigated. Amplicons of the expected size (∼900 bp) were obtained from all 20 isolates. The subsequent sequence analysis identified 3 subtype families and 10 different subtypes. The most common subtype family, IIIb, was identified in 12 isolates, represented by 6 subtypes, 4 new and 2 previously reported. Subtype family IIIe was found in 3 isolates represented by 3 novel, distinct subtypes. Finally, IIIgA31G3R1 was found in 1 human isolate and 4 poultry isolates, all originating from a previously reported C. meleagridis outbreak at a Swedish organic farm.

Unusual cryptosporidiosis cases in Swedish patients: extended molecular characterization of Cryptosporidium viatorum and Cryptosporidium chipmunk genotype I

Most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or Cryptosporidium hominis, but the use of molecular diagnostic methods has revealed that several other less common species or genotypes can also be involved. Here, we describe two unusual causes of cryptosporidiosis, one being the recently described species Cryptosporidium viatorum and the other Cryptosporidium chipmunk genotype I. Two Swedish patients who were infected with C. viatorum had travelled to Kenya and Guatemala, respectively, and two others had been infected with Cryptosporidium chipmunk genotype I in Sweden. None of these four patients were immunocompromised, and all four showed classical symptoms of cryptosporidiosis. We performed extensive molecular characterization, including analysis of four loci. The two C. viatorum isolates were found to differ slightly at the 70-kDa heat shock protein locus, which may indicate a local geographical variation in this species that has previously been described exclusively on the Indian subcontinent.

Low host-specific Enterocytozoon bieneusi genotype BEB6 is common in Swedish lambs

Enterocytozoon bieneusi was found in 49/72 (68%) fecal samples from Swedish lambs while 37 samples from 24 adult sheep were negative. Molecular characterization of the internal transcribed spacer (ITS) revealed three genotypes: BEB6, OEB1, and OEB2, the latter two of which were novel and all three of which belonged to a group of genotypes (Group 2) of probably limited zoonotic importance. Our observations suggest that E. bieneusi is common in young Swedish sheep and add support to emerging evidence that the common BEB6 genotype, originally thought to be adapted to cattle, is capable of infecting a variety of hosts, suggesting low host specificity.

Genomic variation in IbA10G2 and other patient derived Cryptosporidium hominis subtypes.

ABSTRACT In order to improve genotyping and epidemiological analysis of Cryptosporidium spp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 different Cryptosporidium hominis patient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17× and 490× coverage. Sequence homology-based functional annotation identified several genes of interest, including the gene encoding Cryptosporidium oocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to the Cryptosporidium parvum reference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in the C. hominis tree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencing Cryptosporidium directly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes of Cryptosporidium from human fecal samples, while alluding to the potential for a higher degree of genotyping within Cryptosporidium epidemiology.

Possible zoonotic transmission of Cryptosporidium felis in a household.

In humans, the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low, and most of the confirmed cases have been observed in immunocompromised patients. Both anthroponotic and zoonotic transmission routes have been suggested. Here, we report a case of suspected zoonotic transmission of C. felis from a cat to a human. The cat developed diarrhea several months before such symptoms were displayed by its owner, a 37-year-old immunocompetent woman. The presence of identical C. felis SSU rRNA, HSP70, and COWP gene sequences was verified in both hosts. In conclusion, it is highly probable that the cat was the initial source of infection and not the opposite. Our results show that Cryptosporidium infection can be transmitted from pets to humans and that molecular analysis is needed to confirm the identity of the oocysts.

It's a dirty job--A robust method for the purification and de novo genome assembly of Cryptosporidium from clinical material.

We have developed a novel strategy for the purification of Cryptosporidium oocysts from clinical samples using IMS and PCR amplification of target DNA to facilitate uniform coverage genome sequencing and de novo assembly. Our procedure could also be used for other microbial pathogens from clinical specimens.

Improving the genotyping resolution of Cryptosporidium hominis subtype IbA10G2 using one step PCR-based amplicon sequencing

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.

Rapid diagnostic tests relying on antigen detection from stool as an efficient point of care testing strategy for giardiasis and cryptosporidiosis? Evaluation of a new immunochromatographic duplex assay

Microscopy is the gold standard for the diagnosis of gastrointestinal parasites but is time-consuming and dependent on operator skills. Rapid diagnostic tests represent alternative methods but most evaluations have been conducted on a limited number of samples preventing their implementation in the clinical setting. We evaluated a new CE-IVD marked immunochromatographic assay (Crypto/Giardia K-SeT®, Coris Bioconcept) for the detection of G. intestinalis and Cryptosporidium spp. in 2 phases (retrospective and prospective) on a set of 482 stool samples including rare Cryptosporidium species. Besides G. intestinalis, this test could represent a rapid and reliable alternative to the modified Ziehl-Neelsen staining for the diagnosis of cryptosporidiosis (sensitivity/specificity were 89.2%/99.3% and 86.7%/100% for G. intestinalis and Cryptosporidium resp.), reducing diagnostic delays. Such strategy would also be time-saving by avoiding wet mount microscopy and concentrations steps, being particularly appropriate for laboratories having little expertise in microscopy or not able to implement molecular diagnostic methods.