Cryptosporidium parvum causes gastroenteritis epidemics in the Nablus region of Palestine

A total of 30 faecal samples collected from individuals admitted to a local hospital in Nablus city in Palestine with gastroenteritis symptoms, plus five faecal samples from healthy individuals living in the same area were screened for the presence of Cryptosporidium spp. by microscopic analysis using malachite green negative staining. Molecular techniques were used to confirm the microscopic identification. All 30 samples from individuals with gastroenteritis symptoms were positive by both techniques. No other parasites were found in the faecal material of patients or healthy individuals. To explore the source of the outbreak, water was collected from various reservoirs and springs that supply the city with drinking water. Al‐Qaryoon water spring was found to be contaminated with Cryptosporidium using both microscopic and molecular analysis. No other water resources were found to be contaminated. Genotyping analysis of Cryptosporidium oocysts using PCR‐RFLP technique identified the parasite as C. parvum.     

Association of Interleukin-1β-31C/T, -511T/C and -3954C/T Single Nucleotide Polymorphism and Their Blood Plasma Level in Acquired Aplastic Anemia

Aplastic anemia (AA) is an immune-mediated disorder in which hematopoietic stem and progenitor cells are targeted by a number of cellular and molecular pathways. This case control study aims to investigate the association of interleukin-1beta (IL-1β) gene polymorphisms, (IL-1β-31, IL-1β-511 and IL-1β-3954) and their plasma levels with acquired AA. Genotyping was done by Restricted Fragment Length Polymorphism (PCR–RFLP) method and IL-1β plasma levels were evaluated in peripheral blood using ELISA. Increased level of IL-1β was reported to be significant in cases as compared to controls. The susceptibility of developing AA was higher in the cases for IL-1β-3954 genotype. IL-1β-511 genotype showed significant association with the severity groups of AA. No significant association was noticed in responder versus non-responder group. Plasma level of IL-1β gene was found to be significantly higher in severe and very-severe group of AA versus control group. Our findings suggest that IL-1β gene and its genotypes might be involved in the pathophysiology of AA and play a central role in the etiopathogenesis of AA.     

Identification of Old World Leishmania species by PCR–RFLP of the 7 spliced leader RNA gene and reverse dot blot assay

The aim of the study was to assess the 7SL RNA PCR followed by restriction fragment length polymorphism (RFLP) and reverse dot blot (RDB) assays for use in identification of Old World Leishmania species. Species‐specific RFLP patterns were obtained for Leishmania major, Leishmania tropica and the Leishmania donovani complex when the 7SL RNA PCR product was digested with the restriction enzyme BsuRI, an isoschizomer of HaeIII. For the RDB assay, biotin‐labelled 7SL RNA amplicons were hybridized to Leishmania genus‐specific and species‐specific oligonucleotide probes immobilized onto a membrane. The Old World Leishmania species could be distinguished by using five probes: one that was a genus‐specific probe and hybridized to all Leishmania species (Lc), two that were specific for L. major (Lm1 and Lm2), one that was specific for L. tropica (Lt) and one that detected both L. major and L. tropica (Lmt). The PCR–RDB was 10 times more sensitive than 7SL PCR and can detect <1 parasite. In addition, the identification of species was easier and more reliable than with 7SL PCR–RFLP. 7SL PCR–RFLP detected parasites in 50 of 57 clinical samples, whereas PCR–RDB detected 53 and 55 were detected by amplification of kinetoplast (k) DNA. The 7SL RNA PCR has proven useful for direct diagnosis of Old World leishmaniasis, especially when combined with the RBD assay for species identification.     

Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP

Cannabis sativa subspecies, known as industrial hemp (C. sativa sativa) and marijuana (C. sativa indica) show no evident morphological distinctions, but they contain different levels of psychoactive Δ-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp. C. sativa subspecies differ in sequence of tetrahydrocannabinolic acid (THCA) synthase gene, responsible for THC production, and only one active copy of the gene, distinctive for marijuana, is capable of producing THC in concentration more then 0,3% in dried plants, usually punishable by the law.Twenty different samples of marijuana that contain THC in concentration more then 0,3% and three varieties of industrial hemp were analyzed for presence of an active copy of THCA synthase gene using in-house developed restriction fragment length polymorphism (RFLP) method All twenty samples of marijuana were positive for the active copy of THCA synthase gene, 16 of them heterozygous. All three varieties of industrial hemp were homozygous for inactive copy.An algorithm for the fast and accurate forensic analysis of samples suspected to be marijuana was constructed, answering the question if an analyzed sample is capable of producing THC in concentrations higher than 0.3%.     

Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis

BackgoundConventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification of invasive fungal disease (IFD).MethodsWe evaluated 16 clinical fungal species using a combination of PCR assays with 3 different restriction endonucleases targeting various internal transcribed spacer (ITS) regions and high resolution melting analysis (HRMA). Serial samples from 75 patients suspected to have IFD were analyzed for clinical verification.ResultsWe have designed a universal PCR capable of amplifying a portion of the 18S rRNA gene of 16 clinically important fungal species. The restriction patterns of most PCR products generated by EcoRI or double digested by ClaI and AvaI were different, except Aspergillus niger and Aspergillus flavus had a similar pattern, and Aspergillus fumigatus and Aspergillus terreus had a similar pattern. All these species had a unique melting curve shape using the HRMA. Both HRMA and universal PCR had adequate sensitivity, and all sixteen reference fungal species can be clearly distinguished by the universal PCR-RFLP-HRMA assay. With a reference library of 176 clinically relevant fungal strains, and 75 clinical samples from patients with suspicious IFD were tested, our assay identified 100% and 61.1% of isolates from the reference library and clinical samples, respectively.ConclusionsUniversal PCR and RFLP coupled with HRMA could be a highly discriminative and useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of invasive fungal disease.     

Loss of heterozygosity of the APC gene in oral squamous cell carcinoma

The aim of this study was to investigate loss of heterozygosity (LOH) of the APC tumor suppressor gene loci, using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) in 40 cases of oral squamous cell carcinoma (OSCC). Observed informativity was 72.5% for APC exon 11 and 82.5% for APC exon 15. LOH at APC exon 11 was observed in 2 (6.9%) of 29 informative cases, and no LOH was observed for APC exon 15. Our results suggest that inactivation of the APC gene plays a minor role in the carcinogenesis of OSCC.     

Genetic analysis of the hypervariable region of the human astrovirus nsP1a coding region: design of a new RFLP typing method

Human astroviruses (HAstV) are causative agents of viral gastroenteritis worldwide. A hypervariable region (HVR) is located close to the C-terminus of the nsP1a, and recent data support the involvement of the HVR-containing nonstructural protein in viral RNA replication processes, suggesting a correlation between variability in this region and pathogenic properties. The HVR of the C-terminal nsP1a coding region of 104 wild-type and reference isolates of HAstV was sequenced. A phylogenetic analysis was performed to identify different genotypes, and a restriction fragment length polymorphism (RFLP) method was designed. An extensive nucleotide and deduced amino acid sequence variability was observed, as well as many insertions and deletions that retained the reading frame. The resultant phylogenetic tree supported the subdivision of HAstV into the two previously described major genetic groups, genogroup A and B, and the identification of 12 genotypes (9 within genogroup A, and 3 within genogroup B), which could be identified by RFLP. A correlation analysis was performed between genotype information and viral load using information from 35 clinical samples. Significant differences were observed between the viral load in clinical samples and certain HAstV genotypes that belonged to the same serotype, confirming the influence of C-terminal nsP1a variability on the viral replication phenotype. The use of the new RFLP typing method based on the HVR of the C-terminal nsP1a coding region by diagnosticians would help to understand the relationship between different genotypes and the severity of the gastroenteritis.     

Genetic Variability and Linkage Disequilibrium Within the DP Region in the CEPH Families

ABSTRACT: A PCR-based SSO-assay has been developed to characterize the allelic polymorphism at the HLA-DPA1 locus. To validate the performance of this assay, 77 samples were typed side by side in a blinded fashion by the SSO assay and sequencing-based typing (SBT); 100% concordance was seen between the two methods. To address questions of genetic variability and linkage disequilibrium within the class II region, 478 members of the 37 original Caucasian Centre d’Etude du Polymorphisme Humain (CEPH) families were typed for DPA1 using the SSO assay providing information on 247 independent chromosomes. Six of the eight known DPA1 alleles were detected in this population; DPA1*0103 was the most frequent allele. Analysis of the distribution of allele and haplotype frequencies using the homozygosity statistic suggests that balancing selection does not appear to be acting on the DPA1 locus nor on the functional DP heterodimer in this population. Family data permits the unambiguous assignment of haplotypes. Of the 247 independent chromosomes analyzed, 24 distinct DPA1-DPB1 haplotypes were identified with DPA1*0103-DPB1*0401 being the most common. Twelve of the 18 DPB1 alleles identified in this population have an exclusive association with one DPA1 allele. Of the remaining six DPB1 alleles, four are present at a frequency of >3% and show preferential association with just one DPA1 allele. Calculation of the normalized disequilibrium parameter (D′) shows 13 DP haplotypes to be in significant positive disequilibrium. These data suggest there is strong linkage disequilibrium between the DPA1 and DPB1 loci in this Caucasoid population and provide a basis with which to study linkage disequilibrium in other ethnic groups as well as analyze the evolutionary forces which govern allelic and haplotypic variation.     

Non-Mendelian and skewed segregation of DNA markers in wide crosses of the bean rust fungus,Uromyces appendiculatus

The inheritance of DNA markers was investigated in 27 F₂ progeny from a single F₁ hybrid derived from a wide cross inUromyces appendiculatus. This cross was unusual because asexual spores were used to fertilize sexual fruiting structures. Sixty percent of the DNA markers failed to segregate according to simple Mendelian ratios. Segregation bias was evident, in that F₂ progeny inherited on average 91 % of maternal bands and 52% of paternal bands, which deviates significantly from the expected value for each of 75% for dominant markers. Because of these distortions, linkage mapping was not possible with this population. Evaluation of two F₁s from a second wide cross, reciprocals obtained by normal fertilization, also showed non-Mendelian inheritance of one of three co-dominant RFLPs and five of six isozyme markers, indicating that the method of crossing was probably not responsible for the abnormal segregation patterns in the first cross. Either genetic incompatibility, similar to that of an interspecific cross, or selection of particular genotypes could explain the genetic anomalies reported here.     

High-throughput epidemiologic typing in clinical microbiology

Mapping, and ultimately preventing, the dissemination of infectious agents is an important topic in public health. Newly developed molecular–microbiological methods have contributed significantly to recent advances in the efficient tracking of the nosocomial and environmental spread of microbial pathogens. Not only has the application of novel technologies led to improved understanding of microbial epidemiology, but the concepts of population structure and dynamics of many of the medically significant microorganisms have advanced significantly also. Currently, genetic identification of microbes is also within the reach of clinical microbiology laboratory professionals including those without specialized technology research interests. This review summarizes the possibilities for high-throughput molecular–microbiological typing in adequately equipped medical microbiology laboratories from both clinical and fundamental research perspectives. First, the development and application of methods for large-scale comparative typing of serially isolated microbial strains are discussed. The outcome of studies employing these methods allows for long-term epidemiologic surveillance of infectious diseases. Second, recent methods enable an almost nucleotide-by-nucleotide genetic comparison of smaller numbers of strains, thereby facilitating the identification of the genetic basis of, for instance, medically relevant microbiological traits. Whereas the first approach provides insights into the dynamic spread of infectious agents, the second provides insights into intragenomic dynamics and genetic functionality. The current state of technology is summarized, and future perspectives are sketched.