Traditional Typing Methods Research Articles

Whole-genome sequencing to detect recent transmission of Mycobacterium tuberculosis in settings with a high burden of tuberculosis

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been used to trace the transmission of M. tuberculosis, the causative agent of tuberculosis (TB). Previously published studies using WGS were conducted in developed countries with a low TB burden. We sought to evaluate the relative usefulness of traditional VNTR and SNP typing methods, WGS and epidemiological investigations to study the recent transmission of M.tuberculosis in a high TB burden country. We conducted epidemiological investigations of 42 TB patients whose M.tuberculosis isolates were classified into three clusters based on variable-number tandem repeat (VNTR) typing. We applied WGS to 32 (76.2%) of the 42 strains and calculated the pairwise genomic distances between strains within each cluster. Eighteen (56.3%) of the 32 strains had genomic differences ≥100 SNPs with every other strain, suggesting that direct transmission did not likely occurred. Ten strains were grouped into four WGS-based clusters with genomic distances ≤5 SNPs within each cluster, and confirmed epidemiological links were identified in two of these clusters. Our results indicate that WGS provides reliable resolution for tracing the transmission of M.tuberculosis in high TB burden settings. The high resolution of WGS is particularly useful to confirm or exclude the possibility of direct transmission events defined by traditional typing methods.

Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

Bluetongue (BT) is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV) serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT)) are slow (taking weeks, depend on availability of reference virus-strains or antisera) and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2) encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype) were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h) and reliable RT–PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype) were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT–PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

Widespread, focal copy number variations (CNV) and whole chromosome aneuploidies in Trypanosoma cruzi strains revealed by array comparative genomic hybridization

BackgroundTrypanosoma cruzi is a protozoan parasite and the etiologic agent of Chagas disease, an important public health problem in Latin America. T. cruzi is diploid, almost exclusively asexual, and displays an extraordinarily diverse population structure both genetically and phenotypically. Yet, to date the genotypic diversity of T. cruzi and its relationship, if any, to biological diversity have not been studied at the whole genome level.ResultsIn this study, we used whole genome oligonucleotide tiling arrays to compare gene content in biologically disparate T. cruzi strains by comparative genomic hybridization (CGH). We observed that T. cruzi strains display widespread and focal copy number variations (CNV) and a substantially greater level of diversity than can be adequately defined by the current genetic typing methods. As expected, CNV were particularly frequent in gene family-rich regions containing mucins and trans-sialidases but were also evident in core genes. Gene groups that showed little variation in copy numbers among the strains tested included those encoding protein kinases and ribosomal proteins, suggesting these loci were less permissive to CNV. Moreover, frequent variation in chromosome copy numbers were observed, and chromosome-specific CNV signatures were shared by genetically divergent T. cruzi strains.ConclusionsThe large number of CNV, over 4,000, reported here uphold at a whole genome level the long held paradigm of extraordinary genome plasticity among T. cruzi strains. Moreover, the fact that these heritable markers do not parse T. cruzi strains along the same lines as traditional typing methods is strongly suggestive of genetic exchange playing a major role in T. cruzi population structure and biology.

Genotypic Characterization Of Vibrio Cholerae Isolates Using Several DNA Fingerprint Techniques

Serious pandemics of cholera have occurred throughout the known history of mankind, especially in India, which is a motherland for cholera disease. For the last 20 years several DNA-based typing methods have been employed to study the clonal relatedness between Vibrio cholerae isolates irrespective of their geographical locations. Traditional typing methods, such as biochemical tests, phage typing, serotyping, biotyping and antimicrobial susceptibility tests, have produced reliable and informative data regarding V. cholerae for a long time. Gradually molecular typing techniques have taken the place of traditional typing methods because they produce the same results upon repeat testing of V. cholerae strain. In this article we focus on the discriminatory power of different DNA fingerprint techniques that are generally used to know the homogeneity and heterogeneity among different V. cholerae isolates.

Optical genetic mapping defines regions of chromosomal variation in serovars of S. enterica subsp. enterica of concern for human and animal health

Infections involving Salmonella enterica subsp. enterica serovars have serious animal and human health implications; causing gastroenteritis in humans and clinical symptoms, such as diarrhoea and abortion, in livestock. In this study an optical genetic mapping technique was used to screen 20 field isolate strains from four serovars implicated in disease outbreaks. The technique was able to distinguish between the serovars and the available sequenced strains and group them in agreement with similar data from microarrays and PFGE. The optical maps revealed variation in genome maps associated with antimicrobial resistance and prophage content in S. Typhimurium, and separated the S. Newport strains into two clear geographical lineages defined by the presence of prophage sequences. The technique was also able to detect novel insertions that may have had effects on the central metabolism of some strains. Overall optical mapping allowed a greater level of differentiation of genomic content and spatial information than more traditional typing methods.

Compound Evolutionary History of the Rhesus Macaque Mhc Class I B Region Revealed by Microsatellite Analysis and Localization of Retroviral Sequences

In humans, the single polymorphic B locus of the major histocompatibility complex is linked to the microsatellite MIB. In rhesus macaques, however, haplotypes are characterized by the presence of unique combinations of multiple B genes, which may display different levels of polymorphism. The aim of the study was to shed light on the evolutionary history of this highly complex region. First, the robustness of the microsatellite MIB-linked to almost half of the B genes in rhesus macaques (Mamu-B)–for accurate B haplotyping was studied. Based on the physical map of an established haplotype comprising 7 MIB loci, each located next to a certain Mamu-B gene, two MIB loci, MIB1 and MIB6, were investigated in a panel of MHC homozygous monkeys. MIB1 revealed a complex genotyping pattern, whereas MIB6 analysis resulted in the detection of one or no amplicon. Both patterns are specific for a given B haplotype, show Mendelian segregation, and even allow a more precise haplotype definition than do traditional typing methods. Second, a search was performed for retroelements that may have played a role in duplication processes as observed in the macaque B region. This resulted in the description of two types of duplicons. One basic unit comprises an expressed Mamu-B gene, adjacent to an HERV16 copy closely linked to MIB. The second type of duplicon comprises a Mamu-B (pseudo)gene, linked to a truncated HERV16 structure lacking its MIB segment. Such truncation seems to coincide with the loss of B gene transcription. Subsequent to the duplication processes, recombination between MIB and Mamu-B loci appears to have occurred, resulting in a hyperplastic B region. Thus, analysis of MIB in addition to B loci allows deciphering of the compound evolutionary history of the class I B region in Old World monkeys.

A real‐time PCR approach for rapid high resolution subtyping of HLA‐DRB1*04

We present a real‐time PCR approach for the identification and subtyping of HLA‐DR4 alleles. The technique, which uses sequence‐specific primers and probes in conjunction with real‐time PCR for the detection and differentiation of target alleles, is rapid, involves minimal hands‐on time, and is inexpensive compared to existing methods. Further, there is no post‐PCR handling, so the risk of contamination is avoided. We have validated the assay using 44 blinded and 56 unblinded samples, which were identified with 100% accuracy, sensitivity, and specificity. We demonstrate the applicability of this assay as an alternative approach to traditional HLA typing methods.

Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp.

The morbidity and mortality associated with Vibrio-mediated waterborne diseases necessitates the development of sensitive detection technologies that are able to elucidate the identity, potential pathogenicity, susceptibility, and viability of contaminating bacteria in a timely manner. For this purpose, we have designed a single multiplex PCR assay to simultaneously amplify 95 diagnostic regions (encompassing species/serogroup-specific, antimicrobial resistance, and known toxin markers) and combined it with a long oligonucleotide microarray to create a platform capable of rapidly detecting and discriminating the major human pathogenic species from the genus Vibrio: V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus. We were able to validate this strategy by testing 100 geographically and temporally distributed isolates and observed an excellent concordance between species- and serotype-level microarray-based identification and traditional typing methods. In addition to accurate identification, the microarray simultaneously provided evidence of antibiotic resistance genes and mobile genetic elements, such as sulfamethoxazole-trimethoprim constins and class I integrons, and common toxin (ctxAB, rtxA, hap, hlyA, tl, tdh, trh, vvhA, vlly, and vmhA) and pathogenicity (tcpA, type III secretion system) genes that are associated with pathogenic Vibrio. The versatility of this method was further underscored by its ability to detect the expression of known toxin and virulence genes from potentially harmful viable but nonculturable organisms. The results suggest that this molecular identification method provides rapid and definitive information that would be of value in epidemiological, environmental, and health risk assessment surveillance.

Rapid combined characterization of microorganism and host genotypes using a single technology.

Genetic information is becoming increasingly important in diagnosis and prognosis of infectious diseases. In this study we investigated the possibility of using a single technology, the Pyrosequencing trade mark technology (Biotage AB, Uppsala, Sweden), to gather several kinds of important genetic information from the human pathogen Helicobacter pylori, as well as from the carrier of the H. pylori infection. DNA from 87 clinical isolates of H. pylori, 50 isolates from H. pylori-infected transgenic mice and nine gastric biopsies from H. pylori-infected patients was analyzed for targets in the 16S rRNA, 23S rRNA and cytotoxin associated gene A (cagA) genes to determine species identity, clarithromycin susceptibility and virulence level, respectively. In addition, three single nucleotide polymorphisms in the human interleukin-1B (IL-1B) gene, reported to affect the risk of developing gastric cancer, were analyzed in the gastric biopsy samples. All DNA targets were processed and analyzed in parallel, enabling convenient genetic characterization of both pathogen and host. All genotypes were easily and accurately assigned. In the 16S rRNA analysis, 99.83% of the bases were correctly called. We conclude that genetic analysis using Pyrosequencing trade mark technology was nonlaborious, and gave highly accurate data for different kinds of target. We therefore believe that this technology has the potential to complement or in the future substitute the time-consuming traditional microbial identification and typing methods, as well as enabling rapid typing of relevant host genetic markers.

Biotyping, serotyping and genotyping of aeromonads from environmental and clinical samples

Pulsed-field gel electrophoresis (PFGE) and biochemical–serological assays were used to characterize environmental and clinical aeromonads. On the basis of their biochemical characteristics, 31 strains were assigned to one of the recognized groups or species within the Aeromonas genus and 11 different serogroups were detected. Low correlation between molecular and traditional typing methods was observed. The results obtained showed that the genomic analysis performed by PFGE can be a more effective means for distinguishing between Aeromonas isolates than conventional biochemical methods.

Molecular epidemiology in the care of patients.

Several different epidemiologic typing methods have been applied in studies of microbial pathogens. These methods include the more traditional nonmolecular approaches as well as the more sophisticated molecular typing methods. Application of traditional epidemiologic typing methods, such as antibiogram, serotyping, biotyping, and phage typing, have occasionally been useful in describing the epidemiology of infectious diseases. However, these methods have generally been considered to be too variable, labor intensive, and slow to be of practical value in epidemiologic investigations. In response to these limitations, several techniques have been adopted from the molecular biology field for use as epidemiologic typing methods and have been applied in studies of bacteria, fungi, viruses, and protozoa. The most widely used molecular typing methods are the DNA-based methods, such as plasmid profiling, restriction endonuclease analysis of plasmid and genomic DNA, Southern hybridization analysis using specific DNA probes, and chromosomal DNA profiling using either pulsed-field gel electrophoresis or polymerase chain reaction-based methods. The various molecular typing methods may be applied to the investigation of outbreaks of infections or may be used in the context of epidemiologic surveillance. For outbreak investigation, typing methods are used to compare isolates from a suspected outbreak to delineate clonally related and unrelated strains with the goal of short-term control of transmission. In the context of epidemiologic surveillance, molecular typing methods may be used to monitor geographic spread and prevalence shifts of epidemic and endemic clones with the goal of long-term evaluation of preventive strategies or for the detection and monitoring of emerging and reemerging infections. The specific typing method selected may vary with the task at hand; however, the typing studies must always be used to supplement, rather than replace, careful epidemiologic investigation.

Determination of the prevalence of feline blood types in the UK.

The efficacy and diagnostic accuracy of a new desk-top feline blood typing kit was evaluated by comparing the results of the kit with traditional blood typing methods on 35 feline blood samples. The kit was then used to blood type 139 non-pedigree cats from Scotland and the north of England and 207 pedigree cats from throughout the UK. Of the non-pedigree cats, 87.1 per cent were type A, 7.9 per cent were type B and 5.0 per cent were type AB, while of the pedigree cats, 54.6 per cent were type A, 40.1 per cent were type B and 5.3 per cent were type AB. The majority (121 out of 207) of these pedigree cats were British shorthaired, of which 39.7 per cent were type A, 58.7 per cent were type B and 1.6 per cent were type AB. No cats were identified that failed to express the type A and/or type B antigens. The prevalence of type AB cats appears to be higher in this study than previously reported. The prevalence of blood types within specific pedigree breeds in the UK appears to vary from that reported elsewhere.

High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing.

High-resolution HLA-DRB typing is required for bone marrow transplantation between unrelated donors and recipients and also for identification of novel HLA-DRB alleles. Here we describe a method for the unambiguous identification of HLA-DRB alleles using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The highly variable second exon of all HLA-DRB1, -DRB3, -DRB4, -DRB5, -DRB6 and -DRB7 alleles was amplified using a single pair of generic DRB-specific primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and the sequences of these alleles were determined using fluorescent-based sequencing and allele-assignment software. The validity of this typing procedure was confirmed by identification of HLA-DRB alleles for 17 individuals previously characterized by PCR-SSP and/or cloning and sequencing techniques. We identified 34 different HLA-DRB alleles in these 17 unrelated individuals. Importantly, our analysis revealed HLA-DRB1 alleles which had not been identified using the PCR-SSP typing technique. Additionally, alleles from the HLA-DRB3, -DRB4 and -DRB5 loci were identified. Whereas traditional HLA-DRB typing methods provide limited information or require the use of multiple oligonucleotide primers or probes, our technique provides a reliable, specific and relatively rapid way of identifying all HLA-DRB alleles for high-resolution tissue typing.

Differentiation ofCampylobacter jejuniandCampylobacter coliby polymerase chain reaction

A multiplex PCR assay was developed using two primer sets for the identification and differentiation ofCampylobacter coliandCampylobacter jejuni. Primer Set I amplifies a 460-bp fragment present inC. coliandC. jejuni. Set II amplifies a 160-bp target unique toC. jejuni. When the assay was performed on reference strains, amplification ofC. coliyielded only the 460-bp fragment. Amplification ofC. jejunigenerated both the 160- and 460-bp fragments.Campylobacterfield strains (n=85) isolated from raw poultry were identified by PCR and by conventional biochemical methods. Species determination by the two methods agreed for 83 of the 85 isolates examined. By PCR, 23 were identified asC. coliand 62 asC. jejuni. One isolate was unidentifiable by biochemical testing. The PCR assay identified this isolate asC. coli. In addition, one strain which was identified asC. coliby biochemical testing was determined to beC. jejuniby PCR. The PCR assay offers an alternative to traditional biochemical typing methods for the identification and differentiation ofC. coliandC. jejuniisolated from poultry. It is accurate, simple to perform, and can be completed within 8 h.

Identification of new mamu-DRB alleles using DGGE and direct sequencing.

Rhesus macaques represent important animal models for biomedical research. The ability to identify macaque major histocompatibility complex (Mhc) alleles is crucial for fully understanding these models of autoimmune and infectious disease. Here we describe a rapid and unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. Importantly, our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different Mhc-DRB lineages. Interestingly, two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. This new technique should also contribute to ongoing studies of Mhc function and evolution in many different species of nonhuman primates.

Discriminatory power and usefulness of pulsed-field gel electrophoresis in epidemiological studies of Pseudomonas aeruginosa

We assessed the discriminatory power of pulsed-field gel electrophoresis (PFGE) for the analysis of DNA restriction fragment length polymorphism (RFLP) in Pseudomonas aeruginosa. We determined DraI PFGE-RFLP of DNA of unrelated clinical and environmental strains, and clinical strains isolated from two intensive care units of the Besançon University Hospital. The typeability and reproducibility was 100%. The discriminatory index was 0·998, and the DNA patterns were stable in vitro and in vivo. There was a very low correlation between PFGE-RFLP and traditional typing methods. The typeability, reproducibility, the high discriminatory power and the stability of PFGE-RFLP make this a valuable method to be used in conjunction with serotyping. Further standardization and quantitative interpretation are possible and should lead to this technique becoming a library typing system.

Restriction Fragment Length Polymorphism (RFLP) Typing of DNA Extracted from Nasal Secretions

The restriction fragment length polymorphism (RFLP) analysis of blood, semen, and other body fluids, has become increasingly important in violent criminal cases. The identification of additional tissues suitable for comparison with suspected donors has obvious potential benefit. One type of tissue, that found in nasal secretions, has previously received little attention with regards to genetic analysis. We collected blood and nasal secretion samples from eight individuals, subjected them to traditional RFLP typing methods, and analyzed the results using probes for loci D2S44, D1S7, D10S28, D4S139, and D17S79. All nasal samples provided high DNA yields and hybridization results that matched the corresponding blood standards. Thus, nasal secretions are shown to have potentially significant evidentiary value.

An epidemic methicillin-resistant strain of Staphylococcus aureus in Spain.

During 1990, a strain of methicillin-resistant Staphylococcus aureus became epidemic in Spain and spread in a manner analogous to that of EMRSA-1 in England. Isolates of this strain produced little protein A and were resistant to a number of antibiotics including ciprofloxacin. Beta-lactamase production was encoded by a c. 39 kb plasmid, which also conferred resistance to mercury, cadmium, ethidium bromide and propamidine isethionate. Investigation showed that two variants, separable by supplementary and Fisk phage typing, were circulating. The B variant appeared to spread more readily than the A variant. The opportunity was taken to compare the discriminatory power of traditional typing methods with molecular techniques. The discriminatory power of the molecular techniques used only reached the same level as the traditional methods when double enzyme digestion of total cellular DNA by EcoR I and Cla I was performed.