SARS-CoV-2 variants of concern surveillance including Omicron using RT-PCR-based genotyping offers comparable performance to whole genome sequencing.

Known SARS-CoV-2 variants of concern (VOCs) can be detected and differentiated using an RT-PCR-based genotyping approach, which offers quicker time to result, lower cost, higher flexibility, and use of the same laboratory instrumentation for detection of SARS-CoV-2 when compared with whole genome sequencing (WGS). In the current study, we demonstrate how we applied a genotyping approach for identification of all VOCs and that such technique can offer comparable performance to WGS for identification of known SARS-CoV-2 VOCs, including more recent strains, Omicron BA.1 and BA.2.

Targeted Sanger sequencing to recover key mutations in SARS-CoV-2 variant genome assemblies produced by next-generation sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is adaptively evolving to ensure its persistence within human hosts. It is therefore necessary to continuously monitor the emergence and prevalence of novel variants that arise. Importantly, some mutations have been associated with both molecular diagnostic failures and reduced or abrogated next-generation sequencing (NGS) read coverage in some genomic regions. Such impacts are particularly problematic when they occur in genomic regions such as those that encode the spike (S) protein, which are crucial for identifying and tracking the prevalence and dissemination dynamics of concerning viral variants. Targeted Sanger sequencing presents a fast and cost-effective means to accurately extend the coverage of whole-genome sequences. We designed a custom set of primers to amplify a 401 bp segment of the receptor-binding domain (RBD) (between positions 22698 and 23098 relative to the Wuhan-Hu-1 reference). We then designed a Sanger sequencing wet-laboratory protocol. We applied the primer set and wet-laboratory protocol to sequence 222 samples that were missing positions with key mutations K417N, E484K, and N501Y due to poor coverage after NGS sequencing. Finally, we developed SeqPatcher, a Python-based computational tool to analyse the trace files yielded by Sanger sequencing to generate consensus sequences, or take preanalysed consensus sequences in fasta format, and merge them with their corresponding whole-genome assemblies. We successfully sequenced 153 samples of 222 (69 %) using Sanger sequencing and confirmed the occurrence of key beta variant mutations (K417N, E484K, N501Y) in the S genes of 142 of 153 (93 %) samples. Additionally, one sample had the Y508F mutation and four samples the S477N. Samples with RT-PCR Ct scores ranging from 13.85 to 37.47 (mean=25.70) could be Sanger sequenced efficiently. These results show that our method and pipeline can be used to improve the quality of whole-genome assemblies produced using NGS and can be used with any pairs of the most used NGS and Sanger sequencing platforms.

Sensitivity of plasma BRAFmutant and NRASmutant cell-free DNA assays to detect metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression.

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores <5 cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p = 0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27%-58%; P < 0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non-RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non-RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non-RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.

Using Whole Genome Analysis to Examine Recombination across Diverse Sequence Types of Staphylococcus aureus.

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organism's phylogeny and, in particular, the role of recombination, is important both to understand the overall spread of virulent lineages and to characterize outbreaks. To further elucidate the phylogeny of S. aureus, 35 diverse strains were sequenced using whole genome sequencing. In addition, 29 publicly available whole genome sequences were included to create a single nucleotide polymorphism (SNP)-based phylogenetic tree encompassing 11 distinct lineages. All strains of a particular sequence type fell into the same clade with clear groupings of the major clonal complexes of CC8, CC5, CC30, CC45 and CC1. Using a novel analysis method, we plotted the homoplasy density and SNP density across the whole genome and found evidence of recombination throughout the entire chromosome, but when we examined individual clonal lineages we found very little recombination. However, when we analyzed three branches of multiple lineages, we saw intermediate and differing levels of recombination between them. These data demonstrate that in S. aureus, recombination occurs across major lineages that subsequently expand in a clonal manner. Estimated mutation rates for the CC8 and CC5 lineages were different from each other. While the CC8 lineage rate was similar to previous studies, the CC5 lineage was 100-fold greater. Fifty known virulence genes were screened in all genomes in silico to determine their distribution across major clades. Thirty-three genes were present variably across clades, most of which were not constrained by ancestry, indicating horizontal gene transfer or gene loss.

Genomic characterization of Salmonella Cerro ST367, an emerging Salmonella subtype in cattle in the United States.

BACKGROUND: Within the last decade, Salmonella enterica subsp. enterica serovar Cerro (S. Cerro) has become one of the most common serovars isolated from cattle and dairy farm environments in the northeastern US. The fact that this serovar is commonly isolated from subclinically infected cattle and is rarely associated with human disease, despite its frequent isolation from cattle, has led to the hypothesis that this emerging serovar may be characterized by reduced virulence. We applied comparative and population genomic approaches to (i) characterize the evolution of this recently emerged serovar and to (ii) gain a better understanding of genomic features that could explain some of the unique epidemiological features associated with this serovar. RESULTS: In addition to generating a de novo draft genome for one Salmonella Cerro strain, we also generated whole genome sequence data for 26 additional S. Cerro isolates, including 16 from cattle operations in New York (NY) state, 2 from human clinical cases from NY in 2008, and 8 from diverse animal sources (7 from Washington state and 1 from Florida). All isolates sequenced in this study represent sequence type ST367. Population genomic analysis showed that isolates from the NY cattle operations form a well-supported clade within S. Cerro ST367 (designated here "NY bovine clade"), distinct from isolates from Washington state, Florida and the human clinical cases. A molecular clock analysis indicates that the most recent common ancestor of the NY bovine clade dates back to 1998, supporting the recent emergence of this clone.Comparative genomic analyses revealed several relevant genomic features of S. Cerro ST367, that may be responsible for reduced virulence of S. Cerro, including an insertion creating a premature stop codon in sopA. In addition, patterns of gene deletion in S. Cerro ST367 further support adaptation of this clone to a unique ecological or host related niche. CONCLUSIONS: Our results indicate that the increase in prevalence of S. Cerro ST367 is caused by a highly clonal subpopulation and that S. Cerro ST367 is characterized by unique genomic deletions that may indicate adaptation to specific ecological niches and possibly reduced virulence in some hosts.

Progress toward an efficient panel of SNPs for ancestry inference.

Many panels of ancestry informative single nucleotide polymorphisms have been proposed in recent years for various purposes including detecting stratification in biomedical studies and determining an individual's ancestry in a forensic context. All of the panels have limitations in their generality and efficiency for routine forensic work. Some panels have used only a few populations to validate them. Some panels are based on very large numbers of SNPs thereby limiting the ability of others to test different populations. We have been working toward an efficient and globally useful panel of ancestry informative markers that is comprised of a small number of highly informative SNPs. We have developed a panel of 55 SNPs analyzed on 73 populations from around the world. We present the details of the panel and discuss its strengths and limitations.

Comparative analysis of genome sequences covering the seven cronobacter species.

BACKGROUND: Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes. CONCLUSIONS/SIGNIFICANCE: Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.

Validation of the Applied Biosystems MicroSEQ real-time PCR system for detection of E. coli O157:H7 in food.

Modern molecular methods offer the advantages of simplicity and short time-to-results compared to traditional culture methods. We describe the validation of a new Real-Time PCR method to detect E. coli O157:H7 in five food matrixes. The complete system consists of the MicroSEQ E. coli O157:H7 Detection Kit, sample preparation (two sample preparation methods, the PrepSEQ Nucleic Acid Extraction Kit and the PrepSEQ Rapid Spin Sample Preparation Kit, were validated), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. The test method was compared to the U.S. Department of Agriculture Microbiology Laboratory Guidebook 5.04 reference method for detecting E. coli O157:H7 in 25 g and 375 g ground beef and beef trim, and to the ISO 16654 reference method for detecting E. coli O157:H7 in 25 g spinach, orange juice, and apple juice. The MicroSEQ E. coli O157:H7 Detection Kit showed equivalent detection compared to the corresponding reference method based on Mantel-Haenszel Chi-square statistics for all matrixes tested. An independent validation confirmed these findings on ground beef. The MicroSEQ kit detected all 51 E. coli O157:H7 strains tested and showed good discrimination against an exclusivity panel of 30 strains.

Evaluation of applied biosystems MicroSEQ real-time PCR system for detection of Listeria spp. in food and environmental samples.

A complete system for real-time PCR detection of Listeria species was validated in five food matrixes and five environmental surfaces, namely, hot dogs, roast beef, lox (smoked salmon), pasteurized whole cow's milk, dry infant formula, stainless steel, plastic cutting board, ceramic tile, rubber sheets, and sealed concrete. The system consists of the MicroSEQ Listeria spp. Detection Kit, two sample preparation kits (PrepSEQ Nucleic Acid Extraction Kit and PrepSEQ Rapid Spin Sample Preparation Kit), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and the RapidFinderTM Express v1.1 Software for data analysis. The test method was compared to the ISO 11290-1 reference method using an unpaired study design. The MicroSEQ Listeria spp. Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all matrixes except hot dogs. For hot dogs, the MicroSEQ method detected more positives than the reference method for the low- and high-level inoculations, with all of the presumptive positives confirmed by the reference method. An independent validation study confirmed these findings on lox and stainless steel surface. The MicroSEQ kit detected all 50 Listeria strains tested and none of the 31 nontarget bacteria strains.

Opportunities and challenges associated with clinical diagnostic genome sequencing: a report of the Association for Molecular Pathology.

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.

Identification and characterization of novel Salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission.

The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB); this plasmid type was found in four genomes representing different Salmonella serovars and contained a virulence gene array that had not been previously identified. A Salmonella Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3), and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE) encoding a type IVb pilus operon in three non-typhoidal Salmonella serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in Salmonella, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among Salmonella serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits.

Mini-haplotypes as lineage informative SNPs and ancestry inference SNPs.

We propose that haplotyped loci with high heterozygosity can be useful in human identification, especially within families, if recombination is very low among the sites. Three or more SNPs extending over small molecular intervals (<10 KB) can be identified in the human genome to define miniature haplotypes with moderate levels of linkage disequilibrium. Properly selected, these mini-haplotypes (or minihaps) consist of multiple haplotype lineages (alleles) that have evolved from the ancestral human haplotype but show no evidence of recurring recombination, allowing each distinct haplotype to be equated with an allele, all copies of which are essentially identical by descent. Historic recombinants, representing rare events that have drifted to common frequencies over many generations, can be identified in some cases, they do not equate to frequently recurring recombination. We have identified examples in our data collected on various projects and present eight such mini-haplotypes comprised of informative SNPs. We also discuss the ideal characteristics and advantages of minihaps for human familial identification and ancestry inference, and compare them to other types of forensic markers in use and/or that have been proposed. We expect that it is possible to carry out a systematic search and identify a useful panel of mini-haplotypes, with even better properties than the examples presented here.

Expanding data and resources for forensic use of SNPs in individual identification.

The potential value of SNPs for individual identification has been recognized by many researchers and different panels have been proposed. Here we present a new interface in the ALFRED database to access compendia of allele frequencies for several published panels of markers for forensic uses. One of those is our panel of individual identification SNPs (IISNPs) based on samples of 44 populations originating from many parts of the world. Here we also present additional data and additional statistical analyses that continue to support the value of our panel of IISNPs as a universal panel. We also describe initial developments of multiplex methods and various robustness analyses for our 45 marker IISNP panel.

Escherichia coli serotype O55:H7 diversity supports parallel acquisition of bacteriophage at Shiga toxin phage insertion sites during evolution of the O157:H7 lineage.

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models.

Rapid detection of Salmonella in pet food: design and evaluation of integrated methods based on real-time PCR detection.

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead-based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix-associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.

Dominance of multidrug resistant CC271 clones in macrolide-resistant streptococcus pneumoniae in Arizona.

BACKGROUND: Rates of resistance to macrolide antibiotics in Streptococcus pneumoniae are rising around the world due to the spread of mobile genetic elements harboring mef(E) and erm(B) genes and post-vaccine clonal expansion of strains that carry them. RESULTS: Characterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant. The largest portion of the macrolide-resistant population, 52%, is dual mef(E)/erm(B)-positive. All dual-positive isolates are multidrug-resistant clonal lineages of Taiwan19F-14, mostly multilocus sequence type 320, carrying the recently described transposon Tn2010. The remainder of the macrolide resistant S. pneumoniae collection includes 31% mef(E)-positive, and 9% erm(B)-positive strains. CONCLUSIONS: The dual-positive, multidrug-resistant S. pneumoniae clones have likely expanded by switching to non-vaccine serotypes after the heptavalent pneumococcal conjugate vaccine release, and their success limits therapy options. This upsurge could have a considerable clinical impact in Arizona.

A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture.

Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100- to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity. Analysis of 60 blood culture bottles indicated that these latter two methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected 2 hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples. These findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples.

Evaluation of applied biosystems MicroSeq real-time PCR system for detection of Listeria monocytogenes in food. Performance Tested Method 011002.

Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. This report describes the validation of a new Real-Time PCR method to detect Listeria monocytogenes in nine different food matrixes. The complete system consists of the MicroSEQ L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. Two sample preparation methods were validated: the PrepSEQ Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. The test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. The MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. The MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.

A whole-genome single nucleotide polymorphism-based approach to trace and identify outbreaks linked to a common Salmonella enterica subsp. enterica serovar Montevideo pulsed-field gel electrophoresis type.

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods.