Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma.

Dogs across the globe are afflicted by diverse blood- and vector-borne bacteria (VBB), many of which cause severe disease and can be fatal. Diagnosis of VBB infections can be challenging due to the low concentration of bacteria in the blood, the frequent occurrence of coinfections, and the wide range of known, emerging, and potentially novel VBB species encounterable. Therefore, there is a need for diagnostics that address these challenges by being both sensitive and capable of detecting all VBB simultaneously. We detail the first employment of a nanopore-based sequencing methodology conducted on the Oxford Nanopore Technologies (ONT) MinION device to accurately elucidate the "hemobacteriome" from canine blood through sequencing of the full-length 16S rRNA gene. We detected a diverse range of important canine VBB, including Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Bartonella clarridgeiae, "Candidatus Mycoplasma haematoparvum", a novel species of hemotropic mycoplasma, and Wolbachia endosymbionts of filarial worms, indicative of filariasis. Our nanopore-based protocol was equivalent in sensitivity to both quantitative PCR (qPCR) and Illumina sequencing when benchmarked against these methods, achieving high agreement as defined by the kappa statistics (k > 0.81) for three key VBB. Utilizing the ability of the ONT' MinION device to sequence long read lengths provides an excellent alternative diagnostic method by which the hemobacteriome can be accurately characterized to the species level in a way previously unachievable using short reads. We envision our method to be translatable to multiple contexts, such as the detection of VBB in other vertebrate hosts, including humans, while the small size of the MinION device is highly amenable to field use. IMPORTANCE Blood- and vector-borne bacteria (VBB) can cause severe pathology and even be lethal for dogs in many regions across the globe. Accurate characterization of all the bacterial pathogens infecting a canine host is critical, as coinfections are common and emerging and novel pathogens that may go undetected by traditional diagnostics frequently arise. Deep sequencing using devices from Oxford Nanopore Technologies (ONT) provides a solution, as the long read lengths achievable provide species-level taxonomic identification of pathogens that previous short-read technologies could not accomplish. We developed a protocol using ONT' MinION sequencer to accurately detect and classify a wide spectrum of VBB from canine blood at a sensitivity comparable to that of regularly used diagnostics, such as qPCR. This protocol demonstrates great potential for use in biosurveillance and biosecurity operations for the detection of VBB in a range of vertebrate hosts, while the MinION sequencer's portability allows this method to be used easily in the field.

The Healthy Human Blood Microbiome: Fact or Fiction?

The blood that flows perpetually through our veins and arteries performs numerous functions essential to our survival. Besides distributing oxygen, this vast circulatory system facilitates nutrient transport, deters infection and dispenses heat throughout our bodies. Since human blood has traditionally been considered to be an entirely sterile environment, comprising only blood-cells, platelets and plasma, the detection of microbes in blood was consistently interpreted as an indication of infection. However, although a contentious concept, evidence for the existence of a healthy human blood-microbiome is steadily accumulating. While the origins, identities and functions of these unanticipated micro-organisms remain to be elucidated, information on blood-borne microbial phylogeny is gradually increasing. Given recent advances in microbial-hematology, we review current literature concerning the composition and origin of the human blood-microbiome, focusing on bacteria and their role in the configuration of both the diseased and healthy human blood-microbiomes. Specifically, we explore the ways in which dysbiosis in the supposedly innocuous blood-borne bacterial microbiome may stimulate pathogenesis. In addition to exploring the relationship between blood-borne bacteria and the development of complex disorders, we also address the matter of contamination, citing the influence of contaminants on the interpretation of blood-derived microbial datasets and urging the routine analysis of laboratory controls to ascertain the taxonomic and metabolic characteristics of environmentally-derived contaminant-taxa.

Retrospective investigation of 9 years of data on needlestick and sharps injuries: Effect of a hospital infection control committee.

BACKGROUND: The risk of occupational transmission of bloodborne pathogens to health care workers is primarily associated with needlestick and sharps injuries (NSIs). However, most NSIs are not reported, and most health care workers are not aware of postexposure procedures. METHODS: Data for NSIs reported in our hospital between 2008 and 2016 were reviewed retrospectively. RESULTS: A total of 546 staff members reported NSIs. Of these, 376 (68.9%) were women. NSIs were more commonly reported by trainee nurses (243 [44.5%]), followed by nurses (121 [22.2%]), cleaning staff (108 [19.8%]), and doctors (49 [9%]). The rate of postexposure interventions was 13% in 2008 and 92.6% in 2016 (P < .0001; χ2 = 82.866). NSI rates also show that the number of applications with NSIs increased over the years. When occupational blood exposure was examined, the number of bloodborne pathogens was 50 (9.3%) cases of hepatitis B virus, 30 (5.6%) cases of hepatitis C virus, 3 cases of Crimean-Congo hemorrhagic fever, 1 case of HIV, and 2 cases of hepatitis B virus and hepatitis C virus coinfection. DISCUSSION: Over the years, the increase in both the appropriate intervention rate and the number of reports to the hospital infection control committee after NSIs shows that regular training regarding NSIs is effective. CONCLUSIONS: Hospital infection control committees may play a more active role in raising awareness in this regard and thus reducing the rate of unreported NSIs.

Antimicrobial susceptibility among gram-positive and gram-negative blood-borne pathogens collected between 2012-2016 as part of the Tigecycline Evaluation and Surveillance Trial.

Background: Antimicrobial activity of tigecycline and comparator agents was assessedin vitroagainst 27857 isolates source from blood samples collected between 2012 and 2016 as part of the Tigecycline Evaluation and Surveillance Trial (TEST). Methods: The broth microdilution methods was used to determine  minimum inhibitory concentrations (MIC) of blood-borne isolates according to guildlines of the Clinical and Laboratory Standards Institute (CLSI). Antimicrobial susceptibility breakpoints from CLSI guidelines were used as standards to determine susceptibility against comparator agents, whereas tigecycline breakpoints were provided by the US Food and Drug Administration (FDA). Results: More than 91% Enterobacteriaceae isolates, belonging to Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacaeandSerratia marcescens, were susceptible to amikacin, meropenem, and tigecycline. Meropenem resistance was observed in 8% ofK.pneumoniae isolates worldwide. Extended-spectrum ß-lactamase (ESBL) was produced in 15.9 and 20.9%E.coli and K.pneumoniaeisolates, respectively. MIC90 of tigecycline against Acinetobacter baumannii was 2 µg/ml.  The highest proportion of susceptible A.baumanniiisolates was 70.8% for minocycline. Among P.aeruginose  isolates worldwide, 71.1-94.9% were susceptible to six antibiotics. Almost all Staphylococcus aureusisolates were susceptible to linezolid(100%), vancomycin(100%), and tigecycline (99.9%). The proportion of methicillin-resistant S.aureus (MRSA) was 33.0% among S.aureusisolates worldwide; it was highest in Asia with 46.6%, followed by North America and Latin America with 37.7 and 34.2%, respectively. Vancomycin-resistant (VR) isolates represented 1.4% ofEnterococcus faecalis (VR.E.faecalis) and 27.6% of Enterococcus faecium(VR.E.faecium). Highest percentages of VR.E.faeciumwere found in North America and Latin America, with 61.6 and 58.1% of the isolates, respectively. Production of penicillin-resistant Streptococcus pneumoniae(PRSP) represented 9.0% of S. pneumoniae isolates worldwide; the PRSP proportion was 25.8% in Asia, 13.0% in Africa, and 11.8% in Latin America. Conclusions: In our study, tigecycline was the only antibiotic that was active against over 90% of all major blood-borne pathogens. A global comparison revealed that antimicrobial resistance was higher in Africa, Asia and Latin America than in Europe and North America.

Prevalence, genetic diversity and potential clinical impact of blood-borne and enteric protozoan parasites in native mammals from northern Australia.

A molecular survey was conducted to provide baseline information on the prevalence, genetic diversity and potential clinical impacts of blood-borne and enteric protozoans in native wild mammals from the Northern Territory (NT). A total of 209 blood and 167 faecal samples were collected from four target species; the northern brown bandicoot (Isoodon macrourus), common brushtail possum (Trichosurus vulpecula), northern quoll (Dasyurus hallucatus) and brush-tailed rabbit-rat (Conilurus penicillatus). Blood samples were screened by PCR at the 18S rRNA gene for trypanosomes, piroplasms and haemogregarines, with faecal samples tested for Cryptosporidium spp. at the 18S rRNA locus, and for Giardia spp. at the glutamate dehydrogenase (gdh) and 18S rRNA loci. The potential clinical impact was investigated by associating clinical, haematological and biochemical parameters with presence or absence of infection. Overall, 22.5% (95% CI: 17.0-28.8%) of the animals tested were positive for haemoprotozoans. Trypanosomes were found in 26.6% (95% CI: 18.7-35.7%) of the bandicoots and were identified as Trypanosoma vegrandis G6, except for one unique genotype, most similar to T. vegrandis G3 (genetic distance=7%). The prevalence of trypanosomes in possums was 23.7% (95% CI: 11.4-40.2%), and the genotypes identified clustered within the T. noyesi clade. The presence of Babesia sp. and Hepatozoon sp. was confirmed in bandicoots only, both at a prevalence of 9.7% (95% CI: 2.7-9.2%). The total prevalence of intestinal protozoan parasites observed was relatively low (3%; 95% CI: 1.0-6.9%). No evidence of clinical disease associated with protozoan parasitic infection was observed, however bandicoots positive for Trypanosoma exhibited a significantly lower packed cell volume (PCV) compared to negative bandicoots (p=0.046). To the authors' knowledge, this is the first research conducted in the NT to characterise protozoan parasites in threatened native mammals using both molecular and morphological tools; and to assess the potential clinical impacts of these agents. The absence of clear signs of major morbidity in infected animals seems to exclude a direct association between infections with these agents and possible population decline events in northern Australian native mammals. However until the cause(s) of population decline are ascertained for each individual mammal species, further studies are required. The outcome of the present investigation may be used to inform wildlife conservation and zoonotic disease programs.

Actuación ante una lesión percutanea contaminada con sangre / Managemetn of blood-contaminated percutaneous injury

Analizamos en este artículo las normas fundamentales para la prevención de las punciones accidentales por parte de los trabajadores sanitarios, así como los pasos a seguir una vez que se ha producido la punción accidental

In the current paper we have assessed the main guidelines for prevention of accidental puntures by helthcare workers as well s the recommended steps to follow once the punture has occurred

Suspension bead array of the single-stranded multiplex polymerase chain reaction amplicons for enhanced identification and quantification of multiple pathogens.

Rapid identification of single and multiple infectious agents is vital in clinical settings and during biothreat attack. This study assesses the assay of single-stranded multiplex polymerase chain reaction (PCR) amplicons by suspension bead array (SSMP-SBA) for multiple pathogens identification in a single-tube reaction. A 15-plex assay for identification of 11 highly infectious pathogens was developed to evaluate the performance of SSMP-SBA. Pathogen-specific amplicons were obtained by sequential amplification of genomic DNAs using gene-specific primers tagged with artificial unique sequences and unique primers of which the reverse primer was modified by biotin and phosphorothioate. The SSMP products generated by T7 exonuclease-mediated DNA hydrolysis were hybridized to 15 sets of beads coupled with gene-specific and control oligonucleotide probes for pathogen identification and quantification by flow cytometry. This method was validated via assessment of 57 reference strains and one clinical bacterial isolate. All 11 pathogens can be detected by the 15-plex SSMP-SBA assay, and this design significantly enhanced the signal-to-noise ratio and improved the assay performance. This assay achieves similar sensitivity to our in-house real-time PCR system with the limit of detection equivalent to 5-100 genome copies and a linear dynamic range crossing three to five logs. In the validation assay, a 100% accuracy rate was achieved when the pathogens were among the target species. Notably, the species of pathogens were accurately identified from the samples with multiple infections. SSMP-SBA presents superior performance with multiplexing capability in a single-tube reaction and provides a new approach for detection and species identification of multiple pathogen infections.

Identifying and classifying bloodstream infections among hemodialysis patients.

Bloodstream infections (BSIs) are a prominent clinical problem in patients undergoing hemodialysis. These infections appear to be more common among patients who have a central line as their dialysis access and can be associated with substantial morbidity and mortality. Accurately diagnosing BSIs clearly influences patient management, but is also an important part of an infection prevention program; particularly as facility BSI rates are becoming a recognized quality measure for which dialysis facilities might be held accountable. Blood cultures remain the gold standard for diagnosing BSIs and a number of practices can affect the sensitivity and specificity of this important laboratory test. Optimizing the collection of blood cultures can assist providers with interpretation of positive blood cultures and can help minimize the impact of false-positive and false-negative cultures. This review will describe differences between BSI definitions, examine the use of blood cultures to identify these infections including the use of recommended best practices to maximize culture yield, and highlight characteristics that can assist in the clinical interpretation of positive blood cultures.

Managing bloodstream infections in patients who have short-term central venous catheters.

Catheter-related bloodstream infections can be complicated to manage, but a growing body of evidence supports specific recommendations. In 2009, the Infectious Diseases Society of America published updated guidelines for the diagnosis and management of all intravascular catheter-related infections. Here we provide a focused review on the management of bloodstream infections in adult patients with short-term (not surgically implanted and not tunneled) central venous catheters, including peripherally inserted central catheters. This review should serve as a ready reference for providers (eg, hospitalists, surgeons, physician assistants, nurse practitioners, intensivists) managing adult patients with short-term central venous catheters in place.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification.

Until recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for the identification of microorganisms remained confined to research laboratories. In the last 2 years, the availability of relatively simple to use MALDI-TOF MS devices, which can be utilized in clinical microbiology laboratories, has changed the laboratory workflows for the identification of pathogens. Recently, the first prospective studies regarding the performance in routine bacterial identification showed that MALDI-TOF MS is a fast, reliable and cost-effective technique that has the potential to replace and/or complement conventional phenotypic identification for most bacterial strains isolated in clinical microbiology laboratories. For routine bacterial isolates, correct identification by MALDI-TOF MS at the species level was obtained in 84.1-93.6% of instances. In one of these studies, a protein extraction step clearly improved the overall valid identification yield, from 70.3% to 93.2%. This review focuses on the current state of use of MALDI-TOF MS for the identification of routine bacterial isolates and on the main difficulties that may lead to erroneous or doubtful identifications.

Demographic profile of blood donors at three major Brazilian blood centers: results from the International REDS-II study, 2007 to 2008.

BACKGROUND: The profile of blood donors changed dramatically in Brazil over the past 20 years, from remunerated to nonremunerated and then from replacement to community donors. Donor demographic data from three major blood centers establish current donation profiles in Brazil, serving as baseline for future analyses and tracking longitudinal changes in donor characteristics. STUDY DESIGN AND METHODS: Data were extracted from the blood center, compiled in a data warehouse, and analyzed. Population data were obtained from the Brazilian census. RESULTS: During 2007 to 2008, there were 615,379 blood donations from 410,423 donors. A total of 426,142 (69.2%) were from repeat (Rpt) donors and 189,237 (30.8%) were from first-time (FT) donors. Twenty percent of FT donors returned to donate in the period. FT donors were more likely to be younger, and Rpt donors were more likely to be community donors. All were predominantly male. Replacement donors still represent 50% of FT and 30% of Rpt donors. The mean percentage of the potentially general population who were donors was approximately 1.2% for the three centers (0.7, 1.5, and 3.1%). Adjusting for the catchment's area, the first two were 2.1 and 1.6%. CONCLUSIONS: Donors in the three Brazilian centers tended to be younger with a higher proportion of males than in the general population. Donation rates were lower than desirable. There were substantial differences in sex, age, and community/replacement status by center. Studies on the safety, donation frequencies, and motivations of donors are in progress to orient efforts to enhance the availability of blood.

Detection and hazard assessment of pathogenic microorganisms in medical wastes.

This study was undertaken to investigate the types and concentrations of microbial agents in various medical wastes as well as to characterize their survivals in these wastes at different temperatures for microbial risk assessment. Medical wastes collected from 5 major hospitals in South Korea were classified and stored at three different temperatures (-20, 6, and 30 degrees C). Presence of various microorganisms such as pathogenic viruses and bacteria were investigated by both cultivation and by (RT)-PCR assays. A number of (opportunistic) pathogenic bacteria, including Pseudomonas spp., Lactobacillus spp., Staphylococcus spp., Micrococcus spp., Kocuria spp., Brevibacillus spp., Microbacterium oxydans, and Propionibacterium acnes, were identified from the various medical wastes. In addition, pathogenic viruses such as noroviruses and hepatitis B virus were also detected in one of the human tissue wastes. Commonly identified bacterial and viral pathogens such as Pseudomonas spp., Corynebacterium diphtheriae, Escherichia coli, Staphylococcus spp., and respiratory synctial virus (RSV) were inoculated into either gauzes or diapers, and their survivals were characterized. Viral agents such as RSV showed poor survival in most environmental conditions, and demonstrated that various pathogens could be present in medical wastes but that the associated health risk appeared to be low. However, medical waste should be carefully controlled and monitored to prevent nosocomial infection associated with the exposure to these wastes.

Evidence-based semiquantitative methodology for prioritization of foodborne zoonoses.

OBJECTIVES: To prioritize an extended list of food- and water-borne zoonoses to allow food safety authorities to focus on the most relevant hazards in the food chain. METHODS: An evidence-based semiquantitative methodology was developed. Scores were given by 35 scientific experts in the field of animal and public health, food, and clinical microbiology and epidemiology to 51 zoonotic agents according to five criteria related to public health (severity and occurrence in humans), animal health (severity of disease coupled with economic consequences and occurrence in animals), and food (occurrence in food). The scoring procedure was standardized and evidence-based as experts were provided, for each zoonotic agent, a same set of up-to-date help information data related to the five criteria. Independently, the relative importance of the five criteria was weighted by seven food chain risk managers. The zoonotic agents were ranked based on overall weighted scores and were grouped in four statistically different levels of importance. RESULTS: The following foodborne zoonotic pathogens were classified as "most important": Salmonella spp., Campylobacter spp., Listeria monocytogenes, and verocytotoxigenic Escherichia coli. A second group of "significant importance" included Toxoplasma gondii, the agent of bovine spongiform encephalopathy, Clostridium botulinum, Staphylococcus aureus, Cryptosporidium parvum, Mycobacterium bovis, Echinococcus granulosus, Streptococcus spp., Echinococcus multilocularis, Yersinia enterocolitica, Mycobacterium avium, Fasciola hepatica, Giardia intestinalis, and Rotavirus. CONCLUSIONS: This methodology allowed to rank 51 zoonotic agents with objectivity and taking account of a combined input from risk assessors and risk managers. APPLICATIONS: These results support food safety policy makers to establish the multiannual monitoring program of foodborne zoonoses. They also enable to identify knowledge gaps on specific zoonotic agents and to formulate key research questions. Principally, this method of prioritization is of general interest as it can be applied for any other ranking exercise and in any country.

[Needlestick injuries]. / Krankenhaushygiene--Nadelstichverletzungen.

Needlestick injuries (NSI) occur very frequently in the health-care field and represent a very serious risk for health-care workers. It is estimated that 500,000 NSI occur per year in Germany. They are the most frequent cause of infections with blood-borne pathogens. Thus prevention is of decisive importance. A simple reporting procedure and low-threshold care system after NSI would certainly help to reduce the risks and the number of undetected cases.

Multiplexed identification of blood-borne bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay.

We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity.

Infection risks following accidental exposure to blood or body fluids in health care workers: a review of pathogens transmitted in published cases.

Hospital staff and all other human or veterinary health care workers, including laboratory, research, emergency service, or cleaning personnel are exposed to the risk of occupational infection following accidental exposure to blood or body fluids (BBF) contaminated with a virus, a bacteria, a parasite, or a yeast. The human immunodeficiency virus (HIV) or those of hepatitis B (HBV) or C (HCV) account for most of this risk in France and worldwide. Many other pathogens, however, have been responsible for occupational infections in health care workers following exposure to BBF, some with unfavorable prognosis. In developed countries, a growing number of workers are referred to clinicians responsible for the evaluation of occupational infection risks following accidental exposure. Although their principal task remains the evaluation of the risks of HIV, HBV, or HCV transmission and the possible usefulness of postexposure prophylaxis, these experts are also responsible for evaluating risks of occupational infection with other emergent or more rare pathogens and their possible timely prevention. The determinants of the risks of infection and the characteristics of described cases are discussed in this article.

Seroprevalence of human immunodeficiency virus, hepatitis B and C viruses and syphilis infections among blood donors at the Muhimbili National Hospital in Dar es Salaam, Tanzania.

BACKGROUND: According to the latest Tanzanian National AIDS Control Programme (NACP) report a total of 147,271 individuals donated blood during the year 2002. However, blood safety remains an issue of major concern in transfusion medicine in Tanzania where national blood transfusion services and policies, appropriate infrastructure, trained personnel and financial resources are inadequate. Most of the donated blood is screened for HIV alone. METHODS: We determined among blood donors at Muhimbili National Hospital (MNH), the seroprevalence of human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg) and syphilis by donor type, sex and age and to determine association, if any, in the occurrence of the pathogens. The sample included 1599 consecutive donors, 1424(89.1%) males and 175 (10.9%) females, who donated blood between April 2004 and May, 2005. Most of them 1125 (70.4%) were replacement donors and a few 474 (29.6%) voluntary donors. Their age (in years) ranged from 16 to 69, and most (72.2%) were between 20-39 years. RESULTS: Two hundred and fifty four (15.9%) of the donated blood had serological evidence of infection with at least one pathogen and 28 (1.8%) had multiple infections. The current seroprevalence of HIV, HBsAg, HCV and syphilis among blood donors at MNH in Dar es Salaam was found to be 3.8%, 8.8%, 1.5% and 4.7%, respectively. Respective seroprevalences among HIV seronegative blood donors were 8.7% for HBV, 1.6% for HCV and 4.6% for syphilis. The differences in the prevalence of HIV and syphilis infections between replacement and voluntary donors were statistically significant (P < 0.05). Syphilis was the only infection that occurred more frequently among HIV infected (12.1%) than non-infected (4.6%) blood donors (P < 0.05), and whose prevalence increased with age (X2 = 58.5 df = 5, P < 0.001). There were no significant sex differences in the occurrence of pathogens. Finally, there were significant associations in the occurrence of HBsAg and syphilis (OR = 2.2, 95% CI 1.1.-4.2) and HIV and syphilis (OR = 2.2, 95% CI 1.0-5.3). CONCLUSION: The high (15.9%) seroprevalence of blood-borne infections in blood donated at MNH calls for routine screening of blood donors for HBV, HCV, HIV and syphilis and for strict selection criteria of donors, with emphasis on getting young voluntary donors and for establishment of strict guidelines for blood transfusions.