Helicobacter cinaedi is a relatively recently identified bacterium, but it is recognized as an increasingly important cause of disease in humans. This article summarizes methods for its detection and identification as well as routes of infection and treatment.
by Prof. Yoshiaki Kawamura, Dr Tatsuya Okamoto, Dr Shigemoto Fujii and Prof. Takaaki Akaike
What is Helicobacter cinaedi?
Within the genus Helicobacter, 33 species to date have been proposed and validated, but only 7 species have been isolated from human clinical specimens (Table 1). H. pylori, classified as a ‘gastric Helicobacter species’, is a well-known member of Helicobacter, but some less well-known ‘enterohepatic Helicobacter species’, such as H. cinaedi, H. bilis, H. canadensis, H. canis, H. fennelliae, and H. pullorum, have also been isolated from human clinical specimens.
H. cinaedi was first isolated as a Campylobacter-like organism type 1 (CLO-1) in 1984 from rectal swabs from homosexual men displaying intestinal symptoms [1] and the following year the organism was named ‘Campylobacter cinaedi’; however, it was subsequently reclassified as Helicobacter [2]. During the last two decades, there have been many reports of the isolation of H. cinaedi from blood or intestinal tract of human immunodeficiency virus-infected or immunocompromised patients, but, recently, increasing numbers of infections have also been reported in immunocompetent patients.
In Japan, the isolation of H. cinaedi was first reported in 2003; since then, its isolation has been reported from patients regardless of gender and within a wide age range, from the newborn to the elderly, in many hospitals throughout the country. We have experienced many cases of H. cinaedi cellulitis and bacteremia in both immunocompromised and immunocompetent subjects in hospitals. This microorganism should, therefore, be considered a causative agent of nosocomial infection [3].
Illnesses caused by H. cinaedi
Clinical symptoms of H. cinaedi infection include fever, diarrhoea, abdominal pain, gastroenteritis, proctitis, cellulitis, erysipelas, arthritis, meningitis, and bacteremia. In contrast to other Helicobacter species, numerous reports have causally linked H. cinaedi infection with bacteremia, which contributes to this organism’s strong vascular invasion ability. In many cases of H. cinaedi bacteremia, the main symptom is fever accompanied by arthritis and cellulitis at various sites. In addition to these sites providing a source of primary infection, the resultant bacteremia can serve as a source of secondary infections; thus, all these various symptoms are clinically important.
In our experience, at various times after orthopedic surgery (range, 8–113 days; mean, 29 days), some patients had a sudden onset of local flat cellulitis (salmon-pink in colour) at different sites on the operated side, along with fever and an increase in C-reactive protein level (Fig. 1) [3]. Cellulitis was often multifocal without wound infection. Many of these patients had been treated for fracture and were immunocompetent.
In recent years, we have demonstrated the potential association of H. cinaedi with atrial arrhythmias and atherosclerosis [4]. This could be through bacterial translocation of H. cinaedi from the intestinal tract into the blood stream. The possible cause-and-effect relationship between H. cinaedi and vascular diseases may warrant further epidemiological study on proatherosclerotic effect of H. cinaedi infection.
The virulence factor of H. cinaedi is largely unknown. The complete genome sequence of a human clinical isolate was announced in 2012 [5] and revealed that the organism holds a Type VI secretion system, which is expected to be related to its virulence, together with two known virulence factors, cytolethal distending toxin and alkyl hydroperoxide reductase.
Detection, cultivation and identification of H. cinaedi
It is well known that H. cinaedi is a fastidious and slow-growing organism, and that detection and cultivation are extremely difficult. In many cases, H. cinaedi is first detected from a blood culture using an automatic blood culture system. It is generally noted that 4–10 days (average 5.6 days, in our experience) are needed for a positive result in the culture bottle of an automatic blood culture system, such as the BACTEC system (Becton Dickinson) using an aerobic bottle. Therefore, when the culture test using this system is terminated within 3–4 days, the bacterial growth may be still below its detection limit. Information on the detection of H. cinaedi using the BacT/ALERT system (Biomérieux) is scanty. In our experience, the VersaTREK system (Thermo Scientific) is superior for the detection of this microorganism.
Both H. cinaedi and H. pylori are members of the genus Helicobacter; however, the former is extremely difficult to culture. H. cinaedi isolates essentially require microaerobic conditions (5–10% O2) and a high level of humidity. Often a blood agar plate stored in a refrigerator for a few days may fail to support the growth of H. cinaedi because of low water content. Use of fresh medium is strongly recommended. It is established that H. cinaedi growth is accelerated by adding hydrogen gas (5–10%) to microaerobic conditions. The culture success rate can be improved by using a gas mixture such as 6% O2, 7% H2, 7% CO2, and 80% N2 at the initial culture from the clinical specimen or in the culture bottle. Unfortunately, many of the commercially available microaerobic gas-generating packs, such as the GasPak system (Becton Dickinson), deoxidize and generate CO2 but do not generate hydrogen gas; therefore, in some cases H. cinaedi does not grow, or growth is inadequate.
H. cinaedi cultured on an agar plate grows in a film, which is difficult to identify visually. Therefore, the culture should be carefully checked on the plate.
The biochemical identification of this organism is problematic owing to unstable phenotypic reactions. In many cases commercially available identification kits do not produce reliable results. Therefore, identification based on nucleotide sequence or species-specific polymerase chain reaction (PCR) has been used. We have developed a nested PCR system with high specificity and sensitivity (approximately 102 CFU/ml) for detecting H. cinaedi [6]. We have also established an immunological diagnosis method (antibody detecting test) with high specificity to detect the exposure history of H. cinaedi [7].
Antimicrobial therapy and prognosis
To date, antimicrobial susceptibility testing for H. cinaedi has mainly used the agar-dilution method, but this method is too cumbersome for routine use in hospital laboratories. A broth microdilution method for antimicrobial susceptibility testing of H. cinaedi, which can be performed easily, has been developed by our research group [8].
H. cinaedi strains generally show low minimum inhibitory concentration (MIC) values for carbapenems, aminoglycosides, and tetracycline (MIC90 = 1 µg/ml for imipenem/cilastatin, gentamicin, and tetracycline). In contrast, H. cinaedi has well-known resistance to macrolides, with especially high MIC values (MIC90 >64 µg/ml for erythromycin). Recently in Japan and elsewhere, H. cinaedi isolates have shown high resistance to quinolones (MIC90 = 64 µg/ml for ciprofloxacin and levofloxacin) due to point mutation(s) of DNA gyrase genes [8].
Symptoms caused by H. cinaedi, such as fever or cellulitis, usually resolve after 2 to 3 days of drug therapy, but the Centers for Disease Control and Prevention recommended long-term therapy for about 2 to 6 weeks, rather than short-term therapy for only 10 days [9]. Prognosis is generally good, but it is noteworthy that, depending on the study, about 30–60% of patients have recurrent symptoms. Unfortunately, there are no guidelines for the treatment of H. cinaedi infections, including the clinical breakpoints of antimicrobial agents. The MIC values described above are based on our data.
Infection route
H. cinaedi has been found in a wide range of animals including cats, dogs, hamsters, rats, and foxes. There have been many reports of zoonotic transmission vectors, but no reports of the simultaneous isolation of H. cinaedi from human patients and the animals that they have been in close contact with. It is noteworthy that H. cinaedi isolates from human, dog, and hamster formed a distinct ribotype pattern group by host source [10].
Epidemiological analysis methods, such as pulse-field gel electrophoresis, randomly amplified polymorphic DNA, and multilocus sequence typing, have been proposed for H. cinaedi isolates [3, 11]. As described above, we developed a nested PCR system and immunological diagnosis method. Using these methods, we tested many healthy hospital employees (doctors, nurses, staff members, etc.) and found that some currently uninfected individuals had previously had H. cinaedi infections, indicating that there could be asymptomatic carriers with intestinal colonization of H. cinaedi. Our study also suggested that occurrence of such asymptomatic carriers may be related to nosocomial infection.
However, the complete route of infection route, including nosocomial transmission, of H. cinaedi remains unclear.
Summary
A full understanding of H. cinaedi infection remains elusive; however, some features and the clinical relevance of this infection have become increasingly recognized recently. To detect and isolate H. cinaedi from human blood samples using an automated blood culture system, a long-term incubation (up to 10 days) is needed and further skillful culture techniques are required. In many clinical laboratories, however, appropriate culture for isolation of this bacteria might not performed, which may lead to false-negative findings for H. cinaedi. As H. cinaedi was considered not to cause acute severe disease, it seems that its importance may have not been recognized clinically. However, we now know that this microorganism likely causes nosocomial infections that are difficult to eradicate and have a high incidence of recurrence. In recent years, a possible association with chronic illnesses such as arrhythmia and arteriosclerosis has been reported, and therefore we will need to carefully monitor and ascertain trends in H. cinaedi infections.
References
1. Fennell CL, et al. E. Characterization of Campylobacter-like organisms isolated from homosexual men. J Infect Dis. 1984; 149: 58–66.
2. Vandamme P, et al. Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. Int J Syst Bacteriol. 1991; 41: 88–103.
3. Kitamura T, et al. Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery. J Clin Microbiol. 2007; 45: 31–38.
4. Khan S, et al. Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses. Sci Reports 2014; In press.
5. Goto T, et al. Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia. J Bacteriol. 2012; 194: 3744–3745.
6. Oyama K, et al. Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR. J Clin Microbiol. 2012; 50: 3893–3900.
7. Iwashita H, et al. Identification of the major antigenic protein of Helicobacter cinaedi and its immunogenicity in humans with H. cinaedi infections. Clin Vaccine Immunol. 2008; 15: 513–521.
8. Tomida J, et al. Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi. Microbiol Immunol. 2013; 57: 353–358.
9. Kiehlbauch JA, et al. Helicobacter cinaedi-associated bacteremia and cellulitis in immunocompromised patients. Ann Intern Med. 1994; 121: 90–93.
10. Kiehlbauch JA, et al. Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals. J Clin Microbiol. 1995; 33: 2940–2947.
11. Rimbara E, et al. Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from seven hospitals in Japan. J Clin Microbiol. 2012; 50: 2553–2560.
12. Solnick JV, Schauer DB. Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases Clin Microbiol Rev. 2001; 14: 59–97.
The authors
Yoshiaki Kawamura1 PhD; Tatsuya Okamoto2 MD, PhD; Shigemoto Fujii3 PhD; Takaaki Akaike3* MD, PhD
1Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Nagoya, Japan
2Intensive Care Unit, National Center for Global Health and Medicine, Tokyo, Japan
3Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Japan
*Corresponding author
E-mail: takaike@med.tohoku.ac.jp
The use of liquid chromatography-tandem mass spectrometry for clinical analysis is on the increase. This article describes what it is, why it can offer significant improvements over traditional assays and the limitations to be aware of.
by Dr N. Homer
Introduction
Clinical biochemistry laboratories frequently use radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) for analysis of blood and urine. However, these techniques are plagued by issues of cross-reactivity and are only suited to look at one analyte at a time [1]. The use of mass spectrometry (MS) techniques has increased since 2007 when the American Endocrine Society recognized the importance of tandem MS and issued a statement recommending the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of endogenous steroid hormones over more traditional technologies such as immunoassays [2]. This has led to the widespread adoption of LC-MS/MS in clinical biochemistry laboratories, in direct response to this recommendation.
What is liquid chromatography-mass spectrometry?
Mass spectrometry is a technique that measures charged molecules or ions in the gaseous state. Samples are introduced into an ion source, ionized and then separated in a mass analyser according to their mass-to-charge ratio (m/z) and then characterized by their relative abundances. Coupled to chromatographic separation techniques such as gas chromatography (GC) or liquid chromatography (LC), MS is considered to be the ‘gold standard’ for validation of quantitative analytical assays. An overview of how a typical chromatograph-mass spectrometer is set up is shown in Figure 1.
Following separation by a chromatography system the sample is introduced into an ion source at the front end of the mass spectrometer. Ionization modes include atmospheric pressure ionization (API), such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption/ionization (MALDI). ESI is most typically used to ionize the biomolecules encountered in clinical samples.
Once ionized the mass analyser separates the ions according to their m/z. Mass analysers include magnetic or electric sectors, time-of-flight (ToF) tubes, quadrupoles and two-dimensional and three-dimensional ion traps. Softer ionization techniques, which generally leave the molecule intact, such as ESI, have led to the use of quadrupole mass analysers. These consist of four parallel rods or poles, generally of hyperbolic cross-section, through which ions are passed and separated.
Tandem mass spectrometry (often termed MS/MS) technology increases the specificity of MS significantly. There are a number of modes that a tandem mass spectrometer can be operated under, depending on the requirement of the experiment (Fig. 2). Tandem MS requires two or more mass analysers to be placed in sequence and the ions are fragmented in a collision cell to give structural information. Trace analysis of complex biological matrices is ideally suited to tandem MS instruments, operated in selected reaction monitoring (SRM) mode. In addition, linear ion traps as the third mass analyser are also increasing in popularity as they offer additional structural identification and specificity.
Sample preparation and liquid chromatography method development
Clinical samples are complex biological matrices and contain interferences that can lead to so-called matrix effects within the mass spectrometer. For validated assays, samples are prepared by addition of an internal standard followed by extraction to remove as much of the interferences as possible. The internal standard is either a closely related analogue of the compound of interest, or a stable isotope labelled version of the compound, enriched with at least two atoms of 13C, 2H or 15N.
Sample preparation methods commonly applied to clinical samples include protein precipitation with an organic solvent, liquid–liquid extraction (LLE) or solid-phase extraction (SPE). If sample clean-up is not sufficient it can lead to matrix effects, including ion suppression of the analyte, usually observed as a loss of response. This affects the detection limits, accuracy and precision of the assay. Various ion suppression tests have been developed and these are an important part of the method validation set-up required for clinical MS assays. The two most effective ways of avoiding ion suppression are improved sample extraction and optimized chromatographic selectivity.
On-line multidimensional chromatography technology allows an unextracted sample to be introduced into the chromatography apparatus and can lead to faster analysis. These systems generally consist of multi-channel switching valves, on-line SPE cartridges and analytical columns ahead of ESI-LC-MS/MS. Steroids and isoprostanes are often analysed in this manner [3, 4].
Liquid chromatography
Once prepared, a sample is introduced into the LC system which consists of a pump and an analytical column. The purpose of the chromatography system is to separate the components of the sample as much as possible, before introduction into the mass spectrometer. Analytical LC columns are stainless steel tubes that are packed with tiny silica beads. The type of LC used in clinical analysis is usually reversed-phase chromatography as the silica beads are generally chemically modified. Typically, samples are introduced onto the column in a highly aqueous phase, the analytes associate with the chemically-modified packed silica beads and are washed off the column with a high organic solvent such as methanol or acetonitrile.
Once the ionization and mass spectrometer parameters have been optimized, much of the method development falls to the chromatography and the importance of this stage should not be underestimated. It is imperative that co-eluting compounds do not interfere with the analytical peaks of interest. In recent years there has been a trend for fast analysis in LC-MS/MS; however, this does not always give a robust assay. In addition, it is important to be aware of isobaric compounds (same mass) and [M+2] isotopomers. An example of isotopomers is that of the stress hormone cortisol (m/z 363) and its inactive form cortisone (m/z 361). In an LC-MS/MS assay for cortisol it is essential to have two separate peaks for cortisol and cortisone, otherwise the risk of isotopomers of cortisone contributing to cortisol would lead to an over-estimation of cortisol in the sample (Fig. 3).
There are a number of parameters that can be altered in LC and these in turn alter the selectivity of the column, that is the order and rate at which the components elute. Parameters that can be influenced include column temperature, mobile phase pH, composition and flow rate, column dimensions, column particle size and the nature of course the chemical modification of the particles too.
Analytical LC column technology is continuously improving. The better the resolution, which is simply how well separated each peak is, the better the assay. Sub-2 µm particles have been introduced in the past decade, which generate sharp peaks and excellent resolution with improved capacity over the more traditional 3–5 µm particles. However, the smaller particle size leads to high backpressure and requires specific LC pumps that can withstand these ultra-high pressures (UHPLC, ultra-high performance LC). To reduce the need for new instrumentation, LC columns packed with fused-core particles ~2.5 µm have been developed to allow separation comparable to sub-2 µm particles. The backpressure generated by these fused-core particles is significantly less than the sub-2 µm particle columns and exclude the requirement of high-pressure capable LC pumps and fittings.
Considerations when establishing an LC-MS/MS clinical biochemistry method
As with all techniques, there are drawbacks to LC-MS/MS. The instrumentation and software can be complex and requires regular maintenance, although manufacturers are addressing this perception by introducing simpler software interfaces with dedicated instrument support for method development and even fool-proof methods, guaranteed by the provider. Also, some compounds are not amenable to ionization due to their chemical nature, but chemical modification before analysis can improve the chance of ionization efficiency, so all is not lost.
Summary
The benefits that MS offers over other traditional assay techniques have seen an increase in the number of assays using this methodology. The analysis of steroid hormones by MS is a well-documented area. Other commonly encountered uses include newborn screening for congenital metabolic diseases such as aminoacidopathies and fatty acid oxidation disorders, multi-analyte therapeutic drug monitoring, oncology drugs, anti-virals, toxicants and drugs of abuse screening and analysis of endogenous peptides [3, 4, 5].
One area that is continuing to gain interest in clinical research is high-resolution MS (HRMS) [5]. This allows for accurate mass determination over a defined mass range, which differs from the targeted analysis approach used by triple quadrupole MS. With technological improvement in the linear range of HRMS instruments to match that of triple quadrupoles, it seems likely that the benefits of HRMS will also be exploited by the clinical biochemistry field, in addition to LC-MS/MS analysis.
The range of clinical applications of MS outlined is broad and constantly expanding. Much research is being conducted in the pioneering fields of proteomics and metabolomics. In recent years the emergence of imaging mass spectrometry also offers exciting possibilities for the future and there is no doubt that MS will continue to feature heavily in the clinical biochemistry laboratory and function as an important clinical research tool.
References
1. Penning TM, et al. Liquid chromatography-mass spectrometry (LC-MS) of steroid hormone metabolites and its applications. J Ster Biochem Mol Biol. 2010; 121: 546–555.
2. Rosner W, et al. Position statement: utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab. 2007; 92: 405–413.
3. Shushan B. A review of clinical diagnostic applications of liquid chromatography-tandem mass spectrometry. Mass Spectrom Rev. 2010; 29: 930–944.
4. Chace DH, et al. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clin Chem. 2003; 49: 1797–1817.
5. Jiwan J-LH, et al. HPLC-high resolution mass spectrometry in clinical laboratory? Clin Biochem. 2011; 44: 136–147.
The author
Natalie Homer PhD
CRF Mass Spectrometry Core Laboratory, Queen’s Medical Research Institute, University of Edinburgh
E-mail: n.z.m.homer@ed.ac.uk
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