Extraction of nucleic acids from patient samples is an essential step for downstream molecular studies such as quantitative and qualitative PCR. The size of the DNA fragments present in samples can influence extraction efficiency, especially observed in circulating cell-free DNA (cfDNA). Further work is necessary to determine the impact of cfDNA extraction on clinical virology and microbiology testing.
by Dr Kimberly Starr and Dr Linda Cook
Introduction
After sample collection, the next important step in the detection of infectious agents in most patient-derived samples is the extraction of DNA or RNA to remove proteins, lipids, other cellular components, and PCR inhibitors to create a ‘PCR-friendly’ eluate solution. First, the sample is mixed with a lysis buffer and then DNA is purified from the resulting solution by silica-coated filtration membranes or magnetic beads that bind nucleic acid and allow subsequent washing and elution steps to be performed. Extraction methods can range from small-scale manual methods to large-scale fully-automated extraction instruments. For implementation of automated platforms several factors require consideration, including capacity, target range, efficiency, cost, physical footprint, level of automation, and processing time. The variety of instrumentation and extraction methods available contribute to the differences in extraction efficiency that may have downstream consequences when quantifying DNA or RNA in bacteria, fungi, parasites, and viruses. The performance of different kits even on the same instrument can further contribute to variation in efficiency [1]. Inter-laboratory variation as a result of extraction efficiency can affect patient care and reproducibility of testing results, especially for patients who are monitored over a long period with a quantitative test.
Extraction method comparisons
In a study comparing the bacterial DNA quantity and quality extracted from stool, Claassen et al. found DNA yield and purity varied between five commonly used extraction kits [2]. This is the case for fungi as well where extraction of nucleic acid from Aspergillus fumigatus is the main limiting factor for successful Aspergillus PCR from clinical specimens. Perry et al. found differences in reproducibility of DNA extraction at low levels (101 cells/mL) in EDTA whole blood among the four extraction instruments they tested [3]. The same can be seen in parasitic infections, demonstrated by Yera et al., which showed that DNA extraction procedures led to variations in detecting low concentrations of Toxoplasma gondii tachyzoites in amniotic fluid samples, a difference that could affect early diagnosis of congenital toxoplasmosis [4].
Other studies have evaluated extraction systems for human immunodeficiency virus (HIV) [5–8], hepatitis B virus (HBV) [9, 10], Cytomegalovirus (CMV) [11], enterovirus [12], norovirus [13], and HSV [14]. Essentially all published extraction comparison studies have seen quantitative differences in results across the different systems evaluated, sometimes with quantitative differences significantly more than 1 log.
Cell-free DNA measurements
Another level of complexity is added when the size of the nucleic acid to be isolated varies. It is known that nucleic acids fragment during the extraction process, but recent studies have demonstrated that nucleic acids may be a variety of sizes in the initial sample, especially in blood. Cell-free circulating DNA (cfDNA) in blood coming from cellular breakdown was first described by Mandel and Metais in 1948 [15]. The size of cfDNA fragments described is approximately 167 bp, equivalent to the size of chromatosome DNA and similar to post-apoptosis DNA fragments. In the last 20 years, there has been increased interest in measuring and quantifying cfDNA in a variety of cancers. Key observations from these studies are: (1) The concentration in plasma/serum is very low, 10–100 ng/mL. Thus, many studies have focused on identifying extraction methods to maximize cfDNA yield. (2) Sample collection tubes with cell-stabilizing reagents to prevent contamination of plasma with cellular DNA can increase the purity and yield of cfDNA. (3) Use of generic DNA extraction methods can cause further fragmentation of cfDNA and decrease yields compared to cfDNA-specific extraction methods. Recently, extraction instrument manufacturers have introduced cfDNA isolation kits and instruments. These kits utilize higher input volumes of 1.0–5.0 mL, and optimized temperatures or buffer conditions to improve yields. cfDNA kits from several manufacturers have been shown to have better performance in several studies. Four excellent reviews describing the technical aspects of cfDNA extraction and comparison of cfDNA extraction methods have been published [16–19].
Our DNA fragment extraction study
To better understand how DNA fragment size may impact viral infectious disease test results, we designed a study [20] comparing extraction yields for differently sized DNA fragments across 11 commercially available extraction methods commonly used in clinical laboratories, and also compared the performance of four new cfDNA extraction methods. Artificially constructed DNA fragments with sizes ranging from 50 to 1,500 bp were extracted and tested by droplet digital PCR to determine the DNA fragment yield across methods. We found a wide range of extraction yields across both extraction methods and instruments, with the 50 and 100 bp fragment sizes showing especially inconsistent quantitative results and poor yields of less than 20%. Figure 1 shows the yield results of two representative methods and one cfDNA method. Two of the methods designed to extract cfDNA gave the highest yields for the 50 and 100 bp fragments but overall yields were poor. We also observed the lowest variability across methods for the larger sized fragments at higher concentrations. Overall, we saw the most variability for the smallest sized fragments and observed variability dependent on concentration.
Results from our study demonstrate significant differences in fragment extraction yields and overall poor yields of the small artificial DNA fragments even at high concentrations in essentially all routinely used methods. Two of the four cfDNA methods showed improved (although still low) yield of smaller fragments. Further studies are necessary to determine the cause of this significant difference in yields. We speculate as the field moves toward more next generation sequencing approaches, these differences in extraction efficiency and quantification of small cfDNAs will become more widely described.
A critical next step is to determine if viral cfDNA exists in patients with a variety of infectious diseases and if their measurement has clinical relevance. Further studies should focus on identifying which viruses or other infectious agents have cfDNA and then methods to extract and evaluate this cfDNA must be significantly improved. To date, only cfDNA associated with Epstein-Barr virus (EBV) has been extensively studied and hints of cfDNA importance in CMV disease have been seen.
cfDNA in EBV
As early as 2003, Chan et al. described the differential detection of EBV by PCR depending on the size of the PCR amplicon, demonstrating that an assay with an 82 bp amplicon detected 7.5 times more EBV in plasma that a 181 bp amplicon assay [21]. Many additional studies in nasopharyngeal carcinoma have confirmed the excellent utility of measuring the quantity of this small EBV-associated cfDNA for monitoring of therapy response, prediction of recurrence, and monitoring at-risk populations.
Two recent large studies have shown that plasma levels of EBV are the most useful sample type for testing EBV infected patients [22, 23] but cfDNA was not specifically identified in these studies. A study by Lit et al. in EBV-associated lymphoma patients demonstrated EBV cfDNA [24] and noted that the subset of patients with ‘active’ disease had a relative predominance of cfDNA compared to predominantly larger cell-associated EBV DNA seen in cases of inactive disease or remission. Thus, measurement of both EBV cfDNA as well as larger EBV DNA fragments may be important in clinical testing and it may be necessary to distinguish the size of EBV in the plasma. Further studies are necessary to determine how useful detection of cfDNA may be in all EBV-associated malignancies and infections.
cfDNA in CMV
Published data hints that fragmented DNA may also be important for CMV PCR quantitation. In one study, Boom et al. fractionated CMV DNA in plasma and whole blood from three renal transplant cases with primary CMV infection and measured the quantities present with two PCR amplicons sized 578 bp and 134 bp [25]. They demonstrated that CMV DNA was predominantly less than 2000 bp and detected many small sized fragments only with the 134 bp amplicon PCR. Habbal et al. also studied 17 different CMV primer sets and demonstrated that the two of the four primer sets with the smallest amplicons (<100 bp) were the most sensitive for detection of cultured CMV strains [26]. Tong et al., found that among 20 solid organ transplant recipients, 10 had exclusively free CMV DNA, while the remaining 10 had predominantly free CMV DNA with a small percentage of encapsulated-virion DNA present [27]. In addition, they compared results for two assays with small amplicon sizes of 81 and 138 bp and found a 2.6-fold higher level with the smaller amplicon, suggesting CMV DNA present in these clinical samples was very small (<138 bp). It appears critical to use a high-yield small CMV DNA fragment extraction method as well as a small CMV PCR amplicon assay to maximize CMV detection of CMV. Incorporating these two elements into clinical CMV PCR assays could decrease assay variability and decrease inter-lab variability.
cfDNA in other viruses
There is evidence that cfDNA may be useful in infections and malignancies associated with viruses other than EBV and CMV. A recent study by Chesnais et al. mimicked detection of genetic mutations in pre-term children by using CCF from maternal plasma and demonstrated the potential of this technology to detect multiple viruses present in low levels in mothers or pre-term babies [28]. In addition, case reports for Kaposi’s sarcoma and BKPyV-associated bladder cancer (virus-associated cancers) suggest utility of quantitative measurements of cfDNA containing HHV8 (human herpes virus 8, also known as Kaposi’s sarcoma-associated herpesvirus) or BK virus, respectively, in tumor detection and therapeutic monitoring. Further studies are necessary in these two diseases as well as other infectious diseases to evaluate the clinical utility of cfDNA measurements.
References
1. McCulloch E, Ramage G, Jones B, Warn P, Kirkpatrick WR, Patterson TF, et al. Don’t throw your blood clots away: use of blood clot may improve sensitivity of PCR diagnosis in invasive aspergillosis. J Clin Pathol 2009; 62(6): 539–541.
2. Claassen S, du Toit E, Kaba M, Moodley C, Zar HJ, Nicol MP. A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. J Microbiol Methods 2013; 94(2): 103–110.
3. Perry MD, White PL, Barnes RA. Comparison of four automated nucleic acid extraction platforms for the recovery of DNA from Aspergillus fumigatus. J Med Microbiol 2014; 63(Pt 9): 1160–1166.
4. Yera H, Filisetti D, Bastien P, Ancelle T, Thulliez P, Delhaes L. Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid. J Clin Microbiol 2009; 47(12): 3881–3886.
5. Cornelissen M, Gall A, Vink M, Zorgdrager F, Binter S, Edwards S, et al. From clinical sample to complete genome: comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing. Virus Res 2017; 239: 10–16.
6. Alp A, Hascelik G. Comparison of 3 nucleic acid isolation methods for the quantification of HIV-1 RNA by Cobas Taqman real-time polymerase chain reaction system. Diagn Microbiol Infect Dis 2009; 63(4): 365–371.
7. Stevens W, Horsfield P, Scott LE. Evaluation of the performance of the automated NucliSENS easyMAG and EasyQ systems versus the Roche AmpliPrep-AMPLICOR combination for high-throughput monitoring of human immunodeficiency virus load. J Clin Microbiol 2007; 45(4): 1244–1249.
8. Swanson P, Holzmayer V, Huang S, Hay P, Adebiyi A, Rice P, et al. Performance of the automated Abbott RealTime HIV-1 assay on a genetically diverse panel of specimens from London: comparison to VERSANT HIV-1 RNA 3.0, AMPLICOR HIV-1 MONITOR v1.5, and LCx HIV RNA Quantitative assays. J Virol Methods 2006; 137(2): 184–192.
9. Kang SH, Lee EH, Park G, Jang SJ, Moon DS. Comparison of MagNA Pure 96, Chemagic MSM1, and QIAamp MinElute for hepatitis B virus nucleic acid extraction. Ann Clin Lab Sci 2012; 42(4): 370–374.
10. Pyne MT, Vest L, Clement J, Lee J, Rosvall JR, Luk K, et al. Comparison of three Roche hepatitis B virus viral load assay formats. J Clin Microbiol 2012; 50(7): 2337–2342.
11. Bravo D, Clari MA, Costa E, Munoz-Cobo B, Solano C, Jose Remigia M, et al. Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients. J Clin Microbiol 2011; 49(8): 2899–2904.
12. Shulman LM, Hindiyeh M, Muhsen K, Cohen D, Mendelson E, Sofer D. Evaluation of four different systems for extraction of RNA from stool suspensions using MS-2 coliphage as an exogenous control for RT-PCR inhibition. PLoS One 2012; 7(7): e39455.
13. Verheyen J, Kaiser R, Bozic M, Timmen-Wego M, Maier BK, Kessler HH. Extraction of viral nucleic acids: comparison of five automated nucleic acid extraction platforms. J Clin Virol 2012; 54(3): 255–259.
14. Espy MJ, Rys PN, Wold AD, Uhl JR, Sloan LM, Jenkins GD, et al. Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 methods. J Clin Microbiol 2001; 39(6): 2233–2236.
15. Mandel P, Metais P. Les acides nucleiques du plasma sanguin chez l’homme. C R Seances Soc Biol Fil 1948; 142(3–4): 241–243 (in French).
16. Devonshire AS, Whale AS, Gutteridge A, Jones G, Cowen S, Foy CA, et al. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem 2014; 406(26): 6499–6512.
17. Fong SL, Zhang JT, Lim CK, Eu KW, Liu Y. Comparison of 7 methods for extracting cell-free DNA from serum samples of colorectal cancer patients. Clin Chem 2009; 55(3): 587–589.
18. Perez-Barrios C, Nieto-Alcolado I, Torrente M, Jimenez-Sanchez C, Calvo V, Gutierrez-Sanz L, et al. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing. Transl Lung Cancer Res 2016; 5(6): 665–672.
19. Sorber L, Zwaenepoel K, Deschoolmeester V, Roeyen G, Lardon F, Rolfo C, et al. A comparison of cell-free DNA isolation kits: isolation and quantification of cell-free DNA in plasma. J Mol Diagn 2017; 19(1): 162–168.
20. Cook L, Starr K, Boonyaratanakornkit J, Hayden R, Caliendo AM. Does size matter? Comparison of extraction yield for different-sized DNA fragments by 7 different routine and 4 new circulating cell-free extraction methods. J Clin Microbiol 2018; 56(12): pii: e01061-18.
21. Chan KC, Zhang J, Chan AT, Lei KI, Leung SF, Chan LY, et al. Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients. Cancer Res 2003; 63(9): 2028–2032.
22. Ruf S, Behnke-Hall K, Gruhn B, Bauer J, Horn M, Beck J, et al. Comparison of six different specimen types for Epstein-Barr viral load quantification in peripheral blood of pediatric patients after heart transplantation or after allogeneic hematopoietic stem cell transplantation. J Clin Virol 2012; 53(3): 186–194.
23. Kanakry JA, Hegde AM, Durand CM, Massie AB, Greer AE, Ambinder RF, et al. The clinical significance of EBV DNA in the plasma and peripheral blood mononuclear cells of patients with or without EBV diseases. Blood 2016; 127(16): 2007–2017.
24. Lit LC, Chan KC, Leung SF, Lei KI, Chan LY, Chow KC, et al. Distribution of cell-free and cell-associated Epstein-Barr virus (EBV) DNA in the blood of patients with nasopharyngeal carcinoma and EBV-associated lymphoma. Clin Chem 2004; 50(10): 1842–1845.
25. Boom R, Sol CJ, Schuurman T, Van Breda A, Weel JF, Beld M, et al. Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented. J Clin Microbiol 2002; 40(11): 4105–4113.
26. Habbal W, Monem F, Gartner BC. Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive? J Med Microbiol 2009; 58(Pt 7): 878–883.
27. Tong Y, Pang XL, Mabilangan C, Preiksaitis JK. Determination of the biological form of human cytomegalovirus DNA in the plasma of solid-organ transplant recipients. J Infect Dis 2017; 215(7): 1094–1101.
28. Chesnais V, Ott A, Chaplais E, Gabillard S, Pallares D, Vauloup-Fellous C, et al. Using massively parallel shotgun sequencing of maternal plasmatic cell-free DNA for cytomegalovirus DNA detection during pregnancy: a proof of concept study. Sci Rep 2018; 8(1): 4321.
The authors
Kimberly Starr1 PhD and Linda Cook*2,3 PhD, D(ABMLI)
1Clinical Microbiology Division, Department of Laboratory Medicine, University of Washington Medicine, Seattle, WA, USA
2Clinical Virology Division, Department of Laboratory Medicine, University of Washington Medicine, Seattle, WA, USA
3Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
*Corresponding author
E-mail: lincook@uw.edu
Hemochromatosis: more common than first thought
, /in Featured Articles /by 3wmediaHereditary hemochromatosis type 1 is a disease of iron overload caused predominantly by a mutation in the homeostatic iron regulator (HFE) gene, p.Cyst282Tyr (p.C282Y). The incidence of the mutation is most common in people of northern European descent – with 1 in 8 people being carriers, making it the most common genetic condition in this population. Approximately 1 in 150 people are homozygotes, although a previous study suggested that only about 1% of homozygotes went on to develop “frank clinical hemochromatosis” involving liver disease. The overload of iron results in iron deposition in the liver, pancreas and joints, causing liver disease (cirrhosis and cancer), fatigue, diabetes and arthritis. Diagnosis if often missed or delayed because of the insidious onset of symptoms that often only become apparent later in life and which can easily be attributed to other causes. Currently, if hemochromatosis is suspected, diagnosis is made by testing for high blood iron levels. Genetic screening is limited only to close family members of hemochromatosis patients because of the suggestion of low general penetrance of the disease. The damaging effects of iron overload can be easily prevented if the disease is diagnosed early enough, largely by withdrawing blood on a regular basis. However, a recent study by Pilling et al. of nearly 500 000 UK Biobank volunteers is changing the way we think about the condition (Pilling LC, et al. Common conditions associated with hereditary haemochromatosis genetic variants: cohort study in UK Biobank. BMJ 2019; 364: k5222). This study involved a far larger number of people than previous studies, as well as involving older people – important for monitoring a disease where the effects are cumulative. The authors found a much higher prevalence of hemochromatosis and associated conditions than expected. Of the p.C282Y homozygous participants, 21.7% of men and 9.8% of women were eventually diagnosed with hemochromatosis. The results of this study have prompted the UK National Screening Committee to announce that it will review the evidence for hemochromatosis screening at its next routine review. However, in the meantime, we are actually in the fortunate position that this disease is easy to test for and easy to treat. No new methodology is needed, but simply a change in pathway, as advocated by Dr Ted Fitzsimons (consultant hematologist at Gartnavel Hospital, Glasgow, UK): if the results of a serum ferritin test are high and the patient is of northern European descent, the blood iron levels should automatically be tested. If this result is also high, then the patient should be screened for hemochromatosis. Many people have a lot to gain from this simple change.
Rapid Fosfomycin/E. coli NP test: a new technique for the rapid detection of fosfomycin-resistant E. coli isolates
, /in Featured Articles /by 3wmediaFosfomycin is a broad-spectrum antibiotic used as empirical treatment for uncomplicated urinary tract infections (UTIs), of which Escherichia coli is the most common cause. To rapidly detect fosfomycin-resistant E. coli isolates and consequently improve patients’ treatment and management, we have developed the Rapid Fosfomycin/E. coli NP test, a rapid, easy-to-perform, specific and sensitive diagnostic test.
by Dr Linda Mueller, Dr Laurent Poirel and Prof. Patrice Nordmann
Introduction
Fosfomycin, a phosphonic acid-derived bactericidal antibiotic discovered in 1969, is now of renewed interest, especially for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. This antibiotic is water-soluble and has a low molecular weight, allowing high diffusion at the tissue level [1]. Its features such as broad-spectrum activity, safety and efficacy make fosfomycin as one of the first-line antibiotics used for uncomplicated urinary tract infections (UTIs) treatment [2]. More than 75% of UTIs are due to Escherichia coli [3].
Fosfomycin enters the bacterial cell by the transport proteins GlpT (glycerol-3-phosphate transporter) and UhpT (hexose-6-phosphat:phosphate antiporter); once in the cytosol it binds and inactivates MurA (UDP-N-acetylglucosamine enolpyruvyl transferase), the enzyme involved in the first step of peptidoglycan biosynthesis. Hence, it inhibits bacterial cell wall synthesis [4].
Because of its unique structure and mechanism of action, cross-resistance with fosfomycin and other bacterial agents has not been observed. Fosfomycin as a single agent works well for treating most of UTIs. Additionally, synergistic effects of fosfomycin with several unrelated molecules, such as gentamicin, carbapenems, aztreonam and aminoglycosides, have been observed when treating clinically-relevant MDR Gram-negative bacteria [5].
One of the main concerns with antibiotic resistance in E. coli corresponds to the acquisition of extended-spectrum β-lactamases (ESBL) leading to resistance to expanded-spectrum cephalosporins. ESBL-producing E. coli are mostly community-acquired and may represent 10 to 20% of E. coli isolates in several countries including in the US [6]. Those strains are often co-resistant to several aminoglycosides, to trimethoprim, cotrimoxazole and fluoroquinolones, leaving few therapeutic options available including fosfomycin [7].
Both wild-type susceptible E. coli and ESBL-producing E. coli show an overall high susceptibility rate to fosfomycin (>90%) [8]. However, a Spanish study monitoring fosfomycin resistance during 5 years, showed an increased use of fosfomycin [from 0.122 defined daily dose per 1000 inhabitants per day (DID) in 2004 to 0.191 DID in 2008] and an increased fosfomycin resistance rate in E.coli (from 1.6% to 3.8%) as well as in ESBL-producing E. coli (from 2.2% to 21.7%) [9].
The mechanisms of resistance to fosfomycin described in E. coli are either non-transferable or transferable. The non-transferable and chromosome-encoded resistance involve reduced permeability, resulting from mutations in glpT and uhpT genes, encoding for fosfomycin transporters, and amino acid mutations in the active site of the MurA target. Plasmid-mediated fosfomycin resistance mechanisms in E. coli correspond to production of fosfomycin-inactivating metallo-enzymes (encoded by the fosA genes) [10]. Among the plasmid-borne fosA variants described so far, fosA3 remains the most widespread resistance determinant among both human and animal isolates, those latter being either recovered from pets or livestock [11, 12]. Moreover, a study performed in Taiwan reported the transmission of FosA3-producing E. coli between companion animals and respective owners [13]. Importantly, the fosA3 gene is often identified onto conjugative plasmids along with CTX-M-type ESBL encoding genes, thus leading to acquired resistance to both fosfomycin and broad-spectrum cephalosporins [14, 15]. As fosfomycin is being used as an empiric treatment against UTIs, it was of great interest to develop a rapid test to evaluate the efficacy of this antibiotic.
Rapid Fosfomycin/E. coli NP test
Currently the reference technique recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) to evaluate fosfomycin susceptibility is agar dilution, a fastidious technique requiring 18±2 h to get the results [16]. According to EUCAST, an isolate of E. coli is categorized as susceptible or as resistant when minimum inhibitory concentrations (MICs) are ≤32 and >32 mg/L, respectively.
Alternatively, disk diffusion and gradient strips, although exhibiting some discrepancies with the reference agar dilution method, might be used [17]. To accelerate the process of fosfomycin resistance detection, we have developed the Rapid Fosfomycin/E. coli NP test that allows detection of resistance within 1 h 30 min of fosfomycin-resistant E. coli isolated from culture plates.
This user-friendly technique is based on carbohydrate hydrolysis, detecting bacterial growth of fosfomycin-resistant isolates in the presence of a defined concentration (40 mg/L) of fosfomycin. Of note, fosfomycin-resistant isolates are detected independently of the molecular mechanism of resistance.
Briefly, the technique includes the preparation of a bacterial suspension (109 CFU/mL; 3–3.5 McFarland) that is poured on a 96-well polystyrene microplate. This culture is made in the Rapid Fosfomycin NP solution supplemented with 25 mg/L glucose-6-phosphate with or without 40 mg/L fosfomycin. The plate is incubated for 1 h 30 min at 35±2 °C and colour changes are detected by visual inspected. Fosfomycin-resistant isolates grow in the presence and absence of fosfomycin, triggering a colour switch from orange to yellow in both wells, a test result which is, therefore, considered as positive (Fig. 1). When dealing with fosfomycin-susceptible isolates, the well supplemented with fosfomycin does not exhibit any bacterial growth and remains orange; the test is, therefore, considered as negative. This test was evaluated with 100 strains including 22 fosfomycin-resistant isolates. It showed a sensitivity and a specificity of 100% and 98.7% respectively.
Conclusion
The Rapid Fosfomycin/E. coli NP test is rapid (1 h 30 min), specific (98.7%) and sensitive (100%). It is easy to perform, cost-effective, and may be used worldwide, regardless of the technical capabilities of the lab. Ongoing work aims to evaluate its performances directly from urine samples, which would represent significant added-value in terms of diagnostic rapidity.
The speed of this test allows a saving of at least 16 h when compared to the traditional agar dilution method. It is a potentially useful clinical test for first-step screening of fosfomycin resistance in E. coli.
Even though a low level of resistance to fosfomycin is currently observed among E. coli, the fact that we usually observe an increased fosfomycin clinical use, meaning an increased selective pressure, argues for a likely increased occurrence of fosfomycin-resistant isolates in the future. Since the principle of this test is based on a rapid culture, it may be used to detect any fosfomycin resistance trait that may be either chromosomally or plasmid-encoded. Fosfomycin is an old antibiotic that is very useful for the treatment of uncomplicated UTIs. On the one hand, even after extensive use for such an indication, the prevalence of resistance remains low, likely due to the fitness cost of the chromosomal mutations needed for acquired resistance, and also as a consequence of a high urinary drug concentration. On the other hand, the worldwide spread of fosfomycin-modifying enzymes should be monitored, as the biological cost of this emerging mechanism of resistance is much lower than that induced by chromosomal mutations [18] and the co-occurrence of fosA-like genes on plasmids with other resistance genes is commonly observed, meaning that co-selection can occur quite frequently.
References
1. Dijkmans AC, Zacarias NVO, Burggraaf J, Mouton JW, Wilms EB, van Nieuwkoop C, et al. Fosfomycin: pharmacological, clinical and future perspectives. Antibiotics (Basel) 2017; 6(4): pii: E24.
2. Gupta K, Hooton TM, Naber KG, Wullt B, Colgan R, Miller LG, et al. International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases. Clin Infect Dis 2011; 52(5): e103–120.
3. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 2015; 13(5): 269–284.
4. Castaneda-Garcia A, Blazquez J, Rodriguez-Rojas A. Molecular mechanisms and clinical impact of acquired and intrinsic fosfomycin resistance. Antibiotics (Basel) 2013; 2(2): 217–236.
5. Falagas ME, Vouloumanou EK, Samonis G, Vardakas KZ. Fosfomycin. Clin Microbiol Rev 2016; 29(2): 321–347.
6. Castanheira M, Farrell SE, Krause KM, Jones RN, Sader HS. Contemporary diversity of beta-lactamases among Enterobacteriaceae in the nine U.S. census regions and ceftazidime-avibactam activity tested against isolates producing the most prevalent beta-lactamase groups. Antimicrob Agents Chemother 2014; 58(2): 833–838.
7. Wiedemann B, Heisig A, Heisig P. Uncomplicated urinary tract infections and antibiotic resistance-epidemiological and mechanistic aspects. Antibiotics (Basel) 2014; 3(3): 341–352.
8. Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum β-lactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect Dis 2010; 10: 4–-50.
9. Oteo J, Orden B, Bautista V, Cuevas O, Arroyo M, Martinez-Ruiz R, et al. CTX-M-15-producing urinary Escherichia coli O25b-ST131-phylogroup B2 has acquired resistance to fosfomycin. J Antimicrob Chemother 2009; 64(4): 712–717.
10. Silver LL. Fosfomycin: mechanism and resistance. Cold Spring Harb Perspect Med 2017; 7(2): pii: a025262.
11. Alrowais H, McElheny CL, Spychala CN, Sastry S, Guo Q, Butt AA, et al. Fosfomycin resistance in Escherichia coli, Pennsylvania, USA. Emerg Infect Dis 2015; 21(11): 2045–2047.
12. Xie M, Lin D, Chen K, Chan EW, Yao W, Chen S. Molecular characterization of Escherichia coli strains isolated from retail meat that harbor blaCTX-M and fosA3 genes. Antimicrob Agents Chemother 2016; 60(4): 2450–2455.
13. Yao H, Wu D, Lei L, Shen Z, Wang Y, Liao K. The detection of fosfomycin resistance genes in Enterobacteriaceae from pets and their owners. Vet Microbiol 2016; 193: 67–71.
14. Benzerara Y, Gallah S, Hommeril B, Genel N, Decre D, Rottman M, et al. Emergence of plasmid-mediated fosfomycin-resistance genes among Escherichia coli isolates, France. Emerg Infect Dis 2017; 23(9): 1564–1567.
15. Yang X, Liu W, Liu Y, Wang J, Lv L, Chen X, et al. F33: A-: B-, IncHI2/ST3, and IncI1/ST71 plasmids drive the dissemination of fosA3 and bla CTX-M-55/-14/-65 in Escherichia coli from chickens in China. Front Microbiol 2014; 5: 688.
16. Performance standards for antimicrobial susceptibility testing, 28th edn. Clinical and Laboratory Standards Institute (CLSI) document M100-S28 2018.
17. Hirsch EB, Raux BR, Zucchi PC, Kim Y, McCoy C, Kirby JE, et al. Activity of fosfomycin and comparison of several susceptibility testing methods against contemporary urine isolates. Int J Antimicrob Agents 2015; 46(6): 642–647.
18. Cattoir V, Guérin F. How is fosfomycin resistance developed in Escherichia coli? Future Microbiol 2018; 13(16): 1693–1696.
The authors
Linda Mueller*1,2 PhD; Laurent Poirel1,2,3 PhD; Patrice Nordmann1,2,3,4 MD, PhD
1Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland
2Swiss National Reference Center for Emerging Antibiotic Resistance (NARA), University of Fribourg, Fribourg, Switzerland
3INSERM European Unit (IAME, France),University of Fribourg, Fribourg, Switzerland
4University Hospital Center and University of Lausanne, Lausanne, Switzerland
*Corresponding author
E-mail: Linda.mueller@unifr.ch
Effect of DNA extraction on molecular testing in the clinical laboratory
, /in Featured Articles /by 3wmediaExtraction of nucleic acids from patient samples is an essential step for downstream molecular studies such as quantitative and qualitative PCR. The size of the DNA fragments present in samples can influence extraction efficiency, especially observed in circulating cell-free DNA (cfDNA). Further work is necessary to determine the impact of cfDNA extraction on clinical virology and microbiology testing.
by Dr Kimberly Starr and Dr Linda Cook
Introduction
After sample collection, the next important step in the detection of infectious agents in most patient-derived samples is the extraction of DNA or RNA to remove proteins, lipids, other cellular components, and PCR inhibitors to create a ‘PCR-friendly’ eluate solution. First, the sample is mixed with a lysis buffer and then DNA is purified from the resulting solution by silica-coated filtration membranes or magnetic beads that bind nucleic acid and allow subsequent washing and elution steps to be performed. Extraction methods can range from small-scale manual methods to large-scale fully-automated extraction instruments. For implementation of automated platforms several factors require consideration, including capacity, target range, efficiency, cost, physical footprint, level of automation, and processing time. The variety of instrumentation and extraction methods available contribute to the differences in extraction efficiency that may have downstream consequences when quantifying DNA or RNA in bacteria, fungi, parasites, and viruses. The performance of different kits even on the same instrument can further contribute to variation in efficiency [1]. Inter-laboratory variation as a result of extraction efficiency can affect patient care and reproducibility of testing results, especially for patients who are monitored over a long period with a quantitative test.
Extraction method comparisons
In a study comparing the bacterial DNA quantity and quality extracted from stool, Claassen et al. found DNA yield and purity varied between five commonly used extraction kits [2]. This is the case for fungi as well where extraction of nucleic acid from Aspergillus fumigatus is the main limiting factor for successful Aspergillus PCR from clinical specimens. Perry et al. found differences in reproducibility of DNA extraction at low levels (101 cells/mL) in EDTA whole blood among the four extraction instruments they tested [3]. The same can be seen in parasitic infections, demonstrated by Yera et al., which showed that DNA extraction procedures led to variations in detecting low concentrations of Toxoplasma gondii tachyzoites in amniotic fluid samples, a difference that could affect early diagnosis of congenital toxoplasmosis [4].
Other studies have evaluated extraction systems for human immunodeficiency virus (HIV) [5–8], hepatitis B virus (HBV) [9, 10], Cytomegalovirus (CMV) [11], enterovirus [12], norovirus [13], and HSV [14]. Essentially all published extraction comparison studies have seen quantitative differences in results across the different systems evaluated, sometimes with quantitative differences significantly more than 1 log.
Cell-free DNA measurements
Another level of complexity is added when the size of the nucleic acid to be isolated varies. It is known that nucleic acids fragment during the extraction process, but recent studies have demonstrated that nucleic acids may be a variety of sizes in the initial sample, especially in blood. Cell-free circulating DNA (cfDNA) in blood coming from cellular breakdown was first described by Mandel and Metais in 1948 [15]. The size of cfDNA fragments described is approximately 167 bp, equivalent to the size of chromatosome DNA and similar to post-apoptosis DNA fragments. In the last 20 years, there has been increased interest in measuring and quantifying cfDNA in a variety of cancers. Key observations from these studies are: (1) The concentration in plasma/serum is very low, 10–100 ng/mL. Thus, many studies have focused on identifying extraction methods to maximize cfDNA yield. (2) Sample collection tubes with cell-stabilizing reagents to prevent contamination of plasma with cellular DNA can increase the purity and yield of cfDNA. (3) Use of generic DNA extraction methods can cause further fragmentation of cfDNA and decrease yields compared to cfDNA-specific extraction methods. Recently, extraction instrument manufacturers have introduced cfDNA isolation kits and instruments. These kits utilize higher input volumes of 1.0–5.0 mL, and optimized temperatures or buffer conditions to improve yields. cfDNA kits from several manufacturers have been shown to have better performance in several studies. Four excellent reviews describing the technical aspects of cfDNA extraction and comparison of cfDNA extraction methods have been published [16–19].
Our DNA fragment extraction study
To better understand how DNA fragment size may impact viral infectious disease test results, we designed a study [20] comparing extraction yields for differently sized DNA fragments across 11 commercially available extraction methods commonly used in clinical laboratories, and also compared the performance of four new cfDNA extraction methods. Artificially constructed DNA fragments with sizes ranging from 50 to 1,500 bp were extracted and tested by droplet digital PCR to determine the DNA fragment yield across methods. We found a wide range of extraction yields across both extraction methods and instruments, with the 50 and 100 bp fragment sizes showing especially inconsistent quantitative results and poor yields of less than 20%. Figure 1 shows the yield results of two representative methods and one cfDNA method. Two of the methods designed to extract cfDNA gave the highest yields for the 50 and 100 bp fragments but overall yields were poor. We also observed the lowest variability across methods for the larger sized fragments at higher concentrations. Overall, we saw the most variability for the smallest sized fragments and observed variability dependent on concentration.
Results from our study demonstrate significant differences in fragment extraction yields and overall poor yields of the small artificial DNA fragments even at high concentrations in essentially all routinely used methods. Two of the four cfDNA methods showed improved (although still low) yield of smaller fragments. Further studies are necessary to determine the cause of this significant difference in yields. We speculate as the field moves toward more next generation sequencing approaches, these differences in extraction efficiency and quantification of small cfDNAs will become more widely described.
A critical next step is to determine if viral cfDNA exists in patients with a variety of infectious diseases and if their measurement has clinical relevance. Further studies should focus on identifying which viruses or other infectious agents have cfDNA and then methods to extract and evaluate this cfDNA must be significantly improved. To date, only cfDNA associated with Epstein-Barr virus (EBV) has been extensively studied and hints of cfDNA importance in CMV disease have been seen.
cfDNA in EBV
As early as 2003, Chan et al. described the differential detection of EBV by PCR depending on the size of the PCR amplicon, demonstrating that an assay with an 82 bp amplicon detected 7.5 times more EBV in plasma that a 181 bp amplicon assay [21]. Many additional studies in nasopharyngeal carcinoma have confirmed the excellent utility of measuring the quantity of this small EBV-associated cfDNA for monitoring of therapy response, prediction of recurrence, and monitoring at-risk populations.
Two recent large studies have shown that plasma levels of EBV are the most useful sample type for testing EBV infected patients [22, 23] but cfDNA was not specifically identified in these studies. A study by Lit et al. in EBV-associated lymphoma patients demonstrated EBV cfDNA [24] and noted that the subset of patients with ‘active’ disease had a relative predominance of cfDNA compared to predominantly larger cell-associated EBV DNA seen in cases of inactive disease or remission. Thus, measurement of both EBV cfDNA as well as larger EBV DNA fragments may be important in clinical testing and it may be necessary to distinguish the size of EBV in the plasma. Further studies are necessary to determine how useful detection of cfDNA may be in all EBV-associated malignancies and infections.
cfDNA in CMV
Published data hints that fragmented DNA may also be important for CMV PCR quantitation. In one study, Boom et al. fractionated CMV DNA in plasma and whole blood from three renal transplant cases with primary CMV infection and measured the quantities present with two PCR amplicons sized 578 bp and 134 bp [25]. They demonstrated that CMV DNA was predominantly less than 2000 bp and detected many small sized fragments only with the 134 bp amplicon PCR. Habbal et al. also studied 17 different CMV primer sets and demonstrated that the two of the four primer sets with the smallest amplicons (<100 bp) were the most sensitive for detection of cultured CMV strains [26]. Tong et al., found that among 20 solid organ transplant recipients, 10 had exclusively free CMV DNA, while the remaining 10 had predominantly free CMV DNA with a small percentage of encapsulated-virion DNA present [27]. In addition, they compared results for two assays with small amplicon sizes of 81 and 138 bp and found a 2.6-fold higher level with the smaller amplicon, suggesting CMV DNA present in these clinical samples was very small (<138 bp). It appears critical to use a high-yield small CMV DNA fragment extraction method as well as a small CMV PCR amplicon assay to maximize CMV detection of CMV. Incorporating these two elements into clinical CMV PCR assays could decrease assay variability and decrease inter-lab variability.
cfDNA in other viruses
There is evidence that cfDNA may be useful in infections and malignancies associated with viruses other than EBV and CMV. A recent study by Chesnais et al. mimicked detection of genetic mutations in pre-term children by using CCF from maternal plasma and demonstrated the potential of this technology to detect multiple viruses present in low levels in mothers or pre-term babies [28]. In addition, case reports for Kaposi’s sarcoma and BKPyV-associated bladder cancer (virus-associated cancers) suggest utility of quantitative measurements of cfDNA containing HHV8 (human herpes virus 8, also known as Kaposi’s sarcoma-associated herpesvirus) or BK virus, respectively, in tumor detection and therapeutic monitoring. Further studies are necessary in these two diseases as well as other infectious diseases to evaluate the clinical utility of cfDNA measurements.
References
1. McCulloch E, Ramage G, Jones B, Warn P, Kirkpatrick WR, Patterson TF, et al. Don’t throw your blood clots away: use of blood clot may improve sensitivity of PCR diagnosis in invasive aspergillosis. J Clin Pathol 2009; 62(6): 539–541.
2. Claassen S, du Toit E, Kaba M, Moodley C, Zar HJ, Nicol MP. A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. J Microbiol Methods 2013; 94(2): 103–110.
3. Perry MD, White PL, Barnes RA. Comparison of four automated nucleic acid extraction platforms for the recovery of DNA from Aspergillus fumigatus. J Med Microbiol 2014; 63(Pt 9): 1160–1166.
4. Yera H, Filisetti D, Bastien P, Ancelle T, Thulliez P, Delhaes L. Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid. J Clin Microbiol 2009; 47(12): 3881–3886.
5. Cornelissen M, Gall A, Vink M, Zorgdrager F, Binter S, Edwards S, et al. From clinical sample to complete genome: comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing. Virus Res 2017; 239: 10–16.
6. Alp A, Hascelik G. Comparison of 3 nucleic acid isolation methods for the quantification of HIV-1 RNA by Cobas Taqman real-time polymerase chain reaction system. Diagn Microbiol Infect Dis 2009; 63(4): 365–371.
7. Stevens W, Horsfield P, Scott LE. Evaluation of the performance of the automated NucliSENS easyMAG and EasyQ systems versus the Roche AmpliPrep-AMPLICOR combination for high-throughput monitoring of human immunodeficiency virus load. J Clin Microbiol 2007; 45(4): 1244–1249.
8. Swanson P, Holzmayer V, Huang S, Hay P, Adebiyi A, Rice P, et al. Performance of the automated Abbott RealTime HIV-1 assay on a genetically diverse panel of specimens from London: comparison to VERSANT HIV-1 RNA 3.0, AMPLICOR HIV-1 MONITOR v1.5, and LCx HIV RNA Quantitative assays. J Virol Methods 2006; 137(2): 184–192.
9. Kang SH, Lee EH, Park G, Jang SJ, Moon DS. Comparison of MagNA Pure 96, Chemagic MSM1, and QIAamp MinElute for hepatitis B virus nucleic acid extraction. Ann Clin Lab Sci 2012; 42(4): 370–374.
10. Pyne MT, Vest L, Clement J, Lee J, Rosvall JR, Luk K, et al. Comparison of three Roche hepatitis B virus viral load assay formats. J Clin Microbiol 2012; 50(7): 2337–2342.
11. Bravo D, Clari MA, Costa E, Munoz-Cobo B, Solano C, Jose Remigia M, et al. Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients. J Clin Microbiol 2011; 49(8): 2899–2904.
12. Shulman LM, Hindiyeh M, Muhsen K, Cohen D, Mendelson E, Sofer D. Evaluation of four different systems for extraction of RNA from stool suspensions using MS-2 coliphage as an exogenous control for RT-PCR inhibition. PLoS One 2012; 7(7): e39455.
13. Verheyen J, Kaiser R, Bozic M, Timmen-Wego M, Maier BK, Kessler HH. Extraction of viral nucleic acids: comparison of five automated nucleic acid extraction platforms. J Clin Virol 2012; 54(3): 255–259.
14. Espy MJ, Rys PN, Wold AD, Uhl JR, Sloan LM, Jenkins GD, et al. Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 methods. J Clin Microbiol 2001; 39(6): 2233–2236.
15. Mandel P, Metais P. Les acides nucleiques du plasma sanguin chez l’homme. C R Seances Soc Biol Fil 1948; 142(3–4): 241–243 (in French).
16. Devonshire AS, Whale AS, Gutteridge A, Jones G, Cowen S, Foy CA, et al. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem 2014; 406(26): 6499–6512.
17. Fong SL, Zhang JT, Lim CK, Eu KW, Liu Y. Comparison of 7 methods for extracting cell-free DNA from serum samples of colorectal cancer patients. Clin Chem 2009; 55(3): 587–589.
18. Perez-Barrios C, Nieto-Alcolado I, Torrente M, Jimenez-Sanchez C, Calvo V, Gutierrez-Sanz L, et al. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing. Transl Lung Cancer Res 2016; 5(6): 665–672.
19. Sorber L, Zwaenepoel K, Deschoolmeester V, Roeyen G, Lardon F, Rolfo C, et al. A comparison of cell-free DNA isolation kits: isolation and quantification of cell-free DNA in plasma. J Mol Diagn 2017; 19(1): 162–168.
20. Cook L, Starr K, Boonyaratanakornkit J, Hayden R, Caliendo AM. Does size matter? Comparison of extraction yield for different-sized DNA fragments by 7 different routine and 4 new circulating cell-free extraction methods. J Clin Microbiol 2018; 56(12): pii: e01061-18.
21. Chan KC, Zhang J, Chan AT, Lei KI, Leung SF, Chan LY, et al. Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients. Cancer Res 2003; 63(9): 2028–2032.
22. Ruf S, Behnke-Hall K, Gruhn B, Bauer J, Horn M, Beck J, et al. Comparison of six different specimen types for Epstein-Barr viral load quantification in peripheral blood of pediatric patients after heart transplantation or after allogeneic hematopoietic stem cell transplantation. J Clin Virol 2012; 53(3): 186–194.
23. Kanakry JA, Hegde AM, Durand CM, Massie AB, Greer AE, Ambinder RF, et al. The clinical significance of EBV DNA in the plasma and peripheral blood mononuclear cells of patients with or without EBV diseases. Blood 2016; 127(16): 2007–2017.
24. Lit LC, Chan KC, Leung SF, Lei KI, Chan LY, Chow KC, et al. Distribution of cell-free and cell-associated Epstein-Barr virus (EBV) DNA in the blood of patients with nasopharyngeal carcinoma and EBV-associated lymphoma. Clin Chem 2004; 50(10): 1842–1845.
25. Boom R, Sol CJ, Schuurman T, Van Breda A, Weel JF, Beld M, et al. Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented. J Clin Microbiol 2002; 40(11): 4105–4113.
26. Habbal W, Monem F, Gartner BC. Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive? J Med Microbiol 2009; 58(Pt 7): 878–883.
27. Tong Y, Pang XL, Mabilangan C, Preiksaitis JK. Determination of the biological form of human cytomegalovirus DNA in the plasma of solid-organ transplant recipients. J Infect Dis 2017; 215(7): 1094–1101.
28. Chesnais V, Ott A, Chaplais E, Gabillard S, Pallares D, Vauloup-Fellous C, et al. Using massively parallel shotgun sequencing of maternal plasmatic cell-free DNA for cytomegalovirus DNA detection during pregnancy: a proof of concept study. Sci Rep 2018; 8(1): 4321.
The authors
Kimberly Starr1 PhD and Linda Cook*2,3 PhD, D(ABMLI)
1Clinical Microbiology Division, Department of Laboratory Medicine, University of Washington Medicine, Seattle, WA, USA
2Clinical Virology Division, Department of Laboratory Medicine, University of Washington Medicine, Seattle, WA, USA
3Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
*Corresponding author
E-mail: lincook@uw.edu
Colistin resistance detection in Acinetobacter baumannii by mass spectrometry of microbial lipids
, /in Featured Articles /by 3wmediaAcinetobacter baumannii is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections with colistin can result in emergence of colistin-resistant strains. This occurs via modifications of the phosphate moieties of lipopolysaccharide-derived lipid A, which are readily identified by mass spectrometry (MS). In this article, we describe colistin susceptibility determinations by lipid MS of A. baumannii and our recent study in which we correlate MS results with traditional antimicrobial susceptibility testing of clinical isolates.
by Dr Lisa M. Leung, Dr Robert A. Myers, Dr Yohei Doi and Prof. Robert K. Ernst
Background
Colistin resistance in Gram-negative pathogens
Multidrug-resistant, Gram-negative bacterial pathogens continue to pose serious threats to public health. Carbapenem-resistant Enterobacteriaceae (CRE), Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB) are given the highest global priority among drug-resistant organisms by organizations, such as the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) [1]. Carbapenem-resistant infections can be treated with colistin, a last resort antibiotic of the polymyxin class, leading to an increase in colistin resistance and resulting in devastating consequences as it is one of the last remaining effective antimicrobials [2]. Furthermore, discovery of a plasmid-mediated colistin resistance gene, mcr, has intensified this urgency given the potential for rapid and widespread dissemination of colistin-resistant bacteria across the globe [3, 4]. Therefore, the WHO and CDC have prioritized development of novel diagnostics and therapeutics to address the global threat of pathogens, such as multidrug-resistant A. baumannii [5].
A novel diagnostic approach is proposed
In elucidating the mechanism of colistin resistance, researchers analysed microbial glycolipids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These findings contributed to determination of the resistance mechanism in A. baumannii, via addition of phosphoethanolamine onto the terminal phosphate moieties of the lipopolysaccharide (LPS)-derived lipid A (LA), decreasing the electronegativity of the membrane and, subsequently, the binding affinity of colistin [6]. These modifications create unique features on the resultant mass spectra of colistin-resistant strains that can be used as a diagnostic marker. Our group has published proof-of-concept studies utilizing this platform in the identification of the ESKAPE pathogens [7], as well as elucidation of colistin susceptibility in organisms such as Klebsiella pneumoniae [8], E. coli, and P. aeruginosa [9]. Protein-based microbial identification using MALDI-TOF MS is a simple and effective means of identifying causative agents although it still faces challenges, such as identification of closely related organisms (Candida or Shigella subspecies), antimicrobial susceptibility determination, or identification of organisms in polymicrobial or biologically relevant samples (urine, blood or wound effluent) [10]. Therefore, we offered this novel platform as an alternative and complementary approach to strengthen the overall diagnostic power of MALDI-TOF MS and continue to demonstrate its capability in our latest study detecting colistin resistance in A. baumannii [11].
Methods and results
Overview of clinical data
In this study, we prospectively collected A. baumannii complex clinical isolates from a hospital system in Pennsylvania between 2014 and 2016, a total of 451 isolates from 284 patients. Among the 284 unique isolates from each patient, 73.6% (209 isolates) were determined to be A. baumannii, 18.7% (53 isolates) Acinetobacter pittii, 3.5% (10 isolates) Acinetobacter nosocomialis, and 1.8% (5 isolates) Acinetobacter calcoaceticus. The remaining <1% were identified as the following Acinetobacter genospecies that do not belong to the A. baumannii complex: Acinetobacter radioresistens (2 isolates), Acinetobacter guillouliae (1 isolate), and Acinetobacter junii (1 isolate). Three isolates (0.7%) could not be reliably identified. All isolates were evaluated for colistin resistance using standard minimum inhibitory concentration (MIC) testing by both agar dilution and broth microdilution in accordance with the clinical breakpoint provided by the EUCAST [12]. Of the 451 clinical isolates, 394 isolates from 249 patients were found to be susceptible to colistin (≤2 µg/mL), and a total of 39 isolates (8.6%) from 20 patients were identified as resistant (>2 µg/mL).
The colistin-resistant A. baumannii mass spectrum
All strains were cultured overnight and subjected to a hot ammonium isobutyrate reaction to extract cellular lipids. Extracts were analysed by MALDI-TOF in negative ion mode using a Bruker microflex LRF MALDI-TOF mass spectrometer operated in reflectron mode and using norharmane as a matrix. Ions most often observed in the mass spectra were m/z 1404, 1728, and 1910; these have been previously characterized [6], with m/z 1910 representing the full bis-phosphorylated, hepta-acylated lipid A structure (Fig. 1). Resistant isolates were defined by the presence of an ion at m/z 2033, representing the addition of a phosphoethanolamine moiety to one of the phosphate moieties of the m/z 1910 structure (∆m/z=123) (Fig. 1). Determination of resistance was made by observing this ion in acquired mass spectra for each sample above a signal-to-noise ratio of 3. Of the 451 clinical isolates, 397 were determined to be susceptible to colistin (i.e. lacking an ion at m/z 2033), whereas 54 (12.0%) showed the presence of the m/z 2033 and were classified as resistant.
Differentiation of the A. baumannii complex
Differences were observed between spectra collected from the A. baumannii complex isolates, A. baumannii, A. pittii, and A. nosocomialis (Fig. 2). In general, an ion at m/z 1882 displayed higher signal intensity in A. pittii and A. nosocomialis isolates, about 80% relative intensity to the base peak at m/z 1910 compared to about 10% for A. baumannii, which may indicate differences in relative abundances of specific LPS structures. This ion most likely results from an exchange of a shorter chain fatty acyl group (C2H4, ∆m/z=28) from one of the acyl chains of the base structure at m/z 1910, although this structure is inferred and further analyses would need to be conducted for positive structural determinations. In addition, A. pittii and A. nosocomialis isolates showed prominent novel ions at m/z 1866 and 1894, indicating differences in hydroxylation events (∆m/z=16) from ions at m/z 1882 and 1910, respectively, potentially representing the addition of a hydroxyl moiety to one of the attached fatty acyls of lipid A. Among the 39 colistin-resistant isolates, only one was identified as non-baumannii (A. nosocomialis). This means that non-baumannii isolates occur at a lower incidence among resistant isolates (3.1%), as compared to their incidence among Acinetobacter isolates in general (19.7%) indicating a higher resistance rate of A. baumannii versus non-baumannii complex isolates in this study.
MIC versus MS
Discordant results between MIC and MS findings were resolved by multiple-replicate retesting to confirm susceptibility profiles, and final determinations were compared. Of the 451 total isolates used in our study, 394 isolates from 249 patients were determined to be susceptible by both MIC and MS and 39 isolates from 20 patients were determined to be resistant, giving a specificity of 94.0% and a sensitivity of 92.9%. Three isolates were determined to be resistant by MIC yet susceptible by MS and 15 isolates were found to be resistant by MS but susceptible by MIC. When considering only the first isolates isolated from the 284 patients in our study, sensitivity and specificity values change slightly – to 83.3% and 97.4%, respectively. Thirty-nine isolates were subjected to multiple-replicate retesting based on discordant results between agar dilution and broth microdilution methods, MIC and MS results, or both. Of the 33 isolates that underwent MIC retesting, 26 (or 89.7%) gave different susceptibility profiles, 25 went from resistant to susceptible and one was classified as indeterminate. Of the 26 isolates that underwent MS retesting, only three (11.5%) saw a change in their susceptibility profiles; two went from resistant to susceptible and one from susceptible to resistant. Although there was a high association between susceptibility determinations by MIC and MS overall, the positive predictive value was calculated as 72.2% (negative predictive value=99.2%). This is largely owing to the 15 isolates where resistance-associated ions were observed in the mass spectra, but which were determined susceptible by MIC. Chromosomally-mediated colistin resistance in Acinetobacter species is due to overexpression of LPS-modifying genes; therefore, modification of LPS will vary over time. It is presently unclear whether this ‘resistant’ profile is a valid determination of resistance or whether this isolate would present as a resistant infection in a clinical scenario.
Conclusion
A. baumannii, a prevalent, Gram-negative coccobacillus pathogen, poses a significant challenge to clinicians due to the incidence of hospital-acquired and drug-resistant infections. Close monitoring of this pathogen and other A. baumannii complex organisms is considered of critical importance to public health organizations. Here, we surveyed 451 Acinetobacter isolates prospectively collected from patients at a major Pennsylvania health system over a 3-year period. We determined colistin resistance by MIC testing, as well as by MALDI-TOF MS. As in previous studies of colistin-resistant K. pneumoniae, P. aeruginosa, and A. baumannii [6, 8, 13], the data showed a strong association between resistant MIC determinations and the observation of higher m/z ions by MS consistent with modification to LA and previously demonstrated to confer resistance. A. nosocomialis, A. pittii, and A. calcoaceticus, along with A. baumannii are collectively identified as the A. baumannii complex organisms. In our prospective study, we found that A. baumannii isolates were the predominant species within the A. baumannii complex, yet represented a smaller proportion (73.6%) than what has previously been observed [14]. We also demonstrated that a lipid MS profile offers another diagnostic tool for differentiation and accurate surveillance of these pathogens. Furthermore, the finding of resistance ions among a resistant A. nosocomialis isolate demonstrates that A. baumannii complex organisms likely achieve colistin resistance via the same LPS-modifying mechanism (Fig. 2). Overall, we conclude that glycolipid MS profiling can effectively detect colistin resistance in A. baumannii and has the potential to direct antimicrobial stewardship in the clinic, further validating our recently introduced diagnostic platform [7].
References
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11. Leung LM, McElheny CL, Gardner FM, Chandler CE, Bowler SL, Mettus RT, et al. A prospective study of Acinetobacter baumannii complex isolates and colistin susceptibility monitoring by mass spectrometry of microbial membrane glycolipids. J Clin Microbiol 2019; 57(3): pii: e01100-18.
12. Recommendations for MIC determination of colistin (polymyxin E ); as recommended by the joint CLSI-EUCAST Polymyxin Breakpoints Working Group. EUCAST 2016 (http://www.bioconnections.co.uk/files/merlin/Recommendations_for_MIC_determination_of_colistin_March_2016.pdf).
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The authors
Lisa M. Leung1,2 PhD, Robert A. Myers3 PhD, Yohei Doi4 MD, and Robert K. Ernst*2 PhD
1Divisions of Molecular Biology and Microbiology, Maryland Department of Health Laboratories Administration, Baltimore, MD, USA
2Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD, USA
3Maryland Department of Health Laboratories Administration, Baltimore, MD, USA
4Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
*Corresponding author
E-mail: rkernst@umaryland.edu
Automated RPR Syphilis Analyzer – ChemWell® RPR
, /in Featured Articles /by 3wmediaScientific literature review: Clinical microbiology and virology
, /in Featured Articles /by 3wmediaDetection of viruses in clinical samples by use of metagenomic sequencing and targeted sequence capture
Wylie KM, Wylie TN, Buller R, Herter B, Cannella MT, Storch GA. J Clin Microbiol 2018; 56(12): pii: e01123-1
Metagenomic shotgun sequencing (MSS) is a revolutionary approach to viral diagnostic testing that allows simultaneous detection of a broad range of viruses, detailed taxonomic assignment, and detection of mutations associated with antiviral drug resistance. To enhance sensitivity for virus detection, we previously developed ViroCap, a targeted sequence capture panel designed to enrich nucleic acid from a comprehensive set of eukaryotic viruses prior to sequencing. To demonstrate the utility of MSS with targeted sequence capture for detecting clinically important viruses and characterizing clinically important viral features, we used ViroCap to analyse clinical samples from a diagnostic virology laboratory containing a broad range of medically relevant viruses. From 26 samples, MSS with ViroCap detected all of the expected viruses and 30 additional viruses. Comparing sequencing after capture enrichment with standard MSS, we detected 13 viruses only with capture enrichment and observed a consistent increase in the number and percentage of viral sequence reads as well as the breadth and depth of coverage of the viral genomes. Compared with clinical testing, MSS enhanced taxonomic assignment for 15 viruses, and codons associated with antiviral drug resistance in influenza A virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV) could be analysed. Overall, in clinical samples, MSS with targeted sequence capture provides enhanced virus detection and information of clinical and epidemiologic relevance compared with clinical testing and MSS without targeted sequence capture.
Sonication versus tissue sampling for diagnosis of prosthetic joint and other orthopedic device-related infections
Dudareva M, Barrett L, Figtree M, Scarborough M, Watanabe M, et al. J Clin Microbiol 2018; 56(12): pii: e00688-18
Current guidelines recommend collection of multiple tissue samples for diagnosis of prosthetic joint infections (PJI). Sonication of explanted devices has been proposed as a potentially simpler alternative; however, reported microbiological yield varies. We evaluated sonication for diagnosis of PJI and other orthopedic device-related infections (DRI) at the Oxford Bone Infection Unit between October 2012 and August 2016. We compared the performance of paired tissue and sonication cultures against a ‘gold standard’ of published clinical and composite clinical and microbiological definitions of infection. We analysed explanted devices and a median of five tissue specimens from 505 procedures. Among clinically infected cases the sensitivity of tissue and sonication culture was 69% (95% confidence interval, 63 to 75) and 57% (50 to 63), respectively (P<0.0001). Tissue culture was more sensitive than sonication for both PJI and other DRI, irrespective of the infection definition used. Tissue culture yield was higher for all subgroups except less virulent infections, among which tissue and sonication culture yield were similar. The combined sensitivity of tissue and sonication culture was 76% (70 to 81) and increased with the number of tissue specimens obtained. Tissue culture specificity was 97% (94 to 99), compared with 94% (90 to 97) for sonication (P=0.052) and 93% (89 to 96) for the two methods combined. Tissue culture is more sensitive and may be more specific than sonication for diagnosis of orthopedic DRI in our setting. Variable methodology and case mix may explain reported differences between centres in the relative yield of tissue and sonication culture. Culture yield was highest for both methods combined.
MODS-Wayne, a colorimetric adaptation of the microscopic-observation drug susceptibility (MODS) assay for detection of Mycobacterium tuberculosis pyrazinamide resistance from sputum samples
Alcántara R, Fuentes P, Antiparra R, Santos M, Gilman RH, et al. J Clin Microbiol 2019; 57(2): pii: e01162-18
Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a colour change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/mL, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.
Barriers and facilitators and the need for a clinical guideline for microbiological diagnostic testing in the hospital: a qualitative and quantitative study
Bogers SJ, van Daalen FV, Kuil SD, de Jong MD, Geerlings SE. Eur J Clin Microbiol Infect Dis 2019; doi: 10.1007/s10096-019-03516-z [Epub ahead of print]
The appropriate use of microbiological investigations is an important cornerstone of antibiotic stewardship programmes, but receives relatively limited attention. This study aimed to identify influencing factors in performing microbiological diagnostic tests and to assess the need for a clinical guideline. We performed a qualitative (focus group) and quantitative (online questionnaire survey) study among medical specialists and residents to identify physicians’ considerations in performing microbiological diagnostic tests and to assess the need for a diagnostic guideline. The questionnaire consisted of 14 statements, divided into three categories: knowledge, influencing factors and presence of guidelines. The questionnaire was sent to physicians of the departments of internal medicine, intensive care, pediatrics and pulmonology in five hospitals in the Netherlands. Sub-analyses for medical specialists versus residents and for pediatric versus non-pediatric departments were performed. We included 187 completed questionnaires in our analyses. The physicians reported having adequate knowledge on methods, time-to-result and accuracy, but inadequate knowledge on costs of the tests. Patients’ clinical condition, comorbidity, local guidelines and accuracy of tests were appraised as the four most important influencing factors to perform tests. Over 70% (132/187) of physicians reported being interested in a guideline for microbiological diagnostic testing. Fifteen physicians (8.0%) provided additional comments. This study identifies the influencing factors to microbiological testing and shows the demand for a clinical guideline among physicians. IMPORTANCE: Microbiological diagnostic tests are an important cornerstone within antibiotic stewardship programmes. These programmes aim to ameliorate the appropriate use of antibiotics and thus improve clinical outcomes of infectious diseases, whilst reducing the emergence of antimicrobial resistance. However, inappropriate microbiological testing is a widely recognized problem, and influencing factors to testing have not been studied in the past. Our research shows the demand for a clinical guideline among physicians, and it identifies their influencing factors to testing. These results can be used to create a clinical guideline for microbiological diagnostic testing, thus supporting antibiotic stewardship programmes and reducing antimicrobial resistance.
Understanding and overcoming the pitfalls and biases of next-generation sequencing (NGS) methods for use in the routine clinical microbiological diagnostic laboratory
Boers SA, Jansen R, Hays JP. Eur J Clin Microbiol Infect Dis 2019; doi: 10.1007/s10096-019-03520-3 [Epub ahead of print]
Recent advancements in next-generation sequencing (NGS) have provided the foundation for modern studies into the composition of microbial communities. The use of these NGS methods allows for the detection and identification of (‘difficult-to-culture’) microorganisms using a culture-independent strategy. In the field of routine clinical diagnostics, however, the application of NGS is currently limited to microbial strain typing for epidemiological purposes only, even though the implementation of NGS for microbial community analysis may yield clinically important information. This lack of NGS implementation is due to many different factors, including issues relating to NGS method standardization and result reproducibility. In this review article, the authors provide a general introduction to the most widely used NGS methods currently available (i.e. targeted amplicon sequencing and shotgun metagenomics) and the strengths and weaknesses of each method is discussed. The focus of the publication then shifts toward 16S rRNA gene NGS methods, which are currently the most cost-effective and widely used NGS methods for research purposes, and are therefore more likely to be successfully implemented into routine clinical diagnostics in the short term. In this respect, the experimental pitfalls and biases created at each step of the 16S rRNA gene NGS workflow are explained, as well as their potential solutions. Finally, a novel diagnostic microbiota profiling platform (‘MYcrobiota’) is introduced, which was developed by the authors by taking into consideration the pitfalls, biases, and solutions explained in this article. The development of the MYcrobiota, and future NGS methodologies, will help pave the way toward the successful implementation of NGS methodologies into routine clinical diagnostics.
A pan-genotypic Hepatitis C Virus NS5A amplification method for reliable genotyping and resistance testing
Walker A, Ennker KS, Kaiser R, Lübke N, Timm J. J Clin Virol 2019; 113: 8–13
BACKGROUND: Chronic infection with the Hepatitis C Virus (HCV) is associated with the risk of progressive liver disease. Although, HCV treatment options and viral cure rates have tremendously increased over the last decade, all currently licensed combination therapies contain inhibitors of the replication complex NS5A. Resistance-associated substitutions (RAS) in NS5A can limit the efficacy of therapy; however, resistance testing is routinely not recommended for all patients. Notably, pan-genotypic combinations have been approved; however, the correct identification of the HCV genotype is still required for treatment decisions and is a good predictor for treatment success.
OBJECTIVE: The aim of this study was the establishment of a pan-genotypic NS5A amplification method for reliable genotyping and simultaneous resistance testing in a fast and cheap routine diagnostic setup.
STUDY DESIGN: Pan-genotypic degenerated nested PCR primer were designed and tested in 262 HCV-patients. The collection included samples from genotypes 1–7 and the median viral load was 1.07×106 IU/mL (range 248–21×106 IU/mL).
RESULTS: Amplification of the expected 747 bp fragment was successful in 257 of 262 (98.1%) samples including samples <1000 IU/mL. The direct comparison of the genotype information obtained with core sequencing to those obtained by NS5A prediction showed high concordance (97.3%) and discrepancies occurred only for relatively rare subtypes. Resistance analysis using Geno2Pheno[HCV] showed NS5A-RAS in 23 of 257 (8.9%) of samples.
CONCLUSIONS: We successfully developed a routine diagnostic method for pan-genotypic amplification of NS5A. This amplicon can be used for simultaneous genotyping and resistance testing for enhancing and improving routine HCV diagnostic.
Impact of multiplex molecular assay turn-around-time on antibiotic utilization and clinical management of hospitalized children with acute respiratory tract infections
Lee BR, Hassan F, Jackson MA, Selvarangan R. J Clin Virol 2019; 110: 11–16
BACKGROUND: Empiric antibiotic treatment is common among children with acute respiratory tract infections (ARTI), despite infections being predominately viral. The use of molecular respiratory panel assays has become increasingly common for medical care of patients with ARTIs.
STUDY DESIGN: This was a 6-year retrospective, single-centred study of pediatric inpatients who tested positive for an ARTI respiratory pathogen. We examined the relationship between clinical outcomes and whether the patient was tested using the Luminex Respiratory Viral Panel ([RVP]; in-use: Dec 2009 – Jul 2012) or Biofire Respiratory Pathogen Panel ([RP]; in-use Aug 2012 – Jun 2016). The prevalence and duration of pre-test empiric antibiotics, post-test oseltamivir administration to influenza patients, chest X-rays and length of stay between the two assays was compared.
RESULTS: A total of 5142 patients (1264 RVP; 3878 RP) were included. The median laboratory turn-around-time for RP was significantly shorter than RVP (1.4 vs 27.1 h, respectively; P<0.001). Patients tested with RP were less likely to receive empiric antibiotics (OR: 0.45; P<0.001; 95% CI: 0.39, 0.52) and had a shorter duration of empiric broad-spectrum antibiotics (6.4 h vs 32.9 h; P<0.001) compared to RVP patients. RP influenza patients had increased oseltamivir use post- test compared to RVP influenza patients (OR: 13.56; P<0.001; 95% CI: 7.29, 25.20).
CONCLUSIONS: Rapid molecular testing positively impacts patient management of ARTIs. Adopting assays with a shorter turn-around-time improves decision making by decreasing empirical antibiotic use and duration, decreasing chest X-rays, increasing timely oseltamivir administration, and reducing length of stay.
Practical guidance for clinical microbiology laboratories: viruses causing acute respiratory tract infections
Charlton CL, Babady E, Ginocchio CC, Hatchette TF, Jerris RC, et al. Clin Microbiol Rev 2018; 32(1): pii: e00042-18
Respiratory viral infections are associated with a wide range of acute syndromes and infectious disease processes in children and adults worldwide. Many viruses are implicated in these infections, and these viruses are spread largely via respiratory means between humans but also occasionally from animals to humans. This article is an American Society for Microbiology (ASM)-sponsored Practical Guidance for Clinical Microbiology (PGCM) document identifying best practices for diagnosis and characterization of viruses that cause acute respiratory infections and replaces the most recent prior version of the ASM-sponsored Cumitech 21 document, Laboratory Diagnosis of Viral Respiratory Disease, published in 1986. The scope of the original document was quite broad, with an emphasis on clinical diagnosis of a wide variety of infectious agents and laboratory focus on antigen detection and viral culture. The new PGCM document is designed to be used by laboratorians in a wide variety of diagnostic and public health microbiology/virology laboratory settings worldwide. The article provides guidance to a rapidly changing field of diagnostics and outlines the epidemiology and clinical impact of acute respiratory viral infections, including preferred methods of specimen collection and current methods for diagnosis and characterization of viral pathogens causing acute respiratory tract infections. Compared to the case in 1986, molecular techniques are now the preferred diagnostic approaches for the detection of acute respiratory viruses, and they allow for automation, high-throughput workflows, and near-patient testing. These changes require quality assurance programs to prevent laboratory contamination as well as strong preanalytical screening approaches to utilize laboratory resources appropriately. Appropriate guidance from laboratorians to stakeholders will allow for appropriate specimen collection, as well as correct test ordering that will quickly identify highly transmissible emerging pathogens.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the rapid detection of antimicrobial resistance mechanisms and beyond
Oviaño M, Bou G. Clin Microbiol Rev 2018; 32(1): pii: e00037-18
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied in recent years for first-line identification of pathogens in clinical microbiology because it is simple to use, rapid, and accurate and has economic benefits in hospital management. The range of clinical applications of MALDI-TOF MS for bacterial isolates is increasing constantly, from species identification to the two most promising applications in the near future: detection of antimicrobial resistance and strain typing for epidemiological studies. The aim of this review is to outline the contribution of previous MALDI-TOF MS studies in relation to detection of antimicrobial resistance and to discuss potential future challenges in this field. Three main approaches are ready (or almost ready) for clinical use, including the detection of antibiotic modifications due to the enzymatic activity of bacteria, the detection of antimicrobial resistance by analysis of the peak patterns of bacteria or mass peak profiles, and the detection of resistance by semiquantification of bacterial growth in the presence of a given antibiotic. This review provides an expert guide for MALDI-TOF MS users to new approaches in the field of antimicrobial resistance detection, especially possible applications as a routine diagnostic tool in microbiology laboratories.
Detection of urinary microRNAs as biomarkers of diabetic kidney disease
, /in Featured Articles /by 3wmediaCurrent measures for diagnosis and therapy of chronic kidney disease are limited. Better biomarkers are required to improve treatment by directing therapeutic intervention, tracking responses to therapy and providing greater understanding of the underlying mechanisms driving renal disease progression. We describe here the development of microRNAs as biomarkers for diabetic kidney disease, the most common etiology leading to chronic kidney disease and end-stage renal failure.
by Dr Tanya A. Smith, Dr Kate Simpson, Prof Donald J. Fraser and Dr Timothy Bowen
Diabetes, complications and biomarkers
Diabetes is a major global health challenge, with 23.1 million cases diagnosed in the US alone [1]. As described below, our laboratory is currently developing urinary microRNAs as biomarkers for diabetic kidney disease. These transcripts may also have utility as biomarkers for other complications of type 2 diabetes mellitus including diabetic retinopathy, neuropathy, cardiovascular disease, stroke, ulceration and amputation [2].
Diabetic kidney disease
Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease in the United States. Clinical presentation is characterized by proteinuria, hypertension, and progressive reduction in kidney function. DKD is a progressive condition associated with around 35% of patients with type 1 and type 2 diabetes mellitus [3]. A highly significant public health concern, DKD is currently managed by targeting cardiovascular risk reduction, blood pressure management, glycemic control (hemoglobin A1c concentration), nutritional counselling, weight loss, smoking cessation, and pharmacological inhibition of the renin–angiotensin system using angiotensin-converting enzyme inhibitors or angiotensin-2 receptor blockers [4].
Despite the stabilization of the incidence of diabetes over the past 15 years, the United States Renal Data System has demonstrated increased prevalence of end-stage renal disease attributed to diabetes. However, the disease burden is such that patients often do not survive to end-stage renal disease. There is a broad spectrum of cardiovascular complications associated with DKD of which the underlying etiology remains unclear. Cardiovascular disease is the leading cause of death in this patient group, manifesting as cerebral vascular event, sudden cardiac death, myocardial infarction and diabetic cardiomyopathy. It is, therefore, essential to identify and treat patients before irreversible organ damage to reduce the medical and economic burden of disease [4].
Existing DKD biomarkers
DKD is associated with both glomerular hyperfiltration leading to progressive albuminuria, and declining glomerular filtration rate.
Albuminuria
Proteinuria is a biomarker used widely as a proxy to assess the integrity of the glomerular filtration barrier (for detailed glomerular and nephronal physiology see [5]). Quantification of urinary albuminuria excretion is a non-invasive and inexpensive method to monitor disease. Microalbuminuria is currently the primary predictive clinical DKD marker and occurs when urinary albuminuria excretion rate reaches 30–300 mg/24 h, macroalbuminuria is reached when this rate exceeds 300 mg/24 h. In the presence of diabetes mellitus, confirmation of microalbuminuria in two separate samples taken 3–6 months apart is diagnostic of DKD. Screening for albuminuria is more commonly performed using urinary albumin-to-creatinine ratio on an isolated urine sample, and is defined as >30 mg/g.
However, albuminuria is a non-specific biomarker measurable only after kidney injury has occurred and correlates poorly with clinical disease. In addition, albuminuria may be a transient DKD feature, or may occur only when widespread glomerular damage is already present [6, 7]. Recent reports have noted that up to 25% of patients with type 2 diabetes mellitus and diminished kidney function have little or no proteinuria, despite having biopsy-proven DKD [8]. There is, therefore, a need to find sensitive and specific biomarkers to predict DKD susceptibility and progression.
Estimated glomerular filtration rate
The Kidney Disease: Improving Global Outcomes (KDIGO) [4] classification is directed at adults and children over the age of 2 years old with evidence of kidney disease. Glomerular filtration rate (GFR) is considered the best measure of kidney function. Normal GFR is quantified as 100–150 ml/min and can be determined by creatinine clearance or an estimated GFR (eGFR) calculation basis on serum creatinine, age, sex and ethnicity (Table 1).
Histological features of renal biopsies, eGFR and DKD
Histological features (see [5]) correlate with functional alterations in DKD. The Renal Pathological Society system, based on glomerular changes observed in the development of DKD, groups both type 1 and type 2 diabetes mellitus patients into the four classes described below [9].
Class I: Glomerular basement membrane thickening: isolated glomerular basement membrane thickening and only mild, non-specific changes by light microscopy that do not meet the criteria of classes II–IV.
Class II: Mesangial expansion, mild (class IIa) or severe (class IIb). Glomeruli classified as mild or severe mesangial expansion but without nodular sclerosis (Kimmelstiel–Wilson lesions) or global glomerulosclerosis in >50% of glomeruli.
Class III: Nodular sclerosis (Kimmelstiel–Wilson lesions): at least one glomerulus with nodular increase in mesangial matrix (Kimmelstiel–Wilson) without changes described in class IV.
Class IV: Advanced diabetic glomerulosclerosis. Over 50% global glomerulosclerosis with other clinical or pathologic evidence showing that sclerosis is attributable to DKD.
The need for newer biomarkers
Current biomarkers do not relate well to the above pathological classification. Many potential novel biomarkers have been tested in an attempt to improve our ability to discern underlying renal pathology non-invasively, with the aim of guiding therapy. These include urinary transferrin, serum osteopontin, urinary retinol-binding protein (RBP), serum interleukin-18, serum cystatin C, serum resistin, serum TNF-α, serum interleukin-6 and urinary neutrophil gelatinase-associated lipocalin (NGAL) [reviewed in 6]. In patients with albuminuria these markers increase significantly, but their relationships with histopathological changes, eGFR, HBA1C and blood pressure is complex.
Detection and identification of microRNAs in body fluids as kidney disease biomarkers
Members of the short single-stranded endogenous RNA transcript family known as microRNAs (miRNAs) modulate the expression of most mammalian protein coding genes, thereby influencing developmental and metabolic processes, and disease phenotypes [10]. Disease-associated changes in miRNA expression profiles have been observed in cancer, cardiovascular disease, diabetes and chronic kidney disease that is treated by dialysis or transplantation [reviewed in 11–14].
To date, the majority of miRNA biomarker analyses have focused on detection of circulating transcripts [11, 13]. By contrast, the adoption into existing treatment pathways of a miRNA biomarker test on biofluid samples that can be obtained without venipuncture promises attractive reductions in time and cost [15].
We have developed RT-qPCR-based methods for precise quantification of miRNAs in urine, peritoneal dialysis effluent and renal transplantation perfusate [15–19]. The robust recovery of miRNAs from these complex analytical matrices highlights their potential utility both as non-invasive biomarkers of occurrence and/or progression of kidney disease, and as potential targets for therapeutic intervention. We have shown association of increased miR-21 with peritoneal fibrosis [17] and transplantation outcomes [18, 19]. Analysis of the renal transplantation perfusate with which the organ is supplied between donor and recipient also identified elevated miR-21 [18].
Utility of urinary miR-29b, miR-126 and miR-155 to test for DKD
Disease biomarkers are useful only when they can inform our potential to change patient treatment. The US Food and Drug Administration recommends that a reduction in eGFR of 40% over 2–3 years is a broadly acceptable effective surrogate for confirmation of CKD [20]. However, since eGFR decline is typically very gradual over the first decade or so of disease and more rapid thereafter, a biomarker that can differentiate between later stages of CKD maybe more cost-effective in detecting quantifiable responses to therapy in clinical trials [20, 21].
We have recently shown association of elevated urinary miR-29b, miR-126 and miR-155 detection predominantly in patients with type 2 diabetes mellitus and DKD [15]. We observed upregulation of these three miRNAs in two disease cohorts, obtaining an area under the curve of 0.8 in combined receiver operating characteristic curve analysis [15]. Our markers are clustered in late-stage disease (Fig. 1) and at an 80% relative quantification threshold for each miRNA, identified 48% of DKD patients with a 3.6% false positive detection rate [15]. We are currently investigating the significance of this apparent DKD patient stratification.
Utility of urinary miR-29b, miR-126 and miR-155 to investigate DKD mechanisms
We detected increased miR-29b and miR-126 in conditioned medium from cultured glomerular endothelial cells exposed to disease-related cytokines transforming growth factor-β1 and tumour necrosis factor-α, respectively [15]. It is thus conceivable that miRNAs may travel down the nephron [5] to mediate disease-related and functional effects [22]. Our data also included evidence for decreased urinary miR-192 in DKD [15], supporting our previous finding showing downregulated miR-192 expression in renal biopsies from DKD patients [23].
Conclusion
DKD is one of the most important global health challenges. Existing biomarkers provide a non-invasive approach to diagnosis and, in late-stage disease, identify the extent of kidney damage. However, there is a lack of non-invasive measures of active disease processes. New biomarkers are, therefore, required to measure risk of progressive kidney damage and to measure responses to treatment in the individual. Successful development of such biomarkers would help to individualize treatment using existing approaches, and would greatly accelerate testing of new treatments. MicroRNAs tested in urine show promise in this area.
Acknowledgments
Supported by the National Institute for Health Research Invention for Innovation (i4i) Programme grant II-LA-0712-20003 and Kidney Research UK Project grant award RP44/2014. The Wales Kidney Research Unit is funded by core support from Health and Care Research Wales.
Disclosure
TB and DF are inventors for patent WO/2017/129977 Chronic Kidney Disease Diagnostic.
References
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The authors
Tanya A. Smith MB ChB; Kate Simpson PhD; Donald J. Fraser MB ChB, PhD; Timothy Bowen* PhD
Wales Kidney Research Unit, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
*Corresponding author
E-mail: bowent@cardiff.ac.uk
Scientific literature review: Diabetes
, /in Featured Articles /by 3wmediaSkin autofluorescence predicts incident type 2 diabetes, cardiovascular disease and mortality in the general population
van Waateringe RP, Fokkens BT, Slagter SN, van der Klauw MM, van Vliet-Ostaptchouk JV, et al. Diabetologia 2019; 62(2): 269–280
AIMS/HYPOTHESIS: Earlier studies have shown that skin autofluorescence measured with an AGE reader estimates the accumulation of AGEs in the skin, which increases with ageing and is associated with the metabolic syndrome and type 2 diabetes. In the present study, we examined whether the measurement of skin autofluorescence can predict 4-year risk of incident type 2 diabetes, cardiovascular disease (CVD) and mortality in the general population.
METHODS: For this prospective analysis, we included 72 880 participants of the Dutch Lifelines Cohort Study, who underwent baseline investigations between 2007 and 2013, had validated baseline skin autofluorescence values available and were not known to have diabetes or CVD. Individuals were diagnosed with incident type 2 diabetes by self-report or by a fasting blood glucose ≥7.0 mmol/L or HbA1c ≥48 mmol/mol (≥6.5%) at follow-up. Participants were diagnosed as having incident CVD (myocardial infarction, coronary interventions, cerebrovascular accident, transient ischaemic attack, intermittent claudication or vascular surgery) by self-report. Mortality was ascertained using the Municipal Personal Records Database.
RESULTS: After a median follow-up of 4 years (range 0.5–10 years), 1056 participants (1.4%) had developed type 2 diabetes, 1258 individuals (1.7%) were diagnosed with CVD, while 928 (1.3%) had died. Baseline skin autofluorescence was elevated in participants with incident type 2 diabetes and/or CVD and in those who had died (all P< 0.001), compared with individuals who survived and remained free of the two diseases. Skin autofluorescence predicted the development of type 2 diabetes, CVD and mortality, independent of several traditional risk factors, such as the metabolic syndrome, glucose and HbA1c.
CONCLUSIONS/INTERPRETATION: The non-invasive skin autofluorescence measurement is of clinical value for screening for future risk of type 2 diabetes, CVD and mortality, independent of glycaemic measures and the metabolic syndrome.
A renal genetic risk score (GRS) is associated with kidney dysfunction in people with type 2 diabetes
Zusi C, Trombetta M, Bonetti S, Dauriz M, Boselli ML, et al. Diabetes Res Clin Pract 2018; 144: 137–143
This study aims to investigate whether renal and cardiovascular phenotypes in Italian patients with type 2 diabetes (T2D) could be influenced by a number of disease risk SNPs recently found in genome-wide association studies (GWAS). In 1591 Italian subjects with T2D: (1) 47SNPs associated to kidney function and/or chronic kidney disease (CKD) and 49SNPs associated to cardiovascular disease (CVD) risk were genotyped; (2) urinary albumin/creatinine (A/C) ratio, glomerular filtration rate (eGFR) and lipid profile were assessed; (3) a standard electrocardiogram was performed; (4) two genotype risk scores (GRS) were computed (a renal GRS calculated selecting 39 SNPs associated with intermediate traits of kidney damage and a cardiovascular GRS determined selecting 42 SNPs associated to CVD risk phenotypes). After correction for multiple comparisons, the renal GRS was not associated to A/C ratio (P=0.33), but it was significantly related to decreased eGFR (P=0.005). No association between the cardiovascular GRS and electrocardiogram was detected. Thus, in Italian patients with T2D a renal GRS might predict the decline in glomerular function, suggesting that the clock of diabetes associated CKD starts ticking long before hyperglycemia. Our data support the feasibility of gene-based prediction of complications in people with T2D.
Protein markers and risk of type 2 diabetes and prediabetes: a targeted proteomics approach in the KORA F4/FF4 study
Huth C, von Toerne C, Schederecker F, de Las Heras Gala T, Herder C, et al. Eur J Epidemiol 2018: doi: 10.1007/s10654-018-0475-8 [Epub ahead of print]
The objective of the present study was to identify proteins that contribute to pathophysiology and allow prediction of incident type 2 diabetes or incident prediabetes. We quantified 14 candidate proteins using targeted mass spectrometry in plasma samples of the prospective, population-based German KORA F4/FF4 study (6.5-year follow-up). 892 participants aged 42–81 years were selected using a case-cohort design, including 123 persons with incident type 2 diabetes and 255 persons with incident WHO-defined prediabetes. Prospective associations between protein levels and diabetes, prediabetes as well as continuous fasting and 2 h glucose, fasting insulin and insulin resistance were investigated using regression models adjusted for established risk factors. The best predictive panel of proteins on top of a non-invasive risk factor model or on top of HbA1c, age and sex was selected. Mannan-binding lectin serine peptidase (MASP) levels were positively associated with both incident type 2 diabetes and prediabetes. Adiponectin was inversely associated with incident type 2 diabetes. MASP, adiponectin, apolipoprotein A-IV, apolipoprotein C-II, C-reactive protein, and glycosylphosphatidylinositol specific phospholipase D1 were associated with individual continuous outcomes. The combination of MASP, apolipoprotein E (apoE) and adiponectin improved diabetes prediction on top of both reference models, while prediabetes prediction was improved by MASP plus CRP on top of the HbA1c model. In conclusion, our mass spectrometric approach revealed a novel association of MASP with incident type 2 diabetes and incident prediabetes. In combination, MASP, adiponectin and apoE improved type 2 diabetes prediction beyond non-invasive risk factors or HbA1c, age and sex.
Association between circulating tumor necrosis factor-related biomarkers and estimated glomerular filtration rate in type 2 diabetes.
Kamei N, Yamashita M, Nishizaki Y, Yanagisawa N, Nojiri S, et al. Sci Rep 2018; 8(1): 15302
Chronic inflammation plays a crucial role in the development/progression of diabetic kidney disease. The involvement of tumor necrosis factor (TNF)-related biomarkers [TNFα, progranulin (PGRN), TNF receptors (TNFR1 and TNFR2)] and uric acid (UA) in renal function decline was investigated in patients with type 2 diabetes (T2D). Serum TNF-related biomarkers and UA levels were measured in 594 Japanese patients with T2D and an eGFR ≥30 mL/min/1.73 m2. Four TNF-related biomarkers and UA were negatively associated with estimated glomerular filtration rate (eGFR). In a logistic multivariate model, each TNF-related biomarker and UA was associated with lower eGFR (eGFR <60 mL/min/1.73 m2) after adjustment for relevant covariates (basic model). Furthermore, UA and TNF-related biomarkers other than PGRN added a significant benefit for the risk factors of lower eGFR when measured together with a basic model (UA, ΔAUC, 0.049, P<0.001; TNFα, ΔAUC, 0.022, P=0.007; TNFR1, ΔAUC, 0.064, P<0.001; TNFR2, ΔAUC, 0.052, P<0.001) in receiver operating characteristic curve analysis. TNFR ligands were associated with lower eGFR, but the associations were not as strong as those with TNFRs or UA in patients with T2D and an eGFR ≥30 mL/min/1.73 m2.
Plasma endostatin predicts kidney outcomes in patients with type 2 diabetes
Chauhan K, Verghese DA, Rao V, Chan L, Parikh CR, et al. Kidney Int 2019; 95(2): 439–446
Novel biomarkers are needed to predict kidney function decline in patients with type 2 diabetes, especially those with preserved glomerular filtration rate (GFR). There are limited data on the association of markers of endothelial dysfunction with longitudinal GFR decline. We used banked specimens from a nested case-control study in the Action to Control Cardiovascular Disease (ACCORD) trial (n=187 cases; 187 controls) and from a diverse contemporary cohort of type 2 diabetic patients from the Mount Sinai BioMe Biobank (n=871) to assess the association of plasma endostatin and kidney outcomes. We measured plasma endostatin at enrolment and examined its association with a composite kidney outcome of sustained 40% decline in estimated GFR or end-stage renal disease. Baseline plasma endostatin levels were higher in participants with the composite outcome. Each log2 increment in plasma endostatin was associated with approximately 2.5-fold higher risk of the kidney outcome (adjusted odds ratio [OR] 2.5; 95% confidence interval [CI] 1.5–4.3 in ACCORD and adjusted hazard ratio [HR] 2.6; 95% CI 1.8-3.8 in BioMe). Participants in the highest versus lowest quartile of plasma endostatin had approximately fourfold higher risk for the kidney outcome (adjusted OR 3.6; 95% CI 1.8-7.3 in ACCORD and adjusted HR 4.4; 95% CI 2.3-8.5 in BioMe). The AUC for the kidney outcome improved from 0.74 to 0.77 in BioMe with the addition of endostatin to a base clinical model. Plasma endostatin was strongly associated with kidney outcomes in type 2 diabetics with preserved eGFR and improved risk discrimination over traditional predictors
Relation of serum and urine renal biomarkers to cardiovascular risk in patients with type 2 diabetes mellitus and recent acute coronary syndromes (from the EXAMINE Trial)
Vaduganathan M, White WB, Charytan DM, Morrow DA, Liu Y, et al. Am J Cardiol 2019; 123(3): 382–391
A deeper understanding of the interplay between the renal axis and cardiovascular (CV) disease is needed in type 2 diabetes mellitus (T2DM). We aimed to explore the prognostic value of a comprehensive panel of renal biomarkers in patients with T2DM at high CV risk. We evaluated the prognostic performance of both serum (Cystatin C) and urine renal biomarkers (neutrophil gelatinase-associated lipocalin, kidney injury molecule-1 protein, and indices of urinary protein excretion) in 5380 patients with T2DM and recent acute coronary syndromes in the EXAMINE trial. Patients requiring dialysis within 14 days were excluded. Single- and multimarker covariate-adjusted Cox proportional hazards models were developed to predict times to events. Primary endpoint was composite nonfatal myocardial infarction, nonfatal stroke, or CV death. Median age was 61 years, 68% were men, and mean baseline estimated glomerular filtration rate (eGFR) was 74 mL/min/1.73 m2. During median follow-up of 18 months, 621 (11.5%) experienced the primary endpoint and 326 (6.1%) patients had died. All renal biomarkers were robustly associated with adverse CV events in step-wise fashion, independent of baseline eGFR. However, in the multimarker prediction model, only Cystatin C (per 1 SD) was associated with the primary endpoint (hazard ratio [HR] 1.28 [1.14 to 1.45]; P≤0.001), death (HR 1.51 [1.30 to 1.74]; P≤0.001), and heart failure hospitalization (HR 1.20 [0.96 to 1.49]; P=0.11). Association between Cystatin C and the primary endpoint was similar in baseline eGFR above and below 60 mL/min/1.73 m2 (Pinteraction >0.05). In conclusion, serum and urine renal biomarkers, when tested alone, independently predict long-term adverse CV events in high-risk patients with T2DM. In an integrative panel of renal biomarkers, only serum Cystatin C remained independently associated with subsequent CV risk. Renal biomarkers informing various aspects of kidney function may further our understanding of the complex interplay between diabetic kidney disease and CV disease.
A plasma circulating miRNAs profile predicts type 2 diabetes mellitus and prediabetes: from the CORDIOPREV study
Jiménez-Lucena R, Camargo A, Alcalá-Diaz JF, Romero-Baldonado C, Luque RM, et al. Exp Mol Med 2018; 50(12): 168
We aimed to explore whether changes in circulating levels of miRNAs according to type 2 diabetes mellitus (T2DM) or prediabetes status could be used as biomarkers to evaluate the risk of developing the disease. The study included 462 patients without T2DM at baseline from the CORDIOPREV trial. After a median follow-up of 60 months, 107 of the subjects developed T2DM, 30 developed prediabetes, 223 maintained prediabetes and 78 remained disease-free. Plasma levels of four miRNAs related to insulin signalling and beta-cell function were measured by RT-PCR. We analysed the relationship between miRNAs levels and insulin signalling and release indexes at baseline and after the follow-up period. The risk of developing disease based on tertiles (T1-T2-T3) of baseline miRNAs levels was evaluated by Cox analysis. Thus, we observed higher miR-150 and miR-30a-5p and lower miR-15a and miR-375 baseline levels in subjects with T2DM than in disease-free subjects. Patients with high miR-150 and miR-30a-5p baseline levels had lower disposition index (P=0.047 and P=0.007, respectively). The higher risk of disease was associated with high levels (T3) of miR-150 and miR-30a-5p (HRT3-T1 = 4.218 and HRT3-T1=2.527, respectively) and low levels (T1) of miR-15a and miR-375 (HRT1-T3 = 3.269 and HRT1-T3=1.604, respectively). In conclusion, our study showed that deregulated plasma levels of miR-150, miR-30a-5p, miR-15a, and miR-375 were observed years before the onset of T2DM and pre-DM and could be used to evaluate the risk of developing the disease, which may improve prediction and prevention among individuals at high risk for T2DM.
Emerging biomarkers, tools, and treatments for diabetic polyneuropathy
Bönhof GJ, Herder C, Strom A, Papanas N, Roden M, Ziegler D. Endocr Rev. 2019; 40(1): 153–192
Diabetic neuropathy, with its major clinical sequels, notably neuropathic pain, foot ulcers, and autonomic dysfunction, is associated with substantial morbidity, increased risk of mortality, and reduced quality of life. Despite its major clinical impact, diabetic neuropathy remains underdiagnosed and undertreated. Moreover, the evidence supporting a benefit for causal treatment is weak at least in patients with type 2 diabetes, and current pharmacotherapy is largely limited to symptomatic treatment options. Thus, a better understanding of the underlying pathophysiology is mandatory for translation into new diagnostic and treatment approaches. Improved knowledge about pathogenic pathways implicated in the development of diabetic neuropathy could lead to novel diagnostic techniques that have the potential of improving the early detection of neuropathy in diabetes and prediabetes to eventually embark on new treatment strategies. In this review, we first provide an overview on the current clinical aspects and illustrate the pathogenetic concepts of (pre)diabetic neuropathy. We then describe the biomarkers emerging from these concepts and novel diagnostic tools and appraise their utility in the early detection and prediction of predominantly distal sensorimotor polyneuropathy. Finally, we discuss the evidence for and limitations of the current and novel therapy options with particular emphasis on lifestyle modification and pathogenesis-derived treatment approaches. Altogether, recent years have brought forth a multitude of emerging biomarkers reflecting different pathogenic pathways such as oxidative stress and inflammation and diagnostic tools for an early detection and prediction of (pre)diabetic neuropathy. Ultimately, these insights should culminate in improving our therapeutic armamentarium against this common and debilitating or even life-threatening condition.
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