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
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, /in Featured Articles /by 3wmediaHemochromatosis: 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.
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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].
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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