Diagnostic Microbiology and Infectious Disease 75 (2013) 135–138

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Diagnostic Microbiology and Infectious Disease

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ELISA and immuno–polymerase chain reaction assays for the sensitive detection ofmelioidosis☆,☆☆

Alanna Cooper a, Natasha L. Williams b, Jodie L. Morris b, Robert E. Norton c, Natkunam Ketheesan b,Patrick M. Schaeffer a,d,⁎a School of Pharmacy and Molecular Sciences, James Cook University, Douglas QLD 4811, Australiab Infectious Diseases and Immunopathogenesis Research Graduate School of Veterinary and Biomedical Sciences, James Cook University, Douglas QLD 4811, Australiac Pathology Queensland, The Townsville Hospital, Douglas QLD 4811, Australiad Comparative Genomics Centre, James Cook University, Douglas QLD 4811, Australia

☆ Conflict of interest: The authors declare that they h☆☆ Support: This work was supported by grants from thResearch Council (Australia), Queensland Tropical HealFund (NIRAP, Queensland, Australia).

⁎ Corresponding author. Tel.: +61-7-4781-6388; faxE-mail address: patrick.schaeffer@jcu.edu.au (P.M. S

0732-8893/$ – see front matter © 2013 Elsevier Inc. Alhttp://dx.doi.org/10.1016/j.diagmicrobio.2012.10.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 September 2012Received in revised form 17 October 2012Accepted 17 October 2012Available online 21 November 2012

Keywords:MelioidosisBurkholderia pseudomalleiSerologyIndirect haemagglutination assayImmuno-diagnosticsTus-Ter-LockImmuno-PCRLipopolysaccharide

Melioidosis is caused by the Gram-negative bacterium Burkholderia pseudomallei. The gold standard fordiagnosis is culture, which requires at least 3–4 days to obtain a result, hindering successful treatment of acutedisease. An indirect haemagglutination assay (IHA) is often used but lacks sensitivity. Approximately half ofpatients later confirmed culture positive are not detected by IHA at presentation and a subset of patientspersistently continue to be IHA negative. More rapid and reliable serologic testing for melioidosis is essentialand will improve diagnosis and patient outcome. We have developed an ELISA and a quantitative immuno-polymerase chain reaction assay capable of detecting melioidosis-specific antibodies and demonstrate theirvalidity with IHA-negative sera from patients with melioidosis. These new sensitive assays are based upon asecreted antigenic fraction from B. pseudomallei and will be ideal for the diagnosis of melioidosis in patients innonendemic regions returning from endemic tropical areas and for seroepidemiologic surveys.

ave no conflict of interest.e National Health andMedicalth Alliance, and Smart Futures

: +61-7-4781-6078.chaeffer).

l rights reserved.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Melioidosis is caused by the Gram-negative bacillus Burkholderiapseudomallei, an environmental saprophyte endemic in tropical areas,typically between latitudes 20°N and 20°S (Inglis and Sagripanti,2006). B. pseudomallei is also classified as a category B biothreat agent(Rotz et al., 2002). In the case of acute infections, bacterial sepsis candevelop in a few days and requires immediate treatment with thecorrect antibiotics as this bacterium is highly drug resistant (White,2003). Death usually follows within a few days if proper treatment isnot rapidly applied. When correct antibiotics are administered,patient survival levels are still only approx. 50%. Melioidosis hasvariable presentations that often mimic other infectious diseases(White, 2003) further complicating the diagnosis of this disease.

The current gold standard for diagnosis is culture, whichoften requires enrichment followed by several days’ incubation

(Limmathurotsakul et al., 2010). This can delay the administrationof antibiotics and can result in death if bacterial sepsis hasdeveloped. Unfortunately, the use of serologic techniques isproblematic in some endemic areas due to high backgroundseropositivity in the healthy population (Limmathurotsakul andPeacock, 2011). However, in northern Queensland the seropreva-lence of antibodies to B. pseudomallei is relatively low at approx-imately 2.5% (Lazzaroni et al., 2008), making serology a potentiallyadvantageous addition to culture in the diagnosis of melioidosis inthis region. Additionally, serology would be very effective indiagnosing travelers and defence personnel returning from endemictropical areas.

The most common serologic test used is the indirect haemagglu-tination assay (IHA) (Ashdown, 1987). A titre of 1:40 or greater isconsidered to be reactive according to Australian diagnostic standards(Ashdown and Guard, 1984). The pattern of IHA responses varies withapproximately half of patients, later confirmed culture positive, notdetected by IHA at presentation, and a subset of patients found to bepersistently IHA negative (Harris et al., 2009). The use of isolates fromculture-positive IHA-negative patients as antigen in IHA does notimprove sensitivity, indicating these patients do not developantibodies that bind to the epitopes adsorbed on the sheeperythrocytes used in IHA (Harris et al., 2011). However, the same

136 A. Cooper et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 135–138

patients have been demonstrated to have specific immunity to B.pseudomallei (Harris et al., 2011).

We hypothesized that antigens secreted at early stages of infectionmay contain epitopes that are not present in IHA antigenicpreparations. Therefore, using the secreted antigenic fractions inassays would increase the proportion of patients diagnosed byserology. It was also hypothesized that using more sensitivemethodologies such as ELISA and immuno–polymerase chain reaction(PCR) in combination with early-stage secreted antigens wouldincrease the detection of antibodies specific for B. pseudomallei.

Here we describe the development of 2 new indirect immunoas-says derived from the ELISA and quantitative immuno-PCR (qIPCR)platforms (Morin et al., 2010, 2011). These assays are based upon anantigenic fraction from K96243 B. pseudomallei isolate, obtained witha novel extraction technique and are capable of detecting antibodiesin patient sera previously found to be IHA-negative.

2. Material and methods

2.1. Isolation of antigenic fraction

Antigenic fractions were isolated by streaking glycerol stocks ofK96243 B. pseudomallei isolate and DH12S E. coli onto separate LB agarplates and incubating at 37 °C for 24 h. Overnight cultures (5mL)wereinoculated into separate 1-L flasks with 200 mL sterile LB media andincubated at 37 °C with shaking at 150 rpm until log phase wasreached. Bacteria were pelleted and culture supernatants wereremoved and passed through a 0.2-μm filter. Antigenic fractionswere extracted from the culture supernatant by ammonium sulphate((NH4)2SO4) precipitation at a concentration of 0.5 g/mL. Pellets wereresuspended in 10 mmol/L phosphate buffer (pH 7.4; 2 mL per 25 mLculture supernatant) and stored at −20 °C for later use. The proteincontent of antigenic fractions was determined by Bradford assay. Thelipopolysaccharide (LPS) content of antigenic fractions was deter-mined using a phenol-sulphuric acid total carbohydrate quantificationmethod (Fox and Robyt, 1991).

2.2. Sera

IHA-positive patients and patients with persistently IHA-nonreac-tive sera who had culture-proven melioidosis were requested toprovide serial blood samples. Ethical approval for collection of serawas obtained from the Townsville Health Service District EthicsCommittee (nos. 2502 and 7104). A total of 10 serum samples from 3IHA-positive and 3 IHA-negative melioidosis patients were testedalong with sera from 6 healthy controls. For 3 of the patients, multiplesamples (presentation and follow-up sera) were available. Serumsamples were initially provided and tested in blind fashion, thenidentified and confirmed subsequently.

2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining were performed by combining precipitat-ed secreted fractions 1:1 with 2× Laemli buffer (50 mmol/L Tris-HCl[pH 6.8], 2% SDS, 10% glycerol, 0.01% bromophenol blue) andheating at 95 °C for 5 min, then fractionating on a 10% acrylamidestacking gel at 150 V for 45 min. SDS-PAGE–fractionated secretedfraction samples were stained using a standard silver stainingmethod (Mortz et al., 2001). The LPS content of SDS-PAGE–fractionated secreted fraction samples was visualized by stainingwith a modified silver staining method, specific for LPS (Fomsgaardet al., 1990).

2.4. Immunoblotting

Immunoblotting was performed by transferring samples ontopolyvinylidene difluoride (PVDF) membrane pre-wet with methanolfor 30 s (Biorad, Australia) via semi-dry electroblotting at 15 V for 25min. Following blocking with 5% skim milk in phosphate-bufferedsaline (PBS; pH 7.4) at room temperature (RT) for 1h, blots wereprobed with either pooled human serum from 10 culture-confirmedmelioidosis patients or pooled human serum from 10 healthy controlsat RT for 1 h. Serumwas diluted 1:100 in 1% skimmilk in PBS (pH 7.4).After washing 3 times for 5 min, PBS (pH 7.4)–0.05% Tween-20 (PBS-T) blots were probed with peroxidase-conjugated protein G (Sigma,Australia) diluted 1:5000 in 1% skimmilk in PBS (pH 7.4) for 1 h at RT.PVDF membranes were washed again 3 times with PBS-T anddeveloped with 5 mL SIGMAFAST™ (Sigma, Australia) 3,3′-diamino-benzidine/H2O2 solution for 10 min.

2.5. Indirect peroxidase-conjugated protein G ELISA: G-peroxidase ELISA

G-peroxidase ELISA was performed in Nunc Maxisorp 96-wellround-bottom immunoplates (Nunc, Germany), coated with 50 μLantigenic fraction at 2 μg/mL total carbohydrate content in 100 mmol/L carbonate/bicarbonate buffer (pH 9.6) overnight at 4 °C. Wells wereblocked with 50 μL 1% bovine serum albumin (BSA) in binding andwash (BW) buffer (20 mmol/L Tris [pH 8], 150 mmol/L NaCl, 0.005%Tween-20) at RT for 1 h. Human serum (50 μL) was applied at 1:200 inBW and incubated at RT for 1 h. Positive and negative control serawere included. After washing 3 times with BW, 50 μL peroxidase-conjugated protein G (Sigma, Australia) diluted 1:5000 in BW wasapplied for 1 h at RT. Wells were washed again 3 times with BW anddeveloped with 3,3′,5,5′ tetramethylbenzidine for 5 min. Opticaldensities (OD) were measured at 450 nm with a Bio-strategy VersaMax microplate reader and corrected by subtracting backgroundvalues, which were obtained by omitting serum. All samples weretested in duplicate.

2.6. Indirect TT-lock qIPCR

qIPCR was performed using a modification of the indirect TT-lockqIPCR (Morin et al., 2010). Nunc Maxisorp 96-well round-bottomimmunoplates (Nunc) were coated with antigenic fraction as forELISA. Wells were blocked with 50 μL 1% BSA in BW buffer at RT for 1h. Human serumwas applied at 1:800 in BW and incubated at RT for 1h. Positive and negative control sera were included. After washing 3times with BW, 50 μL pre-assembled G-Tus detection device at 0.4nmol/L in 1% BSA in BWwas applied to wells and incubated at RT for 1h. After washing 5 times with BW, 50 μL amplification primers (JCU39and JCU40) were applied to wells at 0.5 μmol/L and incubated at RT for1 h. qPCR was performed as described previously (Morin et al., 2010).Cycle thresholds were measured and corrected by subtracting thebackground cycle threshold (ΔCt), which was obtained by omittingserum. All samples were tested in duplicate.

3. Results

3.1. Isolation of secreted antigens

Silver staining following SDS-PAGE indicated that the secretion ofproteins into the culture supernatant was lower in B. pseudomalleicompared to E. coli at log phase (Fig. 1A). Immunoblotting withpooled confirmed-melioidosis positive control sera revealed severalimmunogenic bands in a ladder pattern characteristic of LPS in the B.pseudomallei secreted fraction (Fig. 1B). In a similar experiment usinga pool of negative control sera, no bands were detected in the B.pseudomallei secreted fraction (data not shown). This was not thecase for the E. coli secreted fractions. Three bands were detected in

Fig. 1. Silver staining (A) and immunoblotting (B) of B. pseudomallei K96243 and E. coliDH12s secreted fractions. Lanes 1 and 2 represent B. pseudomallei and E. coli secretedfractions precipitated with 0.5 g/mL (NH4)2SO4, respectively. Immunoblot shown (B)was probed with confirmed positive pooled sera from melioidosis patients.

Fig. 2. Comparison between indirect G-peroxidase ELISA (A) and indirect TT-lock qIPCR(B) for detection of IgG against K96243 B. pseudomallei antigenic fraction in melioidosispatient sera. Diagrams to the right of each graph represent the methodology for eachassay. For the ELISA and qIPCR, an OD450 ratio between the sample and negative controlof N1.5 and a ΔCt of N2.0, respectively, were considered to be seropositive. Thedifference between melioidosis patient sera (n=10) and negative control sera (n=6)in OD450 and ΔCt was statistically significant (P b 0.05).

Table 1Serologic assay results for culture-confirmed melioidosis patients.

Status Patient no. Sample no. IHAa ELISAb qIPCRc

IHA positive 1 1 320 2.3 2.42 2560 8.8 4.03 320 7.0 4.5

2 1 160 7.4 3.52 640 9.2 3.5

3 1 1280 14.2 5.2IHA negative 4 1 Negative 2.5 3.2

2 Negative 4.3 3.75 1 Negative 1.6 2.36 1 Negative 2.0 2.5

Negative control 1 1 Negative 0.9 1.62 1 Negative 0.8 1.43 1 Negative 0.9 1.64 1 Negative 1.0 1.65 1 Negative 0.9 1.36 1 Negative 0.8 1.4

Values for ELISA represent the OD450 ratio between each sample and the negativecontrol. Values for qIPCR indicate cycles above background (i.e., cycle threshold osample subtracted from the cycle threshold of background).

a Indirect haemagglutination assay (IHA of ≥40 is considered positive).b Indirect G-peroxidase ELISA against K96243 secreted fraction using protein G-

peroxidase.c Indirect TT-lock qIPCR against K96243 secreted fraction.

137A. Cooper et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 135–138

f

the E. coli fractions with both melioidosis-positive sera (cf. lane2 inFig. 1B) and negative control sera (data not shown). The nature ofthese cross-reacting antigenic molecules in E. coli is unknown, andthey do not correspond to any of the B. pseudomallei bands. Thiscould be the result of a previous or co-infection by E. coli of some ofthe negative and positive control patients. Nevertheless, this doesnot represent a specificity issue in our assays, as our B. pseudomalleiantigenic fractions have not been produced in E. coli and therefore donot contain these molecules.

3.2. Detection of B. pseudomallei K96243 antigens by indirectG-peroxidase ELISA and TT-lock qIPCR

Both systems were based on the detection of IgG by protein Gconjugated to a signal generation system consisting of either theenzymatic activity of peroxidase or a quantitative PCR read-out in thecase of Tus and the TT-lock-T DNA probe (Morin et al., 2010, 2011).Serum dilutions of 1:200 and 1:800 were found to be optimal fordetection of antibodies by G-peroxidase ELISA and TT-lock qIPCR,respectively, via checkerboard ELISA with positive and negativecontrol sera. An OD450 ratio between the sample and negative controlof N1.5 and a qPCR ΔCt of N2.0 were considered to be seropositive forthe G-peroxidase ELISA and TT-lock qIPCR, respectively. Cut-off valueswere set at 2 SDs above the OD450 and ΔCt of the negative control ineach assay. All patient sera, including that collected at presentationand in subsequent follow-up collections, reacted against the B.pseudomallei antigenic fraction in both the G-peroxidase ELISA(Fig. 2A) and TT-lock qIPCR format (Fig. 2B). Individual negativecontrol sera from 6 healthy controls did not react against the B.pseudomallei antigenic fraction in either assay. A summary of results,including IHA results (performed by Pathology Queensland, TheTownsville Hospital) for each patient, is provided in Table 1.Importantly, patient 4 was IHA negative at the 2 time points tested,and patients 5 and 6 were persistently IHA negative.

4. Discussion

The relatively poor sensitivity of IHA has led to the development ofmore sensitive and specific assays to detect antibodies to B.pseudomallei. Protein-based EIAs have been found to generally havegreater sensitivity and specificity than IHA (Felgner et al., 2009;Chantratita et al., 2007). However, many seroreactive proteins in

138 A. Cooper et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 135–138

B. pseudomallei have homologs in other bacteria with similar proteinsequences, increasing the possibility of cross-reactivity. LPS andcapsular polysaccharide are considered to be major antigenicmolecules. Interestingly, in a recent report, LPS was not found to beantigenic in mice (Nuti et al., 2011). Several ELISAs have beendeveloped using LPS as antigen, such as a competitive ELISA using amonoclonal antibody (Thepthai et al., 2005) and an indirect ELISA forIgM detection (Anandan et al., 2010).

In our study, relatively few proteins were found to be reactive inthe secreted antigenic fractions of B. pseudomallei K96243. However,reactivity to the LPS was strong (cf. Fig. 1B), confirming its importanceas an antigenic component of B. pseudomallei. Both new immunoassayplatforms target the detection of LPS-specific human IgG and wereable to confirm the diagnosis of all IHA-negative patients included inthis study. The previously described LPS-based immunoassays alsouse B. pseudomallei K96243 isolates (Anandan et al., 2010; Thepthaiet al., 2005). Unfortunately, these IgM-based ELISAs have been foundto have limited clinical utility for the diagnosis of B. pseudomallei(Anandan et al., 2010; Thepthai et al., 2005; Chenthamarakshan et al.,2001). The competitive ELISA relies on a monoclonal antibody thatrecognizes a single epitope on smooth phenotype B. pseudomallei LPS,increasing the possibility of false negatives in patients who haveraised antibodies against other LPS epitopes.

Studies have demonstrated heterogeneity in LPS among B.pseudomallei isolates with 4 phenotypes identified, 3 of which areantigenically distinct (Anuntagool et al., 2006; Tuanyok et al., 2012).Therefore, insufficient coverage of LPS phenotypes in assays couldresult in false negatives, particularly in Australia where atypical LPSphenotypes are more common (Tuanyok et al., 2012). For this reason,future versions of the currently developed ELISA and qIPCR assays willinclude representative LPS from all known LPS phenotypes found inclinical isolates in Australia.

Although the indirect TT-lock qIPCR system is slightly slower thanthe ELISA format, it requires the least amount of serum and would beadvantageous when multiple testing is required for the confirmationof disease. The ability to detect antibodies in patient sera that werepersistently IHA negative is very promising, indicating that bothimmunoassay formats will be ideal for the rapid and reliable diagnosisof melioidosis in patients in nonendemic regions—i.e., travelers anddefence personnel returning from endemic tropical areas—and forseroepidemiologic surveys in endemic areas.

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