# Duplex TaqMan Hydrolysis Probe-Based Molecular Assay for Simultaneous Detection and Differentiation ofBurkholderia pseudomallei andLeptospira spp. DNA

Authors: Mohammad Ridhuan Mohd Ali; Lee Lih Huey; Phiaw Chong Foo; Yuan Xin Goay; Asmaliza S. Ismail; Khairul Mohd Fadzli Mustaffa; Ismail Aziah; Phua Kia Kien; Azian Harun; Nabilah Ismail; Chan Yean Yean
Journal: BioMed Research International
 (2019)

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## Abstract

Melioidosis and leptospirosis, caused by two different bacteria,Burkholderia pseudomallei andLeptospiraspp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection ofB. pseudomallei andLeptospira spp. DNA. The performance of the assay was evaluated on 20B. pseudomallei isolates, 23Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies)Leptospira spp. DNA and 0.2 pg (~ 25.6 copies)B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection ofB. pseudomallei &Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.


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## Body

## 1. Introduction

Burkholderia pseudomallei andLeptospira are two important infectious agents for melioidosis and leptospirosis, respectively [1–3]. The Gram-negativeB. pseudomallei is recognized as CDC Tier 1 select agent and a Category B Priority Pathogen by the National Institute of Allergy and Infectious Diseases (NIAID), in addition to leptospirosis, which has been added to the Emerging Infectious Diseases category (https://www.niaid.nih.gov/research/emerging-infectious-diseases-pathogens). Both organisms are normally found in the soil and freshwater environment [4, 5]. In addition to their ubiquitous habitats, these organisms routinely infect animals, such as cattle, sheep, and horses. Certain animal classes such as rats may asymptomatically carryLeptospira.It is generally accepted that animals are responsible for shedding and maintenance ofLeptospira andB. pseudomallei in the environments, through their urines and faeces [5–7]. Human cases are usually associated with interactions with the contaminated environments [6, 8]. To date, increasing cases of melioidosis and leptospirosis have been reported worldwide, especially in the tropical and subtropical regions [1, 6].Infections byB. pseudomallei andLeptospira portray a very similar spectrum of nonspecific clinical presentations including fever, headache, myalgia, and pneumonia [4, 8]. In animals,B. pseudomallei infections cause pneumonia with lung abscesses, anorexia, and encephalitis [9]. Meanwhile, animal leptospirosis is characterized by abortion, jaundice, and infertility [10]. Several factors, such as bacterial load, underlying medical conditions, and serotypes increase hosts susceptibility to melioidosis and leptospirosis [1, 11–13]. Furthermore, the risk of dual infection is apparent, as several incidences of melioidosis-leptospirosis coinfections were reported previously [14, 15]. It is possible that many cases may be underdiagnosed when only one between the two tests is considered or available [16].Early detection of melioidosis and leptospirosis could significantly increase the chances of survival and reduce potential economic loss [17]. Current gold standard for detectingB. pseudomallei is by the culture method which requires 2-7 days to grow [18]. Meanwhile, leptospiral antibody titer is detected by the microscopic agglutination test (MAT) that usually requires paired sera and is less useful during acute infection [5]. As both diagnostic methods are time-consuming, a more rapid laboratory assay is urgently needed. To date, several molecular assays have been described for detection of individualB. pseudomallei andLeptospira from the clinical specimens [5, 18]. However, to our knowledge, none of the reported assays is able to simultaneously detect and distinguishB. pseudomallei andLeptospirawithin the same reaction tube. In this study, we developed a duplex qPCR that can detectB. pseudomallei andLeptospira DNA and evaluated the assay on selected clinical and environmental isolates.


## 2. Materials and Methods

### 2.1. Microorganism Strains and Growth Conditions

A total of 20B. pseudomallei strains, 23Leptospira strains and 39 other microorganisms isolated from human clinical samples and ATCC strains were used in this study (Table 1). These microorganisms were provided by the Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia; Makmal Kesihatan Awam Kota Bharu; Universiti Putra Malaysia; and Institute for Medical Research. The bacteria were cultured aerobically in nutrient broth overnight at 37°C on a rotating platform of 180 rpm. Meanwhile,Leptospira strains were maintained in EMJH media, incubated at 30°C on rotating platform of 40 rpm, overnight.Entamoeba histolytica DNA was obtained directly from School of Health Sciences, Universiti Sains Malaysia.Table 1
List of organism used for analytical specificity test.
Organism Source No. tested (n) Results in duplex qPCR Aspergillus fumigatus USM, Malaysia 1 Negative Bacillus subtilis USM, Malaysia 1 Negative Burkholderia cepacia USM, Malaysia 6 Negative Burkholderia pseudomallei USM, Malaysia 20 Positive Burkholderia thailandensis USM, Malaysia 1 Negative Campylobacter jejuni USM, Malaysia 1 Negative Candida albicans USM, Malaysia 1 Negative Citrobacter freundii USM, Malaysia 1 Negative Entamoeba histolytica UNAM, Mexico 1 Negative Enterococcus faecalis USM, Malaysia 1 Negative Klebsiella pneumoniae USM, Malaysia 1 Negative Leptospira biflexa serovar Patoc IMR, Malaysia 1 Positive Leptospira biflexa serovar Patoc UPM, Malaysia 1 Positive Leptospira borgpetersenii Celledoni IMR, Malaysia 1 Positive Leptospira borgpetersenii serovar Ballum UPM, Malaysia 1 Positive Leptospira fainei serovar Hurtsbridge IMR, Malaysia 1 Positive Leptospira fainei serovar Hurtsbridge UPM, Malaysia 1 Positive Leptospira interrogansserovar Australis UPM, Malaysia 1 Positive Leptospira interrogansserovar Autumnalis IMR, Malaysia 1 Positive Leptospira interrogansserovar Bataviae IMR, Malaysia 1 Positive Leptospira interrogansserovar Bataviae UPM, Malaysia 1 Positive Leptospira interrogansserovar Canicola UPM, Malaysia 1 Positive Leptospira interrogansserovar Copenhageni IMR, Malaysia 1 Positive Leptospira interrogansserovar Hebdomadis UPM, Malaysia 1 Positive Leptospira interrogansserovar Icterohaemorrhagiae RGA UPM, Malaysia 1 Positive Leptospira interrogansserovar Javanica IMR, Malaysia 1 Positive Leptospira interrogansserovar Pomona IMR, Malaysia 1 Positive Leptospira interrogansserovar Pomona UPM, Malaysia 1 Positive Leptospira interrogansserovar Pyrogenes IMR, Malaysia 1 Positive Leptospira interrogansserovar Pyrogenes UPM, Malaysia 1 Positive Leptospira interrogansserovar Tarassovi IMR, Malaysia 1 Positive Leptospira licerasiae serovar Varillal IMR, Malaysia 1 Positive Leptospira meyeri serovar Semaranga IMR, Malaysia 1 Positive Leptospira wolffii IMR, Malaysia 1 Positive Plasmodium falciparum MKA Kota Bharu 5 Negative Plasmodium knowlesi MKA Kota Bharu 5 Negative Plasmodium vivax MKA Kota Bharu 5 Negative Proteus mirabilis USM, Malaysia 1 Negative Proteus vulgaris USM, Malaysia 1 Negative Salmonella Paratyphi A (ATCC 9150) ATCC, USA 1 Negative Salmonella Paratyphi B (ATCC BAA 1250) ATCC, USA 1 Negative Salmonella Paratyphi C (ATCC 9068) ATCC, USA 1 Negative Salmonella Typhi (ATCC 7251) ATCC, USA 1 Negative Salmonella Typhimurium (ATCC 14028) ATCC, USA 1 Negative Staphylococcus aureus USM, Malaysia 1 Negative Staphylococcus saprophyticus USM, Malaysia 1 Negative


### 2.2. Isolation of Genomic DNA

DNA was extracted from pure bacterial culture using NucleoSpin® Tissue DNA Extraction kit (MACHEREY-NAGEL GmbH & Co. KG, Germany). The extraction procedure was carried out according to the manufacturer instructions with a minor modification on the final elution step, in which the column was incubated at room temperature for 10 minutes prior to centrifugation at 11 000 ×g. Total DNA was quantified using the Eppendorf BioPhotometer (Eppendorf Scientific, Inc., New York, United States) and stored at -20°C until use.


### 2.3. Duplex Real-Time PCR Parameters

The PCR reaction was prepared in a total volume of 20μL, containing 10 μL 2× SsoAdvanced™ Universal Probes Supermix, 1 μL PCR grade distilled water, 0.2 μM primers, 0.1 μM probes, and 8 μL DNA template. Sequences of oligonucleotides used are listed in Table 2. The oligonucleotides were designed for amplification of theorf2 region ofB. pseudomalleitype III secretion system (T3SS) and therrs gene ofLeptospira.Table 2
List of primers and probes used in this study.
Target Type Sequence (5′ →3′) Source Leptospira spp. Forward primer ACTGAGACACGGTCCATACT [19] Reverse primer TAGTTAGCYGGTGCTTTAGGYA Probe FAM-ACGGGAGGCAGC-ZEN-AGTTAAGAATCTTGC-IBFQ  B. pseudomallei Forward primer CCTGGGAGAGCGAGATGTT [20] Reverse primer GCTGGATGAGAAGAAAGTCC Probe TexRed-CCACGCACGGCGGAGATTCT-IBRQAmplifications were conducted using Biorad CFX96 Touch Real-Time PCR Detection System. Thermal cycling condition included an initial denaturation at 95°C for 5minutes, followed by 50 cycles of 95°C for 30 seconds and 61.3°C for 30 seconds. Baseline threshold for the postamplification analysis was set at 50 (forB. pseudomallei) and 25 (forLeptospira). Any Cq value ≤40 is considered positive. All the amplification in this study was carried out in triplicate, unless specified otherwise.


### 2.4. Analytical Sensitivity and Specificity

The analytical sensitivity of the assay was carried out using extractedB. pseudomalleiandL. interrogans gDNA, diluted 10-fold ranging from 10 ng/uL to 1 fg/uL. Two microliters of each diluted gDNA were used in the duplex qPCR. Amount of bacterial DNA in each reaction was calculated based on a formula previously described by Aghamollaeiet al. (2015) [21]. Meanwhile, the assay analytical specificity was determined using 2 μL extracted DNA from other organisms (non-Leptospira DNAs and non-B. pseudomallei), as listed in Table 1. DNA were extracted using NucleoSpin® Tissue extraction kit.


## 2.1. Microorganism Strains and Growth Conditions

A total of 20B. pseudomallei strains, 23Leptospira strains and 39 other microorganisms isolated from human clinical samples and ATCC strains were used in this study (Table 1). These microorganisms were provided by the Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia; Makmal Kesihatan Awam Kota Bharu; Universiti Putra Malaysia; and Institute for Medical Research. The bacteria were cultured aerobically in nutrient broth overnight at 37°C on a rotating platform of 180 rpm. Meanwhile,Leptospira strains were maintained in EMJH media, incubated at 30°C on rotating platform of 40 rpm, overnight.Entamoeba histolytica DNA was obtained directly from School of Health Sciences, Universiti Sains Malaysia.Table 1
List of organism used for analytical specificity test.
Organism Source No. tested (n) Results in duplex qPCR Aspergillus fumigatus USM, Malaysia 1 Negative Bacillus subtilis USM, Malaysia 1 Negative Burkholderia cepacia USM, Malaysia 6 Negative Burkholderia pseudomallei USM, Malaysia 20 Positive Burkholderia thailandensis USM, Malaysia 1 Negative Campylobacter jejuni USM, Malaysia 1 Negative Candida albicans USM, Malaysia 1 Negative Citrobacter freundii USM, Malaysia 1 Negative Entamoeba histolytica UNAM, Mexico 1 Negative Enterococcus faecalis USM, Malaysia 1 Negative Klebsiella pneumoniae USM, Malaysia 1 Negative Leptospira biflexa serovar Patoc IMR, Malaysia 1 Positive Leptospira biflexa serovar Patoc UPM, Malaysia 1 Positive Leptospira borgpetersenii Celledoni IMR, Malaysia 1 Positive Leptospira borgpetersenii serovar Ballum UPM, Malaysia 1 Positive Leptospira fainei serovar Hurtsbridge IMR, Malaysia 1 Positive Leptospira fainei serovar Hurtsbridge UPM, Malaysia 1 Positive Leptospira interrogansserovar Australis UPM, Malaysia 1 Positive Leptospira interrogansserovar Autumnalis IMR, Malaysia 1 Positive Leptospira interrogansserovar Bataviae IMR, Malaysia 1 Positive Leptospira interrogansserovar Bataviae UPM, Malaysia 1 Positive Leptospira interrogansserovar Canicola UPM, Malaysia 1 Positive Leptospira interrogansserovar Copenhageni IMR, Malaysia 1 Positive Leptospira interrogansserovar Hebdomadis UPM, Malaysia 1 Positive Leptospira interrogansserovar Icterohaemorrhagiae RGA UPM, Malaysia 1 Positive Leptospira interrogansserovar Javanica IMR, Malaysia 1 Positive Leptospira interrogansserovar Pomona IMR, Malaysia 1 Positive Leptospira interrogansserovar Pomona UPM, Malaysia 1 Positive Leptospira interrogansserovar Pyrogenes IMR, Malaysia 1 Positive Leptospira interrogansserovar Pyrogenes UPM, Malaysia 1 Positive Leptospira interrogansserovar Tarassovi IMR, Malaysia 1 Positive Leptospira licerasiae serovar Varillal IMR, Malaysia 1 Positive Leptospira meyeri serovar Semaranga IMR, Malaysia 1 Positive Leptospira wolffii IMR, Malaysia 1 Positive Plasmodium falciparum MKA Kota Bharu 5 Negative Plasmodium knowlesi MKA Kota Bharu 5 Negative Plasmodium vivax MKA Kota Bharu 5 Negative Proteus mirabilis USM, Malaysia 1 Negative Proteus vulgaris USM, Malaysia 1 Negative Salmonella Paratyphi A (ATCC 9150) ATCC, USA 1 Negative Salmonella Paratyphi B (ATCC BAA 1250) ATCC, USA 1 Negative Salmonella Paratyphi C (ATCC 9068) ATCC, USA 1 Negative Salmonella Typhi (ATCC 7251) ATCC, USA 1 Negative Salmonella Typhimurium (ATCC 14028) ATCC, USA 1 Negative Staphylococcus aureus USM, Malaysia 1 Negative Staphylococcus saprophyticus USM, Malaysia 1 Negative


## 2.2. Isolation of Genomic DNA

DNA was extracted from pure bacterial culture using NucleoSpin® Tissue DNA Extraction kit (MACHEREY-NAGEL GmbH & Co. KG, Germany). The extraction procedure was carried out according to the manufacturer instructions with a minor modification on the final elution step, in which the column was incubated at room temperature for 10 minutes prior to centrifugation at 11 000 ×g. Total DNA was quantified using the Eppendorf BioPhotometer (Eppendorf Scientific, Inc., New York, United States) and stored at -20°C until use.


## 2.3. Duplex Real-Time PCR Parameters

The PCR reaction was prepared in a total volume of 20μL, containing 10 μL 2× SsoAdvanced™ Universal Probes Supermix, 1 μL PCR grade distilled water, 0.2 μM primers, 0.1 μM probes, and 8 μL DNA template. Sequences of oligonucleotides used are listed in Table 2. The oligonucleotides were designed for amplification of theorf2 region ofB. pseudomalleitype III secretion system (T3SS) and therrs gene ofLeptospira.Table 2
List of primers and probes used in this study.
Target Type Sequence (5′ →3′) Source Leptospira spp. Forward primer ACTGAGACACGGTCCATACT [19] Reverse primer TAGTTAGCYGGTGCTTTAGGYA Probe FAM-ACGGGAGGCAGC-ZEN-AGTTAAGAATCTTGC-IBFQ  B. pseudomallei Forward primer CCTGGGAGAGCGAGATGTT [20] Reverse primer GCTGGATGAGAAGAAAGTCC Probe TexRed-CCACGCACGGCGGAGATTCT-IBRQAmplifications were conducted using Biorad CFX96 Touch Real-Time PCR Detection System. Thermal cycling condition included an initial denaturation at 95°C for 5minutes, followed by 50 cycles of 95°C for 30 seconds and 61.3°C for 30 seconds. Baseline threshold for the postamplification analysis was set at 50 (forB. pseudomallei) and 25 (forLeptospira). Any Cq value ≤40 is considered positive. All the amplification in this study was carried out in triplicate, unless specified otherwise.


## 2.4. Analytical Sensitivity and Specificity

The analytical sensitivity of the assay was carried out using extractedB. pseudomalleiandL. interrogans gDNA, diluted 10-fold ranging from 10 ng/uL to 1 fg/uL. Two microliters of each diluted gDNA were used in the duplex qPCR. Amount of bacterial DNA in each reaction was calculated based on a formula previously described by Aghamollaeiet al. (2015) [21]. Meanwhile, the assay analytical specificity was determined using 2 μL extracted DNA from other organisms (non-Leptospira DNAs and non-B. pseudomallei), as listed in Table 1. DNA were extracted using NucleoSpin® Tissue extraction kit.


## 3. Results and Discussions

Despite the availability of several TaqMan hydrolysis probe-based assays for the detection of eitherLeptospira spp. orB. pseudomallei, none of the reported assays are able to simultaneously detect both organisms within the same reaction [18, 22]. Availability of such diagnostic tool that is able to detect and differentiateB. pseudomallei orLeptospira spp. is crucial as both infections portray similar clinical features and yet require different clinical management. In this study, a duplex qPCR for detection ofB. pseudomallei andLeptospira spp. DNA was evaluated. As shown in Table 3, the developed qPCR was able to amplify 0.02 pg (~ 4 copies)Leptospira spp. DNA and 0.2 pg (~ 25.6 copies)B. pseudomallei DNA, respectively. The sensitivity of the duplex assay for detection ofLeptospira DNA is comparable to other reported leptospiral probe-based assays that detected between 1 and 20 DNA copies per reaction [23–25]. Meanwhile, for theB. pseudomallei detection, the sensitivity was slightly lower than the previously reported assays that amplified 5 and 10 DNA copies per reaction [26–28]. In comparison to the corresponding monoplex assay, the duplex assay had comparable sensitivity forLeptospira, but had a reduced sensitivity forB. pseudomallei target (0.2 pg in duplexversus 0.02 pg in monoplex). Reduced performance of multiplex assay as compared to the monoplex assay has been observed in other molecular studies which are associated with primers competition, primer cross hybridization, and template mispriming [29, 30]. When tested on other microorganisms, no cross amplification was observed (Table 1). Theorf2 region is selected because it is only present inB. pseudomallei[31]. Meanwhile, for the leptospiral target, therrs gene is used because the gene is present in multiple copies perLeptospira genome [32]. As the current panel included limited coverage of organisms, further validation should includeBurkholderia mallei and otherBurkholderia cepacia complex (BCC).Table 3
Analytical sensitivity of the duplex qPCR assays.
Copies number Amount (pg) Duplex qPCR forB. pseudomallei Copies number Amount (pg) Duplex qPCR forLeptospiraspp. Mean Cq SD CV (%) Mean Cq SD CV (%) 2560000 20000 18.41 0.05 0.26 4000000 20000 19.3 0.17 0.86 256000 2000 21.75 0.31 1.43 400000 2000 23.09 0.16 0.67 25600 200 25.34 0.12 0.46 40000 200 27.23 0.04 0.16 2560 20 28.97 0.13 0.44 4000 20 30.67 0.32 1.05 256 2 32.8 0.14 0.44 400 2 34.63 0.08 0.24 25.6 0.2 36.96 1.06 2.88 40 0.2 36.34 0.5 1.37 2.56 0.02 - - - 4 0.02 38.59 1.49 3.87 0.256 0.002 - - - 0.4 0.002 - - -As listed in Table4, the duplex assay had an efficiency of 101.9% for the detection ofLeptospira DNA, comparable to the monoplex assay (100.5%). However, for the detection ofB. pseudomallei DNA, the duplex assay had an efficiency of 86.3%, lower than the monoplex assay (95.9%). The suboptimal efficiency may be attributed to the decreased sensitivity of the assay onB. pseudomallei target. In an ideal condition, PCR efficiency should be 90% and above [33]. Further optimization is necessary in order to increase the assay efficiency, especially for theB. pseudomallei target. Meanwhile, in terms of linearity, the duplex assays (forLeptospira andB. pseudomallei DNA detection) had R2 values of close to 1. Noticeably, at low copy number, the CV values ranged between 2.8 and 3.8% (Table 3).Table 4
PCR efficiency and linearity of the duplex qPCR assays.
Parameter Target Leptospira spp. B. pseudomallei Slope -3.2776 -3.7006 Efficiency 101.9% 86.3% Linearity, R2 0.9837 0.9987Overall, the establishment of a duplex qPCR assay that can detect and differentiateB. pseudomalleiandLeptospiraspp. may help the diagnosis of melioidosis and leptospirosis. However, prior to clinical evaluation, further analytical validation, such as intra- and interassay variation, a wider spectrum of microorganisms for specificity testing, higher number of replicates, and optimization of assays are necessary. In addition, incorporation of internal amplification control should be considered because certain types of clinical samples such as whole blood and urine may cause PCR inhibition.



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*Source: 9451791-2019-07-02.xml*