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Cat-scratch disease Vermeulen, M.J.

2009

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citation for published version (APA) Vermeulen, M. J. (2009). Cat-scratch disease: Diagnostic and clinical aspects of henselae infection.

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Download date: 26. Sep. 2021 Chapter 6

Evaluation of an internally controlled real-time polymerase chain reaction assay targeting the groEL gene for the detection of Bartonella spp. DNA in patients with suspected cat-scratch disease

BM Diederen MJ Vermeulen H Verbakel A van der Zee A Bergmans MF Peeters European Journal of Clinical Microbiology & Infectious Diseases. 2007;26:629-33 110 Chapter 6

ABSTRACT

Bartonella (B.) henselae is the causative agent of cat-scratch disease (CSD), which usually presents as a self-limiting . This study reports the development and evaluation of an internally controlled real-time polymerase chain reaction targeting the groEL gene for detection of Bartonella spp. DNA was extracted using the MagNA Pure system. The lower detection limit was 10–100 fg DNA and the in vitro sensitivity of the assay was not affected by duplexing with an internal control PCR. The real-time PCR assay detected DNA from all five B. henselae strains tested, and from B. birtlesii, B. vinsonii subsp. vinsonii, B. vinsonii subsp. arupensis and B. doshiae. The assay generated negative results with a selection of other , including several Mycobacterium spp., Streptococcus pyogenes and Staphylococcus aureus. Results of real-time PCR in clinical samples were compared with those of a conventional 16S rDNA-based PCR assay. During the period described in the Material and methods section, real-time PCR and conventional 16S PCR were performed on 73 clinical samples. Of these samples, 29 (40%) were found to give positive results and 44 (60%) gave negative results, both by real-time PCR and by conventional PCR, with a 100% agreement between the two tests. The PCR developed in this study is a rapid, sensitive, and simple method for the detection of Bartonella spp. in CSD and is suitable for implementation in the diagnostic laboratory. Validation of groEL real-time PCR 111

INTRODUCTION

Bartonella (B.) henselae is the causative agent of cat-scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy, not only in children but also in adults [1,2]. Clinically, CSD is difficult to distinguish from lymphadenopathy caused by other microbial pathogens, such as atypical mycobacteria. Bacteria within the genus Bartonella are microaerophilic, Gram-negative, fastidious, slow-growing organisms that belong to the α- on the basis of their 16S rDNA sequences [1]. Laboratory methods for the diagnosis of Bartonella infections include isolation of the organisms by culture, serological assays, histopathological examination, and molecular detection of Bartonella DNA in affected tissue [1,3-5]. Routine bacterial culture protocols usually do not allow detection of these organisms. Primary isolates are typically obtained after 12 to 14 days, although prolonged incubation periods of up to 45 days are sometimes necessary [6]. With the Warthin-Starry silver stain, the bacilli can be detected in tissue specimens, but the technique is difficult and the result is not specific for Bartonella. Evaluation of serological tests in earlier studies reported various sensitivities and specificities, depending on study population and definitions of CSD, as well as materials used and techniques [5,7]. Polymerase chain reaction (PCR) offers a rapid and specific means to detect the organism directly from the clinical specimens. Therefore, PCR as a quick and more reliable diagnostic test is being used increasingly [1,3,7-11].

The aim of this study was to evaluate a novel in-house real-time PCR assay with the groEL gene as target sequence for the detection of Bartonella spp. DNA in clinical specimens.

PATIENTS AND METHODS

Bacterial strains The bacterial strains used to test the analytical sensitivity and specificity of the real-time PCR in vitro are presented in Table 1. The B. henselae strains and B. bacilliformis strain are part of the strain collection of the National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands. A39 PCR-AFLP type G is a Bartonella species whose 16S rDNA gene sequence has 99.9% identity with that of B. clarridgeiae (accession number U64691), a species found in cat blood [12,13]. For all other Bartonella spp. presented in Table 1, genomic DNA was kindly provided by Dr. R.J. Birtles (Disease Ecology Unit, Centre for Comparative Infectious Diseases, Faculty of Veterinary Science, University of Liverpool, UK).

A panel of organisms representing commonly isolated pathogenic species (mainly Mycobacterium spp., Streptococcus pyogenes and Staphylococcus aureus) from patients with lymphadenopathy identified by the Laboratory for Medical Microbiology (Tilburg, The Netherlands) were used to evaluate analytical specificity. Because the sequence of the probe is similar to that of several Gramnegative bacteria, including and 112 Chapter 6

Shigella flexneri, the specificity was also verified in vitro with a panel representing bacterial species not commonly isolated from patients with lymphadenopathy. Stock cultures were stored at 70 °C on porous beads and cultured by standard methods.

Collection and processing of clinical samples From August 2002 to December 2002, 73 specimens were obtained from 69 patients suspected of having CSD. The mean age of the patients was 28.0 years (SD 19 years), with 62% of them being male (gender of one patient was unknown). The specimens consisted mainly of pus aspirates from lymph nodes (n = 65) or biopsy material from other organs: eye (n = 2), brain (n = 1), and bone marrow (n = 1). All specimens were stored at -70 °C prior to nucleic acid isolation.

DNA extraction Prior to DNA isolation, tissue samples (biopsy and lymph node material) were processed with MagNALyser (Roche Diagnostics) according to the manufacturer’s instructions. Of the sample, 200 μl was processed in the MagNA Pure LC isolation robot, using the Total Nucleic Acid Isolation Kit (Roche Diagnostics) with an elution volume of 50 μl. Of the eluate, 5 μl was used as a template in the PCR. Conventional end-point PCR and DNA hybridisation results of real-time PCR were compared with those of a conventional 16S rDNA-based PCR assay. The primer pair described by Bergmans et al. was used to amplify a 296 bp fragment of the Bartonella 16S rDNA gene. After amplification, PCR products were subjected to electrophoresis, transferred to a nylon membrane, and hybridised with a B. henselae-specific 5’-biotinylated probe as described previously [3].

Real-time PCR For the detection of Bartonella spp. in clinical samples, a real-time PCR assay was used, targeted at the heat shock protein groEL. Primers Bhe-F1 (5’-ACA GGC TAT TGT CCA AGA AGG TGT A-3’) and Bhe-R1 (5’-TCA ACA GCA GCA TCG ATA CCA -3’) were chosen with Primer-Express (ABI), amplifying an 87 bp fragment. Detection of the fragment in real time was done with a Bartonella specific fluorescent MGB probe (Bhe-1-FAM: 5’-AAA GCC GTT GCT GCA G-MGB 3’). Real-time PCR was performed on an ABIPRISM 7900HT sequence detection system (Applied Biosystems, Foster City, California, USA). DNA was amplified using the following PCR parameters: after 2 min incubation at 50 °C and 10 min denaturation at 95 °C, amplification consisted of 50 cycles of 15 s of denaturation at 95 °C followed by 1 min of annealing and extension at 60 °C.

Internal control real-time PCR The quality of nucleic acid extraction as well as inhibition of the real-time PCR was monitored by amplification of a phocine herpesvirus (PhHV) spike. This method has been described by Niesters, using a Taqman probe in a single tube (duplex) PCR format [14]. Validation of groEL real-time PCR 113

RESULTS

The Bartonella spp. real-time PCR assay detected DNA from all five B. henselae strains and four non-B. henselae species. It amplified DNA from Bartonella birtlesii, Bartonella vinsonii subsp. vinsonii, Bartonella vinsonii subsp. arupensis and Bartonella doshiae. The assay generated negative results with the other bacteria, described in Table 1. To evaluate the sensitivity of the PCR test with internal control, we extracted B. henselae DNA (strain A18) from cultured bacteria, and the DNA concentration was estimated with a spectrophotometer (GeneQuant, Pharmacia Biotech). Serial dilutions were made, and aliquots were extracted and tested in PCR to determine sensitivity. The lower detection limit was 10-100 fg of DNA.

The Bartonella spp. real-time PCR assay was first optimised as a mono-assay. After the duplex assay had been optimised, threefold 100 fg dilution series of B. henselae DNA were analysed by comparison of the monoplex and duplex assays. The mean threshold cycle (Ct) value was 38.8 in monoplex and 38.5 in duplex PCR (R = 0.85, linear regression). A total of 36 stored patient materials was tested. For the detection of B. henselae DNA, the agreement between the monoplex and duplex assays was 100% (21 out of 36 (58%) stored patient materials tested positive). The mean Ct value was 32.4 (range 29.9- 37.9) in monoplex and 32.5 (range 29.8-37.9) in duplex PCR. The Ct values for the detection of B. henselae DNA obtained from testing a series of patient materials in the individual assay and the duplex assay were very similar, indicating similar detection limits. The specificity of the duplex assay was identical to that of the individual assay (data not shown). For the detection of PhHV, the agreement between the monoplex and duplex assays was 92%. In three B. henselae-positive samples PhHv could not be detected by duplex PCR. The mean Ct value was 35.6 (range 29.8-40.7) in monoplex and 36.3 (range 28.6-44.6) in duplex PCR. Samples containing 100 fg B. henselae DNA were assayed in duplo to determine the interassay variability. As determined from the Ct values obtained from ten consecutive runs, the mean inter-run variation was 0.9 for this standard sample (the mean Ct-value was 39.0,range 37.8 to 39.9).

During the period described in the Material and methods section, both real-time PCR and conventional 16S PCR were performed on 73 clinical samples in 12 runs. Of these samples, 29 (40%) gave positive results and 44 (60%) gave negative results, both by real-time PCR and by conventional PCR, with 100% agreement between the two tests. No inhibition of PCR was observed. The average Ct-value was 34.4 in PCR (range 29.8-37.2). 114 Chapter 6

Table 1. Bacterial strains studied.

Bacterial sp. Straina,b Cat isolate A18 (Marseille, AluI RFLP type B)

Cat isolate A29 (Marseille, AluI RFLP type B)

Cat isolate A43 (Houston-I, AluI RFLP type A)

Cat isolate A100 (Marseille, AluI RFLP type B)

91-148 (Marseille, AluI RFLP type B)

Bartonella bacilliformis KC583

Bartonella clarridgeiae

Bartonella quintana Oklahoma strain

Bartonella birtlesii N40

Bartonella tribocorum

Bartonella vinsonii subsp. vinsonii

Bartonella vinsonii subsp. arupensis

Bartonella doshiae

Bartonella elisabethae

Bartonella alsatica

Bartonella taylorii

Bartonella spp Cat isolate A39 (AluI RFLP type G)

Mycobacterium tuberculosis (1 isolate)

Mycobacterium avium (5 isolates)

Mycobacterium kansasii (2 isolates)

Mycobacterium intracellulare (2 isolates)

Mycobacterium gordonae (4 isolates)

Mycobacterium xenopi (3 isolates)

Mycobacterium abscessus (2 isolates)

Mycobacterium fortuitum (1 isolates)

Mycobacterium malmoense (1 isolate)

Mycobacterium africanum (1 isolate)

Streptococcus pyogenes (3 isolates)

Staphylococcus aureus (two clinical isolates, one ATCC 29213)

Escherichia coli (two clinical isolates, one ATCC 25922)

Shigella flexneri (3 isolates)

Streptococcus pneumoniae ATCC49619

Legionella pneumophila ATCC 33623

Bordetella pertussis Tohama strain

Bordetella parapertussis B24

Mycoplasma pneumoniae ATCC15293

Chlamydia pneumoniae ATCCVR1355

Acinetobacter baumannii (1 isolate)

Pseudomonas aeruginosa ATCC27853

a The B. henselae strains and B. bacilliformis strain are part of the strain collection of the National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands [6]. From all other Bartonella spp. presented in table 1, genomic DNA was kindly provided by dr R.J. Birtles (Disease Ecology Unit, Centre for Comparative Infectious Diseases, Faculty of Veterinary Science, University of Liverpool). b If not otherwise specified, non- Bartonella strains represent isolates from the Tilburg Clinical Microbiology Laboratory. Validation of groEL real-time PCR 115

DISCUSSION

The target genes most often used for PCR detection of Bartonella DNA are the 16S rDNA gene, the citrate synthetase gene (gltA), the 16S-23S rRNA intergenic spacer region of Bartonella species (ITS1), the genes encoding the PAP31 and 35 kDa proteins, the riboflavin synthase alpha chain gene (ribC) and the 60 kDa heat shock protein gene (htrA) [1,3,11,15,16]. Few data are available to compare these assays in routine clinical practice. The first primers for molecular identification of the agent of were described by Relman et al. [17]. Using these primers and Southern blot hybridisation of the PCR products, Bergmans et al. [3] found B. henselae DNA in a high percentage of CSD patients. In the absence of a gold standard for diagnosis of CSD, we compared our real-time PCR results of the investigated samples with the results of this well-described and validated primer set [3,7]. The real-time PCR assay showed excellent analytical sensitivity, as shown previously for other PCR methods [3,4,8-11,18,19]. The clinical sensitivity of the assay was not affected by duplexing with an internal control PCR. A PhHV spike was added to the clinical samples prior to DNA isolation and was co-amplified in the Bartonella spp. real-time PCR assay. In this way, control of the DNA isolation procedure, as well as a check for inhibition, was achieved.

No PCR positive results were found among non-Bartonella bacteria tested. However, within the Bartonella genus, the PCR assay described was found to detect four related Bartonella species that are mainly restricted to infected animals. The real-time PCR assay detected DNA from all five B. henselae strains described in the Materials and methods section and from four other Bartonella species. Although Bartonella spp. are considered to be emerging pathogens involved in an increasing number of recognised diseases, only one of the non- B. henselae species that were amplified in real-time PCR has been found to infect humans: B. vinsonii subsp. arupensis, normally isolated from mice, has been shown to be an agent of blood culture-negative endocarditis in humans [20]. B. birtlesii has been isolated from rats, Bartonella vinsonii subsp. vinsonii and B. doshiae have been recovered from voles. Although not likely to occur often, the lack of 100% in vitro specificity for the detection of B. henselae could lead to erroneous clinical interpretations in routine clinical practice, especially in patients with suspected culture-negative endocarditis. Other real-time PCR assays have been developed that target genes specific for B. henselae. The specificity of our assay could be established more firmly by confirmation of B. henselae infection using independent testing for a different B. henselae-specific target or to confirm positive results by sequence analysis. If the initial groEl PCR-positive samples are confirmed by sequencing or an independent B. henselae-specific assay, then no specimens will have false-positive results.

It is important to note that a diagnosis of CSD does not completely eliminate a diagnosis of mycobacteriosis or neoplasm. In a study by Rolain et al. [21], 13 out of 245 patients (6%) with confirmed Bartonella infections had concurrent mycobacteriosis (ten cases) or neo- plasm (three cases). Therefore, histological analysis of lymph node biopsy specimens should be performed routinely, because some patients might have a concurrent malignant disease 116 Chapter 6

or mycobacteriosis. Furthermore, it should be noted that, although a rare occurrence, B. quintana and B. clarridgeiae are responsible for some reported cases of CSD [12,22].

The real-time PCR described here provides a fast, sensitive and specific diagnosis of Bartonella spp. in patients with suspected CSD, in comparison with a more time-consuming conventional end-point PCR and DNA hybridisation. The protocol used enabled us to complete analysis of 26 samples, including controls, in approximately 5 hours, including extraction and assay time, and it is suitable for implementation in a routine diagnostic laboratory.

ACKNOWLEDGEMENTS

Genomic DNA was kindly provided by Dr. R.J. Birtles (Disease Ecology Unit, Centre for Comparative Infectious Diseases, Faculty of Veterinary Science, University of Liverpool, UK). We thank Ingrid Aarts and Caroline de Jong for providing technical support. Validation of groEL real-time PCR 117

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