Introduction

In Africa, relapsing fevers are neglected febrile infections caused by Ornithodoros spp. tick-borne borreliae (Borrelia crocidurae, Borrelia hispanica and Borrelia duttonii) and the Pediculus humanus louse-borne Borrelia recurrentis [1], [2]. Also, poorly characterized, yet uncultured new “Borrelia mvumi” species has been reported in acute patient's blood and Ornithodoros porcinus argasid ticks in Tanzania [3]. The relative geographic specificity of each Borrelia species has been challenged by coexistence of two species in the same region [4]. B. hispanica prevalence was reported to be 20.5% among febrile patients in Northwestern Morocco [5]; the prevalence of cases attributed to B. crocidurae among febrile patients is of 11 per 100 person-years in Senegal [1]. B. duttonii has been documented in Tanzania and B. recurrentis in Ethiopia [5], [6], [7], [8]. Relapsing fevers are of further concern in travelers returning from Africa in Europe [9], [10], [11].

Relapsing fevers are treatable infections but the severity of the disease ranges from asymptomatic to fatal if left untreated [12]. In Rwanda and in Tanzania the investigators found a 30% risk for pregnancy loss and a perinatal mortality rate of 15% [8], [13]. The prognosis depends in part on the causative species with the case-fatality ratio being higher for B. recurrentis infection than for the other infections [12]. However, the vast majority of patients are diagnosed on the basis of non-specific clinical features that overlap with those of malaria [4] and the poorly-sensitive, non-species specific microscopic observation of blood-borne Borrelia [1].

PCR-based tests have been therefore developed to improve the laboratory-based diagnosis of relapsing fevers in Africa [4]. In particular, real-time PCR targeting the 16S rRNA gene or the glpQ gene improved the sensitivity of the diagnosis when compared to microscopy [14], [15]. Also, we previously showed that PCR-sequencing intergenic spacers could be used for genotyping B. crocidurae, B. duttonii and B. recurrentis [16].

Here, we present the development and evaluation of a multiplex, quantitative real-time PCR detecting any relapsing fever Borrelia [14] and specifically B. crocidurae, B. hispanica and B. duttonii/B. recurrentis based on post-genomic analyses [16], [17]

Materials and Methods

Real-time PCR specificity and sensitivity

To assess the specificity of the real-time PCR systems developed herein, DNA extracted from Borrelia burgdorferi, Borrelia hermsii, Borrelia parkeri, Coxiella burnetii, Bartonella henselae, Rickettsia africae, Rickettsia felis and Tropheryma whipplei were incorporated into real-time PCR using the experimental conditions described above. In order to determine sensitivity, a puc 57 plasmid was constructed containing the human albumin gene, a 129-bp recN gene fragment from B. duttonii, a 122-bp recC gene fragment from B. hispanica and a 110-bp glpQ gene fragment from B. crocidurae (Invitrogen, Saint Aubin, France) (Figure 1). Tenfold serial dilutions of this constructed puc 57 plasmid were prepared equivalent to 10⁷ to 10¹ Borrelia organisms.

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Figure 1. Sequence of the chimeric plasmid used as internal control in the real-time PCR.

Yellow characters; B. hispanica recC gene sequence; green characters; B. duttonii recN gene sequence; pink character; B. crocidurae glpQ gene sequence; Red characters; albumine gene sequence; probes are in blue; Colored boxes contain the primer and probe sequences.

https://doi.org/10.1371/journal.pntd.0002042.g001

Results

Species-specific primers and probes sequences

As a specific target for B. crocidurae, we selected glpQ encoding glycerophosphodiester phosphodiesterase that is conserved among relapsing fever borreliae but absent from Lyme disease borreliae and additionally possesses a B. crocidurae specific 4-bp single nucleotide polymorphisms (SNPs). We selected the chromosomal recN gene encoding DNA repair ATPase for B. duttonii/B. recurrentis, that is conserved among the relapsing fever group and absent in the Lyme disease group borreliae, and furthermore exhibits a 5-bp specific SNP. For B. hispanica, we selected recC gene encoding exodeoxyribonuclease V, present in both relapsing fever and Lyme disease group borreliae and exhibiting 4-bp species-specific SNP. One probe specific for each of the three targeted regions was designed to span the region containing the SNPs. Sequences of the primers and probes are given in Table 1. Each set of species-specific primers and probe was first evaluated alone before being incorporated into a multiplex format. There was no difference in the amplification curves when comparing the single-target real-time PCR with multi-target real-time PCR assays. Figure 2 illustrates the results of these two experimental steps and shows that 16SrRNA gene probe labeled with FAM and glpQ gene probe labeled with VIC fluorescent dyes could both be detected in one PCR reaction performed simultanoiusly. Similar results were obtained with recN and recC labeled with VIC and 16SrRNA probe labeled with FAM.

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Figure 2. Fluorescence curves of four Borrelia crocidurae positive blood samples.

Multiplex real-time PCR amplification and detection of the Borrelia genus-specific 16S rRNA gene (probe labeled with FAM fluorescent dye) and the Borrelia crocidurae-specific glpQ gene (probe labeled with VIC fluorescent dye).

https://doi.org/10.1371/journal.pntd.0002042.g002

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Table 1. Primer and probe sequences used for multiplex real-time PCR.

https://doi.org/10.1371/journal.pntd.0002042.t001

Discussion

In this study, all the negative controls remained negative in every real-time PCR experiment. Also, no evidence of PCR inhibition was detected using human blood as confirmed by amplification of the human albumin internal control in every PCR run. Specificity of primers and probes was confirmed by in-silico analyses and reinforced by experimental demonstrations that these assays failed to amplifyother microorganisms responsible for septicemia, including R. felis [21] and T. whipplei [22], all demonstrated to be emerging, highly prevalent pathogens in Africa and in Senegal in particular. Therefore, results reported herein were interpreted as authentic.

In this study, species-specific primers and probes based on the glpQ, recC and recN gene sequences were selected from the alignment of the B. crocidurae, B. duttonii, B. recurrentis and B. burgdorferi reference chromosome genomes [16], [17]. Plasmid sequences were avoided, because of their instability among different strains of the same species, and during replication of the same isolate, with a risk of resulting in false-negative results. This approach proved successful for the differentiation between B. crocidurae, B. duttonii/B. recurrentis and B. hispanica. Despite evident interest in distinguishing B. duttonii and B. recurrentis for accurate epidemiological purposes, discrimination between B. duttonii and B. recurrentis was not possible here in agreement with previously reported very close genetic and genomic proximity of both species [16], [17]. Indeed, genetic and genomic data suggested that B. duttonii and B. recurrentis could be regarded as a single Borrelia species [17]. This limitation may not be problematic as for the routine diagnosis since these two species are respectively transmitted by tick and lice in very different epidemiological contexts [23]. Also, the multiplex real-time PCR proved highly sensitive, detecting 100 copies, that is more sensitive than the 10³–10⁵ borreliae/µL reported for microscopy [14], [24]. Previously, borreliae not detectable by microscopy, were detected by using real-time PCR targeting the fla and the glpQ genes [3], [15]. These assays however could not identify borreliae at the species level [3], [15]. Another real-time PCR assay was devoted to the specific detection of B. recurrentis and Rickettsia prowazekii, as these two pathogens are both transmitted by body lice. This targeted the flagellin gene of B. recurrentis with a sensitivity of 10¹ borreliae [25].

The developed real-time PCR was validated against large number of samples from areas endemic for diverse borreliae causing human infection in Africa, showing 100% sensitivity and specificity except for glpQ gene which had 99% sensitivity due to failure to identify B. crocidurae in one blood specimen. Unfortunately, we could not further analyze this specimen to assess whether this false negative result arose from glpQ mutations or was indicative of a different species or subspecies related to B. crocidurae. Indeed, several recent reports indicate that new relapsing fever Borrelia species are present in Tanzania and South Africa [26], [27]. Therefore careful optimization is required to ensure that the multiplex real-time PCR technique employed will not miss these new species or other Borrelia species. For this, we incorporated the 16S rRNA gene probe in the system to serve as an indicator of the detection of any relapsing fever Borrelia in the specimen. A specimen detected positive by the 16S rRNA gene probe and negative by the species-specific probes would indicate a new Borrelia species. Such samples could be further subjected to in situ typing such as the recently described multiple spacer sequence typing [16].

In conclusion, as detection and identification of these genetically closely related relapsing fever borreliae in Africa remains challenging, the multiplex real-time PCR assay reported herein offers significant improvement over existing procedures for the diagnosis of relapsing fevers in Africa. Importantly, it permits rapid differentiation of relapsing fevers from the clinically similar malaria, that requires drastically different therapeutic management. It is a sensitive and specific technique capable of detection of major Borrelia pathogens in humans, yet will not overlook detection of potentially new species. This multiplex real-time assay being amenable to point-of-care laboratories in Africa [28], it provides an effective solution for enhanced characterization of relapsing fever borreliae in Africa, improving medical management of patients and facilitating epidemiological studies.

Supporting Information

Acknowledgments

The authors thank François Renaud, IRD Montpellier, France for providing them with argasid ticks infected with B. hispanica.