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ABSTRACT Dogs across the globe are afflicted by diverse blood- and vector-borne bacteria (VBB), many of which cause severe disease and can be fatal. Diagnosis of VBB infections can be challenging due to the low concentration of bacteria in the blood, the frequent occurrence of coinfections, and the wide range of known, emerging, and potentially novel VBB species encounterable. Therefore, there is a need for diagnostics that address these challenges by being both sensitive and capable of detecting all VBB simultaneously. We detail the first employment of a nanopore-based sequencing methodology conducted on the Oxford Nanopore Technologies (ONT) MinION device to accurately elucidate the “hemobacteriome” from canine blood through sequencing of the full-length 16S rRNA gene. We detected a diverse range of important canine VBB, including Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Bartonella clarridgeiae, “Candidatus Mycoplasma haematoparvum”, a novel species of hemotropic mycoplasma, and Wolbachia endosymbionts of filarial worms, indicative of filariasis. Our nanopore-based protocol was equivalent in sensitivity to both quantitative PCR (qPCR) and Illumina sequencing when benchmarked against these methods, achieving high agreement as defined by the kappa statistics (k > 0.81) for three key VBB. Utilizing the ability of the ONT’ MinION device to sequence long read lengths provides an excellent alternative diagnostic method by which the hemobacteriome can be accurately characterized to the species level in a way previously unachievable using short reads. We envision our method to be translatable to multiple contexts, such as the detection of VBB in other vertebrate hosts, including humans, while the small size of the MinION device is highly amenable to field use. IMPORTANCE Blood- and vector-borne bacteria (VBB) can cause severe pathology and even be lethal for dogs in many regions across the globe. Accurate characterization of all the bacterial pathogens infecting a canine host is critical, as coinfections are common and emerging and novel pathogens that may go undetected by traditional diagnostics frequently arise. Deep sequencing using devices from Oxford Nanopore Technologies (ONT) provides a solution, as the long read lengths achievable provide species-level taxonomic identification of pathogens that previous short-read technologies could not accomplish. We developed a protocol using ONT’ MinION sequencer to accurately detect and classify a wide spectrum of VBB from canine blood at a sensitivity comparable to that of regularly used diagnostics, such as qPCR. This protocol demonstrates great potential for use in biosurveillance and biosecurity operations for the detection of VBB in a range of vertebrate hosts, while the MinION sequencer’s portability allows this method to be used easily in the field.

ABSTRACT HIV-1 is genetically heterogeneous, having different subtypes and circulating recombinant forms (CRFs). HIV-1 genotyping is used to determine drug resistance profiles and is based on the use of a mixture of consensus and degenerate primers targeting the pol gene. However, the use of this type of primers is associated with either PCR bias or PCR failure. Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) can detect and identify unknown and distantly related gene sequences by PCR. CODEHOPs designed using different HIV-1 subtypes and CRFs were evaluated for HIV-1 genotyping by Sanger and MinION sequencing. A total of 321 plasma samples were used for the validation of CODEHOP-mediated HIV-1 genotyping. CODEHOP-mediated PCR showed 100% sensitivity and specificity, with limits of detection and genotyping below 200 copies/ml. The head-to-head evaluation of CODEHOP-mediated PCR and standard PCR showed 97 to 98% and 82 to 84% PCR success rates, respectively. There was 100% agreement between the CODEHOP and the reference method in the drug resistance profiles determined by Sanger-based sequencing. Using MinION sequencing, the CODEHOP-mediated PCR scheme resulted in better depth of genome coverage and detection of more drug resistance variants in the protease and reverse transcriptase genes than the standard amplification scheme. The overall prevalences of drug resistance mutations were 17.1% in treatment-experienced patients and 1.2% in treatment-naive patients. They were mainly associated with resistance to reverse transcriptase inhibitors and were linked to virological failure and the patient’s treatment history. Findings from this study suggest that the performance of HIV-1 genotyping is improved by using CODEHOP-mediated PCR. IMPORTANCE HIV-1 drug resistance is the main cause of treatment failure. Regular surveillance of resistance-associated mutations in HIV-1 genomes is essential for the optimal management of HIV-1 infections. Due to HIV-1’s genetic diversity, different HIV-1 genotypes are circulating worldwide. Standard primers used in the amplification of HIV-1 RNA have not been designed to cover all HIV-1 genotypes and are the main cause of amplification and drug resistance test failure. In this study, new sets of PCR primers targeting the protease, reverse transcriptase, and integrase genes were designed using the CODEHOP approach. They were compared to primers recommended in part by WHO for drug resistance testing using in-house PCR. Unsuccessful HIV-1 RNA amplification was less likely to occur with CODEHOP primers, leading to fewer test failures and lower cost. Furthermore, CODEHOP primers were more effective than standard primers for the detection of minority resistant variants by MinION sequencing.

Dogs across the globe are afflicted by diverse blood- and vector-borne bacteria (VBB), many of which cause severe disease and can be fatal. Diagnosis of VBB infections can be challenging due to the low concentration of bacteria in the blood, the frequent occurrence of coinfections, and the wide range of known, emerging, and potentially novel VBB species encounterable. Therefore, there is a need for diagnostics that address these challenges by being both sensitive and capable of detecting all VBB simultaneously. We detail the first employment of a nanopore-based sequencing methodology conducted on the Oxford Nanopore Technologies (ONT) MinION device to accurately elucidate the "hemobacteriome" from canine blood through sequencing of the full-length 16S rRNA gene. We detected a diverse range of important canine VBB, including Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Bartonella clarridgeiae, " Candidatus Mycoplasma haematoparvum", a novel species of hemotropic mycoplasma, and Wolbachia endosymbionts of filarial worms, indicative of filariasis. Our nanopore-based protocol was equivalent in sensitivity to both quantitative PCR (qPCR) and Illumina sequencing when benchmarked against these methods, achieving high agreement as defined by the kappa statistics ( k  > 0.81) for three key VBB. Utilizing the ability of the ONT' MinION device to sequence long read lengths provides an excellent alternative diagnostic method by which the hemobacteriome can be accurately characterized to the species level in a way previously unachievable using short reads. We envision our method to be translatable to multiple contexts, such as the detection of VBB in other vertebrate hosts, including humans, while the small size of the MinION device is highly amenable to field use. IMPORTANCE Blood- and vector-borne bacteria (VBB) can cause severe pathology and even be lethal for dogs in many regions across the globe. Accurate characterization of all the bacterial pathogens infecting a canine host is critical, as coinfections are common and emerging and novel pathogens that may go undetected by traditional diagnostics frequently arise. Deep sequencing using devices from Oxford Nanopore Technologies (ONT) provides a solution, as the long read lengths achievable provide species-level taxonomic identification of pathogens that previous short-read technologies could not accomplish. We developed a protocol using ONT' MinION sequencer to accurately detect and classify a wide spectrum of VBB from canine blood at a sensitivity comparable to that of regularly used diagnostics, such as qPCR. This protocol demonstrates great potential for use in biosurveillance and biosecurity operations for the detection of VBB in a range of vertebrate hosts, while the MinION sequencer's portability allows this method to be used easily in the field.

HIV-1 is genetically heterogeneous, having different subtypes and circulating recombinant forms (CRFs). HIV-1 genotyping is used to determine drug resistance profiles and is based on the use of a mixture of consensus and degenerate primers targeting the pol gene. However, the use of this type of primers is associated with either PCR bias or PCR failure. Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) can detect and identify unknown and distantly related gene sequences by PCR. CODEHOPs designed using different HIV-1 subtypes and CRFs were evaluated for HIV-1 genotyping by Sanger and MinION sequencing. A total of 321 plasma samples were used for the validation of CODEHOP-mediated HIV-1 genotyping. CODEHOP-mediated PCR showed 100% sensitivity and specificity, with limits of detection and genotyping below 200 copies/ml. The head-to-head evaluation of CODEHOP-mediated PCR and standard PCR showed 97 to 98% and 82 to 84% PCR success rates, respectively. There was 100% agreement between the CODEHOP and the reference method in the drug resistance profiles determined by Sanger-based sequencing. Using MinION sequencing, the CODEHOP-mediated PCR scheme resulted in better depth of genome coverage and detection of more drug resistance variants in the protease and reverse transcriptase genes than the standard amplification scheme. The overall prevalences of drug resistance mutations were 17.1% in treatment-experienced patients and 1.2% in treatment-naive patients. They were mainly associated with resistance to reverse transcriptase inhibitors and were linked to virological failure and the patient's treatment history. Findings from this study suggest that the performance of HIV-1 genotyping is improved by using CODEHOP-mediated PCR. IMPORTANCE HIV-1 drug resistance is the main cause of treatment failure. Regular surveillance of resistance-associated mutations in HIV-1 genomes is essential for the optimal management of HIV-1 infections. Due to HIV-1's genetic diversity, different HIV-1 genotypes are circulating worldwide. Standard primers used in the amplification of HIV-1 RNA have not been designed to cover all HIV-1 genotypes and are the main cause of amplification and drug resistance test failure. In this study, new sets of PCR primers targeting the protease, reverse transcriptase, and integrase genes were designed using the CODEHOP approach. They were compared to primers recommended in part by WHO for drug resistance testing using in-house PCR. Unsuccessful HIV-1 RNA amplification was less likely to occur with CODEHOP primers, leading to fewer test failures and lower cost. Furthermore, CODEHOP primers were more effective than standard primers for the detection of minority resistant variants by MinION sequencing.