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SUMMARY The Gram-negative bacterium Yersinia pestis is responsible for deadly plague, a zoonotic disease established in stable foci in the Americas, Africa, and Eurasia. Its persistence in the environment relies on the subtle balance between Y. pestis-contaminated soils, burrowing and nonburrowing mammals exhibiting variable degrees of plague susceptibility, and their associated fleas. Transmission from one host to another relies mainly on infected flea bites, inducing typical painful, enlarged lymph nodes referred to as buboes, followed by septicemic dissemination of the pathogen. In contrast, droplet inhalation after close contact with infected mammals induces primary pneumonic plague. Finally, the rarely reported consumption of contaminated raw meat causes pharyngeal and gastrointestinal plague. Point-of-care diagnosis, early antibiotic treatment, and confinement measures contribute to outbreak control despite residual mortality. Mandatory primary prevention relies on the active surveillance of established plague foci and ectoparasite control. Plague is acknowledged to have infected human populations for at least 5,000 years in Eurasia. Y. pestis genomes recovered from affected archaeological sites have suggested clonal evolution from a common ancestor shared with the closely related enteric pathogen Yersinia pseudotuberculosis and have indicated that ymt gene acquisition during the Bronze Age conferred Y. pestis with ectoparasite transmissibility while maintaining its enteric transmissibility. Three historic pandemics, starting in 541 AD and continuing until today, have been described. At present, the third pandemic has become largely quiescent, with hundreds of human cases being reported mainly in a few impoverished African countries, where zoonotic plague is mostly transmitted to people by rodent-associated flea bites.

Objectives Depicting past epidemics currently relies on DNA-based detection of pathogens, an approach limited to pathogens with well-preserved DNA sequences. We used paleoserology as a complementary approach detecting specific antibodies under a mini line-blot format including positive and negative control antigens. Methods Mini line blot assay incorporated skim milk as negative control, Staphylococcus aureus as positive control, and antigens prepared from lice-borne pathogens Rickettsia prowazekii, Borrelia recurrentis, Bartonella quintana, and Yersinia pestis. Paleoserums were extracted from rehydrated dental pulp recovered from buried individuals. Mini line blots observed with the naked eye, were quantified using a scanner and appropriate software. Paleoserology was applied to the indirect detection of lice-borne pathogens in seven skeletons exhumed from a 16th-17th century suspected military burial site (Auxi-le-Château); and 14 civils exhumed from a 5th-13th century burial site (Saint-Mont). Direct detection of pathogens was performed using quantitative real-time PCR. Results In Auxi-le-Château, paleoserology yielded 7/7 interpretable paleoserums including 7/7 positives for B. recurrentis including one also positive for B. quintana. In Saint-Mont, paleoserology yielded 8/14 interpretable paleoserums and none reacted against any of the four pathogens. Antibodies against R. prowazekii and Y. pestis were not detected. The seroprevalence was significantly higher in the military burial site of Auxi-le-Château than in the civil burial site of Saint-Mont. Real-time PCR detection of B. quintana yielded 5/21 positive (3 at Saint-Mont and 2 at Auxi-le-Château) whereas B. recurrentis was not detected. Conclusions Paleoserology unmasked an outbreak of relapsing B. recurrentis fever in one 16th - 17th century military garrison, missed by real-time PCR. Paleoserology offers a new tool for investigating past epidemics, in complement to DNA sequence-based approaches.

International audience; During the two World Wars, Bartonella quintana was responsible for trench fever and is now recognised as an agent of re-emerging infection. Many reports have indicated widespread B. quintana exposure since the 1990s. In order to evaluate its prevalence in ancient populations, we used real-time PCR to detect B. quintana DNA in 400 teeth collected from 145 individuals dating from the 1st to 19th centuries in nine archaeological sites, with the presence of negative controls. Fisher’s exact test was used to compare the prevalence of B. quintana in civil and military populations. B. quintana DNA was confirmed in a total of 28/145 (19.3%) individuals, comprising 78 citizens and 67 soldiers, 20.1% and 17.9% of which were positive for B. quintana bacteraemia, respectively. This study analysed previous studies on these ancient samples and showed that the presence of B. quintana infection followed the course of time in human history; a total of 14/15 sites from five European countries had a positive prevalence. The positive rate in soldiers was higher than those of civilians, with 20% and 18.8%, respectively, in the 18th and 19th centuries, but the difference in frequency was not significant. These results confirmed the role of dental pulp in diagnosing B. quintana bacteraemia in ancient populations and showed the incidence of B. quintana in both civilians and soldiers.

Strains Marseille-P4001 and Marseille-P3668 are new species from the order Bacteroidales isolated from healthy French volunteers. They are anaerobic Gram-negative rod-shaped bacteria. They exhibited 92.68% and 96.68% 16S rRNA sequence identities with Parabacteroides gordonii strain MS-1 and Parabacteroides chinchillae JCM 17104, respectively, the phylogenetically closest species. Their respective draft genomes measured 5.23 Mb and 3.73 Mb with 39.2 mol% and 40.8 mol% of G + C content. Using a taxonogenomics method, we propose here a brief description of Parabacteroides pacaensis sp. nov., strain Marseille-P4001T and Parabacteroides provencensis sp. nov., strain Marseille-P3668T as new bacterial species. Keywords: Culturomics, human gut microbiota, new bacteria, Parabacteroides pacaensis sp. nov., Parabacteroides provencensis sp. nov

Introduction Dental pulp with special structure has become a good reference sample in paleomicrobiology‐related blood‐borne diseases, many pathogens were detected by different methods based on the diagnosis of nucleic acids and proteins. Objectives This review aims to propose the preparation process from ancient teeth collection to organic molecule extraction of dental pulp and summary, analyze the methods that have been applied to detect septicemic pathogens through ancient dental pulps during the past 20 years following the first detection of an ancient microbe. Methods The papers used in this review with two main objectives were obtained from PubMed and Google scholar with combining keywords: “ancient,” “dental pulp,” “teeth,” “anatomy,” “structure,” “collection,” “preservation,” “selection,” “photography,” “radiography,” “contamination,” “decontamination,” “DNA,” “protein,” “extraction,” “bone,” “paleomicrobiology,” “bacteria,” “virus,” “pathogen,” “molecular biology,” “proteomics,” “PCR,” “MALDI‐TOF,” “LC/MS,” “ELISA,” “immunology,” “immunochromatography,” “genome,” “microbiome,” “metagenomics.” Results The analysis of ancient dental pulp should have a careful preparation process with many different steps to give highly accurate results, each step complies with the rules in archaeology and paleomicrobiology. After the collection of organic molecules from dental pulp, they were investigated for pathogen identification based on the analysis of DNA and protein. Actually, DNA approach takes a principal role in diagnosis while the protein approach is more and more used. A total of seven techniques was used and ten bacteria (Yersinia pestis, Bartonella quintana, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi C, Mycobacterium leprae, Mycobacterium tuberculosis, Rickettsia prowazeki, Staphylococcus aureus, Borrelia recurrentis, Bartonella henselae) and one virus (Anelloviridae) were identified. Y. pestis had the most published in quantity and all methods were investigated for this pathogen, S. aureus and B. recurrentis were identified by three different methods and others only by one. The combining methods interestingly increase the positive rate with ELISA, PCR and iPCR in Yersinia pestis diagnosis. Twenty‐seven ancient genomes of Y. pestis and one ancient genome of B. recurrentis were reconstructed. Comparing to the ancient bone, ancient teeth showed more advantage in septicemic diagnosis. Beside pathogen identification, ancient pulp help to distinguish species. Conclusions Dental pulp with specific tissue is a suitable sample for detection of the blood infection in the past through DNA and protein identification with the correct preparation process, furthermore, it helps to more understand the pathogens of historic diseases and epidemics., Schematic cross‐section of a premolar and a mandibular molar representing the different tissues. (1) Enamel, (2) Dentine, (3) Dental pulp, (4) Cementum, (5) Keratinized gum, (6) Blood vessel and nerves, terminale vascularization, (7) Periodontal ligament, (8) Alveolar bone, (9) Nerve and blood supply, (10) Periodontal pocket with dental calculus

Methanogen cultures require hydrogen produced by fermentative bacteria such asBacteroides thetaiotaomicron(biological method). We developed an alternative method for hydrogen production using iron filings and acetic acid with the aim of cultivating methanogens more efficiently and more quickly (chemical method). We developed this new method with a reference strain ofMethanobrevibacter oralis, compared the method to the biological reference method with a reference strain ofMethanobrevibacter smithiiand finally applied the method to 50 saliva samples. Methanogen colonies counted using ImageJ software were identified using epifluorescence optical microscopy, real-time PCR and PCR sequencing. For cultures containing the pure strains ofM. oralisandM. smithii, colonies appeared three days postinoculation with the chemical method versus nine days with the biological method. The average number ofM. smithiicolonies was significantly higher with the chemical method than with the biological method. There was no difference in the delay of observation of the first colonies in the saliva samples between the two methods. However, the average number of colonies was significantly higher when using the biological method than when using the chemical method at six days and nine days postinoculation (Student’s test, p = 0.005 and p = 0.04, respectively). The chemical method made it possible to isolate four strains ofM. oralisand three strains ofM. smithiifrom the 50 saliva samples. Establishing the chemical method will ease the routine isolation and culture of methanogens.

Methanogens, the sole microbes producing methane, are archaea commonly found in human anaerobic microbiota. Methanogens are emerging as opportunistic pathogens associated with dysbiosis and are also detected and cultured in anaerobic abscesses. Their presence in the respiratory tract is yet unknown. As a preliminary answer, prospective investigation of 908 respiratory tract samples using polyphasic approach combining PCR-sequencing, real-time PCR, fluorescent in situ hybridization (FISH), and methanogens culture was carried out. Methanobrevibacter smithii and Methanobrevibacter oralis DNA sequences, were detected in 21/527 (3.9%) sputum samples, 2/188 (1.06%) bronchoalveolar lavages, and none of 193 tracheo-bronchial aspirations. Further, fluorescence in situ hybridization detected methanogens in three sputum investigated specimens with stick morphology suggesting M. oralis and in another one bronchoalveolar lavage sample investigated, diplococal morphology suggesting M. smithii. These observations extend the known territory of methanogens to the respiratory tract and lay the foundations for further interpretation of their detection as pathogens in any future cases of isolation from bronchoalveolar lavages and the lungs.

Strain Marseille-P4122T is a new species from the order Corynebacteriales that was isolated from the dental plaque of a woman with periodontitis. It is a facultative anaerobic Gram-positive rod-shaped bacterium. Strain Marseille-P4122T exhibited a 98.19% sequence identity with Corynebacterium suicordis strain P81/02, the phylogenetically closely related species with standing in nomenclature. The draft genome size of strain Marseille-P4122T is 2.49 Mb with 60.1% G + C content. We propose that strain Marseille-P4122T (=CSURP4122) is the type strain of the new species Corynebacterium dentalis sp. nov. Keywords: bacteria, Corynebacterium dentalis, culturomics, dental plaque, taxono-genomics

Photogrammetry and cascading microscopy investigations of dental pulp specimens collected from 2,000-year-old individuals buried in a Roman necropolis in Besançon, France, revealed unprecedented preserved tissular and cellular morphology. Photogrammetry yielded 3-D images of the smallest archaeological human remains ever recovered. Optical microscopy examinations after standard haematoxylin-phloxine-saffron staining and anti-glycophorin A immunohistochemistry exposed dental pulp cells, in addition erythrocytes were visualised by electron microscopy, which indicated the ancient dental pulp trapped a blood drop. Fluorescence in situ hybridisation applied on red blood cells revealed the louse-borne pathogen Bartonella quintana, a finding confirmed by polymerase chain reaction assays. Through paleohistology and paleocytology, we demonstrate that the ancient dental pulp preserved intact blood cells at the time of the individual’s death, offering an unprecedented opportunity to engage in direct and indirect tests to diagnose pathogens in ancient buried individuals.

Background A few methanogens have been previously recovered from periodontitis lesions, yet their repertoire may not be completed. We recovered a previously unreported methanogen species in this situation. Case presentation A 64-year-old Caucasian woman was diagnosed with chronic, severe generalized periodontitis. In the presence of negative controls, an 18-month culture of periodontal pockets in anaerobe Hungate tube yielded “Methanobrevibacter massiliense” and Pyramidobacter piscolens. Conclusions This case report provides evidence of the symbiotic strategy deployed by the methanogens and the anaerobes, and reports the first culture of a new methanogen, “M. massiliense”. Electronic supplementary material The online version of this article (10.1186/s13104-017-2980-3) contains supplementary material, which is available to authorized users.

International audience

International audience; Trichomonas tenax, an anaerobic protist difficult to cultivate with an unreliable molecular identification, has been suspected of involvement in periodontitis, a multifactorial inflamma-tory dental disease affecting the soft tissue and bone of periodontium. A cohort of 106 peri-odontitis patients classified by stages of severity and 85 healthy adult control patients was constituted. An efficient culture protocol, a new identification tool by real-time qPCR of T. tenax and a Multi-Locus Sequence Typing system (MLST) based on T. tenax NIH4 reference strain were created. Fifty-three strains of Trichomonas sp. were obtained from peri-odontal samples. 37/106 (34.90%) T. tenax from patients with periodontitis and 16/85 (18.80%˚) T. tenax from control patients were detected by culture (p = 0.018). Sixty of the 191 samples were tested positive for T. tenax by qPCR, 24/85 (28%) controls and 36/106 (34%) periodontitis patients (p = 0.089). By combining both results, 45/106 (42.5%) patients were positive by culture and/or PCR, as compared to 24/85 (28.2%) controls (p = 0.042). A link was established between the carriage in patients of Trichomonas tenax and the severity of the disease. Genotyping demonstrates the presence of strain diversity with three major different clusters and a relation between disease strains and the periodontitis severity (p

Bartonella quintanais a facultative intracellular bacterium responsible for relapsing fever, an example of non-sterilizing immunity. The cellular sanctuary ofB. quintanain-between febrile relapses remains unknown but repeated detection ofB. quintanain dental pulp specimens suggested long-term half-life dental pulp stem cells (DPSCs) as candidates. As the capacity of DPSCs to internalize microscopic particles was unknown, we confirmed that DPSCs internalizedB. quintanabacteria: Gimenez staining and fluorescence microscopy localizedB. quintanabacteria inside DPSCs and this internalization did not affect the cellular multiplication of DPSCs during a one-month follow-up despite the increase in the bacterial load.B. quintana-infected DPSCs did not produce Tumor Necrosis Factor-α whereas an important production of Monocytes Chemoattractant Protein-1 was observed. These unprecedented observations suggested the possibility that DPSCs were shelters for the long-term persistence ofB. quintanain the host, warranting further experimental and clinical investigations.

International audience; The oral fluid microbiome comprises an important bacterial diversity, yet the presence of archaea has not been reported so far. In order to quest for the presence of methanogenic archaea (methanogens) in oral fluid, we used a polyphasic approach including PCR-sequencing detection, microscopic observation by fluorescence in-situ hybridization, isolation and culture, molecular identification and genotyping of methanogens in 200 oral fluid specimens. In the presence of negative controls, 64/200 (32%) prospectively analysed oral fluid specimens were PCR-positive for methanogens, all identified as Methanobrevibacter oralis by sequencing. Further, fluorescence in-situ hybridization detected methanogens in 19/48 (39.6%) investigated specimens; with morphology suggesting M. oralis in 10 cases and co-infecting Methanobrevibacter smithii in nine cases. M. oralis was cultured from 46/64 (71.8%) PCR-positive specimens and none of PCR-negative specimens; and one M. smithii isolate was co-cultured with M. oralis in one specimen. Multispacer Sequence Typing found one M. oralis genotype per specimen and a total of five different genotypes with 19/46 (41%) of isolates all belonging to spacer-type four. Statistical analyses showed a significant correlation between the PCR-detection of methanogens in oral fluid and tobacco smoking. These data indicate that M. oralis and M. smithii are oral fluid-borne methanogens in tobacco smokers. Both methanogens could be transmitted during intimate contacts such as mother-to-child contacts and kissing.

Methanogens have already been described in periodontitis but not in peri-implantitis. Thirty peri-implantitis samples and 28 control samples were collected in 28 consenting peri-implantitis patients. PCR-sequencing of the 16S rRNA gene was used as a broad-spectrum screening method and results were further confirmed by real-time quantitative PCR targeting the mcrA genes. Results showed a methanogen community dominated by Methanobrevibacter oralis in 31/58 (51%) samples including 16/28 (57%) control samples and 15/30 (50%) peri-implantitis samples. Methanobrevibacter massiliense was detected in 5/58 (8.6%) samples including 3/28 (1%) control samples and 2/30 (6.7%) peri-implantitis samples. The prevalence of M. oralis or M. massiliense did not significantly differ in peri-implantitis and control samples (exact Fisher test, P = 0.61 and P = 0.67, respectively). Further ponderation of the methanogen load by the real-time quantitative PCR for actin human gene again indicated non-significant difference (Wilcoxon-Mann-Whitney test, P = 0.48 and P = 0.40, respectively). These data show that the prevalence of methanogens does not differ in peri-implantitis lesions and healthy sites, when individuals are their own control. These data do not allow assigning a specific pathogenic role to methanogens in peri-implantitis; methanogens rather are part of the commensal and normal flora of the oral cavity.

The Paleomicrobiology establishes the diagnosis of ancient infectious diseases by studying ancient pathogens. This recent science also analyzes the evolution of these pathogens, virulence, and their adaptation to their habitat and their vectors. The DNA persists a long time after the death of an organism despite the chemical and enzymatic degradation. The possibility of sequencing bacterial, viral, parasitic and archaeal DNA molecules persists over time. Various sources are used for these studies: frozen tissue and particularly human tissue are a exceptional source for the analysis because at very low temperatures, all biological activity is suspended. The coprolites are a source of choice for studying the human microbiome. Other sources, the ancient bones are the most abundant, however, they may contain only small amounts of DNA due to natural leaching. When the use of the tooth is possible, is a particularly interesting source because of its highly mineralized structure, which gives greater persistence than bone. The calcified tartar deposited on teeth is a source of interest for the study of oral microbiome. All these sources are subject to precautions (gloves and masks hat) at the time of sampling to avoid cross contamination and also be listed in the most precise way because they are precious and rare.

International audience; Dental calculus, a material observed in the majority of adults worldwide, emerged as a source for correlating paleomicrobiology with human health and diet. This mini review of 48 articles on the paleomicrobiology of dental calculus over 7550years discloses a secular core microbiota comprising nine bacterial phyla - Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, TM7, Synergistetes, Chloroflexi, Fusobacteria, Spirochetes - and one archaeal phylum Euryarchaeota; and some accessory microbiota that appear and disappear according to time frame. The diet residues and oral microbes, including bacteria, archaea, viruses and fungi, consisting of harmless organisms and pathogens associated with local and systemic infections have been found trapped in ancient dental calculus by morphological approaches, immunolabeling techniques, isotope analyses, fluorescent insitu hybridization, DNA-based approaches, and protein-based approaches. These observations led to correlation of paleomicrobiology, particularly Streptococcus mutans and archaea, with past human health and diet.

International audience; The in-laboratory contamination of the ancient samples hinders the result interpretation of the investigations in the field of paleomicrobiology. We had promoted the dental pulp as a sample that limits the risks of in-laboratory contamination of the ancient material. In this work, we measured the contamination of the dental pulp manipulated according to paleomicrobiology protocol, used as a source of a total DNA for metagenomics. First, total DNA extracted from two dog canines was sequenced using next generation sequencing. This yielded a total of 487,828 trimmed reads with a length of 227 +/- 35 bp. Sequence analysis of the final dataset using Blast algorithm search and stringent thresholds for sequence identity and coverage against a database including both Canis lupus familiaris and Homo sapiens complete genomes showed that 95% of reads were assigned to C familiaris whereas 0.03% was assigned to H. sapiens. In a second step, two teeth collected from two 12th century mammals were manipulated following the same protocol. A total of 13,890 trimmed reads with a 157 +/- 67 bp length yielded 0-0.35% reads assigned to H. sapiens. This study indicates that the dental pulp is a useful for detecting the significant nucleic sequences in both modern and ancient samples. (C) 2017 Elsevier Ltd. All rights reserved.

Peri-implantitis is an infection of the tissue around an implant, resulting in the loss of supporting bone. Risk factors for peri-implantitis consist of a history of periodontitis, dental plaque, poor oral hygiene, smoking, alcohol consumption and diabetes. A clinical diagnosis indicates inflammatory signs including bleeding on probing with or without suppuration and a peri-implant pocket depth ≥5 mm. A radiograph shows images of marginal bone loss ≥2 mm. A differential diagnosis of peri-implant mucositis, occlusal overload, retrograde peri-implantitis and inflammatory implant periapical lesions suggests the appropriate treatment in each case. The non-surgical treatment of peri-implantitis, including a mechanical treatment alone or combined with antiseptics or antibiotics can improve clinical parameters in the short term but residual defects may still persist. Surgical treatment such as guided bone regeneration results in a gain of clinical attachment level and bone reconstruction in the long term. The limited effect of laser-assisted therapy needs to be further evaluated. The concept of prevention based on early detection and regular maintenance plays a principal role in reducing the occurrence of peri-implantitis.

Geminated teeth occur more frequently in the deciduous dentition than in the permanent dentition, with prevalence figures of 0.6% and 0.1%, respectively. Bilateral presentation is rare. A survey of the literature showed that the prevalence estimates for bilateral double teeth range from 0.01% to 0.04% in the deciduous dentition, and 0.05% in the permanent dentition. In this article, we report a rare case of bilateral fusion of the maxillary permanent incisors, discuss possible histogenetic mechanisms related to this anomaly, and explain the treatment management.

Studying ancient infectious diseases is a challenge, as written contemporary descriptions, when available, are often imprecise and do not allow for accurate discrimination among the pathogens endemic at that time. Paleomicrobiology offers a unique access to the history of these infections by identifying precisely the causative agents. Body louse-transmitted infections are amongst the most epidemic diseases in history, especially in war and famine periods. Of these, Bartonella quintana was detected by suicide PCR in 4000-year-old human remains, thus representing the oldest evidence to date of an arthropod-transmitted infection to human beings. This species has also been detected in human specimens from the 11th to 15th, 18th and 19th centuries. In addition, Bartonella henselae, a cat- and flea-associated pathogen, was detected in cat specimens from the 13th to 18th centuries, therefore demonstrating an association of the bacterium and its reservoir for over 800 years. Therefore, pathogenic Bartonella species have been involved in several outbreaks in the past millennia and should systematically be investigated in human remains from suspected epidemics.

Background. Many soldiers in Napoleon's Grand Army died of infectious diseases during its retreat from Russia. Because soldiers were commonly infested with body lice, it has been speculated that louse-borne infectious diseases, such as epidemic typhus (caused by Rickettsia prowazekii), were common. Methods. We investigated this possibility during recent excavations of a mass grave of Napoleon's soldiers in Vilnius, Lithuania. Segments of 5 body lice, identified morphologically and by polymerase chain reaction (PCR) amplification and sequencing, were found in earth from the grave that also contained fragments of soldiers' uniforms. Results. DNA of Bartonella quintana (the agent of trench fever) was identified by PCR and sequencing in 3 of the lice. Similarly, PCR and sequencing of dental pulp from the remains of 35 soldiers revealed DNA of B. quintana in 7 soldiers and DNA of R. prowazekii in 3 other soldiers. Conclusions. Our results show that louse-borne infectious diseases affected nearly one-third of Napoleon's soldiers buried in Vilnius and indicate that these diseases might have been a major factor in the French retreat from Russia.

Bartonella henselae causes chronic bacteremia in cats. To test if B. henselae DNA can be recovered from the dental pulp of cats buried a year previously, we used PCR with primers for a sequence of the conserved groEL to test 104 teeth from 11 cats. Seven of the cats were found positive; canine teeth were more frequently positive than molar teeth. Where PCR sequences could be determined, they were identical to those of B. henselae Marseille (four cats), B. henselae Houston (one cat) or similar to those of B. grahamii (one cat). Our study indicates that dental pulp from the teeth of cats, especially the canine teeth, may be used for the PCR detection of Bartonella in animals buried for a year.

Methanobrevibacter smithii is the main archaea in human, detoxifying molecular hydrogen resulting from anaerobic bacteria fermentations into gaseous methane. Its identification relies on gene sequencing, but no method is available to discriminate among genetic variants of M. smithii. Here, we developed a multispacer sequence typing (MST) for genotyping the genetic variants of M. smithii. Four intergenic spacers recovered from the M. smithii reference genome were PCR amplified and sequenced in three M. smithii reference strains and in a collection of 22 M. smithii isolates from the oral cavity in two individuals and the gut of 10 additional individuals. Sequencing yielded 216 genetic polymorphisms including 89 single nucleotide polymorphisms (41.2 %), 83 insertions (38.4 %), and 44 deletions (20.4 %). Combining these genetic polymorphisms yielded 15 genotypes with an index of discrimination of 0.942 (confidence interval 0.9–0.984; P