Maureen Laroche

Identification of tick species and disseminate pathogen using hemolymph by MALDI-TOF MS.

BACKGROUND Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is increasingly emerging tool for identification of arthropods including tick vectors using whole or body part of specimens. The challenges of the present study were to assess MALDI-TOF MS profiling for the both identification of tick species and Rickettsia spp. in infected ticks using hemolymph as protein mixture. METHODS Firstly, hemolymph protein mixture from legs of 5 tick species, Rhipicephalus sanguineus, Rhipicephalus bursa, Dermacentor marginatus, Hyalomma marginatum rufipes and Amblyomma variegatum infected by Rickettsia africae were submitted to MALDI-TOF MS to assess tick species identification ability. Secondly, hemolymph MS spectra from Rh. sanguineus infected or not by Rickettsia c. conorii were compared to detect protein profiles changes. Finally, leg hemolymph MS spectra from new specimens of the 5 tick species were tested blindly including ticks infected by R. c. conorii. Discriminating mass peaks distinguishing the R. c. conorii infected and non-infected Rh sanguineus were determined. RESULTS Consistent and reproducible MS profiles were obtained into each tick species. Comparison of MS spectra revealed distinct hemolymph protein profiles according to tick species. MS spectra changes were observed between hemolymphs from R. c. conorii-infected and non-infected Rh. sanguineus specimens, revealing 17 discriminating mass peaks. Clustering analysis based on MS protein profiles highlighted that hemolymph samples were grouped according to tick species. All tick hemolymph samples blindly tested against our home-made arthropod MS reference database were correctly identified at the species distinguishing also R. c. conorii-infected from Rickettsia-free Rh. sanguineus specimens. CONCLUSION The present study demonstrated the use of hemolymph MS profiles for dual identification of tick species and associated pathogens. This concomitant identification could be helpful for tick entomological diagnosis, notably for specimens removed directly on patients.

MALDI-TOF MS as an innovative tool for detection of Plasmodium parasites in Anopheles mosquitoes

BackgroundMalaria is still a major public health issue worldwide, and one of the best approaches to fight the disease remains vector control. The current methods for mosquito identification include morphological methods that are generally time-consuming and require expertise, and molecular methods that require laboratory facilities with relatively expensive running costs. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) technology, routinely used for bacterial identification, has recently emerged in the field of entomology. The aim of the present study was to assess whether MALDI-TOF MS could successfully distinguish Anopheles stephensi mosquitoes according to their Plasmodium infection status.MethodsC57BL/6 mice experimentally infected with Plasmodium berghei were exposed to An. stephensi bites. For the determination of An. stephensi infection status, mosquito cephalothoraxes were dissected and submitted to mass spectrometry analyses and DNA amplification for molecular analysis. Spectra were grouped according to mosquitoes’ infection status and spectra quality was validated based on intensity and reproducibility within each group. The in-lab MALDI-TOF MS arthropod reference spectra database, upgraded with representative spectra from both groups (infected/non-infected), was subsequently queried blindly with cephalothorax spectra from specimens of both groups.ResultsThe MALDI TOF MS profiles generated from protein extracts prepared from the cephalothorax of An. stephensi allowed distinction between infected and uninfected mosquitoes. Correct classification was obtained in blind test analysis for (79/80) 98.75% of all mosquitoes tested. Only one of 80 specimens, an infected mosquito, was misclassified in the blind test analysis.ConclusionsMatrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry appears to be a promising, rapid and reliable tool for the epidemiological surveillance of Anopheles vectors, including their identification and their infection status.

Molecular and MALDI-TOF identification of ticks and tick-associated bacteria in Mali

Ticks are considered the second vector of human and animal diseases after mosquitoes. Therefore, identification of ticks and associated pathogens is an important step in the management of these vectors. In recent years, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of arthropods including ticks. The objective of this study was to improve the conditions for the preparation of tick samples for their identification by MALDI-TOF MS from field-collected ethanol-stored Malian samples and to evaluate the capacity of this technology to distinguish infected and uninfected ticks. A total of 1,333 ticks were collected from mammals in three distinct sites from Mali. Morphological identification allowed classification of ticks into 6 species including Amblyomma variegatum, Hyalomma truncatum, Hyalomma marginatum rufipes, Rhipicephalus (Boophilus) microplus, Rhipicephalus evertsi evertsi and Rhipicephalus sanguineus sl. Among those, 471 ticks were randomly selected for molecular and proteomic analyses. Tick legs submitted to MALDI-TOF MS revealed a concordant morpho/molecular identification of 99.6%. The inclusion in our MALDI-TOF MS arthropod database of MS reference spectra from ethanol-preserved tick leg specimens was required to obtain reliable identification. When tested by molecular tools, 76.6%, 37.6%, 20.8% and 1.1% of the specimens tested were positive for Rickettsia spp., Coxiella burnetii, Anaplasmataceae and Borrelia spp., respectively. These results support the fact that MALDI-TOF is a reliable tool for the identification of ticks conserved in alcohol and enhances knowledge about the diversity of tick species and pathogens transmitted by ticks circulating in Mali.

A novel ehrlichial agent detected in tick in French Polynesia

Ticks are hematophageous arthropods that are known to host and transmit miscellaneous pathogens including zoonotic bacteria. The aim of this study was to investigate the presence of tick-associated microorganisms in Tahiti, French Polynesia with molecular tools. A total of 658 ticks from two species including Rhipicephalus sanguineus s.l. and Rh. annulatus were collected with forceps on dogs and cattle respectively, or with a flag on pasture in several locations of Tahiti in 2013. Two Rickettsia belonging to the spotted fever group different from R. conorii and R. massiliae were detected by qPCR in two Rh. sanguineus s.l. ticks, but sequencing failed. A Rh. annulatus tick was found positive for a new ehrlichial agent characterized by amplification and sequencing of fragments of the Anaplasmataceae 23S and Ehrlichia 16S genes. Phylogenetic analyses based on the 23S and 16S sequences reveals that this bacterium is a new genotype, genetically close to Ehrlichia minasensis, a recently described Ehrlichia sp. close to Ehrlichia canis.

Use of eschar swabbing for the molecular diagnosis and genotyping of Orientia tsutsugamushi causing scrub typhus in Quang Nam province, Vietnam

Background Scrub typhus is a rickettsiosis which is caused by Orientia tsutsugamushi and occurs throughout the Asia-Pacific region. Molecular diagnosis of rickettsioses using eschar swabs has recently emerged, and may be very useful for the diagnosis of these diseases in tropical settings. Methodology/Principal findings Quantitative polymerase chain reaction (qPCR) was used to detect O. tsutsugamushi DNA in whole blood and eschar swab specimens of 67 patients who were clinically suspected of scrub typhus in Quang Nam province, Vietnam. Among the 20 patients for whom both eschar and whole blood were obtained, 17 (85%) of the eschar specimens and 5 (25%) of the whole blood specimens tested positive for O. tsutsugamushi. Genetic analysis of the 56-kDa TSA gene sequences demonstrated that the 14 sequences obtained in this study, including 12 eschar swabs and 2 whole blood specimens, were related to 4 groups: Karp, Kawasaki, Gilliam (JG-v and TG-v) and TA716. The majority (9/14; 64.4%) of contemporary O. tsutsugamushi genotypes in Quang Nam province were related to the Karp group. Conclusions These results suggest that polyclonal antigen pools used for serological testing in the future should contain at least Karp, Kawasaki, Gilliam and TA716 antigens for Vietnamese patients, as well as patients who have traveled to Vietnam. qPCR after eschar swabbing should be considered for molecular diagnosis of scrub typhus in endemic patients as well as in travelers, since it is easy to perform and appears very useful for the rapid detection of Orientia tsutsugamushi in the early phase of infection.

Detection of a Potential New Bartonella Species “Candidatus Bartonella rondoniensis” in Human Biting Kissing Bugs (Reduviidae; Triatominae)

Background Among the Reduviidae family, triatomines are giant blood-sucking bugs. They are well known in Central and South America where they transmit Trypanosoma cruzi to mammals, including humans, through their feces. This parasitic protozoan is the causative agent of Chagas disease, a major public health issue in endemic areas. Because of the medical and economic impact of Chagas disease, the presence of other arthropod-borne pathogens in triatomines was rarely investigated. Methodology/Principal findings In this study, seven triatomines species involved in the transmission of T. cruzi were molecularly screened for the presence of known pathogens generally associated with arthropods, such as Rickettsia, Bartonella, Anaplasmataceae, Borrelia species and Coxiella burnetii. Of all included triatomine species, only Eratyrus mucronatus specimens tested positive for Bartonella species for 56% of tested samples. A new genotype of Bartonella spp. was detected in 13/23 Eratyrus mucronatus specimens, an important vector of T. cruzi to humans. This bacterium was further characterized by sequencing fragments of the ftsZ, gltA and rpoB genes. Depending on the targeted gene, this agent shares 84% to 91% of identity with B. bacilliformis, the agent of Carrion’s disease, a deadly sandfly-borne infectious disease endemic in South America. It is also closely related to animal pathogens such as B. bovis and B. chomelii. Conclusions As E. mucronatus is an invasive species that occasionally feeds on humans, the presence of potentially pathogenic Bartonella-infected bugs could present another risk for human health, along with the T. cruzi issue.

Medical Entomology: A Reemerging Field of Research to Better Understand Vector-Borne Infectious Diseases

In the last decade, the Chikungunya and Zika virus outbreaks have turned public attention to the possibility of the expansion of vector-borne infectious diseases worldwide. Medical entomology is focused on the study of arthropods involved in human health. We review here some of the research approaches taken by the medical entomology team of the University Hospital Institute (UHI) Méditerranée Infection of Marseille, France, with the support of recent or representative studies. We propose our approaches to technical innovations in arthropod identification and the detection of microorganisms in arthropods, the use of arthropods as epidemiological or diagnostic tools, entomological investigations around clinical cases or within specific populations, and how we have developed experimental models to decipher the interactions between arthropods, microorganisms, and humans.

Detection of Bartonella spp. in fleas by MALDI-TOF MS

Background Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged in the field of entomology as a promising method for the identification of arthropods and the detection of associated pathogens. Methodology/Principal findings An experimental model of Ctenocephalides felis (cat fleas) infected with Bartonella quintana and Bartonella henselae was developed to evaluate the efficacy of MALDI-TOF MS in distinguishing infected from uninfected fleas, and its ability to distinguish fleas infected with Bartonella quintana from fleas infected with Bartonella henselae. For B. quintana, two groups of fleas received three successive blood meals, infected or not. A total of 140 fleas (100 exposed fleas and 40 control fleas) were engorged on human blood, infected or uninfected with B. quintana. Regarding the second pathogen, two groups of fleas (200 exposed fleas and 40 control fleas) were fed in the same manner with human blood, infected or not with Bartonella henselae. Fleas were dissected longitudinally; one-half was used for assessment of B. quintana and B. henselae infectious status by real-time PCR, and the second half was subjected to MALDI-TOF MS analysis. Comparison of MS spectra from infected fleas and uninfected fleas revealed distinct MS profiles. Blind queries against our MALDI-TOF MS arthropod database, upgraded with reference spectra from B. quintana and B. henselae infected fleas but also non-infected fleas, provided the correct classification for 100% of the different categories of specimens tested on the first model of flea infection with Bartonella quintana. As for Bartonella henselae, 81% of exposed qPCR-positive fleas, 96% of exposed qPCR-negative fleas and 100% of control fleas were correctly identified on the second model of flea infection. MALDI-TOF MS successfully differentiated Bartonella spp.-infected and uninfected fleas and was also able to correctly differentiate fleas infected with Bartonella quintana and fleas infected with Bartonella henselae. MALDI-TOF MS correctly identified flea species as well as their infectious status, consistent with the results of real-time PCR. Conclusions/Significance MALDI-TOF is a promising tool for identification of the infection status of fleas infected with Bartonella spp., which allows new possibilities for fast and accurate diagnosis in medical entomology and vector surveillance.

MALDI-TOF MS identification of ticks of domestic and wild animals in Algeria and molecular detection of associated microorganisms

Recent studies have reported the reliability of MALDI-TOF MS for arthropod identification, including fresh or alcohol-preserved ticks based on leg-derived mass spectra. The aim of this study was to evaluate the performance of MALDI-TOF MS for the identification of alcohol-preserved Algerian ticks collected from different domestic and wild hosts. Secondly, we conducted a molecular survey to detect the presence of bacterial DNA in all ticks that were previously subjected to MALDI-TOF MS. A total of 2635 ixodid and 1401 argasid ticks belonging to 9 distinct species were collected in nine different regions of northeastern Algeria. The legs of 230 specimens were subjected to MALDI-TOF MS assays. Spectral analysis revealed intra-species similarity and inter-species specificity for the MS spectra, which was consistent with the morphological identification. Blind tests against the in-lab database revealed that 93.48% of the tested specimens were correctly identified. The accuracy of the morphological and MALDI-TOF MS identifications was validated by sequencing the 12S ribosomal RNA gene (rRNA) for 33 specimens and all the ticks were correctly identified. The quantitative PCR screening showed that for 219 tested ticks, 15 were positive for Rickettsia spp., 8 for Borrelia spp. and 17 for Anaplasmataceae. The PCR tests were negative for Coxiella burnetii and Bartonella spp. This study supports MALDI-TOF MS being a reliable tool for the identification of arthropods and brings new data that sheds light on tick species diversity and tick-borne diseases in Algeria.

Agents zoonotiques vectorisés étudiés à l’institut Hospitalo-Universitaire Méditerranée Infection

Medical entomology is dedicated to the study of arthropods that are involved with human health but also animal health as many zoonotic diseases have been emerging all around the world for the past few years. We hereby introduce some research fields developed by the Medical Entomology team of the University Hospital Institute Mediterranee Infection, illustrated with recent or emblematic studies: technical innovation for identification of arthropods, the use of arthropods as epidemiological or diagnostic tool, entomological investigations around clinical cases and the development of experimental models for a better understanding of interactions between arthropods, humans and microorganisms.