Mycobacterium leprae (M. leprae) and M. lepromatosis, an obligate intracellular organism, and over 200,000 new cases occur every year. M. leprae parasitizes histiocytes (skin macrophages) and Schwann cells in the peripheral nerves. Although leprosy can be treated by multidrug therapy, some patients relapse or have a prolonged clinical course and/or experience leprosy reaction. These varying outcomes depend on host factors such as immune responses against bacterial components that determine a range of symptoms. To understand these host responses, knowledge of the mechanisms by which M. leprae parasitizes host cells is important. This article describes the characteristics of leprosy through bacteriology, genetics, epidemiology, immunology, animal models, routes of infection, and clinical findings. It also discusses recent diagnostic methods, treatment, and measures according to the World Health Organization (WHO), including prevention. Recently, the antibacterial activities of anti-hyperlipidaemia agents against other pathogens, such as M. tuberculosis and Staphylococcus aureus have been investigated. Our laboratory has been focused on the metabolism of lipids which constitute the cell wall of M. leprae. Our findings may be useful for the development of future treatments.Leprosy is caused by   Mycobacterium leprae (M. leprae), which was discovered by Gerhard Armauer Hansen of Norway in 1873 [M. lepromatosis [Leprosy is caused by  in 1873  and M. lomatosis . Areas homatosis . New lepomatosis , but in omatosis . The WHOomatosis . The goaomatosis .M.leprae is an obligate intracellular organism. This pathogen affects mostly the skin and the peripheral nerves [M. leprae. It is also a major factor that leads to disabling neurological disorders.l nerves . This bal nerves . Skin leDelays in diagnosis and inadequate treatment of leprosy are responsible for a large variety of clinical symptoms. These symptoms frequently cause deformity and disability in perpetuity, often resulting in stigma. In particular, sensory nerve damage results in numbness and analgesia that can cause repeated injuries and subsequent loss of limbs. Motor neuropathy accompanying peripheral neuropathy causes major problems in activities of daily living (ADL) such as hand movement and gait, whereas secondary disuse muscle atrophy further impairs ADL to create a vicious cycle of disease.A leprosy control program led by the WHO focused on early detection of patients and early treatment with antimicrobial agents to control new cases globally . HoweverM. leprae may contribute to clinical manifestations as well as challenges in treatment for leprosy. During infection, M. leprae parasitizes host cells and modifies the host cellular environment to promote its survival. Cell wall components of the bacilli may be important factors for such parasitization, since cell wall components not only elicit host immunity, but can also act as signalling factors that aid its survival [M. leprae has many pseudogenes and is therefore only capable of surviving in host cells, using the functions of host cells for its survival. Further, M. leprae has been shown to survive in soil for more than 46 days [M. leprae infection, colonization and growth are preserved, many other genes have been discarded. Thus, M. leprae carries out various metabolic mechanisms, including lipid metabolism essential for cell wall synthesis, by parasitizing host cell machinery. Survival of the bacteria while immunologically concealed within host cells may require evasion of immune surveillance mechanisms to allow long-term parasitization. In this review, we summarize the pathogenicity of M. leprae, with particular focus on the underlying molecular mechanisms that enable intracellular parasitization.The biological characteristics of survival . M. lepr 46 days . AlthougM.leprae is an obligate intracellular organism and the taxonomic classification of this bacillus comprises the class Schizomycetes, order Actinomycetales, family Mycobacteriaceae, and genus Mycobacterium [M. leprae is an acid-alcohol-fast bacillus, non-motile and microaerophilic. M. leprae mainly infects and invades skin macrophages and Schwann cells in the peripheral nerves to produce a chronic infection in humans. Using the Gram strain, M. leprae tests as invisible, producing negatively stained representations known as “ghosts,” or as rod-shaped Gram-positive bacilli [M. leprae does not become discoloured by acid-alcohol with the Ziehl-Neelsen stain, a red stain that contains fuchsin. Thus, it appears as characteristic acid-alcohol-resistant bacilli using a slit skin smear test [M. leprae multiplies very slowly, requiring 12 to 14 days for generation, compared to the longer time (20 hours) needed by Mycobacterium tuberculosis (M. tuberculosis) [M. leprae requires a low temperature for growth [M. leprae was reported to survive up to 46 days in the environment [acterium ,18. The  bacilli ,20. Due ear test ,20  ,22. Althr growth . Therefoironment ,24–26.M.leprae has a thick cell wall surrounding the plasma membrane that comprises inner and outer layers [M. leprae cell wall includes small amounts of TMM [M. leprae cell wall includes more mycolic acid than that of M. tuberculosis (the ratio of mycolic acid to PGN is 21:10 versus 16:10).r layers   in a glycosidic bond to the constituent phenol [M. leprae promotes neural damage [PGL-I containing trisaccharides are located at the outermost surface and interact with the host cell membrane . The PGLt phenol . PGL-I it phenol ,36. The l damage ,38 by inl damage . Thus, PM. leprae. The immunomodulatory roles of LAM are suppression of T-cell activation [LAM consists of three constituents: a mannosyl-phosphatidyl-myo-inositol (MPI) anchor, a polysaccharide backbone, and capping moieties . Three ttivation , interfetivation , inhibittivation , and indtivation ,47.M. leprae PGN belongs to the chemotype IV group and contains peptide side chains containing diaminopimelic acid (DAP) [Mycobacterium PGN structure, several compositions were identified [N-glycolylmuramic acid (MurNGlyc), cross-links of DAP-DAP, and modifications of the carboxylic acid functional groups of DAP and D-Glu. A number of these attributes are related to PGN of M. leprae [M. leprae PG [M. leprae PGN contains amidated DAP and Gly residues, it may evade host innate immune responses mediated by NOD1 and NOD2 [id (DAP) ,49. Accoentified . These i. leprae , which w. leprae . Howevereprae PG . An immueprae PG . Furthereprae PG ,59. Wheneprae PG . Since Mand NOD2 .M. leprae genome requires it to parasitize host cells for survival. 2001 saw the complete genome sequencing of M. leprae. The 3.3 Mbp genome contains 1,604 genes that encode proteins and the remainder include 1,116 pseudogenes as well as non-coding regions [M. tuberculosis have been lost in M. leprae, and the M. tuberculosis genome is larger than the M. leprae genome [M. leprae all major anabolic pathways are relatively intact. However, the genes encoding the lipolysis pathways used to digest host lipids and fatty acids for energy have been extensively downsized [As mentioned above, the loss of many functional genes from the  regions ,62. The  regions . 50% of e genome . In M. lownsized .M. leprae depends on the host cell lipid metabolism to survive. Of the 24 genes encoding Lip lipolytic enzymes in M. tuberculosis, only ML0119c (lipE), ML0314c (lipU), and ML1899 (lipG) are conserved in M. leprae. Similarly, the central and energy-related metabolic pathways are damaged, and therefore M. leprae cannot use common sources of carbon such as galactose and acetate to produce ATP from NADH oxidation. In addition, all biosynthetic and transport systems including microaerophilic and anaerobic electron transfer, as well as the complementary enzyme groups, have been lost in the M. leprae genome. This loss implies that the catabolic capacity of M. leprae is limited, and the bacterium can only employ a minimal number of carbon sources for growth [M. leprae, but the genes associated with a parasitic lifecycle dependent on the host have been discarded. In particular, M. leprae relies on host genes for various metabolic mechanisms, including those involved in lipid metabolism that is essential for cell wall synthesis. This parasitization of host cell genes could explain the extremely slow growth rate of the bacteria and the difficulties with culture using various types of normal media. Meanwhile, M. leprae is immunologically concealed in host cells, which may facilitate the long-term evasion of host immune surveillance mechanisms.Since the genes that contribute to lipid biosynthesis and metabolism are pseudogenes, r growth ,64,65. GM. leprae pseudogenes arose from stop codon insertions that may have been caused by sigma factor dysfunction or the insertion of transposon-derived repetitive sequences [M. leprae gene structures indicated the existence of multiple stop codon insertions and fragmented ORFs, precluding the translation of these genes into functional proteins [M. leprae genome, namely RLEP (37 copies); REPLEP (15 copies); LEPREP (8 copies); and LEPRPT (5 copies) [M. leprae is largely due to the recombination of these repetitive sequences, which contribute almost 2% of the M. leprae TN genome. RLEP is often present within pseudogenes and at the 3’-termini of genes, and is often utilized as a PCR target to detect M. leprae. These sequences were suggested to be the vestiges of transposons that had lost the ability to go through transposition [Many equences . Investiproteins . Over 26 copies) . The decposition .M. leprae. However, some RNAs encoded by these regions are expressed as RNA, and these RNAs can have variable expression levels after infection and among patients with leprosy [M. leprae genome, Suzuki et al. revealed that many RNAs are transcribed from pseudogenes and untranslated regions [M. leprae pseudogenes is that between one and 40 stop codons are present in-frame. The majority (75%) of pseudogenes that are transcribed do not have a conventional start codon, but 67% contain five or more stop codons [RLEP sequences specific to M. leprae and sequences with no homology to identified functional non-coding RNAs are included in the non-coding regions [There is little detailed information regarding the functional role of pseudogenes and non-coding regions in  leprosy . Among t leprosy ,67. In t regions . Using t regions ,73,74. Ap codons . The sig regions . Althoug regions ,75,76.M. leprae. Globally, approximately 32 STR loci have been employed for the strain typing of M. leprae [M. leprae branches are classified as specific SNP types or subtypes [Genotyping with single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) is useful to elucidate the transmission and origin of . leprae . M. leprsubtypes , includisubtypes ,83, thatsubtypes . Consequsubtypes . Branch subtypes . SNP typsubtypes ,96. Bransubtypes  with zoosubtypes . Branch subtypes ,98. The subtypes .M. leprae to have a long incubation period (as long as 30 years) was demonstrated in a study by Suzuki et al. The authors reported a 31-year-old female chimpanzee who was diagnosed with leprosy in Japan. In 1980, this chimpanzee was 2 years old and was trapped in Sierra Leone, West Africa, then sent to Japan. PCR amplification and direct sequencing were performed for SNP analysis to decide the origin of this case. The genotype of the bacilli had only been identified in West Africa, and had not been detected in Japan. It was classified as the SNP type 4 [M. leprae genomes which were identified from nonhuman primates and humans from around the world have been carried out. These analyses show that an M. leprae strain from a cynomolgus macaque and a human M. leprae strain of New Caledonia are closest in lineage. However, the M. leprae strains found in chimpanzees and sooty mangabeys are closely affiliated with a West African human M. leprae strain [The potential for P type 4 ,101. Nate strain .M. leprae [M. leprae causes the parasite to persist without tissue injury of the peripheral nerves and skin.An excessively strong immune response can also cause damage. Thus, host immune systems influence the clinical presentation of leprosy [M. leprae by humoral immunity is difficult. This contributes to the progression of the disease [The spectrum of clinicopathological manifestations in leprosy arises due to variable host immune responses to . leprae ,103. Imm leprosy ,105. His leprosy ,106 , including Toll-like receptors (TLRs) [M. leprae lipoproteins are ligands of the TLR2/1 heterodimer [M. leprae activates TLR2/1 heterodimers of skin macrophages, stimulating the killing of M. leprae [M. leprae can also activate innate receptors such as TLR4 [M. leprae, PGL-I, dampens TLR4 signalling pathway activity in macrophages by downregulating protein expression of the TIR-domain-containing adapter-inducing interferon-β (TRIF), a TLR4 adaptor.Initial recognition of s (TLRs)  and NLRss (TLRs) . M. leprerodimer ,116 and erodimer ,118. PGNerodimer ,119,120. as TLR4 . HoweverM. leprae also induces IL-8 secretion through the TLR9/NF-κB pathway [M. leprae. NOD2 is known as an another PRR that participates in the detection of M. leprae [In human alveolar epithelial cells,  pathway . IL-8 me. leprae ,123. The. leprae ,114.T-cell responses also lead to M. leprae clearance in the skin lesions [M. leprae-specific humoral immunity has rarely been detected in TT cases [M. leprae in skin lesions cannot be inhibited [M. leprae as well as foamy macrophages in widespread skin lesions can also be seen [The characteristic immune response in patients with TT is the Th1 cytokine response with production of IFN-γ and IL-12, which induces cell-mediated immunity. High  lesions . In TT cTT cases ,127. On nhibited . In LL c be seen   cells in leprosy and there is no consistent theory regarding their roles in leprosy cases. The presence of Tregs in skin lesions in leprosy is known . A relev. leprae ,137. The. leprae . On the . leprae . These fnfection . Howevernfection . During nfection .M. leprae. Macrophages are classified as M1 (pro-inflammatory) and M2 (anti-inflammatory) according to the Th1-Th2 dichotomy. These macrophages differ in their cell surface markers, cytokine secretion and biological functions [M. leprae is phagocytosed by macrophages, a process that is facilitated by the complement receptors CR1 (CD35), CR3 (CD11b/CD18) and CR4 (CD11c/CD18) [M. leprae multiplies in lipid-filled phagosomes in foamy macrophages. On the other hand, in TT lesions, M1 macrophages activate the classical pathway, leading to the induction of IFN-γ, TNF-α, and iNOS [M. leprae [M. leprae infection. Conversely, a predominance of M2 macrophages promotes anti-inflammatory reactions that can be observed in LL lesions. M2 macrophages induce the production of TGF-β, IL-10, fibroblast growth factor (FGF)-β, CD163, CD209, arginase 1, and indoleamine 2, 3-dioxygenase (IDO), which is involved in immunosuppressive reactions and repair of tissue [leprae by CD209+CD163+ macrophages was also demonstrated with LDL in foam cells of LL lesions, suggesting a function for IL-10 derived macrophages in lipid uptake [Macrophages have an important role in leprosy pathogenesis mediate interactions between the host and unctions ,144. A cunctions . M. lepr1c/CD18) . LL skinand iNOS . These c. leprae ,149. A p. leprae . Further. leprae . Therefof tissue . IL-10-pf tissue  are typif tissue . Uptake d uptake .The association of M4 macrophages with the pathogenesis of atherosclerosis was recently acknowledged and studied in relation to leprosy ,157. TheM. bovis Bacille Calmette-Guérin (M. bovis BCG) infection indicated that the tryptophan aspartate-containing coat protein  accumulates on the phagosome membrane and inhibits phagosome-lysosome fusion to enhance intracellular survival [M. leprae suppresses immune responses and can survive in macrophages. We observed that after M. leprae infection, CORO1A was recruited to phagosomal membranes where it suppresses TLR-mediated innate immune activation in cultured macrophages. We also found that the innate immune response, which is activated by TLR2, suppresses CORO1A expression [M. leprae infection also suppresses NF-κB activation [M. leprae in host cells for long periods.On the other hand, studies of survival . Followipression ,161. M. M. leprae in vitro or in animals, and to find an animal model of leprosy to study the pathogenesis and treatment of M. leprae infections. Animals used for this purpose include various birds, mammals, and cold-blooded animals [M. leprae. The high body temperature (≥37°C) in traditional rodent models may also disturb M. leprae proliferation, thereby making it difficult to establish conventional animal models to study M. leprae pathogenesis [M. leprae for cooler areas of the body. Although immunocompetent mice are quite resistant to M. leprae infection, inoculation with 103-104 bacilli into the posterior footpad of BALB/c, B6, or CFW mice resulted in local growth with a plateau at about 105-106 organisms within 4–6 months [6 bacilli into each mouse footpad, the number of bacteria did not increase. This result suggests that proliferation could occur when the inoculum contains few bacilli. Thus, inhibition of bacterial growth was not because of the low temperature of the inoculation site, but because of cellular immunity [T-R)) mice resulted in 10- to 100-fold more growth than that seen in immunocompetent mice.There have been numerous studies aiming to grow  animals . Most atogenesis . An initogenesis , and was6 months . Howeverimmunity ,167. TheM. leprae are assumed to be largely under the control of innate immunity. At the peak or plateau stage of growth, the onset of adaptive cellular immunity takes place, which results in the death of the bacilli. In immunocompetent mice, a moderate granulomatous reaction with a mild lymphocytic infiltrate and a small number of epithelioid cells and histiocytes resembling a TT lesion can be seen. In contrast, M. leprae spontaneously grows in the footpads of athymic nude mice, which lack cell-mediated immune responses, and granuloma formation similar to that seen for LL occurs in athymic nude mice [rnu carrying nude (rnu) and hypertension genes had high IL-10 production and high susceptibility to M. leprae [M. leprae induces leproma patterns in the inoculated and non-inoculated sites. Although this model is not widely used, it may be useful as an animal model for leprosy, particularly LL leprosy. Successful M. leprae growth in this model suggests a possible link between M. leprae growth and a genetic background of hypertension or other abnormalities of systemic metabolism.In the first few months post-infection, host reactions against ude mice . In infeude mice  failed tude mice . Meanwhi. leprae ,173. InoDasypus novemcinctus) is a natural host and reservoir of M. leprae in the United States [M. leprae in the nine-banded armadillo was reported [M. leprae. Thus, the armadillo is useful to understand mechanisms of neuropathy and to investigate new therapeutic interventions [Danio rerio) is a useful model for the study of M. tuberculosis granulomas using Mycobacterium marinum (M. marinum) [M. leprae-induced granulomas and early nerve damage were studied in adult zebrafish [M. leprae and the epidemiology and therapeutic approach to leprosy [M. leprae were established in more than half of sooty mangabey monkeys (Cercocebus atys) and African green monkeys (Chlorocebus aethiops) studied, but experiments with rhesus monkeys (Macaca mulatta) and Cynomolgus monkeys (Macaca fascicularis) were less successful [The nine-banded armadillo  . Recentlebrafish . These a leprosy ,181–183.ccessful ,185.M. leprae’s transmission routes are not completely known, although an increased risk of human-to-human transmission because of close contact with untreated leprosy patients has been noted. The most likely candidate for transmission is the spread through infectious aerosols [M. leprae in placental tissue, congenital transmission of leprosy has not been established [Sciurus vulgaris) were found to harbor M. leprae and M. lepromatosis [aerosols . Althougablished ,187. Mosablished ,188,189.ablished ,97,190. ablished . Recentlomatosis ,193.M. leprae has also been detected in wild chimpanzees (Pan troglodytes) and sooty mangabey monkeys [M. leprae is the causative pathogen. Phylogenomic analysis and comparison to strains from other animals, including humans, allowed the identification of the rare bacterial genotypes 4N/O and 2F among the chimpanzees. There have not been any cases of genotype 2F in West Africa and very few cases in Ethiopia. In West Africa, there was only one case of genotype 4N/O that was found in a human sample. Despite the prime hypothesis that M. leprae infection could occur through chimpanzee-to-human transmission, this phylogenomic analysis suggested a possible distribution of M. leprae in the wild environment. Recently, potential reservoirs have been identified. It is possible that M. leprae could be delivered through tick bites [M. leprae can survive in amoebae and in kissing bugs (Rhodnius prolixus) [M. leprae transmission [ monkeys –194–198.ck bites , and M. rolixus) . Furthersmission ,204.M. leprae from skin biopsies coincided with those identified in environmental samples. The confirmed and hypothetical transmission pathways are shown in Regarding the relationship between the environment and infection, the detection of mRNA indicated the existence of the bacilli in soil and water samples taken in India and Brazil ,206. TheM. leprae infects from the outside of nerves likely through epineural blood vessels and lymphatics [M. leprae can reach the endoneurium via blood vessels and then invade and proliferate within Schwann cells [M. leprae invades Schwann cells through α-dystroglycan (DG) which is present in the G domain of the laminin (LN)-2 α2 chain of the basal lamina [M. leprae interact through the surface moieties of the bacteria, including PGL-I, which has an important role in adhesion and survival within Schwann cells. Histone-like protein (Hlp) [mphatics ,208. M. nn cells . M. leprl lamina ,210. Schin (Hlp)  and LPS-in (Hlp)  also parM. leprae induces neural demyelination by stimulating the production of matrix metalloproteinases (MMPs), C-X-C motif chemokine ligand 10 (CXCL10) and C-C motif chemokine 2 (CCL2), which attract macrophages. PGL-I induces nitric oxide (NO) synthase production in infected macrophages that results in damage to axons caused by mitochondrial injury and the induction of demyelination [M. leprae-induced demyelination depends on pathogen binding to the receptor-type tyrosine kinase ErbB2 on Schwann cells and the resulting activation of Erk1/2 signal transduction pathways through the Ras-Raf-MEK-ERK pathway [M. leprae cell wall in the nerves of patients with leprosy, indicating that complement targets LAM [lination ,213. Nitlination . Also, i pathway ,216. T c pathway ,217 and  pathway ,219. In gets LAM . MAC depgets LAM ,221.M. leprae induces significant changes of the metabolic pathways in Schwann cells, such as inducing insulin-like growth factor 1 (IGF-1) which enables bacteria to survive in host cells [M. leprae upregulates glucose uptake and lipid synthesis while downregulating oxidative stress, apoptosis, and autophagy [M. leprae Man LAM is recognized by CD206, resulting in bacterial invasion, weak activation of PPAR-γ, and upregulation of CD206 expression. M. leprae amplifies CD206/PPAR-γ crosstalk to promote LD formation and phagosome recruitment. Elevated LDs induce PGE2 and IL-10 production [st cells . In addiutophagy . In hostutophagy . Meanwhiutophagy . It has oduction .M.leprae induces the Schwann cell dedifferentiation by promoting early demyelination. The bacteria may further promote the spread of infection by reprogramming Schwann cells to the progenitor/stem cell stage [M. leprae stimulates the expression of multiple immune-related genes related to innate immunity [M. leprae for an extended period showed early demyelination followed by increasing Schwann cell number. Further dedifferentiation results in the loss of cell lineage characteristics and the formation of progenitor/stem cell-like cells (pSLCs) having an expression profile like that of mesenchymal stem cells. These pSLCs can differentiate into fibroblasts and can undergo reprogramming that allows spread of the infection [ll stage . The infimmunity . Schwannnfection ,228.M. leprae has lost approximately 50% of the lipid metabolism-related genes of M. tuberculosis [M. leprae may have also lost the ability to metabolize lipids needed to maintain complex cell walls and as a carbon source. Microscopic analysis of skin biopsies from LL cases showed that phagosomes containing M. leprae co-localized with LDs in foam cells [M. leprae [M. leprae promotes glucose uptake by increasing the expression of the solute carrier family 2 member 1 (SLC2A1). SLC2A1 activates the pentose phosphate pathway and provides TAG synthesis in Schwann cells [M. leprae in skin lesions [M. leprae infection, which is in agreement with in vivo data. These proteins are found in M. leprae-containing phagosomes in THP-1 cells [M. leprae suppresses TAG degradation by inhibiting the revelation of hormone-sensitive lipase (HSL), therefore promoting an intra-host macrophage environment that contains high levels of lipids [de novo pathway that involves the glycerol-3-phosphate pathway [M. leprae infection causes the synthesis of TAG within host cells by inducing expression of GPAT3 among four isoforms (GPAT1–4) [M. leprae infection. These observations indicate that M. leprae changes lipid metabolism of the host cell to sustain TAG accumulation. M. leprae-containing histiocytes and Schwann cells were also reported to be filled with cholesterol [M. leprae-infected primary macrophages [M. leprae survival rate [M. leprae is likely to be associated with lipid metabolism in host cells, especially TAG and cholesterol storage.Mycobacteria cell walls contain a large range of lipids. As stated above, rculosis ,229. Thuam cells . Recentl. leprae . The accnn cells . TAG is nn cells . We prev lesions . The exp-1 cells . On the f lipids . TAG bio pathway . In thatGPAT1–4) . This filesterol ,234, whirophages ,230. In M. leprae causes dramatic changes in host gene expression profiles [M. leprae infection and nuclear translocation in THP-1 cells [M. leprae induced the expression of genes encoding CD36 and the acyl-CoA synthetase long-chain family (ACSL). CD36 mediates the macrophage uptake of oxidized low-density lipoproteins (LDL) and induces the expression of fatty acid-binding protein 4 (FABP4) which promotes foam-cell formation through fatty acid transport. ACSL is associated with de novo TAG synthesis from intracellular fatty acids [M. leprae survival, drugs that target lipid metabolism could be a new therapeutic strategy for leprosy.After infection, profiles ,231,235.-1 cells . Activatty acids ,235. Alsty acids ,237, butM. tuberculosis include alpha-, keto-, and methoxy-mycolic acid based on functional groups within the mero mycolate chain. Meanwhile, M. leprae has alpha- and keto-mycolic acids [Mycobacterium smegmatis (M. smegmatis) synthesizes mycolic acid using an extension reaction from acyl-CoA via fatty acid synthase I and II (FASI and FASII) as well as a pathway that uses TAG-metabolized fatty acids [M. smegmatis stores lipids that could serve as an intracellular fatty acid reservoir for biosynthesis of complex lipids [M. leprae-infected macrophages contains a complex mixture of fatty acids and may produce materials for the synthesis of mycolic acid. Further studies are needed to investigate whether M. leprae synthesizes mycolic acid from host-derived TAGs to escape host-derived exclusion systems and allow intracellular parasitization.As mentioned above, mycolic acid is a cell wall lipid that is unique to mycobacteria and is a key virulence factor . Mycolicic acids . The stric acids . Mycobacty acids . Interesx lipids . As we hAccording to the WHO, there were 202,185 new leprosy cases worldwide in 2019. India had the highest number, followed by Brazil and Indonesia. Of these, 14,981 cases were in children younger than 15 years old. G2D, which is defined as leprosy accompanied by visible deformities due to leprosy neuropathy, was detected in 10,813 cases. The COVID-19 pandemic hampered implementation of the WHO program and there were 37% fewer new cases reported in 2020 than in 2019 , althougLeprosy can be classified into five types: TT, BT, BB, BL and LL by the Ridley-Jopling scale. This classification is based on clinical, immunological and histological differences in the disease . DimorphOn a histological basis, TT is characterized by granulomas with lymphocyte infiltration. These granulomas are composed of epithelioid histiocytes that build multinuclear Langhans giant cells. Erosion of the basal layer of epidermis with lymphocytes can be observed without an intervening clear zone . Nerve bM. leprae DNA about 20 years ago, and field-based assays are now being developed. This technology has come to be used not only for diagnosis, but also for detection of resistant bacteria, identification of the route of infection, evaluation of therapeutic effects, and confirmation of household contacts. It has been verified and reported that multiplex real-time quantitative PCR (qPCR) using two or three genes, namely RLEP, 16S rRNA and superoxide dismutase (sodA), is effective [Recently, the diagnosis of leprosy by polymerase chain reaction (PCR) has become a useful and recommended technique. PCR began to be used for the amplification of ffective .M. leprae can be detected by enzyme-linked immunosorbent assays (ELISAs) and related immunoassays; these have also been used in epidemiological research [M. leprae antigens are useful for diagnosis and evaluation of the effectiveness of antibiotic treatment. Other molecular detection methods for M. leprae include analysis of urine for DNA and protein [The antibodies to PGL-I of research ,255. Immresearch ,257. Eleresearch . It was research ,259. Imm protein  and dete protein ,262. In  protein ,264.Neuropathy is an important clinical manifestation of leprosy and has a large impact on affected individuals. Nerve thickness is a prominent clinical manifestation of leprosy. In the WHO classification , PB is dM. leprae and is a major factor in neurological disorders associated with leprosy. This reaction occurs with a sudden and severe immune response to the degraded leprosy bacillus components, which often appear with antibiotic therapy. Leprosy reactions are classified as type 1 (T1 R) and type 2 . These reactions occur in 30 to 50% of all leprosy patients [Leprosy reaction is the immune response against patients ,272. T1 patients . Neurolopatients . The immpatients  and CD4+patients . The Th1M. leprae [M. leprae itself or to the components of dead bacilli after antimicrobial treatment [M. leprae infection.ENL occurs with neutrophil infiltration and systemic inflammation. Complement activation, immune complex formation, increasing CD4+/CD8+ T cell subset ratios and elevated TNF-α may be seen in the lesions and in the circulation  Figure . In the . leprae ,282. The. leprae . ENL occ. leprae . These ireatment . In factreatment . The maiThe current WHO-recommended MDT strategy for leprosy control is a three-drug combination of rifampicin, dapsone and clofazimine given over a 6-month period for PB leprosy and over a 12-month period for MB leprosy. The standard regimen for adult MB patients is a 12-month course consisting of a once-monthly 600 mg dose of rifampicin, 100 mg dapsone daily, 50 mg clofazimine daily and one 300 mg dose monthly . Adult PLeprosy treatment with antimicrobials began in the early 1940s, by Dr. Guy Henry Faget of the National Hansen’s Disease Center in Carville, Louisiana. Sulfone therapy (Promin) was replaced with dapsone to limit toxicity of the treatment ,288. DaprpoB gene lead to changes in the amino acids lining the rifampin binding pocket and produce changes in the enzyme’s structure and rifampin resistance [rpoB gene occur between the codon positions 410–480 [Rifampicin is an antibiotic that inhibits transcription in bacteria, especially mycobacteria, by impeding the enzymatic activity of the beta-subunit of RNA polymerase (rpoB) via steric hindrance of the 5’-ribonucleotide of the elongating RNA ,293. Missistance . Within  410–480 .p-aminobenzoate (PABA) analog that competes with PABA as a substrate for dihydropteroate synthase (DHPS). It acts to bacteriostatically inhibit nucleic acid (both RNA and DNA) synthesis in pathogens [M. leprae dapsone resistance incorporates mutations found in the folP gene encoding DHPS, causing a reduction in binding affinity between dapsone and the DHPS active site [folP1 gene [M. leprae. Fluoroquinolones target type II DNA topoisomerases, such as DNA gyrase and topoisomerase IV [N-terminus of the DNA gyrase A (Gyr A) due to mutations in the gyrA gene [M. leprae are Ala91Val substituted strains, with limited resistance to Gly89Cys substitutions [Dapsone is a sulpha drug that does not have a sulphonamide structure but is instead a athogens . The mecive site . In the lP1 gene . ClofazilP1 gene . Ofloxacerase IV . FluoroqyrA gene . Two amiitutions .M. leprae drug resistance, in particular to rifampicin, was an issue of deep concern. In 2008, in 18 sentinel countries, global-level drug resistance surveillance was performed to evaluate drug resistance levels and MDT efficacy. This surveillance showed that resistant strains are present in many endemic regions across the world [M. leprae rpoB, folP1, and gyrA were used to gauge resistance to rifampicin, dapsone, and quinolones, respectively [M. leprae DNA in most endemic countries remains challenging. In this regard, development of a rapid, cost-effective point-of-care diagnostic tool is needed.Since the 1970s, rifampicin has been a mainstay of leprosy treatment; however, since the reporting in 1976 of the first case of resistance , resistahe world . Drug-reectively . Althougectively , such seMycobacterium bovis (M. bovis) and Bacille Calmette-Guérin (BCG) has been reported in different countries [Mycobacterium indicus pranii vaccine, which is known as Mycobacterium w (Mw) [M. leprae subunit vaccines have been developed, of which the new subunit recombinant vaccine LepVax has successfully completed Phase 1a clinical trials [The WHO Guidelines Development Group (GDG) analysed evidence regarding vaccines for leprosy . Currentountries . Three mountries ,317,318.ountries . Recentlm w (Mw) ,320. Varl trials .M. leprae. PEP reduces the risk of developing leprosy by about 60% during the first two years after exposure [M. leprae detected in the nasal cavities of asymptomatic people is a risk for the onset of illness in a community over time [Allocation of post-exposure prophylaxis (PEP), consisting of a single dose of rifampicin (SDR), is one of the most promising options for people exposed to exposure . M. leprver time ,328. Thever time . Individver time ,330. Thuver time .M. leprae pathogenicity and difficulties accessing medical treatment that persist in endemic regions, especially those in Southeast Asia, East Africa, and Brazil, which still account for a large proportion of newly reported cases. This review summarized the unique biological properties of M. leprae with a focus on survival within host cells such as macrophages and Schwann cells through modulation of the innate immune response and lipid metabolism. M. leprae has extremely few gene-coding regions, and pseudogenes and non-coding regions consume nearly half its genome. Thus, these bacteria are highly dependent on host cells, particularly for the production of lipids and cell wall components. Alterations in lipid metabolism affect bacterial survival and proliferation. Although WHO-recommended MDT administration has drastically brought down the prevalence of leprosy in the world, the emergence of new cases along with drug-resistant cases at this point of elimination may delay the elimination of leprosy. Further research on the physiological characteristics, particularly the parasitic mechanisms, of M. leprae will contribute to the development of new therapeutic strategies and drugs and may help realize the goal of “Towards zero leprosy.”The Global Leprosy Strategy 2021–2030 “Towards zero leprosy” has been carried out with the goal of targeted reduction of new patients, but a substantial number of infections still occur. This is due to inadequate knowledge about the mechanisms of


Toxoplasma gondii (T. gondii) infects approximately one third of the world’s population. Globally there are an estimated 13.1 million cases of T. gondii co-infection in HIV-infected people with 87.1% of these individuals living in sub-Saharan Africa. The risk of T. gondii infection in HIV-infected women rises significantly with lower CD4+ T cell counts (particularly under 100 cells/μl). Mother-to-child transmission (MTCT) occurs in approximately 30% of cases of maternal T. gondii infection during pregnancy. The global prevalence of latent toxoplasmosis in HIV-infected pregnant women is 47.5% but the overall risk in HIV-infected mothers of MTCT of T. gondii is however, estimated to be low at < 5%. MTCT in HIV-infected mothers not only occurs due to T. gondii primary infection in pregnancy but also due to reactivation. Infants with congenital toxoplasmosis born to HIV-infected mother may have a more rapid onset and greater dissemination of disease thus having potentially devastating effects. This article discusses the key risks for MTCT of T. gondii infection in HIV-infected mothers as well highlighting the many knowledge gaps for which further study is required. Toxoplasma gondii (T. gondii), infects approximately one third of the world´s population and has a worldwide distribution [T. gondii co-infection in HIV-infected people with 87.1% of these individuals living in sub-Saharan Africa [T. gondii acquisition is primarily through consumption of undercooked meat harbouring tissue cysts or contaminated food and water containing oocysts. Despite being preventable, T. gondii infection during pregnancy and subsequent congenital infection continues to devastate lives globally [T. gondii infection in HIV-infected pregnant women are amplified when compared to HIV-negative pregnant women potentially leading to even greater morbidity and mortality in this group of individuals, however, T. gondii infection in HIV-infected pregnant women remains a relatively neglected area of focus or study. This article therefore, aims to discuss the key risks for MTCT of T. gondii infection  as well highlighting the many knowledge gaps in this subject area, which may give impetus for further studies and for the development of strategies to help mitigate the risks from this potentially devastating infection. This may also help HIV-infected pregnant mothers to receive appropriate and informed counselling regarding T. gondii infection in pregnancy.The World Health Organisation (WHO) estimated that at the end of 2019 there were 38 million people living with HIV (81% of whom knew their HIV status) and that 68% of HIV-infected adults were receiving long-term antiretroviral therapy (ART) [n Africa . T. gondglobally . Acute tT. gondii is from acute T. gondii infection during pregnancy [T. gondii infection greater than 3 months prior to conception confers minimal or no risk to the fetus. Transmission of T. gondii through breast milk has not been described in HIV-infected (or in HIV-uninfected) mothers. In pregnancy, maternal parasitaemia and subsequent invasion of the placenta by tachyzoites leads to the parasite entering the foetal circulation [T. gondii infection during pregnancy [T. gondii infection during pregnancy is therefore a major determinant of foetal outcome. A recent study found a global prevalence of acute T. gondii infection in pregnant women (including both HIV-positive and HIV-negative women) of 1.1% (0.6% when the most strict criteria for acute T. gondii infection were used); highest in the Eastern Mediterranean region (2.5%) and lowest in the European region (0.5%) [T. gondii infection was significantly higher in the first trimester (1.7%) compared to the second (1.0%) and third (0.1%) trimesters. The study did not report rates of MTCT/congenital toxoplasmosis or provide specific data regarding HIV-infected pregnant women. They did however estimate that based on an overall MTCT risk of approximately 24% in acute maternal T. gondii infection, annually 201,600 children could be born with congenital toxoplasmosis.The primary overall risk of MTCT of regnancy . Maternaculation . MTCT trregnancy . Foetal regnancy -12. The n (0.5%) . They foT. gondii infection in HIV-infected women rises substantially with CD4+ T lymphocyte counts of <100 cells/µl. The risk of MTCT of this parasite in HIV-infected mothers is not precisely known but is likely to be, at least to some extent, related to the degree of immunosuppression [T. gondii is however, estimated to be low at <5% [T. gondii infection is largely unknown for HIV-infected pregnant women. MTCT typically occurs due to a primary T. gondii infection occurring during pregnancy, however, in HIV-1 infected mothers, reactivation of latent T. gondii infection is also an important cause of congenital infection [3) [T. gondii reactivation in pregnancy in HIV-infected mothers have been reported in the literature [T. gondii infection in pregnancy, however, in HIV-infected mothers who may be highly immunosuppressed, serological markers can be less reliable, further complicated by the fact that T. gondii IgM responses may be diminished in cases reactivation/reinfection when compared to primary infection. Three T. gondii lineages  exist and re-infection with a different strain is another potential cause of congenital infection [T. gondii strains isolated from HIV-infected individuals are type II [The risk of pression . The ovew at <5% . MTCT rinfection . Moreovetion [3) . At leasterature -19 althoT. gondii in this patient population. A Brazilian study of 2421 HIV-negative pregnant women and 168 HIV-positive pregnant women found that although high T. gondii-specific IgG values were seen more frequently in the HIV-positive pregnant women, this was not associated with an increased risk of MCTD of T. gondii [A recent meta-analysis estimating the prevalence of toxoplasmosis in HIV-infected pregnant women found an overall pooled prevalence of latent toxoplasmosis of 47.5% . This st. gondii .T. gondii that are born to HIV-infected mothers versus those born to HIV-negative mothers, however, infants with congenital toxoplasmosis born to HIV-infected mother appear to have a more rapid and disseminated disease process  [T. gondii, with some studies reporting a risk reduction but others showing no benefit [There is a lack of studies comparing outcomes between infants congenitally infected with eizures) . There i benefit . The ris benefit , however benefit .T. gondii during pregnancy amniocentesis is required. In the pre-HAART era amniocentesis was discouraged in HIV-infected mothers due to the risks of MTCT transmission of HIV. A recent large multicentre case series by Florida et al. in which 88 HIV-infected pregnant underwent either amniocentesis (n = 79) or chorionic villous sampling (n = 9) found that no vertical transmission of HIV occurred in the 86 women that were on HAART [T. gondii this risk is altered. Placental invasion by T. gondii results in placental inflammation and whether this potentially renders the placental tissue more permissive to other pathogens such as HIV has not been determined. It has also been hypothesised that HIV-1 infected mothers, due to immune suppression, may in fact mount a diminished placental inflammatory response [et al. studied the effect of placental membrane inflammation due to acute chorioamnionitis in HIV-1 infected mothers in Kenya and found no association between chorioamnionitis and in utero transmission of HIV-1 although there was an association with peripartum HIV-1 transmission [T. gondii infection significantly impacts the transmissibility of HIV to the child.An additional consideration is the fact that for definitive confirmation of MTCT of on HAART . Two cason HAART . Thereforesponse . Mwanyumsmission . Extraposmission . Howeversmission . FurtherT. gondii infection in HIV-infected pregnant mothers and risks of mother MTCT. Greater efforts are needed to educate pregnant women in measures to mitigate the risk of T. gondii acquisition, ensure appropriate screening of HIV-infected pregnant women for T. gondii in pregnancy and provide timely initiation of T. gondii prophylaxis/treatment where indicated. In addition, novel approaches are required to achieve progress in the development of a T. gondii human vaccine.


Candidatus Neoehrlichia. Clinicians should be aware of possible ehrlichiosis even in immunocompetent patients.We describe a case of neoehrlichiosis in an immunocompetent child with acute febrile illness in South Africa. Neoehrlichiosis was diagnosed by PCR on 16S rDNA from bone marrow aspirate. Phylogenetic analysis indicated an organism closely related to  Ehrlichia species and related organisms are obligate intracellular membrane-bound bacteria transmitted via ticks between a range of mammalian animal hosts, including humans. In recent years, taxonomy and phylogeny of these bacterial species has been revised. The family Anaplasmataceae comprises 5 genera: Ehrlichia, Anaplasma, Neorickettsia, Neoehrlichia, and Wolbachia. However, the number of recognized species and candidate species has substantially increased in recent years. We describe a case of neoehrlichiosis in an immunocompetent child in South Africa. A previously healthy 9-year-old boy from Eastern Cape Province, South Africa, was admitted to the hospital with a 3-week history of fever, back pain, and myalgia. Symptoms failed to respond to a course of azithromycin prescribed by his general practitioner for suspected tickbite fever. The child lived on a farm and was exposed to a variety of domestic companion and farm animals, but he had no history of recent tick bites or travel.At admission, results of a physical examination were unremarkable, except for a temperature of >40°C. The patient had no rash, skin lesions, lymphadenopathy, or clinically detectable hepatosplenomegaly. Laboratory investigation results were unremarkable . The patBrucella serologic test results, despite absence of definite exposure history, and began treatment with doxycycline, rifampin, and gentamicin. The patient gradually experienced partial improvement over the next week.Clinicians considered brucellosis because of initial equivocal Brucella spp. was negative, and cultures yielded no growth. However, PCR and sequencing of a 598-bp section of the hypervariable 5 to 8 regions of the 16S rRNA gene .Ehrlichia and Anaplasma spp. by using a PCR targeting the 16S rRNA gene . The consensus sequence was deposited in Genbank (accession no. OP208838). BLASTn (https://blast.ncbi.nlm.nih.gov) homology search results revealed no identical sequences in the public databases. The most closely related sequences, at 96%–98% identity, were various Candidatus Neoehrlichia 16S rDNA sequences, including Candidatus N. mikurensis (strain TK4456R), Candidatus N. lotoris (strain RAC413R), and Candidatus N. arcana (strain HT94R)  using primers fD1 and rP2 (n HT94R) .http://www.clustal.org). We manually examined the alignments and truncated to smallest sequence size, a 1,264-bp Candidatus N. australis sequence, by using BioEdit 7.2.5 (https://bioedit.software.informer.com). We used MEGA X (https://www.megasoftware.net) to determine Kimura 2-parameter plus gamma plus invariable site substitution as the best-fit model. We used it in combination with the bootstrap method using 1,000 replicates to infer a maximum-likelihood phylogenetic tree, which indicated that our obtained sequence clustered with Candidatus N. arcana, although with low bootstrap support (60%)  . The seqCandidatus N. lotoris, 24 bp for Candidatus N. arcana, and 34 bp for Candidatus N. mikurensis. These reference species differ by 46–50 bp from each other. Our findings confirmed that the ehrlichial organism we identified is a member of the family Anaplasmataceae and most closely related to, but distinct from, Candidatus N. lotoris and Candidatus N. arcana. Until appropriate type-material can be deposited and the species is formally described, we will refer to this novel organism as Candidatus Neoehrlichia sp. South Africa (OP208838).We calculated estimated evolutionary divergence by determining the number of nucleotide differences between similar sequences. We removed all ambiguous positions for each sequence pair (pairwise deletion option), which left a total of 1,264 positions in the final dataset. Our obtained sequence differed from GenBank reference strains by 23 bp for Candidatus Neoehrlichia, a sister genus to Anaplasma and Ehrlichia, detected in the bone marrow aspirate of an acutely febrile immunocompetent child from South Africa. Candidatus N. lotoris has been described from raccoons in Georgia, USA, and an associated tick species (Candidatus N. arcana has been described from Ixodes holocyclus ticks in Australia (Candidatus N. mikurensis was described in 2004 (Candidatus N. mikurensis appears to be widely distributed in Europe (Candidatus N. mikurensis has been isolated from dog ticks in Nigeria (Candidatus N. mikurensis is small mammals, and its vectors include Ixodes and Haemaphysalis tick species (We report a novel ehrlichial organism, most closely related to the genus Candidatus N. mikurensis, and clinical findings are usually nonspecific, including fever, headache, and myalgia; however, vasculitis and thrombo-embolic events can occur (Most human neoehrlichia infections described to date have been caused by Candidatus N. lotoris and Candidatus N. mikurensis (Another probable clinical neoehrlichia case from Africa was reported in 2013 in a traveler who returned to Denmark from Tanzania (groEL/groESL and gltA genes. Application of new molecular methods is leading to an explosion in discovery of related novel species or variants, as noted in Australia (Neoehrlichia gene sequences are available, including 16S rRNA or ftsZ, gatB, groEL, and lipA.Ehrlichiae and related organisms are usually detected via molecular methods, including PCR of 16S rRNA, or Ehrlichia spp. are not susceptible to macrolides and our case would not have been recognized if the patient had initially been treated with doxycycline. Although tickborne rickettsial infections are common in South Africa, Anaplasmataceae infections likely are undiagnosed because of lack of awareness among clinicians and unavailability of laboratory tests. Of note, In conclusion, our case represents an expansion of the known range of neoehrlichiosis. Clinicians should be aware of neoehrlichia as a possible tickborne cause of acute febrile illness in Africa.


Hyperolius frogs from East Africa. Unlike many members of the genus which have relatively wide distributions, these species tend to be small-range endemics found in montane and submontane forests. Recent discovery of a golden-hued frog with the clade-specific traits of spines on their gular discs prompted a morphological and genetic exploration of the distinctness of this new lineage and relationships to other members of the clade. Genetic  results resolved many sister-relationships, but deeper nodes in the phylogeny were poorly resolved. A reduced-representation genome-wide Single Nucleotide Polymorphism (SNP) dataset was able to fully resolve the phylogenetic relationships within this clade, placing this new lineage, here named after the mountain range in which is it found–H. ukaguruensis sp. nov., as an early diverging lineage within the group. This new species is distinct from all other spiny-throated reed frogs, necessitating further understanding as a single-mountain endemics vulnerable to habitat loss and potential decline. Morphometric analyses identify clear morphological characteristics that are distinct for the herein described species, most noticeably in that the eyes are significantly smaller than other members of the genus for which we have samples.The spiny-throated reed frog species group is a small radiation of  Hyperolius burgessi, H. davenporti, H. minutissimus, H. ruvuensis, H. spinigularis, H. tanneri, H. ukwiva), but due to the small, mostly montane-restricted ranges typical of these species .Hyperolius spinigularis was described by Stevens [H. burgessi in the East Usambara, Nguru, and Uluguru mountains [Hyperolius spinigularis was also ascribed to populations in the Udzungwa Mountains in Southern Tanzania [H. minutissimus [H. spinigularis is not found within the Udzungwa Mountains, though two populations of H. minutissumus appear to have allopatric distributions within the mountain block [Hyperolius tanneri is found only within the West Usambara Mountains of northern Tanzania [Hyperolius ukwiva from the Rubeho Mountains in central Tanzania [H. davenporti from the Livingstone Mountains of Tanzania near the Malawian border [H. ruvuensis from a Tanzanian coastal lowland forest [As currently recognized,  Stevens  based on Stevens  originalountains . HyperolTanzania , 7, alontissimus . Recent in block . HyperolTanzania . The mosTanzania , H. daven border , and H. d forest .H. ukwiva and H. ruvuensis), have only been observed once (despite attempts to re-survey) and likely to be highly threatened, new spiny-throated reed frog lineages could be of conservation interest. We investigate morphological, coloration, and genetic differences along with spatial distributions of this lineage compared to all existing lineages to describe a new species within the spiny-throated reed frog complex.A recent survey in the Ukaguru Mountains of central Tanzania encountered a phenotypically distinct (golden coloration) spiny-throated reed frog, prompting investigation into the phylogenetic relationships and morphological distinction of this potentially new lineage. As two lineages of this group (In addition to the newly collected specimens (outlined in the sections below), we compiled relevant information for morphological, genetic and genomic comparative analyses across species of this group. Summary information on localities, and sample sizes for morphology and genetics shown in th of February 2019 for a total of approximately 200 person-hours  between 10-12th of February 2019. Four individuals were encountered at the edge of a large wetland on the main road outside of the pine plantation. Searching took place for ~20 person hours at this site over the three nights.Specimens collected were euthanized using an overdose of benzocaine and fixed in 5% formalin, and subsequently stored in 70% ethanol. Prior to fixing, samples of muscle and/or liver were taken from representative individuals and preserved in 95% ethanol, these specimens are listed in All specimens for this study were collected in accordance with animal ethics guidelines established in the institutions of authors (including the University of Cincinnati (IACUC 21-04-21-02 (LPL), the Natural History Museum London, and approved by the Tanzania Wildlife Research Institute (TAWIRI)).Hyperolius minutissimus population (n = 4 new extractions) using the DNeasy blood and tissue kit . Both male and female specimens were assessed to confirm they were conspecific. This is necessary as males and females are sexually dimorphic, requiring genetic confirmation of conspecific status. All other DNA used in this study was previously extracted using these methods and sequenced following the methods below and downloaded from NCBI GenBank [Total DNA was extracted from tissue preserved in 95% EtOH from one male and one female specimen of the newly identified spiny-throated reed frog from the Ukaguru Mountains and of the Sao Hill  GenBank .https://www.geneious.com). See Extraction, amplification and sequencing followed standard protocols . Each ofPhylogenetic relationships and structure within this clade were assessed in two ways. The mitochondrial data (16S), which had high levels of sequence divergence was assessed using phylogenetic tree methods (outlined below). Nuclear genes, which had much lower levels of divergence and may still retain strong signals of incomplete lineage sorting, were assessed as haplotype networks to reveal structure without enforcing unrealistic tree structure for slow evolving genes. A species tree using the StarBeast function in BEAST was also created combining nuclear and mitochondrial loci (see below).H. minutissimus from the Sao Hill locality). The resulting sequences were aligned in MUSCLE v3.7 [16S mtDNA sequences for all known members of the spiny-throated reed frog clade were obtained from GenBank and incorporated in the mitochondrial gene tree analysis along with the four newly-sequenced specimens  and maxitchelli ) and wititchelli , 21 on thttp://tree.bio.ed.ac.uk/software/figtree/).In the MrBayes analyses, two runs with four Markov chains were run for 10 million generations and sampled every 1000 generations with heating parameter of 0.2; discarding the first 25% as burn-in. RAxML was run allowing the bootstrapping to halt automatically under default settings for RAxML-HPC BlackBox. In BEAST2, a relaxed lognormal clock was used to accommodate some potential variation of rates within the group. A Yule tree prior was used with a uniform birth rate, exponential gamma shape, log normal HKY transition/transversion (as identified by jModelTest2), and uniform prior for proportion invariant sites. Other models (Birth-Death) gave identical topologies, but without an explicit expectation of extinctions, the Yule model results are shown. The run consisted of 15 million generations logging every 1000 with the first 10% discarded as burn-in. An assessment of model performance was completed in Tracer v1.7.1  for BEASPairwise population divergence and within population divergence was calculated using MEGA7 . Within-Nuclear loci (POMC and RAG-1) were also downloaded from GenBank and combined with newly sequenced individuals. All loci aligned unambiguously. Divergence in nuclear genes were visualized as unrooted TCS allele networks  using PoH. ruvuensis which only has 16S sequences) was also evaluated. A relative mitochondrial clock rate was set at 5.0, with nuclear clock rates of 1.0 to reflect estimates of relative rates [A “species tree” in StarBeast combining datasets  or from existing, high-quality DNA. The libraries were quantified using a Qubit fluorometer , normalized to 500 ng DNA when possible (>100 ng minimum), and sent to Admira Genomics  for sequencing. The ddRAD-seq library was prepared with EcoRI-MspI restriction enzymes and Illumina HiSeq 2500 150 × 2 pair-ended sequencing.A smaller dataset  was used to create a reduced-representation whole-genome single nucleotide polymorphism (SNP) dataset through double-digest RAD-sequencing (ddRAD-seq)  Files. Ode novo ddRAD-seq assembly and SNP discovery, as well as downstream analyses. As only 1–2 individuals were representative of each population, all individuals were listed as a single population for the R80 method . M & n = 4 were ultimately used to balance the diversity of samples within the dataset and identifying true SNPs. In total, 821,683 loci were generated, composed of 214,643,861 sites and 2,201,442 variant sites. Due to the need for a very complete dataset, only loci present in every population (-p 10) were retained. This yielded a total of 1,909 loci. The mean effective per-sample coverage was 18.3x . The mean number of sites per locus was 257.5. A consistent phasing was found for 95.2% of diploid loci needing phasing.The STACKS pipeline v2.60 –30 was uFinally, only the first SNP of each locus was included in the final dataset to create unlinked loci, crucial for SNAPP  and STRhttps://www.phylo.org) with the GTR site substitution model. MrBayes v 3.2.7 was run for 500,000 MCMC steps, with a burn-in of 25%, and sampling occurred every 500 iterations. Effective chain mixing and effective sample sizes (ESS) across parameters (≥ 100) were assessed in Tracer (v1.7.1). BEAST2 (v. 2.6.0) analyses were conducted in two ways, as a concatenated SNP tree (matching MrBayes methodology) and as a SNAPP tree [https://www.cs.auckland.ac.nz/~remco/DensiTree/). RAxML v 8.2.12 was run using the GTR-GAMMA model of sequence evolution and automatic bootstrapping.MrBayes and RAxML analyses were run on the CIPRES server  was used for population genetic analyses on the phased SNP data. Population clusters (K) between 1–12 were assessed, with 10 iterations each. The burn-in period was 100,000, and the MCMC reps were set at 200,000. We used the admixture model without a population prior. All other parameters were set to default values. The program Structure Harvester v 0.6.94  was usedtimation , which dIndividuals of the new species were all collected from the same locality  in Mamiwa-Kisara North Forest Reserve on the Ukaguru Mountains of Tanzania. Adult (>20mm) specimens of both sexes were measured for 17 standard morphological traits  were taken from [H. ukwiva were available, but photographs of a male taken in the field allow characterization of the gular spines  on log-transformed measurements. As females tend to be larger than males, do not have gular flaps and are represented by lower sample sizes, this PCA was restricted to male specimens only. Morphological traits identified as being potential diagnostic features distinguishing the new species from the rest of the species group were explored further by means of statistical hypothesis testing for both males and females separately. These features were: overall body size , eye size (most important loading of PC2) and gular flap width (most important loading of PC3). Differences in SUL, eye diameter/head width ratio and gular flap width/height ratio across species were therefore tested using univariate ANOVAs and post-hoc Tukey tests. All analyses were performed in R v 3.6.1  on log10http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub: A2AA3A2D-29F8-46A6-A328-88E8C280E110. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central and LOCKSS.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "H. ukaguruensis sp. nov.) represent monophyletic lineages . As the type locality for H. minutissimus (Southern Highlands) was not sampled for genetics or morphology, and no morphological measurements were taken of the Sao Hill H. minutissimus specimens, we refrain from assigning either lineage a new name. We nonetheless treat them as distinct phylogenetic lineages from here on out, referring to these by their localities . All morphological measurements however, correspond to the Uzungwa Scarp lineage.Based on 16S mitochondrial sequences, all previously recognized species and the new species  has support as the sister to the poorly known H. ukwiva from the Rubeho Mountains , a “minutissimus” clade (H. minutissimus populations from two different areas in the Udzungwa Mountains), H. tanneri, and H. ukaguruensis sp. nov. The SNAPP analysis predicted the placement of H. tanneri as basal, with H. ukaguruensis sp. nov. as the next diverging lineage  with posthoc tests showing that male H. ukaguruensis sp. nov. are significantly larger in size (p<0.001) than H. burgessi, H. davenporti, H. ruvuensis and H. spinigularis  and females significantly larger than H. burgessi and H. spinigularis  differences among species of the p<0.001; . It is ivailable . More stp<0.001; , and forH. ukaguruensis sp. nov. being wider than long, significantly more so than H. burgessi, H. davenporti, and H. tanneri  was also significantly different between species , with that of p<0.001; . To summH. tanneri, H. davenporti, and H. burgessi possessing more circular gular shapes (GFW/GFH ~ 1.0) and H. minutissimus, H. ruvuensis, H. spinigularis, and H. ukaguruensis sp. nov. possessing wider oval shaped gular flaps .The new lineage discovered here appears to only occur in the Ukaguru Mountains of Tanzania and not in any other mountain blocks within the East African range of the spiny-throated reed frog clade . No otheH. ukwiva. Genetically it is distinct , and morphologically it is distinct in female eye to head width ratios .Most importantly, this lineage is distinct from its closest relative, Hyperolius ukaguruensis sp. nov. urn:lsid:zoobank.org:act:4166708E-2561-4085-B29A-4FAE83510661Holotype.—BMNH 2022.7682  collected in Mamiwa Kisara Forest Reserve, Ukaguru Mountains, Tanzania  on 19 February 2019 by Lawson, Loader, Lyakurwa, Liedtke.Paratypes.—Male: Field numbers SL_4033–4037, 4059–4063. Female: BMNH 2022.7683, Field numbers SL_4057, 4058. All paratypes were collected in the same small stream as holotype but collected between 19th– 23rd February.Diagnosis.—Horizontal pupil with distinctive gular flap in males. As with most other members of the spiny-throated group , H. ukaguruensis sp. nov. also has the presence of dermal asperities (including the body and chin region) on the ventrum. This trait is unique amongst members of the genus Hyperolius. The presence of asperities on the gular flap diagnoses H. ukaguruensis sp. nov. from H. tanneri, for which they are absent. Hyperolius ukaguruensis sp. nov. primarily has asperities anteriorly positioned (closer to the mouth), which differentiates it from H. spinigularis, H. burgessi and H. davenporti which have an even distribution of dermal asperities on the gular flap. Hyperolius minutissimus and H. ukwiva have a similar distribution of asperities to H. ukaguruensis sp. nov.. Furthermore, in males, H. ukaguruensis sp. nov has a distinctively shaped gular flap, differentiating it from H. davenporti, H. tanneri and H. burgessi , and were much bigger on average than H. minutissimus and H. spinigularis, though overlapped in distributions.Females are larger in 18–24 mm , no femaigularis , Table 3H. ukaguruensis sp. nov. compared to ~0.55–0.60 for all other species.Most notably, the ratio of eye width to head width was very distinct in this group, with relatively “very small eyes”. This should be considered a diagnostic feature in these frogs, with ratios of ~0.35 for H. ukaguruensis sp. nov. has similarities in dorsal patterning to other members of the complex in that individuals have a greenish-brown uniform coloration with either mottled white patterns on the dorsum or distinct white dorsolateral lines extending from a canthal triangle on the snout reaching nearly the groin. Dorsolateral lines contain small black speckles. Hyperolius ukaguruensis sp. nov. has distinct dorsal coloration of this uniform color, however, from other spiny-throated reed frogs. Hyperolius ukaguruensis sp. nov. has golden-green dorsal coloration with flashes of orange on the thighs and feet. Hyperolius spinigularis, H. burgessi, H. davenporti, and H. tanneri have silvery-green tones in their dorsal coloration. Hyperolius ruvuensis also has a brown dorsal color, and has a cream ventral surface and orange flashes on the thighs. Hyperolius ukwiva is described as brown in dorsal coloration, though lack of photos and live specimens makes it difficult to quantify coloration variation. Hyperolius minutissimus individuals also have distinct coloration from other species in this clade, with mottled yellows and green similar to H. pictus coloration, but which do not resemble H. ukaguruensis sp. nov.In coloration, H. ukaguruensis sp. nov., which is bright yellow on many individuals in life. Only H. ukwiva also has a yellow ventral surface.Ventral coloration is also distinct in H. ukaguruensis sp. nov. is also genetically distinct from close relatives, and is minimally 2.9% pairwise divergent from its closest relative (H. ukwiva), based on mtDNA . The veSexual dimorphism.—Females attain a much larger size than the males  where the type series was collected. The species name is a masculine Latin singular adjective in the nominative case.Distribution, Ecology and Conservation.—Hyperolius ukaguruensis sp. nov. is only known from one locality in Mamiwa Kisara North Forest Reserve in the Ukaguru Mountains. Specimens were collected in and around the edge of montane forest in a swamp. Collecting across the Ukaguru Mountains has been patchy [H. ukaguruensis sp. nov. has a localized distribution. Unfortunately, Mamiwa Kisara North Forest Reserve is impacted by anthropogenic impacts–including deforestation. Forest loss has recently been exacerbated by fire which has impacted large patches of forest. Given its small distribution and habitat loss, Hyperolius ukaguruensis sp. nov. is likely to be of high conservation concern [n patchy , so it i concern .Hyperolius ruvuensis, Ruvu South Forest) [The spiny-throated reed frogs show substantial population structure between geographically restricted areas in Tanzania, Malawi, and Mozambique. These species are narrowly distributed endemics , mostly  Forest) , 2. EachHyperolius species do not appear to show as much single-mountain block endemism within the Eastern Arc Mountains and nearby mountain blocks [H. minutissimus [Hyperolius males can when they call at night. Second, these species appear to be habitat specialists, found in dense to semi-dense concentrations in some areas with in-tact habitat. This includes Amani Nature Reserve in the East Usambara Mountains of Tanzania with closed canopy primary forest cover and deep and stable wetlands and not marginal habitats or more seasonal water bodies as seen in other co-distributed species H. substriatus, H. mitchelli, and H. rubrovermiculatus [Hyperolius species [Other co-distributed n blocks , 38–40. iculatus , 38, 41. species .H. burgessi and H. davenporti found in Northern and Southern Tanzania  and the fact that Schiøtz proposed a lineage of H. spinigularis and a lineage of H. minutissimus within the block [H. minutissimus. Samples from Sao Hill (newly included in this paper) and the Uzungwa Scarp population  and Massisiwe village and nearby Uzungwa Scarp were classified as H. spinigularis in [H. minutissimus. These descriptions, in reference to the specimens from the type locality suggest that the Sao Hill collection may reflect true H. minutissimus and the specimens from the Uzungwa scarp represent a new lineage yet to be named. Detailed genetic and morphological evaluations of these lineages are required.A second new lineage was revealed in these analyses, within he block , which wmpled in ) are genmpled in , Table 2et al.’s [H. tanneri was basal to H. spinigularis and H. minutissimus. Portik et al.’s [Hyperolius tanneri as containing loci from both the “spinigularis” and “minutissimus” clades (under K = 3), which might explain why resolving the relationships between the groups has been historically challenging. As we do not expect gene flow between these distinct lineages, this may reflect the short branching times when all three major lineages split. Further clusters (K = 4 and 5) did not show this result, though the implication of a nearly simultaneous split of the three main lineages may mean that incomplete lineage sorting is still in effect despite significant time passing.Lawson et al.’s  “geneticet al.’s  where H.et al.’s  analysisH. tanneri as potentially basal or near basal to the rest of the clade might help clarify the role that the gular spines and lack of “voice” play in evolution within this clade. These are both highly unusual traits for Hyperolius frogs (or any frogs). If H. tanneri is ultimately basal with H. ukaguruensis sp. nov. and H. ruvuensis as the next lineage in the phylogenetic tree, then the spine-free throat of H. tanneri matched with the voiceless nature of those frogs, would imply that loss of calling occurred first, followed by the subsequent synapomorphy of spines on the throat  of other species. Of note, H. minutissimus has a very quiet and high pitched call, which was able to be recorded in a tent [Hyperolius species which have an easily audible call. The cause of a “non-calling” or “quiet” trait in this group of frogs may not be known, or even fully characterized , butng calls –46.H. ruvuensis, was recently described from a single coastal forest in Tanzania yet has not been encountered since 2001 [H. ukaguruensis, also appears to be unique, a habitat specialist, and to have a very narrow range. In naming this H. ukaguruensis sp. nov., we hope to bring awareness to the value of additional biodiversity surveys and careful taxonomic consideration in East Africa to identify the full extent of lineages found there. This is especially critical during the current amphibian extinction crisis of combined threats of habitat destruction, climate change, and chytrid fungus infections [The spiny-throated reed frogs appear especially vulnerable to extinction threat, even for amphibians which are generally declining, due to the combination of being habitat specialists, single locality endemics, and possessing small population sizes . For insfections .S1 File(XLSX)Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 FileAll measurements are recorded in cm.(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.


Cimex lectularius, prevalent in temperate areas, and C. hemipterus, also known as the tropical bed bug. A global increase in infestation has been observed in recent decades. However, data is lacking concerning species distribution, which may be affected by population movements . Moreover, although these insects are not known to harbor and transmit infectious agents to humans, continuous monitoring of their infection status is part of active surveillance. We analyzed bed bug specimens collected from beddings in Mauritania, and assessed MALDI-TOF MS as an identification tool for immature specimens. Moreover, we also conducted bacterial detection in each specimen. Overall, our findings supported MALDI-TOF MS as being a robust tool allowing species level identification of immature Cimex specimens.Bed bugs are hematophagous insects that have been described as a cause of nuisance since ancient times. Two species are known to parasitize humans: Cimex hemipterus  and C. lectularius. Identification of these two species is based on morphological characteristics, and gene sequencing, and may also rely on Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS). The present work aimed to assess whether MALDI-TOF MS was applicable for species level identification of immature stages of Cimex. Arthropods were collected in domestic settings in Nouakchott, Mauritania. Identification used morphological keys and MALDI-TOF MS identification was assessed for immature stages. Quantitative PCR and sequencing assays were used to detect arthropod-associated bacteria in each specimen. A total of 92 arthropods were collected, all morphologically identified as C. hemipterus . A total of 35/45 specimens produced good quality MALDI-TOF MS spectra. Analysis allowed species level identification of all immature C. hemipterus after their spectra were entered into our in-house MALDI-TOF MS arthropod spectra database. Molecular screening allowed detection of Wolbachia DNA in each specimen. These results suggested that MALDI-TOF MS is a reliable tool for species level identification of Cimex specimens, including immature specimens. Future studies should assess this approach on larger panels of immature specimens for different Cimex species and focus on the precise staging of their different immature developmental stages.Human infestations by bed bugs have upsurged globally in recent decades, including in African countries, where recent reports pointed out an increase in infestation. Sympatric dwelling has been described for two species of bed bug parasitizing humans:  Cimex genus are globally distributed hematophagous arthropods, some of which are known to have parasitized humans since ancient times [Cimex lectularius, the common bed bug, and Cimex hemipterus, the tropical bed bug [True bugs of the nt times . Two spe bed bug . Althoug bed bug . Moreove bed bug . Thus, iC. lectularius and C. hemipterus has already been described in several tropical and subtropical areas worldwide, and notably in Eastern and Western Africa, in which sympatric dwelling has previously been described [Cimex sp. fauna in Mauritania, West Africa.The simultaneous presence of escribed ,4,5,6,7.Population movements have an impact on the health and management of health systems, notably concerning vector-borne diseases in the countries of transit and destination . MauritaPrior to any control measures, identification of medically important arthropods at the species level is mandatory. This is generally done by morphological identification, which is helped by the use of magnifying tools, along with dichotomic identification keys . HoweverCimex species [Cimex genus.In recent decades, Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) has been successfully proposed as an identification method, notably concerning medically important arthropods . In part species ,14. HoweCimex sp. specimens, collected in domestic settings in Mauritania. Secondarily, all specimens  were screened for potential arthropod-borne bacteria using molecular methods.The objective of the present work was to assess MALDI-TOF MS as an identification tool over field-collected immature stages of Bed bug sampling was conducted simultaneously in October 2021 in two locations in Western Mauritania, Nouadhibou and Noaukchott . NouadhiCimex adult species [Cimex specimens when the present work was initiated, species level identification of immature stages relied on notable morphological features applicable for adult specimens, including pronotum width, bristle length and density as well as the specimen’s general coloration [Bed bug specimens were collected in beddings in domestic settings in Nouakchott and Nouadhibou, Mauritania . Adult a species . In the loration . Some sploration . Followiloration . SecondaC. hemipterus and C. lectularius, from various geographical origins  [Immature specimens’ heads were incubated at 37 °C overnight in order to facilitate ethanol evaporation . Followi Sweden) . The relCimex specimens was subjected to standard PCR and sequencing using a thermal cycler automaton  with primers targeting the 18S rDNA gene [C. lectularius was used as a positive control. The sequences obtained were assembled and identified, as previously described [The distal part of each bed bug specimen’s abdomen was transferred in a 1.5 mL tube with 180 µL of G2 lysis buffer and 20 µL of proteinase K  and incubated overnight at 56 °C. DNA extraction was performed using an EZ1 DNA Tissue Kit (Qiagen), according to the manufacturer’s protocol, as previously described . The DNADNA gene . The DNAescribed .Borrelia, Rickettsia, Bartonella, Coxiella burnetii and Wolbachia, as previously described [Rickettsia montanensis, Bartonella elizabethae, Coxiella burnetii, and Borrelia crocidurae were used as positive controls, as previously described [Wolbachia detection, a positive DNA extract previously sequenced as Wolbachia pipientis was used as positive control. DNA extracted from a laboratory-bred specimen of Rhipicephalus sanguineus s.l. tick was used as a negative control. When DNA from Wolbachia was detected from a Cimex specimen, bacterial species level identification was performed by gene sequencing, following standard PCR amplification of a fragment of 16SrDNA, as previously described [The DNA extract from each specimen was subjected to five different quantitative PCR (qPCR) assays for the detection of escribed . The reaescribed . Amplifiescribed . For Wolescribed .Cimex hemipterus, including 45 immature stages (49.4%), 32 males (35.1%) and 14 females (15.3%). Approximately half of the specimens were engorged . One specimen could not be morphologically identified due to poor conservation state. Among our study sample, two immature specimens were selected for scanning electron microscopy imaging , and 6 specimens were identified as C. hemipterus with LSVs ranging from 1.58 to 1.786. All these MALDI-TOF MS spectra matched with higher LSV with reference spectra obtained from heads of field-collected adult C. hemipterus originating from Senegal, which were preserved dried (following a quick immersion in 90% EtOH at the moment of collection) [C. hemipterus originating from Kenya and reared in our laboratory. Secondarily, our pre-existing database was upgraded with spectra originating from two of our samples. Querying of our spectra against the upgraded database resulted in more reliable identification scores, with 35/35 (100%) of the specimens identified as C. hemipterus. Precisely, 3/35 specimens retrieved LSVs between 1.967 and 1.990, and the remaining (32/35) specimens were identified with LSVs >2.Among the 45 immature specimens, a total of 43 were submitted to MALDI-TOF MS analysis. Two specimens were selected for electron microscopic study. Among the 43 specimens tested subjected to MALDI-TOF MS analysis, a total of 35 specimens produced good quality spectra . When enC. hemipterus were subjected to molecular identification through sequencing of the 18S rDNA gene: 8/14 because of obtention of low-quality spectra through MALDI-TOF MS analysis, and 6/14 because of obtention of Log Score Values <1.8  after spectra were entered into our in-house arthropod database. BLAST analysis allowed identification of C. hemipterus for all the 14 specimens subjected to molecular identification, with identification percentages ranging from 99.2% to 100% (GenBank: MN056507), confirming the identification.A total of 14 immature specimens morphologically identified as Borrelia, Rickettsia, Bartonella, Coxiella burnetii and Wolbachia. All tested specimens were positive for the Wolbachia qPCR, with Ct values ranging from 29.93 to 37.82 . All DNA samples tested negative for the other bacterial detection assays. Four DNA samples (obtained from two adults and two immature stages) were selected for Wolbachia 16S rDNA sequencing. Following amplification and sequencing, good quality sequences were obtained for the four specimens. BLAST analysis of the four sequences showed that they were 99.2% and 99.7% similar to the corresponding sequences of Wolbachia pipientis (GenBank: MN123078), and other Wolbachia sp., including a sequence obtained from C. lectularius bed bugs originating from Serbia (MH618380.1), and from C. hemipterus from Senegal .All the 92 specimens’ DNA extracts were submitted to specific qPCR assays for detection of Cimex specimens originating from Nouakchott and Nouadhibou, Mauritania. To the best of our knowledge, the present work represents the first proteomic and molecular investigation concerning bed bugs in this West-African country.The present work focused on MALDI-TOF MS identification of field-collected immature Cimex species could act as a key driver for its expansion in this continent [Bed bugs nowadays represent a global nuisance issue, as corroborated by recent scientific literature and rather important media coverage ,20,21. Iontinent ,22,23. Tontinent .C. hemipterus. This species is generally associated with tropical settings, although it has already been reported in sub-tropical and temperate areas [Cimex species parasitizing humans, the geographical distribution of both species could shift in the following years, highlighting the need for deployment of robust species level identification techniques in order to effectively monitor their distribution [In our study sample, all bed bugs specimens were identified as te areas ,7. Takinribution ,7.Cimex species level identification has been assessed in regard to, and successfully applied to, various species of medical and veterinary interest [Cimex specimens, and also that it was applicable for the identification of immature stages of this genus.MALDI-TOF MS has proved to be a robust tool for species level identification of a wide range of arthropods impacting human and animal health ,12. Indeinterest ,13,14. IC. hemipterus and C. lectularius, and the previously published work assessing MALDI-TOF MS identification for adult stages of the Cimex genus, only immature stages were retained for MALDI-TOF MS identification in the present work [Cimex hemipterus species [C. hemipterus collected from Ghana has been recently proposed [C. hemipterus stage, whereas other morphological characters seem to overlap among developmental stages, as assessed by means of Principal Component Analysis (PCA) by the authors [C. hemipterus specimens originating from other geographical areas. Furthermore, the applicability of MALDI-TOF MS to effectively stage immatures should also be assessed, with the help of laboratory-bred colonies, for instance. Ongoing works in our institute are currently investigating MALDI-TOF MS applicability to discriminate the different immature developmental stages of Cimex species.Given the availability of recognized morphological identification criteria between adult stages of ent work ,13,14. I species . Howeverproposed . This st authors . This stC. hemipterus originating from Senegal, even though our arthropod spectra database also contained reference spectra from immature stages of C. hemipterus and C. lectularius. However, previous studies focusing on MALDI-TOF MS identification of adult C. hemipterus specimens used heads as a study compartment, whereas reference spectra for immature stages in our in-house database were obtained from cephalothoraxes [Another interesting part of our results resides in the fact that the spectra obtained from our sample  matched more closely with spectra obtained from field-collected adult specimens of thoraxes ,13. In tthoraxes . The folthoraxes ,25,26. IWolbachia DNA in our study specimens. These bacteria act as endosymbionts for numerous arthropods and have been detected in specimens originating from multiple geographical locations [Wolbachia species in field-collected Cimex specimens originating from Senegal [Wolbachia species in their study sample, which allowed detection of Wolbachia massiliensis and W. pipientis, as well as Wolbachia sp, along with phylogenetic analysis of their likelihood [Wolbachia DNA was detected from all specimens, and sequencing from four of our specimens showed that these sequences were close to the corresponding sequences of W. pipientis, and Wolbachia sp., with similar identification percentages. Even though detection of Wolbachia is common among cimicids, more data is necessary concerning the different species that could be detected in bed bugs to better understand their respective impacts on their arthropod hosts [In the present work, bacterial screening and identification only allowed detection of ocations ,27,28.ThCimex study sample. This data was in accordance with previously published studies assessing infection status with vector-borne bacteria for field-collected Cimex specimens [Cimex specimens for vector-borne infectious agents, such as Borrelia recurrentis, Bartonella quintana and Trypanosoma cruzi, their role as infectious disease vectors in “real life” has never yet been demonstrated [Cimex sp., as well as future studies evaluating their vector competence for other vector-borne microorganisms should enable better knowledge of their potential as infectious disease vectors.No other potentially vector-borne bacterial DNA was detected from our pecimens ,7,13. Innstrated ,29,30. CCimex hemipterus. These results shed a promising light on effective identification and monitoring of Cimex populations. Indeed, MALDI-TOF MS has established itself as a current reference method for identification of medically important microbes in clinical microbiology laboratories [In the end, even taking into account the relative low number of specimens assessed, these results suggest that MALDI-TOF MS could be a robust tool for identification of immature stages of ratories . Besidesratories ,12. The ratories ,33.