Hemidactylus Oken is the second most species-rich genus of geckos with greatest diversity in the tropical regions of the world. Some species of the genus are commensal and widespread; however, there are several endemic lineages with restricted distribution. India is home to at least 35 species, with 20 endemic species and the number is steadily increasing with exploration of new habitats and integrated taxonomic approach including molecular data. We made investigations into the molecular and morphological variation throughout the distribution of Hemidactylus triedrusH. triedrus is a species complex represented by three species, H. triedrus sensu stricto and two undescribed taxa. H. subtriedrus Jerdon, 1854 syn. nov. was found to be morphologically similar to the type specimen of H. triedrus, and genetically embedded in a clade containing H. triedrus sensu stricto and is here treated as a junior synonym of H. triedrus, whereas H. lankae Deraniyagala is referred to as nomen dubium given that the types are presently not traceable and the original description is inadequate in diagnosing the taxon. The populations from western-central India and parts of Pakistan, and from southern Karnataka are distinct and diagnosable, and are herein described as two new species, respectively. Morphological and molecular data support the distinctiveness of the new species. The present work resolves a taxonomic turmoil that lasted over two centuries highlighting the need for studies that integrate morphological and molecular data.The gekkonid lizard genus  Cryptic species are considered to be the worst-case scenario of taxonomic incompleteness. These are discrete species that are difficult, or sometimes impossible, to distinguish morphologically alone and are usually classified as a single species mostly spanning a wide range in its distribution . A substHemidactylus Oken whose range spans from Pakistan, across the Peninsular India down to Sri Lanka . H. subtriedrus appeared on checklists and inventories of reptilian fauna of the Eastern Ghats of Chhattisgarh, Odisha, Telangana and northern Andhra Pradesh .ate form . The maltriedrus . It is l or lost  and hencH. subtriedrus proposed by H. subtriedrus, we collected a male specimen from Nellore district and compared it with the existing specimens in the collection of the museums, which also includes the type of H. triedrus. Additionally, specimens of H. triedrus were collected near Pondicherry and the outskirts of Chennai which might have been the localities from where the type of H. triedrus originated and a few from Maharashtra, Karnataka, Andhra Pradesh and Gujarat. The specimen from Nellore is identical to the specimens collected from Pondicherry and Chennai and match the type of H. triedrus confirming that H. subtriedrus is a synonym of H. triedrus. The synonymy allows us to restrict the type locality of H. triedrus to Nellore and provide a detailed description of the species based on fresh as well as museum material. Restriction of type locality for H. triedrus is here deemed necessary as this group is a species complex and to avoid further taxonomic confusion. The specimen from Nellore agrees in all respect with the available description and the type of the species in accord with the International Code for Zoological Nomenclature  and the forest department of concerned states. Specimens were captured and processed following the guidelines and protocols stated in the collecting permits  and agreements obtained from the competent authorities. All efforts were made to minimize animal suffering. Specimens were captured by hand in field and euthanized with Halothane following standard animal euthanasia guidelines , and fixAll measurements were taken following ® Skyscan 1272 . Head of the specimens were scanned from 16 to 20 min at 15 μm. Volume rendering was performed with CTVox  and images were edited in Adobe Photoshop CS6. Osteological description is based on volume renders retrieved from CTVox following terminology of the skull described by Micro-CT scans were generated for three male specimens using a BrukerInstitutional acronyms used in the manuscript are as follows: BNHS—Bombay Natural History Society, Mumbai; CAS—Californian Academy of Sciences, San Francisco; CES—Centre for Ecological Sciences, Bangalore; CM—Herpetology collection, Carnegie Museum of Natural History, Pittsburgh; MNHN—Muséum national d’Histoire naturelle, Paris; NHM—Natural History Museum, London; NCBS—Collection facility, National Centre for Biological Sciences, Bangalore; USNM—National Museum of Natural History, Washington; WHT—Wildlife Heritage Trust, Colombo; ZSI—Zoological Survey of India, Kolkata. Comparative material examined is listed in ND2) using newly designed primers- Liz_ND2F 5′- ACCTGACTTAGCCTAGAAC-3′, Liz_ND2R 5′- TAGGTTGAGTTGTATAGTGC-3′ and cytochrome b (cyt b) with published primers Cytb1 (5′-CCATCCAACATCTCAGCATGATGAAA-3′) and Cytb2 (5′-ACTGTAGCCCCTCAGAATGATATTTGTCCTCA-3′) for cyt b  and Phosducin (PDC) gene of selected Hemidactylus spp. of the “brookii group” of p-distance” was calculated in MEGA6 for cyt b and ND2    using tho.ac.nz) .http://species.h-its.org/ptp/) following ND2 gene was supplied for the analysis. The outgroup, H. flaviviridis, was excluded from the analysis for optimum results. The analysis was run for 100,000 generations with three chains and 25% of the trees were discarded as burn-in. Results of the analysis are presented in Bayesian Poisson Tree Process (bPTP) based on evolutionary placement algorithm was implemented on the web server , and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. 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. triedrus fell into eight haplotypes, with a haplotype diversity of 0.9242. A sequence from Pakistan, one from an unknown locality from India, and another sequence from the Deccan Traps region was recovered as a single haplotype, Hap_1, which here represents H. sahgali sp. nov. The Sri Lankan population was recovered as a unique haplotype (Hap_2), which was closely related to the central-eastern and eastern coastal population . This cluster of haplotypes includes a sequence from Nellore, and represents H. triedrus sensu stricto, by the virtue of restriction of type locality. The Bangalore population fell into four closely related haplotypes, this population is herein treated as H. whitakeri sp. nov. The 12 sequences of ND2 gene (966 bp) and two nuclear genes, RAG1  and PDC (395 bp) recovered three divergent, well supported clades (bootstrap support 100% + BI support 1), supporting the hypothesis that H. triedrus represents a species complex : Nellore, Sri Potti Sriramulu Nellore District, Andhra Pradesh following the synonymy of H. subtriedrus.Material examined: INDIA – female CAS 104151, Nagercoil, Kanyakumari district, Tamil Nadu; male BNHS 1668 Kalakkad Mundanthurai Tiger Reserve, Tamil Nadu; male BNHS 849 Kanyakumari district, Tamil Nadu; three females BNHS 683, BNHS 683-1, BNHS 1110 Palghat, Kerala; male NCBS AU703, Donthali near Nellore, Sri Potti Sriramulu Nellore District, Andhra Pradesh; male NCBS AU705, (near Pondicherry) Aranya forest and sanctuary, Poothurai Rd, Thiruchitrambalam, Tamil Nadu ; two males NCBS AU706–707, Chengalpettu, Tamil Nadu; male NCBS AU704 near Sri Varahalakshmi Narasimha Swamy Vari Devasthanam, Visakhapatnam, Andhra Pradesh . Sri Lanka: male WHT 2161, Virankattuwa; males CM 67601–67603, Poḷonnaruwa, North Central Province; undetermined sex CM 67801 Tabbowa, Puttalam district, North Western province; male CM 83615 Marichchukkaddi, Northern province; male USNM 120313, Matale District, Central Province; male USNM 254547 & three females USNM 254549–USNM 254551, Trincomalee, Eastern Province; male USNM 254583, Anuradhapura District, North Central District; male USNM 267726, Mannar, Northern Province; females USNM 120311, Rattota, Clodagh Estate, Kandy District, Central Province; three females USNM 254664–USNM 254666 Mundel, three mi from, Nawadamkulama, Puttalam, North Western province; four females USNM 254703, USNM 254727—USNM 254729 Marichchukkaddi, Mannar, Northern Province; female USNM 267725 Mahiyangana, Badulla District, Uva; female USNM 267727 Parayannalankulam Rest House, Mannar, Northern Province.Diagnosis: A medium sized fairly stout gecko, adults ranging 58–76 mm in SVL. Dorsum in a shade of light brown with paired, thin black edged white bands at regular intervals. Dorsal scalation on trunk, granular, intermixed with enlarged, keeled 19–20 trihedral tubercle rows arranged in fairly regular longitudinal series. Seven lamellae under digit I of pes and manus, eight to nine under digit four of manus and pes. An angular series of seven to nine precloacal femoral pores separated at mid-pelvic by a diastema of one to three non-pored scales.Genetic divergence: Genetic divergence within H. triedrus is 1–2% for cyt b, whereas divergence from H. sahgali sp. nov. and H. whitakeri sp. nov. is 14% and 14–16% respectively. Divergence from H. sahgali sp. nov. and H. whitakeri sp. nov. for the gene ND2 is 17–18% each. The two sequences of H. triedrus are 3% divergent from each other for ND2.Description of male NCBS AU703 from Nellore ( Nellore  and 5: T Nellore .A medium sized gecko (SVL 72 mm) with a fairly large head (HL/SVL ratio 0.28), head slightly longer than wide (HW/HL ratio 0.80), head depressed (HH/HL ratio 0.54), distinct from neck –5D; cantBody elongate (TRL/SVL ratio 0.42) and dorsoventrally flattened; lacking distinct ventrolateral furrow; dorsal scalation on trunk granular intermixed with enlarged, keeled, trihedral tubercles, fairly arranged in 19–20 longitudinal rows; dorsal tubercles on mid-dorsum situated in close proximity, slightly depressed, slightly longer (1.4) than wide (1.3); tubercles on the lateral aspect of the trunk larger, slightly spaced out in comparison with tubercles on mid-dorsum; ventral scales on trunk smooth, flat, larger than dorsal scales; mid body scales across belly 30–32; seven (left) and seven (right) femoral pores separated at mid-pelvic region by a diastema of three non-pored scales; size of non-pored scales equal to pored scales .Limbs moderately long, stout; digits dilated, bearing horizontally oriented lamellae on ventral surface; lamellae on basal half of digit I of manus and pes undivided, lamellae on rest other digits divided ; clawed, claw slightly smaller than length of the lamellar region; forelimbs short (FL/SVL ratio 0.14), slightly shorter than hind limbs (CL/SVL ratio 0.15). Terminal phalanx of all digits curved, arising angularly from distal portion of expanded lamellar pad, free portion of phalanx of all digits half to more than half long as the dilated portion. Lamellae beneath the digits, 7-8-8-8-8  on left Tail moderately depressed, oval in cross section, longer than SVL (TL/SVL ratio 1.21), 42.5 mm of the tail regenerated. Caudal segments distinct; pholidosis of original tail dorsum with small, juxtaposed scales intermixed with large keeled trihedral tubercles, scales on regenerated portion of tail homogenous, slightly smaller compared to the scales on original tail and lacking tubercles. First tail segment with a whorl of eight large conical, keeled tubercles, second segment onward, each segment with six tubercles. Ventral aspect with large, broad scales covering about ∼60% of the tail width from base of tail to the tip. A single sub-conical post cloacal spur.Coloration in preservative: Dorsum in a shade of light brown with black edged white bands at regular intervals. The first band is present on the nape and is not complete, interrupted medially at the vertebral region, second pair of the band is present on the intersection of the forelimbs, two pairs on the trunk and the last pair on the intersection of the hindlimbs. The white coloration of the bands is present on the tubercles which is surrounded by black colored scales. A white stripe running from the nares along the canthus rostralis to the ear opening, interrupted by adjoining dark brown coloration posterior to the eye. Limbs much paler nearly whitish pink in color with a few white tubercles. Ventrally off-white with each scale bearing sparse black speckles.Coloration in life: Background coloration on the head, trunk and tail light brown with paired bands from the nape all through the trunk to the tip of the tail. The bands are formed of a central white band edged with blackish brown on its anterior and posterior portion. The white central portion in these bands in mostly restricted to the tubercles which are bright white. Limbs pinkish in shade with a few white colored tubercles. Hind limbs slightly in a shade of brown and are darker compared to the anterior limbs. A yellowish stripe runs from mid canthus rostralis through the supraciliary scales up till the supra-tympanic region. Ventrally white.Variation observed in examined specimens: All examined specimens match the topotype from Nellore except for morphometric data presented in Natural history: A species generally associated with termite mounds. Several individuals of different age classes can be seen occupying a single mound. Individuals can be seen at the entrance of the openings of termite mounds just after dusk and will retreat in the mound with the slightest disturbance. Juveniles when disturbed will attain a posture with their bodies high and the tail is moved slowly in a curling and uncurling manner, likely to draw attention toward the tail. Several individuals were found in a single mound when the termite mound was dug, along with scorpions of the genus Heterometrus sp., and frogs of the genus Uperodon sp. Individuals also seek shelter under boulders, abandoned houses during the day emerging just after dusk. Mostly terrestrial in its habits but will climb trees too. Breeding likely takes places from February to May as hatchlings and eggs have been seen in the months of April–May in Pondicherry and Kanyakumari. Occupies areas that are dry like scrub, dominated with boulders and even in cities closer to the coast. Widely distributed from Visakhapatnam in north to Kanyakumari in south and recorded from elevation ranging from 17 to 1,913 m AMSL. Recorded from the following states/Union territories in India: Kerala, Tamil Nadu, Andhra Pradesh and Pondicherry , collected by Mayuresh Ambekar & Gaurang Gowande on November 20th, 2017.Paratypes: female NCBS AU713 same locality as holotype, collected by Rishikesh Patil and Zeeshan Mirza; two males NCBS AU719 & AU720, GKVK campus, Bangalore Karnataka, India , collected by Akshay Khandekar on April 16th, 2018.Referred material: female BNHS HTK1/2010 & male BNHS HTK2/2015 Kolli hills, Namakkal district, Tamil Nadu; male BNHS 1253 Nilgiri, Tamil Nadu; female BNHS 1827, two males ZSI 5852 & ZSI 5853 Bangalore, Karnataka.Diagnosis: A medium sized fairly stout gecko, adults ranging 45–60 mm in SVL. Dorsum in a shade of light brown with paired, thin black edged white bands at regular intervals. Dorsal scalation on trunk granular, intermixed with enlarged, keeled 16–17 sub-trihedral tubercle rows arranged in fairly regular longitudinal series on dorsum. Seven lamellae (rarely six) under digit I of pes and manus, eight to nine under digit four of manus and pes. An angular series of seven to eight precloacal femoral pores separated at a mid-pelvic by a diastema of three non-pored scales.Hemidactylus whitakeri sp. nov. differs from most congeners in bearing the following set of differing and non-overlapping characters: dorsum with large, keeled sub-trihedral tubercles in 16–17 fairly regular longitudinal rows ; dorsal pattern with fairly distinct bands and dorsal tubercles sub-trihedral ; SVL 45–60 mm ; SVL >80 mm H. maculatus Duméril & Bibron, H. graniticolus Agarwal, Bauer & Giri, H. kangerensis, H. sushilduttai, H. vanam Chaitanya, Lajmi & Giri, H. acanthopholis Mirza & Sanap, and H. hunae Deraniyagala; dorsum in a shade of brown with distinct, regularly spaced transverse bands on dorsum . The new species is most similar to H. triedrus in general appearance, however, differs in bearing 16–17 rows of keeled, sub-trihedral tubercles in fairly longitudinal rows , bands on dorsum thin, paired and usually broken incomplete , 7–8 precloacal femoral pores separated by a diastema of three non-pored scales . Postorbitofrontal slender in anteriorly, gradually widens posterior to the Fronto-Parietal suture as seen in H. sahgali sp. nov. , Frontal much wider posteriorly as in H. sahgali sp. nov. ; quadrate bone moderately robust , surangular slender as in H. triedrus (vs. robust in its width lacking a distinct constriction at the suture between—articular surface and retroarticular process).Genetic divergence: Genetic divergence within H. whitakeri sp. nov is 1–2% for the gene cyt b and 0–6% for ND2, divergence from H. sahgali sp. nov. and H. triedrus is 10–11% and 14–16% respectively for the gene cyt b. Divergence from H. sahgali sp. nov. and H. triedrus for the gene ND2 is 13–14% and 17–18% respectively.Etymology: The specific epithet is a patronym honoring Romulus Earl Whitaker for his valuable contribution toward the study and conservation of reptiles of India.Description of female holotype NCBS AU712,8: The specimen is in good condition, preserved in a linear manner lacking the entire tail (ire tail .A medium sized gecko (SVL 53.7 mm) with a fairly large head (HL/SVL ratio 0.23), head slightly longer than wide (HW/HL ratio 0.88), head depressed (HH/HL ratio 0.61), distinct from neck –8D; cantBody elongate (TRL/SVL ratio 0.50) and dorsoventrally flattened; lacking distinct ventrolateral furrow; dorsal scalation on trunk granular intermixed with enlarged, keeled, sub-trihedral tubercles, fairly arranged in 16–17 longitudinal rows; dorsal tubercles on mid-dorsum situated in close proximity, slightly depressed, slightly longer (1.0) than wide (0.8); tubercles on the lateral aspect of the trunk larger, slightly spaced out in comparison with tubercles on mid-dorsum; ventral scales on trunk smooth, flat, larger than dorsal scales; mid body scales across belly 28–30.Limbs moderately long, stout; digits dilated, bearing horizontally oriented lamellae on ventral surface; lamellae on basal half of digit I of manus and pes undivided, lamellae on rest other digits divided ; clawed, claw slightly smaller than length of the lamellar region; forelimbs short (FL/SVL ratio 0.15), slightly shorter than hind limbs (CL/SVL ratio 0.14). Terminal phalanx of all digits curved, arising angularly from distal portion of expanded lamellar pad, free portion of phalanx of all digits half to more than half long as the dilated portion. Lamellae beneath the digits, left manus 7-9-9-9-9 , right mTail moderately depressed, oval in cross section, longer than SVL (TL/SVL ratio 1.21). Caudal segments distinct; pholidosis of original tail dorsum with small, juxtaposed scales intermixed with large keeled trihedral tubercles, scales on regenerated portion of tail homogenous, slightly smaller compared to the scales on original tail and lacking tubercles. First tail segment with a whorl of eight large conical, keeled tubercles, second segment onward, each segment with six tubercles. Ventral aspect with large, broad scales covering about ∼60% of the tail width from base of tail to the tip. A single rounded post cloacal spur.Variation observed in examined specimens: All examined specimens match the holotype except for morphometric data presented in H. triedrus , collected by Pranav Vaghashiya on May 29, 2017.Paratypes: male BNHS 2496 Bilimora near Chikili, Navsari district, Gujarat  collected by Harshil Patel, Kaushal Patel & Vaibhav Naik on June 3, 2017; female BNHS 2497 Ambapani Village, Tapi district, Gujarat  collected by Harshil Patel & Vaibhav Naik on October 23, 2016; female BNHS 2498 Kangvai Village, Navsari district, Gujarat  collected by Harshil Patel & Parimal Patel on September 8, 2015; BNHS 2499, Bilimora, Navsari district, Gujarat  collected by Harshil Patel, & Vaibhav Naik on May 14, 2017; female NCBS AU709, Saswad, Pune district, Maharashtra , collected by Varun Vaze, Gaurang Gowande & Rishikesh Patil on June 2017.Material examined: India – female CAS 9414, Bhopal; female BNHS 93 Indore, Madhya Pradesh; female CAS/SUR 9630 Bishrampur, Chhattisgarh; male BNHS 94 Dangs, Gujarat; female BNHS 96 Hingolgadh; female BNHS 1478 Toranmal fort, Nandurbar, Maharashtra; male BNHS 1564 Tuljapur, Osmanabad district, Maharashtra; three males NCBS AU710—AU712 & female NCBS AU7013 Lonavala, Pune District, Maharashtra; two males ZSI 21483 & 21486 Pune, Maharashtra. Pakistan: male CAS 101355 Karachi, Sindh province.Diagnosis: A medium sized fairly stout gecko, adults ranging 56–78 mm in SVL. Dorsum in a shade of light brown with paired, broad black edged white bands at regular intervals. Dorsal scalation on trunk, granular, intermixed with enlarged, keeled 15–16 trihedral tubercle rows arranged in fairly regular longitudinal series. Seven to eight lamellae under digit I of pes and manus, 8–10 under digit four of manus and pes. An angular series of 11–15 precloacal femoral pores separated at a mid-pelvic by a diastema of one to three non-pored scales.Hemidactylus sahgali sp. nov. differs from most congeners in bearing the following set of differing and non-overlapping characters: dorsum with large, keeled trihedral tubercles in 16–18 fairly regular longitudinal rows ; dorsal pattern with fairly distinct bands and dorsal tubercles trihedral ; SVL 56–78 mm . The new species is most similar to H. triedrus in general appearance, however, differs in bearing 15–16 rows of keeled, trihedral tubercle in fairly longitudinal rows , broad bands on dorsum complete , 11–15 precloacal femoral pores separated by a diastema of one to three non-pored scales .H. whitakeri sp. nov. , Frontal much wider posteriorly as in H. whitakeri sp. nov. ; quadrate slender and arched (vs. quadrate bone moderately robust and thick in H. triedrus and H. whitakeri sp. nov.), surangular robust in its width lacking a distinct constriction at the suture between—articular surface and retroarticular process (vs. slender in H. triedrus and H. whitakeri sp. nov.).Postorbitofrontal slender in anteriorly, gradually widens posterior to the Fronto-Parietal suture as seen in Genetic divergence: Genetic divergence between populations of within H. sahgali sp. nov is 0–1% for cyt b as well as ND2, divergence from H. whitakeri sp. nov. and H. triedrus is 10–11% and 14% respectively for the gene cyt b; Divergence from H. whitakeri sp. nov. and H. triedrus for the gene ND2 is 13–14% and 17–18% respectively.Etymology: The specific epithet is a patronym honoring Bittu Sahgal, Editor and founder of Sanctuary Asia magazine for his contribution toward conservation of wildlife.Description of holotype male NCBS AU708 (: Holotype in good condition preserved in a linear manner with only posterior part of the tail slightly curved (BS AU708 : Holotypy curved .A large sized gecko (SVL 66.4 mm) with a fairly large head (HL/SVL ratio 0.22), head nearly as long as wide (HW/HL ratio 0.97), head depressed (HH/HL ratio 0.58), distinct from neck  and 10C;Body elongate (TRL/SVL ratio 0.44) and dorsoventrally flattened; lacking distinct ventrolateral furrow; dorsal scalation on trunk granular intermixed with enlarged, keeled, trihedral tubercles, fairly arranged in 15–16 longitudinal rows; dorsal tubercles on mid-dorsum situated in close proximity, slightly depressed, slightly longer (1.4) than wide (1.3); tubercles on the lateral aspect of the trunk larger, slightly spaced out in comparison with tubercles on mid-dorsum; ventral scales on trunk smooth, flat, larger than dorsal scales; mid body scales across belly >30; 14 (left) and 13 (right) femoral pores separated medially by a single non-pored scale; non-pored scales slightly larger than pored scales .Limbs moderately long, stout; digits dilated, bearing horizontally oriented lamellae on ventral surface; lamellae on basal half of digit I of manus and pes undivided, lamellae on rest other digits divided ; clawed, claw slightly smaller than length of the lamellar region; forelimbs short (FL/SVL ratio 0.14), equal in length with the hind limbs (CL/SVL ratio 0.14). Terminal phalanx of all digits curved, arising angularly from distal portion of expanded lamellar pad, free portion of phalanx of all digits half to more than half long as the dilated portion. Lamellae beneath the digits, left manus 8-9-9-9-8, right manus 8-9-9-9-8 ; left peTail moderately depressed, oval in cross section, longer than SVL (TL/SVL ratio 1.12). Caudal segments distinct; pholidosis of original tail dorsum with small, juxtaposed scales intermixed with large keeled trihedral tubercles, scales on regenerated portion of tail homogenous, slightly smaller compared to the scales on original tail and lacking tubercles. First tail segment with a whorl of eight large conical, keeled tubercles, second segment onward, each segment with six tubercles. Ventral aspect with large, broad scales covering about ∼60% of the tail width from base of tail to the tip. One sub-conical post cloacal spur.Coloration in preservative: Dorsum in a shade of dark brown with broad, fairly equidistant bands commencing at the nape to the tip of the tail. A total of five bands present, first narrow band on the nape, second on the intersection of the forelimbs, two on the trunk and the last one on the intersection of the hindlimbs. The bands are off-white with a diffused brown blotches, edged with black on its anterior and posterior ends. Limbs in the same color as the dorsum with a few white tubercles. A faint yellow stripe from the canthus rostralis through the supraciliary scales up till the temporal region. Ventrally off-white with each scale bearing sparse black speckles.Coloration in life: Head, trunk and tail in a shade of brown, trunk coloration darker. Bands on the dorsum dark black edged with yellow borders on the head and trunk. Yellow edging of the bands absent on tail. Legs in a shade of light brown with a few scattered yellow spots, especially on tubercles. A yellowish stripe runs from mid canthus rostralis through the supraciliary scales up till the supra-tympanic region. Ventrally pinkish white.Variation observed in examined specimens: All examined specimens match the holotype except for morphometric data presented in Natural history: A species associated with termite mounds as its related species H. triedrus. Found in dry open scrub areas with boulders. Seen actively moving about on the ground just after dusk. Hatchlings have been seen in the month of May. Distributed throughout the Deccan Traps, its distribution extends beyond the traps north-west into Pakistan. In India, it is recorded from the following states: Maharashtra, Gujarat, Madhya Pradesh, Rajasthan and Chhattisgarh records ranging in elevation from 11 to 1,191 m AMSL. All records of the species from Pakistan are from elevation ranging from 15 to 25 m AMSL.Suggested common name: Sahgal’s termite hill gecko.Hemidactylus reveal a major endemic radiation in South Asia and largely concentrated in India and Sri Lanka  million years ago (Hemidactylus inhabiting Indian peninsula.The divergence dates estimated by previous work show that ears ago  which coears ago  and likeHemidactylus, only two species, H. depressus and H. pieresii bear 15–19 and 17–20 pre-cloacal femoral pores respectively (H. lankae 13–19, falls within the range of H. depressus and H. pieresii. Until recently, H. pieresii was considered a synonym of H. depressus (H. depressus + H. pieresii to his H. lankae. The identity of H. lankae remains unclear as no specimen examined by nomen dubium until specimens that match the description are found.Among Sri Lankan species of the genus ectively . The ranepressus , and henH. maculatus (H. brookii (Cyrtodactylus spp. (Discovery of two new gecko species with such a wide distribution highlights the need for undertaking dedicated taxonomic revision of widespread taxa which likely represent multiple species. This situation parallels with most gekkonid lizards, example aculatus , H. broo brookii  and Cyrtlus spp. , 2016. MA common practice for naturalist and even taxonomists during biodiversity surveys is to overlook subtle difference in commonly distributed species and attribute them to a readily available name when the species in question might not be what it has been considered conspecific with . This pr10.7717/peerj.5341/supp-1Supplemental Information 1Numbers at nodes and tips signify support to species.Click here for additional data file.10.7717/peerj.5341/supp-2Supplemental Information 2Note the 9bp indel from 281bp to 280bp. Alligned with a reference sequence of Gekko gekko.Click here for additional data file.10.7717/peerj.5341/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.5341/supp-4Supplemental Information 4Numbers in bold show sequences generated in the present work. Names within brackets refers to the old taxonomy.Click here for additional data file.10.7717/peerj.5341/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.5341/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.5341/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.5341/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.5341/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.5341/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.5341/supp-11Supplemental Information 11Click here for additional data file.


Objectives:Candida species are a major cause of hospital infections, including ocular candidiasis, but few studies have examined the propensities of specific species to invade the eye or the unique immunological responses induced. This study examined the frequency and characteristics of species-specific Candida eye infections by epidemiology and experiments using a mouse ocular candidiasis model.Methods: We reviewed medical records of candidemia patients from January 2012 to March 2017. We also evaluated ocular fungal burden, inflammatory cytokine and chemokine profiles, and inflammatory cell profiles in mice infected with Candida albicans, Candida glabrata, or Candida parapsilosis.Results: During the study period, 20 ocular candidiasis cases were diagnosed among 99 candidemia patients examined by ophthalmologists. Although C. parapsilosis was the most frequent candidemia pathogen, only C. albicans infection was significantly associated with ocular candidiasis by multivariate analysis. In mice, ocular fungal burden and inflammatory mediators were significantly higher during C. albicans infection, and histopathological analysis revealed invading C. albicans surrounded by inflammatory cells. Ocular neutrophil and inflammatory monocyte numbers were significantly greater during C. albicans infection.Conclusion:Candida albicans is strongly associated with ocular candidiasis due to greater capacity for invasion, induction of inflammatory mediators, and recruitment of neutrophils and inflammatory monocytes. Candida species are among the most common causative pathogens of bloodstream infections in hospitals, and ocular candidiasis is a potentially severe complication that can lead to visual field defects or blindness if appropriate therapy is delayed  were identified. Candidemia was defined by the isolation of any  culture .Candida bloodstream infection examined by ophthalmologists were included in this study. Candidemia cases were excluded from consideration if (1) ophthalmologists could not examine the fundus because of the poor general condition or (2) patients died before the blood cultures were confirmed positive. The medical records of each patient were also reviewed to identify the primary disease, the presence of central venous catheter insertion, neutrophil counts, usage of immunosuppressive agents, presence of pre-treatment antifungal agents, serum 1,3-beta-d-glucan (BDG) values, and 30 day all-cause mortality. Serum BDG was measured using the Wako turbidimetric assay  with positivity cutoff of 11 pg/mL  at 35°C and identified at the species level by a Vitek or Vitek 2 system  for all germ tube-negative Candida yeast in the Toranomon Hospital. The D1/D2 regions and/or internal transcribed spacer regions of the rRNA gene from Candida isolates were sequenced to provide further support for species identity by the National Institute of Infectious Diseases . All Diseases . This reFemale C57BL/6J mice, 7–9 weeks old, were purchased from Japan SLC, Inc.  and maintained under specific pathogen-free conditions at the National Institute of Infectious Diseases in Japan. All experiments were reviewed and approved by the Animal Care and Use Committee of the National Institute of Infectious Diseases. Protocols were designed to minimize animal suffering and limit the numbers used for experiments (Approved Number: 116111).C. albicans (SC5314), Candida glabrata (CBS138), and C. parapsilosis (ATCC22019), and clinically isolated strains of C. albicans (SF-30), C. glabrata (SF-31), and C. parapsilosis (TOR-1) were used for mouse candidiasis model studies. Clinical strains were isolated from blood cultures of candidemia patients. C. albicans and C. parapsilosis were grown at 30°C and C. glabrata at 37°C for 2 days on yeast extract peptone dextrose (YPD) agar. C. albicans and C. parapsilosis were then grown at 30°C and C. glabrata at 37°C in YPD broth for 18–24 h. After incubation, yeasts were collected, washed, and resuspended in sterile PBS at approximately 2.5–3.5 × 106 colony-forming units (CFU)/mL. Mice were injected with 200 μL (approximately 5–7 × 105 CFU/mouse) via the lateral tail vein to cause Candida dissemination.Reference strains of C. albicans, C. glabrata, or C. parapsilosis infected group. The infected mice were euthanatized 3 days after injection, followed by aseptic collection of kidneys and eyeballs. The kidneys and eyeballs were homogenized in sterile PBS, and the homogenates of the kidneys were then serially diluted. Kidney homogenate was plated on YPD agar and eyeball homogenate on YPD agar with antibiotics (penicillin/streptomycin), both at 100 μL per plate. The fungal burden was determined by counting CFUs after 1 day incubation at 30°C. The remaining homogenates were centrifuged and stored at -30°C for cytokine analysis. Cytokines were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits .In this experiment, mice were divided into three groups as follows: C. albicans, C. glabrata, or C. parapsilosis infected group. The eyeballs were carefully enucleated and then washed with sterile PBS to eliminate contaminating peripheral blood. After washing, samples were incubated in RPMI medium with 2% fetal bovine serum and digestive enzymes  under rotation at 37°C for 1 h. After enzyme digestion, the eyeballs were homogenized and filtered to remove undigested particles. The ocular immune cells recovered in the filtrate were blocked with Fc-receptor blocking antibody and stained with fluorochrome-conjugated antibodies (Listed in the Supplementary Table S1). Immune cells were enumerated using the FACSCanto II® flow cytometer (BD Biosciences). Obtained data were analyzed using FlowJo, version 9.5 .In this experiment, mice were divided into four groups as follows: uninfected control, For histopathological analysis, eyeballs were removed 3 days after yeast injection and fixed in 10% formalin, dehydrated with ethanol, and embedded in paraffin following standard procedures. Five micrometer tissue sections were mounted onto glass slides  and stained with hematoxylin and eosin, periodic acid Schiff, or Gomori methenamine silver. After staining, histological examination was performed under light microscopy.post hoc Tukey–Kramer HSD tests for pair-wise comparisons. If the standard deviations differed among groups, means were compared by Kruskal–Wallis test and post hoc Dunn’s test for pair-wise comparisons. Correlations between ocular fungal burden and cytokine/chemokine levels were evaluated by Pearson’s test. A P-value <0.05 (two tailed) was considered significant for all tests. All variables at P < 0.20 by univariate analysis were included in multivariate stepwise logistic regression analysis in epidemiological investigations. Statistical analyses in epidemiological studies were performed with EZR , which is a graphical user interface for R . More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics  followed by atistics . StatistCandida infection at Toranomon Hospital and identified 123 cases of candidemia during the study period, of which 99 were included in this study. Among these 99 patients, C. parapsilosis was the most common infective species, followed by C. albicans and C. glabrata. C albicans and C. parapsilosis co-infection was detected in one patient. Serum BDG was measured during the treatment period in 97 patients (98%).We retrospectively investigated the epidemiological data on Candida species distribution of the patients with ocular candidiasis and non-ocular candidiasis are summarized in Tables 1, 2, respectively. The rate of C. albicans bloodstream infection was significantly higher in the ocular candidiasis group than the non-ocular candidiasis group (76% vs. 13%). In contrast, the rates of hematological malignancies, antifungal agent prophylaxis, and neutropenia were significantly higher in the non-ocular candidiasis group. Serum BDG values did not differ between the entire candidiasis cohort and the subgroup with C. albicans bloodstream infection . In addition, the proportion of patients seropositive for BDG values did not differ between ocular and non-ocular candidiasis groups (75% vs. 58%). Multivariate analysis showed that only C. albicans bloodstream infection was significantly associated with ocular candidiasis , suggesting that ocular candidiasis is most likely to occur during C. albicans bloodstream infection regardless of underlying diseases or comorbidities.Twenty of 99 candidemia patients were diagnosed with ocular candidiasis. The general clinicodemographic characteristics and in vivo experiments using our mouse ocular candidiasis model to compare the propensity for eye infection and severity of ocular inflammation among Candida species following bloodstream infection. Based on our epidemiological data and previous reports, C. albicans, C. glabrata, and C. parapsilosis are the most common Candida species causing in-hospital infection; accordingly, we decided to compare these species using both reference strains and clinical isolated strains. We injected reference strains of C. albicans, C. glabrata, and C. parapsilosis into separate mouse groups as described and then examined the fungal burdens in eyes and kidneys 3 days after injection. The fungal burden in eyes was significantly higher during C. albicans infection compared with non-albicans Candida infection . In the kidneys as well, C. albicans burden was over 100-fold greater than non-albicans Candida burden . Similar results were found using clinically isolated strains .On the basis of the above results, we performed C. albicans invaded the mouse retina in the form of hyphae and also invaded the vitreous body in one sample . In addition, accumulation of inflammatory cells around C. albicans was detected. Conversely, there were no signs of fungal invasion into the eye during infection by C. glabrata or C. parapsilosis . These results suggest that C. albicans can more easily invade the retina and cause profound inflammation compared with non-albicans Candida, indicating that these species-specific propensities for ocular candidiasis are due to differences in invasive capacity.Ocular complications were compared among pathogens by histopathological analysis. Both the reference strain and clinically isolated strain of Candida injection. Ocular concentrations of IL-6, CXCL1/KC, CXCL2/MIP-2, and CCL2/MCP-1 were significantly elevated during C. albicans infection compared with non-albicans Candida infection by the reference strains . Surprisingly, IL-6 was not detected in most eyes following C. parapsilosis injection despite detection of this species in some eye samples. In contrast, the levels of IL-1β, IL-12p40, and TNF-α in ocular homogenate did not differ significantly among the three reference strain infection groups . In the preliminary experiment, these cytokines and chemokines were not elevated in uninfected mouse eyes (data not shown). Ocular concentrations of IL-6, CXCL1/KC, CXCL2/MIP-2, and CCL2/MCP-1 were also higher following injection of the C. albicans clinical strain compared with the other clinical strains . In addition, ocular IL-1β was higher in mice injected with the clinical C. albicans strain than in mice injected with clinically isolated non-albicans Candida. Conversely, ocular TNF-α and IL-12p40 were significantly lower in mice with C. albicans infection compared with those with C. parapsilosis infection .To identify factors inducing ocular inflammation following invasion, we measured several cytokines and chemokines in mouse eye homogenates by ELISA after C. albicans . On the other hand, there were no correlations between ocular C. parapsilosis fungal burden and the levels of these chemokines/cytokines . During infection by the C. glabrata reference strain, ocular IL-6 and MCP-1 were positively correlated with ocular fungal burden; however, the concentrations of these mediators were low and not detected in most infected mice, indicating only mild inflammation induced by C. glabrata . Consistent with the propensity of C. albicans for high ocular fungal burden, invading C. albicans induced severe inflammation by strongly stimulating the release of inflammatory cytokines and neutrophil or monocyte chemotactic factors. Conversely, non-albicans Candida did not exhibit the same invasive capacity or induce the release of inflammatory mediators, especially C. parapsilosis. These differences in the ocular inflammatory cytokine and chemokine profiles between C. albicans and non-albicans Candida likely explain the difference in ocular candidiasis frequency and severity following blood infection.Correlation analysis revealed strong positive correlations between ocular fungal burden and IL-6, CXCL1/KC, CXCL2/MIP-2, and CCL2/MCP-1 after injection of both the reference strain and clinically isolated strain of Candida species by flow cytometry. We infected mice with reference strains of each Candida species and quantified inflammatory cells in the eye after 3 days . The number of CD45+ cells was higher in mice infected by C. albicans compared with non-albicans Candida infection; in fact, there were no differences in CD45+ cell numbers between the non-albicans Candida group and uninfected controls . Among CD45+ cells, the numbers of ocular neutrophils and inflammatory monocytes were significantly elevated in mice infected by C. albicans compared with the non-albicans Candida group and uninfected controls . In addition, the number of resident monocytes was also higher in mouse eyes during C. albicans infection . Conversely, the number of ocular macrophages was slightly lower in mice infected by C. albicans compared with non-albicans Candida and control groups . There was also a difference in neutrophil number between C. parapsilosis and C. glabrata groups, although this was quite small. Collectively, these results suggest that the greater invasive capacity of C. albicans induces a stronger inflammatory cytokine and chemokine response, leading to greater accumulation of neutrophils and inflammatory monocytes in the eye and more severe retinal inflammation .On the basis of the above results, we examined the ocular inflammatory cell profile associated with each Candida species. In our epidemiological investigation, C. albicans tended to cause ocular complications more frequently than non-albicans Candida, in accord with previous reports. Moreover, in our mouse ocular candidiasis model, the fungal burden, inflammatory cytokine and chemokine concentrations, and the numbers of ocular neutrophils and inflammatory monocytes were significantly higher following C. albicans injection compared with non-albicans Candida injections. These results indicate the stronger propensity for ocular candidiasis following C. albicans infection results from greater ocular invasive capacity and concomitantly greater secretion of inflammatory mediators and recruitment of neutrophils and inflammatory monocytes.Although ocular candidiasis is a serious complication of candidemia, there are few previous reports describing the differences in ocular complications among C. albicans was the most frequent pathogen in ocular candidiasis, in accord with previous studies (C. albicans was also the most frequently isolated species from blood cultures (C. parapsilosis was the most frequent pathogen in our investigation; nevertheless, the rate of ocular candidiasis caused by C. albicans was significantly higher than that caused by non-albicans Candida, suggesting that C. albicans has a stronger propensity for eye invasion, colonization, and induction of ocular candidiasis. In addition, serum BDG values were not significantly different between patients with ocular candidiasis and those without ocular candidiasis in our study, whereas previous reports concluded that serum BDG could be the marker of ocular candidiasis (Candida species among sites. In our study, C. parapsilosis was the most frequent pathogen for candidemia, and non-albicans Candida accounted for approximately three quarters of all candidemia cases. The other possibility is the presence of antifungal prophylaxis. A previous report found that serum BDG was significantly lower in patients with breakthrough candidemia (In the epidemiological study,  studies . In thescultures . In contdidiasis . One posndidemia . Given oC. albicans but not the other species test strongly induced inflammatory cytokine or neutrophil chemotactic chemokine release. Neutrophil accumulation was also found in the rabbit ocular candidiasis model, although the factors inducing neutrophil migration were not identified (C. albicans retinal invasion. On the other hand, other inflammatory cytokines, such as IL-12p40, IL-1β, or TNF-α, were not elevated; therefore, the aforementioned mediators appear to have central roles in C. albicans-induced ocular inflammation. Although there are no previous reports describing ocular candidiasis in mice, it was reported that KC/CXCL1, but not IL-6, impacts intraocular inflammation in mice during Bacillus cereus infection (B. cereus intraocular infection. On the other hand, another study reported a substantial role for IL-6 in the retinal inflammatory response in a mouse ocular toxoplasmosis reactivation model (C. albicans infection aside from neutrophil-activating KC/CXCL1. These chemokines and IL-6 may recruit other immune cells as flow cytometry revealed inflammatory monocyte infiltration into mouse eyes. Given our results and previous reports, IL-6, CXCL1, and other neutrophil or monocyte chemotactic factors likely all contribute to inflammation in ocular candidiasis.In mouse experiments, entified . Our mounfection . In thaton model . In our Candida species and the number of strains was limited to three in mouse experiments. Although many Candida species cause bloodstream infections, these are the top three according to previous studies and accounted for over 80% of candidemia cases in our epidemiological study (There are several limitations to this study. First, the epidemiological study was retrospective and the number of cases involved was not large. However, most cases of candidemia were reviewed by experts in infectious diseases, so there were relatively few missing data on ocular candidiasis or serum BDG except for patients in terminal phases. Second, the variety of al study . TherefoC. albicans and non-albicans Candida in mice. Notably, results were in accord with epidemiological investigations in that C. albicans was both the most prevalent causative pathogen in humans and exhibited the greatest ocular invasive capacity in mice. Ocular candidiasis appeared more likely in cases of systemic infection by C. albicans compared with other Candida species, and the inflammation is more severe because of the greater cytokine/chemokine release and concomitant recruitment of neutrophils and monocytes compared with non-albicans Candida infection.In summary, this is the first report describing differences in fungal burden, histopathological manifestations, chemotactic factor profiles, and invading inflammatory cell profiles among ocular candidiasis cases induced by MA and YK designed the experiments. MA performed the experiments, acquired, and analyzed the data. KU, ST, SN, MiK, and YM contributed to the mouse experiments. SO, MuK, HA, and AY contributed to the epidemiological study. SS, MS, and KS contributed to the histopathological study. MA and YK drafted the manuscript. All the authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Listeria monocytogenes, Shigella flexneri, Rickettsia spp., and Burkholderia spp. disseminate within nonphagocytic cells, such as epithelial and endothelial cells, through a process referred to as cell-to-cell spread [A subset of intracellular pathogens, including l spread . These pPathogenic bacteria gain access to the host cell actin assembly machinery through bacterial engulfment in membrane-bound compartments termed primary vacuoles from which the bacteria escape through secretion of bacterial factors that challenge the integrity of the vacuole membrane . This inL. monocytogenes primary vacuole escape in human epithelial cells is facilitated by the production of the pore-forming toxin Listeriolysin O (LLO) [ O (LLO)  and phos O (LLO) .S. flexneri vacuole escape depends on the type-three secretion system (T3SS). Similar to L. monocytogenes LLO, the T3SS translocases, primarily IpaC, contribute to vacuole escape by forming pores in the vacuole membrane [S. flexneri vacuole escape [S. flexneri T3SS effector protein IpgD [Rickettsia spp. produce phospholipases that are implicated in vacuole escape; however, this mechanism is still unclear [Burkholderia spp. escape vacuoles through the activity of the T3SS [membrane ,6. The he escape . The recein IpgD , suggest unclear –11. Burkthe T3SS .Once in the cytosol, intracellular bacteria spread from cell to cell by first acquiring actin-based motility (ABM). In uninfected cells, actin polymerization relies on actin nucleators and their cognate regulators . In infeS. flexneri and L. monocytogenes, the actin-related protein ARP2/3 complex—a critical host cell actin nucleator—is recruited to the bacterial pole. S. flexneri secretes a bacterial autotransporter protein IcsA , whose activity recruits the ARP2/3 nucleation-promoting factor Wiskott−Aldrich Syndrome protein (N-WASP), and consequently ARP2/3, to the bacterial pole [In ial pole ,16.L. monocytogenes expresses a bacterial factor, ActA [By contrast, or, ActA , that reor, ActA  through or, ActA –21 and aRickettsia spp. encode RickA, an N-WASP mimic that recruits ARP2/3 to the bacterial surface and induces ABM [R. rickettsii produce Sca2, which is required for cell-to-cell spread and resembles actin nucleators of the formin family [uces ABM ,23. In an family . While Rn family . RickA in family .Listeria and Rickettsia, B. thailandensis BimA acts as a nucleator that activates Arp2/3. However, orthologs of BimA from pathogenic counterparts B. pseudomallei and B. mallei mimic actin nucleators of the host Ena/VASP family [Similarly to P family . SimilarP family .Cytosolic ABM allows intracellular pathogens to reach the plasma membrane at sites of cell−cell contacts, where they form membrane protrusions that project into adjacent cells . This prL. monocytogenes releases Tuba/N-WASP-mediated tension at cell−cell contacts by secreting Internalin C (InlC), which binds Tuba, thereby displacing N-WASP [R. parkeri effector protein Sca4 has been proposed to release tension by interfering with vinculin−α-catenin interactions, potentially creating unequal actomyosin tension at cell junctions and promoting bacterial spread. This mechanism has been shown to contribute to protrusion resolution [S. flexneri and Burkholderia spp. overcome membrane tension is unknown.g N-WASP . The R. solution . How S. L. monocytogenes cytosolic ABM, membrane protrusion formation relies on the AIP1/CFL1-dependent disassembly machinery [In addition to the ARP2/3-dependent actin assembly machinery required for achinery . The disachinery . Efficieachinery  and formachinery , whose fS. flexneri protrusions. In addition to ARP2/3, the host formins mDia1/2 localize to protrusions and are required for their proper formation [It is presumed that, similar to cytosolic ABM, IcsA and N-WASP/Arp2-3 are responsible for actin polymerization in ormation . Myosin-ormation .Rickettsia spp. ABM relies on the actin cytoskeleton, it was recently observed that R. parkeri protrusions uniquely lack actin tails, suggesting that the R. parkeri protrusion formation may not rely on the forces generated by actin assembly [Although assembly . This poB. pseudomallei and B. thailandensis infection [Burkholderia dissemination [Actin-containing membrane protrusions are formed during nfection ,35, but mination ,35. HoweDuring bacterial spread from cell to cell, membrane protrusions resolve into double membrane vacuoles (DMVs), whose inner and outer membranes are contributed by the primary infected cell and the adjacent cell, respectively . The forL. monocytogenes, the host AIP1/CFL1-dependent disassembly machinery is critical not only for the formation but also for the resolution of protrusions [L. monocytogenes spread in epithelial cells is unclear.In trusions . It was trusions . The bactrusions . An additrusions ; howeverS. flexneri, protrusions are resolved in a two-step process. The collapse of the protrusion neck, presumably due to the disassembly of the actin cytoskeleton network, results in the formation of intermediate structures termed vacuole-like protrusions (VLPs) [In s (VLPs) . Formatis (VLPs) ,39. The s (VLPs) , but theRickettsia effector protein Sca4 contributes to Rickettsia spp. protrusion resolution [Burkholderia spp. have been observed in protrusions, they have not been observed in DMVs [As mentioned above, the solution . Althoug in DMVs ; therefoIn order to resume ABM in the cytosol of adjacent cells, spreading bacterial pathogens must escape from the DMVs formed as a result of protrusion resolution. In contrast with primary vacuole escape, DMV escape is a complex process that requires the destabilization of two membranes .L. monocytogenes accomplishes DMV escape by using pore-forming toxins and enzymes that challenge the integrity of the vacuole membranes [embranes . Similarembranes .S. flexneri mutants lacking functional T3SS are trapped in DMVs, it was proposed that, similar to primary vacuole escape, the T3SS translocases may mediate DMV escape through pore formation [Because ormation . In addiormation . IcsB isormation , althougRickettsia spp. DMV escape is poorly understood, and whether Burkholderia spp. forms DMVs altogether is unknown.The mechanism supporting L. monocytogenes and S. flexneri and are now fairly well understood for Rickettsia spp. and Burkholderia spp. By contrast, further investigation will be required to uncover the mechanisms supporting the formation and resolution of protrusions resolution and DMV escape for most pathogens that spread from cell to cell.In conclusion and as shown in


Pseudomonas aeruginosa colonization in lower respiratory tract (LRT) and reduced P. aeruginosa ventilator-associated pneumonia (VAP) rate. Recent studies also suggested that probiotics could improve lung inflammation in mice infected with P. aeruginosa. It seems that microbiota regulation may be a potential therapy for P. aeruginosa VAP patients. However, we know less about the LRT microbial composition and its correlation with prognosis in P. aeruginosa VAP patients. This study aimed to characterize LRT microbiota in P. aeruginosa VAP patients and explore the relationship between microbiota and patient prognosis.Probiotics could prevent P. aeruginosa VAP patients were evaluated. Clinical pulmonary infection score and the survival of intensive care unit were both the indicators of patient prognosis.Deep endotracheal secretions were sampled from subjects via intubation. Communities were identified by 16S ribosomal RNA gene sequencing. The relationship between microbiota and the prognosis of P. aeruginosa VAP patients was significantly different from non-infected intubation patients, and showed significant individual differences, forming two clusters. According to the predominant phylum of each cluster, these two clusters were named Pro cluster and Fir-Bac cluster respectively. Patients from Pro cluster were dominated by Proteobacteria (adj.P < 0.001), while those from Fir-Bac cluster were dominated by Firmicutes, and Bacteroidetes (both adj.P < 0.001). These two varied clusters (Pro and Fir-Bac cluster) were associated with the patients’ primary disease . The primary disease of the Pro cluster mainly included gastrointestinal disease (63%), and the Fir-Bac cluster was predominantly respiratory disease (89%). During the two-week dynamic observation period, despite the use of antibiotics, the dominant genera and Shannon diversity of the LRT microbiota did not change significantly in patients with P. aeruginosa VAP. In prognostic analysis, we found a significant negative correlation between Lactobacillus and clinical pulmonary infection score on the day of diagnosis (P = 0.014); but we found no significant difference of microbial composition between survivors and non-survivors.In this study, the LRT microbial composition of P. aeruginosa VAP patients, forming two clusters which were associated with the primary diseases of the patients.LRT microbial composition was diversified among The online version of this article (10.1186/s12931-018-0847-3) contains supplementary material, which is available to authorized users. Pseudomonas aeruginosa is one of the most common pathogens causing VAP and is independently associated with increased mortality; in China, it has been staying in the top three pathogens  for non-normally distributed data. Categorical variables were presented as number of subjects and percentages. Figures were created using GraphPad Prism version 6.0.P. aeruginosa VAP patients and 18 control subjects. Sequential samples were obtained from 26 P. aeruginosa VAP patients for dynamics evaluation. Baseline characteristics are listed in Additional file P. aeruginosa VAP patients than for controls (P = 0.009). All P. aeruginosa VAP patients used antibiotics before mechanical ventilation. There was no significant difference in gender, body mass index (BMI), number of smokers, number of common complications including hypertension and diabetes in P. aeruginosa VAP group and control group  by 16S rRNA gene sequencing. Within the 94 samples, 914 OTUs were detected and classified into 19 phyla including 269 genera.One sample collected from P. aeruginosa VAP was significantly different from that in the control group    Fig. .Fig. 1ThP. aeruginosa VAP group. At phylum level, the dominant taxa of patients in the control group and the P. aeruginosa group were both Proteobacteria, Firmicutes, and Bacteroidetes, but the average abundance of Proteobacteria in the P. aeruginosa group was significantly increased (adj.P = 3.02 × 10− 5)  and Lachnospiraceae_incertae_sedis were dominant taxa in the P. aeruginosa group  Fig. . At the oup Fig. .P. aeruginosa VAP group varied; thus, we performed clustering analysis. Samples from P. aeruginosa VAP patients were hierarchically clustered based on similarities in microbial composition (measured by Bray Curtis distance) and visualized as phylum and genus dendrograms with two clusters , whereas the abundance of Pseudomonas was significantly lower than that of Pro group (adj.P < 0.001)  Fig. . Convent2-test P < 0.0001)    (Table To gain insight into correlation between LRT microbiota and severity of pulmonary infection (measured by CPIS) in patients with IS Table . Among tP. aeruginosa VAP on days 1, 7, and 14 . Those patients were all received antibiotics during the two-week observation .40 sequential samples collected from patients with nts Fig. . During P. aeruginosa VAP obtained clinical improvement. No significant taxa was found when we compared the microbial composition between acute phase and clinical improvement phase in those patients . However, of the six patients with P. aeruginosa VAP, abundance of Pseudomonas was decreased in five patients while was increased in one patients (from 21 to 24%)  had significant negative correlation with SOFA score . The LRT microbial composition and Shannon diversity of the survivors and non-survivors showed no significant difference   and the predominant taxa differed by cluster. Fir-Bac cluster was enriched for Lachnospiraceae_incertae_sedis, Bacteroides, Blautia, and Alloprevotella, and Pro cluster was enriched for Proteobacteria including Enterobacteriaceae and Pseudomonas. Lachnospiraceae_incertae_sedis, Bacteroides, and Blautia are the predominant (and harmless) taxa in normal healthy intestines [Enterobacteriaceae and Pseudomonas are common opportunistic pathogens causing lung infections [P. aeruginosa VAP appear to be related to the primary disease. The primary disease in the Pro group was mostly digestive diseases, whereas in the Fir-Bac cluster the patients were mainly respiratory diseases. Previous studies have shown that Lachnospiraceae and Bacteroides are the predominant taxa in fecal samples of patients with respiratory disease such as cystic fibrosis [Enterobacteriaceae increases more frequently in the gut of patients with gastrointestinal diseases [P. aeruginosa VAP patients. However, further analysis of the intestinal microbiota of P. aeruginosa VAP patients is needed.We found testines . Enterobfections . Microbifibrosis , while Ediseases –30. Intediseases . Few studiseases . Thus, oBurkholderia, Pseudomonas, and Enterobacter were positively correlated with CPIS, while Lactobacillus was negatively correlated with CPIS. These results suggested that the LRT microbiota in patients with P. aeruginosa VAP was associated with the severity of pneumonia. We also found that Pseudomonas showed a decreasing trend in the patients that acquired clinical improvement, and Lactobacillus and Bifidobacterium showed an upward trend in those patients. Burkholderia, Pseudomonas, and Enterobacter were common pathogens of hospital-acquired pneumonia [Lactobacillus and Bifidobacterium could inhibit pathogen proliferation, affect the behavior of other taxa, and regulate the host immune response [Lactobacillus and Bifidobacterium have an inhibitory effect on P. aeruginosa, Escherichia coli, Klebsiella pneumonia, and Enterococcus faecalis in vitro [Lactobacillus decreases P. aeruginosa virulence by decreasing the expression of lasI, lasR, rhlI, and rhlR involved in quorum sensing and inhibition of biofilm formation [Bifidobacterium has the ability to inhibit the adhesion of P. aeruginosa to epithelial cells [P. aeruginosa VAP patients. Microbiota regulation may become a potential therapy for P. aeruginosa VAP, however, the issues of delivery and probiotic selection require careful consideration. Although we found LRT microbiota was associated with the severity of pneumonia and early treatment effect, we did not observed difference between survival group and non-survival group. The sampling collection time was far from the death time, ranged from one month to 12 months, which may be one of the reasons that resulted in no significant difference in the composition of the LRT microbial composition between the survivors and non-survivors. On the other hand, we found the SOFA score which was associated with patients’ survival showed little correlation with LRT microbiota in P. aeruginosa VAP patients. It seemed that the LRT microbiota had greater effect on lung than the whole organ system.The CPIS is commonly used to assess the severity of lung infections in patients and the efficacy of antibiotic therapy , 18. In neumonia . Althougneumonia , 32. Lacresponse . Severalin vitro –36. Cotaormation . It has al cells . TherefoP. aeruginosa VAP patient, patients of each cluster showed similar microbial composition. Our findings were similar to previous lung-infection-disease studies which showed the lung dominant taxa were stable following the antibiotic treatment [P. aeruginosa but changed in those not dominated by P. aeruginosa [Pseudomonas, Enterobacteriaceae and Acinetobacter were common antibiotic-tolerant pathogens; it was not surprising that they could exist consistently with antibiotic exposure. The consistent microbial dysbiosis had significant negative impact on patient health, so identifying community member interactions and how to break up the network should be pay attention in future studies.Though antibiotic species and numbers were individual in each reatment , 39. Rogruginosa . The mecP. aeruginosa VAP. Second, we characterized the LRT microbiota using the culture-independent technology, which could identify low-proportion or uncultured taxa; however, we were unable to determine the function of specific species accurately. Further analysis regarding probiotic species and associated mechanisms in P. aeruginosa VAP is needed. Third, in this study, the sample collection time was limited. If we could increase the collecting time points in future, it may be better to reflect the changes of the LRT microbiota of patients with P. aeruginosa VAP.This study had several limitations. First, the sample size was limited. We could not exclude other microbial types that may exist in P. aeruginosa VAP. Importantly, we have found that in those P. aeruginosa VAP patients, the microbial composition of the LRT is diversified; and that difference was associated with the type of primary disease. We also found that the microbial composition of LRT was related to the severity of P. aeruginosa VAP. Therefore, the LRT microbiota may be a potential target for the treatment of P. aeruginosa VAP.In summary, we described the composition and diversity of the LRT microbiota in patients with Additional file 1:Figure S1. Rarefaction curve of samples collected from P. aeruginosa VAP patients and control subjects. Control, control group; PA VAP, Pseudomonas aeruginosa VAP group. Figure S2. The LRT microbiota was different between P. aeruginosa VAP patients and control subjects. The unweighted unifrac distance between the LRT microbiota of each sample was calculated and it was shown as a Pcoa two-dimensional map. Each dot represents one sample. Control, control group; PAVAP, Pseudomonas aeruginosa VAP group. Figure S3. The LRT microbiota and hierarchical clustering of P. aeruginosa VAP patients at genus level. Initial samples were analyzed. The Bray curtis distance matrix (in genus level) was used to construct a dendrogram including 36 P. aeruginosa VAP patients. Clusters were separated by black dotted line originating from the top of the dendrogram. The microbial composition at genus level was visualized as bar charts. Patient number is indicated at the left of each bar. Each bar represents 100% of the OTUs detected per patient; OTUs are color-coded according to genus. LRT lower respiratory tract; VAP, ventilator-associated pneumonia; OTUs, operational taxonomic units. Figure S4. The Shannon diversity between different time points was stable in LRT samples from P. aeruginosa VAP patients. Comparison of the Shannon diversity between day1  and day7 (A), day1 and day14 (B) were conducted by paired non-parametric test (both P > 0.05). Figure S5. The variety of the relative abundance of Pseudomonas, Lactobacillus and Bifidobacterium in acute phase and improvement phase of P. aeruginosa VAP patients. Pseudomonas (A) showed a decreased trend while Lactobacillus (B) and Bifidobacterium (C) showed an increased trend when the P. aeruginosa VAP patients obtained clinical improvement. Figure S6. The LRT microbiota of survivors of P. aeruginosa VAP patients had no significant difference compared to that of the non-survivors. Samples from day1 were collected for analysis. The unweighted unifrac distance between the survivors and non-survivors was calculated and it was shown as a Pcoa two-dimensional map. Each dot represents one sample. Table S1. Baseline characteristics of control subjects and P. aeruginosa VAP patients. Table S2. Conventional culture of endotracheal aspiration samples from P. aeruginosa VAP patients. Table S3. Antibiotic use before one week sampling of P. aeruginosa VAP patients. Table S4. Antibiotics statistics of P. aeruginosa VAP patients before sampling. Table S5. Antibiotic use during the dynamic observation period of P. aeruginosa VAP patients. Table S6. Negative correlation between genus of P. aeruginosa VAP and SOFA score. (ZIP 1314 kb)


Mycobacterium obuense is a pigmented, rapidly growing mycobacterium. Because it has been considered nonpathogenic, M. obuense is being investigated in clinical trials of cancer immunotherapy and bioremediation. We report a case of bacteremia caused by M. obuense in a patient with pneumonia, showing its potential pathogenicity. Mycobacterium obuense, an environmental pigmented mycobacterium, is mobile and easily adaptable to the environment and possesses oxygenases that enable it to degrade organic compounds and dechlorinate methoxychlor-based insecticides (M. obuense has been considered nonpathogenic. We report a case of bacteremia caused by M. obuense.Approximately 75 species of rapidly growing mycobacteria (RGM) have been isolated from soil, animals, and water  of the KDIGO classification  without replacement therapy. He reported taking metformin, amlodipine, furosemide, and iron sulfate.The patient was a farmer; had been in close contact with pigs, sheep, and cows; and reported consuming unpasteurized dairy products. He had a history of diabetes mellitus with chronic kidney disease categorized as stage G4 A3 . After a 7-day incubation period, we detected growth and observed gram-positive bacilli in the gram stain  but were unsuccessful because no species-specific probe is available. We performed amplification and sequencing of the 16S rRNA gene (498 bp) and hsp65 gene (400 bp). Pairwise sequence aligned 100% with 16S rRNA and 99.6% with hsp65 to the sequences of M. obuense strain CIP 106803. (GenBank accession no. AF547954.1).We attempted to identify the bacilli by using GenoType Mycobacterium   (3).The patient completed 2 weeks of intravenous moxifloxacin, amikacin, and clarithromycin and was discharged. He received 4 additional weeks of oral azithromycin and moxifloxacin and experienced complete resolution.M. obuense, first described as a scotochromogenic RGM species isolated primarily from soil >40 years ago in Obu, Japan, is a catalase-positive, peroxidase-negative bacillus that can degrade salicylates, forming a black product  and M. rufum (92.2% identity) (M. chubuense. Although these mycobacteria are considered nonpathogenic, they have homologous genes to mammalian cell entry that encode proteins involved in virulence and cell invasion. In addition, M. obuense contains genes involved in antimicrobial resistance, such as marA, aminoglycoside-resistance protein kinase, β-lactamases, and monooxygenases, which confer M. obuense with intrinsic rifampin resistance (Phylogenetic analysis of M. obuense correctly. Clinicians should avoid discarding RGM or misclassifying these isolates as colonizers until definitive species identification confirms etiology.Establishing etiology in this case was challenging because current nucleic acid probe assays cannot identify M. obuense has been evaluated as an adjuvant immunotherapy in phase I and II trials on patients with melanoma, pancreatic cancer, and colorectal cancer, with promising results. This treatment consists of an intradermal application of a suspension of heat-killed whole cell M. obuense (M. obuense from blood cultures of a patient with community-acquired pneumonia suggests its capacity for virulence and invasiveness in humans. Because M. obuense might become an adjuvant in cancer therapy, researchers should ensure implementation of proper, standardized inactivation protocols.The isolation of Mycobacterium obuense from a patient with bacteremic pneumonia.Results of susceptibility testing and MICs for a variety of antimicrobial drugs. Images of gram stains and agar of


Plasmodium falciparum and Plasmodium vivax infections, varying travel history, and varying disease severity. Variation in immunity in hospital settings is particularly understudied.Naturally acquired immunity to malaria across the globe varies in intensity and protective powers. Many of the studies on immunity are from hyperendemic regions of Africa. In Asia, particularly in India, there are unique opportunities for exploring and understanding malaria immunity relative to host age, co-occurrence of P. falciparum or P. vivax or both species, and with different clinical severity were applied to an established protein array system with over 1000 P. falciparum and P. vivax antigens. Differential binding of patient IgG to different antigens was measured.A US NIH ICEMR (South Asia) team examined the level of immunity in an Indian malaria patient population visiting or admitted to Goa Medical College and Hospital in Goa, India. Sera from 200 patients of different ages, in different seasons, infected with P. vivax than P. falciparum, IgG reactivity towards P. falciparum antigens was very strong and comparable to that seen in regions of the world with high P. falciparum endemicity. Of 248 seropositive P. falciparum antigens, the strongest were VAR, MSP10, HSP70, PTP5, AP2, AMA1, and SYN6. In P. vivax patients, ETRAMPs, MSPs, and ApiAP2, sexual stage antigen s16, RON3 were the strongest IgG binders. Both P. falciparum and P. vivax patients also revealed strong binding to new antigens with unknown functions. Seropositives showed antigens unique to the young  or to non-severe malaria (MSP3.8 and PHIST).Even though Goa itself has much more P. falciparum and P. vivax in a low malaria transmission region with much migration. In addition to markers of transmission, the data points to specific leads for possible protective immunity against severe disease. Several, but not all, key antigens overlap with work from different settings around the globe and from other parts of India. Together, these studies confidently help define antigens with the greatest potential chance of universal application for surveillance and possibly for disease protection, in many different parts of India and the world.Seroreactivity at a major hospital in Southwest India reveals antibody responses to The online version of this article (10.1186/s12936-019-2771-5) contains supplementary material, which is available to authorized users. Plasmodium falciparum dominates in sub-Saharan Africa and accounts for the very high prevalence rates associated with this continent. Plasmodium vivax is mostly prevalent in the American continent and Asia [Nearly half of the world’s population lives in malaria endemic zones. and Asia . Prevaleand Asia .Within India, individuals are exposed to varying levels of endemicity and many develop partial protection against infection –4. This P. falciparum and P. vivax in a cost-effective manner [NAI is mediated by acquired immune mechanisms, primarily directed against blood stages of malaria parasites. Such immunity involves circulating immunoglobulins, antibody-producing plasma cells, and memory B cells –9. Knowle manner , 11. A le manner –19. The e manner , 17, 18.P. vivax and no local P. falciparum. Yet, importantly, asymptomatic carriers revealed higher reactivity to 19 P. falciparum antigens compared to symptomatic patients and they included PHISTc, MSP11, RH2b, RON2, SERA4, two VARs, RESA, ETRAMP5, MSP2, SEMP1, HSP70-x, GEXP18, GCN5, LSA3, ETRAMP14, MSP4, ring infected erythrocyte surface antigen and a conserved protein.Immunity against malaria in India is less well understood compared to in Africa, SE Asia, or neighbouring islands. The effort above with the first protein arrays included a promising start to genome-scale probing of malaria immunity in India. An early comparative analysis of the seroreactivity profile from three sites, Raurkela, Nadiad, and Chennai in India indicated varied immune responses across the epidemiologically diverse endemic settings . RaurkelP. falciparum and P. vivax. Most importantly, Goa Medical College and Hospital offers free health care to all and attracts a very wide spectrum of malaria patients. Many participants involved in the present study were immigrants from different parts of India, with different genetic backgrounds, and different histories of prior exposure to malaria [To assess the generality of the early findings from the studies in India and the world, and to understand and measure the level of exposure in low transmission areas, protein array-based immune surveillance was performed in a low epidemiological setting in Southwest India (Goa). While Goa is considered a non-malaria endemic region of India, it has significant local seasonal transmission of both  malaria . These cP. falciparum and P. vivax antigens of symptomatic malaria patients in Goa and (ii) the relationship of IgG antibody levels of this population in Goa to multiple factors that could contribute to differential immunity. These include their age, disease severity status, and season of sample collection. Such insights can potentially identify markers of exposure and disease severity that would be useful for malaria surveillance. The present results compare favourably with reactivity from non-urban areas of India and to the other ICEMR studies from around the world. This suggests that such antigens may have value as general sero-surveillance tools in very different epidemiological settings.In summary, the present study reveals, (i) seroreactivity profiles against both The present study was a part of the US National Institutes of Health-sponsored Program Project entitled Malaria Evolution in South Asia-International Center of Excellence for Malaria Research (MESA-ICEMR). Samples were collected at Goa Medical College and Hospital (GMC), Bambolim, Goa, India. The sample collection process was approved by the Institutional Review Boards (IRB) of the Division of Microbiology and Infectious Diseases (DMID) at the US National Institute of Allergy and Infectious Diseases (NIAID), GMC, and the University of Washington (UW). Detailed ethics statements have been previously reported . After sP. falciparum, 100 P. vivax, and 4 mixed infections) were analysed by hybridization to protein microarrays. Parasite density (parasites/µL) was determined by microscopy for 199/200 individuals. Patients were further classified based on age , hospitalization status, signs/symptoms of disease severity and time of sample collection , year-round for 4 years 2012–2015). Written informed consent was obtained from all the volunteers during enrolment. A detailed description of the study site, enrolment and sample processing protocols has been published elsewhere [12–2015. P. falciparum and P. vivax proteins was studied using a protein microarray  displaying 515 P. vivax and 500 P. falciparum proteins . At ADI, the proteins were expressed as polypeptide fragments and printed from cell-free in vitro transcription translation (IVTT) reactions by the manufacturing team. A few large proteins such as dynein beta chains, rhoptry neck proteins, NOT family proteins, conserved Plasmodium proteins, reticulocyte binding proteins, erythrocyte membrane proteins (PfEMP1) and a few transcription factors were printed as overlapping peptides or exon fragments. The antigens were down-selected from larger arrays (Pf4528 and Pv4441) based on the seroreactivity of different patient populations from other malaria endemic regions [http://www.plasmodb.org).Twenty microlitre (µL) aliquots of sera from 200 patients were transported to IITB in cryovials labelled with unique numerical codes, one for each patient. Antibody reactivity to  regions . AntigenHybridizations were carried out at the Indian Institute of Technology Bombay (IITB). Each slide was composed of eight microarray pads. Multiple slides were used in each large experiment. Highly reactive sera from a single patient was used on one pad of every slide as a positive control. The rest of the microarray pads were used to probe sera from different patients. To track possible variations during array printing on individual pads on slides, batches of slides, or even assay performance during each experiment, select patient sera were probed across all runs and their signal intensities were compared for quality control and reproducibility. To account for non-specific reactivity of the IVTT expression system, each array pad contained 24 “No DNA-control” reaction spots, lacking a DNA template in the plasmid vector . Normalization was performed as previously described . Median P. falciparum infected and P. vivax infected based on the Plasmodium species detected by microscopy. Analysis of these two populations would provide information on immune status of a patient with respect to responses to infection by each of the two human malaria parasite species seen. In addition, both study groups  were sub-divided into individual age distributions as shown in Fig. P. falciparum and P. vivax groups, respectively, Fig. In order to correlate immune responses with aspects of the local clinical manifestations of malaria, the participant data were grouped into two major classes; P. falciparum and 66 P. vivax) who were all out-patients. It is important to reiterate that not all hospitalized individuals exhibited severe symptoms.Patients were also sub-divided according to the severity of disease, as immune patterns that correlate with low severity might, in principle, give an indication of disease protecting immunity. According to the clinical information that was documented for each patient, 90/200 patients were hospitalized (in-patients), while 110 participants did not require hospitalization (out-patients) [P. falciparum malaria, patients with parasite densities > 10% have severe infection, while there is no parasite density threshold for severe P. vivax malaria. A majority of the patients in this study presented with moderate to low parasite densities  except two with parasite densities of 192,153  and 147,809 parasites/µL , estimated by microscopy. Importantly, there was no correlation between parasite density, hospitalization status and severity, thus making study of immune status important , while a separate group of 68 patients were admitted during the dry season (December to April). In order to eliminate sampling bias, the sample details were kept blind to those handling sera hybridizations or microarray slides until after data analysis.P. falciparum and 100 P. vivax positive patients were probed on a protein array having 500 P. falciparum and 515 P. vivax polypeptides . The magnitude and breadth of the Ab response, determined by the number of features recognized, was greater in case of P. falciparum than P. vivax malaria  were found to be seroreactive in 30–40 patients, while only a few antigens  were seroreactive in more than 80 P. falciparum and 90 P. vivax patients. Two P. falciparum antigens and 8 P. vivax antigens showed lowest reactivity among all other seroreactive antigens. These were reactive in 10–20 P. falciparum and 20–30 P. vivax patients, respectively.As detailed in "ria Fig. a. Two huP. falciparum antigens while one P. vivax patient had high reactivity to 265 out of 515 P. vivax antigens. Despite the large breadth of antibody responses, these patients were not excluded from the study. The gene IDs of the top antigens showing seroreactivity in more than 60% of the patients in both groups are listed in Table The antibody breadth  is shown as a box and whisker plot in Fig. P. falciparum seroreactive antigens indicated that a majority of the seroreactive P. falciparum antigens were conserved Plasmodium proteins (17.9%) , transcription and translation factors (3.5%), proteins involved in ATP-binding (3.9%), ion-binding (3.5%) and proteins involved in ubiquitination (1.5%) also elicited high IgG responses despite their intracellular locations in the nucleus and the cytosol. Antigens with features and functions similar to P. falciparum antigens were also seroreactive in P. vivax patients, along with hypothetical proteins that constituted the major group (33.3%).An analysis of the features/functions of the 248 P. falciparum and P. vivax antigens but all of the patterns are not easy to understand. One hundred and seventeen seroreactive P. falciparum antigens (Sr) from P. falciparum patients were also recognized by P. vivax patients , including non-human infecting parasites. However, 29 P. falciparum antigens did not have orthologs in P. vivax-Sal1 strain. On the contrary, 98 P. falciparum antigens which were reactive only in P. falciparum group (not crossreactive in P. vivax patients) had orthologs in P. vivax . These antigens could represent markers of cumulative exposure. Alternatively, certain 88 P. falciparum antigens were found to be seroreactive in all age-groups; they included transmembrane emp24 domain-containing protein, ApiAP2, several conserved Plasmodium proteins, MSPs, PF70, members of ETRAMP family, liver stage antigens, rhoptry neck proteins, SYN6, PIESP2, members of the PfEMP1 family, enzymes and proteins involved in translation and transcription, rifin and a few cytoskeleton proteins like dynein heavy chain. In case of P. vivax, 41 antigens were seroreactive in all age groups, most of which were hypothetical proteins and merozoite surface proteins, while other hypothetical proteins and other known proteins such as MSPs and HSPs were exclusively seroreactive in older adults . The sample size differed in each age group as follows: The number of falciparum patients were 5, 36, 36 and 10, while vivax patients were 9, 30, 34 and 27 patients in age groups A, B, C and D, respectively. Clearly, the study involved some groups with very small numbers, especially children aged 1 to 5 years. Hence, a comparison of seroreactivity between groups was not performed. Moreover, there was no correlation between the number of seroreactive proteins and patient age, when scatter plots depicted antibody breadth for individual patients  and P. vivax (p = 0.01) study groups, respectively. SAM analysis using Log2 FOC values revealed a list of five candidate P. falciparum antigens with differential seroreactivity among the two P. falciparum sub-groups sensitive to hospitalization . In case of P. vivax, a hypothetical protein and ubiquitin-like protein . It is possible that some of these antigens could offer protection from severe disease, although many more diverse clinical events will have to be characterized to fully test this hypothesis. Several conserved Plasmodium proteins, MSP 3.8, PHIST a, NOT family protein and a few others were found to be seroreactive only in non-severe patients  and non-severe (NS), non-severe falciparum patients displayed significantly higher Ab levels compared to severe falciparum patients . Most of these antigens were seroreactive in both seasons, but a few P. falciparum antigens , had higher Ab responses in select patients during the dry season,  were seroreactive in both wet and dry seasons and 31% were seroreactive only during peak wet season.Goa is characterized by seasonal malaria transmission, yet many clinical cases are also reported during the dry season. To study the dynamics of IgG Ab responses accompanying changes in intensity of malaria incidence and transmission, patients were segregated based on time of the year during sample collection. A marked increase in seroreactivity was observed during the dry season  in on, Fig. . The reaPlasmodium species. To an extent, it is also specific to different life stages of the parasite [Naturally acquired immunity (NIA) depends on previous infection and builds with every pathogen encounter . It is gparasite . To diseparasite . In prinPlasmodium species and vectors, (c) changing climatic patterns that may have an impact on malaria transmission and (d) emergence of anti-malarial drug resistance [P. vivax and 373 P. falciparum antigens. A significant difference in the levels of seroreactivity and breadth of antibody response was observed across the three study sites [P. falciparum, but not P. vivax. These findings suggest that indicators of exposure may vary across diverse endemic settings and there may be a need for establishing pan-specific general antigens that may be of use for surveillance campaigns in India [India contributes significantly to the overall global malaria burden. It is important for the study of malaria immunity due to (a) the diverse eco-epidemiological profiles across the country, (b) co-existence of multiple sistance . India hsistance . As a pady sites . Additioin India . Of partin India .P. vivax (77%), while 21% had P. falciparum malaria. Of these 1000 confirmed malaria cases at GMC, 96 falciparum and 100 vivax patient sera were used in the present serology study.The current study focuses on patients from Goa, a small prosperous coastal state in western India where the epidemiology of malaria includes the presence of migrant workers and transient communities. Goa is classified as a non-endemic region with low transmission intensities where parasitaemia are usually low (< 1%). Using the shared ICEMR protein array platform, the present study describes the profiles of NAI in malaria-positive patients residing in Goa. The primary goal was to explore species-specific and pan-species serological correlates of exposure. Further, differential seroreactivity was identified in severe versus non-severe malaria patients to help identify antigens likely to offer protection from severe manifestations. This cross-sectional serological study was performed using a small subset of the total number of malaria-positive individuals referred to the MESA-ICEMR study team . Of overP. falciparum patients showed a remarkably stronger immune response compared to P. vivax. The reasons for this could be many, the major one being the significantly different biology of the two parasites [P. falciparum 3D7, P. vivax Sal1), allowing the introduction of several important biases. Genetically, P. vivax is extremely diverse and the protein array is made based on a South American Strain. This may explain the lower general reactivity seen in Goa. Overall, 248 P. falciparum and 73 P. vivax seroreactive proteins were identified. Interestingly, most of the top seroreactive antigens of P. falciparum , a hypothetical protein (PVX_118705) and MSP10 could discriminate between naïve and semi-immune individuals [P. vivax recently published using a protein array with 1936 genes encoding P. vivax proteins identified 151 highly seroreactive P. vivax antigens, a few of which  were also recognized in our study [Consistent with previous ICEMR protein array data from around the world, arasites . Secondllatforms , 28, 29.latforms . Similarur study . In concP. falciparum and P. vivax. Individual proteins, or even different peptides in a protein, can elicit varying levels of IgG reactivity. Protein-specific features such as subcellular location, protein abundance, degree of polymorphism and presence of human orthologs may influence the magnitude of antibody responses during natural malaria infections [Importantly, the malaria patients in Goa showed differential levels of IgG reactivity to different polypeptides from the same protein, in both fections . OverallP. falciparum and 73 P. vivax seroreactive antigens identified were understandably either proteins normally exported to the erythrocyte cell surface during the parasite life cycle or present on the merozoite surface. Yet, some nuclear, cytoskeletal and cytoplasmic proteins also triggered high IgG binding in the majority of the infected population. A 1990s study from India, provided early evidence for immune reaction against intracellular proteins [P. falciparum growth in culture and correlated with protection in mice [A majority of the 248 proteins . A diffe in mice . Most imP. falciparum and P. vivax proteins printed on the chip, but it is not that simple. Of 123 P. falciparum antigens exclusively seroreactive in the P. falciparum study group, 93 were found to share orthologs in P. vivax . The biggest benefit is the ease with which antibodies can be stored using dried blood spots, making sample collection and storage relatively simple .To build on present advances, future follow-up studies may improve in four areas: first, more effort is required to enroll children and adolescent patients. Second, longitudinal serology studies will track exposure to malaria, protective immunity, and waning of such immunity after an infection is resolved. Third, more detailed information will be sought on the origin of migrant workers and prior history of infection in their place of origin. Finally, the array platform itself will evolve to test sera against some shorter exon fragments on the array, some full-length proteins, all accepted blood-stage antigens, and antigens from non-erythrocytic stages of malaria parasites to assess their contribution to local transmission.Malaria exposure is largely underestimated in several countries due to limitations in microscopy-based diagnosis. The present study provides insights into antibody-based immune responses against parasite antigens in low transmission settings in India, using data collected over 4 years during both peak and dry malaria seasons. The work complements global efforts by ICEMR programs to compare variations in malaria across the globe, including possible variations in immunity to malaria. The overall immunity patterns in Southwestern India mirror what has been seen in other parts of India and the world. The contrasting immunity in individuals with varying disease presentations at a tertiary care hospital offer powerful new avenues to achieve protective immunity against severe disease.Additional file 1. Patient data for parasite density, hospitalization status and severity.Additional file 2. Seroreactive P. falciparum and P. vivax antigens.Additional file 3. Functional classification of seroreactive P. falciparum and P. vivax antigens.Additional file 4. Details of cross-reactive P. falciparum and P. vivax antigens.Additional file 5. Data for orthology and synteny.Additional file 6. Age-dependent seroreactivity of P. falciparum and P. vivax antigens.Additional file 7. Scatter plots for antibody breadth in patients.