Pathologic Complete Response to Neoadjuvant Chemotherapy and Pembrolizumab in Postpartum High-Risk Basal-Type Breast Cancer.

Neoadjuvant chemoimmunotherapy with pembrolizumab now defines the standard of care for early high-risk triple-negative breast cancer (TNBC). However, the role of pembrolizumab in neoadjuvant therapy (NAT) for estrogen receptor-positive (ER+) breast cancer remains uncertain. A 39-year-old G2P2 female discovered a palpable mass in the right breast while breastfeeding her 7-month-old child, leading to the diagnosis of a high-grade ER+ (80% moderate staining), human epidermal growth factor receptor 2-negative (ErbB2-) invasive ductal carcinoma with axillary nodal involvement. Gene expression profiling with the MammaPrint 70-gene signature and BluePrint 80-gene signature revealed a tumor with high-risk, basal-type biology. The multidisciplinary breast cancer team recommended NAT with pembrolizumab, carboplatin, paclitaxel, doxorubicin, and cyclophosphamide. Within six weeks, the patient exhibited a remarkable response, with no palpable mass or lymph node, and post-treatment examinations confirmed a complete clinical and radiologic response. The patient underwent lumpectomy and sentinel lymph node biopsy, revealing a pathological complete response with minimal ductal carcinoma in situ and negative axillary nodes. Adjuvant radiation therapy was administered, and the patient completed adjuvant pembrolizumab, currently showing no evidence of recurrence. This case underscores the potential benefits of neoadjuvant chemoimmunotherapy for patients with ER+ErbB2- high-risk, basal-type breast cancer. The use of immunotherapy in patients with pregnancy-associated breast cancer remains to be further investigated.

Review of genotyping methods for Yersinia pestis in Madagascar.

BACKGROUND: Plague, a zoonotic disease caused by Yersinia pestis, was responsible for 3 historical human pandemics that killed millions of people. It remains endemic in rodent populations in Africa, Asia, North America, and South America but human plague is rare in most of these locations. However, human plague is still highly prevalent in Madagascar, which typically records a significant part of all annual global cases. This has afforded an opportunity to study contemporary human plague in detail using various typing methods for Y. pestis. AIM: This review aims to summarize the methods that have been used to type Y. pestis in Madagascar along with the major discoveries that have been made using these approaches. METHODS: Pubmed and Google Scholar were used to search for the keywords: "typing Yersinia pestis Madagascar," "evolution Yersinia pestis Madagascar," and "diversity Yersinia pestis Madagascar." Eleven publications were relevant to our topic and further information was retrieved from references cited in those publications. RESULTS: The history of Y. pestis typing in Madagascar can be divided in 2 periods: the pre-genomics and genomics eras. During the pre-genomics era, ribotyping, direct observation of plasmid content and plasmid restriction fragment length polymorphisms (RFLP) were employed but only revealed a limited amount of diversity among Malagasy Y. pestis strains. Extensive diversity only started to be revealed in the genomics era with the use of clustered regularly interspaced palindromic repeats (CRISPR), multiple-locus variable number tandem repeats (VNTR) analysis (MLVA), and single-nucleotide polymorphisms (SNPs) discovered from whole genome sequences. These higher-resolution genotyping methods have made it possible to highlight the distribution and persistence of genotypes in the different plague foci of Madagascar (Mahajanga and the Central and Northern Highlands) by genotyping strains from the same locations across years, to detect transfers between foci, to date the emergence of genotypes, and even to document the transmission of antimicrobial resistant (AMR) strains during a pneumonic plague outbreak. Despite these discoveries, there still remain topics that deserve to be explored, such as the contribution of horizontal gene transfer to the evolution of Malagasy Y. pestis strains and the evolutionary history of Y. pestis in Madagascar. CONCLUSIONS: Genotyping of Y. pestis has yielded important insights on plague in Madagascar, particularly since the advent of whole-genome sequencing (WGS). These include a better understanding of plague persistence in the environment, antimicrobial AMR and multi-drug resistance in Y. pestis, and the person-to-person spread of pneumonic plague. Considering that human plague is still a significant public health threat in Madagascar, these insights can be useful for controlling and preventing human plague in Madagascar and elsewhere, and also are relevant for understanding the historical pandemics and the possible use of Y. pestis as a biological weapon.

Host population structure and rare dispersal events drive leptospirosis transmission patterns among Rattus norvegicus in Boston, Massachusetts, US.

Leptospirosis (caused by pathogenic bacteria in the genus Leptospira ) is prevalent worldwide but more common in tropical and subtropical regions. Transmission can occur following direct exposure to infected urine from reservoir hosts, such as rats, or a urine-contaminated environment, which then can serve as an infection source for additional rats and other mammals, including humans. The brown rat, Rattus norvegicus , is an important reservoir of leptospirosis in urban settings. We investigated leptospirosis among brown rats in Boston, Massachusetts and hypothesized that rat dispersal in this urban setting influences the movement, persistence, and diversity of Leptospira . We analyzed DNA from 328 rat kidney samples collected from 17 sites in Boston over a seven-year period (2016-2022); 59 rats representing 12 of 17 sites were positive for Leptospira . We used 21 neutral microsatellite loci to genotype 311 rats and utilized the resulting data to investigate genetic connectivity among sampling sites. We generated whole genome sequences for 28 Leptospira isolates obtained from frozen and fresh tissue from some of the 59 Leptospira -positive rat kidneys. When isolates were not obtained, we attempted Leptospira genomic DNA capture and enrichment, which yielded 14 additional Leptospira genomes from rats. We also generated an enriched Leptospira genome from a 2018 human case in Boston. We found evidence of high genetic structure and limited dispersal among rat populations that is likely influenced by major roads and/or other unknown dispersal barriers, resulting in distinct rat population groups within the city; at certain sites these groups persisted for multiple years. We identified multiple distinct phylogenetic clades of L. interrogans among rats, with specific clades tightly linked to distinct rat populations. This pattern suggests L. interrogans persists in local rat populations and movement of leptospirosis in this urban rat community is driven by rat dispersal. Finally, our genomic analyses of the 2018 human leptospirosis case in Boston suggests a link to rats as the source. These findings will be useful for guiding rat control and human leptospirosis mitigation efforts in this and other urban settings.

Detection of Leptospira kirschneri in a short-beaked common dolphin (Delphinus delphis delphis) stranded off the coast of southern California, USA.

BACKGROUND: Pathogenic Leptospira species are globally important zoonotic pathogens capable of infecting a wide range of host species. In marine mammals, reports of Leptospira have predominantly been in pinnipeds, with isolated reports of infections in cetaceans. CASE PRESENTATION: On 28 June 2021, a 150.5 cm long female, short-beaked common dolphin (Delphinus delphis delphis) stranded alive on the coast of southern California and subsequently died. Gross necropsy revealed multifocal cortical pallor within the reniculi of the kidney, and lymphoplasmacytic tubulointerstitial nephritis was observed histologically. Immunohistochemistry confirmed Leptospira infection, and PCR followed by lfb1 gene amplicon sequencing suggested that the infecting organism was L.kirschneri. Leptospira DNA capture and enrichment allowed for whole-genome sequencing to be conducted. Phylogenetic analyses confirmed the causative agent was a previously undescribed, divergent lineage of L.kirschneri. CONCLUSIONS: We report the first detection of pathogenic Leptospira in a short-beaked common dolphin, and the first detection in any cetacean in the northeastern Pacific Ocean. Renal lesions were consistent with leptospirosis in other host species, including marine mammals, and were the most significant lesions detected overall, suggesting leptospirosis as the likely cause of death. We identified the cause of the infection as L.kirschneri, a species detected only once before in a marine mammal - a northern elephant seal (Mirounga angustirostris) of the northeastern Pacific. These findings raise questions about the mechanism of transmission, given the obligate marine lifestyle of cetaceans (in contrast to pinnipeds, which spend time on land) and the commonly accepted view that Leptospira are quickly killed by salt water. They also raise important questions regarding the source of infection, and whether it arose from transmission among marine mammals or from terrestrial-to-marine spillover. Moving forward, surveillance and sampling must be expanded to better understand the extent to which Leptospira infections occur in the marine ecosystem and possible epidemiological linkages between and among marine and terrestrial host species. Generating Leptospira genomes from different host species will yield crucial information about possible transmission links, and our study highlights the power of new techniques such as DNA enrichment to illuminate the complex ecology of this important zoonotic pathogen.

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

Metabolite profiling of foslevodopa/foscarbidopa in plasma of healthy human participants by LC-HRMS indicates no major differences compared to administration of levodopa/carbidopa intestinal gel.

Analysis was conducted to compare levodopa/carbidopa pharmacokinetics and drug-related material in plasma of healthy participants after receiving a continuous infusion of Levodopa/Carbidopa Intestinal Gel (LCIG) to a continuous subcutaneous infusion of foslevodopa/foscarbidopa. Study samples were from a randomized, open-label, 2-period crossover study in 20 healthy participants. Participants received either 24-h foslevodopa/foscarbidopa SC infusion to the abdomen or LCIG delivered for 24 h to the jejunum through a nasogastric tube with jejunal extension. Serial blood samples were collected for PK. Comparability of the LD PK parameters between the two treatment regimens was determined. Selected plasma samples were pooled per treatment group and per time point for metabolite profiling. LC-MSn was performed using high-resolution mass spectrometry to identify drug-related material across the dosing regimens and time points. The LD PK parameter central values and 90% confidence intervals following the foslevodopa/foscarbidopa subcutaneous infusion were between 0.8 and 1.25 relative to the LCIG infusion. With LCIG administration, LD, CD, 3-OMD, DHPA, DOPAC, and vanillacetic acid were identified in plasma at early and late time points (0.75 and 24 h); the metabolic profile after administration of foslevodopa/foscarbidopa demonstrated the same drug-related compounds with the exception of the administered foslevodopa. 3-OMD and vanillacetic acid levels increased over time in both treatment regimens. Relative quantification of LC-MS peak areas showed no major differences in the metabolite profiles. These results indicate that neither the addition of monophosphate prodrug moieties nor SC administration affects the circulating metabolite profile of foslevodopa/foscarbidopa compared to LCIG.

A mutation associated with resistance to synthetic pyrethroids is widespread in US populations of the tropical lineage of Rhipicephalus sanguineus s.l.

The brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.), is an important vector for Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever. Current public health prevention and control efforts to protect people involve preventing tick infestations on domestic animals and in and around houses. Primary prevention tools rely on acaricides, often synthetic pyrethroids (SPs); resistance to this chemical class is widespread in ticks and other arthropods. Rhipicephalus sanguineus s.l. is a complex that likely contains multiple unique species and although the distribution of this complex is global, there are differences in morphology, ecology, and perhaps vector competence among these major lineages. Two major lineages within Rh. sanguineus s.l., commonly referred to as temperate and tropical, have been documented from multiple locations in North America, but are thought to occupy different ecological niches. To evaluate potential acaricide resistance and better define the distributions of the tropical and temperate lineages throughout the US and in northern Mexico, we employed a highly multiplexed amplicon sequencing approach to characterize sequence diversity at: 1) three loci within the voltage-gated sodium channel (VGSC) gene, which contains numerous genetic mutations associated with resistance to SPs; 2) a region of the gamma-aminobutyric acid-gated chloride channel gene (GABA-Cl) containing several mutations associated with dieldrin/fipronil resistance in other species; and 3) three mitochondrial genes (COI, 12S, and 16S). We utilized a geographically diverse set of Rh sanguineus s.l. collected from domestic pets in the US in 2013 and a smaller set of ticks collected from canines in Baja California, Mexico in 2021. We determined that a single nucleotide polymorphism (T2134C) in domain III segment 6 of the VGSC, which has previously been associated with SP resistance in Rh. sanguineus s.l., was widespread and abundant in tropical lineage ticks (>50 %) but absent from the temperate lineage, suggesting that resistance to SPs may be common in the tropical lineage. We found evidence of multiple copies of GABA-Cl in ticks from both lineages, with some copies containing mutations associated with fipronil resistance in other species, but the effects of these patterns on fipronil resistance in Rh. sanguineus s.l. are currently unknown. The tropical lineage was abundant and geographically widespread, accounting for 79 % of analyzed ticks and present at 13/14 collection sites. The temperate and tropical lineages co-occurred in four US states, and as far north as New York. None of the ticks we examined were positive for Rickettsia rickettsii or Rickettsia massiliae.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

Theories of Relativistic Dissipative Fluid Dynamics.

Relativistic dissipative fluid dynamics finds widespread applications in high-energy nuclear physics and astrophysics. However, formulating a causal and stable theory of relativistic dissipative fluid dynamics is far from trivial; efforts to accomplish this reach back more than 50 years. In this review, we give an overview of the field and attempt a comparative assessment of (at least most of) the theories for relativistic dissipative fluid dynamics proposed until today and used in applications.

Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017.

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

Mucin-producing urothelial-type adenocarcinoma of the prostate with sarcomatoid features and novel molecular phenotype.

Introduction: Mucin-producing urothelial-type adenocarcinoma of the prostate is a rare tumor that may not elevate serum prostate-specific antigen, creating significant diagnostic and monitoring challenges. We evaluate our case in detail and review prior studies to demonstrate that the pathologic and molecular features of this tumor are distinct from conventional prostate adenocarcinoma. Case presentation: Our patient had a remote history of radiation-treated conventional prostate adenocarcinoma and presented many years later with an abscess-like prostate mass leading to urinary obstruction and hematuria. Biopsy revealed mucin-producing urothelial-type adenocarcinoma of the prostate with concurrent sarcomatoid features. Molecular studies showed a unique phenotype involving alterations in the KRAS, PTEN, RAD21, and TP53 genes. Conclusions: To our knowledge, this is the first report that describes sarcomatoid features and molecular mutations in mucin-producing urothelial-type adenocarcinoma of the prostate.

High-resolution repeat topography of drifting ice floes in the Arctic Ocean from terrestrial laser scanning.

Snow and ice topography impact and are impacted by fluxes of mass, energy, and momentum in Arctic sea ice. We measured the topography on approximately a 0.5 km2 drifting parcel of Arctic sea ice on 42 separate days from 18 October 2019 to 9 May 2020 via Terrestrial Laser Scanning (TLS). These data are aligned into an ice-fixed, lagrangian reference frame such that topographic changes (e.g., snow accumulation) can be observed for time periods of up to six months. Using in-situ measurements, we have validated the vertical accuracy of the alignment to ± 0.011 m. This data collection and processing workflow is the culmination of several prior measurement campaigns and may be generally applied for repeat TLS measurements on drifting sea ice. We present a description of the data, a software package written to process and align these data, and the philosophy of the data processing. These data can be used to investigate snow accumulation and redistribution, ice dynamics, surface roughness, and they can provide valuable context for co-located measurements.

Recommendations on the Use of Multiple Labels in Human Mass Balance Studies.

The administration of radiolabeled drug candidates is considered the gold standard in absorption, distribution, metabolism, and excretion studies for small-molecule drugs since it allows facile and accurate quantification of parent drug, metabolites, and total drug-related material independent of the compound structure. The choice of the position of the radiolabel, typically 14C or 3H, is critical to obtain relevant information. Sometimes, a biotransformation reaction may lead to cleavage of a part of the molecule. As a result, only the radiolabeled portion can be followed, and information on the fate of the nonlabeled metabolite may be lost. Synthesis and administration of two or more radiolabeled versions of the parent drug as a mixture or in separate studies may resolve this issue but comes with additional challenges. In this paper, we address the questions that may be considered to help make the right choice whether to use a single or multiple radiolabel approach and discuss the pros and cons of different multiple-labeling strategies that can be taken as well as alternative methods that allow the nonlabeled part of the molecule to be followed. SIGNIFICANCE STATEMENT: Radiolabeled studies are the gold standard in drug metabolism research, but molecules can undergo cleavage with loss of the label. This often results in discussions around potential use of multiple labels, which seem to be occurring with increased frequency since an increasing proportion of the small-molecule drugs are tending towards larger molecular weights. This review provides insight and decision criteria in considering a multiple-label approach as well as pros and cons of different strategies that can be followed.

Metabolite Bioanalysis in Drug Development: Recommendations from the IQ Consortium Metabolite Bioanalysis Working Group.

The intent of this perspective is to share the recommendations of the International Consortium for Innovation and Quality in Pharmaceutical Development Metabolite Bioanalysis Working Group on the fit-for-purpose metabolite bioanalysis in support of drug development and registration. This report summarizes the considerations for the trigger, timing, and rigor of bioanalysis in the various assessments to address unique challenges due to metabolites, with respect to efficacy and safety, which may arise during drug development from investigational new drug (IND) enabling studies, and phase I, phase II, and phase III clinical trials to regulatory submission. The recommended approaches ensure that important drug metabolites are identified in a timely manner and properly characterized for efficient drug development.

Non-Labeled, Stable Labeled, or Radiolabelled Approaches for Provision of Intravenous Pharmacokinetics in Humans: A Discussion Piece.

A review of the use of microdoses and isotopic microtracers for clinical intravenous pharmacokinetic (i.v. PK) data provision is presented. The extent of application of the varied approaches available and the relative merits of each are highlighted with the aim of assisting practitioners in making informed decisions on the most scientifically appropriate design to adopt for any given new drug in development. It is envisaged that significant efficiencies will be realized as i.v. PK data in humans becomes more routinely available for suitable assets in early development, than has been the case prior to the last decade.

Tamoxifen retinopathy: A comprehensive review.

Tamoxifen is a selective estrogen receptor modulator used mainly for the treatment of breast cancer. Based on the case reports and studies performed to date on the retinal toxicity of tamoxifen, retinopathy appears to occur in as many as 12% of patients taking 20 mg tamoxifen a day for over 2 years. Of this 12%, as many as half develop symptomatic changes in visual acuity. Retinal changes consist primarily of crystalline deposits, cystoid macular edema, hyperreflective deposits in the inner retinal layers, and telangiectasia. Tamoxifen retinopathy is currently managed by discontinuing tamoxifen therapy as the cancer prognosis permits; however, discontinuing therapy demonstrates little to no improvement in visual acuity once visual changes have taken place. Intravitreal injections of steroids or antivascular endothelial growth factor therapy have been performed, but require further studying before conclusions can be made. Until then, optical coherence tomography screening for retinal changes should be performed every 6 months for patients who have been on tamoxifen therapy for 2 years or more. This way, patients can become aware of retinal changes, and their physicians can consider adjusting tamoxifen therapy before they risk developing changes in visual acuity.

Pathogenic Leptospira are widespread in the urban wildlife of southern California.

Leptospirosis, the most widespread zoonotic disease in the world, is broadly understudied in multi-host wildlife systems. Knowledge gaps regarding Leptospira circulation in wildlife, particularly in densely populated areas, contribute to frequent misdiagnoses in humans and domestic animals. We assessed Leptospira prevalence levels and risk factors in five target wildlife species across the greater Los Angeles region: striped skunks (Mephitis mephitis), raccoons (Procyon lotor), coyotes (Canis latrans), Virginia opossums (Didelphis virginiana), and fox squirrels (Sciurus niger). We sampled more than 960 individual animals, including over 700 from target species in the greater Los Angeles region, and an additional 266 sampled opportunistically from other California regions and species. In the five target species seroprevalences ranged from 5 to 60%, and infection prevalences ranged from 0.8 to 15.2% in all except fox squirrels (0%). Leptospira phylogenomics and patterns of serologic reactivity suggest that mainland terrestrial wildlife, particularly mesocarnivores, could be the source of repeated observed introductions of Leptospira into local marine and island ecosystems. Overall, we found evidence of widespread Leptospira exposure in wildlife across Los Angeles and surrounding regions. This indicates exposure risk for humans and domestic animals and highlights that this pathogen can circulate endemically in many wildlife species even in densely populated urban areas.

Importance of CD40/CD40 dyad in the course of infection with Trypanosoma cruzi: Impact of its inhibition.

Chagas heart disease (CHD), caused by the protozoan parasite Trypanosoma cruzi, consists of a progressive myocarditis which may lead to congestive heart failure or sudden death. Previous work from our laboratory has demonstrated that the experimental infection of mice with T. cruzi positively modulates the expression of CD40 by myocardial cells, whose ligation potentiates IFN-γ-induced IL-6 production. Herein, we investigate the role of the CD40/CD40L interaction during T. cruzi infection using a CD40-targeted peptide and evaluating parasitological, histopathological and serological parameters. To reproduce acute and chronic phases of theT. cruzi infection, we used two experimental models: Balb/c mice infected with RA strain of T. cruzi (Balb/c-RA) and C3H/HeN mice infected with Sylvio X-10/4 parasites (C3H/HeN-Sylvio), respectively. Balb/c-RA treated with CD40-tageted peptide since day 0 post infection (pi), were unable to control the acute infection dying within 23-26 days pi with marked tissue damage. In contrast, treatment of C3H/HeN-Sylvio treated with CD40-targeted peptide starting on day 30 pi resulted in amelioration of myocardial and skeletal muscle damage. Altogether, our results indicate a dual role of CD40/CD40L dyad in the control of T.cruzi infection as well as the associated pathology, depending on the timing of treatment initiation.

Knockdown resistance mutations are common and widely distributed in Xenopsylla cheopis fleas that transmit plague in Madagascar.

BACKGROUND: Plague, caused by the bacterium Yersinia pestis, remains an important disease in Madagascar, where the oriental rat flea, Xenopsylla cheopis, is a primary vector. To control fleas, synthetic pyrethroids (SPs) have been used for >20 years, resulting in resistance in many X. cheopis populations. The most common mechanisms of SP resistance are target site mutations in the voltage-gated sodium channel (VGSC) gene. METHODOLOGY/PRINCIPAL FINDINGS: We obtained 25 collections of X. cheopis from 22 locations across Madagascar and performed phenotypic tests to determine resistance to deltamethrin, permethrin, and/or dichlorodiphenyltrichloroethane (DDT). Most populations were resistant to all these insecticides. We sequenced a 535 bp segment of the VGSC gene and identified two different mutations encoding distinct substitutions at amino acid position 1014, which is associated with knockdown resistance (kdr) to SPs in insects. Kdr mutation L1014F occurred in all 25 collections; a rarer mutation, L1014H, was found in 12 collections. There was a significant positive relationship between the frequency of kdr alleles and the proportion of individuals surviving exposure to deltamethrin. Phylogenetic comparisons of 12 VGSC alleles in Madagascar suggested resistant alleles arose from susceptible lineages at least three times. Because genotype can reasonably predict resistance phenotype, we developed a TaqMan PCR assay for the rapid detection of kdr resistance alleles. CONCLUSIONS/SIGNIFICANCE: Our study provides new insights into VGSC mutations in Malagasy populations of X. cheopis and is the first to report a positive correlation between VGSC genotypes and SP resistance phenotypes in fleas. Widespread occurrence of these two SP resistance mutations in X. cheopis populations in Madagascar reduces the viability of these insecticides for flea control. However, the TaqMan assay described here facilitates rapid detection of kdr mutations to inform when use of these insecticides is still warranted to reduce transmission of plague.