Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2024 Update by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM).

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by experts in both adult and pediatric laboratory and clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including arboviral Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also addressed. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

Comparative reservoir competence of Peromyscus leucopus, C57BL/6J, and C3H/HeN for Borrelia burgdorferi B31.

Borrelia burgdorferi, a Lyme disease spirochete, causes a range of acute and chronic maladies in humans. However, a primary vertebrate reservoir in the United States, the white-footed deermouse Peromyscus leucopus, is reported not to have reduced fitness following infection. Although laboratory strains of Mus musculus mice have successfully been leveraged to model acute human Lyme disease, the ability of these rodents to model B. burgdorferi-P. leucopus interactions remains understudied. Here, we compared infection of P. leucopus with B. burgdorferi B31 with infection of the traditional B. burgdorferi murine models-C57BL/6J and C3H/HeN Mus musculus, which develop signs of inflammation akin to human disease. We find that B. burgdorferi was able to reach much higher burdens (10- to 30-times higher) in multiple M. musculus skin sites and that the overall dynamics of infection differed between the two rodent species. We also found that P. leucopus remained transmissive to larval Ixodes scapularis for a far shorter period than either M. musculus strain. In line with these observations, we found that P. leucopus does launch a modest but sustained inflammatory response against B. burgdorferi in the skin, which we hypothesize leads to reduced bacterial viability and rodent-to-tick transmission in these hosts. Similarly, we also observe evidence of inflammation in infected P. leucopus hearts. These observations provide new insight into reservoir species and the B. burgdorferi enzootic cycle.IMPORTANCEA Lyme disease-causing bacteria, Borrelia burgdorferi, must alternate between infecting a vertebrate host-usually rodents or birds-and ticks. In order to be successful in that endeavor, the bacteria must avoid being killed by the vertebrate host before it can infect a new larval tick. In this work, we examine how B. burgdorferi and one of its primary vertebrate reservoirs, Peromyscus leucopus, interact during an experimental infection. We find that B. burgdorferi appears to colonize its natural host less successfully than conventional laboratory mouse models, which aligns with a sustained seemingly anti-bacterial response by P. leucopus against the microbe. These data enhance our understanding of P. leucopus host-pathogen interactions and could potentially serve as a foundation to uncover ways to disrupt the spread of B. burgdorferi in nature.

Host Contributions to the Force of Borrelia burgdorferi and Babesia microti Transmission Differ at Edges of and within a Small Habitat Patch.

In the northeastern United States, the emergence of Lyme disease has been associated, in part, with the increase of small forest patches. Such disturbed habitat is exploited by generalist species, such as white-footed mice, which are considered the host with the greatest reservoir capacity for the agents of Lyme disease (Borrelia burgdorferi sensu stricto) and human babesiosis (Babesia microti). Spatial risk analyses have identified edge habitat as particularly risky. Using a retrotransposon-based quantitative PCR assay for host bloodmeal remnant identification, we directly measured whether the hosts upon which vector ticks fed differed at the edge or within the contiguous small habitat patch. Questing nymphal deer ticks, Ixodes dammini, the northern clade of Ixodes scapularis, were collected from either the edge or within a thicket on Nantucket Island over 3 transmission seasons and tested for evidence of infection as well as bloodmeal hosts. Tick bloodmeal hosts significantly differed by site as well as by year. Mice and deer were identified most often (49.9%), but shrews, rabbits, and birds were also common. Ticks from the edge fed on a greater diversity of hosts than those from the thicket. Surprisingly, mice were not strongly associated with either infection at either sampling site (odds ratio [OR] < 2 for all). Although shrews were not the most common host utilized by ticks, they were highly associated with both infections at both sites (OR = 4.5 and 11.0 for B. burgdorferi and 7.9 and 19.0 for B. microti at the edge and thicket, respectively). We conclude that reservoir hosts may differ in their contributions to infecting ticks between edge and contiguous vegetated patches. IMPORTANCE Habitat fragmentation is thought to be a main factor in the emergence of Lyme disease and other deer tick-transmitted infections. The patchwork of forest and edges promotes altered biodiversity, favoring the abundance of generalist rodents, such as white footed mice, heretofore considered a key tick and reservoir host in the northeastern United States. We used tick bloodmeal analyses to directly identify the hosts from which nymphal deer ticks became infected. We demonstrate that there is considerable microfocality in host contributions to the cohort of infected ticks and that shrews, although they fed fewer ticks than mice, disproportionately influenced the force of pathogen transmission in our site. The venue of transmission of certain deer tick-transmitted agents may comprise a habitat scale of 10 m or fewer and depend on alternative small mammal hosts such as shrews.

Limited Capacity of Deer To Serve as Zooprophylactic Hosts for Borrelia burgdorferi in the Northeastern United States.

Because deer are considered to be incompetent reservoirs of the agent of Lyme disease (Borrelia burgdorferi sensu stricto) in the northeastern United States, they may serve as zooprophylactic or "dilution" hosts if larvae of the deer tick vector (Ixodes dammini, "northern" clade of Ixodes scapularis) frequently feed on them. To determine whether host-seeking nymphal deer ticks commonly feed on deer as larvae, we used a real-time PCR host bloodmeal remnant identification assay to identify the host on which these ticks had fed. Nymphal lone star ticks (Amblyomma americanum) were collected simultaneously in our sites and provided an index of the availability of deer in these sites. At 3 of the 4 sites, Ixodes nymphs had fed as larvae on a variety of hosts, including mice, birds, and shrews, but rarely on deer (<6% for all sites); in contrast, lone star tick nymphs had commonly fed on deer (31 to 78%). Deer were common larval hosts for Ixodes ticks (39% of bloodmeals) in only one site. The prevalence of B. burgdorferi in host-seeking nymphal deer ticks was associated with mouse-fed ticks (P = 0.007), but there was no association with deer-fed ticks (P = 0.5). The diversity and prevalence of hosts that were identified differed between deer ticks and lone star ticks that were collected simultaneously, demonstrating that there is no confounding of host bloodmeal identification by contaminating environmental DNA (eDNA). We conclude that deer were not common hosts for larval deer ticks, thus limiting their zooprophylactic role in our sites. IMPORTANCE Because deer are incompetent reservoirs for B. burgdorferi, their presence may modulate the force of enzootic transmission by serving as zooprophylactic or "dilution" hosts. Such an effect would depend on the extent to which subadult deer ticks feed on other hosts. We used bloodmeal analysis on nymphal deer ticks to identify the host upon which larvae had fed. We found that lone star ticks collected at the same time as deer ticks commonly fed on deer, but deer ticks did not. We conclude that deer are not a preferred host for larval deer ticks and, thus, are not necessarily zooprophylactic.

Powassan Virus Encephalitis Following Brief Attachment of Connecticut Deer Ticks.

BACKGROUND: Powassan virus (POWV) is a tick-transmitted pathogen that may cause severe encephalitis; experimentally, it can be transmitted within just 15 minutes following a tick bite. The deer tick virus subtype of POWV (DTV) is transmitted by the deer tick and is the likely cause of the increase in the number of POWV cases reported in the United States. However, DTV has only been definitively documented in 6 patients by molecular analysis of the virus. METHODS: Two patients from Connecticut with encephalitis, who had a recent deer tick bite, were evaluated by the relevant serologic tests to determine if they had been infected with POWV. Evaluation also included molecular testing of an adult deer tick that had been removed from one of the patients. RESULTS: We documented neuroinvasive POWV infection in 2 children from Connecticut. Based on the results of testing the tick removed from case 2, this patient was infected by DTV, representing the 7th reported case and the first documented case of DTV infection in a child. Of note, the duration of the tick bites in both cases was very short. CONCLUSIONS: We provide the first clinical and epidemiologic evidence that POWV/DTV can be rapidly transmitted to a human host, that is, within hours of tick attachment, which is distinctive when compared to other deer tick-transmitted infections such as Lyme disease.

Retrotransposon-Based Blood Meal Analysis of Nymphal Deer Ticks Demonstrates Spatiotemporal Diversity of Borrelia burgdorferi and Babesia microti Reservoirs.

Deer tick-transmitted Borrelia burgdorferisensu stricto (Lyme disease) and Babesia microti (babesiosis) increasingly burden public health across eastern North America. The white-footed mouse is considered the primary host for subadult deer ticks and the most important reservoir host for these and other disease agents. Local transmission is thought to be modulated by less reservoir-competent hosts, such as deer, diverting ticks from feeding on mice. We measured the proportion of mouse-fed or deer-fed host-seeking nymphs from 4 sites during 2 transmission seasons by blood meal remnant analysis using a new retrotransposon-based quantitative PCR (qPCR) assay. We then determined the host that was associated with the infection status of the tick. During the first year, the proportion of mouse-fed ticks ranged from 17% on mainland sites to 100% on an island, while deer-fed ticks ranged from 4% to 24%. The proportion of ticks feeding on mice and deer was greater from island sites than mainland sites (on average, 92% versus 43%). Mouse-fed ticks decreased significantly during year 2 in 3 of 4 sites (most were <20%), while deer-fed ticks increased for all sites (75% at one site). Overall, ticks were more likely to be infected when they had fed on mice (odds ratio [OR] of 2.4 and 1.6 for Borrelia and Babesia, respectively) and were less likely to be infected if they had fed on deer (OR, 0.8 and 0.4). We conclude that host utilization by deer ticks is characterized by significant spatiotemporal diversity, which may confound efficacy tests of interventions targeting reservoir hosts.IMPORTANCE White-footed mice are thought to be the most important reservoir host for the deer tick-transmitted pathogens that cause Lyme disease and human babesiosis because they are the primary host for immature ticks. Transmission would be reduced, however, if ticks feed on deer, which are not capable of infecting ticks with either pathogen. By directly measuring whether ticks had fed on either mice or deer using a new quantitative PCR (qPCR) assay to detect remnants of host DNA leftover from the larval blood meal, we demonstrate that host utilization by ticks varies significantly over time and space and that mice often feed fewer ticks than expected. This finding has implications for our understanding of the ecology of these diseases and for the efficacy of control measures.

Ecology of Francisella tularensis.

Tularemia is a Holarctic zoonosis caused by the gamma proteobacterium Francisella tularensis and is considered to be a vector-borne disease. In many regions, human risk is associated with the bites of flies, mosquitoes, or ticks. But the biology of the agent is such that risk may be fomite related, and large outbreaks can occur due to inhalation or ingestion of contaminated materials. Such well-documented human risk factors suggest a role for these risk factors in the enzootic cycle as well. Many arthropods support the growth or survival of the agent, but whether arthropods (ticks in particular) are obligately required for the perpetuation of F. tularensis remains to be demonstrated. As with most zoonoses, our knowledge of the ecology of F. tularensis has been driven with the objective of understanding human risk. In this review, we focus on the role of the arthropod in maintaining F. tularensis, particularly with respect to long-term enzootic persistence.

Protective Immunity and New Vaccines for Lyme Disease.

Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.

Early Questing by Lone Star Tick Larvae, New York and Massachusetts, USA, 2018.

Subtropical lone star tick larvae typically emerge in late summer. We found clusters of host-seeking lone star tick larvae during early June 2018 in New York and Massachusetts, USA. Invasion and persistence of this tick in more northern locations may have been promoted by adaptation to an accelerated life cycle.

Blood Smears Have Poor Sensitivity for Confirming Borrelia miyamotoi Disease.

Borrelia miyamotoi disease (BMD) is a newly recognized borreliosis that is cotransmitted by ticks wherever Lyme disease is zoonotic. Unlike Borrelia burgdorferisensu lato, the agent of Lyme disease, B. miyamotoi is closely related to relapsing fever spirochetes, such as Borrelia hermsii Some authors have suggested that the disease caused by B. miyamotoi should be considered a hard-tick-transmitted relapsing fever, and thus, the main mode of confirming a diagnosis for that infection, microscopy to analyze a blood smear, may have clinical utility. To determine whether blood smears may detect B. miyamotoi in the blood of acute BMD patients, we made standard malariological thick smears from anticoagulated blood samples that were previously determined to contain this agent (by PCR) and analyzed them for morphological evidence of spirochetes. Spirochetes were not detected in the blood smears from 20 PCR positive patient blood samples after examination of 100 thick smear fields and only 2 of 20 demonstrated spirochetes when the examination was extended to 300 thick smear fields. Inoculation of severe combined immunodeficient (SCID) mice yielded isolates from 5 of 5 samples, but 0 of 3 BALB/c mice became infected. We conclude that in strong contrast to the diagnosis of typical relapsing fever, microscopy of blood smears is not sensitive enough for confirming a diagnosis of BMD but that SCID mouse inoculation could be a useful complement to PCR.

A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology.

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology.

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

Rapid Detection of Powassan Virus in a Patient With Encephalitis by Metagenomic Sequencing.

We describe a patient with severe and progressive encephalitis of unknown etiology. We performed rapid metagenomic sequencing from cerebrospinal fluid and identified Powassan virus, an emerging tick-borne flavivirus that has been increasingly detected in the United States.

Minimal infectious dose and dynamics of Babesia microti parasitemia in a murine model.

BACKGROUND: Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain. STUDY DESIGN AND METHODS: Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay. RESULTS: In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 107 pRBCs/mL at 2 to 3 weeks or 5 × 108 pRBCs/mL at 6 weeks was observed for DBA/2 and NSG mice, respectively. Protracted fluctuating parasitemia was observed for 8 months in DBA/2 mice, whereas NSG mice exhibited a high-plateau parasitemia. Antibody titers continued to increase until 6 to 18 weeks in DBA/2 mice and remained high through 6 months. This study also investigated the analytical performance of Babesia assays that detect parasite DNA or RNA using a blinded panel. A Babesia assay targeting parasite RNA was approximately 10-fold more sensitive compared to qPCR targeting DNA. CONCLUSION: The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection.

Fatal Deer Tick Virus Infection in Maine.

Deer tick virus (DTV), a genetic variant (lineage II) of Powassan virus, is a rare cause of encephalitis in North America. We report a fatal case of DTV encephalitis following a documented bite from an Ixodes scapularis tick and the erythema migrans rash associated with Lyme disease.

Meningoencephalitis from Borrelia miyamotoi in an immunocompromised patient.

Ixodes ticks serve as vectors for Borrelia burgdorferi, the agent of Lyme disease. Globally, these ticks often concurrently harbor B. miyamotoi, a spirochete that is classified within the relapsing-fever group of spirochetes. Although humans presumably are exposed to B. miyamotoi, there are limited data suggesting disease attributable to it. We report a case of progressive mental deterioration in an older, immunocompromised patient, and even though Koch's postulates were not met, we posit B. miyamotoi as the cause, owing to its direct detection in cerebrospinal fluid (CSF) with the use of microscopy and a polymerase-chain-reaction (PCR) assay. It is likely that B. miyamotoi is an underrecognized cause of disease, especially in sites where Lyme disease is endemic.

Serologic screening of United States blood donors for Babesia microti using an investigational enzyme immunoassay.

BACKGROUND: The tick-borne pathogen Babesia microti has become recognized as the leading infectious risk associated with blood transfusion in the United States, yet no Food and Drug Administration-licensed screening tests are currently available to mitigate this risk. The aim of this study was to evaluate the performance of an investigational enzyme immunoassay (EIA) for B. microti as a screening test applied to endemic and nonendemic blood donor populations. STUDY DESIGN AND METHODS: The study aimed to test 20,000 blood donors from areas of the United States considered endemic for B. microti and 10,000 donors from a nonendemic area with the investigational B. microti EIA. Repeat-reactive samples were retested by polymerase chain reaction (PCR), blood smear, immunofluorescent assay (IFA), and immunoblot assay. In parallel, serum samples from symptomatic patients with confirmed babesiosis were tested by EIA, IFA, and immunoblot assays. RESULTS: A total of 38 of 13,757 (0.28%) of the donors from New York, 7 of 4583 (0.15%) from Minnesota, and 11 of 8363 (0.13%) from New Mexico were found repeat reactive by EIA. Nine of the 56 EIA repeat-reactive donors (eight from New York and one from Minnesota) were positive by PCR. The specificity of the assay in a nonendemic population was 99.93%. Among IFA-positive clinical babesiosis patients, the sensitivity of the assay was 91.1%. CONCLUSION: The B. microti EIA detected PCR-positive, potentially infectious blood donors in an endemic population and exhibited high specificity among uninfected and unexposed individuals. The EIA promises to provide an effective tool for blood donor screening for B. microti in a format amenable to high-throughput and cost-effective screening.

Borrelia miyamotoi Disease in the Northeastern United States: A Case Series.

BACKGROUND: The first recognized cases of Borrelia miyamotoi disease (BMD) in North America were reported in the northeastern United States in 2013. OBJECTIVE: To further describe the clinical spectrum and laboratory findings for BMD. DESIGN: Case series. SETTING: Patients presenting to primary care offices, emergency departments, or urgent care clinics in 2013 and 2014. PARTICIPANTS: Acutely febrile patients from the northeastern United States in whom the treating health care providers suspected and ordered testing for tick-transmitted infections. MEASUREMENTS: Whole-blood polymerase chain reaction (PCR) testing was performed for the presence of specific DNA sequences of common tickborne infections (including BMD). Serologic testing for B. miyamotoi was performed using a recombinant glycerophosphodiester phosphodiesterase (rGlpQ) protein. Clinical records were analyzed to identify the major features of acute disease. RESULTS: Among 11,515 patients tested, 97 BMD cases were identified by PCR. Most of the 51 case patients on whom clinical histories were reviewed presented with high fever, chills, marked headache, and myalgia or arthralgia. Twenty-four percent were hospitalized. Elevated liver enzyme levels, neutropenia, and thrombocytopenia were common. At presentation, 16% of patients with BMD were seropositive for IgG and/or IgM antibody to B. miyamotoi rGlpQ. Most (78%) had seropositive convalescent specimens. Symptoms resolved after treatment with doxycycline, and no chronic sequelae or symptoms were observed. LIMITATION: Findings were based on specimens submitted for testing to a reference laboratory, and medical records of only 51 of the 97 case patients with BMD were reviewed. CONCLUSION: Patients with BMD presented with nonspecific symptoms, including fever, headache, chills, myalgia, and arthralgia. Laboratory confirmation of BMD was possible by PCR on blood from acutely symptomatic patients who were seronegative at presentation. Borrelia miyamotoi disease may be an emerging tickborne infection in the northeastern United States. PRIMARY FUNDING SOURCE: IMUGEN.

Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study.

BACKGROUND: Animal studies suggest that Borrelia burgdorferi, the agent of Lyme disease, may persist after antibiotic therapy and can be detected by various means including xenodiagnosis using the natural tick vector (Ixodes scapularis). No convincing evidence exists for the persistence of viable spirochetes after recommended courses of antibiotic therapy in humans. We determined the safety of using I. scapularis larvae for the xenodiagnosis of B. burgdorferi infection in humans. METHODS: Laboratory-reared larval I. scapularis ticks were placed on 36 subjects and allowed to feed to repletion. Ticks were tested for B. burgdorferi by polymerase chain reaction (PCR), culture, and/or isothermal amplification followed by PCR and electrospray ionization mass spectroscopy. In addition, attempts were made to infect immunodeficient mice by tick bite or inoculation of tick contents. Xenodiagnosis was repeated in 7 individuals. RESULTS: Xenodiagnosis was well tolerated with no severe adverse events. The most common adverse event was mild itching at the tick attachment site. Xenodiagnosis was negative in 16 patients with posttreatment Lyme disease syndrome (PTLDS) and/or high C6 antibody levels and in 5 patients after completing antibiotic therapy for erythema migrans. Xenodiagnosis was positive for B. burgdorferi DNA in a patient with erythema migrans early during therapy and in a patient with PTLDS. There is insufficient evidence, however, to conclude that viable spirochetes were present in either patient. CONCLUSIONS: Xenodiagnosis using Ixodes scapularis larvae was safe and well tolerated. Further studies are needed to determine the sensitivity of xenodiagnosis in patients with Lyme disease and the significance of a positive result. Clinical Trials Registration NCT01143558.