Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

Solar Energy-driven Land-cover Change Could Alter Landscapes Critical to Animal Movement in the Continental United States.

The United States may produce as much as 45% of its electricity using solar energy technology by 2050, which could require more than 40,000 km2 of land to be converted to large-scale solar energy production facilities. Little is known about how such development may impact animal movement. Here, we use five spatially explicit projections of solar energy development through 2050 to assess the extent to which ground-mounted photovoltaic solar energy expansion in the continental United States may impact land-cover and alter areas important for animal movement. Our results suggest that there could be a substantial overlap between solar energy development and land important for animal movement: across projections, 7-17% of total development is expected to occur on land with high value for movement between large protected areas, while 27-33% of total development is expected to occur on land with high value for climate-change-induced migration. We also found substantial variation in the potential overlap of development and land important for movement at the state level. Solar energy development, and the policies that shape it, may align goals for biodiversity and climate change by incorporating the preservation of animal movement as a consideration in the planning process.

Dynamics of SARS-CoV-2 seroassay sensitivity: a systematic review and modelling study.

BackgroundSerological surveys have been the gold standard to estimate numbers of SARS-CoV-2 infections, the dynamics of the epidemic, and disease severity. Serological assays have decaying sensitivity with time that can bias their results, but there is a lack of guidelines to account for this phenomenon for SARS-CoV-2.AimOur goal was to assess the sensitivity decay of seroassays for detecting SARS-CoV-2 infections, the dependence of this decay on assay characteristics, and to provide a simple method to correct for this phenomenon.MethodsWe performed a systematic review and meta-analysis of SARS-CoV-2 serology studies. We included studies testing previously diagnosed, unvaccinated individuals, and excluded studies of cohorts highly unrepresentative of the general population (e.g. hospitalised patients).ResultsOf the 488 screened studies, 76 studies reporting on 50 different seroassays were included in the analysis. Sensitivity decay depended strongly on the antigen and the analytic technique used by the assay, with average sensitivities ranging between 26% and 98% at 6 months after infection, depending on assay characteristics. We found that a third of the included assays departed considerably from manufacturer specifications after 6 months.ConclusionsSeroassay sensitivity decay depends on assay characteristics, and for some types of assays, it can make manufacturer specifications highly unreliable. We provide a tool to correct for this phenomenon and to assess the risk of decay for a given assay. Our analysis can guide the design and interpretation of serosurveys for SARS-CoV-2 and other pathogens and quantify systematic biases in the existing serology literature.

COVID-19 prevalence and mortality in longer-term care facilities.

This essay considers the factors that have contributed to very high COVID-19 mortality in longer-term care facilities (LTCFs). We compare the demographic characteristics of LTCF residents with those of community-dwelling older adults, and then we review the evidence regarding prevalence and infection fatality rates (IFRs), including links to frailty and some comorbidities. Finally, we discuss policy measures that could foster the physical and mental health and well-being of LTCF residents in the present context and in potential future pandemics.

Highly Distorted Chiral Two-Dimensional Tin Iodide Perovskites for Spin Polarized Charge Transport.

Incorporating chiral organic molecules into organic/inorganic hybrid 2D metal-halide perovskites results in a novel family of chiral hybrid semiconductors with unique spin-dependent properties. The embedded chiral organic moieties induce a chiroptical response from the inorganic metal-halide sublattice. However, the structural interplay between the chiral organic molecules and the inorganic sublattice, as well as their synergic effect on the resulting electronic band structure need to be explored in a broader material scope. Here we present three new layered tin iodide perovskites templated by chiral (R/S-)methylbenzylammonium (R/S-MBA), i.e., (R-/S-MBA)2SnI4, and their racemic phase (rac-MBA)2SnI4. These MBA2SnI4 compounds exhibit the largest level of octahedral bond distortion compared to any other reported layered tin iodide perovskite. The incorporation of chiral MBA cations leads to circularly polarized absorption from the inorganic Sn-I sublattice, displaying chiroptical activity in the 300-500 nm wavelength range. The bandgap and chiroptical activity are modulated by alloying Sn with Pb, in the series of (MBA)2Pb1-xSnxI4. Finally, we show that vertical charge transport through oriented (R-/S-MBA)2SnI4 thin films is highly spin-dependent, arising from a chiral-induced spin selectivity (CISS) effect. We demonstrate a spin-polarization in the current-voltage characteristics as high as 94%. Our work shows the tremendous potential of these chiral hybrid semiconductors for controlling both spin and charge degrees of freedom.

Assessing the age specificity of infection fatality rates for COVID-19: systematic review, meta-analysis, and public policy implications.

Determine age-specific infection fatality rates for COVID-19 to inform public health policies and communications that help protect vulnerable age groups. Studies of COVID-19 prevalence were collected by conducting an online search of published articles, preprints, and government reports that were publicly disseminated prior to 18 September 2020. The systematic review encompassed 113 studies, of which 27 studies (covering 34 geographical locations) satisfied the inclusion criteria and were included in the meta-analysis. Age-specific IFRs were computed using the prevalence data in conjunction with reported fatalities 4 weeks after the midpoint date of the study, reflecting typical lags in fatalities and reporting. Meta-regression procedures in Stata were used to analyze the infection fatality rate (IFR) by age. Our analysis finds a exponential relationship between age and IFR for COVID-19. The estimated age-specific IFR is very low for children and younger adults (e.g., 0.002% at age 10 and 0.01% at age 25) but increases progressively to 0.4% at age 55, 1.4% at age 65, 4.6% at age 75, and 15% at age 85. Moreover, our results indicate that about 90% of the variation in population IFR across geographical locations reflects differences in the age composition of the population and the extent to which relatively vulnerable age groups were exposed to the virus. These results indicate that COVID-19 is hazardous not only for the elderly but also for middle-aged adults, for whom the infection fatality rate is two orders of magnitude greater than the annualized risk of a fatal automobile accident and far more dangerous than seasonal influenza. Moreover, the overall IFR for COVID-19 should not be viewed as a fixed parameter but as intrinsically linked to the age-specific pattern of infections. Consequently, public health measures to mitigate infections in older adults could substantially decrease total deaths.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

HIV protease inhibitors block parasite signal peptide peptidases and prevent growth of Babesia microti parasites in erythrocytes.

Malaria and babesiosis are bloodborne protozoan infections for which the emergence of drug-resistant strains poses a threat. Our previous phage display cDNA screens established the essentiality of Plasmodium falciparum signal peptide peptidase (SPP) in asexual development at the blood stage of malaria infection. Given the structural similarities between SPP inhibitors and HIV protease inhibitors, we screened ten HIV protease inhibitors and selected Lopinavir and Atazanavir for their ability to inhibit PfSPP activity. Using a transcription-based assay, we observed that Lopinavir inhibits both parasite-and host-derived SPP activities whereas Atazanavir inhibited only parasite derived SPP activity. Consistent with their inhibitory effect on Plasmodium growth, both Lopinavir and Atazanavir strongly inhibited intraerythrocytic Babesia microti growth ex vivo. Moreover, Lopinavir prevented the steep rise in Babesia microti parasitemia typically observed in rag1-deficient mice. Our data provide first evidence that inhibition of parasite-derived SPPs by HIV protease inhibitors offers a promising therapeutic avenue for the treatment of severe babesiosis and infections caused by other Apicomplexa parasites.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

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.

Expediting red blood cell transfusions by syringing causes significant hemolysis.

BACKGROUND: Techniques commonly used to expedite blood transfusions include pneumatically pressurizing red blood cell (RBC) bags or manual syringing its contents. We compared these techniques on RBC hemolysis using a simulated transfusion model. STUDY DESIGN AND METHODS: Fifteen warmed RBC units that were 12.3 ± 4.3 (95% confidence interval [CI], 10.1-14.5) days old were each subjected to two experimental rapid transfusion techniques. RBCs from each technique were directed through 18- and 22-gauge cannulas attached to blood administration sets. One technique involved RBC bag pressurization to 300 mmHg. The other employed a 20-mL syringe to effect forceful, manual aspiration from the RBC bag followed by forceful, manual RBC injection. The control group was gravity driven without cannulas. Free hemoglobin (Hb) concentrations were measured and percent hemolysis was calculated. RESULTS: Free Hb concentrations and percent hemolysis (median [95% CI]) were similar in the control (0.05 [0.03-0.08] g/dL and 0.13% [0.09%-0.17%], respectively) and pressurized experiments (0.06 [0.05-0.09] g/dL; 0.14% [0.12%-0.22%]), respectively. Syringing resulted in 10-fold higher free Hb concentrations (0.55 [0.38-0.92] g/dL) and percent hemolysis (1.28% [1.03%-2.15%]) than when employing the control (p < 0.0001) or pressurization (p < 0.0001) techniques. Cannula sizes studied did not affect hemolysis. CONCLUSION: Forceful manual syringing caused significant hemolysis and high free Hb concentrations. Pressurizing RBC bags induced no more hemolysis than after gravity-facilitated transfusions. Syringing to expedite RBC transfusions should be avoided in favor of pneumatic RBC bag pressurization.

Reinfusate Heparin Concentrations Produced by Two Autotransfusion Systems.

OBJECTIVES: Cell saver reinfusate ideally should contain low, clinically insignificant heparin concentrations. The American Association of Blood Banks has defined the clinically insignificant threshold as 0.5 IU/mL. Furthermore, there is uncertainty about the meaning of cell saver "heparin elimination rates." These concerns prompted the authors' independent investigation of reinfusate heparin concentrations of devices used in their institution. It was hypothesized that cell saver reinfusates contain clinically insignificant heparin concentrations. DESIGN: Two prospective, pragmatic, sequential, observational, single-center studies. SETTING: University teaching hospital. PARTICIPANTS: A total of 32 and 31 patients for on-pump cardiac surgery were enrolled in the Sorin (Dideco) Electa and Sorin Xtra studies, respectively. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Postcardiac surgery reinfusate heparin concentrations were measured using a modified anti-Xa chromogenic assay. Heparin concentrations above 0.5 IU/mL were present in 56% (95% confidence interval, 35% to 68%) of Sorin Xtra reinfusates. Heparin concentrations in the Sorin (Dideco) Electa reinfusates were lower than recommended in 29 of 32 reinfusates. Only 3 of 32 Sorin (Dideco) Electa reinfusates (9.4%; 95% confidence interval 3.2% to 24%) exhibited heparin concentrations exceeding 0.5 IU/mL. CONCLUSIONS: Sorin (Dideco) Electa reinfusates contained heparin concentrations below the American Association of Blood Banks recommended threshold in 90.6% of cases, while Sorin Xtra reinfusate heparin concentrations exceeded this recommendation in 56% of cases. Measurement of cell saver reinfusate heparin concentrations necessitates the use of a modified chromogenic assay. Studies explicitly should confirm that such a modification was indeed used. Periodic quality control of reinfusate composition is recommended.

Transfusion-transmitted babesiosis: is it time to screen the blood supply?

PURPOSE OF REVIEW: This review summarizes the current status of blood screening to prevent transfusion-transmitted babesiosis (TTB). RECENT FINDINGS: Babesia microti has recently been determined to be the most common transfusion-transmitted pathogen in the United States. Patients who acquire TTB often experience severe illness with an associated mortality rate of about 20%. Recent studies have demonstrated that laboratory screening using B. microti antibody and/or PCR assays can effectively identify infectious blood donors and that this approach may offer a cost- effective means of intervention. Pathogen inactivation methods may offer an alternative solution. None of these methods has yet been licensed by US Food and Drug Administration, however, and current efforts to prevent TTB rely on excluding blood donors who report having had babesiosis. SUMMARY: TTB imposes a significant health burden on the United States population. Further research is needed to better inform decisions on optimal screening strategies and reentry criteria, but given the acute need and the currently available screening tools, initiation of blood donor screening to prevent TTB should be given high priority.

A prospective evaluation of chronic Babesia microti infection in seroreactive blood donors.

BACKGROUND: Babesia microti is the foremost infectious risk to the US blood supply for which a Food and Drug Administration (FDA)-licensed test is unavailable for donation screening. Characterization of the antibody response to B. microti and correlation with parasitemia is necessary to guide screening and donor management policies. STUDY DESIGN AND METHODS: During an FDA licensure trial, blood donors were prospectively screened (July-November 2013) using a B. microti-specific antibody enzyme immunoassay (EIA, Immunetics) in highly endemic (New York [NY]; n = 13,688), moderately endemic (Minnesota [MN]; n = 4583), and nonendemic (New Mexico [NM]; n = 8451) regions. Blood donors with repeat-reactive (RR) results participated in a 12-month prospective cohort study using B. microti EIA, immunofluorescent assay, polymerase chain reaction (PCR), blood smear, and clinical questionnaire. RESULTS: Thirty-seven (61.67%; 24 NY, seven MN, six NM) of 60 eligible RR donors enrolled in the study; 20 of 37 (54%) completed the 12-month follow-up visit of which 15 (75%) were still seroreactive. Nine PCR-positive donors were identified during index screening; five participated in the follow-up study, three were PCR positive at 6 months, and two remained positive at final follow-up (378 and 404 days). Most RR donors displayed low-level seroreactivity that was either stable or waning during follow-up. The level and pattern of reactivity correlated poorly with PCR positivity. CONCLUSION: The findings indicate prolonged seropositivity in blood donors. Although rare, asymptomatic, persistent PCR positivity supports the current policy of indefinite deferral for donors with a history of babesiosis or positive test results. Repeat testing by PCR and serology will be necessary if reinstatement is to be considered.

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.

Comparison of Minimally and More Invasive Methods of Determining Mixed Venous Oxygen Saturation.

OBJECTIVE: To investigate the accuracy of a minimally invasive, 2-step, lookup method for determining mixed venous oxygen saturation compared with conventional techniques. DESIGN: Single-center, prospective, nonrandomized, pilot study. SETTING: Tertiary care hospital, university setting. PARTICIPANTS: Thirteen elective cardiac and vascular surgery patients. INTERVENTIONS: All participants received intra-arterial and pulmonary artery catheters. Minimally invasive oxygen consumption and cardiac output were measured using a metabolic module and lithium-calibrated arterial waveform analysis (LiDCO; LiDCO, London), respectively. For the minimally invasive method, Step 1 involved these minimally invasive measurements, and arterial oxygen content was entered into the Fick equation to calculate mixed venous oxygen content. Step 2 used an oxyhemoglobin curve spreadsheet to look up mixed venous oxygen saturation from the calculated mixed venous oxygen content. The conventional "invasive" technique used pulmonary artery intermittent thermodilution cardiac output, direct sampling of mixed venous and arterial blood, and the "reverse-Fick" method of calculating oxygen consumption. MEASUREMENTS AND MAIN RESULTS: LiDCO overestimated thermodilution cardiac output by 26%. Pulmonary artery catheter-derived oxygen consumption underestimated metabolic module measurements by 27%. Mixed venous oxygen saturation differed between techniques; the calculated values underestimated the direct measurements by between 12% to 26.3%, this difference being statistically significant. CONCLUSION: The magnitude of the differences between the minimally invasive and invasive techniques was too great for the former to act as a surrogate of the latter and could adversely affect clinical decision making.

Determination of Babesia microti seroprevalence in blood donor populations using an investigational enzyme immunoassay.

BACKGROUND: Transfusion-transmitted babesiosis caused by Babesia microti has emerged as a significant risk to the US blood supply. This study estimated the prevalence of B. microti antibodies in blood donors using an investigational enzyme immunoassay (EIA). STUDY DESIGN AND METHODS: A peptide-based EIA that detects both immunoglobulin (Ig)G and IgM antibodies to B. microti was developed and validated. Donor samples randomly selected from areas defined as high-risk endemic, lower-risk endemic, and nonendemic for B. microti were deidentified and tested using the investigational EIA. Samples that were EIA repeat reactive were further tested by B. microti immunofluorescent assay (IFA), polymerase chain reaction (PCR) on red blood cell lysates, and peripheral blood smear examination. A random subset of 1272 samples from high-risk endemic areas was tested by IFA, PCR, and peripheral blood smear in parallel with EIA. RESULTS: Among 15,000 donations tested with the investigational B. microti EIA, EIA repeat-reactive rates were 1.08% (54/5000) in a high-risk endemic area, 0.74% (37/5000) in a lower-risk area, and 0.40% (20/5000) in a nonendemic area. After application of a revised cutoff, these values were reduced to 0.92%, (46/5000), 0.54% (27/5000), and 0.16% (8/5000). Overall concordance between EIA and IFA among donor samples was 99.34%. One seropositive sample was positive by PCR. CONCLUSION: The seroprevalence of B. microti in blood donors in a high-risk area measured by an investigational EIA was approximately 1%. The EIA shows promise as an efficient high-throughput blood donor screening assay for B. microti.

Heparinase thromboelastography compared with activated coagulation time for protamine titration after cardiopulmonary bypass.

OBJECTIVE: The present study is a comparison of two point-of-care (POC) tests as endpoints of protamine titration after CPB. The authors hypothesized that using the heparinase-kaolin thromboelastography (TEG-HK) R-time difference would more readily identify residual heparin necessitating additional protamine than when using activated coagulation time (ACT). The primary endpoint was the between-group difference in protamine dose. Whether this approach would lessen postoperative bleeding and sequelae also was investigated. DESIGN: Single center, blinded, prospective, randomized study. SETTING: University teaching hospital. PARTICIPANTS: Eighty-two adult patients for on-pump coronary artery bypass and/or valve surgery. INTERVENTIONS: Patients were randomized. In the ACT group, protamine was titrated until ACT did not exceed baseline by more than 10%. In the TEG group, a TEG-HK R-time difference less than 20% was targeted. Protamine was repeated to achieve the endpoints. Clinicians in the ACT group were blinded to TEG data and vice versa. MEASUREMENTS AND MAIN RESULTS: There was no between-group difference in total protamine dose (3.9 ± 0.6 and 4.2 ± 0.7; 95% CI of the difference between means: -0.544 to 0.008 mg/kg; p = 0.057) or protamine:heparin ratios (1.3:1 and 1.4:1; 95% CI of the difference between means: -0.05 to 0.03 mg/mg; p = 0.653). In the ACT group, 17% of patients required a second protamine dose, and in the TEG group, 24% of patients required a second protamine dose. No between-group differences in the postoperative transfusion requirements or intensive care unit length of stay were demonstrated. CONCLUSION: No difference was identified in protamine dosing using either ACT or TEG-HK R-time difference as endpoints. Heparinase TEG may be useful for monitoring heparin reversal.

Reply to Shah, J.S.; Ramasamy, R. Target Antigens in Western and Line Immunoblots for Supporting the Diagnosis of Lyme Disease. Comment on "Porwancher et al. Immunoblot Criteria for Diagnosis of Lyme Disease: A Comparison of CDC Criteria to Alternative Interpretive Approaches. Pathogens 2023, 12, 1282".

We are writing in response to comments made by Shah and Ramasamy [...].