Determination of fatty acid and sphingoid base composition of eleven ceramide subclasses in stratum corneum by UHPLC/scheduled-MRM.

INTRODUCTION: Ceramides play a key role in skin barrier function in homeostatic and pathological conditions and can be sampled non-invasively through stratum corneum collection. OBJECTIVES: To develop a novel UHPLC/Scheduled MRM method for the identification and relative distribution of eleven classes of ceramides, which are separated by UHPLC and determined by their specific retention times. The precise composition of the fatty acid and sphingoid base parts of each individual ceramide is determined via mass fragmentation. METHODS: More than 1000 human and pig ceramides were identified. Three human and minipig ceramide classes, CER[AS], CER[NS] and CER[EOS] have been investigated in depth. RESULTS: Sphingoid bases were characterized by a prevalence of chain lengths with sizes from C16 to C22, whereas fatty acids were mainly observed in the range of C22-C26. Overall, the ceramide profiles between human and minipig stratum corneum were similar. Differences in the CER[AS] and CER[NS] classes included a more homogeneous distribution of fatty acids (16-30 carbon atoms) in minipig, whereas in human longer fatty acid chains (> 24 carbon atoms) predominated. CONCLUSION: The method will be useful for the analysis of healthy and pathological skin in various specie, and the measurement of the relative distribution of ceramides as biomarkers for pharmacodynamic studies.

Stage-specific differences in secretory profile of mesenchymal stromal cells (MSCs) subjected to early- vs late-stage OA synovial fluid.

OBJECTIVE: Although, mesenchymal stromal cells (MSCs) are being clinically investigated for their use in osteoarthritis (OA), it is unclear whether their postulated therapeutic properties are equally effective in the early- and late-stages of OA. In this study we investigated MSC cytokine secretion post-exposure to synovial fluid (SF), obtained from early- vs late-stage knee OA patients to justify a potential patient stratification strategy to maximize MSC-mediated treatment effects. METHOD: Subjects were recruited and categorized into early- [Kellgren-Lawrence (KL) grade I/II, n = 12] and late-stage (KL-III/IV, n = 12) knee OA groups. SF samples were obtained, and their proteome was tested using multiplex assays, after 3-days culture, with and without MSCs. SFs cultured without MSCs were used as a baseline to identify MSC-secreted factors into SFs cultured with MSCs. Linear mixed-effect models and non-parametric tests were used to identify alterations in the MSC secretome during exposure to OA SF (3-days). MSCs cultured for 3-days in 0.5% fetal bovine serum (FBS)-supplemented medium were used to compare SF results with culture medium. RESULTS: Following exposure to OA SF, the MSC secretome contained proteins that are involved in tissue repair, angiogenesis, chemotaxis, matrix remodeling and the clotting process. However, chemokine (C-X-C motif) ligand-8 (CXCL8; chemoattractant), interleukin-6 (IL6) and chemokine (C-C motif) ligand 2 (CCL2) were elevated in the MSC-secretome in response to early- vs late-stage OA SF. CONCLUSION: Early- vs late-stage OA SF samples elicit a differential MSC secretome response, arguing for stratification of OA patients to maximize MSC-mediated therapeutic effects.

Phase transitions in biogenic amorphous calcium carbonate.

Crystalline biominerals do not resemble faceted crystals. Current explanations for this property involve formation via amorphous phases. Using X-ray absorption near-edge structure (XANES) spectroscopy and photoelectron emission microscopy (PEEM), here we examine forming spicules in embryos of Strongylocentrotus purpuratus sea urchins, and observe a sequence of three mineral phases: hydrated amorphous calcium carbonate (ACC · H(2)O) → dehydrated amorphous calcium carbonate (ACC) → calcite. Unexpectedly, we find ACC · H(2)O-rich nanoparticles that persist after the surrounding mineral has dehydrated and crystallized. Protein matrix components occluded within the mineral must inhibit ACC · H(2)O dehydration. We devised an in vitro, also using XANES-PEEM, assay to identify spicule proteins that may play a role in stabilizing various mineral phases, and found that the most abundant occluded matrix protein in the sea urchin spicules, SM50, stabilizes ACC · H(2)O in vitro.

Measurement of c-axis angular orientation in calcite (CaCO3) nanocrystals using X-ray absorption spectroscopy.

We demonstrate that the ability to manipulate the polarization of synchrotron radiation can be exploited to enhance the capabilities of X-ray absorption near-edge structure (XANES) spectroscopy, to include linear dichroism effects. By acquiring spectra at the same photon energies but different polarizations, and using a photoelectron emission spectromicroscope (PEEM), one can quantitatively determine the angular orientation of micro- and nanocrystals with a spatial resolution down to 10 nm. XANES-PEEM instruments are already present at most synchrotrons, hence these methods are readily available. The methods are demonstrated here on geologic calcite (CaCO(3)) and used to investigate the prismatic layer of a mollusk shell, Pinctada fucata. These XANES-PEEM data reveal multiply oriented nanocrystals within calcite prisms, previously thought to be monocrystalline. The subdivision into multiply oriented nanocrystals, spread by more than 50°, may explain the excellent mechanical properties of the prismatic layer, known for decades but never explained.

Mark-specific hazard ratio model with multivariate continuous marks: an application to vaccine efficacy.

In randomized placebo-controlled preventive HIV vaccine efficacy trials, an objective is to evaluate the relationship between vaccine efficacy to prevent infection and genetic distances of the exposing HIV strains to the multiple HIV sequences included in the vaccine construct, where the set of genetic distances is considered as the continuous multivariate "mark" observed in infected subjects only. This research develops a multivariate mark-specific hazard ratio model in the competing risks failure time analysis framework for the assessment of mark-specific vaccine efficacy. It allows improved efficiency of estimation by employing the semiparametric method of maximum profile likelihood estimation in the vaccine-to-placebo mark density ratio model. The model also enables the use of a more efficient estimation method for the overall log hazard ratio in the Cox model. In addition, we propose testing procedures to evaluate two relevant hypotheses concerning mark-specific vaccine efficacy. The asymptotic properties and finite-sample performance of the inferential procedures are investigated. Finally, we apply the proposed methods to data collected in the Thai RV144 HIV vaccine efficacy trial.

Transformation and crystallization energetics of synthetic and biogenic amorphous calcium carbonate.

Amorphous calcium carbonate (ACC) is a metastable phase often observed during low temperature inorganic synthesis and biomineralization. ACC transforms with aging or heating into a less hydrated form, and with time crystallizes to calcite or aragonite. The energetics of transformation and crystallization of synthetic and biogenic (extracted from California purple sea urchin larval spicules, Strongylocentrotus purpuratus) ACC were studied using isothermal acid solution calorimetry and differential scanning calorimetry. Transformation and crystallization of ACC can follow an energetically downhill sequence: more metastable hydrated ACC → less metastable hydrated ACC ⇒ anhydrous ACC ∼ biogenic anhydrous ACC ⇒ vaterite → aragonite → calcite. In a given reaction sequence, not all these phases need to occur. The transformations involve a series of ordering, dehydration, and crystallization processes, each lowering the enthalpy (and free energy) of the system, with crystallization of the dehydrated amorphous material lowering the enthalpy the most. ACC is much more metastable with respect to calcite than the crystalline polymorphs vaterite or aragonite. The anhydrous ACC is less metastable than the hydrated, implying that the structural reorganization during dehydration is exothermic and irreversible. Dehydrated synthetic and anhydrous biogenic ACC are similar in enthalpy. The transformation sequence observed in biomineralization could be mainly energetically driven; the first phase deposited is hydrated ACC, which then converts to anhydrous ACC, and finally crystallizes to calcite. The initial formation of ACC may be a first step in the precipitation of calcite under a wide variety of conditions, including geological CO(2) sequestration.

Long-term effects of breast aging in patients undergoing explantation: analysis of breast aesthetics from before augmentation to after explantation.

PURPOSE: Although most patients with implants have an uneventful course, some will require explantation. Moreover, women's breasts and their perception of their body habitus change with time. This study covering greater than a 32-year period will address the reconstruction options available after breast implant explantation. METHODS: Augmentation mammoplasty was performed on 42 patients who subsequently underwent explantation. The following data were recorded: age at time of implantation and explantation, length of implant, type, reason for explantation, and decision after explantation. Recommendations were made based on patient preferences, degree of ptosis, clinical history, opinions regarding scars, and breast contour. Reconstruction options were categorized into none, mastopexy, capsulectomy and reaugmentation with saline implants, and mastopexy with immediate or delayed augmentation. RESULTS: The average age of patients at implantation was 32.3 years, 46.8 years at explantation, with a length of implantation of 14.4 years. Thirty-six (86%) of 42 patients received explantation for capsular contracture, 7 (17%) for negative publicity of silicone implants, 7 (17%) for change in body habitus and perception of implants, 6 (14%) for rupture, 5 (12%) for ptosis, and 1 (2.4%) each for synmastia, breast cancer, and painful implants. Sixteen (38%) patients underwent mastopexy after explantation, 15 (36%) underwent no reconstruction after explantation, 6 (14%) with mastopexy and reaugmentation (2 immediate and 4 delayed), 4 (9.5%) with implant exchange, and 1 (2.4%) with mastectomy and reconstruction. All patients demonstrated satisfactory to excellent results. CONCLUSIONS: This study provides long-term results of augmentation mammoplasty by a single surgeon (G.P.G.) evaluating available options and reasonable expectations after explantation. Although most of the augmentation patients have a good outcome, some require removal of implants for a variety of reasons and long-term satisfactory options do exist after explantation.

Improving assessment of alcohol treatment barriers among Latino and White adults with an alcohol use disorder: Development of the barriers to specialty alcohol treatment scale.

INTRODUCTION: The present study's aims were two-fold. First, we sought to validate a novel measure to assess barriers to specialty alcohol treatment among White and Latino individuals with an alcohol use disorder (AUD): The Barriers to Specialty Alcohol Treatment (BSAT) scale. Second, we sought to demonstrate that the BSAT scale could be used to explain Latino-White disparities in barriers to alcohol treatment. METHODS: In 2021, we recruited an online national sample of 1200 White and Latino adults with a recent AUD. Participants completed an online questionnaire that included the BSAT items. Confirmatory and exploratory factor analyses were conducted to validate the BSAT. Multiple group analyses across race/ethnicity and language were also performed using the final model. RESULTS: The final model consisted of 36 items across 7 factors that reflect barriers related to low problem recognition, recovery goals, low perceived treatment efficacy, cultural factors, immigration-related concerns, low perceived social support, and logistical barriers. The final model's factor structure and factor loadings held up across race/ethnicity and language. The top endorsed barriers were low problem recognition, recovery goals, low perceived social support, logistical issues, and low perceived treatment efficacy. Compared to Whites, Latinos were more likely to report perceived lack of social support, logistical barriers, low perceived treatment efficacy, cultural barriers, and immigration-related concerns as barriers. CONCLUSION: Findings provide empirical support for the validity of the BSAT scale, which offers improved measurement of specialty alcohol treatment barriers and can be used to explore Latino-White disparities in a future study.

Allospecific CD154 + T-cytotoxic memory cells as potential surrogate for rejection risk in pediatric intestine transplantation.

Clinical end-points dictate large trial enrollments and exclude children with the rare intestine transplant procedure (ITx), who experience higher drug-related morbidity. We evaluate the novel rejection-risk parameter, allo-(antigen)-specific CD154 + TcMs (i) as surrogates for ACR using Prentice's criteria, (ii) for association with immunosuppression targets to determine Fleming's surrogate end-point designation, and (iii) as time-to-event end-point in a simulated comparison of alemtuzumab (NCT#01208337, n = 14) and rabbit anti-human thymocyte globulin (rATG, n = 16) among 30 children with ITx. CD154 + TcM were measured in MLR before, and at 1-60 and 61-200 days after ITx (NCT#01163578). CD154 + TcM correlate significantly with rejection severity (Spearman r = 0.685, p = 2.03E-5) and associate with biopsy-proven ITx rejection with sensitivity/specificity of 94%/84% [corrected] independent of immunosuppressant. Previously stated sensitivity of 90% is incorrect. [corrected]. The rejection-risk threshold of CD154 + TcM resolves rapidly in 200-day follow-up (46 ± 20 vs. 158 ± 59 days, p = 0.009, K-M) with alemtuzumab, which demonstrates lower 90-day ACR incidence (50% vs. 69%, p=NS, Fisher's exact), and is associated with accelerated prednisone minimization to ≤2.5 mg/day, compared with rATG (120 ± 28 vs. 180 ± 30 days, p = 0.027, K-M). As a surrogate end-point, time-to-rejection-risk resolution measured with CD154 + TcM portends 50% reduction in sample sizes in a simulated trial of alemtuzumab vs. rATG. Rejection-risk assessment with CD154 + TcM may enable informed immunosuppression minimization, and preliminary efficacy comparisons in pediatric ITx.

The grinding tip of the sea urchin tooth exhibits exquisite control over calcite crystal orientation and Mg distribution.

The sea urchin tooth is a remarkable grinding tool. Even though the tooth is composed almost entirely of calcite, it is used to grind holes into a rocky substrate itself often composed of calcite. Here, we use 3 complementary high-resolution tools to probe aspects of the structure of the grinding tip: X-ray photoelectron emission spectromicroscopy (X-PEEM), X-ray microdiffraction, and NanoSIMS. We confirm that the needles and plates are aligned and show here that even the high Mg polycrystalline matrix constituents are aligned with the other 2 structural elements when imaged at 20-nm resolution. Furthermore, we show that the entire tooth is composed of 2 cooriented polycrystalline blocks that differ in their orientations by only a few degrees. A unique feature of the grinding tip is that the structural elements from each coaligned block interdigitate. This interdigitation may influence the fracture process by creating a corrugated grinding surface. We also show that the overall Mg content of the tooth structural elements increases toward the grinding tip. This probably contributes to the increasing hardness of the tooth from the periphery to the tip. Clearly the formation of the tooth, and the tooth tip in particular, is amazingly well controlled. The improved understanding of these structural features could lead to the design of better mechanical grinding and cutting tools.

An Ad5-vectored HIV-1 vaccine elicits cell-mediated immunity but does not affect disease progression in HIV-1-infected male subjects: results from a randomized placebo-controlled trial (the Step study).

BACKGROUND: The Step study was a randomized trial to determine whether an adenovirus type 5 (Ad5) vector vaccine, which elicits T cell immunity, can lead to control of human immunodeficiency virus (HIV) replication in participants who became HIV-infected after vaccination. METHODS: We evaluated the effect of the vaccine on trends in HIV viral load, CD4+ T cell counts, time to initiation of antiretroviral therapy (ART), and AIDS-free survival in 87 male participants who became infected with HIV during the Step study and who had a median of 24 months of post-infection follow-up. RESULTS: There was no overall effect of vaccine on mean log(10) viral load (estimated difference between groups, -0.11; P = .47). In a subset of subjects with protective HLA types (B27, B57, B58), mean HIV-1 RNA level over time was lower among vaccine recipients. There was no significant difference in CD4+ T cell counts, time to ART initiation, or in AIDS-free survival between HIV-1-infected subjects who received vaccine versus those who received placebo. CONCLUSIONS: HIV RNA levels, CD4+ T cell counts, time to initiation of ART, and AIDS-free survival were similar in vaccine and placebo recipients. There may have been a favorable effect of vaccine on HIV-1 RNA levels in participants with HLA types associated with better control of HIV-1.

Meteorological data rescue: Citizen science lessons learned from Southern Weather Discovery.

Daily weather reconstructions (called "reanalyses") can help improve our understanding of meteorology and long-term climate changes. Adding undigitized historical weather observations to the datasets that underpin reanalyses is desirable; however, time requirements to capture those data from a range of archives is usually limited. Southern Weather Discovery is a citizen science data rescue project that recovered tabulated handwritten meteorological observations from ship log books and land-based stations spanning New Zealand, the Southern Ocean, and Antarctica. We describe the Zooniverse-hosted Southern Weather Discovery campaign, highlight promotion tactics, and replicate keying levels needed to obtain 100% complete transcribed datasets with minimal type 1 and type 2 transcription errors. Rescued weather observations can augment optical character recognition (OCR) text recognition libraries. Closer links between citizen science data rescue and OCR-based scientific data capture will accelerate weather reconstruction improvements, which can be harnessed to mitigate impacts on communities and infrastructure from weather extremes.

Molecular aspects of biomineralization of the echinoderm endoskeleton.

Echinoderms possess a rigid endoskeleton composed of calcite and small amounts of occluded organic matrix proteins. The test (i.e., the shell-like structure of adults), spines, pedicellariae, tube feet, and teeth of adults, as well as delicate endoskeletal spicules found in larvae of some classes, are the main skeletal structures. They have been intensively studied for insight into the mechanisms of biomineralization. Recent work on characterization of the mineral phase and occluded proteins in embryonic skeletal spicules shows that these simple-looking structures contain scores of different proteins, and that the mineral phase is composed of amorphous calcium carbonate (ACC), which then transforms to an anhydrous ACC and eventually to calcite. Likewise, the adult tooth shows a similar transition from hydrated ACC to anhydrous ACC to calcite during its formation, and a similar transition is likely occurring during adult spine regeneration. We speculate that: (1) the ACC precursor is a general strategy employed in biomineralization in echinoderms, (2) the numerous occluded proteins play a role in post-secretion formation of the mature biomineralized structure, and (3) proteins with "multi-valent" intrinsically disordered domains are important for formation of occluded matrix structures, and regulation of crucial matrix-mineral interactions, such as ACC to calcite transitions and polymorph selection.

Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule.

Sea urchin larval spicules transform amorphous calcium carbonate (ACC) into calcite single crystals. The mechanism of transformation is enigmatic: the transforming spicule displays both amorphous and crystalline properties, with no defined crystallization front. Here, we use X-ray photoelectron emission spectromicroscopy with probing size of 40-200 nm. We resolve 3 distinct mineral phases: An initial short-lived, presumably hydrated ACC phase, followed by an intermediate transient form of ACC, and finally the biogenic crystalline calcite phase. The amorphous and crystalline phases are juxtaposed, often appearing in adjacent sites at a scale of tens of nanometers. We propose that the amorphous-crystal transformation propagates in a tortuous path through preexisting 40- to 100-nm amorphous units, via a secondary nucleation mechanism.

Asprich peptides are occluded in calcite and permanently disorder biomineral crystals.

Macromolecules are a minority but important component of the minerals formed by living organisms, or biominerals. The role these macromolecules play at the early stages of biomineral formation, as well as their long-term and long-range effects on the mature biomineral, is poorly understood. A 42-amino acid peptide, asp2, was derived from the Asprich family of proteins. In this study we present X-ray absorption near-edge structure spectroscopy and X-ray photoelectron emission microscopy data from the asp2 peptide, the calcite (CaCO(3)) crystals, and the peptide + crystal composites. The results clearly show that asp2 is occluded in fully formed biomineral crystals and slightly but permanently disorders the crystal structure at short- and long-range distances.

Nacre protein fragment templates lamellar aragonite growth.

Proteins play a major role in the formation of all biominerals. In mollusk shell nacre, complex mixtures and assemblies of proteins and polysaccharides were shown to induce aragonite formation, rather than the thermodynamically favored calcite (both aragonite and calcite are CaCO(3) polymorphs). Here we used N16N, a single 30 amino acid-protein fragment originally inspired by the mineral binding site of N16, a protein in the nacre layer of the Japanese pearl oysters (Pinctada fucata). In a calcite growth solution this short peptide induces in vitro biomineralization. This model biomineral was analyzed using X-ray PhotoElectron Emission spectroMicroscopy (X-PEEM) and found to be strikingly similar to natural nacre: lamellar aragonite with interspersed N16N layers. This and other findings combined suggest a hypothetical scenario in which in vivo three proteins (N16, Pif80, and Pif97) and a polysaccharide (chitin) work in concert to form lamellar nacre.

Evaluation of a risk assessment questionnaire to assist hepatitis C screening in the general population.

Many individuals with hepatitis C virus (HCV) infection are undiagnosed. This study evaluates a risk assessment questionnaire, developed for use online to target blood-screening for HCV. Two hundred and eighty-nine patients with known HCV status completed a written questionnaire on prominent HCV risk factors. Questionnaires generated advice to seek testing if at least one risk factor was reported. Agreement of the testing advice with the HCV status of respondents was evaluated. Subsequently, we validated our questionnaire among 985 patients of an outpatient clinic for sexually transmitted infections. The post-test-probability-of-disease (PTPD) and diagnostic gain (PTPD minus prior probability of disease) were calculated. The questionnaire's sensitivity and specificity were 84.6% and 63.8%, respectively, and higher in the STI clinic patients. The PTPD of positive testing advice was 72.5% given HCV prevalence of 53.0%, yielding a diagnostic gain of 19.5%. Applying the estimated prevalence in the general Dutch population (0.1-0.4%), and the anticipated prevalence in the online project (1.0-6.0%), yielded diagnostic gains of 0.13-0.53% and 1.3-7.0%, respectively. We conclude that our questionnaire succeeded in selecting at-risk individuals as its testing advice agreed well with the HCV status. We suggest that the questionnaire be used online as a selection tool for HCV blood-screening in the general population.

Macroscopic and microvascular blood supply of the canine common calcaneal tendon.

Chronic tendinopathy injuries to the canine common calcaneal tendon are relatively common in large breed dogs and typically affect the distal portion of the tendon. In humans, poor blood supply, biomechanical faults, poor training methods and fluoroquinolone administration have all been linked with the development of Achilles tendinopathy. The most common sites for Achilles tendinopathy in humans seem to correspond with areas of poor blood supply within the tendon. The aim of this study was to evaluate the blood supply of the canine common calcaneal (Achilles) tendon to determine if variations occur along the tendon. The null hypothesis was that there would be no difference in the microvascular blood supply at varying points along the tendon. Paired pelvic limbs were collected from 12 large breed dog cadavers. A 50% barium sulphate and 50% saline solution was infused into the femoral artery of one limb from each dog and radiographs were taken to outline the blood supply to the common calcaneal tendon. Indian ink was infused into the contralateral limb. The common calcaneal tendon was removed, fixed and sectioned at 1 cm intervals, from calcaneal insertion to musculotendinous junction. The ink-filled arteries and arterioles in each section were counted. Radiographs revealed fine branches from the caudal saphenous artery entering the mid-body of the tendon along its cranial border. The musculotendinous junction had additional branches from the gastrocnemius muscles. Distally, vessels radiated proximally from the calcaneus 2 to 3 cm into the tendon. Mean total vessel counts at the insertion (138.54 +/- SD 31.06) were significantly higher than all other sections (p <0.001). The mid-body had significantly lower total vessel counts. When the cross sectional area of the tendon was taken into account, only the insertion had a significantly higher mean vessel count/cm2 than the mid-body of the tendon. There were no other significant differences in mean vessel count/cm2. Areas of poorer blood supply did not correspond with the most commonly reported site for chronic common calcanean tendinopathies, suggesting that inherent poor blood supply at the site of injury may not play a role in the pathogenesis. Atraumatic handling and minimal manipulation should be used during the surgical approach and debridement to preserve the remaining blood supply in ruptured tendons.

General regression model for the subdistribution of a competing risk under left-truncation and right-censoring.

Left-truncation poses extra challenges for the analysis of complex time-to-event data. We propose a general semiparametric regression model for left-truncated and right-censored competing risks data that is based on a novel weighted conditional likelihood function. Targeting the subdistribution hazard, our parameter estimates are directly interpretable with regard to the cumulative incidence function. We compare different weights from recent literature and develop a heuristic interpretation from a cure model perspective that is based on pseudo risk sets. Our approach accommodates external time-dependent covariate effects on the subdistribution hazard. We establish consistency and asymptotic normality of the estimators and propose a sandwich estimator of the variance. In comprehensive simulation studies we demonstrate solid performance of the proposed method. Comparing the sandwich estimator with the inverse Fisher information matrix, we observe a bias for the inverse Fisher information matrix and diminished coverage probabilities in settings with a higher percentage of left-truncation. To illustrate the practical utility of the proposed method, we study its application to a large HIV vaccine efficacy trial dataset.

Loa loa filariasis in a tropical savanna area: report of one case in Ouagadougou.

Loa loa filariasis is usually found in the forest areas of Central and West Africa. We report a case that was diagnosed in Ouagadougou (Burkina Faso), a savanna area. The patient lived in Gabon but was visiting his family in Ouagadougou. He complained of fatigue, fever, itchy legs with scratch marks, and intermittent edema of the legs. A blood smear was first examined for malaria parasites, but Loa loa microfilariae were observed. Laboratory tests showed hypereosinophilia (30%). Transient angioedema (Calabar edema) was observed. Loa loa filariasis was diagnosed based on these findings. There were no other laboratory test abnormalities, and ophthalmological examination was normal. The patient received a single dose of ivermectin at 200 µg/kg. After 1 month, the patient's course was favorable and a control blood smear was negative.