Inherited disorders of hemoglobin: A review of old and new diagnostic methods.

The genetic regulation of hemoglobin is complex and there are a number of genetic abnormalities that result in clinically important hemoglobin disorders. Here, we review the molecular pathophysiology of hemoglobin disorders and review both old and new methods of diagnosing these disorders. Timely diagnosis of hemoglobinopathies in infants is essential to coordinate optimal life-saving interventions, and accurate identification of carriers of deleterious mutations allows for genetic counseling and informed family planning. The initial laboratory workup of inherited disorders of hemoglobin should include a complete blood count (CBC) and peripheral blood smear, followed by carefully selected tests based on clinical suspicion and available methodology. We discuss the utility and limitations of the various methodologies to fractionate hemoglobin, including cellulose acetate and citrate agar hemoglobin electrophoresis, isoelectric focusing, high-resolution high-performance liquid chromatography, and capillary zone electrophoresis. Recognizing that most of the global burden of hemoglobin disorders exists in low- and middle-income countries, we review the increasingly available array of point-of-care-tests (POCT), which have an increasingly important role in expanding early diagnosis programs to address the global burden of sickle cell disease, including Sickle SCAN, HemoTypeSC, Gazelle Hb Variant, and Smart LifeLC. A comprehensive understanding of the molecular pathophysiology of hemoglobin and the globin genes, as well as a clear understanding of the utility and limitations of currently available diagnostic tests, is essential in reducing global disease burden.

Distribution and expression of the aac(6')-Im (aacA16) aminoglycoside resistance gene.

BACKGROUND: The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. OBJECTIVES: To identify sources for the aac(6')-Im fragment found in A. baumannii. METHODS: MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. RESULTS: Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different. CONCLUSIONS: The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.

Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy.

A protracted outbreak of New Delhi metallo-ß-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Saving lives through early diagnosis: the promise and role of point of care testing for sickle cell disease.

Sickle cell disease (SCD) is a devastating and under-recognised global child health issue affecting over 300,000 infants annually, with the highest prevalence in India and sub-Saharan Africa. Most affected infants born in low- and middle-income countries (LMIC) lack access to SCD testing and die from complications in the first years of life without a formal diagnosis. The majority of deaths are preventable with early diagnosis and provision of inexpensive interventions. Despite global recognition of the urgent need, expansion of SCD newborn screening (NBS) programmes beyond the pilot stage has been obstructed by a dependence on an expensive and logistically challenging centralised laboratory testing model. Recently, several point-of-care tests (POCT) for SCD have been developed with promising field validation studies. Here, we summarise the state of POCT for SCD, review barriers and unanswered questions, and discuss optimal strategies for utilising POCT to address the growing global burden of SCD. There is an urgent need to prospectively evaluate the ability of POCT to reduce the morbidity and high early mortality of SCD. To impact a sustainable reduction to this end, it is essential to link a diagnosis with comprehensive SCD care, including wide and affordable access to affordable hydroxycarbamide therapy.

Hydroxyurea for Children with Sickle Cell Anemia in Sub-Saharan Africa.

BACKGROUND: Hydroxyurea is an effective treatment for sickle cell anemia, but few studies have been conducted in sub-Saharan Africa, where the burden is greatest. Coexisting conditions such as malnutrition and malaria may affect the feasibility, safety, and benefits of hydroxyurea in low-resource settings. METHODS: We enrolled children 1 to 10 years of age with sickle cell anemia in four sub-Saharan countries. Children received hydroxyurea at a dose of 15 to 20 mg per kilogram of body weight per day for 6 months, followed by dose escalation. The end points assessed feasibility (enrollment, retention, and adherence), safety (dose levels, toxic effects, and malaria), and benefits (laboratory variables, sickle cell-related events, transfusions, and survival). RESULTS: A total of 635 children were fully enrolled; 606 children completed screening and began receiving hydroxyurea at a mean (±SD) dose of 17.5±1.8 mg per kilogram per day. The retention rate was 94.2% at 3 years of treatment. Hydroxyurea therapy led to significant increases in both the hemoglobin and fetal hemoglobin levels. Dose-limiting toxic events regarding laboratory variables occurred in 5.1% of the participants, which was below the protocol-specified threshold for safety. During the treatment phase, 20.6 dose-limiting toxic effects per 100 patient-years occurred, as compared with 20.7 events per 100 patient-years before treatment. As compared with the pretreatment period, the rates of clinical adverse events decreased with hydroxyurea use, including rates of vaso-occlusive pain (98.3 vs. 44.6 events per 100 patient-years; incidence rate ratio, 0.45; 95% confidence interval [CI], 0.37 to 0.56), nonmalaria infection (142.5 vs. 90.0 events per 100 patient-years; incidence rate ratio, 0.62; 95% CI, 0.53 to 0.72), malaria (46.9 vs. 22.9 events per 100 patient-years; incidence rate ratio, 0.49; 95% CI, 0.37 to 0.66), transfusion (43.3 vs. 14.2 events per 100 patient-years; incidence rate ratio, 0.33; 95% CI, 0.23 to 0.47), and death (3.6 vs. 1.1 deaths per 100 patient-years; incidence rate ratio, 0.30; 95% CI, 0.10 to 0.88). CONCLUSIONS: Hydroxyurea treatment was feasible and safe in children with sickle cell anemia living in sub-Saharan Africa. Hydroxyurea use reduced the incidence of vaso-occlusive events, infections, malaria, transfusions, and death, which supports the need for wider access to treatment. (Funded by the National Heart, Lung, and Blood Institute and others; REACH ClinicalTrials.gov number, NCT01966731 .).

Complete genome of the extensively antibiotic-resistant GC1 Acinetobacter baumannii isolate MRSN 56 reveals a novel route to fluoroquinolone resistance.

OBJECTIVES: To examine the causes of antibiotic resistance in the extensively resistant global clone 1 (GC1) Acinetobacter baumannii isolate MRSN 56 recovered at a US military treatment facility. METHODS: MRSN 56 was sequenced using MinION (Oxford Nanopore) and the reads combined with available Illumina MiSeq data using Unicycler. Acquired resistance genes were identified using ABRicate and their environment examined. ISAba1 and ISAba125 copies were located. RESULTS: MRSN 56 is ST1IP:ST231Ox:KL1:OCL1 and the complete genome includes four small plasmids, none of which carry resistance genes. The acquired resistance genes were found at four locations in the chromosome in addition to AbaR28 (aphA1, aacC1, aadA1, sul1) in comM. Tn2006 (oxa23, carbapenem resistance) was both in AbaR4 and alone elsewhere. Two copies of Tn7 (dfrA1, sat, aadA1) were identified. One was associated with a 22 852 bp adjacent segment [tetA(B), sul2] derived from the AbGRI1 island, and this novel configuration was designated Tn7+. Tn7+ was incorporated in the position preferred by Tn7, downstream of glmS, by transposition using a sequence in AbGRI1 resembling the Tn7 terminal inverted repeats. Tn7 was found at a secondary site. Fluoroquinolone resistance appears to involve a mutation in gyrA combined with inactivation by ISAba1 of the marR gene in the mar operon and constitutive expression of marA from the promoter internal to ISAba1. CONCLUSIONS: MRSN 56 represents a new sublineage of GC1 lineage 1 with novel features that had not been detected previously. The involvement of the mar operon in fluoroquinolone resistance has not been noted previously.

Early hydroxyurea use is neuroprotective in children with sickle cell anemia.

Children with sickle cell disease (SCD) who began hydroyxurea before age five years scored no differently on a measure of cognitive funciton than age, sex, and race-matched unaffected peers.

Impact of hydroxyurea dose and adherence on hematologic outcomes for children with sickle cell anemia.

BACKGROUND: Hydroxyurea is the primary treatment for sickle cell anemia (SCA), yet real-world implementation in high-income settings is suboptimal. Variation in prescribed hydroxyurea dose and patient adherence in these settings can both affect actual exposure to hydroxyurea. Quantifying the contributions of hydroxyurea dose and medication adherence to the relationship between hydroxyurea exposure and hematologic parameters could inform strategies to optimize exposure and improve outcomes. PROCEDURE: We evaluated the relationship between hydroxyurea exposure, defined by average prescribed dose and adherence, and hematologic parameters using data from children with SCA who were enrolled in two prospective hydroxyurea adherence studies. Hydroxyurea adherence was assessed by video directly observed therapy or electronic pill bottle and medication administration record. Average prescribed dose was abstracted from prescriptions in patients' electronic medical record. Participants with a hydroxyurea exposure >20 mg/kg/day and ≤20 mg/kg/day were included in the higher and lower exposure groups, respectively. RESULTS: Forty-five participants were included in the analysis (56% male; median age 12 years [range 2-19]; 98% Black). Higher exposed participants (n = 23) were prescribed a higher dose (27.2 vs. 24.4 mg/kg/day, p = .002) and had better adherence (0.92 vs. 0.71, p ≤ .001) compared to lower exposed participants (n = 22). Higher exposure was associated with higher fetal hemoglobin (p = .04) and mean corpuscular volume (p = .02). CONCLUSIONS: Higher hydroxyurea exposure is associated with improved hematologic parameters in the high-income setting and is affected by both prescribed dose and adherence. Future studies are needed to optimize both adherence and hydroxyurea prescribing and confirm that increasing exposure improves clinical outcomes in this setting.

Early initiation of hydroxyurea (hydroxycarbamide) using individualised, pharmacokinetics-guided dosing can produce sustained and nearly pancellular expression of fetal haemoglobin in children with sickle cell anaemia.

Hydroxyurea (hydroxycarbamide) is an effective treatment for sickle cell anaemia (SCA), but clinical responses depend primarily upon the degree of fetal haemoglobin (HbF) induction and the heterogeneity of HbF expression across erythrocytes. The number and characteristics of HbF-containing cells (F-cells) are not assessed by traditional HbF measurements. Conventional hydroxyurea dosing (e.g. fixed doses or low starting doses with stepwise escalation) produces a moderate heterocellular HbF induction, but haemolysis and clinical complications continue. Robust, pancellular HbF induction is needed to minimise or fully inhibit polymerisation of sickle haemoglobin. We treated children with hydroxyurea using an individualised, pharmacokinetics-guided regimen starting at predicted maximum tolerated dose (MTD). We observed sustained HbF induction (mean >30%) for up to 6 years, which was not dependent on genetic determinants of HbF expression. Nearly 70% of patients had ≥80% F-cells (near-pancellular), and almost half had ≥90% F-cells (pancellular). The mean HbF/F-cell content was ~12 pg. Earlier age of initiation and better medication adherence were associated with high F-cell responses. In summary, early initiation of hydroxyurea using pharmacokinetics-guided starting doses at predicted MTD can achieve sustained near-pancellular or pancellular HbF expression and should be considered an achievable goal for children with SCA treated with hydroxyurea at optimal doses. Clinical trial registration number: NCT02286154 (clinicaltrials.gov).

Academic Challenges and School Service Utilization in Children with Sickle Cell Disease.

OBJECTIVES: To describe the academic concerns and risk strata of children with sickle cell disease (SCD) as identified through a parent-directed screening tool and to compare the rates of these concerns with actual school service utilization in the clinic population. STUDY DESIGN: We completed a retrospective review of patients with SCD referred to the school intervention program during the 2017-2018 and 2018-2019 school years because of a school-related concern raised by parents or noted by the clinical team. All parents completed the Brief School Needs Inventory (BSNI), a validated parent-response tool used to stratify academic risk. Rates of special education services, grade retention, and results from neuropsychologic testing were captured. Clinical history, the use of disease-modifying therapy, and results from laboratory and neuroimaging studies were also obtained. Descriptive statistics were performed to examine demographic information, clinical history, and BSNI results. RESULTS: In total, 137 unique patients (age range, 14 months to 19 years) completed the BSNI during the study period, for 181 events. According to BSNI risk-stratification, 45% of patients were deemed low, 36% moderate, and 19% high academic risk. Over one-half of parents were concerned about their ability to advocate for their child's needs. Despite legal qualification for a Section 504 accommodation plan, only 20% had established plans. Academic concerns were common with 31% of children reporting an individualized education program and 20% with grade retention/remediation. CONCLUSIONS: Concerns for academic challenges remain high among parents of children with SCD; however, school service utilization remains disproportionately low attributable to numerous reasons.

Hydroyxurea improves cerebral oxygen saturation in children with sickle cell anemia.

Neurologic complications are common in patients with sickle cell anemia (SCA), but conventional tools such as MRI and transcranial Doppler ultrasonography (TCD) do not fully assess cerebrovascular pathology. Cerebral tissue oximetry measures mixed oxygen saturation in the frontal lobes (SCT O2 ) and provides early prognostic information about tissue at risk of ischemic injury. Untreated patients with SCA have significantly lower SCT O2 than healthy controls that declines with age. Hydroxyurea is effective in preventing many SCA-related complications, but the degree to which it preserves normal neurophysiology is unclear. We analyzed participants enrolled in the Therapeutic Response Evaluation and Adherence Trial (TREAT, NCT02286154), which enrolled participants initiating hydroxyurea using individualized dosing (new cohort) and those previously taking hydroxyurea (old cohort) and was designed to monitor the long-term benefits of hydroxyurea. Cerebral oximetry was performed at baseline and annually. For the new cohort (median starting age = 12 months, n = 55), mean baseline SCT O2 was normal before starting hydroxyurea (mean 65%, 95% CI 58-72%) and significantly increased after 2 years (mean 72%, 95% CI 65-79%, p < .001). The SCT O2 for patients receiving long-term hydroxyurea (median age = 9.6 years) was normal at study entry (mean 66%, 95% CI 58-74%) and remained stable across 2 years. Both cohorts had significantly higher SCT O2 than published data from predominantly untreated SCA patients. Cerebral oximetry is a non-invasive method to assess cerebrovascular pathology that complements conventional imaging. Our results indicate that hydroxyurea suggests protection against neurophysiologic changes seen in untreated SCA.

How I approach disease-modifying therapy in children with sickle cell disease in an era of novel therapies.

Finally,after decades of stagnation, the therapeutic landscape for sickle cell disease (SCD) is changing with an increasing number of novel therapeutics. Hydroxyurea remains the primary disease-modifying therapy and, when started early in life with maintenance of an optimal dose, can reduce many SCD-related complications. To complement hydroxyurea, there are a growing number of pharmacologic options with additional efforts focused on the development and optimization of curative therapies. Here, we review current treatment options and provide recommendations as to how to approach the treatment of children and adolescents within this evolving therapeutic landscape to allow for full and healthy lives.

A retrospective study of the safety and efficacy of low molecular weight iron dextran for children with iron deficiency anemia.

BACKGROUND: Iron deficiency anemia (IDA) affects millions of children worldwide. Oral iron replacement is effective but often poorly tolerated. Intravenous iron has been demonstrated to have utility in all ages, but pediatric use remains limited. Low molecular weight iron dextran (LMWID) has a dosing range capable of replacing iron deficits in a single infusion and has been evaluated in small pediatric cohorts, but additional safety and efficacy data are limited. Here, we evaluate the safety and efficacy of LMWID in association with an electronic medical record (EMR)-based effort to optimize dosing. PROCEDURE: A retrospective IRB-approved investigation of LMWID utilization at a tertiary pediatric hospital between January 1, 2016 and March 31, 2020 was undertaken to evaluate the therapeutic efficacy and frequency/severity of infusion-related adverse event (AE) in children and adolescents receiving LMWID. Patient demographics and LMWID dosing characteristics were collected, and primary outcome measures included laboratory response and the incidence/severity of any infusion-related events. The utilization of an EMR-based nomogram for LMWID dosing was also evaluated. RESULTS: A total of 254 infusions for 191 patients were included (ages 0.7-20.9 years), most with IDA. LMWID replaced at least 75% of the estimated iron deficit in a single infusion for 76% of patients. The mean hemoglobin and ferritin increases were 2.1 g/dl and >100 ng/ml, respectively. Infusion-related AEs were rare, occurring in only 12/254 (4.7%) of infusions and 67% during the test dose; each rapidly resolved without long-term sequelae. No AEs occurred in those <10 years of age. Premedication use markedly decreased with nomogram use without a change in AE rate. CONCLUSIONS: In a large institutional cohort, LMWID was well tolerated in children and adolescents, with most patients having their total iron deficits relieved in a single infusion. These data support expanded use of LMWID in the management of pediatric iron deficiency.

Implementation of near-universal hydroxyurea uptake among children with sickle cell anemia: A single-center experience.

BACKGROUND: Without early initiation of disease-modifying therapy, the acute and chronic complications of sickle cell anemia (SCA) begin early in childhood and progress throughout life. Hydroxyurea is a safe and effective medication that reduces or prevents most SCA-related complications. Despite recommendations to prescribe hydroxyurea for all children with SCA as young as 9 months, utilization remains low. PROCEDURE: We completed a retrospective review of hydroxyurea-prescribing practices and associated clinical outcomes at our institution over a 10-year period before and after the 2014 National Heart, Lung, and Blood Institute (NHLBI) recommendations to use hydroxyurea for all children with SCA. RESULTS: Hydroxyurea use more than doubled within our pediatric SCA population from 43% in 2010 to 95% in 2019. The age of hydroxyurea initiation was significantly younger during 2014-2019 compared to 2010-2013 (median 2 years vs. 6 years, p ≤ .001). With this change in clinical practice, nearly all (69/71 = 97%) children born after 2013 received disease-modifying therapy by the end of 2019, primarily hydroxyurea (93%). Concurrently, the number of SCA-related admissions significantly decreased from 67/100 patient-years in 2010 to 39/100 patient-years in 2019 (p < .001). CONCLUSION: The early and universal prescription of hydroxyurea for children with SCA is the standard of care. Here, we demonstrate that a careful and deliberate commitment to follow this guideline in clinical practice is feasible and results in measurable improvements in clinical outcomes. Our approach and improved outcomes can serve as a model for other programs to expand their hydroxyurea use for more children with SCA.

AnemoCheck-LRS: an optimized, color-based point-of-care test to identify severe anemia in limited-resource settings.

BACKGROUND: Severe anemia is common and frequently fatal for hospitalized patients in limited-resource settings. Lack of access to low-cost, accurate, and rapid diagnosis of anemia impedes the delivery of life-saving care and appropriate use of the limited blood supply. The WHO Haemoglobin Colour Scale (HCS) is a simple low-cost test but frequently inaccurate. AnemoCheck-LRS (limited-resource settings) is a rapid, inexpensive, color-based point-of-care (POC) test optimized to diagnose severe anemia. METHODS: Deidentified whole blood samples were diluted with plasma to create variable hemoglobin (Hb) concentrations, with most in the severe (≤ 7 g/dL) or profound (≤ 5 g/dL) anemia range. Each sample was tested with AnemoCheck-LRS and WHO HCS independently by three readers and compared to Hb measured by an electronic POC test (HemoCue 201+) and commercial hematology analyzer. RESULTS: For 570 evaluations within the limits of detection of AnemoCheck-LRS (Hb ≤ 8 g/dL), the average difference between AnemoCheck-LRS and measured Hb was 0.5 ± 0.4 g/dL. In contrast, the WHO HCS overestimated Hb with an absolute difference of 4.9 ± 1.3 g/dL for samples within its detection range (Hb 4-14 g/dL, n = 405). AnemoCheck-LRS was much more sensitive (92%) for the diagnosis of profound anemia than WHO HCS (22%). CONCLUSIONS: AnemoCheck-LRS is a rapid, inexpensive, and accurate POC test for anemia. AnemoCheck-LRS is more accurate than WHO HCS for detection of low Hb levels, severe anemia that may require blood transfusion. AnemoCheck-LRS should be tested prospectively in limited-resource settings where severe anemia is common, to determine its utility as a screening tool to identify patients who may require transfusion.

Non-transfusion-dependent ß-Thalassemia Because of a Single ß-Thalassemia Mutation and Coinherited α-Globin Gene Triplication: Need for Increased Awareness to Prevent Incorrect and Delayed Diagnosis.

The thalassemias are genetically complex and usually autosomal recessive. We describe 5 unrelated individuals with non-transfusion-dependent ß-thalassemia (NTDT), some with apparently dominant transmission, because of a single ß-thalassemia mutation coinherited with a triplicated α-globin locus. Each had an initial, incorrect diagnosis of ß-thalassemia trait. The correct diagnosis of NTDT was made at a mean of 7 years of age. Despite reports of this compound genotype causing NTDT, it remains unfamiliar to many clinicians. To increase awareness, we highlight its varied and sometimes subtle clinical and laboratory features and the need for comprehensive genetic testing for timely and correct diagnosis.

Identification of the OXA-48 Carbapenemase Family by Use of Tryptic Peptides and Liquid Chromatography-Tandem Mass Spectrometry.

Phenotypic detection of the OXA-48-type class D ß-lactamases in Enterobacteriaceae is challenging. We describe a rapid (less than 90 min) assay for the identification of OXA-48 family carbapenemases in subcultured bacterial isolates based on a genoproteomic approach. Following in silico trypsin digestion to ascertain theoretical core peptides common to the OXA-48 family, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data-dependent acquisition was used to identify candidate peptide markers. Two peptides were selected based on performance characteristics: ANQAFLPASTFK, a core peptide common to all 12 OXA-48 family ß-lactamase members, and YSVVPVYQEFAR, a highly specific peptide common to 11 of 12 OXA-48 family proteins providing the basis for an LC-MS/MS multiple reaction monitoring assay. An accuracy assessment was performed that included 98 isolates, 26 of which were OXA-48 positive. Two additional specificity assessments were performed including a mixture of isolates positive for OXA-48, KPC, NDM, VIM, and IMP carbapenemases. A combination of expert rules and expert judgment was applied by blinded operators to identify positive isolates. All isolates containing an OXA-48 family carbapenemase across all three test sets were correctly identified with no false positives, demonstrating 100% sensitivity (95% confidence interval [CI], 91.2% to 100%) and 100% specificity (95% CI, 96.2% to 100%) for the assay. These findings provide a framework for an LC-MS/MS-based method for the direct detection of OXA-48 family carbapenemases from cultured isolates that may have utility in predicting carbapenem resistance and tracking hospital outbreaks of OXA-48-carrying organisms.

Rapid detection of colistin resistance protein MCR-1 by LC-MS/MS.

BACKGROUND: Colistin (polymyxin E) and polymixin B are important bactericidal antibiotics used in the treatment of serious infections caused by multi-drug resistant Gram-negative organisms. Transferrable plasmid-mediated colistin resistance, conferred by the product of the mcr-1 gene, has emerged as a global healthcare threat. Consequently, the rapid detection of the MCR-1 protein in clinical bacterial isolates has become increasingly important. We used a genoproteomic approach to identify unique peptides of the MCR-1 protein that could be detected rapidly by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: MCR-1 tryptic peptides that were efficiently ionized and readily detectable were characterized in a set of mcr-1-containing isolates with triple quadrupole LC-MS. Three optimal peptides were selected for the development of a rapid multiple reaction monitoring LC-MS/MS assay for the MCR-1 protein. To investigate the feasibility of rapid detection of the MCR-1 protein in bacterial isolates using this assay, a blinded 99-sample test set was built that included three additional mcr-1-containing clinical isolates tested in triplicate (9 samples) and 90 negative control isolates. RESULTS: All of the mcr-1-containing isolates in the test set were accurately identified with no false positive detections by three independent, blinded operators, yielding an overall performance of 100% sensitivity and specificity for multiple operators. Among the three peptides tested in this study, the best performing was DTFPQLAK. The isolate-to-result time for the assay as implemented is less than 90 min. CONCLUSIONS: This work demonstrates the feasibility of rapid detection of the MCR-1 protein in bacterial isolates by LC-MS/MS.

Robust clinical and laboratory response to hydroxyurea using pharmacokinetically guided dosing for young children with sickle cell anemia.

Hydroxyurea is FDA-approved and now increasingly used for children with sickle cell anemia (SCA), but dosing strategies, pharmacokinetic (PK) profiles, and treatment responses for individual patients are highly variable. Typical weight-based dosing with step-wise escalation to maximum tolerated dose (MTD) leads to predictable laboratory and clinical benefits, but often takes 6 to 12 months to achieve. The Therapeutic Response Evaluation and Adherence Trial (TREAT, NCT02286154) was a single-center study designed to prospectively validate a novel personalized PK-guided hydroxyurea dosing strategy with a primary endpoint of time to MTD. Enrolled participants received a single oral 20 mg/kg dose of hydroxyurea, followed by a sparse PK sampling approach with three samples collected over three hours. Analysis of individual PK data into a population PK model generated a starting dose that targets the MTD. The TREAT cohort (n = 50) was young, starting hydroxyurea at a median age of 11 months (IQR 9-26 months), and PK-guided starting doses were high (27.7 ± 4.9 mg/kg/d). Time to MTD was 4.8 months (IQR 3.3-9.3), significantly shorter than comparison studies (p < 0.0001), thus meeting the primary endpoint. More remarkably, the laboratory response for participants starting with a PK-guided dose was quite robust, achieving higher hemoglobin (10.1 ± 1.3 g/dL) and HbF (33.3 ± 9.1%) levels than traditional dosing. Though higher than traditional dosing, PK-guided doses were safe without excess hematologic toxicities. Our data suggest early initiation of hydroxyurea, using a personalized dosing strategy for children with SCA, provides laboratory and clinical response beyond what has been seen historically, with traditional weight-based dosing.

Hereditary elliptocytosis-associated alpha-spectrin mutation p.L155dup as a modifier of sickle cell disease severity.

The broad phenotypic variability among individuals with sickle cell disease (SCD) suggests the presence of modifying factors. We identified two unrelated SCD patients with unusually severe clinical and laboratory phenotype that were found to carry the hereditary elliptocytosis-associated alpha-spectrin mutation c.460_462dupTTG (p.L155dup), a mutation enriched due to positive selective pressure of malaria, similar to the SCD globin mutations. A high index of suspicion for additional hematologic abnormalities may be indicated for challenging patients with SCD. These cases highlight the validity of specialized testing such as ektacytometry and next-generation sequencing for patients and family members to assess genotype/phenotype correlations.