Differentiation of hypervirulent and classical Klebsiella pneumoniae with acquired drug resistance.

Distinguishing hypervirulent (hvKp) from classical Klebsiella pneumoniae (cKp) strains is important for clinical care, surveillance, and research. Some combinations of iucA, iroB, peg-344, rmpA, and rmpA2 are most commonly used, but it is unclear what combination of genotypic or phenotypic markers (e.g., siderophore concentration, mucoviscosity) most accurately predicts the hypervirulent phenotype. Furthermore, acquisition of antimicrobial resistance may affect virulence and confound identification. Therefore, 49 K. pneumoniae strains that possessed some combinations of iucA, iroB, peg-344, rmpA, and rmpA2 and had acquired resistance were assembled and categorized as hypervirulent hvKp (hvKp) (N = 16) or cKp (N = 33) via a murine infection model. Biomarker number, siderophore production, mucoviscosity, virulence plasmid's Mash/Jaccard distances to the canonical pLVPK, and Kleborate virulence score were measured and evaluated to accurately differentiate these pathotypes. Both stepwise logistic regression and a CART model were used to determine which variable was most predictive of the strain cohorts. The biomarker count alone was the strongest predictor for both analyses. For logistic regression, the area under the curve for biomarker count was 0.962 (P = 0.004). The CART model generated the classification rule that a biomarker count = 5 would classify the strain as hvKP, resulting in a sensitivity for predicting hvKP of 94% (15/16), a specificity of 94% (31/33), and an overall accuracy of 94% (46/49). Although a count of ≥4 was 100% (16/16) sensitive for predicting hvKP, the specificity and accuracy decreased to 76% (25/33) and 84% (41/49), respectively. These findings can be used to inform the identification of hvKp.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a concerning pathogen that can cause life-threatening infections in otherwise healthy individuals. Importantly, although strains of hvKp have been acquiring antimicrobial resistance, the effect on virulence is unclear. Therefore, it is of critical importance to determine whether a given antimicrobial resistant K. pneumoniae isolate is hypervirulent. This report determined which combination of genotypic and phenotypic markers could most accurately identify hvKp strains with acquired resistance. Both logistic regression and a machine-learning prediction model demonstrated that biomarker count alone was the strongest predictor. The presence of all five of the biomarkers iucA, iroB, peg-344, rmpA, and rmpA2 was most accurate (94%); the presence of ≥4 of these biomarkers was most sensitive (100%). Accurately identifying hvKp is vital for surveillance and research, and the availability of biomarker data could alert the clinician that hvKp is a consideration, which, in turn, would assist in optimizing patient care.

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.

Differentiation of hypervirulent and classical Klebsiella pneumoniae with acquired drug resistance.

Distinguishing hypervirulent (hvKp) from classical Klebsiella pneumoniae (cKp) strains is important for clinical care, surveillance, and research. Some combination of iucA, iroB, peg-344, rmpA, and rmpA2 are most commonly used, but it is unclear what combination of genotypic or phenotypic markers (e.g. siderophore concentration, mucoviscosity) most accurately predicts the hypervirulent phenotype. Further, acquisition of antimicrobial resistance may affect virulence and confound identification. Therefore, 49 K. pneumoniae strains that possessed some combination of iucA, iroB, peg-344, rmpA, and rmpA2 and had acquired resistance were assembled and categorized as hypervirulent hvKp (hvKp) (N=16) or cKp (N=33) via a murine infection model. Biomarker number, siderophore production, mucoviscosity, virulence plasmid's Mash/Jaccard distances to the canonical pLVPK, and Kleborate virulence score were measured and evaluated to accurately differentiate these pathotypes. Both stepwise logistic regression and a CART model were used to determine which variable was most predictive of the strain cohorts. The biomarker count alone was the strongest predictor for both analyses. For logistic regression the area under the curve for biomarker count was 0.962 (P = 0.004). The CART model generated the classification rule that a biomarker count = 5 would classify the strain as hvKP, resulting in a sensitivity for predicting hvKP of 94% (15/16), a specificity of 94% (31/33), and an overall accuracy of 94% (46/49). Although a count of ≥ 4 was 100% (16/16) sensitive for predicting hvKP, the specificity and accuracy decreased to 76% (25/33) and 84% (41/49) respectively. These findings can be used to inform the identification of hvKp. Importance: Hypervirulent Klebsiella pneumoniae (hvKp) is a concerning pathogen that can cause life-threatening infections in otherwise healthy individuals. Importantly, although strains of hvKp have been acquiring antimicrobial resistance, the effect on virulence is unclear. Therefore, it is of critical importance to determine whether a given antimicrobial resistant K. pneumoniae isolate is hypervirulent. This report determined which combination of genotypic and phenotypic markers could most accurately identify hvKp strains with acquired resistance. Both logistic regression and a machine-learning prediction model demonstrated that biomarker count alone was the strongest predictor. The presence of all 5 of the biomarkers iucA, iroB, peg-344, rmpA, and rmpA2 was most accurate (94%); the presence of ≥ 4 of these biomarkers was most sensitive (100%). Accurately identifying hvKp is vital for surveillance and research, and the availability of biomarker data could alert the clinician that hvKp is a consideration, which in turn would assist in optimizing patient care.

Early diagnosis of sickle cell disease at birth hospitals and vaccination centers in Angola using point-of-care tests.

Sickle cell disease (SCD) is a life-threatening blood disorder affecting >500 000 infants annually, mostly in sub-Saharan Africa. Most infants do not have access to an early diagnosis and die early from treatable complications of SCD. Universal newborn screening (NBS) is not yet available in any African country for a variety of reasons, including lack of laboratory capacity, difficulty in tracking affected infants, and the relatively short stay of mothers and newborns at maternity hospitals. Several point-of-care (POC) tests for SCD have been recently developed and validated, but the 2 most well-established tests (Sickle SCAN and HemoTypeSC) have not been rigorously compared with one another. In this study, we aimed to evaluate and compare these 2 POC tests to screen infants aged ≤6 months in Luanda, Angola. Challenging the traditional NBS paradigm, we performed testing not only at maternity centers, but also at vaccination centers across Luanda. We enrolled 2000 babies and performed 1000 tests with each POC test. Both tests demonstrated diagnostic accuracy, with 98.3% of Sickle SCAN results and 95.3% of HemoTypeSC results aligning with the gold standard isoelectric focusing hemoglobin pattern. When the result was provided at the POC, 92% of infants were linked to SCD care compared with 56% in the pilot Angolan NBS program, which used centralized laboratory testing. This study demonstrates the real-world feasibility and accuracy of POC tests to screen infants for SCD in Angola. This study also suggests that including vaccination centers may improve the capture rate for early infant SCD screening programs.

Prevalence of Iron Deficiency and Iron-Deficiency Anemia in US Females Aged 12-21 Years, 2003-2020.

This study examines prevalence of iron deficiency among females aged 12 to 21 years to inform future screening strategies for iron deficiency and iron-deficiency anemia.

Rethinking Care Models for Young Adults With Sickle Cell Disease.

This Viewpoint discusses developing new sickle cell disease adult care models to support specialized health care homes that are patient-focused and antiracist, rather than an approach focused mainly on financial incentives.

Hydroxyurea for children with sickle cell disease in sub-Saharan Africa: A summary of the evidence, opportunities, and challenges.

Sickle cell disease (SCD) is a common and life-threatening inherited blood disorder that affects more than 300,000 newborns per year. Because of the origins of the sickle gene mutation as a protective mechanism against malaria for those with sickle cell trait, more than 90% of annual SCD births are in sub-Saharan Africa (sSA). Over the past several decades, there have been many important advances in the care of individuals with SCD, including early diagnosis through newborn screening programs (NBS), prophylactic penicillin, the development of vaccines to prevent invasive bacterial infections, and the emergence of hydroxyurea as the primary disease-modifying pharmacologic therapy. These relatively simple and inexpensive interventions have significantly reduced the morbidity and mortality of sickle cell anemia (SCA) such that individuals with SCD can live longer and more complete lives. Unfortunately, although these interventions are relatively inexpensive and evidence-based, they are only readily available for affected individuals living in high-income settings, representing <5% of the global SCD population. In sSA, where >90% of the global SCD burden exists, SCD still results in early mortality with 50-90% of infants likely dying before reaching 5 years of age. Recently, there are an increasing number of efforts in many African countries to prioritize SCA with pilot NBS programs, improved diagnostics, and expanded SCD education for health care professionals and the general public. It is essential to include access to hydroxyurea as a core component of any SCD care program, but there are still many barriers that prevent the widespread global use of hydroxyurea. Here, we summarize what we know about SCD and hydroxyurea use in Africa and discuss a strategy to respond to what we consider to be a public health imperative to maximize access to and appropriate use of hydroxyurea for all individuals with SCD using innovative dosing and monitoring strategies.

Design of a Bacteriophage Cocktail Active against Shigella Species and Testing of Its Therapeutic Potential in Galleria mellonella.

Shigellosis is a leading global cause of diarrheal disease and travelers' diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival.

IS26-mediated plasmid reshuffling results in convergence of toxin-antitoxin systems but loss of resistance genes in XDR Klebsiella pneumoniae from a chronic infection.

Carbapenem-resistant Enterobacterales pose an urgent threat to human health worldwide. Klebsiella pneumoniae sequence type (ST) 14, initially identified in the Middle East and South-Asia and co-harbouring the carbapenemase genes bla OXA-232 and bla NDM-1, is now emerging globally. One such strain was detected in the USA in 2013 from a patient initially treated in India that also carried armA, a 16S rRNA methyltransferase that confers resistance to all clinically relevant aminoglycosides. Genetic and phenotypic changes were observed in 14 serial isolates collected from this chronically infected patient. The index isolate carried five plasmids, including an IncFIB-IncHI1B (harbouring armA and bla NDM-1), an IncFIA (bla CTX-M-15) and a ColE-like (bla OXA-232), and was extensively resistant to antibiotics. Four years later, a subsequent isolate had accumulated 34 variants, including a loss-of-function mutation in romA, resulting in tigecycline non-susceptibility. Importantly, this isolate now only carried two plasmids, including a large mosaic molecule made of fragments, all harbouring distinct toxin-antitoxin systems, from three of the canonical plasmids. Of the original acquired antibiotic resistance genes, this isolate only retained bla CTX-M-15, and as a result susceptibility to the carbapenems and amikacin was restored. Long-read sequencing of a subset of five representative isolates, collected between 2013 and 2017, allowed for the elucidation of the complex plasmid patterns and revealed the role of IS26-mediated plasmid reshuffling in the evolution of this clone. Such investigations of the mechanisms underlying plasmid stability, together with global and local surveillance programmes, are key to a better understanding of plasmid host range and dissemination.

Mechanisms of IS26-Mediated Amplification of the aphA1 Gene Leading to Tobramycin Resistance in an Acinetobacter baumannii Isolate.

Enhanced levels of resistance to antibiotics arising from amplification of an antibiotic resistance gene that impact therapeutic options are increasingly observed. Amplification can also disclose novel phenotypes leading to treatment failure. However, the mechanism is poorly understood. Here, the route to amplification of the aphA1 kanamycin and neomycin resistance gene during tobramycin treatment of an Acinetobacter baumannii clinical isolate, leading to tobramycin resistance and treatment failure, was investigated. In the tobramycin-susceptible parent isolate, MRSN56, a single copy of aphA1 is present in the pseudocompound transposon PTn6020, bounded by directly oriented copies of IS26. For two clinical resistant isolates, new long-read sequence data were combined with available short-read data to complete the genomes. Comparison to the completed genome of MRSN56 revealed that, in both cases, IS26 had generated a circular translocatable unit (TU) containing PTn6020 and additional adjacent DNA. In one case, this TU was reincorporated into the second product generated by the deletion that formed the TU via the targeted conservative route and amplified about 7 times. In the second case, the TU was incorporated at a new location via the copy-in route and amplified about 65 times. Experimental amplification ex vivo by subjecting MRSN56 to tobramycin selection pressure yielded different TUs, which were incorporated at either the original location or a new location and amplified many times. The outcomes suggest that when IS26 is involved, amplification occurs via rolling circle replication of a newly formed TU coupled to the IS26-mediated TU formation or reincorporation step. IMPORTANCE Heteroresistance, a significant issue that is known to impact antibiotic treatment outcomes, is caused by the presence of spontaneously arising cells with elevated levels of resistance to therapeutically important antibiotics in a population of susceptible cells. Gene amplification is one well-documented cause of heteroresistance, but precisely how extensive amplification occurs is not understood. Here, we establish the case for the direct involvement of IS26 activity in the amplification of the aphA1 gene to disclose resistance to tobramycin. The aphA1 gene is usually found associated with IS26 in Gram-negative pathogens and is commonly found in extensively resistant Acinetobacter baumannii strains. IS26 and related IS cause adjacent deletions, forming a nonreplicating circular molecule known as a translocatable unit (TU), and amplification via a rolling circle mechanism appears to be coupled to either IS26-mediated TU formation or reincorporation. Related IS found in Gram-positive pathogens may play a similar role.

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.

Identification and Characterization of vB_PreP_EPr2, a Lytic Bacteriophage of Pan-Drug Resistant Providencia rettgeri.

Providencia rettgeri is an emerging opportunistic Gram-negative pathogen with reports of increasing antibiotic resistance. Pan-drug resistant (PDR) P. rettgeri infections are a growing concern, demonstrating a need for the development of alternative treatment options which is fueling a renewed interest in bacteriophage (phage) therapy. Here, we identify and characterize phage vB_PreP_EPr2 (EPr2) with lytic activity against PDR P. rettgeri MRSN 845308, a clinical isolate that carries multiple antibiotic resistance genes. EPr2 was isolated from an environmental water sample and belongs to the family Autographiviridae, subfamily Studiervirinae and genus Kayfunavirus, with a genome size of 41,261 base pairs. Additional phenotypic characterization showed an optimal MOI of 1 and a burst size of 12.3 ± 3.4 PFU per bacterium. EPr2 was determined to have a narrow host range against a panel of clinical P. rettgeri strains. Despite this fact, EPr2 is a promising lytic phage with potential for use as an alternative therapeutic for treatment of PDR P. rettgeri infections.

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.

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.

Enablers and barriers to newborn screening for sickle cell disease in Africa: results from a qualitative study involving programmes in six countries.

OBJECTIVES: Given the fundamental role of newborn bloodspot screening (NBS) to enable prompt diagnosis and optimal clinical management of individuals with sickle cell disease (SCD), we sought to systematically assess enablers and barriers to implementation of NBS programmes for SCD in Africa using established qualitative research methods. SETTING: Childbirth centres and NBS laboratories from six countries in East, West and Southern Africa. PARTICIPANTS: Eight programme leaders involved with establishing and operating NBS programmes for SCD in Angola, Democratic Republic of Congo, Ghana, Liberia, Nigeria and Tanzania. PRIMARY AND SECONDARY OUTCOME MEASURES: Data obtained through a structured, phased interview approach were analysed using a combination of inductive and deductive codes and used to determine primary themes related to the implementation and sustainability of SCD NBS programmes. RESULTS: Four primary themes emerged from the analysis relating to governance (eg, pragmatic considerations when deploying overcommitted clinical staff to perform NBS), technical (eg, design and execution of operational processes), cultural (eg, variability of knowledge and perceptions of community-based staff) and financial (eg, issues that can arise when external funding may effectively preclude government inputs) aspects. Key learnings included perceived factors that contribute to long-term NBS programme sustainability. CONCLUSIONS: The establishment of enduring NBS programmes is a proven approach to improving the health of populations with SCD. Organising such programmes in Africa is feasible, but initial implementation does not assure sustainability. Our analysis suggests that future programmes should prioritise government partner participation and funding from the earliest stages of programme development.

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.