Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi.

BACKGROUND: Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(-NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology. RESULTS: Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids. CONCLUSION: Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.

Analysis of sulfur and nitrogen odorants using solid-phase microextraction and GC-MS.

An analytical method involving solid-phase micro-extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was applied to analyze biosolids odors. A selective ion monitoring (SIM)-based MS method was developed, using SPME injections of odorant standards under the full-scan mode to select the quantification and confirmation ions for each odorant. The odorants analyzed in this study include: dimethylsulfide, dimethyldisulfide, methyl mercaptan, hydrogen sulfide, carbon disulfide, trimethylamine and dimethylamine. We have used this method to quantify parts-per-billion levels of odorant vapors produced during anaerobic incubation of digested wastewater sludge. Important considerations for expedient and accurate calibration under static and dynamic flow conditions are discussed. The SPME-GC-MS method may give a positive intercept in the calibration curve, especially under static sampling conditions, which sets a practical detection limit for odor analysis.

Cardiac and noncardiac malformations: observations in a population-based study.

A regional case-control study of congenital cardiovascular malformations (CCVMs) searches for all live-born infants in the community in whom the cardiac diagnosis has been confirmed by echocardiography, cardiac catheterization, surgery, or autopsy. Their families are studied in comparison to those of a representative sample of resident live-born infants. Detailed descriptions of noncardiac abnormalities are obtained from physician reports and maternal interviews expanded by medical record and death certificate data. Among 1,494 cases and 1,572 controls, chromosomal abnormalities, syndromes, heritable disorders, and suspect syndromes occurred with an overwhelming excess in cases (chromosomes, P less than 10(-4); syndromes/heritable disorders, P less than .005). Abnormalities affecting chromosomes 13, 18, and 21 constituted 93% of the cytogenetic defects. Syndromes and heritable disorders were of 39 types. Nonsyndromic abnormalities were three times more frequent in cases than in controls (P less than .005). Case excesses occurred for central nervous system malformations, eye disorders, major abdominal wall defects, and abnormalities of the alimentary and urinary tracts. Severe anomalies frequent among cases were those which also occur in certain recognized syndromes, and it is suggested that paired combinations of cardiac and other midline anomalies may represent "formes frustes" of syndromes with similar though variable phenotypic expressions. Cleft lip and palate, inguinal hernia, and lower limb anomalies occurred with equal frequency, suggesting their association with CCVMs by chance alone.

Congenital heart disease: prevalence at livebirth. The Baltimore-Washington Infant Study.

The Baltimore-Washington Infant Study is a regional epidemiologic study of congenital heart disease. Among Infants born in the study area in 1981 and 1982, 664 had a diagnosis of congenital heart disease confirmed in the first year of life by echocardiography, cardiac catheterization, cardiac surgery, or autopsy. The prevalence rate was 3.7/1,000 livebirths for all cases and 2.4/1,000 livebirths for cases confirmed by invasive methods only. Diagnosis-specific prevalence rates of congenital heart disease are compared with those of eight previous case series. Changing diagnostic categorizations in the time span covered and methodological differences resulted in great variation of the data. However, the data of the New England Infant Cardiac Program which used the same case discovery methods showed similar occurrences of major morphologic abnormalities, suggesting that these are stable basic estimates in the eastern United States. For all case series, the rate of confirmed congenital heart disease was approximately 4/1,000 livebirths over the 40-year time span.

Hematologic disorders and congenital cardiovascular malformations: converging lines of research.

In a population-based study on congenital cardiovascular malformations (CCVM), the occurrence of heritable coagulopathies among case parents and not among controls raised the possibility of an etiologic association of CCVM with blood disorders. The literature was searched for evidence that such an association could be biologically plausible. Reported embryologic and clinical data provided confirmatory findings. The heart and blood arise from common angiogenic cells; endothelial cells, the first components of the primitive heart, synthesize coagulation factors; resultant osmotic alterations of embryonic fluids could alter early cardiac morphogenesis. Bleeding diatheses are common in cyanotic and acyanotic patients with CCVM and hemostatic disorders have been reported in some families. CCVM and blood disorders are joint components of several malformation syndromes. The hypothesis of an etiologic relationship between HBD and CCVM needs to be tested in multiple research areas. Future experimental studies should be based on current theories of cardiac morphogenesis to include investigations of embryonic blood in genetic blood disorders. Clinical studies should clarify hematologic alterations in CCVM probands and their families.

Incidence of acute rheumatic fever. A suburban community hospital experience during the 1970s.

The incidence of acute rheumatic fever has declined remarkably in the past three decades, but the disease has not been eradicated, as some physicians believe. This study documents the diagnosis of the disease in 23 middle-class children from Fairfax County, Virginia, during the 11-year-period from 1970 through 1980. This county has one of the highest median family incomes in the United States. The annual age-adjusted incidence rate of initial attacks of acute rheumatic fever per 100,000 Fairfax County children declined from 3.0 in 1970 to 0.5 in 1980. In six of the 23 children, carditis accompanied by monarticular arthritis or arthralgia was present. Another seven children had carditis plus polyarthritis. Two patients had Sydenham's chorea. None of the children had erythema marginatum or rheumatoid nodules. In two children, symptoms of acute rheumatic fever developed two weeks after they finished a 10-day course of penicillin for Group A streptococcal pharyngitis. The remaining 21 children had not been considered ill enough to be brought to medical care prior to development of symptoms of acute rheumatic fever.

Successful treatment of supraventricular tachycardia with metoprolol, a cardioselective beta blocker.

Paroxysmal supraventricular tachycardia is a common problem in infancy and childhood. Past treatment has included digitalis and nonselective beta blockers (propranolol). We describe a new mode of therapy with a cardioselective beta blocker (metoprolol) that may be of use in patients resistant to standard therapy.

Echocardiographic measurements in normal subjects. Growth-related changes that occur between infancy and early adulthood.

Echocardiographic measurements of the left ventricular dimensions and wall thicknesses at end diastole and end systole, aortic root and left atrial dimensions, mitral valve E-F slope, left ventricular ejection fraction, percent fractional shortening of the left ventricular internal dimension, estimated left ventricular mass and percentage systolic thickening of the ventricular septum and left ventricular free wall were obtained in 105 normal subjects ranging from one day to 23 years of age. Each parameter was found to follow a linear regression upon one of three functions of the body surface area. The internal dimensions of left ventricle, the left atrium, and the aortic root, and the mitral valve E-F slope varied in a linear relation to the cube root of the body surface area. Thickness of the ventricular septum and left ventricular free wall varied in a linear relation to the square root of the body surface area. Estimated left ventricular mass varied linearly with the direct measurement of body surface area. Ejection fraction, percent fractional shortening of the left ventricle and percent systolic thickening of the ventricular septum and left ventricular free wall were independent of body surface area despite a marked increase in the size of the left ventricle during normal growth and development.

The postpericardiotomy and similar syndromes.

A syndrome of pericardial and often pleural reaction with effusions and with fever occurs in three different settings. It bears the descriptive term of the condition which it follows: the postpericardiotomy syndrome, the postmyocardial infarction syndrome, and the postpericardial trauma syndrome. The manifestations, incidence, theories of etiology, treatment, and possibility of prevention were discussed. The syndrome is probably due to an immunologic response with an associated viral illness, since in a prospective study of children undergoing cardiac surgery in which the pericardium was entered, we found that the development of heart-reactive antibody in high titer and a significant rise in antiviral antibody occurred in patients with clinically evident syndrome.