Detection and Strain Typing of Ancient Mycobacterium leprae from a Medieval Leprosy Hospital

Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.     

Osteological,Biomolecular and Geochemical Examination of an Early Anglo-Saxon Case of Lepromatous Leprosy

We have examined a 5th to 6th century inhumation from Great Chesterford, Essex, UK. The incomplete remains are those of a young male, aged around 21–35 years at death. The remains show osteological evidence of lepromatous leprosy (LL) and this was confirmed by lipid biomarker analysis and ancient DNA (aDNA) analysis, which provided evidence for both multi-copy and single copy loci from the Mycobacterium leprae genome. Genotyping showed the strain belonged to the 3I lineage, but the Great Chesterford isolate appeared to be ancestral to 3I strains found in later medieval cases in southern Britain and also continental Europe. While a number of contemporaneous cases exist, at present, this case of leprosy is the earliest radiocarbon dated case in Britain confirmed by both aDNA and lipid biomarkers. Importantly, Strontium and Oxygen isotope analysis suggest that the individual is likely to have originated from outside Britain. This potentially sheds light on the origins of the strain in Britain and its subsequent spread to other parts of the world, including the Americas where the 3I lineage of M. leprae is still found in some southern states of America.     

Distance to Care,Facility Delivery and Early Neonatal Mortality in Malawi and Zambia

Background

Globally, approximately 3 million babies die annually within their first month. Access to adequate care at birth is needed to reduce newborn as well as maternal deaths. We explore the influence of distance to delivery care and of level of care on early neonatal mortality in rural Zambia and Malawi, the influence of distance (and level of care) on facility delivery, and the influence of facility delivery on early neonatal mortality.

Methods and Findings

National Health Facility Censuses were used to classify the level of obstetric care for 1131 Zambian and 446 Malawian delivery facilities. Straight-line distances to facilities were calculated for 3771 newborns in the 2007 Zambia DHS and 8842 newborns in the 2004 Malawi DHS. There was no association between distance to care and early neonatal mortality in Malawi (OR 0.97, 95%CI 0.58–1.60), while in Zambia, further distance (per 10 km) was associated with lower mortality (OR 0.55, 95%CI 0.35–0.87). The level of care provided in the closest facility showed no association with early neonatal mortality in either Malawi (OR 1.02, 95%CI 0.90–1.16) or Zambia (OR 1.02, 95%CI 0.82–1.26). In both countries, distance to care was strongly associated with facility use for delivery (Malawi: OR 0.35 per 10km, 95%CI 0.26–0.46). All results are adjusted for available confounders. Early neonatal mortality did not differ by frequency of facility delivery in the community.

Conclusions

While better geographic access and higher level of care were associated with more frequent facility delivery, there was no association with lower early neonatal mortality. This could be due to low quality of care for newborns at health facilities, but differential underreporting of early neonatal deaths in the DHS is an alternative explanation. Improved data sources are needed to monitor progress in the provision of obstetric and newborn care and its impact on mortality.     

Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean

Background

Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship.

Methodology/Principal Findings

We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9250-8160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex.

Conclusions/Significance

Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.     

Going Wireless: Fe(III) Oxide Reduction without Pili by Geobacter sulfurreducens Strain JS-1

Previous studies have suggested that the conductive pili of Geobacter sulfurreducens are essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every 3 days, the wild-type strain outcompeted the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA being continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron shuttle-producing Fe(III) reducers in many anaerobic soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current, consistent with the concept that long-range electron transport through G. sulfurreducens biofilms is more effective via pili.     

Where There Is No Toilet: Water and Sanitation Environments of Domestic and Facility Births in Tanzania

Background

Inadequate water and sanitation during childbirth are likely to lead to poor maternal and newborn outcomes. This paper uses existing data sources to assess the water and sanitation (WATSAN) environment surrounding births in Tanzania in order to interrogate whether such estimates could be useful for guiding research, policy and monitoring initiatives.

Methods

We used the most recent Tanzania Demographic and Health Survey (DHS) to characterise the delivery location of births occurring between 2005 and 2010. Births occurring in domestic environments were characterised as WATSAN-safe if the home fulfilled international definitions of improved water and improved sanitation access. We used the 2006 Service Provision Assessment survey to characterise the WATSAN environment of facilities that conduct deliveries. We combined estimates from both surveys to describe the proportion of all births occurring in WATSAN-safe environments and conducted an equity analysis based on DHS wealth quintiles and eight geographic zones.

Results

42.9% (95% confidence interval: 41.6%–44.2%) of all births occurred in the woman''s home. Among these, only 1.5% (95% confidence interval: 1.2%–2.0%) were estimated to have taken place in WATSAN-safe conditions. 74% of all health facilities conducted deliveries. Among these, only 44% of facilities overall and 24% of facility delivery rooms were WATSAN-safe. Combining the estimates, we showed that 30.5% of all births in Tanzania took place in a WATSAN-safe environment (range of uncertainty 25%–42%). Large wealth-based inequalities existed in the proportion of births occurring in domestic environments based on wealth quintile and geographical zone.

Conclusion

Existing data sources can be useful in national monitoring and prioritisation of interventions to improve poor WATSAN environments during childbirth. However, a better conceptual understanding of potentially harmful exposures and better data are needed in order to devise and apply more empirical definitions of WATSAN-safe environments, both at home and in facilities.     

A finite-element model of tracheal collapse

The trachea has been approximated by an appropriate finite-element model. The three-dimensional equilibrium problems set by the tracheal deformation under various stresses have been solved using a convenient augmented Lagrangian functional. The dimensions were obtained from human tracheae. Mechanical constants for the anatomic components were calculated from the stress-strain relationships. The compressive narrowing is essentially due to the invagination of the posterior membrane in the tracheal lumen for transmural pressures down to -7 kPa. A surface of contact between the membranous wall and the lateral walls appears when the transmural pressure equals -6 kPa. The transmural pressure-area relationship is sigmoidal with a compliance equal to 0.08 kPa-1 for a transmural pressure of -2 kPa. The tracheal collapse is greater when the material constants of the membranous wall decrease or when the tracheal segment is subjected to a longitudinal tension. A slight flexion of the trachea induces an asymmetric deformation.     

Dependence of central airway resistance on frequency and tidal volume: a model study

The resistance of a hollow cast of human central airways was measured during true sinusoidal airflow oscillations over a wide range of frequencies (0.5-40 Hz) and for various flow amplitudes up to 8 l/s. Pressure and flow were measured in the trachea with high-performance transducers, digitized and averaged over 100 cycles. Data were studied at two points in the flow cycle: at peak inspiratory and expiratory flows and in the two neighborhoods around zero flow where airway resistance (Rv approximately equal to o) was taken as the average slope of the pressure-flow (P-V) curve in each zone. When data obtained near peak flow were plotted in terms of dimensionless pressure drop vs. peak Reynolds number (Rem) and compared with steady-state data, we found no difference up to 2 Hz as previously reported (Isabey and Chang, J. Appl. Physiol. 51: 1338-1348, 1981), a slight decay in pressure drop between 4 and 8 Hz, a frequency-dependent increase in peak flow resistance at high frequencies (10-40 Hz) governed by the Strouhal number alpha 2/Rem beyond alpha 2/Rem = 0.5. On the other hand RV approximately equal to o was found to increase relative to steady state as local acceleration increases, e.g., as peak flow increases at a fixed frequency; this differs from the classical linear theory of oscillatory flow in a long straight tube. To explain these results, we had to use, as in our previous study, an alternative expression for the Strouhal number, i.e., epsilon = L X A X (dV/dt)/V2 (where L and A are the length and cross-sectional area of the trachea and V is a constant flow range over which resistance around flow reversal was computed), which accurately reflects the ratio of local acceleration [d(V/A)/dt)] to convective acceleration [(V/A)2/L] in developing branching flow. Finally, to delineate the regions of dominance of each of the dimensionless parameters, we compiled frequency-tidal volume diagrams for peak flows as well as for reversal. Epsilon, which is negligible near peak flows, appeared to govern the oscillatory P-V relationship near flow reversal in a transitional region of the diagram located between regions of steadiness, or moderate unsteadiness, and a region of dominant unsteadiness governed by alpha.