Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping

Background

Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage.

Methods

A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates.

Results

Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently identified serotypes were 11A, 13, 15B/15C, 16F and 10A.

Conclusion

The rmPCR assay performed well for the 21 serotypes/-groups included in the assay. However, in our study setting, a large proportion of serotypes were not detected by rmPCR. The sequetyping assay performed well, but did misassign specific serotypes. It may be useful for regions where vaccine serotypes are less common, however confirmatory testing is advisable.     

Avoiding bias in calculations of relative growth rate

In classical growth analysis, relative growth rate (RGR) is calculated as RGR = (ln W2 - ln W1)/(t2 - t1), where W1 and W2 are plant dry weights at times t1 and t2. Since RGR is usually calculated using destructive harvests of several individuals, an obvious approach is to substitute W1 and W2 with sample means W1 and W2. Here we demonstrate that this approach yields a biased estimate of RGR whenever the variance of the natural logarithm-transformed plant weight changes through time. This bias increases with an increase in the variance in RGR, in the length of the interval between harvests, or in sample size. The bias can be avoided by using the formula RGR = (ln W2 - ln W1)/(t2 - t1),where ln W1 and ln W2 are the means of the natural logarithm-transformed plant weights.     

Convenient fluorescence-based methods to measure membrane potential and intracellular pH in the Archaeon Methanobacterium thermoautotrophicum

New and improved methods to determine the membrane potential (Delta Psi) and the Delta pH in methanogenic archaea were developed and tested in Methanobacterium thermoautotrophicum strain Delta H. The Delta pH measurements took advantage of the pH-dependent fluorescence properties of coenzyme F(420), the major intracellular electron carrier in the organism. The protonophore p-nitrophenol did not show any interference with the F(420) fluorescence spectra and was therefore suitable to equalize internal and external pH. The method developed allowed the determination of the intracellular pH with an error of less than 0.05 pH units.Membrane potentials could easily be assessed using the fluorescent probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)) with an accuracy of approximately 10 mV.Both methods were tested with cell suspensions of M. thermoautrophicum incubated at medium pH values between 5.5 and 8. It was found that Delta Psi and Delta pH values remained constant under these conditions. Membrane potentials were about -160 mV and Delta pH was kept at 0.35 pH units (inside minus outside) resulting in a total proton motive force of about -180 mV (inside negative).     

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

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Successional shifts in tree demographic strategies in wet and dry Neotropical forests

Aim

Tropical forest succession and associated changes in community composition are driven by species demographic rates, but how demographic strategies shift during succession remains unclear. Our goal was to identify generalities in demographic trade-offs and successional shifts in demographic strategies across Neotropical forests that cover a large rainfall gradient and to test whether the current conceptual model of tropical forest succession applies to wet and dry forests.

Location

Mexico and Central America.

Time period

1985–2018.

Major taxa studied

Trees.

Methods

We used repeated forest inventory data from two wet and two dry forests to quantify demographic rates of 781 tree species. For each forest, we explored the main demographic trade-offs and assigned tree species to five demographic groups by performing a weighted principal components analysis to account for differences in sample size. We aggregated the basal area and abundance across demographic groups to identify successional shifts in demographic strategies over the entire successional gradient from very young (<5 years) to old-growth forests.

Results

Across all forests, we found two demographic trade-offs, namely the growth–survival trade-off and the stature–recruitment trade-off, enabling the data-driven assignment of species to five demographic strategies. Fast species dominated early in succession and were then replaced by long-lived pioneers in three forests. Intermediate and slow species increased in basal area over succession in all forests, but, in contrast to the current conceptual model, long-lived pioneers continued to dominate until the old-growth stage in all forests. The basal area of short-lived breeders was low across all successional stages.

Main conclusions

The current conceptual model of Neotropical forest succession should be revised to incorporate the dominance of long-lived pioneers in late-successional and old-growth forests. Moreover, the definition of consistent demographic strategies that show clear dominance shifts across succession substantially improves the mechanistic understanding and predictability of Neotropical forest succession.     

Warming,drought, and disturbances lead to shifts in functional composition: A millennial-scale analysis for Amazonian and Andean sites

Tropical forests are changing in composition and productivity, probably in response to changes in climate and disturbances. The responses to these multiple environmental drivers, and the mechanisms underlying the changes, remain largely unknown. Here, we use a functional trait approach on timescales of 10,000 years to assess how climate and disturbances influence the community-mean adult height, leaf area, seed mass, and wood density for eight lowland and highland forest landscapes. To do so, we combine data of eight fossil pollen records with functional traits and proxies for climate (temperature, precipitation, and El Niño frequency) and disturbances (fire and general disturbances). We found that temperature and disturbances were the most important drivers of changes in functional composition. Increased water availability (high precipitation and low El Niño frequency) generally led to more acquisitive trait composition (large leaves and soft wood). In lowland forests, warmer climates decreased community-mean height probably because of increased water stress, whereas in highland forests warmer climates increased height probably because of upslope migration of taller species. Disturbance increased the abundance of acquisitive, disturbance-adapted taxa with small seeds for quick colonization of disturbed sites, large leaves for light capture, and soft wood to attain fast height growth. Fire had weak effects on lowland forests but led to more stress-adapted taxa that are tall with fast life cycles and small seeds that can quickly colonize burned sites. Site-specific analyses were largely in line with cross-site analyses, except for varying site-level effects of El Niño frequency and fire activity, possibly because regional patterns in El Niño are not a good predictor of local changes, and charcoal abundances do not reflect fire intensity or severity. With future global changes, tropical Amazonian and Andean forests may transition toward shorter, drought- and disturbance-adapted forests in the lowlands but taller forests in the highlands.     

Photosynthetic induction responses of two rainforest tree species in relation to light environment

Photosynthetic induction of in situ saplings of two Costa Rican rainforest tree species wre compared in relation to their light environment, using infrared gas analysis and hemispherical photography. The species studied were Dipteryx panamensis, a climax species found in bright microsites, and Cecropia obtusifolia, a pioneer species. In the morning, when leaves were most responsive, induction time necessary to reach 90% of the lightsaturated rate of photosynthesis was on average 16 min for Dipteryx and 10 min for Cecropia. However, induction times for both species increased in the afternoon resulting in shorter daily average induction times for Dipteryx than for Cecropia. Dipteryx also maintained higher levels of induction for a longer period under low light conditions than did Cecropia. The two species differed in the way they adjusted to light availability. Dipteryx saplings growing in shady sites had faster rates of induction than saplings growing in bright sites, with no difference in light-saturated photosynthetic rate. In contrast, Cecropia saplings growing in bright sites had higher light-saturated photosynthetic rates than saplings growing in shady sites, with no difference in rates of induction. Dipteryx appears to exploit temporal variation in light availability by refining the quickness of the induction response to the light environment, while Cecropia adjusts its scale of exploitation by realizing a higher lightsaturated photosynthetic rate in sites of higher light.     

Relationships among ecologically important dimensions of plant trait variation in seven neotropical forests

BACKGROUND AND AIMS: When ecologically important plant traits are correlated they may be said to constitute an ecological 'strategy' dimension. Through identifying these dimensions and understanding their inter-relationships we gain insight into why particular trait combinations are favoured over others and into the implications of trait differences among species. Here we investigated relationships among several traits, and thus the strategy dimensions they represented, across 2134 woody species from seven Neotropical forests. METHODS: Six traits were studied: specific leaf area (SLA), the average size of leaves, seed and fruit, typical maximum plant height, and wood density (WD). Trait relationships were quantified across species at each individual forest as well as across the dataset as a whole. 'Phylogenetic' analyses were used to test for correlations among evolutionary trait-divergences and to ascertain whether interspecific relationships were biased by strong taxonomic patterning in the traits. KEY RESULTS: The interspecific and phylogenetic analyses yielded congruent results. Seed and fruit size were expected, and confirmed, to be tightly related. As expected, plant height was correlated with each of seed and fruit size, albeit weakly. Weak support was found for an expected positive relationship between leaf and fruit size. The prediction that SLA and WD would be negatively correlated was not supported. Otherwise the traits were predicted to be largely unrelated, being representatives of putatively independent strategy dimensions. This was indeed the case, although WD was consistently, negatively related to leaf size. CONCLUSIONS: The dimensions represented by SLA, seed/fruit size and leaf size were essentially independent and thus conveyed largely independent information about plant strategies. To a lesser extent the same was true for plant height and WD. Our tentative explanation for negative WD-leaf size relationships, now also known from other habitats, is that the traits are indirectly linked via plant hydraulics.