Identification and Epidemiological Typing of Campylobacter strains isolated from Patients in Finland

C. jejuni constitutes the majority of Campylobacter strains isolated from patients in Finland, and C. coli strains are also reported. To improve the species identification, a combination of phenotype- and genotype-based methods was applied. Standardising the cell suspension turbidity in the hippurate hydrolysis test enabled the reliable identification of hippurate-positive Campylobacter strains as C. jejuni. The detection of species-specific genes by PCR showed that about 30% of the hippurate-negative strains were C. jejuni. Three typing methods, serotyping, PCR-RFLP analysis of LOS biosynthesis genes and pulsed-field gel electrophoresis (PFGE) were evaluated as epidemiological typing tools for C. jejuni. The high number of non-typeable strains lowered the discriminatory ability of serotyping. PCR-RFLP typing offered high discrimination for both serotypeable and non-typeable strains, but the correlation between serotypes and RFLP-types was not high enough to enable its use for molecular serotyping of non-typeable strains. PFGE was a highly discriminative typing method. Although the use of two restriction enzymes generally increases the discriminatory ability, KpnI alone offered almost as high discrimination as the use of SmaI and KpnI. The characteristic seasonal distribution of Campylobacter infections with a peak in summer and low incidence in winter was mainly due to domestically acquired infections. Of the C. jejuni strains, 41% were of domestic origin compared to only 17% of the C. coli strains. Serotypes Pen 12, Pen 6,7 and Pen 27 were significantly associated with domestic C. jejuni infections, Pen 1,44, Pen 3 and Pen 37 with travel-related infections. Pen 2 and Pen 4-complex were common both in domestic and travel-related infections. Serotype Pen 2 was less common among patients 60 years or older than in younger patients, more prevalent in Western Finland than in other parts of the country and more prevalent than other serotypes in winter. The source of Pen 2 infections may be related to cattle, since Pen 2 is the most common serotype in isolates from Finnish cattle. PFGE subtypes among isolates from patients and chickens during the summer 2003 and from cattle during the whole year were compared. The analysis of indistinguishable SmaI/KpnI subtypes suggested that up to 31% of the human infections may have been mediated by chickens and 19% by cattle. Human strains isolated during two one-year sampling periods were studied by PFGE. Of the domestic strains, 69% belonged to SmaI subtypes found during both sampling periods. Four SmaI subtypes accounted for 45% of the domestic strains, further typing of these subtypes by KpnI revealed six temporally persistent SmaI/KpnI subtypes. They were only occasionally identified in travel-related strains, and therefore, can be considered to be national subtypes. Each subtype was associated with a serotype: Pen 2, Pen 12, Pen 27, Pen 4-complex, Pen 41, and Pen 57. Five of these subtypes were identified in cattle (S5/K27, S7/K1, S7/K2, S7/K5 and S64/K19), and two in chickens (S7/K1 and S64/K19) with a temporal association with human infections in 2003. Cattle are more likely potential sources of these persistent subtypes, since long-term excretion of Campylobacter strains by cattle has been reported.

Molecular methods for the epidemiological analysis of methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae are major health problems worldwide, both found in symptomless carriage but also causing even life-threatening infections. The aim of this thesis was to characterise MRSA and S. pneumoniae in detail by using several molecular typing methods for various epidemiological purposes: clonality analysis, epidemiological surveillance, outbreak investigation, and virulence factor analysis. The characteristics of MRSA isolates from the strain collection of the Finnish National Infectious Disease Register (NIDR) and pneumococcal isolates collected from military recruits and children with acute otitis media (AOM) were analysed using various typing techniques. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and the detection of Panton-Valentine leukocidin (PVL) genes were performed for MRSA isolates. Pneumococcal isolates were analysed using antimicrobial susceptibility testing, serotyping, MLST, and by detecting pilus islet 1 (PI-1) and 2 (PI-2) genes. Several international community- and hospital-associated MRSA clones were recognised in Finland. The genetic diversity among MRSA FIN-4 isolates and among FIN-16 isolates was low. Overall, MRSA blood isolates from 1997 to 2006 were genetically diverse. spa typing was found to be a highly discriminatory, rapid and accurate typing method and it also qualifies as the primary typing method in countries with a long history of PFGE-based MRSA strain nomenclature. However, additional typing by another method, e.g. PFGE, is needed in certain situations to be able to provide adequate discrimination for epidemiological surveillance and outbreak investigation. An outbreak of pneumonia was associated with one pneumococcal strain among military recruits, previously healthy young men living in a crowded setting. The pneumococcal carriage rate after the outbreak was found to be exceptionally high. PI-1 genes were detected at a rather low prevalence among pneumococcal isolates from children with AOM. However, the study demonstrated that PI-1 has existed among pneumococcal isolates prior to pneumococcal conjugate vaccine and the increased antimicrobial resistance era. Moreover, PI-1 was found to associate with the serotype rather than the genotype. This study adds to our understanding of the molecular epidemiology of MRSA strains in Finland and the importance of an appropriate genotyping method to be able to perform high-level laboratory-based surveillance of MRSA. Epidemiological and molecular analyses of S. pneumoniae add to our knowledge of the characteristics of pneumococcal strains in Finland.

Molecular and epidemiological aspects of Streptococcus pyogenes disease in Finland: Severe infections and bacterial, non-necrotizing cellulitis

Streptococcus pyogenes (group A streptococcus) is an important human pathogen, causing a wide array of infections ranging in severity. The majority of S. pyogenes infections are mild upper respiratory tract or skin infections. Severe, invasive infections, such as bacteraemia, are relatively rare, but constitute a major global burden with a high mortality. Certain streptococcal types are associated with a more severe disease and higher mortality. Bacterial, non-necrotizing cellulitis and erysipelas are localised infections of the skin, and although they are usually not life-threatening, they have a tendency to recur and therefore cause substantial morbidity. Despite several efforts aimed at developing an effective and safe vaccine against S. pyogenes infections, no vaccine is yet available. In this study, the epidemiology of invasive S. pyogenes infections in Finland was described over a decade of national, population-based surveillance. Recent trends in incidence, outcome and bacterial types were investigated. The beta-haemolytic streptococci causing cellulitis and erysipelas infections in Finland were studied in a case-control study. Bacterial isolates were characterised using both conventional and molecular typing methods, such as the emm typing, which is the most widely used typing method for beta-haemolytic streptococci. The incidence of invasive S. pyogenes disease has had an increasing trend during the past ten years in Finland, especially from 2006 onwards. Age- and sex-specific differences in the incidence rate were identified, with men having a higher incidence than women, especially among persons aged 45-64 years. In contrast, more infections occurred in women aged 25-34 years than men. Seasonal patterns with occasional peaks during the midsummer and midwinter were observed. Differences in the predisposing factors and underlying conditions of patients may contribute to these distinctions. Case fatality associated with invasive S. pyogenes infections peaked in 2005 (12%) but remained at a reasonably low level (8% overall during 2004-2007) compared to that of other developed countries (mostly exceeding 10%). Changes in the prevalent emm types were associated with the observed increases in incidence and case fatality. In the case-control study, acute bacterial non-necrotizing cellulitis was caused predominantly by Streptococcus dysgalactiae subsp. equisimilis, instead of S. pyogenes. The recurrent nature of cellulitis became evident. This study adds to our understanding of S. pyogenes infections in Finland and provides a basis for comparison to other countries and future trends. emm type surveillance and outcome analyses remain important for detecting such changes in type distribution that might lead to increases in incidence and case fatality. Bacterial characterisation serves as a basis for disease pathogenesis studies and vaccine development.

Foodborne Yersinia : Identification and Molecular Epidemiology of Isolates from Human Infections

Yersinia enterocolitica and Yersinia pseudotuberculosis are among the major enteropathogenic bacteria causing infections in humans in many industrialized countries. In Finland, Y. pseudotuberculosis has caused 10 outbreaks among humans during 1997-2008. Some of these outbreaks have been very extensive involving over 400 cases; mainly children attending schools and day-care. Y. enterocolitica, on the contrary, has caused mainly a large number of sporadic human infections in Finland. Y. pseudotuberculosis is widespread in nature, causing infections in a variety of domestic and wild animals. Foodborne transmission of human infections has long been suspected, however, attempts to trace the pathogen have been unsuccessful before this study that epidemiologically linked Y. pseudotuberculosis to a specific food item. Furthermore, due to modern food distribution systems, foodborne outbreaks usually involve many geographically separate infection clusters difficult to identify as part of the same outbreak. Among pathogenic Y. enterocolitica, the global predominance of one genetically homogeneous type (bioserotype 4/O:3) is a challenge to the development of genetic typing methods discriminatory enough for epidemiological purposes, for example, for tracing back to the sources of infections. Furthermore, the diagnostics of Y. enterocolitica infections is hampered because clinical laboratories easily misidentify some other members of the Yersinia species (Y. enterocolitica–like species) as Y. enterocolitica. This results in misleading information on the prevalence and clinical significance of various Yersinia isolates. The aim of this study was to develop and optimize molecular typing methods to be used in epidemiological investigations of Y. enterocolitica and Y. pseudotuberculosis, particularly in active surveillance and outbreak investigations of Y. pseudotuberculosis isolates. The aim was also to develop a simplified set of phenotypic tests that could be used in routine diagnostic laboratories for the correct identification of Y. enterocolitica and Y. enterocolitica –like species. A PFGE method designed here for typing of Y. pseudotuberculosis was efficient in linking the geographically dispersed and apparently unrelated Y. pseudotuberculosis infections as parts of the same outbreak. It proved to be useful in active laboratory-based surveillance of Y. pseudotuberculosis outbreaks. Throughout the study period, information about the diversity of genotypes among outbreak and non-outbreak related strains of human origin was obtained. Also, to our knowledge, this was the first study to epidemiologically link a Y. pseudotuberculosis outbreak of human illnesses to a specific food item, iceberg lettuce. A novel epidemiological typing method based on the use of a repeated genomic region (YeO:3RS) as a probe was developed for the detection and differentiation between strains of Y. enterocolitica subspecies palearctica. This method was able to increase the discrimination in a set of 106 previously PFGE typed Finnish Y. enterocolitica bioserotype 4/O:3 strains among which two main PFGE genotypes had prevailed. The developed simplified method was a more reliable tool than the commercially available biochemical test kits for differentiation between Y. enterocolitica and Y. enterocolitica –like species. In Finland, the methods developed for Y. enterocolitica and Y. pseudotuberculosis have been used to improve the identification protocols and in subsequent outbreak investigations.

Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

Univariate, bivariate, and multivariate methods in corpus-based lexicography : A study of synonymy

In this dissertation, I present an overall methodological framework for studying linguistic alternations, focusing specifically on lexical variation in denoting a single meaning, that is, synonymy. As the practical example, I employ the synonymous set of the four most common Finnish verbs denoting THINK, namely ajatella, miettiä, pohtia and harkita ‘think, reflect, ponder, consider’. As a continuation to previous work, I describe in considerable detail the extension of statistical methods from dichotomous linguistic settings (e.g., Gries 2003; Bresnan et al. 2007) to polytomous ones, that is, concerning more than two possible alternative outcomes. The applied statistical methods are arranged into a succession of stages with increasing complexity, proceeding from univariate via bivariate to multivariate techniques in the end. As the central multivariate method, I argue for the use of polytomous logistic regression and demonstrate its practical implementation to the studied phenomenon, thus extending the work by Bresnan et al. (2007), who applied simple (binary) logistic regression to a dichotomous structural alternation in English. The results of the various statistical analyses confirm that a wide range of contextual features across different categories are indeed associated with the use and selection of the selected think lexemes; however, a substantial part of these features are not exemplified in current Finnish lexicographical descriptions. The multivariate analysis results indicate that the semantic classifications of syntactic argument types are on the average the most distinctive feature category, followed by overall semantic characterizations of the verb chains, and then syntactic argument types alone, with morphological features pertaining to the verb chain and extra-linguistic features relegated to the last position. In terms of overall performance of the multivariate analysis and modeling, the prediction accuracy seems to reach a ceiling at a Recall rate of roughly two-thirds of the sentences in the research corpus. The analysis of these results suggests a limit to what can be explained and determined within the immediate sentential context and applying the conventional descriptive and analytical apparatus based on currently available linguistic theories and models. The results also support Bresnan’s (2007) and others’ (e.g., Bod et al. 2003) probabilistic view of the relationship between linguistic usage and the underlying linguistic system, in which only a minority of linguistic choices are categorical, given the known context – represented as a feature cluster – that can be analytically grasped and identified. Instead, most contexts exhibit degrees of variation as to their outcomes, resulting in proportionate choices over longer stretches of usage in texts or speech.

The epidemiology of severe Streptococcus pyogenes disease in Europe

Diseases caused by the Lancefield group A streptococcus, Streptococcus pyogenes, are amongst the most challenging to clinicians and public health specialists alike. Although severe infections caused by S. pyogenes are relatively uncommon, affecting around 3 per 100,000 of the population per annum in developed countries, the case fatality is high relative to many other infections. Despite a long scientific tradition of studying their occurrence and characteristics, many aspects of their epidemiology remain poorly understood, and potential control measures undefined. Epidemiological studies can play an important role in identifying host, pathogen and environmental factors associated with risk of disease, manifestation of particular syndromes or poor survival. This can be of value in targeting prevention activities, as well directing further basic research, potentially paving the way for the identification of novel therapeutic targets. The formation of a European network, Strep-EURO, provided an opportunity to explore epidemiological patterns across Europe. Funded by the Fifth Framework Programme of the European Commission s Directorate-General for Research (QLK2.CT.2002.01398), the Strep-EURO network was launched in September 2002. Twelve participants across eleven countries took part, led by the University of Lund in Sweden. Cases were defined as patients with S. pyogenes isolated from a normally sterile site, or non-sterile site in combination with clinical signs of streptococcal toxic shock syndrome (STSS). All participating countries undertook prospective enhanced surveillance between 1st January 2003 and 31st December 2004 to identify cases diagnosed during this period. A standardised surveillance dataset was defined, comprising demographic, clinical and risk factor information collected through a questionnaire. Isolates were collected by the national reference laboratories and characterised according to their M protein using conventional serological and emm gene typing. Descriptive statistics and multivariable analyses were undertaken to compare characteristics of cases between countries and identify factors associated with increased risk of death or development of STSS. Crude and age-adjusted rates of infection were calculated for each country where a catchment population could be defined. The project succeeded in establishing the first European surveillance network for severe S. pyogenes infections, with 5522 cases identified over the two years. Analysis of data gathered in the eleven countries yielded important new information on the epidemiology of severe S. pyogenes infections in Europe during the 2000s. Comprehensive epidemiological data on these infections were obtained for the first time from France, Greece and Romania. Incidence estimates identified a general north-south gradient, from high to low. Remarkably similar age-standardised rates were observed among the three Nordic participants, between 2.2 and 2.3 per 100,000 population. Rates in the UK were higher still, 2.9/100,000, elevated by an upsurge in drug injectors. Rates from these northern countries were reasonably close to those observed in the USA and Australia during this period. In contrast, rates of reports in the more central and southern countries (Czech Republic, Romania, Cyprus and Italy) were substantially lower, 0.3 to 1.5 per 100,000 population, a likely reflection of poorer uptake of microbiological diagnostic methods within these countries. Analysis of project data brought some new insights into risk factors for severe S. pyogenes infection, especially the importance of injecting drug users in the UK, with infections in this group fundamentally reshaping the epidemiology of these infections during this period. Several novel findings arose through this work, including the high degree of congruence in seasonal patterns between countries and the seasonal changes in case fatality rates. Elderly patients, those with compromised immune systems, those who developed STSS and those infected with an emm/M78, emm/M5, emm/M3 or emm/M1 were found to be most likely to die as a result of their infection, whereas those diagnosed with cellulitis, septic arthritis, puerperal sepsis or with non-focal infection were associated with low risk of death, as were infections occurring during October. Analysis of augmented data from the UK found use of NSAIDs to be significantly associated with development of STSS, adding further fuel to the debate surrounding the role of NSAIDs in the development of severe disease. As a largely community-acquired infection, occurring sporadically and diffusely throughout the population, opportunities for control of severe infections caused by S. pyogenes remain limited, primarily involving contact chemoprophylaxis where clusters arise. Analysis of UK Strep-EURO data were used to quantify the risk to household contacts of cases, forming the basis of national guidance on the management of infection. Vaccines currently under development could offer a more effective control programme in future. Surveillance of invasive infections caused by S. pyogenes is of considerable public health importance as a means of identifying long and short-term trends in incidence, allowing the need for, or impact of, public health measures to be evaluated. As a dynamic pathogen co-existing among a dynamic population, new opportunities for exploitation of its human host are likely to arise periodically, and as such continued monitoring remains essential.

Genotyping for genetic association studies: Methods and applications

In this thesis, two separate single nucleotide polymorphism (SNP) genotyping techniques were set up at the Finnish Genome Center, pooled genotyping was evaluated as a screening method for large-scale association studies, and finally, the former approaches were used to identify genetic factors predisposing to two distinct complex diseases by utilizing large epidemiological cohorts and also taking environmental factors into account. The first genotyping platform was based on traditional but improved restriction-fragment-length-polymorphism (RFLP) utilizing 384-microtiter well plates, multiplexing, small reaction volumes (5 µl), and automated genotype calling. We participated in the development of the second genotyping method, based on single nucleotide primer extension (SNuPeTM by Amersham Biosciences), by carrying out the alpha- and beta tests for the chemistry and the allele-calling software. Both techniques proved to be accurate, reliable, and suitable for projects with thousands of samples and tens of markers. Pooled genotyping (genotyping of pooled instead of individual DNA samples) was evaluated with Sequenom s MassArray MALDI-TOF, in addition to SNuPeTM and PCR-RFLP techniques. We used MassArray mainly as a point of comparison, because it is known to be well suited for pooled genotyping. All three methods were shown to be accurate, the standard deviations between measurements being 0.017 for the MassArray, 0.022 for the PCR-RFLP, and 0.026 for the SNuPeTM. The largest source of error in the process of pooled genotyping was shown to be the volumetric error, i.e., the preparation of pools. We also demonstrated that it would have been possible to narrow down the genetic locus underlying congenital chloride diarrhea (CLD), an autosomal recessive disorder, by using the pooling technique instead of genotyping individual samples. Although the approach seems to be well suited for traditional case-control studies, it is difficult to apply if any kind of stratification based on environmental factors is needed. Therefore we chose to continue with individual genotyping in the following association studies. Samples in the two separate large epidemiological cohorts were genotyped with the PCR-RFLP and SNuPeTM techniques. The first of these association studies concerned various pregnancy complications among 100,000 consecutive pregnancies in Finland, of which we genotyped 2292 patients and controls, in addition to a population sample of 644 blood donors, with 7 polymorphisms in the potentially thrombotic genes. In this thesis, the analysis of a sub-study of pregnancy-related venous thromboses was included. We showed that the impact of factor V Leiden polymorphism on pregnancy-related venous thrombosis, but not the other tested polymorphisms, was fairly large (odds ratio 11.6; 95% CI 3.6-33.6), and increased multiplicatively when combined with other risk factors such as obesity or advanced age. Owing to our study design, we were also able to estimate the risks at the population level. The second epidemiological cohort was the Helsinki Birth Cohort of men and women who were born during 1924-1933 in Helsinki. The aim was to identify genetic factors that might modify the well known link between small birth size and adult metabolic diseases, such as type 2 diabetes and impaired glucose tolerance. Among ~500 individuals with detailed birth measurements and current metabolic profile, we found that an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene was associated with the duration of gestation, and weight and length at birth. Interestingly, the ACE insertion allele was also associated with higher indices of insulin secretion (p=0.0004) in adult life, but only among individuals who were born small (those among the lowest third of birth weight). Likewise, low birth weight was associated with higher indices of insulin secretion (p=0.003), but only among carriers of the ACE insertion allele. The association with birth measurements was also found with a common haplotype of the glucocorticoid receptor (GR) gene. Furthermore, the association between short length at birth and adult impaired glucose tolerance was confined to carriers of this haplotype (p=0.007). These associations exemplify the interaction between environmental factors and genotype, which, possibly due to altered gene expression, predisposes to complex metabolic diseases. Indeed, we showed that the common GR gene haplotype associated with reduced mRNA expression in thymus of three individuals (p=0.0002).

Variation in tracheid cross-sectional dimensions and wood viscoelasticity extent and control methods

Printing papers have been the main product of the Finnish paper industry. To improve properties and economy of printing papers, controlling of tracheid cross-sectional dimensions and wood viscoelasticity are examined in this study. Controlling is understood as any procedure which yields raw material classes with distinct properties and small internal variation. Tracheid cross-sectional dimensions, i.e., cell wall thickness and radial and tangential diameters can be controlled with methods such as sorting wood into pulpwood and sawmill chips, sorting of logs according to tree social status and fractionation of fibres. These control methods were analysed in this study with simulations, which were based on measured tracheid cross-sectional dimensions. A SilviScan device was used to measure the data set from five Norway spruce (Picea abies) and five Scots pine (Pinus sylvestris) trunks. The simulation results indicate that the sawmill chips and top pulpwood assortments have quite similar cross-sectional dimensions. Norway spruce and Scots pine are on average also relatively similar in their cross-sectional dimensions. The distributions of these species are somewhat different, but from a practical point of view, the differences are probably of minor importance. The controlling of tracheid cross-sectional dimensions can be done most efficiently with methods that can separate fibres into earlywood and latewood. Sorting of logs or partitioning of logs into juvenile and mature wood were markedly less efficient control methods than fractionation of fibres. Wood viscoelasticity affects energy consumption in mechanical pulping, and is thus an interesting control target when improving energy efficiency of the process. A literature study was made to evaluate the possibility of using viscoelasticity in controlling. The study indicates that there is considerable variation in viscoelastic properties within tree species, but unfortunately, the viscoelastic properties of important raw material lots such as top pulpwood or sawmill chips are not known. Viscoelastic properties of wood depend mainly on lignin, but also on microfibrillar angle, width of cellulose crystals and tracheid cross-sectional dimensions.

From a tree to a stand in Finnish boreal forests: biomass estimation and comparison of methods

There is an increasing need to compare the results obtained with different methods of estimation of tree biomass in order to reduce the uncertainty in the assessment of forest biomass carbon. In this study, tree biomass was investigated in a 30-year-old Scots pine (Pinus sylvestris) (Young-Stand) and a 130-year-old mixed Norway spruce (Picea abies)-Scots pine stand (Mature-Stand) located in southern Finland (61º50' N, 24º22' E). In particular, a comparison of the results of different estimation methods was conducted to assess the reliability and suitability of their applications. For the trees in Mature-Stand, annual stem biomass increment fluctuated following a sigmoid equation, and the fitting curves reached a maximum level (from about 1 kg/yr for understorey spruce to 7 kg/yr for dominant pine) when the trees were 100 years old. Tree biomass was estimated to be about 70 Mg/ha in Young-Stand and about 220 Mg/ha in Mature-Stand. In the region (58.00-62.13 ºN, 14-34 ºE, ≤ 300 m a.s.l.) surrounding the study stands, the tree biomass accumulation in Norway spruce and Scots pine stands followed a sigmoid equation with stand age, with a maximum of 230 Mg/ha at the age of 140 years. In Mature-Stand, lichen biomass on the trees was 1.63 Mg/ha with more than half of the biomass occurring on dead branches, and the standing crop of litter lichen on the ground was about 0.09 Mg/ha. There were substantial differences among the results estimated by different methods in the stands. These results imply that a possible estimation error should be taken into account when calculating tree biomass in a stand with an indirect approach.