P-20DIAGNOSTIC ACCURACY OF CONVENTIONAL SMEARS AND THINPREP SAMPLES IN CERVICAL SCREENING

Thyroid fine needle aspiration (FNA) is the first choice procedure for differentiating between benign and malignant/suspicious lesions. Despite being highly sensitive and specific, it unfortunately has high inadequacy rates, with false-negatives reported in most series. The fundamental contribution of the aspirator's skill, experience and expertise to inadequacy rates is well documented, differing between both individuals and aspirator groups. We performed a retrospective audit comparing inadequacy rates of cytopathologists and clinicians for all thyroid FNAs performed from 2002–2005 within the Hammersmith Hospitals Trust. A crude cost-effectiveness was estimated, and using histological data where available, positive and negative powers (65.5% and 87.9% respectively), sensitivity (61.5%) and specificity (89.7%) were calculated. Pathologists were found to have significantly ( P  = 0.001) lower inadequacy rates (2.8%) than clinicians (16.1%) and were also more cost-effective (non-significant), with micropapillary carcinomas and lymphomas being identified as the main sources of false-negatives. We thus propose updated protocols to reduce inadequacy and false-negative rates, improve thyroid cancer diagnosis and the quality of patient care within our centre.     

Factors contributing to false‐negative and potential false‐negative cytology reports in SurePath™ liquid‐based cervical cytology

N. Gupta, D. John, N. Dudding, J. Crossley and J. H. F. Smith
Factors contributing to false‐negative and potential false‐negative cytology reports in SurePath ? liquid‐based cervical cytology Objectives: The characteristics of false‐negative conventional cervical cytology smears have been well documented, but there is limited literature available for liquid‐based cytology (LBC), especially SurePath? samples. We aimed to assess the characteristics of false‐negative SurePath LBC samples. Methods: Over a period of 5 years, an audit of false‐negative reports in SurePath cervical cytology was undertaken. In a workload of 183, 112 samples, 481 (0.3%) false negatives were identified using two routes: those detected by routine laboratory internal quality control (rapid pre‐screening) (n = 463) and those reported as normal (true false negatives) with concurrent high‐grade cervical histology (n = 18). Ninety‐five false‐negative cases with a subsequent biopsy reported as at least cervical intraepithelial neoplasia grade 2 (CIN2+) were reviewed for a number of different cytomorphological features. Results: Of 95 samples with subsequent CIN2+, 30.5% predominately contained microbiopsies/hyperchromatic crowded cell groups (HCGs), 27.3% sparse dyskarytotic cells, 4.2% pale cell dyskaryosis, 6.3% small dyskaryotic cells; 3.2% were misinterpreted cells, 8.4% contained other distracting cells, 7.4% were low contrast, 5.3% were unexplained and 7.4% were true negatives. The mean number of microbiopsies/HCGs in that category was 4.6. The mean number of abnormal cells in the sparse dyskaryotic cell category was 13.8. Conclusions: Microbiopsies/HCGs were the commonest reason for false negatives. They were usually present in sufficient numbers to be detected but interpretation could be problematic. Dispersed single abnormal cells were usually not identified because of their scarcity or the presence of distracters.     

O-9AUDIT ON BK VIRUS DETECTION IN RENAL TRANSPLANT RECIPIENTS

Introduction and Aims:  The sensitivity of bronchial cytology (brushings, washings) is in the region of 70–80%. Therefore a significant number of false negatives occur. The main aims of this study were to: (i) To identify negative bronchial washings and brushings performed in 2004 with malignant follow-up and (ii) To ascertain the cause of the false negative result where possible. 56 patients with negative bronchial cytology and subsequent malignant follow-up on a pulmonary specimen were identified. These cases formed the basis of the study.
Results:  In our series peripheral tumour location and specimen inadequacy accounted for the majority of false negative results. Malignant cells were missed in a minority of cases (5 of 56). An explanation for failure of diagnosis in 4 of the 5 cases was paucity of malignant cells. In one case of small cell carcinoma, malignant cells were abundant, but were not recognized.
Recommendations:  What can be done to reduce the false negative rate?
(1) In cases where radiology has indicated that the tumour is peripherally located patients could proceed directly to fine needle aspiration.
(2) Improved communication between clinicians and pathologists is required in relation to: (a) specimen adequacy and (b) degree of clinical suspicion.     

Posters. P13 Cytohistological correlation in 212 FNAs of thyroid: analysis of discordant cases

Aim To detect major pitafalls in thyroid FNA and to confirm its in a clinical sensitivity and specificity. Methods A total of 9251 fine needle aspirations biopsy carried out at Bellaria Hospital in Bologna from 1991 to 2000 by a pathologist in the FNA Clinic or by a clinician under ultrasonic guidance using a small needle (25–27 G); at least two passes have been made for each nodule. The specimen was considered satisfactory if at least five groups of follicular cells with at least 10 cells each, were seen. The cytological results were tiered in a four categories classification: inadequate, negative, suspicious and positive. Cyto‐histological correlations were available in 212 cases: 127 benign lesions and 85 malignant lesions. An analysis of false positive cases and false negative cases was performed and discordant case reviewed according to the flowing criteria: architecture, cellularity, colloid, pseudoinclusions, nuclear groovings, chromatin pattern, nuclear membrane, cytoplasm, naked nuclei and lymphocytes. Results Diagnostic distribution in 9251 FNAs from the thyroid: 88.6% negatives, 2.8 suspicious, 2.4% positives and 6.2% inadequates. Specificity was 85.8% and sensitivity was 78.8%. Among the 18 false negative cases eight were papillary microcarcinomas, four papillary carcinomas, five follicular carcinoma and one a Hurtle cell carcinoma. Four false positive cases were found: three reported as papillary carcinomas and one as carcinoma NOS. Review of false positives showed that in three cases the colloid was fluid, in three cases nuclear grooving was rare or absent, in two cases degenerative vacuoles at MGG were interpreted as nuclear inclusions and in three cases benign naked nuclei were present in the background. Review of false negatives confirmed lack of malignant features in 13 (eight papillary microcarcinomas and five follicular carcinomas), five were interpretation errors (three papillary carcinomas, one follicular, one Hurtle cell). Conclusion FNAC of the thyroid is a sensitive and specific method of assessment for thyroid nodules but false negative and false positive cases do occur. Use of all and only few criteria enhances diagnostic accuracy.     

Search and foraging behaviors from movement data: A comparison of methods

Search behavior is often used as a proxy for foraging effort within studies of animal movement, despite it being only one part of the foraging process, which also includes prey capture. While methods for validating prey capture exist, many studies rely solely on behavioral annotation of animal movement data to identify search and infer prey capture attempts. However, the degree to which search correlates with prey capture is largely untested. This study applied seven behavioral annotation methods to identify search behavior from GPS tracks of northern gannets (Morus bassanus), and compared outputs to the occurrence of dives recorded by simultaneously deployed time–depth recorders. We tested how behavioral annotation methods vary in their ability to identify search behavior leading to dive events. There was considerable variation in the number of dives occurring within search areas across methods. Hidden Markov models proved to be the most successful, with 81% of all dives occurring within areas identified as search. k‐Means clustering and first passage time had the highest rates of dives occurring outside identified search behavior. First passage time and hidden Markov models had the lowest rates of false positives, identifying fewer search areas with no dives. All behavioral annotation methods had advantages and drawbacks in terms of the complexity of analysis and ability to reflect prey capture events while minimizing the number of false positives and false negatives. We used these results, with consideration of analytical difficulty, to provide advice on the most appropriate methods for use where prey capture behavior is not available. This study highlights a need to critically assess and carefully choose a behavioral annotation method suitable for the research question being addressed, or resulting species management frameworks established.     

Evaluating next‐generation sequencing (NGS) methods for routine monitoring of wild bees: Metabarcoding,mitogenomics or NGS barcoding

Implementing cost‐effective monitoring programs for wild bees remains challenging due to the high costs of sampling and specimen identification. To reduce costs, next‐generation sequencing (NGS)‐based methods have lately been suggested as alternatives to morphology‐based identifications. To provide a comprehensive presentation of the advantages and weaknesses of different NGS‐based identification methods, we assessed three of the most promising ones, namely metabarcoding, mitogenomics and NGS barcoding. Using a regular monitoring data set (723 specimens identified using morphology), we found that NGS barcoding performed best for both species presence/absence and abundance data, producing only few false positives (3.4%) and no false negatives. In contrast, the proportion of false positives and false negatives was higher using metabarcoding and mitogenomics. Although strong correlations were found between biomass and read numbers, abundance estimates significantly skewed the communities' composition in these two techniques. NGS barcoding recovered the same ecological patterns as morphology. Ecological conclusions based on metabarcoding and mitogenomics were similar to those based on morphology when using presence/absence data, but different when using abundance data. In terms of workload and cost, we show that metabarcoding and NGS barcoding can compete with morphology, but not mitogenomics which was consistently more expensive. Based on these results, we advocate that NGS barcoding is currently the seemliest NGS method for monitoring of wild bees. Furthermore, this method has the advantage of potentially linking DNA sequences with preserved voucher specimens, which enable morphological re‐examination and will thus produce verifiable records which can be fed into faunistic databases.     

Identification of genetic variants using bar-coded multiplexed sequencing

We developed a generalized framework for multiplexed resequencing of targeted human genome regions on the Illumina Genome Analyzer using degenerate indexed DNA bar codes ligated to fragmented DNA before sequencing. Using this method, we simultaneously sequenced the DNA of multiple HapMap individuals at several Encyclopedia of DNA Elements (ENCODE) regions. We then evaluated the use of Bayes factors for discovering and genotyping polymorphisms. For polymorphisms that were either previously identified within the Single Nucleotide Polymorphism database (dbSNP) or visually evident upon re-inspection of archived ENCODE traces, we observed a false positive rate of 11.3% using strict thresholds for predicting variants and 69.6% for lax thresholds. Conversely, false negative rates were 10.8-90.8%, with false negatives at stricter cut-offs occurring at lower coverage (<10 aligned reads). These results suggest that >90% of genetic variants are discoverable using multiplexed sequencing provided sufficient coverage at the polymorphic base.     

Comparison of the validity of preimplantation genetic diagnosis for embryo chromosomal anomalies by fluorescence in situ hybridization on one or two blastomeres

Is it necessary to analyze two blastomeres in preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH) or is one blastomere enough, as suggested by some teams? We analyzed the sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), false positives (FP), false negatives (FN), and the efficiency (Eff) of FISH performed on one (Group I) or two (Group II) blastomeres. Ninety embryos were analyzed (day 3), 19 blastocysts were replaced (day 5), 64 embryos were reanalyzed (day 5), (Group I = 23; Group II = 41). No differences were observed between the two groups for all of the parameters considered, but one false negative was observed in Group I. Furthermore, two embryos from Group II, which had a discordant diagnosis at PGD (one blastomere being normal and one abnormal), were read as abnormal after reanalysis. The accidental biopsy of the normal blastomere could have lead to the selection of these 2 embryos for transfer, causing a misdiagnosis rate of 4.8%. We conclude that embryo reanalysis is a useful tool to test the reliability of PGD in each laboratory: that PGD on two blastomeres is safer because the practice of PGD on one blastomere can result in a false-negative misdiagnosis.     

The effects of alignment error and alignment filtering on the sitewise detection of positive selection

When detecting positive selection in proteins, the prevalence of errors resulting from misalignment and the ability of alignment filters to mitigate such errors are not well understood, but filters are commonly applied to try to avoid false positive results. Focusing on the sitewise detection of positive selection across a wide range of divergence levels and indel rates, we performed simulation experiments to quantify the false positives and false negatives introduced by alignment error and the ability of alignment filters to improve performance. We found that some aligners led to many false positives, whereas others resulted in very few. False negatives were a problem for all aligners, increasing with sequence divergence. Of the aligners tested, PRANK's codon-based alignments consistently performed the best and ClustalW performed the worst. Of the filters tested, GUIDANCE performed the best and Gblocks performed the worst. Although some filters showed good ability to reduce the error rates from ClustalW and MAFFT alignments, none were found to substantially improve the performance of PRANK alignments under most conditions. Our results revealed distinct trends in error rates and power levels for aligners and filters within a biologically plausible parameter space. With the best aligner, a low false positive rate was maintained even with extremely divergent indel-prone sequences. Controls using the true alignment and an optimal filtering method suggested that performance improvements could be gained by improving aligners or filters to reduce the prevalence of false negatives, especially at higher divergence levels and indel rates.     

Replication levels,false presences and the estimation of the presence/absence from eDNA metabarcoding data

Environmental DNA (eDNA) metabarcoding is increasingly used to study the present and past biodiversity. eDNA analyses often rely on amplification of very small quantities or degraded DNA. To avoid missing detection of taxa that are actually present (false negatives), multiple extractions and amplifications of the same samples are often performed. However, the level of replication needed for reliable estimates of the presence/absence patterns remains an unaddressed topic. Furthermore, degraded DNA and PCR/sequencing errors might produce false positives. We used simulations and empirical data to evaluate the level of replication required for accurate detection of targeted taxa in different contexts and to assess the performance of methods used to reduce the risk of false detections. Furthermore, we evaluated whether statistical approaches developed to estimate occupancy in the presence of observational errors can successfully estimate true prevalence, detection probability and false‐positive rates. Replications reduced the rate of false negatives; the optimal level of replication was strongly dependent on the detection probability of taxa. Occupancy models successfully estimated true prevalence, detection probability and false‐positive rates, but their performance increased with the number of replicates. At least eight PCR replicates should be performed if detection probability is not high, such as in ancient DNA studies. Multiple DNA extractions from the same sample yielded consistent results; in some cases, collecting multiple samples from the same locality allowed detecting more species. The optimal level of replication for accurate species detection strongly varies among studies and could be explicitly estimated to improve the reliability of results.     

The consequences of using indirect signs that decay to determine species' occupancy

Statistical models of species' distributions rely on data on species' occupancy, or use, of sites across space and/or time. For rare or cryptic species, indirect signs, such as dung, may be the only realistic means of determining their occupancy status across broad spatial extents. However, the consequences of sign decay for errors in estimates of occupancy have not previously been considered. If signs decay very rapidly, then false‐negative errors may occur because signs at an occupied site have decayed by the time it is surveyed. On the other hand, if signs decay very slowly, false‐positive errors may occur because signs remain present at sites that are no longer occupied. We addressed this issue by quantifying, as functions of sign decay and accumulation rates: 1) the false‐negative error rate due to sign decay and, 2) the expected time interval prior to a survey within which signs indicate the species was present; as this time interval increases, false‐positives become more likely. We then applied this to the specific example of koala Phascolarctos cinereus occupancy derived from faecal pellet surveys using data on faecal pellet decay rates. We show that there is a clear trade‐off between false‐negative error rates and the potential for false‐positive errors. For the koala case study, false‐negative errors were low on average and the expected time interval prior to surveys that detected pellets indicate the species was present within less than 2–3 yr. However, these quantities showed quite substantial spatial variation that could lead to biased parameter estimates for distribution models based on faecal pellet surveys. This highlights the importance of observation errors arising from sign decay and we suggest some modifications to existing methods to deal with this issue.     

Quantitative and qualitative assessment of pollen DNA metabarcoding using constructed species mixtures

Pollen DNA metabarcoding—marker‐based genetic identification of potentially mixed‐species pollen samples—has applications across a variety of fields. While basic species‐level pollen identification using standard DNA barcode markers is established, the extent to which metabarcoding (a) correctly assigns species identities to mixes (qualitative matching) and (b) generates sequence reads proportionally to their relative abundance in a sample (quantitative matching) is unclear, as these have not been assessed relative to known standards. We tested the quantitative and qualitative robustness of metabarcoding in constructed pollen mixtures varying in species richness (1–9 species), taxonomic relatedness (within genera to across class) and rarity (5%–100% of grains), using Illumina MiSeq with the markers rbcL and ITS2. Qualitatively, species composition determinations were largely correct, but false positives and negatives occurred. False negatives were typically driven by lack of a barcode gap or rarity in a sample. Species richness and taxonomic relatedness, however, did not strongly impact correct determinations. False positives were likely driven by contamination, chimeric sequences and/or misidentification by the bioinformatics pipeline. Quantitatively, the proportion of reads for each species was only weakly correlated with its relative abundance, in contrast to suggestions from some other studies. Quantitative mismatches are not correctable by consistent scaling factors, but instead are context‐dependent on the other species present in a sample. Together, our results show that metabarcoding is largely robust for determining pollen presence/absence but that sequence reads should not be used to infer relative abundance of pollen grains.     

Diagnostic pitfalls in the evaluation of fine needle aspiration cytology of the thyroid: correlation with histopathology in 260 cases

Objectives:  Fine needle aspiration cytology (FNAC) of the thyroid is a non-invasive, cost-effective screening procedure that is valuable for distinguishing neoplastic lesions from non-neoplastic nodules. The aim of this study was to determine the diagnostic accuracy of FNACs performed at our institution by correlating FNAC results with histopathological diagnoses.
Methods:  Two hundred and seventy-one aspiration cytology specimens followed by thyroidectomy were included in the study, and the results of 260 adequate FNACs were compared with their histological diagnoses.
Results:  The sensitivity and specificity of thyroid FNAC for detecting neoplasia were 92.6% and 91.6%, respectively. There were 15 (5.7%) false positives and six (2.3%) false negatives.
Conclusions:  The results showed that follicular cells that exhibit some of the features of papillary carcinoma could be observed in a cytology slide of Hashimoto's thyroiditis, leading to a diagnostic pitfall. In addition, cellularity and overlapping cytological criteria in hyperplasia might lead to a false diagnosis.     

How do steppe plants follow their optimal environmental conditions or persist under suboptimal conditions? The differing strategies of annuals and perennials

For a species to be able to respond to environmental change, it must either succeed in following its optimal environmental conditions or in persisting under suboptimal conditions, but we know very little about what controls these capacities. We parameterized species distribution models (SDMs) for 135 plant species from the Algerian steppes. We interpreted low false‐positive rates as reflecting a high capacity to follow optimal environmental conditions and high false‐negative rates as a high capacity to persist under suboptimal environmental conditions. We also measured functional traits in the field and built a unique plant trait database for the North‐African steppe. For both perennial and annual species, we explored how these two capacities can be explained by species traits and whether relevant trait values reflect species strategies or biases in SDMs. We found low false‐positive rates in species with small seeds, flowers attracting specialist pollinators, and specialized distributions (among annuals and perennials), low root:shoot ratios, wide root‐systems, and large leaves (perennials only) (R² = .52–58). We found high false‐negative rates in species with marginal environmental distribution (among annuals and perennials), small seeds, relatively deep roots, and specialized distributions (annuals) or large leaves, wide root‐systems, and monocarpic life cycle (perennials) (R² = .38 for annuals and 0.65 for perennials). Overall, relevant traits are rarely indicative of the possible biases of SDMs, but rather reflect the species' reproductive strategy, dispersal ability, stress tolerance, and pollination strategies. Our results suggest that wide undirected dispersal in annual species and efficient resource acquisition in perennial species favor both capacities, whereas short life spans in perennial species favor persistence in suboptimal environmental conditions and flowers attracting specialist pollinators in perennial and annual species favor following optimal environmental conditions. Species that neither follow nor persist will be at risk under future environmental change.     

Implications of False Negative and False Positive Diagnosis in Lymph Node Staging of NSCLC by Means of 18F-FDG PET/CT

Background

Integrated ¹⁸F-fluorodeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT) is widely performed in hilar and mediastinal lymph node (HMLN) staging of non-small cell lung cancer (NSCLC). However, the diagnostic efficiency of PET/CT remains controversial. This retrospective study is to evaluate the accuracy of PET/CT and the characteristics of false negatives and false positives to improve specificity and sensitivity.

Methods

219 NSCLC patients with systematic lymph node dissection or sampling underwent preoperative PET/CT scan. Nodal uptake with a maximum standardized uptake value (SUVmax) >2.5 was interpreted as PET/CT positive. The results of PET/CT were compared with the histopathological findings. The receiver operating characteristic (ROC) curve was generated to determine the diagnostic efficiency of PET/CT. Univariate and multivariate analysis were conducted to detect risk factors of false negatives and false positives.

Results

The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of PET/ CT in detecting HMLN metastases were 74.2% (49/66), 73.2% (112/153), 54.4% (49/90), 86.8% (112/129), and 73.5% (161/219). The ROC curve had an area under curve (AUC) of 0.791 (95% CI 0.723-0.860). The incidence of false negative HMLN metastases was 13.2% (17 of 129 patients). Factors that are significantly associated with false negatives are: concurrent lung disease or diabetes (p<0.001), non-adenocarcinoma (p<0.001), and SUVmax of primary tumor >4.0 (p=0.009). Postoperatively, 45.5% (41/90) patients were confirmed as false positive cases. The univariate analysis indicated age > 65 years old (p=0.009), well differentiation (p=0.002), and SUVmax of primary tumor ≦4.0 (p=0.007) as risk factors for false positive uptake.

Conclusion

The SUVmax of HMLN is a predictor of malignancy. Lymph node staging using PET/CT is far from equal to pathological staging account of some risk factors. This study may provide some aids to pre-therapy evaluation and decision-making.     

S100B and brain natriuretic peptide predict functional neurological outcome after intracerebral haemorrhage

Colorimetric test strip assays are a convenient and inexpensive means for the determination of cotinine in human urine because they can be performed in a nonlaboratory environment using a trained technician. Four hundred human urine samples were separated into four categories: (1) heavy smokers (>20 cigarettes smoked per day), (2) light smokers (<20 cigarettes smoked per day), (3) non-smokers, and (4) vegetarian non-smokers. Samples were evaluated by a gas chromatography/mass selective detector (GC/MSD) method as a reference and using NicCheck I? (DynaGen, Inc.). Colour intensity can range from 0 (no colour) to 14 (deep pink). Qualitative values were assigned as negative (0), low (1-6) and high (7-14). Comparison of the test strip and GC/MSD results showed: (1) 43 (10.75%) false negatives using the criterion of a GC/MSD cotinine level above 200 ng ml^-1 and test strip reading of 0, (2) 31 (7.75%) false positives using the criterion of a GC/MSD cotinine level below 1 ng ml^-1 and a test strip reading of 1 or greater, and (3) no correlation between the test strip and GC/MSD results (r = 0.597, p < 0.05). The fact that the colorimetric reaction is sensitive to many nicotine metabolites and/or heterocyclic amine structures whereas the GC/MSD method measures nicotine and cotinine selectively might explain the false positive results. False negative results were likely to be due to a lack of sensitivity of the test strip.     

Posters. P11 Positive predictive values for high and low‐grade cytological abnormalities as surrogates for specificity and sensitivity

Introduction Positive predictive value (PPV), measuring the percentage of moderate dyskaryosis or worse confirmed as CIN2 or worse, is used as a measure of accuracy in cervical screening. However, it relates more to specificity than sensitivity because the denominator includes false positives rather than false negatives. Low values reflect over‐reporting of high‐grade dyskaryosis but high values may reflect under‐reporting. Sensitivity is impossible to measure from correlation of cytology with outcome because women with negative cytology are rarely referred for colposcopy. Rates of CIN3 resulting from referrals for low‐grade cytology may be used as a surrogate for sensitivity, as high values may reflect under‐reporting (ref). Study design Outcome of colposcopy referrals was monitored during a period of 4 years, using a fail‐safe database. Results PPV at Guy's & St Thomas rose from 54% in 1998/1999 to 69% in 2001/2002. The former was below the NHSCSP recommended range. During the same period of time CIN1 rates for moderate dyskaryosis fell from 37% to 24%, reflecting the main source of discrepancy. While specificity increased (as reflected by increasing PPV) sensitivity remained constant in that CIN3 rates for mild dyskaryosis and borderline remained below 6%: average rates in England have fallen over the last 3 years and were 7.4% in 2000/2001 (ref). CIN2 rates for mild dyskaryosis also remained constant at 11% to 12%. Conclusion Correlation of biopsy results with high‐ and low‐grade cytological abnormalities is a useful method of monitoring accuracy of cytology reporting, and can be used to measure over‐ and under‐reporting as surrogates for specificity and sensitivity.     

One‐locus‐several‐primers: A strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies

In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets’ complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the “one‐locus‐several‐primer‐sets” (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey–predator interactions.     

Factors influencing detection of nesting crested caracaras

Management of crested caracaras (Caracara cheriway), focuses on nests identified during surveys. If no nests are found, management can be suspended. Thus, false negatives can have substantial consequences. We surveyed 49 breeding territories to assess factors with the potential to cause false negatives in detecting nests of crested caracaras and in observing adult birds. The probability that a nest would be detected on any given visit increased by about 0.5% for each hour of observer experience up to about 70 hours (our maximum). Experience did not affect the probability of observing an adult. The probability of detecting a caracara nest or observing an adult caracara decreased by 2.0–3.5% each hour after sunrise that a visit began. If visibility during any portion of a visit was obscured by fog or rain, the probability of detecting a nest decreased by as much as 60%, and the probability of observing an adult caracara decreased by about 50%. We provide a tool managers can use to calculate the likelihood of successful surveys. We recommend that managers disregard negative results from surveys conducted under conditions that are unlikely to yield positive results, and repeat those surveys under better conditions. © 2011 The Wildlife Society.