The Incidence of AIDS-Defining Illnesses at a Current CD4 Count >=200 Cells/ L in the Post-Combination Antiretroviral Therapy Era

Background. Few studies consider the incidence of individual AIDS-defining illnesses (ADIs) at higher CD4 counts, relevant on a population level for monitoring and resource allocation. Methods. Individuals from the Collaboration of Observational HIV Epidemiological Research Europe (COHERE) aged ≥14 years with ≥1 CD4 count of ≥200 µL between 1998 and 2010 were included. Incidence rates (per 1000 person-years of follow-up [PYFU]) were calculated for each ADI within different CD4 strata; Poisson regression, using generalized estimating equations and robust standard errors, was used to model rates of ADIs with current CD4 ≥500/µL. Results. A total of 12 135 ADIs occurred at a CD4 count of ≥200 cells/µL among 207 539 persons with 1 154 803 PYFU. Incidence rates declined from 20.5 per 1000 PYFU (95% confidence interval [CI], 20.0–21.1 per 1000 PYFU) with current CD4 200–349 cells/µL to 4.1 per 1000 PYFU (95% CI, 3.6–4.6 per 1000 PYFU) with current CD4 ≥ 1000 cells/µL. Persons with a current CD4 of 500–749 cells/µL had a significantly higher rate of ADIs (adjusted incidence rate ratio [aIRR], 1.20; 95% CI, 1.10–1.32), whereas those with a current CD4 of ≥1000 cells/µL had a similar rate (aIRR, 0.92; 95% CI, .79–1.07), compared to a current CD4 of 750–999 cells/µL. Results were consistent in persons with high or low viral load. Findings were stronger for malignant ADIs (aIRR, 1.52; 95% CI, 1.25–1.86) than for nonmalignant ADIs (aIRR, 1.12; 95% CI, 1.01–1.25), comparing persons with a current CD4 of 500–749 cells/µL to 750–999 cells/µL. Discussion. The incidence of ADIs was higher in individuals with a current CD4 count of 500–749 cells/µL compared to those with a CD4 count of 750–999 cells/µL, but did not decrease further at higher CD4 counts. Results were similar in patients virologically suppressed on combination antiretroviral therapy, suggesting that immune reconstitution is not complete until the CD4 increases to >750 cells/µL.

Prospective, paired crossover comparison of multiple, single-needle plateletpheresis procedures with the Amicus and Trima Accel cell separators

BACKGROUND: The Baxter Amicus Version 2.51 (A) and the Gambro BCT Trima Accel Version 5.0 (T) cell separators may produce multiple platelet (PLT) concentrates within a single donation. STUDY DESIGN AND METHODS: The single-needle multiple plateletpheresis procedures of the two devices were compared in a prospective, randomized, paired crossover study in 60 donors. The 120 donations were compared for donor comfort, collection efficiency, residual white blood cell (WBC) count, and (in selected patients) corrected count increment (CCI). RESULTS: The mean PLT yield and the resultant mean number of units per donation were significantly lower for A (6.06 x 10(11) vs. 7.48 x 10(11) and 2.57 vs. 3.19, respectively, both p < 0.001), in spite of a longer apheresis duration (89 min vs. 79 min; p < 0.001). This resulted in a higher collection rate of T (5.68 x 10(11) PLTs/hr vs. 4.10 x 10(11) PLTs/hr, p < 0.001). Residual WBC count of every unit was fewer than 5 x 10(6), but significantly fewer A-PLT donations contained more than 10(5) WBCs per unit (1 vs. 9, p = 0.008). Although the ACD-A consumption was slightly higher for A (489 mL vs. 469 mL, p = 0.04), a trend to a higher frequency of side effects was found for T (42.4% vs. 23.7%, p = 0.06). The 1-hour CCIs of 33 transfused A-PLT units were comparable with those of 43 T-PLT units (11.8 vs. 13.9, p = 0.480). CONCLUSIONS: Both cell separators showed safe collections of up to 4 PLT units per donation with adequate CCI. T produced a higher PLT yield despite shorter apheresis duration, but with slightly higher residual WBC counts and a trend to a higher side-effect frequency.

Prognosis of HIV-1-infected patients up to 5 years after initiation of HAART: collaborative analysis of prospective studies

OBJECTIVE: To estimate the prognosis over 5 years of HIV-1-infected, treatment-naive patients starting HAART, taking into account the immunological and virological response to therapy. DESIGN: A collaborative analysis of data from 12 cohorts in Europe and North America on 20,379 adults who started HAART between 1995 and 2003. METHODS: Parametric survival models were used to predict the cumulative incidence at 5 years of a new AIDS-defining event or death, and death alone, first from the start of HAART and second from 6 months after the start of HAART. Data were analysed by intention-to-continue-treatment, ignoring treatment changes and interruptions. RESULTS: During 61 798 person-years of follow-up, 1005 patients died and an additional 1303 developed AIDS. A total of 10 046 (49%) patients started HAART either with a CD4 cell count of less than 200 cells/microl or with a diagnosis of AIDS. The 5-year risk of AIDS or death (death alone) from the start of HAART ranged from 5.6 to 77% (1.8-65%), depending on age, CD4 cell count, HIV-1-RNA level, clinical stage, and history of injection drug use. From 6 months the corresponding figures were 4.1-99% for AIDS or death and 1.3-96% for death alone. CONCLUSION: On the basis of data collected routinely in HIV care, prognostic models with high discriminatory power over 5 years were developed for patients starting HAART in industrialized countries. A risk calculator that produces estimates for progression rates at years 1 to 5 after starting HAART is available from www.art-cohort-collaboration.org.

Treatment of patients with the hypereosinophilic syndrome with mepolizumab

BACKGROUND: The hypereosinophilic syndrome is a group of diseases characterized by persistent blood eosinophilia, defined as more than 1500 cells per microliter with end-organ involvement and no recognized secondary cause. Although most patients have a response to corticosteroids, side effects are common and can lead to considerable morbidity. METHODS: We conducted an international, randomized, double-blind, placebo-controlled trial evaluating the safety and efficacy of an anti-interleukin-5 monoclonal antibody, mepolizumab, in patients with the hypereosinophilic syndrome. Patients were negative for the FIP1L1-PDGFRA fusion gene and required prednisone monotherapy, 20 to 60 mg per day, to maintain a stable clinical status and a blood eosinophil count of less than 1000 per microliter. Patients received either intravenous mepolizumab or placebo while the prednisone dose was tapered. The primary end point was the reduction of the prednisone dose to 10 mg or less per day for 8 or more consecutive weeks. RESULTS: The primary end point was reached in 84% of patients in the mepolizumab group, as compared with 43% of patients in the placebo group (hazard ratio, 2.90; 95% confidence interval [CI], 1.59 to 5.26; P<0.001) with no increase in clinical activity of the hypereosinophilic syndrome. A blood eosinophil count of less than 600 per microliter for 8 or more consecutive weeks was achieved in 95% of patients receiving mepolizumab, as compared with 45% of patients receiving placebo (hazard ratio, 3.53; 95% CI, 1.94 to 6.45; P<0.001). Serious adverse events occurred in seven patients receiving mepolizumab (14 events, including one death; mean [+/-SD] duration of exposure, 6.7+/-1.9 months) and in five patients receiving placebo (7 events; mean duration of exposure, 4.3+/-2.6 months). CONCLUSIONS: Our study shows that treatment with mepolizumab, an agent designed to target eosinophils, can result in corticosteroid-sparing for patients negative for FIP1L1-PDGFRA who have the hypereosinophilic syndrome. (ClinicalTrials.gov number, NCT00086658 [ClinicalTrials.gov].).

Relation of morning serum cortisol to prothrombotic activity in women with stable coronary artery disease

BACKGROUND: Increased circulating cortisol levels have been associated with severity of atherosclerosis. Low-grade systemic thrombogenicity plays a major role in the initiation and progression of coronary disease. We hypothesized a direct relationship between cortisol and hemostasis factors related to a prothrombotic state in coronary artery disease. METHODS: We measured morning serum cortisol and activated clotting factor VII, fibrinogen, von Willebrand factor antigen, and plasminogen activator inhibitor-1 activity in 285 women (56 +/- 7 years) between 3 and 6 months after an acute coronary event. To test whether the relationship between cortisol and hemostasis factors would be independent, statistical adjustment was made for demographic, biomedical, life style, and psychosocial variables. RESULTS: Higher serum cortisol levels predicted higher fibrinogen (beta = .17, P = .001) and higher von Willebrand factor (beta = .16, P = .008), all independently of covariates, including C-reactive protein, which was also an independent predictor of fibrinogen (beta = .20, P = .001) and von Willebrand factor (beta = .16, P = .004). Higher levels of vital exhaustion were associated with higher levels of activated clotting factor VII independently of covariates and depression (beta = .18, P = .045). Cortisol showed crude correlations with vital exhaustion (r = .14, P = .022) and with depression (r = .13, P = .043) but did not mediate the relationship between psychosocial variables and hemostatic factors. CONCLUSIONS: Morning serum cortisol showed a modest but independent association with prothrombotic activity in women with coronary artery disease suggesting that increased cortisol levels might contribute to atherosclerosis via eliciting a hypercoagulable state.

Mortality of HIV-infected patients starting antiretroviral therapy in sub-Saharan Africa: comparison with HIV-unrelated mortality

BACKGROUND: Mortality in HIV-infected patients who have access to highly active antiretroviral therapy (ART) has declined in sub-Saharan Africa, but it is unclear how mortality compares to the non-HIV-infected population. We compared mortality rates observed in HIV-1-infected patients starting ART with non-HIV-related background mortality in four countries in sub-Saharan Africa. METHODS AND FINDINGS: Patients enrolled in antiretroviral treatment programmes in Côte d'Ivoire, Malawi, South Africa, and Zimbabwe were included. We calculated excess mortality rates and standardised mortality ratios (SMRs) with 95% confidence intervals (CIs). Expected numbers of deaths were obtained using estimates of age-, sex-, and country-specific, HIV-unrelated, mortality rates from the Global Burden of Disease project. Among 13,249 eligible patients 1,177 deaths were recorded during 14,695 person-years of follow-up. The median age was 34 y, 8,831 (67%) patients were female, and 10,811 of 12,720 patients (85%) with information on clinical stage had advanced disease when starting ART. The excess mortality rate was 17.5 (95% CI 14.5-21.1) per 100 person-years SMR in patients who started ART with a CD4 cell count of less than 25 cells/microl and World Health Organization (WHO) stage III/IV, compared to 1.00 (0.55-1.81) per 100 person-years in patients who started with 200 cells/microl or above with WHO stage I/II. The corresponding SMRs were 47.1 (39.1-56.6) and 3.44 (1.91-6.17). Among patients who started ART with 200 cells/microl or above in WHO stage I/II and survived the first year of ART, the excess mortality rate was 0.27 (0.08-0.94) per 100 person-years and the SMR was 1.14 (0.47-2.77). CONCLUSIONS: Mortality of HIV-infected patients treated with combination ART in sub-Saharan Africa continues to be higher than in the general population, but for some patients excess mortality is moderate and reaches that of the general population in the second year of ART. Much of the excess mortality might be prevented by timely initiation of ART.

CD4(+) T cell count decreases by ethnicity among untreated patients with HIV infection in South Africa and Switzerland

BACKGROUND: Estimates of the decrease in CD4(+) cell counts in untreated patients with human immunodeficiency virus (HIV) infection are important for patient care and public health. We analyzed CD4(+) cell count decreases in the Cape Town AIDS Cohort and the Swiss HIV Cohort Study. METHODS: We used mixed-effects models and joint models that allowed for the correlation between CD4(+) cell count decreases and survival and stratified analyses by the initial cell count (50-199, 200-349, 350-499, and 500-750 cells/microL). Results are presented as the mean decrease in CD4(+) cell count with 95% confidence intervals (CIs) during the first year after the initial CD4(+) cell count. RESULTS: A total of 784 South African (629 nonwhite) and 2030 Swiss (218 nonwhite) patients with HIV infection contributed 13,388 CD4(+) cell counts. Decreases in CD4(+) cell count were steeper in white patients, patients with higher initial CD4(+) cell counts, and older patients. Decreases ranged from a mean of 38 cells/microL (95% CI, 24-54 cells/microL) in nonwhite patients from the Swiss HIV Cohort Study 15-39 years of age with an initial CD4(+) cell count of 200-349 cells/microL to a mean of 210 cells/microL (95% CI, 143-268 cells/microL) in white patients in the Cape Town AIDS Cohort > or =40 years of age with an initial CD4(+) cell count of 500-750 cells/microL. CONCLUSIONS: Among both patients from Switzerland and patients from South Africa, CD4(+) cell count decreases were greater in white patients with HIV infection than they were in nonwhite patients with HIV infection.

Timing of initiation of antiretroviral therapy in AIDS-free HIV-1-infected patients: a collaborative analysis of 18 HIV cohort studies

BACKGROUND: The CD4 cell count at which combination antiretroviral therapy should be started is a central, unresolved issue in the care of HIV-1-infected patients. In the absence of randomised trials, we examined this question in prospective cohort studies. METHODS: We analysed data from 18 cohort studies of patients with HIV. Antiretroviral-naive patients from 15 of these studies were eligible for inclusion if they had started combination antiretroviral therapy (while AIDS-free, with a CD4 cell count less than 550 cells per microL, and with no history of injecting drug use) on or after Jan 1, 1998. We used data from patients followed up in seven of the cohorts in the era before the introduction of combination therapy (1989-95) to estimate distributions of lead times (from the first CD4 cell count measurement in an upper range to the upper threshold of a lower range) and unseen AIDS and death events (occurring before the upper threshold of a lower CD4 cell count range is reached) in the absence of treatment. These estimations were used to impute completed datasets in which lead times and unseen AIDS and death events were added to data for treated patients in deferred therapy groups. We compared the effect of deferred initiation of combination therapy with immediate initiation on rates of AIDS and death, and on death alone, in adjacent CD4 cell count ranges of width 100 cells per microL. FINDINGS: Data were obtained for 21 247 patients who were followed up during the era before the introduction of combination therapy and 24 444 patients who were followed up from the start of treatment. Deferring combination therapy until a CD4 cell count of 251-350 cells per microL was associated with higher rates of AIDS and death than starting therapy in the range 351-450 cells per microL (hazard ratio [HR] 1.28, 95% CI 1.04-1.57). The adverse effect of deferring treatment increased with decreasing CD4 cell count threshold. Deferred initiation of combination therapy was also associated with higher mortality rates, although effects on mortality were less marked than effects on AIDS and death (HR 1.13, 0.80-1.60, for deferred initiation of treatment at CD4 cell count 251-350 cells per microL compared with initiation at 351-450 cells per microL). INTERPRETATION: Our results suggest that 350 cells per microL should be the minimum threshold for initiation of antiretroviral therapy, and should help to guide physicians and patients in deciding when to start treatment.

Dynamics of vessel wall changes following the implantation of the Absorb everolimus-eluting bioresorbable vascular scaffold: a multi-imaging modality study at 6, 12, 24 and 36 months

Aims: To assess observations with multimodality imaging of the Absorb bioresorbable everolimus-eluting vascular scaffold performed in two consecutive cohorts of patients who were serially investigated either at 6 and 24 months or at 12 and 36 months. Methods and results: In the ABSORB multicentre single-arm trial, 45 patients (cohort B1) and 56 patients (cohort B2) underwent serial invasive imaging, specifically quantitative coronary angiography (QCA), intravascular ultrasound (IVUS), radiofrequency backscattering (IVUS-VH) and optical coherence tomography (OCT). Between one and three years, late luminal loss remained unchanged (6 months: 0.19 mm, 1 year: 0.27 mm, 2 years: 0.27 mm, 3 years: 0.29 mm) and the in-segment angiographic restenosis rate for the entire cohort B (n=101) at three years was 6%. On IVUS, mean lumen, scaffold, plaque and vessel area showed enlargement up to two years. Mean lumen and scaffold area remained stable between two and three years whereas significant reduction in plaque behind the struts occurred with a trend toward adaptive restrictive remodelling of EEM. Hyperechogenicity of the vessel wall, a surrogate of the bioresorption process, decreased from 23.1% to 10.4% with a reduction of radiofrequency backscattering for dense calcium and necrotic core. At three years, the count of strut cores detected on OCT increased significantly, probably reflecting the dismantling of the scaffold; 98% of struts were covered. In the entire cohort B (n=101), the three-year major adverse cardiac event rate was 10.0% without any scaffold thrombosis. Conclusions: The current investigation demonstrated the dynamics of vessel wall changes after implantation of a bioresorbable scaffold, resulting at three years in stable luminal dimensions, a low restenosis rate and a low clinical major adverse cardiac events rate.

When to start antiretroviral therapy in children aged 2-5 years: a collaborative causal modelling analysis of cohort studies from southern Africa

BACKGROUND There is limited evidence on the optimal timing of antiretroviral therapy (ART) initiation in children 2-5 y of age. We conducted a causal modelling analysis using the International Epidemiologic Databases to Evaluate AIDS-Southern Africa (IeDEA-SA) collaborative dataset to determine the difference in mortality when starting ART in children aged 2-5 y immediately (irrespective of CD4 criteria), as recommended in the World Health Organization (WHO) 2013 guidelines, compared to deferring to lower CD4 thresholds, for example, the WHO 2010 recommended threshold of CD4 count <750 cells/mm(3) or CD4 percentage (CD4%) <25%. METHODS AND FINDINGS ART-naïve children enrolling in HIV care at IeDEA-SA sites who were between 24 and 59 mo of age at first visit and with ≥1 visit prior to ART initiation and ≥1 follow-up visit were included. We estimated mortality for ART initiation at different CD4 thresholds for up to 3 y using g-computation, adjusting for measured time-dependent confounding of CD4 percent, CD4 count, and weight-for-age z-score. Confidence intervals were constructed using bootstrapping. The median (first; third quartile) age at first visit of 2,934 children (51% male) included in the analysis was 3.3 y (2.6; 4.1), with a median (first; third quartile) CD4 count of 592 cells/mm(3) (356; 895) and median (first; third quartile) CD4% of 16% (10%; 23%). The estimated cumulative mortality after 3 y for ART initiation at different CD4 thresholds ranged from 3.4% (95% CI: 2.1-6.5) (no ART) to 2.1% (95% CI: 1.3%-3.5%) (ART irrespective of CD4 value). Estimated mortality was overall higher when initiating ART at lower CD4 values or not at all. There was no mortality difference between starting ART immediately, irrespective of CD4 value, and ART initiation at the WHO 2010 recommended threshold of CD4 count <750 cells/mm(3) or CD4% <25%, with mortality estimates of 2.1% (95% CI: 1.3%-3.5%) and 2.2% (95% CI: 1.4%-3.5%) after 3 y, respectively. The analysis was limited by loss to follow-up and the unavailability of WHO staging data. CONCLUSIONS The results indicate no mortality difference for up to 3 y between ART initiation irrespective of CD4 value and ART initiation at a threshold of CD4 count <750 cells/mm(3) or CD4% <25%, but there are overall higher point estimates for mortality when ART is initiated at lower CD4 values. Please see later in the article for the Editors' Summary.

Characterization of new staphylococcal cassette chromosome mec (SCCmec) and topoisomerase genes in fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius

Fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius isolates harbor two new staphylococcal cassette chromosome mec (SCCmec) elements that belong to class A, allotype 3 (SCCmec II-III), and to the new allotype 5 (SCCmec VII). Analysis of the complete nucleotide sequences of the topoisomerase loci gyrB/gyrA and grlB/grlA revealed mutations involved in fluoroquinolone resistance.

Intracranial Aneurysm Rupture Is Predicted by Measures of Solar Activity.

OBJECTIVE The cause precipitating intracranial aneurysm rupture remains unknown in many cases. It has been observed that aneurysm ruptures are clustered in time, but the trigger mechanism remains obscure. Because solar activity has been associated with cardiovascular mortality and morbidity, we decided to study its association to aneurysm rupture in the Swiss population. METHODS Patient data were extracted from the Swiss SOS database, at time of analysis covering 918 consecutive patients with angiography-proven aneurysmal subarachnoid hemorrhage treated at 7 Swiss neurovascular centers between January 1, 2009, and December 31, 2011. The daily rupture frequency (RF) was correlated to the absolute amount and the change in various parameters of interest representing continuous measurements of solar activity (radioflux [F10.7 index], solar proton flux, solar flare occurrence, planetary K-index/planetary A-index, Space Environment Services Center [SESC] sunspot number and sunspot area) using Poisson regression analysis. RESULTS During the period of interest, there were 517 days without recorded aneurysm rupture. There were 398, 139, 27, 12, 1, and 1 days with 1, 2, 3, 4, 5, and 6 ruptures per day. Poisson regression analysis demonstrated a significant correlation of F10.7 index and RF (incidence rate ratio [IRR] = 1.006303; standard error (SE) 0.0013201; 95% confidence interval (CI) 1.003719-1.008894; P < 0.001), according to which every 1-unit increase of the F10.7 index increased the count for an aneurysm to rupture by 0.63%. A likewise statistically significant relationship of both the SESC sunspot number (IRR 1.003413; SE 0.0007913; 95% CI 1.001864-1.004965; P < 0.001) and the sunspot area (IRR 1.000419; SE 0.0000866; 95% CI 1.000249-1.000589; P < 0.001) emerged. All other variables analyzed showed no significant correlation with RF. CONCLUSIONS We found greater radioflux, SESC sunspot number, and sunspot area to be associated with an increased count of aneurysm rupture. The clinical meaningfulness of this statistical association must be interpreted carefully and future studies are warranted to rule out a type-1 error.

The Silencing of N-myc Downstream-Regulated Gene-1 in an Orthotopic Pancreatic Cancer Model Leads to More Aggressive Tumor Growth and Metastases

BACKGROUND: The understanding of molecular mechanisms leading to poor prognosis in pancreatic cancer may help develop treatment options. N-myc downstream-regulated gene-1 (NDRG1) has been correlated to better prognosis in pancreatic cancer. Therefore, we thought to analyze how the loss of NDRG1 affects progression in an orthotopic xenograft animal model of recurrence. METHODS: Capan-1 cells were silenced for NDRG1 (C(sil)) or transfected with scrambled shRNA (C(scr)) and compared for anchorage-dependent and anchorage-independent growth, invasion and tube formation in vitro. In an orthotopic xenograft model of recurrence tumors were grown in the pancreatic tail. The effect of NDRG1 silencing was evaluated on tumor size and metastasis. RESULTS: The silencing of NDRG1 in Capan-1 cells leads to more aggressive tumor growth and metastasis. We found faster cell growth, double count of invaded cells and 1.8-fold increase in tube formation in vitro. In vivo local tumors were 5.9-fold larger (p = 0.006) and the number of metastases was higher in animals with tumors silenced for NDRG1 primarily (3 vs. 1.1; p = 0.005) and at recurrence (3.3 vs. 0.9; p = 0.015). CONCLUSION: NDRG1 may be an interesting therapeutic target as its silencing in human pancreatic cancer cells leads to a phenotype with more aggressive tumor growth and metastasis.

CD41 T cell recovery during suppression of HIV replication: an international comparison of the immunological efficacy of antiretroviral therapy in North America, Asia and Africa.

BACKGROUND Even among HIV-infected patients who fully suppress plasma HIV RNA replication on antiretroviral therapy, genetic (e.g. CCL3L1 copy number), viral (e.g. tropism) and environmental (e.g. chronic exposure to microbial antigens) factors influence CD4 recovery. These factors differ markedly around the world and therefore the expected CD4 recovery during HIV RNA suppression may differ globally. METHODS We evaluated HIV-infected adults from North America, West Africa, East Africa, Southern Africa and Asia starting non-nucleoside reverse transcriptase inhibitorbased regimens containing efavirenz or nevirapine, who achieved at least one HIV RNA level <500/ml in the first year of therapy and observed CD4 changes during HIV RNA suppression. We used a piecewise linear regression to estimate the influence of region of residence on CD4 recovery, adjusting for socio-demographic and clinical characteristics. We observed 28 217 patients from 105 cohorts over 37 825 person-years. RESULTS After adjustment, patients from East Africa showed diminished CD4 recovery as compared with other regions. Three years after antiretroviral therapy initiation, the mean CD4 count for a prototypical patient with a pre-therapy CD4 count of 150/ml was 529/ml [95% confidence interval (CI): 517–541] in North America, 494/ml (95% CI: 429–559) in West Africa, 515/ml (95% CI: 508–522) in Southern Africa, 503/ml (95% CI: 478–528) in Asia and 437/ml (95% CI: 425–449) in East Africa. CONCLUSIONS CD4 recovery during HIV RNA suppression is diminished in East Africa as compared with other regions of the world, and observed differences are large enough to potentially influence clinical outcomes. Epidemiological analyses on a global scale can identify macroscopic effects unobservable at the clinical, national or individual regional level.

Minimum count sums for charcoalconcentration estimates in pollen slides: accuracy and potential errors

Charcoal particles in pollen slides are often abundant, and thus analysts are faced with the problem of setting the minimum counting sum as small as possible in order to save time. We analysed the reliability of charcoal-concentration estimates based on different counting sums, using simulated low-to high-count samples. Bootstrap simulations indicate that the variability of inferred charcoal concentrations increases progressively with decreasing sums. Below 200 items (i.e., the sum of charcoal particles and exotic marker grains), reconstructed fire incidence is either too high or too low. Statistical comparisons show that the means of bootstrap simulations stabilize after 200 counts. Moreover, a count of 200-300 items is sufficient to produce a charcoal-concentration estimate with less than+5% error if compared with high-count samples of 1000 items for charcoal/marker grain ratios 0.1-0.91. If, however, this ratio is extremely high or low (> 0.91 or < 0.1) and if such samples are frequent, we suggest that marker grains are reduced or added prior to new sample processing.