Comparative ecotoxicity of single and binary mixtures exposures of cadmium and nickel on growth and biomarkers of Lemna gibba.

The aim of the present study was to investigate the toxicity effects of cadmium-nickel (Cd-Ni) after single and mixtures exposures over the macrophyte Lemna gibba. Effects were assessed on growth, as frond number and fresh weight and biochemical parameters, such as total protein content and activity of antioxidant enzymes. Plants were exposed to single Cd and Ni in concentrations that ranged between 0.13-33 mg/L and 0.18 and 11.82 mg/L, respectively. For binary mixtures, individual metal IC50 values were used for selection of the evaluated concentrations. The experimental design consisted in three different ratios based on the concept of toxic units (TU), each ratio was evaluated by five different concentrations. Both single and mixture treatments were performed for 7 days following the conditions according to OECD (2006). Single and mixture exposures affected plant growth and the biomarkers of the antioxidant response. Growth parameters showed a differential sensitivity after individual metal exposures. Cd was more toxic for L. gibba plants when fresh weight was considered, but on the contrary, considering frond number, Ni was the most toxic metal. IC50-7d, based on growth rate calculated on frond number were 17.8 and 2.47 mg/L, and on fresh weight were 1.08 and 3.89 mg/L, for Cd and Ni respectively. LOEC values for Cd were obtained at 2.06 and 1.03 mg/L, for frond number and fresh weight, respectively; while for Ni, these values were 0.92 and 11.82 mg/L. The three evaluated ratios for binary mixtures resulted in a high toxicity considering the same response variables in single metal exposures. Ratio 1 (2/3 TU Cd-1/3 TU Ni) was the most toxic considering both frond number and fresh weight, showing percentage inhibition of growth rates of 96 and 90%, respectively for the highest concentration. A modification of the protein content was observed in single, but especially in the mixture treatments. The activity of catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APOX; EC 1.11.1.11) and guaiacol peroxidase (GPOX; EC 1.11.1.7) was also affected in single and mixtures assays. APOX and GPOX showed a higher increase of its activities respect the controls after mixture treatments than for single metal treatments. Such optimization of the antioxidant system could be one of the causes of the antagonistic toxicity observed in mixture exposures. Concentration addition (CA) reference model, based on frond number, in Cd-Ni mixtures was not a good predictor to evaluate toxicity from dissolved metal concentration since the results showed that toxicity was less than additive, with an average of ΣTU = 2.17. The observed antagonisms resulted to be stronger in mixtures with higher metal concentrations.

Comparative ecotoxicity of single and binary mixtures exposures of cadmium and zinc on growth and biomarkers of Lemna gibba.

In the present study, single and mixture effects of cadmium (Cd) and zinc (Zn) on Lemna gibba were analyzed and compared using growth parameters, based on frond number and fresh weight, and biochemical parameters, such as pigment, protein content and activity of antioxidant enzymes. Plants were exposed for 7 days to these metals in nutrient solution. Single and mixture exposures affected plant growth and the biomarkers of the antioxidant response. Considering the growth parameters, Cd was found to be much more toxic than Zn. IC50-7d, based on growth rate calculated on frond number, were 17.8 and 76.73 mg/L, and on fresh weight were 1.08 and 76.93 mg/L, for Cd and Zn respectively. For Cd, LOEC values were obtained at 2.06 and 1.03 mg/L, for frond number and fresh weight respectively; while for Zn, at 20.1 and 74.6 mg/L. A high toxicity effect, considering the same response variables, was observed in plants exposed to the mixtures. Three fixed ratios, based on toxic units (TU) were assayed, ratio 1: 2/3 Cd-1/3 Zn, ratio 2: 1/2 Cd-1/2 Zn and ratio 3: 1/3 Cd-2/3 Zn. Ratio 3 (where Zn was added in higher proportion) was the less toxic. All concentrations of Ratio 1 and 2 significantly inhibited plant growth, showing a 100% inhibition of growth rate at the highest concentrations when based on frond number. Catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APOX; EC 1.11.1.11) and guaiacol peroxidase (GPOX; EC 1.11.1.7) activities in single metals assays were higher than controls. In mixture tests, the activity of APOX and GPOX was significantly stimulated in plants exposed to all evaluated combinations, while CAT was mainly stimulated in Ratio 3. It was observed that the activity of the enzymes was increased in the mixtures compared with similar concentrations evaluated individually. APOX activity was observed to fit the CA model and following a concentration-response pattern. The response of this antioxidant enzyme could serve as a sensitive stressor biomarker for Cd-Zn interactions. Frond number in Cd-Zn mixtures was not well predicted from dissolved metal concentration in solution using concentration addition (CA) as reference model, as results showed that toxicity was more than additive, with an average of ΣTU = 0.75. This synergistic effect was observed up to 50 mg Zn/L in the mixture, but when it was present in higher concentrations a less than additive effect was observed, indicating a protective effect of Zn. A synergistic and dose-ratio deviations from CA model were also observed.

Comparative ecotoxicity of single and binary mixtures exposures of nickel and zinc on growth and biomarkers of Lemna gibba.

The aim of this study was to compare the ecotoxicity of nickel (Ni) and zinc (Zn) assayed as single and as binary mixture. In addition, how were affected the population growth rates and oxidative stress biomarkers, comparing single to binary exposures. The toxicity tests were performed on Lemna gibba using a 7-day test. All calculations were made using measured total dissolved metal concentrations. IC50-7d, based on growth rate calculated on frond number and fresh weight, were 2.47/3.89 mg/L, and 76.73/76.93 mg/L, for Ni and Zn, respectively. Single metals affected plant growth following a non-linear concentration-response relationship. LOEC values for each metal were obtained at 0.92 and 20.1 mg/L for Ni and Zn, respectively. Biomarkers of the antioxidant response like Catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APOX; EC 1.11.1.11) and guaiacol peroxidase (GPOX; EC 1.11.1.7) activities in single metals assays were higher than controls, but when similar concentrations were added as mixtures, that increase was reduced and inhibition with respect to the control was observed for GPOX. APOX showed the highest activity. The concentration addition (CA) approach was evaluated and resulted in a correct predictor of Ni-Zn mixture toxicity on Lemna gibba. This was made comparing the EC50 and LOEC, measured taking the growth rate as endpoint, with those expected values according to the CA model. However, the measured biomarkers indicating a positive response to free radicals did not fit to concentration addition model when assayed in the binary mixture. Also, the main activity response of these was observed within a range of concentrations below the LOEC values for the mixture.

[Conjunctival keratoacanthoma: diagnosis, treatment and monitoring by conjunctival impression cytology]. / Queratoacantoma conjuntival. Diagnóstico, tratamiento y control mediante citología de impresión conjuntival.

CASE REPORT: We present a case report of a 28-year-old patient with a lesion that is compatible both clinically and histopathologically with conjunctival keratoacanthoma. The treatment given was complete excision and 0.04% mitomycin C eye drops in the postoperative period. The outcome was a complete clinical remission during the follow-up period (6 months). DISCUSSION: It is important to make a correct differential diagnosis between keratoacanthoma and squamous cell carcinoma, as well as carrying out close monitoring after surgery due to the possibility of relapse and conversion to squamous cell carcinoma. For this reason, we propose the use of conjunctival impression cytology as a non-invasive method for monitoring such patients.

Queratoacantoma conjuntival. Diagnóstico, tratamiento y control mediante citología de impresión conjuntival / Conjunctival keratoacanthoma. Diagnosis, treatment and monitoring by conjunctival impression cytology

Caso clínico: Se presenta el caso de un paciente de 28 años con una lesión compatible clínica e histopatológicamente con queratoacantoma conjuntival. Fue tratado mediante excisión completa, y colirio de mitomicina C al 0,04% en el posoperatorio. El resultado ha sido la remisión clínica completa durante el tiempo de seguimiento (6 meses). Discusión: Es importante hacer un correcto diagnóstico diferencial entre queratoacantoma y carcinoma de células escamosas, así como un estrecho seguimiento postoperatorio por la posibilidad de recidiva o conversión a carcinoma de células escamosas. Para ello proponemos la citología de impresión conjuntival como un método no invasivo para el seguimiento de estos pacientes(AU)

Case report: We present a case report of a 28-year-old patient with a lesion that is compatible both clinically and histopathologically with conjunctival keratoacanthoma. The treatment given was complete excision and 0.04% mitomycin C eye drops in the postoperative period. The outcome was a complete clinical remission during the follow-up period (6 months). Discussion: It is important to make a correct differential diagnosis between keratoacanthoma and squamous cell carcinoma, as well as carrying out close monitoring after surgery due to the possibility of relapse and conversion to squamous cell carcinoma. For this reason, we propose the use of conjunctival impression cytology as a non-invasive method for monitoring such patients(AU)

Positron annihilation and relaxation dynamics from dielectric spectroscopy and nuclear magnetic resonance: cis-trans-1,4-poly(butadiene).

We report a joint analysis of positron annihilation lifetime spectroscopy (PALS), dielectric spectroscopy (BDS), and nuclear magnetic resonance (NMR) on cis-trans-1,4-poly(butadiene) (c-t-1,4-PBD). Phenomenological analysis of the orthopositronium lifetime τ(3)-T dependence by linear fitting reveals four characteristic PALS temperatures: T(b1)(G)=0.63T(g)(PALS), T(g)(PALS), T(b1)(L)=1.22T(g)(PALS), and T(b2)(L)=1.52T(g)(PALS). Slight bend effects in the glassy and supercooled liquid states are related to the fast or slow secondary ß process, from neutron scattering, respectively, the latter being connected with the trans-isomers. In addition, the first bend effect in the supercooled liquid coincides with a deviation of the slow effective secondary ß(eff) relaxation related to the cis-isomers from low-T Arrhenius behavior to non-Arrhenius one and correlates with the onset of the primary α process from BDS. The second plateau effect in the liquid state occurs when τ(3) becomes commensurable with the structural relaxation time τ(α)(T(b2)). It is also approximately related to its crossover from non-Arrhenius to Arrhenius regime in the combined BDS and NMR data. Finally, the combined BDS and NMR structural relaxation data, when analyzed in terms of the two-order parameter (TOP) model, suggest the influence of solidlike domains on both the annihilation behavior and the local and segmental chain mobility in the supercooled liquid. All these findings indicate the influence of the dynamic heterogeneity in both the primary and secondary relaxations due to the cis-trans isomerism in c-t-1,4-PBD and their impact into the PALS response.

Genotoxicity of aqueous elutions of industrial soils.

The genotoxicity of industrial soils was evaluated. A single cell electrophoresis assay or comet assay, using eleocyte cells of Eisenia foetida, was performed to assess the genotoxicity of aqueous elutions. These were obtained from industrial soils containing metals. All soil samples meet the environmental quality guidelines for metal concentrations. However, elutions have produced genotoxic effects at dilutions as low as 6%. Total metal concentrations for each aqueous elution could express synergistic effects of these compounds.

Risk assessment of domestic and industrial effluents unloaded into a freshwater environment.

An ecotoxicologic study was performed to assess the environmental status of the Lujan River. It is an important freshwater system in the northeast of Buenos Aires Province, Argentina. Surface waters (SWs) and liquids effluents (LEs), before they reached the river, and sediments were assessed via acute toxicity screening using a battery of tests with native species. Additionally, the presence, in each LE and SW sample, of bioaccumulatable compounds was checked by SPME extraction and gas chromatograph-MS determination. An environmental risk assessment of each LE was carried out via toxic units and assessment factors approach and through extrapolation methods. Hazardous concentrations for each LE were compared with their river effluent concentrations. Ninety-one percent (91%) of the total toxic load of the river was due to 4 of 11 LEs (37%) evaluated. Although SW samples were not toxic, a real environmental risk was found for this freshwater environment. Sediment toxicity was found to be related to the proximity to pipe discharges. Bioaccumulatable compounds were found in SWs and in LEs. Esters of phthalic acids, morpholine, hydroquinone, and nonylphenol were found throughout the river at different sample sites and in different months during the 1-year sampling program.

[Multicenter cohort of patients with HIV infection in the Madrid south-eastern metropolitan crown (COMESEM): basis, organization and initial results]. / Cohorte multicéntrica de pacientes con infección VIH de la corona metropolitana sudeste de Madrid (COMESEM): fundamentos, organización y resultados iniciales.

BACKGROUND: In order to evaluate changes related to the effect of new anti-retroviral agents and preventive programs, cohort studies of patients with HIV in our environment are needed. METHODS: Cohort study of patients diagnosed of HIV infection in the five hospitals included in the Madrid metropolitan south-eastern crown (COMESEM), which attend a population of 1,300,000 inhabitants. RESULTS: A total of 5,532 patients had been recruited until August 2001 (with a follow-up of 34,227 patients-year). The male/female ratio was 3/1. As for the transmission mechanism, 72.9% were parenteral drug users (PDU), 13.7% heterosexuals (HTX) and 8% of males having sex with males (MSM). The maximal figure of diagnosis per year corresponded to 1991 for PDU, 1993 for MSM and 1995 for heterosexuals. A decline in the number of diagnosed patients was observed for all groups, but MSM showed an increase in the year 2001 for the first time. Notably, among HTX, a growing and sustained trend was observed of patients not born in Spain (0% in 1993 and 50% in 2001; p < 0.001). CONCLUSIONS: Establishing a large cohort of HIV infected patients based upon medium-sized hospitals is possible. The analysis of data derived from this cohort allows the early detection of changes in the clinical and epidemiological profile of HIV infection.

Cohorte multicéntrica de pacientes con infección VIH de la corona metropolitana sudeste de Madrid(COMESEM): fundamentos, organización y resultados iniciales / Multicenter cohrt of patients with HIV infection in the Madrid south-eastern metropolitan crown (COMESEN): basis organization and initial results

Fundamento. Se necesitan estudios de cohorte de pacientes con infección del virus de la inmunodeficiencia humana (VIH) en nuestro medio para evaluar los cambios relacionados con el efecto de los nuevos antirretrovíricos y los programas de prevención. Métodos. Estudio de cohorte de todos los pacientes diagnosticados de infección VIH en los 5 hospitales de la corona metropolitana sudeste de Madrid (COMESEM) que atienden a una población de 1.300.000 habitantes. Resultados. Hasta agosto de 2001 se han reclutado 5.532 pacientes (con un seguimiento de 34.227 pacientes año). Hay 3 varones por cada mujer. Por vía de transmisión, el 72,9 por ciento eran usuarios de drogas por vía parenteral (UDVP), el 13,7 por ciento heterosexuales (HTX) y el 8 por ciento hombres que mantienen relaciones sexuales con hombres (HSH). El máximo de diagnósticos por año correspondió a 1991 para los UDVP, 1993 para los HSH y 1995 para los heterosexuales. En todos los grupos se ha observado un declive en el número de pacientes diagnosticados, pero los HSH en el año 2001 muestran un incremento por vez primera. Entre los HTX destaca la tendencia creciente y mantenida de pacientes no nacidos en España (0 por ciento en 1993 y 50 por ciento en 2001; p < 0,001).Conclusiones. Es posible establecer una cohorte amplia de pacientes con infección VIH basándose en hospitales de tamaño medio. El análisis de los datos derivados de esta cohorte permite objetivar cambios en el perfil clínico-epidemiológico de la infección VIH de manera precoz (AU)

[A user-friendly scale for the short and long term outcome of bipolar disorder: the CGI-BP-M]. / Escala sencilla de evaluación del curso del trastorno bipolar: CGI-BP-M.

UNLABELLED: Bipolar disorder is a cyclic and polymorphic disease. Patients may show manic, hipomanic, depressive or mixed symptoms, and they may be in partial or complete remission. For this reason, the assessment of the course, severity and outcome of the disorder is very complex. Most of the available psychometric instruments have been designed for the assessment of acute episodes of specific polarity. METHODS: We present the modified version of the Clinical Global Impression for Bipolar Disorder (CGI-BP-M) a condensed version of the CGI-BP, which is also an adaptation of the CGI for bipolar patients. RESULTS: The CGI-BP-M takes only a few minutes to complete, and it has proved to be useful in the assessment of the short and long-term efficacy of several treatments such as olanzapine and quetiapine. Significant improvements in several subescales of the CGI-BP-M after treatment with these compounds are shown. CONCLUSIONS: The CGI-BP-M, a user-friendly scale for the assessment of manic, hypomanic, depressive or mixed symptoms, and long-term outcome of bipolar disorder, is a useful tool for the assessment of the efficacy of several treatments.

Escala sencilla de evaluación del curso del trastorno bipolar: CGI-BP-M / A user-friendly scale for the short and long term outcome of bipolar disorder: the CGI-BP-M

El trastorno bipolar se caracteriza por su curso clínico y su gran polimorfismo. Los pacientes pueden presentar episodios maníacos, hipomaníacos, depresivos o mixtos, con períodos de remisión parcial o completa. Por este motivo, la evaluación del curso, gravedad y pronóstico de la enfermedad es enormemente compleja. La mayor parte de instrumentos psicométricos disponibles están diseñados para la evaluación de síntomas agudos de un tipo específico de episodio. Material y método. Se presenta la 'escala de impresión clínica global para el trastorno bipolar modificada' (CGIBP-M), que es una versión reducida diseñada por los autores a partir de la CGI-BP, que a su vez es una adaptación de la CGI para su uso específico en pacientes bipolares. Resultados. La CGI-BP-M exige sólo unos minutos y se ha demostrado útil en la evaluación de la eficacia de diversos tratamientos como la olanzapina y la quetiapina, tanto a corto como a largo plazo, mostrando reducciones significativas en las puntuaciones de gravedad sintomática para diversas subescalas tras el tratamiento. Conclusiones. La CGI-BP-M, una escala sencilla de evaluación de la enfermedad de los síntomas maníacos, hipomaníacos, depresivos y mixtos, y de la evolución a largo plazo del trastorno bipolar, es un instrumento útil para la evaluación de la eficacia de nuevos tratamientos (AU)

Heat waves in Madrid 1986-1997: effects on the health of the elderly.

OBJECTIVE: The objective of this paper is to analyse and quantify the effects exerted on summer mortality by extremes of heat, particularly among persons aged 65-74 and 75 years and over, groups in which mortality is higher. METHODS: The study included the period from 1 January 1986 to 31 December 1997, for all people aged over 65 years resident in Madrid, based on mortality due to all causes except accidents (ICD-9 codes 1-799), and circulatory (390-459) and respiratory (460-487) causes. Meteorological variables analysed were: daily maximum temperature, daily minimum temperature and relative humidity. To control the effect of air pollution on mortality we considered the daily mean values of sulphur dioxide (SO2), total suspended particulate (TSP), nitric oxides (NOx), nitrogen dioxide (NO2) and tropospheric ozone (O3). Univariate and multivariate ARIMA models were used. Box-Jenkins pre-whitening was performed. RESULTS: The results yielded by this study indicate a mortality increase up to 28.4% for every degree the temperature rises above 36.5 degrees C, with particular effect in women over the age of 75 years and circulatory-cause mortality. The first heat wave that leads to the greatest effects on mortality, due to the higher number of susceptible people and the duration of the heat wave, show an exponential growth in mortality. Furthermore, low relative humidity enhances the effects of high temperature, linking dryness to air pollutants, ozone in particular. CONCLUSIONS: Since a warmer climate is predicted in the future, the incidence of heat wave should increase, and more comprehensive measures, both medical and social, should be adopted to prevent the effects of extreme heat on the population, particularly the elderly.

Concordance degrees in macrozoobenthic monitoring programmes using different sampling methods and taxonomic resolution levels.

In summer 1997, an intensive survey on the hard bottoms of the 'Abra de Bilbao' (N. Iberian Peninsula) was carried out in the context of the macrozoobenthic monitoring programmes developed to assess the biological recovery of the area. Three types of measurements (abundance, biomass and cover) were used to describe and compare the structure and composition of the communities at three littoral zones: subtidal, lower intertidal and upper intertidal. In addition, several taxonomic aggregation levels of data were successfully applied. The main objective of this paper is to explore the relative effect caused on the results of such programmes by earlier decisions concerning the type of measurement and the taxonomic resolution level to be applied. A 'second stage' multivariate procedure of analysis has been perforrmed based on the previously obtained sampling site ordinations. The measurement type chosen has been found to have a greater effect on the results than the taxonomic resolution used. Moreover, it is suggested that analyses based on abundance data usually lose more information when taxonomic resolution decreases than those based on biomass or cover estimates. The highest concordance among the different analyses has been found in the subtidal zone, which is considered the most appropriate habitat for the development of benthic monitoring programmes.

A model for forecasting emergency hospital admissions: effect of environmental variables.

This study modeled patterns and trends in emergency hospital admissions at a hospital in Madrid, Spain. The purpose was to quantify qualitative associations that have been detected between such admissions and a number of environmental variables. The following data were used: unscheduled daily emergency hospital admissions, Madrid air pollution data, and meteorological data. Time-series analysis was performed, with Box-Jenkins modeling. A multivariate model was constructed, incorporating the different causes of admissions and the respective environmental variables. Statistically significant associations were found between hospital admissions and other variables, indicating relationships with temperature, relative humidity, and mean daily tropospheric ozone concentrations. Whereas the effect of heat on admissions was short term, that of cold was in evidence from the second week. The association with ozone showed a seven-day lag and basically manifested itself as an influence on admissions for circulatory disease.

Relationship between atmospheric pressure and mortality in the Madrid Autonomous Region: a time-series study.

This study set out to determine the possible influence of variations in atmospheric pressure on mortality in the Madrid Autonomous Region (MAR), taking into account the possible confounding effect of other atmospheric variables. The study was based on daily mortality data from the MAR Revenue and Excise Authority, meteorological data from Getafe Observatory and air pollution data from the Madrid Municipal Automatic Air Pollution Monitoring Grid. A time-series analysis was performed, using Box-Jenkins modelling and controlling for the respective confounding variables. Furthermore, the different variables studied (pressure, temperature, pollutants, etc.) were used to produce a multivariate model of the different causes of mortality. A significant association was found between anticyclonic trend and mortality with circulatory causes in the medium-long term and anticyclonic trend and mortality with respiratory causes in the long term.

Diagnóstico prenatal de las triploidias / Prenatal diagnosis of triploidies

Exponemos dos casos de triploidias recogidos en nuestro medio, y detectados previamente por las anomalías morfológicas apreciadas en el estudio ultrasonográfico: hidrocefalia grave, gastrosquisis, oligoamnios y retraso de crecimiento intraútero severo. En ningún caso hubo cambios degenerativos molares placentarios, que son más frecuentes cuando las dos series de cromosomas son de origen paterno. Aunque la mayoría de los casos conducen el aborto precoz, a veces pueden llegar a vivir unas pocas semanas si el juego extra cromosómico es de origen materno. En ambos la confirmación del diagnóstico depende del cariotipo fetal obtenido por amniocentesis (AU)

A randomized trial of the discontinuation of primary and secondary prophylaxis against Pneumocystis carinii pneumonia after highly active antiretroviral therapy in patients with HIV infection. Grupo de Estudio del SIDA 04/98.

BACKGROUND: Prophylaxis against Pneumocystis carinii pneumonia is indicated in patients with human immunodeficiency virus (HIV) infection who have less than 200 CD4 cells per cubic millimeter and in those with a history of P. carinii pneumonia. However, it is not clear whether prophylaxis can be safely discontinued after CD4 cell counts increase in response to highly active antiretroviral therapy. METHODS: We conducted a randomized trial of the discontinuation of primary or secondary prophylaxis against P. carinii pneumonia in HIV-infected patients with a sustained response to antiviral therapy, defined by a CD4 cell count of 200 or more per cubic millimeter and plasma HIV type 1 (HIV-1) RNA level of less than 5000 copies per milliliter for at least three months. Prophylactic treatment was restarted if the CD4 cell count declined to less than 200 per cubic millimeter. RESULTS: The 474 patients receiving primary prophylaxis had a median CD4 cell count at entry of 342 per cubic millimeter, and 38 percent had detectable HIV-1 RNA. After a median follow-up period of 20 months (758 person-years), there had been no episodes of P. carinii pneumonia in the 240 patients who discontinued prophylaxis (95 percent confidence interval, 0 to 0.85 episode per 100 person-years). For the 113 patients receiving secondary prophylaxis, the median CD4 cell count at entry was 355 per cubic millimeter, and 24 percent had detectable HIV-1 RNA. After a median follow-up period of 12 months (123 person-years), there had been no episodes of P. carinii pneumonia in the 60 patients who discontinued prophylaxis (95 percent confidence interval, 0 to 4.5 episodes per 100 person-years). CONCLUSIONS: In HIV-infected patients receiving highly active antiretroviral therapy, primary and secondary prophylaxis against P. carinii pneumonia can be safely discontinued after the CD4 cell count has increased to 200 or more per cubic millimeter for more than three months.