Size-fractioned ultrafine particles and black carbon associated with autonomic dysfunction in subjects with diabetes or impaired glucose tolerance in Shanghai, China
2015

Yitong Sun
Equal contributors

Department of Occupational & Environmental Health Sciences
Institute of Environmental Medicine
Ministry of Education
Key Laboratory of Molecular Cardiovascular Sciences
Peking University School of Public Health
100191BeijingChina

†
Xiaoming Song
Equal contributors

Department of Occupational & Environmental Health Sciences
Institute of Environmental Medicine
Ministry of Education
Key Laboratory of Molecular Cardiovascular Sciences
Peking University School of Public Health
100191BeijingChina

†
Yiqun Han
Yunfang Ji
Shuna Gao
Yu Shang
Shou-En Lu
Tong Zhu
Wei Huang firstname.lastname@example.com
Department of Occupational & Environmental Health Sciences
Institute of Environmental Medicine
Ministry of Education
Key Laboratory of Molecular Cardiovascular Sciences
Peking University School of Public Health
100191BeijingChina

Size-fractioned ultrafine particles and black carbon associated with autonomic dysfunction in subjects with diabetes or impaired glucose tolerance in Shanghai, China

Particle and Fibre Toxicology
128201510.1186/s12989-015-0084-6RESEARCH Open Access *Correspondence: Full list of author information is available at the end of the article to the data made available in this article, unless otherwise stated. Sun et al. Particle and Fibre Toxicology (2015) 12:8 Page 2 of 11Ultrafine particulatesBlack carbonTraffic pollutionHeart rate variabilityDiabetesImpaired glucose tolerance
Background: Particles in smaller size fractions, such as ultrafine particles (UFPs) (with diameter less than 100 nm), has become of significant cardiovascular health concerns. However, the biological plausibility underlying potential relationship between UFPs and cardiovascular outcomes is less studied. Methods: Fifty-three subjects living in Shanghai with type-2 diabetes (T2D) or impaired glucose tolerance (IGT) were followed for autonomic dysfunctions with three repeated measurements in 2010. Minute-to-minute concentrations of ambient particles in small size-fractions (5-560 nm), black carbon (BC), sulfur dioxide (SO 2 ), nitrogen dioxide (NO 2 ), carbon monoxide (CO), and ozone (O 3 ) were monitored using a central monitoring laboratory equipped with real-time air monitors close to residential area of the subjects. Generalized linear mixed models, with adjustment for individual risk factors, were applied to assess the effects of air pollution on autonomic dysfunctions in subjects. Results: Our study showed that significant reduction in the standard deviation of all NN intervals (SDNN) ranging from 3.4% to 8.1% were associated with interquartile range (IQR) increase of number concentration of particles (PNC) in size fractions <100 nm, and reduction from 1.3% to 4.6% with particles of diameter 100-200 nm, in subjects with diabetes or glucose tolerance. Increased exposure to traffic-related pollutants BC, NO 2 and CO, and combustion pollutant SO 2 , were also significantly associated with HRV reductions. However, no effect was observed for particles in size fraction of 200-560 nm and O 3 . Diabetic risk factor and gender appeared to have significant interactions on autonomic dysfunction associated with UFPs and traffic pollution exposures in certain time-window. Conclusions: Our results suggest that underlying diabetes or impaired glucose tolerance may confer reduced autonomic function of heart due to traffic pollution exposure.

Background

Association between particulate matter, with diameter less than 10 and 2.5 μm (PM 10 and PM 2.5 ), and cardiovascular diseases (CVD) has been supported by numerous epidemiologic studies [1]. Particles in smaller size fractions, such as ultrafine particles (UFPs) (with diameter less than 100 nm), have have been reported that may contribute to increasing cardiovascular morbidity [2][3][4]. Particles emitted from transportation and industry combustion have been found dominating the number and surface concentrations of ambient particulate matters in small size fractions [4][5][6], and carrying large amount of adsorbed or condensed toxic air pollutants (oxidant gases, organic compounds, and transition metals) [7]. Studies reported that UFPs can potentially lead to acute cardiovascular responses [8][9][10][11]. However, compared to ambient PM 2.5 and PM 10 , underlying biological plausibility of relationship between UFPs and cardiovascular morbidity has been far less studied.

Epidemiologic study findings suggested altered heart rate variability (HRV) as a possible mechanism linking ambient PM exposure to coronary heart disease events in subjects with preexisting cardiometabolic diseases [12][13][14][15][16]. Reduced HRV has been found associated with increasing risk of first cardiovascular event in population without known cardiovascular diseases [17], and associated with the development of coronary heart disease in individuals with diabetes [18]. The reduction of HRV derived from electrocardiographic (ECG) monitor recordings were found inversely associated with exposure to ambient PM originated from traffic and combustion sources [19,20]. Recent studies also reported the associations between HRV reduction and exposure to particles in smaller size including UFPs and particles in accumulation mode (with diameter less than 1 μm) [21,22], and among persons with cardiometabolic diseases or with metabolic syndromes [12,13,23,24]. However, the susceptibility of persons with cardiometabolic disease to the adverse effects of particulate matter is still uncertain, despite being biologically plausible.

Because of the increasing prevalence of diabetes and its association with an increasing risk of CVD, it is important to determine whether diabetes or impaired glucose tolerance may modify PM-HRV association. Some evidence suggests that ambient particles in small size fractions may play an important role in previously reported associations between traffic exposure and acute cardiovascular effects [4,25,26]. In this study, we assessed the role of exposure to particles in small size fractions 5-560 nm and black carbon (as a proxy for traffic related pollutant exposure) as cardiovascular risk factors in a group of subjects with diabetes or impaired glucose tolerance. We further examined if the association would be modified by diabetic state, obesity and gender.

Results and discussion

Results

The demographic and clinical characteristics of our study subjects are summarized in Table 1. The age of subjects ranged from 51 to 68 years old. There was no significant difference in gender ratio between type-2 diabetes (T2D) and impaired glucose tolerance (IGT) groups, or between obese and non-obese groups (p > 0.1). No subject had severe cardiovascular diseases, except 68% of the subjects were with mild hypertension. All subjects with T2D were on regular medications (i.e. biguanides or sulfonylureas), and/or with insulin treatment; however, the subjects with IGT did not take regular medication, except one subject was taking biguanides regularly controlling for blood glucose level.

Summary statistics of particle number concentrations (PNC) in six aggregated size fractions, BC, gaseous pollutants, and meteorologic parameters are summarized in Table 2. Within all the size fractions of particles, which are those of diameters from 5 nm to 560 nm, particles of size less than 200 nm dominated the number concentration, and ones of size 50-560 nm contributed the most of the surface concentration and the mass concentration (see Additional file 1).

All metrics of concentrations of particles in the size fraction 50-100 nm and 100-200 nm were highly correlated with BC, NO 2 , and CO with correlation coefficients ranging from 0.55 to 0.78 (Table 3 and Additional file 1). The total number and surface area concentrations of particles were also highly correlated with BC and NO 2 . Particles in all size fractions of measurement were inversely correlated with O 3 , temperature and relative humidity (RH).  SD for standard deviation and IQR for interquatile range. The observations of all the pollutants and meteorologic parameters were obtained in 1 minute interval covering the whole period of each visit in April, June and September.

In Figure 1, we presented the adjusted percent changes of standard deviation of all NN intervals (SDNN) per interquartile range (IQR) increase of number concentrations of particles in all size fractions, BC, and gaseous pollutants in various time windows of pollutant exposure, controlling age, gender, body mass index (BMI), time of day, day of week, temperature, RH and visit. Greater reductions in SDNN were associated with increase in exposure to particles in smaller size fractions <100 nm, followed by particles in 100-200 nm. Increased exposure to traffic-related pollutants BC, NO 2 and CO, and combustion pollutant SO 2 , were also significantly associated with SDNN reductions. Overall, the inverse associations were the strongest for 4-hour average air pollutants exposure. No effect was observed for particles in size fraction of 200-560 nm and O 3 . Similar association patterns are observed for other HRV measures, including the root mean square of successive differences between adjacent normal cycles (rMSSD), low frequency (LF) (0.04-0.15 Hz) and high frequency (HF) (0.15-0.4 Hz), whereas the magnitude of reduction for frequency-domain measure LF and HF were greater (see Additional file 2).

To control the confounding effects of highly correlated pollutants, we compared the estimated effects of prior 4-hour average exposure to all size-fractioned particles, BC, O 3 , and NO 2 in single-and two-pollutant models (Table 4). In two-pollutant models with adjustment for BC, the magnitude of effect estimates remained similar for number concentrations of particles of size 10 to 20 nm (PNC 10−20 ), NO 2 , and CO as the magnitude in single-pollutant models. The magnitude of effects was reduced for PNC 20−50 , PNC 50−100 and PNC 5−560 , whereas the effects became insignificant for PNC 100−200 and PNC 200−560 . With adjustment for BC, the associations with surface area concentrations of particles (PSC), and mass concentrations of particles (PMC), remained significant for particles in size-fraction 10-20 nm and 20-50 nm only. In two-pollutant models with adjustment for NO 2 , the associations between SDNN and all measured pollutants, except BC, remained robust and consistent as assessed in single-pollutant models. We did not observe confounding effects of exposure to O 3 (results summarized in Additional file 3).

Between subjects with T2D and in prediabetic state with IGT, effects assessed at 4-and 24-hour average exposure were presented in Figures 2 and 3. For prior 4-hour average air pollution exposure, we did not observe significant heterogeneity in effect estimates (with p values for interaction >0.05); however, effects in subjects with IGT were significantly stronger with prior 24-hour average exposure (with p values for interaction <0.05). Figure 4 shows the interaction effect of obesity on associations between SDNN and 4-hour average of air pollution exposure. Subjects with BMI>25 kg/m 2 were found of greater SDNN reduction associated with exposure to air pollution, and the risk was even higher in subjects with BMI>28 kg/m 2 (with p values for interaction <0.05). However, we did not find significant interaction of central  adiposity in female nor in male subjects, though subjects with lower waist to hip ratio appeared in greater SDNN reduction associated with exposure to air pollution (with p values for interaction >0.05, data in Additional file 4). The correlation coefficients between obesity index BMI and central adiposity index waist to hip ratio (WHr) ranged 0.27 to 0.45 in our subjects.

In the regression analyses of 4-hour average exposure stratified by gender, with controlling for same covariates in main models, we observed significant heterogeneity of gender (with p values for interaction <0.05) ( Figure 5). Per IQR increase in 4-hour average of PNC 5−560 (10,300 cm 3 ), we observed -12.0% (95% CI, -14.1% to -9.8%) in SDNN in female, and -4.2% (95% CI, -6.4% to -1.9%) in male, with p value for interactions <0.05. The modifiction effect of gender were significant when exposure to particles in size fraction <200 nm, NO 2 , SO 2 , or CO was considered, and marginally significant on BC exposure.

Discussion

In our study, we observed significant inverse associations between autonomic dysfunctions in subjects with diabetes  and impaired glucose tolerance and exposure to ambient particles in small size fractions (diameter less than 200 nm), BC, NO 2 (traffic pollution proxy) and CO. The associations were stronger in subjects with obesity and in female. However, the interaction effect of diabetic states was not consistent over different averaging hours. Our findings suggest that possible cardiovascular effects associated with traffic pollution exposure may be mediated, in part, through altered autonomic function, among persons with diabetes or impaired glucose tolerance. Our findings have broad clinical and public health implications for air pollution induced cardiometabolic risks.

UFPs are an important component of combustionrelated or secondary aerosol-related air pollution with large amount of toxic air pollutants and chemicals adsorbed on its surfaces. With its high lung deposition efficiency in the lungs, UFPs can enter into pulmonary interstitial sites and through circulation system  reaching other target sites. The hypothesized intermediary pathophysiologic pathways associated with UFP exposures include release of proinflammatory mediators or vasculoactive molecules from lung-based cells, perturbation of systemic autonomic nerve system balance or heart rhythm, and potentially translocation of PM or PM constituents into circulation system [27]. Thus, we focused on examining cardiovascular risk associated with exposure to particles in small size fractions (between 5 to 560 nm) including UFPs, and traffic pollution markers (BC, NO 2 , and CO). Our overall results support the hypothesis that greater cardiovascular responses are associated with UFPs than with larger particles, and are consistent with findings of autonomic, vascular and thrombotic effects of UFPs inhalation observed in susceptible and healthy subjects [21,22,25,28,29].

However, the associations between UFPs exposures and acute changes in HRV reported in previous studies are not consistent and the directions and magnitudes of the associations varied across study locations. Chan et al. reported significant inverse relationship between hours prior average UFP exposures in HRV in a panel of healthy and elderly patients, with stronger effects in elderly subjects in Taiwan [30]. Weichenthal et al. reported significant reduction in HF and the proportion of NN50 divided by total number of NNs (pNN50) within hours immediately following UFP exposures in healthy cyclists in traffic in Ottawa, Canada [21].

Park et al. observed inverse association between ambient UFP exposures over 48-hour moving averages and HRV in elderly male subjects in Boston, USA, though the associations were not statistically significant [23].

In more recent studies, Schneider et al. did not observe significant associations between fixed-site ambient UFP measures and HRV over up to 47 lag hours in elderly men with coronary artery disease in Erfurt, Germany [14]; whereas Rich et al. reported decreased rMSSD associated with UFP exposures at significant level, but not with particles in accumulation mode (100-500 nm) and PM 2.5 , in a cardiac panel in rehabilitation in Rochester, USA [22]. Vora et al. lately reported that inhalation of elemental carbon ultrafine particles can alter heart rate and HRV in subjects with T2D [31]. It is possible that other pathophysiological factors such as activation of the renin-angiotensin system, oxidative stress, and inflammatory status in subjects with cardiovascular morbidities may have modulated the association between air pollution exposure and autonomic nervous system dysfunction.

Pathophysiologically, autonomic nervous system dysfunction also relates to insulin resistance [32,33], and abnormal glucose metabolism [34,35]. Park et al. reported significant HRV reduction in subjects with metabolic syndrome associated with exposure to 2-day average PM 2.5 in six US communities in the Multi-Ethnic Study of Atherosclerosis (MESA) study, whereas no significant change was found among subjects without metabolic syndrome [13]. Whitsel et al. observed stronger inverse PM 10 -HRV associations in subjects with diabetes, and diabetic subjects of higher insulin and insulin resistance level were of statistically greater HRV reduction [12]. Recently, Min et al. observed a significant reduction in HRV related to exposure to carbon monoxide (CO) among persons with metabolic syndrome but not among persons without metabolic syndrome [36]. In our analysis, we examined the roles of diabetic state in modifying autonomic dysfunctions associated with ambient pollution. We observed significantly stronger inverse associations with 24-hour prior exposure to UFPs and traffic pollutants in subjects with IGT who did not receive regular medication to control for glucose level, but not in T2D subjects. However, with 4-hour prior exposure, the interaction of diabetic state between subjects with T2D and with IGT was not significant. The differential responses observed may be partially explained by insulin resistance or glucose metabolism altered by medication use in subjects.

As an important risk factors of metabolic syndrome, obesity is closely correlated with increased systemic inflammation and oxidative stress that are cardiovascular risk factors [37,38]. There is a growing body of evidence that T2D is a chronic inflammatory state aggravated by factors that promote inflammation at the level of vasculature and adipose tissue [39,40], and increased oxidative stress to adipocytes is central to the pathogenesis of cardiovascular disease in metabolic syndrome [41]. Air pollution may also promote T2D by increasing adipose inflammation and insulin resistance. Sun et al. recently found that PM 2.5 exposure exaggerates whole-body insulin resistance and adiposity inflammation in mice [42]. Consistent with previous studies [20,24], obesity modified air pollution attributed cardiac autonomic function in our subjects. However, we did not observe interaction of abdominal fat accumulation. It seems plausible to hypothesize that adipose tissues located at different sites of body may have different metabolic properties in predisposing reduced skeletal muscle glucose utilization and insulin resistance, thus to pose differential mechanisms in regulation and/or impairments in sympathetic nervous system function associated with air pollution exposure.

Previous research suggested that gender could modify the cardiovascular risk and air pollution associations; however, the findings are heterogeneous [43,44]. Recent studies reported higher traffic pollution associated with cardiometabolic risks in females. Brook et al. found an association between modeled NO 2 exposure and T2D prevalence among female patients, but not among male patients, of two respiratory health clinics in Canada [45]. A study by Kramer et al. supported the plausibility of oxidative stress and inflammation as a biological mechanism for the relation between air pollution and T2D, by showing that females with high C3c blood levels (a marker for subclinical inflammation) were more susceptible for particulate matter related excess risk of diabetes than were females with low C3c levels. This prospective study furthermore found a relation between traffic-related particulate matter and incident T2D among elderly women in Germany [46]. In addition, an American study found an association with distance to road among women, while no strong evidence of an association with particulate matter exposure was observed [47]. Consistent with our previous analysis [20], we observed greater HRV declines associated with exposures to traffic-related pollutants BC and gaseous pollutants in female elderly Chinese. Yet the prevalence of obesity and distribution of adipose tissue differ by age and gender which could modify cardiac responses pathophysiologically, the mechanisms for age, obesity and gender specific interactions of air pollution associated cardiovascular effects are not yet clear and deserve further investigation.

Several study limitations should also be noted in interpreting our findings. First, we only investigated subjects with T2D and IGT, and had no normal glucose tolerant or lean control subjects. Second, we did not collect individual data, such as insulin level and insulin resistance, homeostasis, systemic inflammation and oxidative stress, which might have limited our capability further examining the biological plausibility of diabetes in modifying UFPs and traffic pollution associated cardiovascular dysfunctions. Third, the fixed-location monitoring data was used in this study, and the lack of information on personal exposure to ambient pollution may have resulted in potential exposure misclassification errors and may have biased the effect estimates toward null. Fourth, we did not control subjects' activity during observation, which may affect the results of frequency-domain measurement of HRV. And lastly, we did not obtain real-time minute-to-minute PM 2.5 concentrations due to operational errors of the monitoring instruments installed in the monitoring laboratory. Thus we were not able to examine the commonly studied associations between autonomic dysfunction and exposure to criteria pollutant PM 2.5 , which has limited the generalization of our results in broader context of adverse effects of ambient fine particulates.

In summary, we observed autonomic dysfunction associated with increased exposure to ambient UFPs and traffic-related pollutants in previous hours among persons with diabetes or impaired glucose tolerance, and the magnitude of estimated effects were significantly stronger in obese persons and in females. Our results suggest that underlying metabolic condition may confer susceptibility of traffic pollution on the heart.

Materials and methods

Study design and subjects

This panel study was conducted in April, June and September of 2010. We recruited 53 elderly subjects with diagnosed type-2 diabetes or impaired glucose tolerance through an existing diabetes cohort followed by Luwan District Center for Disease Control (CDC) and Prevention in Shanghai. The diabetes cohort was designed to allow randomized or controlled evaluation of behavior and dietary modification on progression and risk factors of T2D. Since 2004, this cohort has enrolled approximately 4,000 individuals with diagnosed T2D or at risk, but free of clinically apparent cardiovascular diseases at baseline examination. At the enrollment, the disease state of each individual was identified by standardized medical record review and physician adjudication. During the follow-up, fasting plasma samples were measured every six months.

All the subjects were retired elderly living within one kilometer from Luwan CDC in central Shanghai. The information on subjects' age, gender, weight, height, smoking status and medical history was obtained through administered baseline questionnaire interview during recruitment process. Our subjects received three repeated measurements of 24-hour ambulatory ECG during study period. The Institutional Review Board of Peking University Health Science Center approved study protocol and written consent form. Informed consent was obtained from each participant before the study participation.

Individual metabolic abnormality measurement

Our subjects had fasting blood samples drawn and measured for plasma glucose, gycosylated hemoglobin, triglyceride, and cholesterol in routine follow-up in April and October in 2010. Each participant was scheduled arriving the check-up room in Luwan CDC at the same time of a weekday morning for all visits. At each visit, technical staff measured body weight, height, waist and hip circumferences, and blood pressure of each subject, and recorded 24-hour activity of the subjects prior to the visit through questionnaire survey. We used clinical cutoff points for diabetic states: T2D was defined as fasting blood glucose ≥ 126 mg/dL.

HRV measures

After physical examination at each visit, trained personnel placed 7 bipolar leads of a ECG monitor on the participant, following standard protocol. HRV were derived from 24-h ECG recordings of subjects, under normal daily activity conditions, using a 3-channel Holter monitoring system (Model MGY H7, DM Software Inc., USA).

The ECG digital recordings were reviewed and processed by trained cardiologists. Mean heart rate and various measure of HRV were calculated for each 24-Hour session using PC-based software (Holter System Version 12.Net for Windows, DM Software). Each 5-minute segment of normal to normal intervals of the heart beat was used to calculate the HRV index that included timedomain measurements of SDNN and rMSSD, as well as frequency-domain measurements of LF and HF.

Air pollution measures

Air pollution concentrations and meteorological data were monitored continuously using instruments equipped in a laboratory in Luwan CDC. The repeatability, accuracy, and predictive validity of the air pollution measurements conducted by the laboratory have been described previously [48].

Minute-to-minute number concentration of particles in size between 5 to 560 nm was monitored by a Fast Mobility Particle Sizer Spectrometer (FMPS Model 3091, Thermo, USA). Number-size distributions were converted to particle number concentrations, particle surface area concentrations, and particle mass concentrations, assuming spherical particles and 1 g/cm 3 as default density parameter. In this analyses, we calculated 5-minute mean concentration for particles in 5-10 nm, 10-20 nm, 20-50 nm, 50-100 nm, 100-200 nm, 200-560 nm size fractions. Total number, surface, and area concentrations of particles were computed as the sum of all fractions (5-560 nm).

Statistical analysis

We calculated descriptive statistics for each air pollution measurement. Spearman correlations were calculated across minute-to-minute data of air pollutants and meteorologic parameters. All HRV variables were logarithmically transformed using base 10 due to right skewed distributions.

We first built basic generalized linear mixed models with each HRV metric as the dependent variable. Based on the assessment of Akaikes information Criterion (AIC), we chose to model the temperature and RH using natural splines with degree of freedom up to 3, and modeled time of the day using quadratic polynomial functions. The first-order autoregressive model (AR1) was chosen to account for intra-subject correlation within each visit between repeated measures after comparison of several covariance structures, based on the criteria of minimizing AIC. The effect of uncontrolled features of subjects was modeled by random coefficient relying on subjects' id. Models were also adjusted for potential confounders such as day of week, age, gender, BMI and visit.

Single-pollutant models were then developed by including pollutant variables to examine the associations between air pollution exposure and HRV measures. The effects of particles in all size-fractions and other pollutants under 1-, 4-, 12-, 18-, and 24-hour moving averages were examined. Due to relatively high correlation between concentration of particles and other air pollutants, we conducted two-pollutant analyses by including the second pollutant of prior 4-hour average exposure to examine the robustness of the estimates associated with particles.

To evaluate potential effect modification by status of disease and other factors, we conducted the analysis of SDNN with each air pollutant using the 4-hour averaging exposure, stratified by diabetic state (T2D vs. IGT), obesity (Group 1, BMI≤25; Group 2, 25≤ BMI ≤28; Group 3, 28≤BMI), central adiposity (WHr), and gender, controlling the same covariates as in main models. We examined the heterogeneity in the association estimates across strata at the .05 significance level.

All effect estimates were expressed as percent change in each HRV variable for interquartile range (IQR) increase of the corresponding moving average of each pollutant, calculated as [ 10 (β×IQR) − 1] ×100, with 95% confidence intervals (CI) {10 [IQR×(β∓1.96×SE)] − 1} × 100, where β and SE are the estimated regression coefficient and its standard error. All analyses were conducted in SAS statistical software (Version 9.2; SAS Institute; Cary, NC).

Additional files

Additional file 1: Description and correlations. This file contains 2 sheets. One is named 'description_of_pollutants', which is the extension of Table 2, with basic descriptive statistics of surface concentration (PSC) and mass concentration (PMC) of particles. The numbers after PSC or PMC indicate the size fraction of particles. Another sheet is named 'correlations_among_pollutants', which is an extension of Table 3, with correlations between surface or mass concentration of particles in each size fraction, and BC, gaseous pollutants and meteorologic parameters. T for temperature and * indicates correlation coefficients >0.6.

Additional file 2: Per IQR change single pollutant. This file contains 26 sheets. The name of the sheet gives the pollutant whose effect is shown in it, and the numbers after PNC, PMC or PSC indicate the size fraction of particles. Each sheet shows the percent change of all 5 HRV metrics, SDNN, rMSSD, LF, HF and LF/HF, in response to the IQR increases in 1-. 4-, 12-, 18-, and 24-hour moving averaged exposure to air pollutants. The column 'Yvar' indicates the HRV metric and the second column label the moving average. The column 'Per IQR ↑' shows the percent change of the HRV metric, and the columns '95% CI-Low' and '95% CI-High' give the lower and upper bounds of 95% confidence interval.

Additional file 3: Per IQR change two pollutant. This file contains 1 sheet. It shows the effects of different metrics of particles on SDNN when another gaseous pollutant or BC is also included in the model. The time window for all pollutants is 4 hours prior to the HRV observation. The column 'Confound Pol' indicates which pollutant is included in the model to detect confounding effect. The column 'Effect Pol' indicates which pollutant is potentially confounded. The numbers after PNC, PMC or PSC indicate the size fraction of particles. The column 'Per IQR ↑' shows the percent change of SDNN, and the columns '95% CI-Low' and '95% CI-High' give the lower and upper bounds of 95% confidence interval.

Additional file 4: Per IQR change stratified. This file contains 5 sheets. Each sheet shows the modification effect of a factor discussed in this study. The ratio of waist to hip circumference (WHr) is analyzed within each gender due to different classification criteria. We only considered the effect on SDNN and pollutants under prior 4-hour moving average for gender, BMI and WHr. For the modification effect of diabetic state, we analyzed both 4-hour and 24-hour time window. The group label is given in the first row of each sheet. The numbers after PNC, PMC or PSC indicate the size fraction of particles. The column 'Per IQR ↑' shows the percent change of SDNN within each group, and the columns '95% CI-Low' and '95% CI-High' give the lower and upper bounds of 95% confidence interval.

Competing interests

The authors declare that they have no competing interests.

Figure 1
1Results of single-pollutant mixed-effects model. It shows the changes in SDNN per IQR increase in different proceeding moving average exposures to ambient pollutants in the single-pollutant mixed-effects model.

Figure 2
2Stratified analysis by diabetic states. It shows estimates for adjusted percent changes in SDNN per IQR increase in 4-hour average exposures to different air pollutants in IGT and T2D groups.

Figure 3
3Stratified analysis by diabetic states. It shows estimates for adjusted percent changes in SDNN per IQR increase in 24-hour average exposures to different air pollutants in IGT and T2D groups.

Figure 4
4Stratified analysis by obesity. It shows estimates for adjusted percent changes in SDNN per IQR increase in 4-hour average exposures to different air pollutants in three groups stratified based on BMI.

Figure 5
5Stratified analysis by gender. It shows estimates for adjusted percent changes in SDNN per IQR increase in 4-hour average exposures to different air pollutants in male and female groups.

Table 1
1Demographic characteristics for IGT and T2D subjectsIGT
T2D

Number
17
36

Age (year)
60.1 (1.0)
59.7 (0.6)

BMI (kg/m 2 )
25.3 (0.7)
26.7 (0.7)

Gender (Female/Male)
10/7
17/19

Weight (kg)
69.0 (1.8)
73.4 (2.0)

Height (cm)
165.2 (1.4)
165.4 (1.1)

Waist (cm)
92.2 (2.7)
92.8 (1.7)

Hip circumference (cm)
101.0 (2.0)
100.6 (1.3)

Waist/hip circumference
0.91 (0.01)
0.92 (0.01)

SDNN (ms)
119.6 (7.5)
108.6 (4.4)

rMSSD (ms)
23.8 (3.2)
19.8 (0.9)

LF (ms 2 )
305.2 (62.5)
263.7 (25.5)

HF (ms 2 )
160.7 (62.0)
95.9 (10.2)

Hypertension
8
28

Table 2
2Summary statistics of exposure and meteorologic parametersMean (SD)

Table 3
3Correlations between number concentration of particles in each size fraction, and BC, gaseous pollutants and meteorologic parametersBC
NO 2
CO
SO 2
O 3
T
RH
Air pressure

PNC 5−560
0.52
0.65*
0.45
0.48
-0.1
-0.13
-0.14
0.18

PNC 5−10
-0.06
0.16
-0.05
0.09
0.08
-0.2
-0.15
0.34

PNC 10−20
0.13
0.35
0.08
0.1
0.02
-0.08
-0.19
0.25

PNC 20−50
0.35
0.54
0.36
0.38
-0.14
-0.04
-0.14
0.21

PNC 50−100
0.75*
0.78*
0.65*
0.58
-0.28
-0.12
-0.07
0.04

PNC 100−200
0.72*
0.7*
0.56
0.6*
-0.07
-0.2
-0.09
0.15

PNC 200−560
0.37
0.36
0.3
0.31
-0.05
-0.19
0
0.18

Table 4
4Percent change in SDNN per IQR increases in proceeding 4-hour moving average exposures to ambient pollutants estimated in single-, and two-pollutant mixed-effects modelsSingle-pollutant
Two-pollutant

Adj. for BC
Adj. for NO 2
Adj. for O 3
Adj. for CO

BC
-6.14 (-8.12,-4.11)
-6.14 (-8.12,-4.12)
1.09 (-1.72,3.98)
-5.18 (-7.31,-3)
0.07 (-2.7,2.91)

NO 2
-8.98 (-10.72,-7.18)
-9.56 (-11.85,-7.2)
-8.97 (-10.72,-7.18)
-9.33 (-11.34,-7.27)
-6.43 (-8.64,-4.17)

CO
-7.33 (-8.91,-5.73)
-7.37 (-9.43,-5.25)
-7.05 (-8.95,-5.11)
-6.68 (-8.52,-4.81)
-4.22 (-6.19,-2.21)

SO 2
-4.36 (-5.85,-2.86)
-2.91 (-4.66,-1.13)
-0.56 (-2.38,1.30)
-3.24 (-4.83,-1.62)
-1.25 (-3.02,0.55)

O 3
1.55 (-0.13,3.27)
0.51 (-1.22,2.27)
1.55 (-0.14,3.28)
0.94 (-0.77,2.68)
-1.73 (-3.51,0.09)

PNC 5−560
-7.89 (-9.69,-6.07)
-6.82 (-8.87,-4.72)
-7.73 (-9.57,-5.85)
-7.47 (-9.65,-5.24)
-4.54 (-6.82,-2.21)

PNC 10−20
-7.0 (-8.88,-5.08)
-7.05 (-8.92,-5.14)
-7.21 (-9.14,-5.24)
-6.73 (-8.65,-4.77)
-6.18 (-8.1,-4.21)

PNC 20−50
-6.57 (-8.07,-5.04)
-5.77 (-7.34,-4.17)
-6.36 (-7.92,-4.77)
-6.07 (-7.77,-4.33)
-4.32 (-6.07,-2.53)

PNC 50−100
-5.37 (-7.34,-3.35)
-2.76 (-5.29,-0.16)
-5.65 (-7.69,-3.56)
-3.49 (-6.03,-0.89)
1.52 (-1.32,4.45)

PNC 100−200
-2.98 (-4.63,-1.3)
2.53 (-0.29,5.42)
-2.53 (-4.26,-0.77)
0.3 (-1.84,2.49)
2.83 (0.71,4.99)

PNC 200−560
-0.45 (-2.43,1.56)
5.15 (2.53,7.84)
0.09 (-1.96,2.18)
3.25 (0.97,5.59)
4.46 (2.22,6.75)

T for temperature and * indicates correlation coefficients >0.6.
AcknowledgementsThis study was supported by Ministry of Science and Technology of ChinaAuthors' contributions YTS, YQH, YFJ, SNG and YS collected sample and measurement data, YTS, XMS and WH analyzed the data and drafted the manuscript, SL supervised data analysis, WH and TZ designed and managed the study. All authors read and approved the final manuscript.
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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. M Wang, T Zhu, J Zheng, R Zhang, S Zhang, X Xie, Atmos Chem Phys. 921Use of a mobile laboratory to evaluate changes in on-road air pollutants during the beijing 2008 summer olympics. Submit your manuscript at www.biomedcentral.com/submitWang M, Zhu T, Zheng J, Zhang R, Zhang S, Xie X, et al. Use of a mobile laboratory to evaluate changes in on-road air pollutants during the beijing 2008 summer olympics. Atmos Chem Phys. 2009;9(21):8247-63. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


Mononeuritis multiplex: an uncommon neurological manifestation of cytomegalovirus reactivation in an HIV-infected patient

Pedro Palma
Andreia Costa
Raquel Duro
Nélia Neves
Cândida Abreu
António Sarmento
Mononeuritis multiplex: an uncommon neurological manifestation of cytomegalovirus reactivation in an HIV-infected patient
10.1186/s12879-018-3501-2C A S E R E P O R T Open Access
Background: Cytomegalovirus (CMV) reactivation with neurological involvement in patients with acquired immunodeficiency syndrome (AIDS) is increasingly rare since the introduction of antiretroviral therapy (ART). Manifestations include encephalitis, myelitis, polyradiculopathy and, less commonly, mononeuritis multiplex (MNM). We report a case of disseminated CMV disease with gastrointestinal and peripheral and central nervous system involvement in a patient with AIDS, manifesting primarily as MNM. Case presentation: A 31-year old woman with AIDS presented with a clinical picture of MNM. Electromyography confirmed the clinical findings. CMV DNA was detected in cerebrospinal fluid (CSF) and blood. Gastrointestinal involvement was histologically documented. HIV RNA was also detected in CSF and brain MRI was consistent with HIV encephalopathy. A diagnosis of disseminated CMV disease (with esophagitis, colitis, encephalitis and MNM) and HIV encephalopathy was made. Treatment consisted of ganciclovir and foscarnet, followed by maintenance therapy with valganciclovir. Evolution was favorable and valganciclovir was stopped after sustained immune recovery following ART initiation. Conclusion: We discuss the diagnostic approach to CMV neurological disease, with a focus on MNM and CMV encephalitis. Combination therapy with ganciclovir and foscarnet should be considered for all forms of neurological involvement, although available data are scarce. Since there is significant overlap between CMV encephalitis and HIV encephalopathy, ART drugs with higher CSF penetration may have to be considered. ART and immune recovery are essential to improve outcomes.

Background

Mononeuritis Multiplex (MNM) is an uncommon form of peripheral neuropathy, usually presenting with motor and sensory symptoms in an asymmetric pattern involving two or more peripheral nerves [1,2]. In HIV-infected patients with MNM, two etiological mechanisms have been described: autoimmune, typically a limited form in patients without acquired immunodeficiency syndrome (AIDS); and cytomegalovirus (CMV) reactivation, a generalized form in patients with AIDS [2,3]. CMV reactivation in HIV patients with advanced disease most frequently presents as retinitis, colitis, and esophagitis. Neurological involvement is less common and can manifest as encephalitis, myelitis, polyradiculopathy, and mononeuritis multiplex. In the pre-antiretroviral therapy (ART) era, where up to 40% of HIV-infected patients with advanced disease developed CMV disease [4,5], CMV MNM was already considered uncommon [6,7]. A more frequent type of neurological disease, CMV encephalitis, was reported in 1% of cases [6,8]. Autopsy studies, however, recognized CMV infection of the central nervous system (CNS) in 12 to 28% of patients with AIDS [6,9]. Similarly to other opportunistic infections, since the introduction of ART the incidence of CMV disease decreased considerably, with recently reported incidence rates of < 0.50 per 100 person-years [10,11].

We describe a case of disseminated CMV disease in a patient with AIDS, manifesting primarily as MNM. We focus on the neurological involvement of CMV and discuss the diagnostic challenges and treatment approach of this increasingly uncommon manifestation.

Case report

A 31-year-old white woman presented with severe burning pain with tingling sensation and asymmetric weakness of the lower limbs that, over a six-month period, gradually worsened and progressed to involve the upper limbs; she was then unable to walk or eat alone. She had been diagnosed with HIV infection five years earlier at another tertiary care hospital but refused follow-up.

On admission, she was undernourished and her neurological examination revealed: lethargy, disorientation and psychomotor slowing; asymmetrically diminished motor strength (Medical Research Council Scale) in the four limbs (grade 2/5 in right upper limb extension; grade 3/5 in bilateral lower limb extension; grade 4/5 in the remaining); symmetrical deep tendon reflexes apart from absent right brachioradialis and bilateral patellar reflexes; impaired pin-prick sensibility in the right ulnar and radial distribution. The remainder physical examination was unremarkable.

Brain MRI ( Fig. 1) was consistent with HIV encephalopathy and electromyography (EMG) with the diagnosis of MNM (severe confluent multifocal demyelination and axonal loss in both upper and lower limbs).

CD4 cell count was 75 cells/μL (8%) and HIV RNA was 633000 copies/mL. CMV DNA in blood was 64000 copies/mL; CMV antigen was negative. CMV IgG antibodies were positive and IgM antibodies negative; furthermore, electronic medical records from five years earlier confirmed prior CMV IgG seropositivity, suggesting CMV reactivation. Cerebrospinal fluid (CSF) analysis revealed 7 cells, protein of 1.19 g/dL and glucose of 49 mg/dL; negative bacterial and fungal cultures; positive CMV DNA (14400 cp/mL) and HIV RNA (184222 cp/mL). Gastrointestinal involvement (esophageal and colonic) by disseminated CMV disease was histologically documented, even though the patient reported no related symptoms; retinal involvement was excluded.

With disseminated CMV disease as the most likely cause of MNM, she started IV ganciclovir (5 mg/kg every 12 h). Due to no significant clinical improvement, treatment was intensified 7 days later with the association of IV foscarnet (90 mg/kg every 12 h). Combination therapy was maintained for three weeks, after which CMV DNA became undetectable in blood (she refused a new lumbar puncture) and oral valganciclovir 900 mg/day was started as maintenance therapy. She initiated ART with emtricitabine/tenofovir and dolutegravir following two weeks of combination therapy targeted at CMV disease and slow, but obvious, clinical response. Her neurological symptoms, both neurocognitive and motor, gradually improved and she began a rehabilitation program. She was transferred to a rehabilitation center and discharged after two months.

Follow up at three months of ART initiation showed CD4 cell count improvement (313 cells/μL (27%)) and undetectable HIV RNA. After 6 months of sustained immune recovery (CD4 cell count 722 cells/μL (24%)) and virologic suppression, maintenance therapy with valganciclovir was stopped.

At 12 months of initial symptoms, she showed no neurocognitive impairment and was able to return to her normal daily activities. Motor strength improved globally to normality besides grade 4/5 in right-hand fingers extension and in the lower limb extension; pin-prick sensibility remained impaired in the right upper limb. She reported neuropathic pain in the lower limbs, which was managed with pregabalin 225 mg twice daily. EMG was consistent with nerve regeneration in the upper limbs and, to a lesser extent, in the right lower limb.

Discussion and conclusions

In this patient, the evidence of disseminated CMV disease and the presentation with asymmetric neurological signs, with EMG findings consistent with MNM, was sufficient for the clinical diagnosis of CMV MNM.

CMV mononeuritis multiplex can be extensive, involving several limbs or cranial nerves. Both a classic presentation with painful, progressive, multifocal deficits and a more rapidly progressive syndrome, involving multiple nerve distributions, have been described in patients with advanced AIDS [7]. EMG is key in the confirmation of clinical findings, typically showing a neuropathy with multifocal demyelination and axonal loss [2,7,12]. While not necessary for a clinical diagnosis of CMV MNM, polymerase chain reaction (PCR) detection of CMV DNA in CSF has been shown to correlate with all forms of CMV neurological disease (sensibility and specificity of > 90%) [13,14], even in cases of isolated peripheral neuropathy [14]. PCR of plasma or whole blood is also valuable as it can be used as a sensitive "surrogate marker" of CMV reactivation and subsequent disease and is less prone to false negatives compared to antigenemia assays [15,16]. Moreover, serial quantitative PCR testing of specimens is helpful to monitor response to treatment [15]. Nerve biopsy may provide additional confirmation as CMV has been demonstrated in macrophages, fibroblasts, and endoneurial cells in the superficial nerves of patients with MNM [17]. In our patient, CMV DNA detection in the CSF coupled with signs of altered mental status suggested a more extensive neurologic involvement of CMV disease. However, simultaneous CMV encephalitis and HIV encephalopathy were considered since HIV RNA was also detected in CSF and MRI findings were consistent with the latter.

The diagnosis of CMV encephalitis can be particularly challenging. Patients often present with progressive altered mental status which may be difficult to distinguish from HIV encephalopathy [6,8,9]. In contrast to HIV encephalopathy, CMV encephalitis has a more rapid onset (mean onset of less than four weeks) and symptoms of delirium, confusion, apathy, and withdrawal are more frequent [6,8]. Yet, other non-distinguishing neurological manifestations, such as forgetfulness, memory impairment, and psychomotor slowing, are also common [6]. A more distinct type of CMV encephalitis characterized by ventriculoencephalitis has also been described and presents with rapidly progressive confusion and lethargy [6,8], with variably associated radiculopathy and cranial nerve deficits [8]. MRI may show multiple hypertense foci distributed widely in the brain or periventricular enhancement on T2-weighted images, although these findings are inconsistently present and largely non-specific [18,19]. Conversely, HIV encephalopathy typically shows widespread hyperintense lesions on T2-weighted/FLAIR, localized bilaterally in the deep white matter [18], a picture that more closely resembled the MRI findings in our patient. Similarly to CMV neurological disease, HIV RNA detection in CSF correlates significantly (albeit weakly) with the presence of HIV encephalopathy in untreated patients, an association not seen in patients on ART [20,21]. However, CNS opportunistic infections may also increase intrathecal HIV replication [22].

Treatment of all forms of CMV neurologic disease is similar, although data are scarce. Most authors recommend ganciclovir or foscarnet, or a combination of both drugs. Combination therapy may be considered in those previously treated with CMV-directed drugs (hence with risk of drug-resistant virus) and in patients with disease progression under monotherapy [8]. Foscarnet levels in CSF vary widely, achieving 0 to 3.4 times of plasma concentration (mean values of 23%) [23]. A study of the pharmacokinetics of ganciclovir in plasma and CSF in a nonhuman primate model showed that the drug penetrates into the CSF following IV administration (CSF to plasma area under the curve of 15.5%+/− 7.1%) [24]. Therefore, another rationale for combination therapy use is to increase CSF penetration due to the variable levels achieved by either drug alone. Despite being relatively well tolerated, this regimen may be limited by increased side effects, particularly bone marrow suppression with ganciclovir and nephrotoxicity with foscarnet [23,25]. There are no data on CSF penetration of cidofovir and therefore it should be avoided [8,25]. Optimal duration of initial therapy is unknown and should be guided by an improvement of clinical symptoms [6,8,9].

Our patient was treated with combination therapy considering the severity of the disease, extensive neurological involvement and slow initial improvement with ganciclovir alone.

Additionally, as with other opportunistic infections, initiation of ART and reversal of profound immunodeficiency is considered essential to successful management [26,27]. Optimal timing for ART initiation is not well defined and the decision to start ART early in the course of CMV disease must be weighed against the risk of CMV immune reconstitution inflammatory syndrome (IRIS). Nonetheless, data on CMV IRIS presenting as neurological disease are limited: a case of CMV encephalitis was reported in an HIV-infected patient with CMV colitis on ART and valganciclovir (although with adherence issues), where IRIS may have played a role [28]. Reports of immune recovery uveitis following early ART initiation in patients with CMV retinitis are more commonly described throughout the literature [29][30][31]. Considering the extensive involvement of CMV disease in our patient, ART was initiated after an obvious clinical improvement with combination therapy for CMV was observed.

Optimization of ART using drugs that penetrate effectively into the CSF has been shown to improve the outcome of patients with neurological symptoms and detectable HIV viral loads in the CSF [32,33]. The possibility of associated HIV encephalopathy motivated our choice to include dolutegravir in the ART regimen since concentrations in the CSF have been shown to be similar to unbound plasma concentrations, achieving therapeutic levels in the CNS [34].

This patient outcome was favorable: clinical improvement was obvious, CMV viremia was cleared and no significant side effects were observed. In the pre-ART era, outcomes of CMV-related neurological disease were generally poor, with a reported median survival of less than three months [6,9]. Furthermore, CMV MNM was described with simultaneous encephalitis, polyradiculopathy or retinitis leading to death within a few days or weeks if left untreated [6]. The improved survival of HIV-infected patients and decreasing incidence of CMV disease in recent years denotes that prognosis is dependent on adequate antiretroviral treatment and immune reconstitution. The impact of the choice of ART regimen on the patient neurological improvement (besides the obvious benefit of immune recovery) is difficult to ascertain. CSF evaluation at follow-up might have been valuable to determine the effect in CSF HIV viral load since CNS compartmentalization of HIV has also been reported [32].

Duration of maintenance therapy for CMV following neurological disease is debatable. However, considering both the risk of recrudescence of a serious disease and the risk of IRIS, awaiting immune reconstitution (i.e., a sustained rise of CD4 cell count ≥100-150/μL and undetectable HIV RNA for more than 6 months) may be wise before stopping maintenance therapy [26,27].

In conclusion, CMV neurological disease is increasingly uncommon since the introduction of ART. This patient with AIDS presented predominantly with a clinical picture of MNM, that harbored a disseminated CMV disease. The widespread availability of highly sensitive and specific PCR techniques can significantly shorten the time to diagnosis of CMV reactivation. While the spectrum of neurological involvement of CMV disease in HIV-infected patients can be diverse, it is associated with high morbimortality and risk of rapid progression if left untreated. Since CSF penetration of the available antivirals varies widely, combination therapy with ganciclovir and foscarnet should be considered. The risk of IRIS should be weighed in when deciding the timing of ART initiation and the overlap in manifestations of CMV and HIV infection of the CNS may also be a factor during the selection of ART regimens, favoring drugs with higher CSF penetration efficacy. Nonetheless, ART initiation and subsequent immune recovery were essential for the favorable outcome reported, which contrasted vastly from the natural course of CMV neurological disease in the pre-ART era.

Fig. 1
1Brain MRI. Axial T1 (a-b), T2/FLAIR (c-d), DWI (e-f) and ADCmap (g-h). Bilateral relatively symmetric periventricular and deep white matter T2/FLAIR hyperintensity predominantly in the posterior supratentorial area. Areas of restricted diffusion were not observed

Abbreviations AIDS: Acquired immunodeficiency syndrome; ART: Antiretroviral therapy; CMV: Cytomegalovirus; CNS: Central nervous system; CSF: Cerebrospinal fluid; EMG: Electromyography; HIV: Human deficiency virus; IRIS: Immune reconstitution inflammatory syndrome; MNM: Mononeuritis multiplex; PCR: Polymerase chain reaction
AcknowledgmentsNot applicable.FundingNot applicable.Availability of data and materialsData sharing is not applicable to this article as no datasets were generated or analyzed during the current study.Authors' contributions PP, AC, RD, and NN were responsible for the patient's care and management during hospitalization and follow-up after discharge, collected all significant clinical information, and drafted this manuscript. CA and AS reviewed, redrafted and given significant contribution to the final version. All authors read and approved the final manuscript.Ethics approval and consent to participate Not applicable.Consent for publicationWritten informed consent was obtained from the patient for publication of data and images contained in this case report. A copy of the written consent is available for review by the Editor of this journal.Competing interestsThe authors declare that they have no competing interests.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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