Early diagnosis along with new drugs targeted to cancer receptors and immunocheckpoints have improved breast cancer survival. However, full remission remains elusive for metastatic breast cancer due to dose-limiting toxicities of heavily used, highly potent drug combinations such as gemcitabine and paclitaxel. Therefore, novel strategies that lower the effective dose and improve safety margins could enhance the effect of these drug combinations. To this end, we developed and evaluated a novel drug combination of gemcitabine and paclitaxel (GT). Leveraging a simple and scalable drug-combination nanoparticle platform (DcNP), we successfully prepared an injectable GT combination in DcNP (GT DcNP). Compared to a Cremophor EL/ethanol assisted drug suspension in buffer (CrEL), GT DcNP exhibits about 56-fold and 8.6-fold increases in plasma drug exposure  and apparent half-life of gemcitabine respectively, and a 2.9-fold increase of AUC for paclitaxel. Using 4T1 as a syngeneic model for breast cancer metastasis, we found that a single GT (20/2 mg/kg) dose in DcNP nearly eliminated colonization in the lungs. This effect was not achievable by a CrEL drug combination at a 5-fold higher dose . A dose-response study indicates that GT DcNP provided a therapeutic index of ~15.8. Collectively, these data suggest that GT DcNP could be effective against advancing metastatic breast cancer with a margin of safety. As the DcNP formulation is intentionally designed to be simple, scalable, and long-acting, it may be suitable for clinical development to find effective treatment against metastatic breast cancer. Breast cancer is a leading cause of death in women in the US and worldwide. Estimates suggest that in 2019 over 270,000 people will be newly diagnosed and that 42,000 people will die of the disease in the US alone . Collectively, if one can co-deliver an effective drug combination to advancing metastatic breast cancer cells, a lower overall dose would be needed to overcome dose-limiting toxicities.In developing long-acting (LA) and combination anti-retroviral (drug) therapeutics or cART, our laboratory has discovered a simple, scalable technology that enables the incorporation of 2–3 HIV drugs that are water insoluble such as lopinavir, ritonavir, atazanavir, and efavirenz together with water soluble tenofovir and lamivudine –11. This2000) (GMP grade) were purchased from Corden Pharma . Paclitaxel (>99.5%), gemcitabine free form (>99%), and gemcitabine hydrochloride (>99%) were purchased from LC Laboratories . All other chemicals and reagents were analytical grade or higher. 4T1 cell line transfected and verified to express luciferase and green fluorescence protein (GFP) (referred to as 4T1-luc) was kindly provided by Stanley Riddell laboratory [1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and N-(carbonylmethoxypolyethyleneglycol with MW = 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt  were prepared aseptically as follows: Lipid excipients and drugs were solubilized together in ethanol at 60°C. Ethanol was removed by controlled solvent evaporation at 60°C, followed by vacuum desiccation to remove residual solvent. The dry film was rehydrated to 100 mM lipids in 0.45% NaCl with 20 mM NaHCO3 buffer at 60°C for 2 h. Particle size was reduced at ~40°C using a bath sonicator  . GT DcNP formulations were stored at room temperature for further use. Particle size was determined by a NICOMP 380 ZLS . Drug extraction with acetonitrile followed by HPLC were used to quantify drugs in formulations. Drug association was measured by dialysis (6–8 kDa) of DcNP against 0.9% NaCl 20 mM NaHCO3 buffer for 4 h and quantification by HPLC.DcNP composed of DSPC and DSPE-mPEGPaclitaxel was dissolved in ethanol (20 mg/mL) and diluted with an equal volume of Cremophor EL . The solution was then diluted 10× with a premade PBS solution of gemcitabine  (12.65 mg/mL). Final concentrations of drugs were 10/1 mg/mL GT. CrEL drug suspensions were used within the same day of preparation due to instability.All animal studies were conducted in accordance with University of Washington Institute of Animal Care and Use Committee (IACUC) approved protocols (protocol number 2372–06). The UW IACUC has specifically approved this study. Isoflurane was used for anesthesia during live animal imaging. 5–6 week-old female BALB/c mice were purchased from The Jackson Laboratory  and housed in an animal research facility for at least one week before use.2 overdose followed by cervical dislocation as the secondary method of euthanasia. Drugs in plasma were extracted and analyzed by LC-MS/MS as described below.Mice were administered with either a CrEL drug combination or GT DcNP intravenously with doses of 50/5 mg/kg GT. Blood was collected through retro-orbital bleeding at 5, 60, 120, 360 min for CrEL GT and 5, 60, 120, 360, 1440, 4320 min (72 h) for GT DcNP. Each group had three animals and each animal was bled once only. Retro-orbital blood collection in this study was a terminal procedure and animals were under anesthesia at the time of bleeding. After blood collection, mice were euthanized by COA liquid-liquid extraction was used to extract drugs from plasma or tissue homogenates. 50 μL of sample were transferred into 1.5 mL tubes with or without dilution by blank matrix to an appropriate concentration range. Samples were spiked by internal standards (see below) followed by the addition of acetonitrile. Samples were then vortexed and centrifuged at 4°C for 15 minutes at 14000 rpm. The supernatant was then removed and dried under nitrogen at 40°C. The dried samples were reconstituted in 20% methanol and 80% water in 50 μL.Drugs were quantified by a Shimadzu HPLC system coupled to a 3200 QTRAP mass spectrometer . The HPLC system consisted of two Shimadzu LC-20A pumps, a DGU-20A5 degasser, and a Shimadzu SIL-20AC HT autosampler. The mass spectrometer was equipped with an electrospray ionization (ESI) TurboIonSpray source. The system was operated with Analyst software, version 1.5.2 .813CH12ClF2N15N2O4) (m/z 267.067→115.100) was used as an internal standard for gemcitabine; docetaxel (m/z 830.312→549.3) was used as an internal standard for paclitaxel.Chromatographic separation of drugs was achieved using a Synergi column  with an inline C8 guard column (4.0 × 2.0 mm) . An ammonium acetate buffer/reagent alcohol gradient was used to separate components. Analytes were monitored using multiple-reaction monitoring for positive ions. The following ion transitions were monitored: gemcitabine, m/z 264.066→112.000; paclitaxel, m/z 854.266→286.200; a stable labeled isotope (C5 cells) suspended in a 100 μL ice-cold HBSS suspension was intravenously inoculated through mouse tail veins. Mice were monitored for a two-week period. Bioluminescence of 4T1-luc from living mice was examined by a XENOGEN IVIS 200 imaging system . Mice received 150 mg/kg D-luciferin through intraperitoneal injections 10~15 min before imaging. The bioluminescence imaging parameters for living mice were set as follows: field of view, 12; excitation filter, closed; emission filter, open; exposure time, 120 sec; binning factor, 4; f/stop, 2. Total 4T1-luc bioluminescence emission from living mice was integrated using Live Image software .Six-week-old, female BALB/c mice were used in this study. 4T1 cells were transfected with luciferase and green fluorescence protein (GFP) (4T1-luc); thus 4T1 growth could be monitored based on that bioluminescence . 4T1-luc5 4T1-luc cells IV in 100 μL HBSS on day 0. Three hours later, mice were given a single administration of saline, a CrEL drug combination, or GT DcNP through IV injections (n = 8–15). The GT doses were 50/5 mg/kg for CrEL and DcNP formulations. On day 14, mice were euthanized immediately after live imaging and lungs were collected and placed in 12-well plates to quantify luminescence images. The images were acquired by a Xenogen IVIS-200. The bioluminescence imaging parameters for living mice were set as follows: field of view, 24; excitation filter, closed; emission filter, open; exposure time, 180 sec; binning factor, 4; f/stop, 2. The imaging parameters for lungs were set as follows: field of view, 10; excitation filter, closed; emission filter, open; exposure time, 30 sec; binning factor, 4; f/stop, 2. Bioluminescence intensity from living mice and lungs was integrated using Live Image software. Mouse lung tissue was fixed in formalin and stored in 70% EtOH before being embedded in paraffin blocks. GFP staining of thin sections (5 μm) was carried out by UW histology and imaging core.Six-week-old, female BALB/c mice were inoculated with 2 × 105 4T1-luc cells in 100 μL HBSS through IV injections on day 0. Three hours later, mice received a single administration of saline or a different dosage of GT DcNP through IV injections (n = 8–15). The dosages for DcNP were 0.125/0.0125, 1.25/0.125, 5/0.5, 10/1, and 20/2 mg/kg GT, respectively. Mouse behavior and overall health conditions were observed on a daily basis and body weight was measured every 2 days. On day 14, bioluminescence from living mice was examined with the IVIS imaging system as described above. Lung metastasis was detected by isolating lungs and imaging with IVIS as described above. After euthanasia, all organs were collected and visually examined for apparent toxicity.Six-week-old, female BALB/c mice were inoculated with 2 × 10Data were presented as mean ± standard error of the mean (SEM). The number of mice in all groups ranges from 8 to 15. Students’ t-tests were performed for two groups, and the statistical significance was evaluated using one-way ANOVA for multiple groups. A P-value of <0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism (Version 7.0).2000 (9:1 m/m), a stable and scalable DcNP with approximately 60 nm diameter can be made. We have tested at least 4 batches of DcNP preparation and verified that this process is reproducible, that DcNP products are stable, and that they can be scaled-up for the in vivo studies described. As the resulting GT DcNP product is less than 200 nm in diameter and stable in suspension (for at least 3 months and amenable for sterilization by 0.2 μm filtration), it is suitable for IV administration. Since the current clinical dose for GT is approximately 10:1 (w/w) , we used the DcNP with a similar drug ratio for the studies in mice described below.We first determined whether a water soluble gemcitabine (LogP = -1.5) and insoluble paclitaxel (LogP = 3) could be integrated into a drug combination particle in suspension presented as an injectable dosage form. Employing a composition of lipid excipients previously reported to enable stabilization and formulation of 3-to-4 HIV drugs that exhibit disparate hydrophobicity in each set of DcNP , we testmax and an ~8.7-fold increase in apparent half-life. At a 10-fold lower dose than gemcitabine, paclitaxel in the fixed dose combination (5 mg/kg) given in GT DcNP exhibited an ~21% decrease in Cmax but a similar half-life (1.97 vs 1.81 h). Due to the higher persistence of paclitaxel in DcNP, the overall AUC enhancement is about 2.9 fold  mg/kg IV dose, gemcitabine in DcNP exhibits about 56-fold higher exposure (AUC) and 8.6-fold longer apparent half-life than an equivalent CrEL drug combination dosage in mice . The dra2.9 fold . Collect3 4T1-luc cells in the inoculum per mouse). Bioluminescence (of 4T1-luc), body weight, and general behavior were monitored over two weeks. We found that bioluminescence signals increased exponentially from days 10 to 13 (from 0.5 to 3.5 × 105 photo counts). Furthermore, the body weight of mice gradually declined (~10% from day 10 to day 13) at a higher inoculum dose in mice corresponding to the exponential increase in the lung bioluminescence intensity. We found, with 200 × 103 cells, that about 20~30 tumor nodules and 2.0~3.0 × 105 photon counts of bioluminescence could be detected in the mouse lungs—showing that colonies establish and grow over time in these tissues with acceptable weight and overall health for interventional studies. Thus, 200 × 103 4T1-luc cells in the inoculum was used for the following studies.To determine whether enhanced GT exposure can translate into improvements in inhibition of metastatic tumor establishment and growth, we first evaluated if 4T1 inoculated intravenously into BALB/c mice could model hematogenous metastasis. 4T1 introduced into the blood have recently been shown to establish and grow in the lungs as nodules, and at a much faster rate than in the lymph nodes and the sinuses . In addi3 cell inoculation number. Results indicate that the model is highly reproducible and reliable with 100% tumor uptake and no significant difference between the mean bioluminescence (p = 0.0681 by one-way ANOVA). The rapid and aggressive 4T1 tumor growth at this dose has limited our ability to keep untreated mice for up to14 days. The effectiveness studies in following sections were determined using a 200 × 103 4T1 cell inoculation while carrying saline controls for each set of experiments or replications.It is well known that animal studies are highly variable due to individual differences, batch variances, etc. To verify the reproducibility of this model, we repeated our study five times with a total of 21 mice and compared lung bioluminescence intensities using a 200 × 10To determine the effects of enhanced GT exposure when presented in DcNP dosage form, we first used 50/5 mg/kg (GT) based on the current clinical (surface area converted to weight based) dose. Mice were first inoculated with 4T1 cells and given a single IV dose of GT either in CrEL or DcNP form. We intentionally chose identical GT doses (50/5 mg/kg) for the two formulations to evaluate DcNP effect on this treatment model (and without using dose compensations to match plasma drug exposures between the two formulations). The short interval between cell inoculation and GT administration (3h) was also purposefully designed, as the goals of this study were examining the clearance of advancing cancer cells in blood and eliminating formation of lung metastasis nodules. Tumor nodule formation was monitored over 14 days. As shown in To further characterize lung metastasis and treatments at the microscopic level, lung sections were examined with GFP immunohistochemistry given GFP is expressed by 4T1-luc, and the microstructures were evaluated in comparison to controls . First, 50s) for GT in DcNP fixed dose combinations were determined to be 1.655/0.1655 and 2.958/0.2958 mg/kg based on luminescence intensity and nodule count respectively (50 was determined to be 36.48/3.648 mg/kg GT. Using the mid-point of effective dose and toxic (weight-loss) doses, and the average therapeutic index, the ratio of toxicity-to-effective dose is estimated to be about 15.8 for GT DcNP. Taken together, dose-ranging studies indicate that the effective dose range is lower and well-separated from the toxicity range for GT combination by about 16 fold when given in DcNP dosage form. These data also confirmed that a single IV dose may clear a significant burden of invasive 4T1 with a sufficient margin of safety.To confirm the initial results of the GT fixed-ratio combination in DcNP on 4T1 nodules in the lungs and establish preliminary dose-dependent effects, we performed a dose-finding study. Within the range of a single IV dose of DcNP carrying 0.125 to 50 mg/kg gemcitabine and 10/1 w/w paclitaxel (0.0125 to 5 mg/kg), we followed the growth (based in 4T1-luc bioluminescence intensity) of tumor nodules as therapeutic outcome measures. No clinical behavioral or hematological effects were notable for animals in all treatment groups for the 14-day study. However, a reproducible measure is the weight loss detected within 4–6 days after dosing . Thus, wectively . Based oin vivo effectiveness. Due to high variance of the animal models, we carried out at least two independent effectiveness studies for each DcNP dose reported in p = 0.0854 for replication factor and p<0.0001 for dose factor). Our results verified that the DcNPs produced in different batches exhibit similar characteristics—nearly identical mean particle size, drug loading and association efficiency, and more importantly, the ability to inhibit 4T1 metastasis in mice; thus these results were reproducible and consistent.Due to inherent challenges in translating nanomedicine into clinical applications, it is important to validate the reproducibility of our GT DcNP regarding physicochemical properties and With early detection and targeted therapies, breast cancer survival rates have increased , even asFor metastatic breast cancer treatment, combination therapy has been proven more effective than monotherapy . However2 GT, equivalent to ~35/3.5 mg/kg [The current human GT combination is given in a sequence with IV infusions of paclitaxel followed by gemcitabine (after 2–3 h) at doses of ~1250/175 mg/m.5 mg/kg , 7. Sequ.5 mg/kg . Such en.5 mg/kg . RegardlThe therapeutic effects mediated by DcNP on GT were evaluated in 4T1 inoculated systematically to produce the lung metastasis model. This model is immunocompetent and relevant to human disease, where immune contribution is important. A genomic profiling study revealed a high consistency between lung metastases from orthotopic (mammary fat pad) and IV inoculation models, demonstrating that this approach mimics the spread of metastatic breast cancer cells from the primary tumor site . This moLimited by safety concerns and the short half-lives of most current chemotherapeutic drugs, repeated dosing regimens are typically used in humans. Single agent drug resistance, and cumulative drug toxicity for some drugs could limit treatment options for metastatic diseases. In mouse model of this study, cancer cells progress into the lungs from the blood to cause mortality in only ~14 days. In this time window, the highest achievable single dose of the CrEL drug combination (limited by paclitaxel solubility) inhibited the process marginally. For 80~90% inhibition of lung metastasis of 4T1 within a similar timeframe, multiple dosing was required in previous studies –27. EvenThe ability of DcNP to enhance GT combination also opens other opportunities to enhance cancer chemotherapy. As lymphatic cancer invasion is believed to be an early site for metastasis, it is possible that DcNP loaded drugs may provide additional benefits. With at least 4 sets of HIV drug combinations formulated in DcNP, all drugs have been given subcutaneously in DcNP dosage for each composition into lymphatics in their first-pass –11, 29. In summary, we have developed a simple, stable, and scalable GT DcNP dosage suitable for IV administration that transforms short-acting gemcitabine into a long-acting variation. We have also enhanced the pharmacokinetic profile of both drugs. The enhanced GT pharmacokinetic profile provided by DcNP dosage form parallels the GT effect against 4T1 metastatic breast cancer. A single GT DcNP injection completely inhibited lung metastasis in mice at levels that cannot be achieved with a CrEL dosage form at equal or higher doses. The enhanced dose-response was observed with a significant margin of safety. With the flexibility of the DcNP platform and impact on GT pharmacokinetics, pharmacodynamics, and safety windows, this approach could be generally applicable for use with other drug combinations intended to treat a number of cancer types, especially for the metastatic disease stage. The ability to transform current short-acting drugs into long-acting forms with preferential uptake could also accelerate clinical translation of the drug combinations in DcNP dosage form.S1 FigOn day 0 GT in CrEL suspension or DcNP at 1.25/0.125, 10/1, or 50/5 mg/kg IV doses, and the 4T1 inoculated mice were monitored over 14 days. Each treatment group contains 8–15 mice and the data presented are mean ± SEM. In the group of 50/5 mg/kg of DcNP treated mice, some animals, due to clinical necessity, were sacrificed ahead of schedule. The remaining animals in the high dose group recovered and by day 14 appeared to exhibit body weight higher than at entry. In comparison, the saline placebo treated animals exhibit significant (15%) weight loss by day 14 due to rapid growth of lung metastatic nodules. The same trend is seen in the group treated with GT in CrEL suspensions (at two lower doses—10/1 and 1.25/0.125 mg/kg).(TIF)Click here for additional data file.


Lymphovascular invasion (LVI) has never been revealed by preoperative scans. It is necessary to use digital mammography in predicting LVI in patients with breast cancer preoperatively.n = 42) and the LVI negative group (n = 80). Independent t-test and χ2 test were performed.Overall 122 cases of invasive ductal carcinoma diagnosed between May 2017 and September 2018 were enrolled and assigned into the LVI positive group (P = 0.012). Differences in interstitial edema (P = 0.013) and skin thickening (P = 0.000) were statistically significant between the two groups. Multiple factor analysis showed that there were three independent risk factors for LVI: interstitial edema , blurring of subcutaneous fat  and skin thickening .Difference in Ki-67 between the two groups was statistically significant (Interstitial edema, blurring of subcutaneous fat, and skin thickening are independent risk factors for LVI. The specificity of LVI prediction is as high as 98.8% when the three are used together. Breast cancer metastasizes through lymphatic and blood vessels, which makes the patients susceptible to distant metastasis, postoperative recurrence and even death. Lymphovascular invasion (LVI) has never been revealed by preoperative scans. Diagnostic histopathology is needed to reveal LVI of cancer cells. Preoperative identification of LVI can help to predict the prognosis of patients with breast cancer, especially those with axillary node-negative breast cancer, and to develop adjuvant treatment plans in clinical settings , 2.Digital mammography is one of the important imaging tools for breast cancer screening and diagnosis. There have been many reports on prediction of axillary lymph node metastasis –5 and onIt is necessary to use digital mammography, a more readily available imaging tool, in preoperative prediction of LVI in patients with breast cancer, and to evaluate the predictive specificity of the imaging findings and certain biomarkers detected by immunohistochemistry so as to better foresee disease progression and develop targeted treatment plans.This single-center retrospective study enrolled 122 cases of invasive ductal carcinoma diagnosed between May 2017 and September 2018. Since the data were obtained from a picture archiving and communication system, patients’ informed consent was not required for this study. The inclusion criteria were: (1) modified radical mastectomy or breast-conserving surgery + axillary lymph node dissection; (2) diagnosis of invasive breast cancer confirmed by routine histopathological and immunohistochemical examinations; (3) no previous history of breast tumors or primary tumors in other locations; and (4) LVI status confirmed by immunohistochemistry. Patients were excluded if they had obscured lesions revealed by digital mammograms, were breastfeeding, had underwent lumbar puncture or radiotherapy prior to diagnosis, and had incomplete clinical data.n = 42) and the LVI negative group (n = 80).Mammographic lesions were confirmed either by core needle biopsies or by surgical pathology. All the patients underwent digital mammography preoperatively. All of them were female and aged between 26 and 77 with a median age of 45. They were assigned into the LVI positive group (GE (Senographe DS) and IMS (GIOTTO IMAGE) systems were used. Four standard body positions were imaged under automatic exposure conditions, and they were the right craniocaudal (RCC) view, left craniocaudal (LCC) view, right mediolateral oblique (RMLO) view and left mediolateral oblique (LMLO) view. When lesions were found in the axillary tail of Spence or near the cleavage, amplified imaging in these locations was done to reveal the lesions fully. The flat-panel detector of internal and external obliques was parallel to the pectoralis major muscle. According to the patients’ body shape, the projecting angle ranged from 40° to 65° and was usually 60°. The glands were fully unfolded, and the skin folds below the breasts and upper abdomen were within the mammographic field.n < 5). Those cases were excluded to avoid inaccurate statistical results. And the lesion type was classified as mass and calcification only.The imaging characteristics of breast cancer lesions were described according to the 5th edition of ACR BI-RADS® Atlas published in 2013 [n = 0), nipple discharge was not included as a variable. Thickening of the skin means that the affected breast has localized or diffuse thickening of the skin greater than 2 mm in thickness. Interstitial edema and subcutaneous fat layer blurring are caused by the filling and dilation of lymphatic vessels and blood vessels in the breast, while the performance of the whole breast including subcutaneous fat layer is blurring, and multiple cord shadows are seen.Associated features included nipple discharge, interstitial edema, blurring of subcutaneous fat layer, skin thickening, and axillary adenopathy . Due to its absence in the LVI negative group  immunohistochemical method were performed. DAB detection systems were used. A score of 3 and more indicated Her-2 overexpression. Positive FISH result of Her-2 gene amplification also indicated overexpression when the score was 2 and more. Ki67 proliferative index (PI) of <10% indicated low expression level, while that of > 30% indicated high expression level. Ki67 PI of 10 to 30% indicated intermediate expression level .The gold standard of this study was that LVI was defined as the intravenous tumor emboli and lymphatic tumor emboli detected by immunohistochemistry. These two kinds of tumor emboli were clinically referred to as intralymphovascular tumor emboli due to the difficulty of distinguishing them with pathological sections. The specimens were read by a senior pathologist who had been working on breast cancer for 21 years.P < 0.05 indicated statistically significant difference. The count data whose values were 0 were excluded from statistical analysis and listed only in table(s).The data were analyzed using SPSS Version 19.0. The quantitative data were expressed as means ± SDs. The independent t-test was done for intergroup comparisons. The count data were expressed as frequencies or rates, and the χ2 test or Fisher’s method was performed. P = 0.012), while no statistically significant differences were observed in the other factors mentioned.Table P = 0.013, Figs. P = 0.000, Figs. P = 0.431), radiological diameter (P = 0.094), mass number (P = 0.746), lesion number (P = 0.8855), location (P = 0.879), mass shape (P = 0.160), mass margin (P = 0.088), boundary (P = 0.714), calcification (P = 0.279), subcutaneous fat (P = 0.697), nipple retraction (P = 0.073) and axillary adenopathy (P = 0.166).Details are shown in Table P = 0.045), subcutaneous fat  and skin thickening .Risk factor analysis results are shown in Table Table LVI or intralymphovascular tumor emboli is closely related to the adverse outcome of many malignant tumors –17. As aThere are no statistically significant differences in age, childbearing history, miscarriage history, family history and other medical history between the LVI positive group and the LVI negative group. Nulliparity, miscarriage, and family history of breast cancer are not associated with increase of LVI occurrence.P = 0.012). Some literature have confirmed that Ki-67 is associated with tumor differentiation, LVI, metastasis, and recurrence [CA153 is a tumor marker first discovered in breast cancer cells. Its specificity is relatively high in diagnosis of breast cancer. Diagnostic sensitivity of CA153 increases from 66 to 80% as breast cancer advances . Howevercurrence –24.P = 0.013 and 0.000, respectively). In addition, multivariate analysis demonstrates that interstitial edema, blurring of subcutaneous fat, and skin thickening are independent risk factors for LVI . In clinical work-up, physicians should be highly alerted about LVI occurrence when digital mammography reveals the above three phenomena. Even if lymph node metastasis is negative in sentinel lymph node and axillary lymph node biopsies, there is possibility that breast cancer cells has infiltrated surrounding vessels but not metastasized upwards to the axillary lymph nodes yet. Besides, modification of postoperative adjuvant therapy should be considered to reduce risk of recurrence and distant metastasis, and thus to prolong survival.There are no statistically significant differences between the two groups in mammographic density, location, number, radiological diameter, shape, margin, boundary, calcification. Therefore, the above factors cannot be used to predict LVI. However, intergroup differences in interstitial edema and skin thickening are statistically significant (P = 0.166). One possible explanation might be that digital mammography failed to reveal axillary lymph nodes. In addition, an enlarged (> 1 cm) or complete lymph node does not always suggest metastasis. There is possibility of reactive hyperplasia.We believed that the axillary lymph nodes shown on digital mammography could be used to predict LVI. However, there are no differences between the two groups in axillary lymph nodes . When the three imaging features are applied together, the specificity of LVI prediction is as high as 98.8%.There is statistically significant difference in Ki-67 between the LVI positive group and the LVI negative group. Interstitial edema, blurring of subcutaneous fat and skin thickening are independent risk factors for LVI (


Aedes aegypti and Aedes albopictus mosquitoes. Because both Aedes species are peri-domestic and container-breeding mosquitoes, dengue surveillance should begin at the local level—where a variety of local factors may increase the risk of transmission. Dengue has been endemic in Colombia for decades and is notably hyperendemic in the city of Cali. For this study, we use weekly cases of DENF in Cali, Colombia, from 2015 to 2016 and develop space–time conditional autoregressive models to quantify how DENF risk is influenced by socioeconomic, environmental, and accessibility risk factors, and lagged weather variables. Our models identify high-risk neighborhoods for DENF throughout Cali. Statistical inference is drawn under Bayesian paradigm using Markov chain Monte Carlo techniques. The results provide detailed insight about the spatial heterogeneity of DENF risk and the associated risk factors  at a fine level, informing public health officials to motivate at-risk neighborhoods to take an active role in vector surveillance and control, and improving educational and surveillance resources throughout the city of Cali.Vector-borne diseases affect more than 1 billion people a year worldwide, causing more than 1 million deaths, and cost hundreds of billions of dollars in societal costs. Mosquitoes are the most common vectors responsible for transmitting a variety of arboviruses. Dengue fever (DENF) has been responsible for nearly 400 million infections annually. Dengue fever is primarily transmitted by female  Dengue fever (DENF) is a mosquito-borne disease that is responsible for most of the global burden of arboviruses.3 More than 40% of humans are at risk of transmission, with incidence rising 30-fold in the last 50 years, and it is estimated that there are approximately 390 million DENF infections annually.4 Dengue fever is primarily transmitted by the Aedes aegypti and Aedes albopictus mosquitoes.6 Both species are container-breeding mosquitoes that have become prolific in urban areas because of the widespread availability of breeding habitats.7Vector-borne diseases (VBDs), more specifically mosquito-borne arboviruses, are responsible for 1 billion infectious disease cases each year, globally.8 The incubation period ranges from 3 to 14 days after being bit by an infected mosquito, and symptoms can last from 2 to 7 days9; however, approximately 80% of infected individuals are asymptomatic. Infection from one serotype will result in lifelong immunity to that serotype; however, secondary infection with another serotype can lead to severe forms of DENF,10 such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue hemorrhagic fever and DSS primarily not only affect pediatric patients but also have been found among adults ; mortality from dengue is highest among children and those who have experienced DSS.11Dengue is a flavivirus that causes DENF, and there are four serotypes that follow the human cycle.12 ultimately reducing the economic and public health burdens in at-risk locations around the world.13 Approaches and advances in geographic information science and spatial epidemiology play critical roles in DENF surveillance—such as tracking diffusion and cyclic patterns, detecting clusters, mapping disease rates and risk,14 and understanding the place-based determinants of disease transmission.15 Dengue fever risks and rates will vary by place, and covariate data are needed to identify significant variables responsible for observable spatial patterns.16It is critical to implement surveillance strategies that can improve the understanding of DENF transmission. Improving DENF surveillance can facilitate the timely reporting of disease cases, reduce underreporting, inform policy-makers, increase disease awareness, and define funding and research priorities,17 Therefore, more small-area studies in spatial epidemiology are required to effectively uncover the spatial and temporal heterogeneity of DENF rates across urban landscapes at these fine levels of granularity. For example, education, income, age, access to care, and quality of prevention strategies are known to strongly influence an individual’s susceptibility to VBDs.19 Likewise, the dynamics of how temperature, precipitation, and humidity affect vector abundance and DENF transmission are critical to developing and implementing effective sub-seasonal risk model20 and long-term mitigation in response to climate change.21Therefore, it is critical to examine the social, economic, environmental, biological, and institutional factors that may affect DENF prevalence in a particular area. Urban regions are highly complex, and neighborhoods are the scale that public health departments most effectively operate.Aedes habitats) contained in surrounding locations . Conditional autoregressive models can be fitted to data under Bayesian paradigm  using Bayesian hierarchical models (BHMs), which are widely used techniques in geography and public health to model spatial and spatiotemporal data.22 In short, BHMs can model complicated spatial and space–time processes by conditionally modeling the variations in data, the process, and unknown parameters.23 The temporal extension, space-time (ST)-CAR, can estimate the value of a variable  at a particular location and time, which will be related to current and past values of the surrounding locations and time periods, essentially testing for spatiotemporal dependence. ST-CAR models have been used to study the effect of air pollution on human health,24 substance abuse, and its relationship with child abuse,25 influenza,26 and DENF.29Conditional autoregressive (CAR) models can be used to examine how DENF risk is influenced by socioeconomic, environmental, and accessibility risk factors, and lagged weather variables. For example, a particular location may be influenced by DENF rates and a variety of explanatory variables  model which identified six significant socioeconomic and environmental independent variables (including proximity to tire shops and population density) of DENF rates in Cali, Colombia, at the neighborhood level. However, the explanatory power of GWR and its temporal extension—GTWR32—is limited. Both CAR and ST-CAR models produce model-based estimates and inference derived from varying effects via spatial random fields—for example, borrowing strength from neighborhood spatial and temporal proximity, whereas GWR models allow the covariates to vary in space (and time in the case of GTWR), but inference is ad hoc. In other words, ST-CAR models can estimate spatially and temporally varying associations between dependent  and independent variables based on locally weighted regressions in both geographic and attribute spaces, whereas GTWR can only produce local estimates in geographic space. It is therefore worthwhile to use ST-CAR modeling in small-area DENF studies at fine temporal scales.More research is necessary to examine the local variations in DENF transmission dynamics at very fine spatial and temporal scales. Delmelle et al.This study uses a ST-CAR modeling approach to examine the influence of socioeconomic, environmental, weather, and climatic variables on DENF outbreaks in Cali, Colombia, at the neighborhood and weekly levels between 2015 and 2016. The approach can determine if DENF rates and covariates in one neighborhood are influenced by rates and covariates in surrounding neighborhoods and time periods. We also estimate disease risks using temporally lagged weather variables. Our modeling approach is capable of identifying regions with high-risk clusters at the neighborhood level. The results provide detailed insight about the spatial heterogeneity of disease risk and the associated risk factors at a fine level, informing public health officials to motivate at-risk neighborhoods to take an active role in vector surveillance and control, and improving educational and surveillance resources throughout the city of Cali.The remainder of this article is as follows: Section 2 provides information about Cali, the DENF cases, and candidate-independent variables; the technique to select the lagged weather variables; and the ST-CAR modeling approach. Section 3 provides the modeling results, including maps of the estimated DENF rates by week for each neighborhood in Cali. Section 4 discusses key findings, strengths, limitations, and avenues of future research. Section 5 summarizes findings with concluding remarks.2). The city comprises 340 neighborhoods (Spanish: barrios), which are classified by socioeconomic stratum (ranging from 1 to 6), where a ranking of 1–2 is low, 3–4 is middle, and 5–6 is high. The classifications are defined by the external physical characteristics of the dwelling, its immediate surroundings, and its urban context. For example, urban context includes variables such as poverty, social deviation, urban decay, industry, and commercial; immediate surroundings include access roads, and sidewalks; and the characteristics of the dwelling include front lawn, garage, façade material, door material, front of the house dimensions, and windows (income is not considered). This stratification is only applied to residential constructions.332. Some of the smaller neighborhoods are in the city core, which is where the city was founded. The largest neighborhood is to the south and corresponds to newer developments, and is an area that houses three of the largest universities in Cali.Cali is the second largest city in Colombia and the third most populous, with an estimated 2010 population of 2.3 million —representing the density of points for each layer. Kernel density estimation was also computed to produce the density of trees. Zonal statistics in ArcGIS 10.6 was used to summarize the average KDE for each neighborhood in Cali.The socioeconomic and demographic data were provided by the Colombian census , including population density, age, race, households with sewer and water access, educational attainment, employment status, and socioeconomic stratum, among others. The last national census occurred in 2005, whereas the new 2018 census has yet to be released. The location of healthcare centers and the environmental variables were provided by the city of Cali (2010 data)—green zones, rivers, tire shops, water pumps, cemeteries, and plant nurseries, which were geocoded as point layers with the exception of green zones (area: polygons) and rivers (lines). The environmental variables are included as potential Aedes survival, abundance, and behavior, including viral transmission rates34 . All weekly weather variables were computed following the methods outlined in the study by Eastin et al.,20 and summary statistics for the 2014–2016 period are provided in Finally, the weather variables selected for evaluation  are cons rates34 . Given on = 36), we used VIF36 testing and a PCA37 in Stata. We did not include all variables in the PCA because we wanted to interpret particular independent variables of DENF individually, which is important for potential decision-making. For example, identifying the effects of individual Aedes habitats  can provide more intuitive results than grouping them in a PCA. As a result, five variables were selected for subsequent modeling with a VIF value < 3: population density, tree density, relative proximity to rivers, relative proximity to tire shops, and relative proximity to plant nurseries.Because of the large number of socioeconomic and demographic variables, and environmental variables (The PCA’s purpose is to reduce and simplify the variables into new variables (components) that explain a large degree of variation without collinearity between the components. Principal component (PC) 1 is strongly associated with (correlation close to 0.5 and greater) neighborhood strata, individuals who are employed, retired persons, married individuals, empty households, and individuals with high education. PC2 mainly explains occupied households, individuals who do housework, and individuals who are students. PC3 explains the combined effect from disabled individuals but was excluded from further analysis, and it was included as a separate variable in further VIF testing with the remaining covariates. PC1 can be interpreted as employed, higher-income people who are likely to be older and married because of the large coefficients of retired and married individuals. PC2 can be interpreted as people who are likely to spend more time at home than those in PC1 because they are either students or do housework for a living. PC2 probably also captures younger individuals because of the inclusion of students.34 .First, a Poisson GLM is computed to detect significant effects of independent variables on a dependent variable (DENF risk), and the presence of spatiotemporal autocorrelation in the residuals. The Poisson GLM is defined as54 was then computed to detect spatial autocorrelation of the Poisson GLM residuals for each time period. Essentially, the Global Moran’s I test determines if there is evidence of unexplained spatial autocorrelation in the residuals, and if positive spatial autocorrelation is detected, then the assumption of independence is not valid for the data, and spatiotemporal autocorrelation should be considered when estimating covariate effects on the dependent variable. The global Moran’s I index ranges from −1 to 1, whereas −1 indicates strong negative spatial autocorrelation, 0 indicates complete spatial randomness, and one indicates strong positive spatial autocorrelation, and the statistic is defined asn is the total number of neighborhoods, wij is the spatial weight between neighborhood i and l, i, and l. The Moran’s I tests were conducted in RStudio 1.2.5 with R version 3.6 .Global Moran’s I25 is defined using a Poisson data model (for case/population data). The model “represents the spatiotemporal pattern in the mean response with a single set of spatially and temporally autocorrelated random effects. The effects follow a multivariate autoregressive process of order 1.54 In other words, when going from 1 week to another , it will yield an effect on the dependent variable (DENF risk). Therefore, this model examines linear trends which can be interpreted as how DENF risk is influenced across time.Next, a BHMN nonoverlapping areal units  indexed by W = i and k are spatially adjacent, and 0 otherwise. Because it is unknown where a person was infected, the abovementioned adjacency matrix is a proxy for Aedes’ maximum range—because they do not fly more than 400 m from where they emerged as larvae.10 A temporal weight matrix can also be defined as D = t − j = 1, and 0 otherwise. The first part of the model is defined asi at week j, i at week j, and i at week j. p for neighborhood i and week j. The parameter β is an associated p × 1 vector of regression parameters, which can come from the initial Poisson GLM in K × 1 column vector of offsets (expected DENF cases) for week ji and week j. A variety of spatiotemporal structures can be fit for 55).j. Temporal autocorrelation is enforced because I is a N × N identity matrix and “1” is a vector of ones (N × 1). The α controls the temporal autocorrelation, where 0 is temporally independent and 1 is strongly temporally dependent. The CAR priors also include weakly informative hyperpriors , which are the three parameters defined in the following text:j + 1), it yields an effect on the dependent variable (DENF risk), which is influenced by spatially and temporally dependent covariates. In other words, DENF risk in a target neighborhood is influenced by current and past values of DENF risk and covariates at surrounding neighborhoods and time periods (which is a process that evolves over time). Conceptually, a spatial example would suggest that a neighborhood with a low risk of DENF would have an increased risk of DENF if an adjacent neighborhood reported a high risk of DENF (dependence/autocorrelation).It is assumed that the estimated effect on DENF risk in the ST-CAR model is not specific to a particular week, but a process that is influenced by the covariate data across the weeks . Suppose a study region is divided into a collection of 56 Markov chain Monte Carlo is a popular technique for Bayesian inference when posterior distributions are not available in closed forms. We used MCMC to generate samples from the posterior distributions induced by our ST-CAR models and used them for parameter and density estimations. For this study, we selected 220,000 MCMC samples; initial 20,000 samples are removed as burn-in, and the thinning parameter is set to 10 , which thins the samples to reduce autocorrelation of the Markov chain. As a result, 20,000 samples are used for statistical inference. The deviance information criterion (DIC) was used for model assessment and comparison, where lower values of the DIC indicate a better model fit. The results of two models are presented in Section 3: model 1 (DENF with no lags) and model 2 (DENF with lagged weather variables). Using CARBayesST24 in R, the two Poisson GLM models (DENF with no lags and DENF with lags) were each fitted to the ST-CAR model described earlier.Statistical inference is derived from Markov chain Monte Carlo (MCMC) simulations.The following subsections contain summaries of model results, relative risk estimates for each independent variable, and mapping the posterior estimates of DENF rates in Cali for particular time periods .When comparing the six temporal cross sections, July 2016 experienced the highest DENF risk  after accounting for the 14 covariates (including the lagged weather variables). Interestingly, some locations with high rates of DENF are classified as middle- (3 or 4) or high-strata neighborhoods (5 or 6). These middle- and high-strata neighborhoods are adjacent to low-strata neighborhoods (1 or 2), which suggests that there is spatiotemporal dependence between them. In other words, there is evidence that middle- and high-strata neighborhoods are at higher risk when surrounded by lower-strata neighborhoods after accounting for the covariates in the models. Some of the highest proportion of cases were not only observed in the eastern portions of Cali (now shown here) but also include some of the highest and most densely populated neighborhoods of the city. After computing rates per 1,000 persons , the eas22 Independent variables were used to characterize large-scale variations, whereas spatiotemporal autocorrelation was used to characterize small-scale variations. The lagged independent variables modified the large-scale variations, and, for these data, it seems they have no effect on small-scale variations. The predictive power of the models remains the same, very close DIC values  and small-scale variations .lues see . That isFirst, there is very strong evidence that there are both spatial and temporal dependences between DENF risk and the significant covariates for adjacent neighborhoods in Cali. Although DENF had a much lower temporal autocorrelation , this can be explained by the distribution of cases between 2015 and 2016—there were three distinct peaks, but DENF remains a persistent threat because of the four serotypes of the virus. The very strong spatial autocorrelation values  for DENF suggest that outbreaks in adjacent neighborhoods are strongly related .30 who suggest that in the southern part of Cali, houses are typically bigger with relatively larger yards, which may provide a suitable habitat for Aedes to breed. Neighborhoods with a high proportion of people in the PC1 category  are also typically adjacent to middle- or lower-strata neighborhoods (with the exception of the extreme south), which also may increase the risk of disease due to the very strong evidence of space–time dependence between the locations, as suggested by the models. An increased risk of DENF was reported for neighborhoods with higher proportion of individuals in the PC2 category . This finding corroborates with strong evidence that Aedes proliferates in and around homes,59 and it has also been found that cases can substantially decline if Aedes trap interventions are put in place in at-risk communities.60When examining the results of the three socioeconomic covariates , the increased risk of DENF for PC1 is an interesting finding. This corroborates with Delmelle et al.,62 Furthermore, there is evidence that shows that low-density areas with poor infrastructure may have increased Aedes presence63 and DENF’s complex immunology, and the herd immunity resulting after infection from the viruses64 may have contributed to these patterns. Among other factors, high density of populations may not necessarily be a main risk factor of VBD transmission.65 Although increases in population and urbanization will undoubtedly increase the risk of VBD transmission, the true effect of population density may vary at fine spatial levels . Public health authorities typically carry out fumigation and education programs in neighborhoods with higher population densities, which may result in a lack of targeted interventions in less dense areas with Aedes populations . Vector control efforts in Cali include storm sewer control, spraying, use of guppy fish, visits to health facilities and homes, and community educational campaigns.35Another unexpected result was the negative relationship between population density and DENF. One explanation can be that some of the densely populated neighborhoods in the eastern part of Cali have a high concentration of Afro-Colombian population, which has been suggested to be less susceptible to the viruses.Aedes; thus, neighborhoods within close proximity are generally at a higher risk of disease transmission. Tree density may also be a significant risk factor of Aedes presence because studies have shown that high tree shade density stimulates breeding, and tree holes are suitable water containers where Aedes have been found in abundance.67Closer proximity to plant nurseries, tire shops, and higher tree density all exhibited an increased risk of DENF. Plant nurseries and tire shops are common breeding grounds of Aedes require stagnant water as a breeding ground; therefore, the flowing water of a river or ravine would prove to be an unsuitable habitat for the mosquitoes. Although flooding events during the rainy season could create stagnant water sources surrounding the rivers, however, floods may also act as a disruptive force on Aedes habitat by flushing out their breeding sites and eggs.69 Further research using remote-sensing techniques  could provide insight to areas prone to stagnant water.Closer proximity to rivers and ravines  resulted in a significantly lower risk of DENF. Aedes life cycle . Moreover, because the optimal lags were different among the weather covariates, our results imply that individual covariates impact different stages of the vector life cycle see . Given tfe cycle  and 5 arfe cycle .Variables with an optimal 5-week lag  are most influential on larval development. Both DTRmax and WarmD exhibit the expected positive relationship  and largThe short-lived rain showers also induce evaporative cooling that lowers the daily minimum temperature and increases the daily temperature range , leading to slightly cooler, but above-average daily mean temperatures . Overall, regular rainfall combined with average- to above-average temperatures produces numerous stagnant pools within a favorable thermal environment for prolific larval development.Aedes feeding.Variables with an optimal 3-week lag (RainT and RHrng) are most influential on the gonotrophic cycle. Both RainT and RHrng exhibit the expected relationships  with simFinally, variables with an optimal 2-week lag (CoolD) are most influential on the gonotrophic cycle and extrinsic incubation. Specifically, CoolD exhibits the expected negative relationship , but its20). Careful monitoring of weather conditions within this time window could optimally inform any sub-seasonal DENF mitigation efforts. Moreover, it should be noted that a moderate El Niño occurred during our 2015–2016 study period,70 and there is strong evidence that major DENF epidemics are more severe during El Niño events.71 Therefore, any sub-seasonal mitigation efforts should also account for inter-seasonal climate variability.Overall, such consistent multi-lag relationships reinforce the idea that DENF outbreak dynamics are dependent on a complex combination of weather conditions 2–5 weeks prior  but is currently unavailable. Using 2005 and 2010 data for this study will bias the results, but the neighborhood classifications (strata) mostly remained unchanged. The uncertainty resulting from using outdated census data is a common limitation found in many studies in Latin America and developing countries. Several articles examining patterns of DENF in Colombia have recognized outdated population census being a critical issue that could affect the findings of their research.78 Until more recent census are available, different population growth models could address this issue, or relying on disaggregated population projection, using dasymetric mapping.80 Third, including vector surveillance data (presence/absence) in each neighborhood would improve the accuracy of the relative risk estimates.17 Fourth, the spatial weight matrix only considered adjacent neighborhoods as “neighbors”; we recognize that individual activity spaces expand far beyond locations nearby their home. Future research can implement different spatial and temporal weight matrices for sensitivity analysis purposes. Fifth, the weather conditions and severity of outbreaks for DENF varied between 2015 and 2016, which may have affected the model results. Future work can disaggregate the years and run two separate models for further examination. Sixth, the space–time patterns of the DENF outbreaks did not exhibit much seasonality, which is likely because of only using 2 years of data. Further work can use 5–10 years of DENF and weather data to detect potential seasonal patterns of the epidemics. Finally, weather variability is represented by a single weather station . Future work would benefit from multiple weather stations that can document spatial variability across the region.Despite the strengths and contributions of this research, there are notable limitations and areas of future work that are worth discussing. First, the underreporting of cases and unmatched addresses during the geocoding process likely undermines the true burden of DENF. Underreporting is also a major issue in the low-strata neighborhoods and also not uniform across the city, whereas surveillance systems can improve the identification of risk factors throughout the city.Aedes), and further investigation can develop a multivariate space–time (MVST) CAR model to examine which neighborhoods are at higher risk for one disease, two diseases, or all three concurrently. Multivariate space–time approach in CAR modeling is still in its infancy stages,24 and the development of such models can more accurately examine and compare the co-occurrence of diseases transmitted by the same vector. A long-term goal is to develop a multiparameter early warning system (EWS) that informs public health officials and motivates effective vector surveillance and control measures. The space–time models and methods described herein represent progress toward that goal. However, effective EWS development would require larger and more comprehensive databases  than those currently available. As noted earlier, an EWS system would benefit from longitudinal information regarding socioeconomic, demographic, and environmental variables combined with spatial information regarding weather variability across the region.Chikungunya and Zika are transmitted by the same vector  should start at the neighborhood and community levels.Supplemental materials


P = 0.02), orthostatic dysregulation (P = 0.005), and motion sickness scores (P = 0.03) were all significantly higher after the earthquakes. A subset of participants underwent clinical equilibrium testing, showing significant deteriorations in the equilibrium test results (stabilometry: P = 0.01), cervical vestibular-evoked myogenic potentials (P = 0.04), and head-up tilt (P = 0.03) after the earthquake. The findings of this study also suggest that earthquake-induced disequilibrium may be further influenced by physical stressors, including sensory disruptions induced by earthquake vibrations, changes in the living conditions, and autonomic stress. This study increases our understanding of human equilibrium in response to natural disasters. Moreover, these findings will facilitate the management of dizziness experienced during or after such disasters. Future studies should identify strategies for mitigating autonomic dysfunction to prevent post-earthquake dizziness.This retrospective chart review aimed to examine both the vestibular function and causes of dizziness experienced by individuals following a series of major earthquakes and repetitive aftershocks. All patients with balance disorders who experienced the 2016 Kumamoto earthquakes and their aftershocks completed questionnaires relevant to balance disorders and were enrolled in this study after providing informed consent. There were 2.8 times more patients with balance disorders post the earthquake. Anxiety ( Major earthquakes are associated with an increased prevalence of psychiatric morbidities , 2, sleeAlthough several reports have described post-earthquake dizziness –7, the cThe patient records were reviewed between and data collected from 21 otolaryngology clinics and hospitals in Kumamoto prefecture April 1st and July 21st, 2016 were extracted. Data were analyzed between 2017 and 2019. Inclusion criteria were a cumulative total patients with new-onset and worsened balance disorders, who visited otolaryngology clinics or hospitals in Kumamoto prefecture between April 1st and July 21st, 2016, were aged between 10 and 100 years, and could describe their symptoms and experiences following the Kumamoto earthquakes. The exclusion criterion was individuals who had not visited an otolaryngology clinic or hospital due to balance disorders. Participants who reported vertigo/dizziness before major earthquakes and visited otolaryngology clinic or hospital after major earthquakes were classified as experiencing balance disorders in the pre-earthquake period.The study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Kumamoto University Hospital . All participants enrolled in the study provided written informed consent.Data, including sex, age, symptoms, history of vertigo/dizziness, floor location during the earthquake, evacuation methods, and diagnosis, which were obtained on the day of ear, nose, and throat (ENT) consultation between April 1st and July 21st, 2016, were collected from hospital/clinic medical records and analyzed. Clinical diagnosis of balance disorders was based on the diagnostic criteria published by the Japan society for equilibrium research , 11. AllMethodological details and criteria regarding the questionnaires and tests are described in The normal range of the CP% was defined as <20% . The HUTThe primary outcome was the occurrence of post-earthquake dizziness , and the secondary outcomes were psychological stress and vestibular and autonomic dysfunction.post hoc Dunnett's test to avoid an inflated Type I error rate because data were normally distributed. Regarding secondary outcomes, since the data were also normally distributed, both one-way ANOVA and post hoc Tukey–Kramer tests were used to investigate the ratio of patients with ERD who were evacuated to a shelter to avoid an inflated Type I error rate. Residual plots were used to confirm the correctness of the assumptions made for both primary and secondary outcomes. Missing values were imputed by random forest method. There were no outliers in the analysis of either the primary or the secondary outcomes. For non-parametric analysis of subjective variables, which were not normally distributed, Wilcoxon signed-rank tests were used to investigate changes in the pre- and post-earthquake questionnaire scores and the equilibrium test results in patients with ERD who underwent equilibrium testing before and after the earthquake. Fisher's exact test was used to compare pre- and post-earthquake HUT and nystagmus scores with an abnormal distribution in patients who underwent equilibrium testing before and after the earthquake. Bland-Altman analysis with test-retest intervals was performed to discern test-retest effects from clinical changes. Statistical significance was set at P < 0.05. Evaluations were determined as “not applicable” if the calculated sample size after data collection was found to be insufficient for statistical analysis. All statistical analyses were performed with EZR , which is a graphical user interface for R  and a modified version of R commander designed to add statistical functions frequently used in biostatistics.Power and sample size calculations were conducted before and after data collection using the PS software  . RegardiP < 0.001; A cumulative total of 575 patients  diagnosed with balance disorders after the Kumamoto earthquakes participated in this study. The median (interquartile range) age in this cohort was 63.5 (16-93) years in men and 59.0 (10-95) years in women. The proportion of patients with balance disorders was significantly higher during the 0–2-week , 2–4-week , and 4–6-week  post-earthquake periods than the 2-week pre-earthquake period  , sudden deafness with vertigo , vertebrobasilar insufficiency , psychogenic vertigo , and vestibular neuritis  during the post-earthquake period than during the pre-earthquake period , VBI , OH , or psychogenic vertigo  during the 0–2-week post-earthquake period than during the 2-week pre-earthquake period , SDV , psychogenic vertigo , and VN  than in those with other balance disorders  and DHI sub-scores for physical , emotional , and functional  subscales after the earthquake compared to those before the earthquake. Similarly, the scores for patient anxiety  and depression  components of HADS, OD , motion sickness , and ESS  were found to be higher after the earthquake than those before  (P = 0.03) and positive rate of nystagmus   . The rat< 0.001) .This study examined the occurrence and possible mechanisms of ERD after major earthquakes. The results showed that the prevalence of balance disorders increased significantly after the major earthquakes when compared to before the earthquakes. The equilibrium test and questionnaire-based survey results also indicated that vestibular functions, especially otolith and autonomic functions, were deteriorated after the major Kumamoto earthquakes and its repetitive aftershocks. Moreover, these results suggested that psychological factors might be a cause for the increase in the prevalence of balance disorders in the patients enrolled in this study. These findings suggest that exposure to major earthquakes and aftershocks induce post-earthquake balance disorders due to sensory conflicts mediated by vestibular dysfunction, autonomic dysfunction, and/or psychological factors, which is congruent with previous findings , 32, 33.One of the main strengths of this study is the use of objective equilibrium testing. In the 41 patients who underwent pre- and post-earthquake equilibrium testing, stabilometry results indicated that the static equilibrium significantly deteriorated during the post-earthquake period compared to that of the pre-earthquake period. In patients with post-earthquake ERD, I also observed a significant deterioration of cVEMP responses. The prevalence of nystagmus was also higher in patients with post-earthquake ERD than in patients with pre-earthquake ERD. A previous study had also reported that healthy individuals who repeatedly experience aftershocks presented with greater equilibrium dysfunction than those who had rarely experienced aftershocks . OverallLarge earthquakes and the physiological and psychological stress they elicit can worsen cardiovascular function , 39, whiDifferent studies have reported a strong relationship between autonomic dysfunction and balance disorders , 31. GenI observed that equilibrium dysfunction was augmented by psychological stress, as previously reported , 46 in bThis study has several limitations. First, the sample size of participants who performed the equilibrium test was small. Moreover, ocular VEMP test and video Hit impulse test were not performed because of the breakdown of the measurement instruments due to this major earthquake. Besides, several participating ENT clinics did not have access to these instruments. Examining equilibrium dysfunction in individuals with ERD will enable identification of the causes of post-earthquake dizziness. Conducting equilibrium tests during the post-earthquake period is necessary to increase the sample size and enhance the study's validity. Second, psychological stress, such as post traumatic stress, was not fully investigated; investigating the impact of post-disaster psychological stress on healthy individuals with ERD would help verify the causes of post-earthquake dizziness. Third, the influence of age and sex was not assessed in this study. Future studies should determine whether age or sex might affect the risk of balance disorder after major earthquakes.In conclusion, exposure to major earthquakes and aftershocks induced post-earthquake dizziness in a significant percentage of individuals. My results indicate that post-earthquake dizziness may be due to sensory conflicts mediated by vestibular dysfunction, autonomic dysfunction, and/or psychological factors. My findings will facilitate the management of dizziness experienced during or after such high magnitude earthquakes, which occur frequently in Japan. Future studies should assess whether earthquakes with less magnitude may affect human sense of balance.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Kumamoto University. The patients/participants provided their written informed consent to participate in this study.TM designed and conceptualized the study, acquired and analyzed the data, and drafted the manuscript for intellectual content.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While emerging studies have demonstrated the benefit of exercise in Substance UseDisorder (SUD) recovery outcomes, lack of motivation to engage in exercise hasbeen indicated as one of many perceived barriers that contribute to lowrecruitment and adherence rates in SUD treatment. The current study aimed toexplore participants’ perceptions of attending a supervised exercise program while intreatment for SUD. A total of 109 participants were recruited to a 14-weekexercise training program and 61 chose to participate in, and completed, a raceat the close of the program. Interviews were conducted during weeks 6 through 14and data were examined using Thematic Analysis. Three main themes wereidentified: (1) pushing forward recovery through running, (2) gaining a sense ofachievement by crossing the finish line, and (3) building a sense of belongingin the program. Implications for SUD recovery programs are discussed. Results suggested that participants who received theintervention experienced improvement in the physical health ,psychological health , and domains of quality of life  relative to controls.14 Exercise influences many of the same signal molecules and neuroanatomicalstructures that mediate the effects of substance use, consistent with the theory that itcan serve as a healthy, non-drug source of reinforcement to decrease substance use.15 The effects of exercise on health related outcomes among clients diagnosed SUDshave supported that exercise is a feasible and safe adjunct treatment for SUDs withbenefits to physical, psychological, behavioral, neurological, and overall personalhealth.1618A lifestyle-based adjunct to traditional treatment for people experiencing SUDs isinvolvement in physical activity. Health benefits of regular physical activity isevident, including reduced risk of various chronic diseases, improved concentration,elevated mood, and better sleep quality.13 In addition, low adherence to exercise programs might undermine their effectiveness.16 Other most commonly reported barriers to engaging in exercise programs amongpeople with SUDs included cost, transportation, and equipment.19 Although many residential SUD recovery centers are equipped with fitnessfacilities, the use rate is low. Flemmen and Wang indicated that SUD patients haveimpaired endurance and muscular strength compared with an age and sex matched referencegroup, and suffer higher risk of developing other chronic illness and early death.20 Findings of another study demonstrated that participants in SUD treatment exhibitlow rates of physical activity and low motivation to engage in physical activity;however, participants indicated a tendency to attend if an exercise program was offeredduring their substance abuse treatment in the recovery facility.21 These findings suggested the importance of increasing motivation to engage inphysical activity for people in recovery, especially by providing guided and scheduledexercise programs in hope of enhancing participants’ engagement in physicalactivity.However, Linke and Ussher reported that recruitment of SUD patients to an exerciseprogram is challenging.In view of the complexity of SUD and recovery, it might be crucial to provide activitiesthat can serve as positive coping responses to substance use, and coping strategies thatclients can sustain in their life to help manage their SUDs. There is limited researchthat has explored participants’ perceptions in exercise programs during their recovery.In this study, we implemented a supervised group exercise program, which was composed ofboot camp workouts, walking/running practices and a race event, in a treatment facilityfor SUD. We investigated the perceptions of people experiencing SUDs in a residentialrecovery center during the 14-week supervised group exercise program.22This qualitative study investigated SUD patients’ perceptions of participation in asupervised walking/running exercise program. Thematic Analysis was utilized for thisstudy to acquire knowledge about participants’ experiences.n = 109) were recruited from a treatment facilityfor recovery in a mid-sized city in the Midwest, USA. Inclusion criteriaincluded: resided in the recovery center, older than 18 years old, and providedconsent to participate in the walking/running training program. The recoverycenter serves populations who are economically disadvantaged, low-income,unemployed, underemployed, and dislocated. There were 54% females, and 46%males. The racial makeup of these participants was 63% Caucasian, 28% AfricanAmerican, 3% Hispanic, and the remaining 6% of participants were marked unknownor other. Participants’ age group was made up of 21% 20 to 29 years, 28% 30 to39 years, 15% 40 to 49 years, 25% 50 to 59 years, and 11% 60 to 69 years.Participants were impacted by frequent and severe use of substances includingalcohol, cocaine, heroin etc. The majority (80%) of people who have accessedrecovery services at the treatment center had opioid use disorders. Other lifeexperiences reported by participants included homelessness, incarceration,prostitution, and unhealthy domestic issues. A typical stay lasted for12 months; however, the length of stay was determined by the participant’sindividual goals and treatment plan. To provide a safe and healthy livingenvironment that promotes recovery from SUD, the supervised residential recoverycenter provided participants with treatment components including meal andshelter services, individual and group counseling, educational and vocationaltraining classes, financial counseling, parenting skills training, and aftercareservices. Participants were required compliance with all prescribed medicationto remain in the program. Random and routine drug screens, alcohol screens witha Breathalyzer unit, and room searches were also conducted.Participants (This 14-week exercise program included four weeks of boot camp (once weekly),nine weeks of walking/running (twice weekly), and the 14th week included therace see ,which wBoot camp workout was delivered once a week  at the facilityfor the first four weeks of the program. It included intense body movement suchas aerobic, strength training, and speed element in each session. Each boot campworkout lasted for one hour and thirty minutes. Walking/running practices lastedfrom weeks five to fourteen and were held twice weekly outside of the facility.A total of 18 practice sessions were scheduled and implemented. Eachwalking/running session lasted from the range of 30 to 90 minutes . In the beginning of each training,participants were guided to stretch their body to warm up beforewalking/running. In the beginning of the program, participants were encouragedto select a type of race they expect to train for as their goal. Volunteers werethen partnered with individuals or a group of participants for the rest of thetraining sessions. During the practice time, the goal was to keep participantsengaging in body movement. In each practice, the staff announced the aimeddistance of walking/running for each type of race event; participants wereencouraged to walk and/or run for the planned distance or time. Participants canwalk first to warm up and start to run when they feel comfortable. The staff andvolunteers, who were experienced in instructing fitness classes and runningregularly, led both boot camp and walking/running sessions.Several events such as athlete of the week, participation of a local runningevent, cheer up day, pre-race lunch, and post-race celebration were implementedalong with the walking/running practices. To recognize participants’ engagementin the exercise practice, athlete of the week is selected and voted byvolunteers or endorsed by their running coach or program director. Joining alocal running event while in training offered a tune-up run for theparticipants. Participants experienced the race atmosphere where other runnersand community members greeted them. On the cheer up day, live music, food, andthe mascot of a professional sports team were there to cheer participants.Pre-race lunch was scheduled one week prior to the race. Participants andvolunteers gathered together to celebrate how far they have trained for the racewhile in recovery. Finally, after the race day, family, volunteers and communitymembers were all invited to join the post-race celebration.To get a complete sense of how participants experience the program, interviewswere conducted beginning in week six and ending after the race. Forty-nineparticipants completed interviews. During training weeks six through 13, 24individual interviews were completed. After the race, an additional 25participants were interviewed. The interviewer asked the participant anopen-ended question: describe your experiences in the exercise program.Interviews were conducted in a quiet space inside or outside of the treatmentcenter. Each interview period ranged from 15 to 30 minutes. Interview videoswere then transcribed into texts by the authors.22 Initially, two researchers read the texts and coded them independentlyusing open coding.23 The purpose of this step was to simplify the data and allow for emergentcategories to be identified.25 Any disagreement aboutcoding was discussed until consensus was reached over the course of severalface-to-face meetings. Codes were then combined into themes, which involvedfinding general patterns among specific information.27The data analysis was conducted based on a six-phase process: familiarizationwith the data, coding, generating initial themes, reviewing themes, defining andnaming themes, and producing the report.28 To achieve these criteria, the authors reviewed transcripts andinterviews several times to gain a deeper understanding of the phenomenon andprovoke meaningful themes. The codes and themes were then reviewed by experts inqualitative studies to evaluate the analysis method and provide feedback toensure its validity. The summary of the results were returned to interviewees,and they were asked to review if authors’ interpretation matched theirperceptions and experiences.Lincoln and Guba argued that trustworthiness of a study involves establishingcredibility, transferability, dependability, and confirmability.The research was approved by the university’s research review board (ID:20132115). The program manager introduced the researcher to participants in oneof the group meetings. The researchers reiterated the research procedure andobtained informed consent from all participants. Each participant voluntarilyagreed to be interviewed.The purpose of this study was to explore perceptions of participants in a supervisedgroup exercise program for people with SUD. This program was 14 weeks in length and61  out of 109 participants ultimately chose to participatein, and completed, the race at the close of the program. After analyzing interviewdata about participants’ perceptions of participating in the walking/runningtraining program on their recovery, the researchers identified three main themes:(1) pushed forward recovery through running, (2) gained a sense of accomplishment bycrossing the finish line, and (3) experienced a sense of belonging in the programsee . FindingIn the program, participants engaged in running and walking instead of drug useas a part of their recovery journey. Participants reported that walking/runningpractices helped them “tough through” the recovery by reducing cravings todrugs, and improving overall personal health such as weight control, feelinggood, and being fit.“Running is the one thing I look forward to the most everyweek. I’ve traded my addiction that brought me here for runningand healing. However, the most important thing for me is to pushthrough the pain on the inside, and that’s what I’ve dedicatedmyself to now. Running helps me do that.”Several participants indicated that running served as a positive copingstrategy and helped them “tough through” the pain of SUD recovery. Inreferring to positive coping via running, one participant said,Most participants expressed how they reduced cravings to drugs via running.For example, one participant stated, “I had experienced the urge to usedrugs, but that urge went away while running.” Another participant describedhow the experience of running helps with recovery, “There is no drugs, noglass of wine that ever makes me feel like I feel every single time that Iget out and run with the program.” One participant acknowledged how runninghas transformed negative coping  into a positivecoping, “I don’t know what I am doing. My thinking is always negative. WhenI start to run, I know I can do something.”Participants noted another benefit by involving in running/walking is theimprovement of overall personal health such as managing weight, quittingsmoking, and eating healthier. One participant stated, “I weighed 350 poundswhen I came to the (treatment center). I’m 220 now.” Another participantagreed that, “It (the program) inspires me to quit smoking, eat healthier,and just kind of stay more fit. It is like general better life all over.”Several participants mentioned that the walking/running training programplayed an important role in helping them during their SUD recovery. As aparticipant shared that, “I will stay in the treatment longer if I get tocontinue to run or walk with a walking/running training program.” Runningmay have facilitated attempts to rebuild their lives during rehab andsustain a positive lifestyle during their recovery. Many participantsreported that they hoped to continue this  after thecompletion of the walking/running training program and to participate inother races.n = 13) stated that they gained asense of achievement and completed something they never imagined when crossingthe finish line. One participant in recovery said, “crossing the finish line isthe best accomplishment I have ever had.” Another participant noted, afterreceiving the medal at the race, “this was the first time in my life that I sawan entire process through from start to finish. I couldn’t have done it withoutthis team.”For many of those participants, achieving a goal (in the race) might be the firstsuccess they had experienced in a long time. In this study, all participants whostarted the race completed the race. In reflecting on the feelings of thisexperience, many completers . Yeah, it actually took me two months to settle in.But I had the urge to run. It wasn’t so much to the program. Itwas me trying to settle down in some place where I haven’t donein a long time.”Participants experienced that walking and running practice in the programhelped them find inner peace and feel settled down during recovery. Oneparticipant noted, “I chased drugs and alcohol because I was empty inside.It wasn’t until I started the program that I found purpose. When I’mrunning, I get an overwhelming sense of peace.” Another participantdescribed that,“everything has gotten in a circle. I have got everything Ineed when I came here. That is family and community. Iabsolutely love these people that the Lord has placed in my lifeat this time in this journey. I realized I need thesepeople.”Several participants reflected on how their dedication and faith in Godhelped them face the pain and fear. One participant was significantlyinvolved in drugs and just recently released from a local prison beforeparticipating in the walking/running training program. This participantexpressed his desire to build healthy relationships, be free from anxiety,and be able to renew confidence because of his “rededicated to God”. Anotherparticipant noted, before his first ever trial of a full walking/running inhis life, “God said, ‘you think you can do the half , Ithink you can do the full .’ I’m nervous, yes, but I’m notfearful. I know this is going to happen.” Another participant shared that,“today due to the help of my friends in the walking/runningtraining program. Man, look, I got to walk. I don’t know how farI have walked today. But I am blessed to have done it. I have somany friends here. You know, I can’t do it. But so many peoplehere always drive me through that nothing can take medown.”Encouragement and company received from volunteers have motivatedparticipants to stay in the training program. One participant lived under abridge, never changed his clothes, was shot twice, was hospitalized for ninedays by a snakebite, lost a toe to frostbite and had a heart attack. Henoted that,The support from volunteers in the walking/running training program is a bigpart of his hope. Another participant noted that, “I never thought I coulddo something like this. I feel so good. The volunteers here have given meeven more motivation to do what I need to succeed.”In the first week of boot camp, groups were doing leg stretching exercises.One participant remembered that she said, “I can’t do this.” A volunteer infront of her said, “Yes, you can.” From that moment, they formed a wonderfulfriendship as they trained together. Another participant shared that, “oneyear ago today, I was strung out on heroin, was homeless and felt worthless.This program has helped me because now I don’t need a drink or heroin. Thesevolunteers make me feel like I belong.” Another participant was addicted tomethamphetamine and felt about giving up before joining the walking/runningtraining program who shared how this program helped him heal, “I feel likemy life is coming together. . . . The walking/running training programvolunteers have stuck with me every step of the way. They just encourage meto keep going, and I love them like my own family.” Several participantswere not feeling well physically, but they battled on because therelationships they built with volunteers motivated them. One participantdescribed that, “my leg is killing me, but I don’t want to stop because Idon’t want to let the volunteers down.”“For a long time, I saw help as a sign of weakness. To havethe volunteers come beside me, cheer me on, and give meencouragement. . .embracing volunteer is a good thing. Itdoesn’t make me weak; it doesn’t make me seem I am afraid of anymatter. Having these people come beside me is definitely ablessing in my life.”Relationships established between participants and volunteers seems to be thekey factor to transforming lives. One participant shared that, “I want to beAthlete of the Week so bad. I know I can do it now. I didn’t think I couldat the beginning, but (the volunteer) believes in me. So, I know there mustbe something good in me!” Another participant described that,I came to (the treatment facility) to heal. Heal from poorchoices; heal from being rejected by my father when I was alittle boy, and the devastation me and my family went through;heal from hating myself and then hating others because of thelies I believed. This  isopening my eyes to my life and the words that are on our shirts,‘Let us run with endurance the race God has set before us.’ Ijust want to heal and for the pain to go away. Thank you to allthe volunteers that care about us to come out here in the coldand do this.”“Most participants in the program expressed how appreciative they were thatthose volunteers would show up on the practice for them no matter what kindof weather conditions. One participant stated,Participants perceived that through commitment, discipline, teamwork anddetermination, they had gained more hope in their race to rebuild theirlives.The purpose of this program was to explore participants’ experiences in thewalking/running training program during their recovery from SUD. Of the 109recruited to the program, 61 started as well as completed the race at the close ofthe program. Participants reported enhanced recovery experiences by participating inthe exercise program during the treatment. Specifically, results indicated thatparticipants experienced running/walking as a helpful coping mechanism duringrecovery, gained a sense of achievement by crossing the finish line, and developed asense of belonging in the program. Through involvement in walking or running, theprogram may cultivate a sense of hope for people recovering from SUD. Overall, thewalking and running training program seemed to be a promising adjunct treatment tohelp participants transform their lifestyles during SUD recovery.29 This study added to theliterature by describing participant experiences of an adjunct exercise program formanaging SUDs.The walking and running training program was designed to facilitate recovery throughparticipant adoption of healthy lifestyle-based coping strategies . Results of this study indicated thatparticipants showed positive recovery experience through exercise training.Researchers, for example Weinstock et al. and Stoutenberg et al, have argued thebenefits  ofengaging in exercise for clients during their recovery.17 Exercise as a stress relief strategy, a healthy coping mechanism, canfunction as a positive reinforcer that stimulates the brain’s reward pathway andmood-boosting neurochemicals. Exercise can also buffer against substance use byenhancing prefrontal cortex function.15 Results of this program are consistent with the findings of past research.30 For instance, Muller et al. surveyed 591 patients enrolled in the SUDtreatment facility. The study found that most respondents reported poor or very poorquality of life; among women, depression was strongly associated with poor and verypoor quality of life; among men, physical inactivity was associated with very poorquality of life, as was reporting eating most meals alone. This study concluded thatSUD treatment facilities should provide activities that improve physical health,mental health, and social support in the intervention.30 Positive and lasting relationships between participation of regular exerciseand various types of health benefits have been well chronicled. Results of theprogram support previous work suggesting that people with SUDs can receive physicaland psychological benefits from group exercise.14 It is critical to ensure improved access to such evidence-based adjuncttreatment services, and integrated with mainstream healthcare for those at risk foror affected by SUDs.Results of the program indicated the importance of incorporation of alternatedbehavior  to replace substance use in the cycle of relapse.Re-exposure to substance use associated cues, such as using substances when facingstressful situations, can cause even individuals with years of recovery to relapse.Perceived stress and feeling loss of control can also lead to relapse. Physicalactivity has been linked to reduced depression and improved tolerance of stress,which may in turn facilitate recovery from SUD.16 In this walking/running training program, participants selected a goal  for the race (self-efficacy), could be nominated for theathlete of the week and recognized before each practice for the efforts they madetoward reaching their goals ,and teamed up running with volunteers (modeling) throughout the training and duringthe race.31 The dynamic and reciprocal interaction of participants (person), exercise ascoping response (behavior), and exercise program context and volunteer support(environment) were utilized in hope to sustain participants’ motivation in thewalking and running practices.Attrition rate is a threat to the effectiveness of exercise adjuncts to treatment.High attrition rates usually prohibit participants from obtaining the benefit ofregular exercise involvement.32 outlines the power of choice in illness self-management. When the stimuli arepresented , the individual recognizes the stimuli and affective responses to thestimuli; then choose the coping responses. The coping responses determine theoutcomes. Considering the evidence that physical activity may positively impact themental and physical wellness among people with substance abuse problems, futurestudies should continue examining the effect of applying exercise as a copingresponse in substance use prevention and treatment. Future research could considerexamining other forms of low-impact physical activity  as anadjunct to SUD treatment.This program allows participants to choose the type of race they plan to complete.Then a training plan was designed to facilitate participants to reach their goal.Future programs could include components such as goal setting that allowsparticipants to establish their competence and confidence toward the goal. Forexample, encouraging participants to walk before running, or inviting participantsto run for two blocks instead of two miles in the beginning of the training. TheSelf-Regulation Model33 The findings indicated the importance of positive lifestyles , increased self-control , and enhanced social capital during recovery. Establishing recovery capital should be a focus during treatment,in hopes that clients adopt those resources or healthy lifestyles while returning to society.34 This also indicated adaptive continuing care for people who are recoveringfrom SUD is necessary.35 If participants can stay in the healthy environment provided by treatmentfacility and continue receiving appropriate treatment services, they might be morelikely to experience positive outcomes.2Recovery capital is comprised of four domains  of resources and relationships that could be utilized during recovery.36 Early substance use is a primary risk factor for SUD, future programs mightconsider including component of exercise in the prevention of substance useinitiation and examine the impact of exercise-based program on broader populations.Further research is required to strengthen these conclusions and to inform policyand healthcare systems.The cost expenses of substance use on the healthcare and crime-related costs are farabove the expenditure on prevention.Health professionals can encourage clients to engage in any physical activitiesthat clients might be interested in, or search physical activity based programor events at local area, in hopes of enabling clients to experience the benefits of exercise during their recovery. Following the physicalactivity engagement, support groups can be conducted to empower participants toadopt healthy and positive lifestyles that push them to avoid relapse. Althoughfurther research is needed to determine the therapeutic effects of usingexercise in SUD, it is evident that performing physical activity could improvemood and reduce cravings for drugs. Health professionals could also considerutilizing other forms of physical activity requiring little to no specificequipment  as an adjunct to SUD treatment.Findings of this study indicated that participants perceived positive benefits  to their overall health and recovery whileattending the walking/running program. The study evidenced the influence ofutilizing exercise as an adjunct treatment on SUD recovery via participants’perceptions and provided implications for SUD treatment services. More studies ofholistic interventions are needed in SUD studies to inform the treatmentpractices.Whether participants involved in exercise activities other than the scheduledpractices during the program implementation is unknown. Relatedly, participantssimultaneously receive services like counseling or medication while participating inthe walking and running training program. The data were also limited to participantswho continued attending the training; thus, the results could be biased. Severaladditional limitations include participant’s self-reported assessment, lack ofattendance records; the effects of timing of interviews could have on the interviewresponses, given that the data were collected during the course of the practice orwith an immediate follow up after the end of the race. Future research is needed todetermine whether behavior change was sustained over time.