*Review* **A Systematic Review and Meta-Analysis of Enzyme Replacement Therapy in Late-Onset Pompe Disease**

**Alícia Dorneles Dornelles 1,2, Ana Paula Pedroso Junges 2,3, Tiago Veiga Pereira 4,5, Bárbara Corrêa Krug 6, Candice Beatriz Treter Gonçalves 6, Juan Clinton Llerena, Jr. 7, Priya Sunil Kishnani 8, Haliton Alves de Oliveira, Jr. 9 and Ida Vanessa Doederlein Schwartz 1,2,3,6,10,\***

**Abstract:** Pompe disease (PD) is a glycogen storage disorder caused by deficient activity of acid alpha-glucosidase (GAA). We sought to review the latest available evidence on the safety and efficacy of recombinant human GAA enzyme replacement therapy (ERT) for late-onset PD (LOPD). Methods: We systematically searched the MEDLINE (via PubMed), Embase, and Cochrane databases for prospective clinical studies evaluating ERT for LOPD on pre-specified outcomes. A meta-analysis was also performed. Results: Of 1601 articles identified, 22 were included. Studies were heterogeneous and with very low certainty of evidence for most outcomes. The following outcomes showed improvements associated with GAA ERT, over a mean follow-up of 32.5 months: distance walked in the 6-min walking test (6MWT) (mean change 35.7 m (95% confidence interval [CI] 7.78, 63.75)), physical domain of the SF-36 quality of life (QOL) questionnaire (mean change 1.96 (95% CI 0.33, 3.59)), and time on ventilation (TOV) (mean change −2.64 h (95% CI −5.28, 0.00)). There were no differences between the pre- and post-ERT period for functional vital capacity (FVC), Walton and Gardner-Medwin Scale score, upper-limb strength, or total SF-36 QOL score. Adverse events (AEs) after ERT were mild in most cases. Conclusion: Considering the limitations imposed by the rarity of PD, our data suggest that GAA ERT improves 6MWT, physical QOL, and TOV in LOPD patients. ERT was safe in the studied population. PROSPERO register: 135102.

**Keywords:** glycogen storage disease type II; alpha-glucosidase; Pompe disease; enzyme replacement therapy

#### **1. Introduction**

Pompe disease (PD), or type II glycogenosis, is a rare genetic disease characterized by progressive neuromuscular involvement, often fatal in severe forms [1]. It is caused by deficient activity of acid alpha-glucosidase (also known as acid maltase), a lysosomal enzyme encoded by the *GAA* gene that breaks down glycogen into glucose [1]. This

**Citation:** Dornelles, A.D.; Junges, A.P.P.; Pereira, T.V.; Krug, B.C.; Gonçalves, C.B.T.; Llerena, J.C., Jr.; Kishnani, P.S.; de Oliveira, H.A., Jr.; Schwartz, I.V.D. A Systematic Review and Meta-Analysis of Enzyme Replacement Therapy in Late-Onset Pompe Disease. *J. Clin. Med.* **2021**, *10*, 4828. https://doi.org/10.3390/ jcm10214828

Academic Editors: Sylvia Lee-Huang, Karolina M. Stepien, Christian J. Hendriksz and Gregory M. Pastores

Received: 1 September 2021 Accepted: 12 October 2021 Published: 21 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

deficient activity, caused by biallelic pathogenic variants in *GAA*, leads to lysosomal glycogen accumulation in the skeletal and cardiac muscles, hindering cell function and ultimately destroying cells by hypertrophy and lysosome rupture [1–6].

Residual enzyme activity correlates positively with age at disease onset and inversely with the rate of disease progression, which allows PD to be classified according to the age of onset, cardiac involvement, and speed of progression. When clinical onset occurs before the age of 12 months and cardiomyopathy is present, it is known as infantile-onset PD (IOPD); all other forms are referred to as late-onset PD (LOPD) [1,7]. LOPD occurs on a spectrum; patients may present through the second year of life without cardiomyopathy, in childhood, adolescence, or at any point in adult life [1,7]. Although the overall severity of involvement is variable, life expectancy is generally shorter than in healthy individuals [1,2,8].

The overall incidence of PD worldwide is around 1 in 40,000 newborns (NBs). The incidence is higher in African Americans (1/12,000 NBs) and lower in Chinese (1/40,000 to 1/50,000 NBs) individuals [1–3]. After the inclusion of PD in the newborn screening programs of some countries, more reliable estimates of its incidence have emerged: 1/26,319 NBs in Illinois (USA) [9], 1/17,134 NBs in Pennsylvania (USA) [10], 1/10,152 NBs in Missouri (USA) [11], and 1/34,402 NBs in the Asian population of Japan [12].

There is no curative treatment for PD. Currently available treatment options are designed to address the mutant protein and consist of enzyme replacement therapy (ERT) with alglucosidase alfa (Myozyme™), a form of human acid alpha-glucosidase (GAA) produced by recombinant DNA technology in Chinese hamster ovary cells [2]. The recommended dosage regimen of alglucosidase alfa is 20 mg/kg body weight, administered every 2 weeks by intravenous (IV) infusion [8].

Two previous systematic reviews aimed to assess the effectiveness, safety, and appropriate dose regimen of enzyme replacement therapy (ERT) for treating LOPD; however, both have limitations in important domains. One, which included a meta-analysis, evaluated survival, vital capacity, and performance in the 6-min walking test (6MWT), reporting improvements in all outcomes [13]. The second, which did not perform a meta-analysis, showed improvement in the 6MWT but failed to include relevant prospective cohort studies [14]. Therefore, the impact of alglucosidase alfa treatment on key outcomes, such as quality of life (QOL) and time on ventilatory support (TOV), is still unclear. A further, significant knowledge gap that still remains is the ideal timing of ERT initiation. Within this context, the present systematic review with meta-analysis was designed to evaluate the effects of alglucosidase alfa ERT in LOPD.

#### **2. Methodology**

This study aimed to review the latest available evidence on the effects of alglucosidase alfa ERT in LOPD and its safety. To guide the literature search, a structured PICO question was formulated as follows: "Is the use of alglucosidase alfa as effective and safe as ERT in patients with PD?". The systematic review is reported as proposed by the PRISMA Guidelines [15] and has been registered in the PROSPERO database (123700).

#### *2.1. Information Sources and Search Strategy*

The MEDLINE (via PubMed), Embase, and Cochrane Central Register of Controlled Trials databases were searched for studies published before 30 May 2021. The search strategies are shown in Table 1; for the Cochrane Library, we used both strategies combined.

**Table 1.** Database search queries.

#### *2.2. Eligibility Criteria and Study Selection*

We planned to include only randomized clinical trials (RCT) and observational comparative studies in which ERT with alglucosidase alfa was used for the treatment of patients with LOPD. Other prospective study designs were included (open-label and nonrandomized trials, controlled or otherwise, including quasi-experimental designs) if the sample size was ≥5. In vitro studies or animal models, reviews, expert opinions, and retrospective studies were excluded. Unpublished work was covered by the identification of conference abstracts containing data deemed to be of interest. The final published articles were then included when available.

Studies that did not evaluate at least one of the eight outcomes of interest, defined a priori by a team of experts, were excluded. These outcomes were QOL, functional capacity (6MWT, forced vital capacity (FVC), and Walton and Gardner-Medwin Scale (WGMS) score), survival, TOV in hours/day, muscle strength, sleep quality, swallowing, and safety. For FVC, an increase of at least 10% after the intervention was considered a clinically relevant improvement [16,17]. For 6MWT, an increase of at least 26 m was considered a clinically significant change, as recommended by Schrover et al. for muscular diseases [18]. The WGMS is a scale that evaluates functional activity on a point system ranging from 0 = normal to 10 = bedridden.

The selection stage was performed independently by two investigators (APPJ, CG), who assessed the abstracts retrieved during the search for eligibility. Decisions were compared, and articles deemed relevant were forwarded to two other investigators (ADD, BK) who, independently, using standardized data collection forms, extracted information on the characteristics of these studies (design, randomization methods, population of participants, interventions, and outcomes). The two investigators then took part in a consensus meeting. Any disagreement that remained was addressed by the intervention of a third investigator (IVDS). Finally, the references of the selected articles were handsearched for potentially relevant studies not identified by the previous search strategies. When such information could not be retrieved, an email was sent to authors requesting non-reported data.

#### *2.3. Data Collection*

Studies with overlapping data were excluded from meta-analyses. In these cases, the study with the largest sample (or, if both studies had the same sample size, that with the longest follow-up) was retained for analysis.

#### *2.4. Statistical Analysis*

We summarized results using mean changes from baseline with 95% confidence intervals (CIs) for continuous outcomes. To incorporate follow-up time, we used incidence rates (IRs) with 95% CIs to summarize events. To facilitate interpretation, we standardized all IR estimates in events per 100 person-years. Study-specific mean changes were combined through an inverse-variance random-effects model with the restricted maximum-likelihood estimator (REML) of between-study variance (τ 2) for continuous variables. Events were combined with a generalized linear mixed model (GLMM) [19], in which a random intercept logistic regression model was fitted (log transformation) with a maximum-likelihood (ML) estimator. Sparse data were naturally taken into account in the GLMM, and no continuity corrections were used. The random-effects model was used for the primary analysis, but summary estimates obtained with a fixed-effects model (inverse-variance) were presented as a sensitivity analysis.

When not directly reported, mean change from baseline and standard error estimates were approximated based on reported statistics (95% CI, *p*-values, median, and interquartile range). We imputed standard deviations for baseline changes, assuming a correlation of 0.7 between baseline and follow-up scores. When only the median and interquartile range were informed, we used an approximate Bayesian computation (ABC) model to estimate means and standard deviations [20]. We employed clinically plausible ranges for the prior [~uniform (0,100)] distributions derived from studies reporting complete information and the opinion of specialists. Statistical heterogeneity was tested with Cochran's Q test and quantified with the I2 metric. Cochran's Q was considered statistically significant for heterogeneity if *p* < 0.10 [21]. No threshold for statistical significance was used for the evaluation of clinical variables. Analyses were performed with Stata (version 16, StataCorp, College Station, TX, USA) and R (version 3.2.3, R Core Team, The R Foundation, Vienna, Austria).

#### *2.5. Evaluation of the Quality of Included Studies*

The quality of included studies was evaluated with tools appropriate for the study designs: Risk of Bias tool (RoB) 2.0 for RCTs and Risk Of Bias In Non-randomized Studies of Interventions tool (ROBINS-I) for non-randomized studies of interventions (NRSI) [22,23]. Certainty of evidence of outcomes defined a priori was evaluated according to GRADE criteria [24–26]. Assessment of certainty of evidence for outcomes was performed independently by two investigators (ADD, HAOJ).

#### **3. Results**

The broad search strategy retrieved 1601 references (768 from MEDLINE, 833 from EMBASE, none from the Cochrane Library), of which 242 were duplicated. The titles and abstracts of 1359 references were read, and 33 publications were selected for full-text evaluation. Of these, 22 were selected for eligibility, and 11 were excluded. A flow diagram of evidence selection is shown in Figure 1. Ultimately, 22 studies were identified for LOPD, including one RCT.

**Figure 1.** PRISMA flow diagram of search results.

Studies that evaluated the outcomes defined a priori are described in Table 2. Our search did not retrieve any articles evaluating sleep quality, survival, or swallowing disorder that matched the inclusion criteria; therefore, these outcomes could not be evaluated.

**Table 2.** Outcomes of interest defined a priori and studies that met the inclusion criteria.

FVC = forced vital capacity. 6MWT = 6-min walking test. WGMS = Walton and Gardner-Medwin Scale.

#### *3.1. Characteristics of Included Studies*

All included studies and their characteristics are described in Table 3. Only one double-blind RCT was identified and included [36].

**Table 3.** Included studies and their characteristics.

**Table 3.** *Cont.*

NRSI = non-randomized study of interventions. N/A = not available. LOTS = late-onset treatment study. All studies used alglucosidase alfa IV 20 mg/kg/biweekly, except those marked with \*, in which dosage was not specified.

#### *3.2. Assessment of Functional Capacity*

#### 3.2.1. Forced Vital Capacity

Based on data from 15 studies (participants = 348; mean follow-up = 36.8 mo; Table 2 [13,27–40]), there was no evidence of improvement in FVC during the performance of spirometry in the sitting, supine, or orthostatic positions (within-group mean change: 0.41% (95% CI: −0.3 to 1.12%)), as shown in Figure 2 and Supplementary Table S1. There was low heterogeneity between studies and very low certainty of evidence (Supplementary Table S2).

#### 3.2.2. Six-Minute Walking Test

Performance on the 6MWT was evaluated in 14 studies before and after ERT (participants = 348; mean follow-up = 36.8 mo), as shown in Table 2 [13,27–31,34–39,41,42] and Supplementary Table S3. Van der Ploeg et al. (2010) [36] included the same population as van der Ploeg et al. (2012) [29] and was thus excluded; Kuperus et al. [39] measured the outcome as median and, therefore, was excluded from meta-analysis as well. Despite the considerable heterogeneity between studies and very low certainty of evidence (Supplementary Table S4), there was evidence of clinically significant improvement after treatment (within-group mean change: 35.7 m (95% CI: 7.78 to 63.75); Figure 3).

**Figure 2.** Evaluation of forced vital capacity (% of predicted) in upright position in patients with late-onset Pompe disease

on enzyme replacement therapy with alglucosidase alfa. Weights are inverse-variance weights and are proportional to the contribution of each study to the summary estimate. I2 is the fraction of variance that is due to statistical heterogeneity and not chance. Tau2 denotes the between-study variance.

**Figure 3.** Evaluation of 6-min walking test performance (distance walked in meters) in patients with late-onset Pompe disease on enzyme replacement therapy with alglucosidase alfa. Weights are inverse-variance weights and are proportional to the contribution of each study to the summary estimate. I2 is the fraction of variance that is due to statistical heterogeneity and not chance. Tau2 denotes the between-study variance.

#### 3.2.3. Walton and Gardner-Medwin Scale (WGMS)

Six studies evaluated WGMS scores, as shown in Table 2 [13,27,30,31,40,43] (results shown in Supplementary Table S5). The data suggest that ERT had no effect on the WGMS score in any of the included studies. Certainty of evidence was very low (Supplementary Table S6), mainly due to the imprecision of the results of the included studies.

#### *3.3. Upper-Limb Strength*

Nine studies evaluated strength in the upper limbs, but did so very heterogeneously (Supplementary Table S7). Although several studies carried out strength assessment according to the Medical Research Council (MRC) scale [13,30,33,46], they reported different methods of calculating it, evaluated different muscle groups, and some did not evaluate upper and lower limbs separately, limiting the comparability of this outcome. Thereby, muscle strength was evaluated through a meta-analysis in relation to two variables: handheld dynamometry and the Quick Motor Function Test. Handheld dynamometry was evaluated in 2 of 9 studies, without significant differences (mean change 244.05 (95% CI −151.18, 639.27)) (Figure 4), with considerable heterogeneity between studies; the Quick Motor Function Test was evaluated in 3 of 9 studies, without a significant difference (mean change 7.85 (95% CI −2.48, 18.18)) (Figure 5), with substantial heterogeneity between studies and very low certainty of evidence (Supplementary Table S8).

**Figure 4.** Evaluation of handheld dynamometry in patients with late-onset Pompe disease on enzyme replacement therapy with alglucosidase alfa. Weights are inverse-variance weights and are proportional to the contribution of each study to the summary estimate. I2 is the fraction of variance that is due to statistical heterogeneity and not chance. Tau2 denotes the between-study variance.

**Figure 5.** Evaluation of the Quick Motor Function Test in patients with late-onset Pompe disease on enzyme replacement therapy with alglucosidase alfa. Weights are inverse-variance weights and are proportional to the contribution of each study to the summary estimate. I2 is the fraction of variance that is due to statistical heterogeneity and not chance. Tau2 denotes the between-study variance.

#### *3.4. Quality of Life*

Six studies evaluated this outcome; the meta-analysis of their results is shown in Figure 6 and detailed in Supplementary Table S9. The instrument most commonly used to assess QOL was the Medical Outcome Study 36-item Short Form Health Survey, or SF-36 questionnaire [13,30,36,43,47]. The SF-36 is a generic instrument that has been widely used, has been translated into many languages, and has been shown to have good reliability and validity. Strothotte et al. [13] was excluded from the meta-analysis due to incomplete data available, and van der Ploeg et al. (2016) was excluded due to the use of the Pediatric Quality of Life Inventory™ (PedsQL) [28].

There were no differences in overall QOL (mean change 7.05 (95% CI −7.30, 21.41)) or in the mental component of the SF-36 (mean change 5.37 (95% CI −4.04, 14.78)), with considerable heterogeneity. However, there was a difference in the physical component (mean change 1.96 (95% CI 0.33, 3.59)) (Figure 6), with substantial heterogeneity and very low certainty of evidence (Supplementary Table S10).

**Figure 6.** Evaluation of the quality of life of patients with late-onset Pompe disease on enzyme replacement therapy with alglucosidase alfa. Weights are inverse-variance weights and are proportional to the contribution of each study to the summary estimate. I2 is the fraction of variance that is due to statistical heterogeneity and not chance. Tau2 denotes the between-study variance.

#### *3.5. Time on Ventilation*

Six studies evaluated this outcome in LOPD; a synthesis of their results is shown in Figure 7 and Supplementary Table S11. Two studies [30,33] were not included in the meta-analysis due to a lack of data. There was weak evidence indicating that ERT, on average, is associated with a positive effect on TOV, despite substantial heterogeneity between studies and low certainty of evidence (Supplementary Table S12) (mean change −2.64 h (95% CI −5.28, 0.00)).

*3.6. Safety*

#### 3.6.1. Adverse Events

Several studies have assessed the safety profile of ERT concerning the presence of adverse events (AEs) and infusion-associated reactions (IARs), as shown in Table 4 and Supplementary Table S13 [13,28–31,33,34,36,39,40,42–45]. The reported AEs are tachycardia, desaturation, malaise, chills, facial erythema, erythema at the enzyme infusion site, urticarial reactions, hyperhidrosis, chest discomfort, vomiting, systemic arterial hypertension, flu-like symptoms, pruritus, bronchospasm, and hyperthermia. Certainty of evidence is presented in Supplementary Table S14.

**Figure 7.** Evaluation of the time on ventilation (h) of patients with late-onset Pompe disease on enzyme replacement therapy with alglucosidase alfa. Weights are inverse-variance weights and are proportional to the contribution of each study to the summary estimate. I2 is the fraction of variance that is due to statistical heterogeneity and not chance. Tau2 denotes the between-study variance.

**Table 4.** Summary estimates for incidence of safety outcomes of enzyme replacement therapy for patients with late-onset Pompe disease.

AE = adverse event. SAE = serious adverse event. IAR = infusion-associated reaction. AB+ = presence of anti-alglucosidase alfa antibodies. PQ denotes the *p*-value for Cochran's Q test. I2 is the fraction of variance that is due to statistical heterogeneity and not chance. 95% CI = 95% confidence interval. IR = incidence rate (expressed in number of events per 100 person-years).

> In the study by van der Ploeg et al. (2016) [28], there was only one severe AE (not specified), unrelated to treatment. Mild and moderate AEs occurred in 35.5% of patients, with 25% experiencing IARs. The incidence of IARs reported by de Vries et al. (2017) [45] was 18% (13/73 patients), the most common being malaise, chills, and hyperthermia. In another study by the same author [33], 12/69 patients (17%) developed an infusion reaction; however, only three patients remained symptomatic after administration of antihistamines and corticosteroids.

> Angelini et al. (2012) [31] reported the following adverse reactions to ERT, occurring in 4/74 patients (6%) and considered of moderate intensity: facial erythema, erythema at the infusion site, flu-like symptoms, generalized pruritus, and bronchospasm (also described by Bembi et al. [42]). The symptoms were controlled with antihistamines. In another study by Orlikowski et al. with five patients, 58 mild to moderate AEs were described after starting treatment, including erythema and hyperthermia.

> In the LOTS RCT, the ERT group and the placebo group had similar frequencies of severe AEs, treatment-related events, and infusion reactions (Supplementary Table S13). The treatment group had a higher frequency of mild to moderate AEs, which did not prevent the continuation of treatment. Urticariform reactions (also described by Bembi et al. [42]), hyperhidrosis, chest discomfort, flushing, vomiting, and increased blood pressure occurred in up to 8% of patients treated with alglucosidase alfa, and were not reported in the placebo

group. These findings were corroborated by an extension study with the same population (LOTS Extension) [29]. In addition, Forsha et al. [44], using data obtained in the LOTS study, evaluated only the safety of ERT regarding cardiovascular events that occurred after the initiation of alglucosidase alfa, and found no significant difference between the treatment and placebo groups in change in ejection fraction (*p* = 0.8), PR interval (*p* = 0.71), ventricular mass (*p* = 0.71), or QRS duration (*p* = 0.67).

In the study by Kuperus et al. [39], 19 patients (22%) had at least one AE, all of which were controlled by a reduced infusion rate or premedication (antihistamines or corticosteroids). ERT was discontinued in four patients, but in only one for safety reasons (a patient with a history of autoimmune diseases and drug allergies before treatment developed multiple IARs). AEs have also been described in the study by Strothotte et al. [13], not included in meta-analysis due to incomplete data, and by Regnery et al. [30]: erythema, tachycardia, desaturation, rash, and pruritus, with no deaths occurring during the 12 and 36 months, respectively, of these studies. In the study by Vianello et al. [40], no AE has been described. In the study by van Capelle et al. [34], five patients were treated with ERT for 3 years and no AEs were observed in any of the 390 total intravenous infusions.

#### 3.6.2. Mortality

Data on mortality were described in nine studies (Table 4 and Supplementary Table S15); however, only five described the occurrence of the event [31,33,36,39,43]. In the study conducted by Orlikowski et al. [43] with five patients, there was one death due to tracheal hemorrhage not related to treatment. In the study with the highest overall mortality rate, Kuperus et al. [39], 1/19 patients died of respiratory failure at 56 years of age, 1.1 years after discontinuing ERT for personal reasons. Another 6/19 patients on ERT died, though no deaths were considered treatment-related. The incidence rate of death (events per person-year) across all included studies was 4.4 events per 1000 person-years (95% CI 1.5 to 12.8).

#### 3.6.3. Anti-Alglucosidase Alfa Antibodies

Data on anti-alglucosidase alfa antibody (Ab) titers for patients receiving ERT are shown in Table 5. Although most patients presented with elevated Ab titers, few showed a reduction in response to treatment or higher incidence of AEs. The most in-depth evaluation of Ab titers for patients receiving ERT was by de Vries et al. (2017) [45], and this study will therefore be described in greater detail. The patients were divided into three groups, according to their respective Ab titers: the first, of 16 patients, corresponded to high titers (>1: 31,250); the second, of 29 patients, corresponded to moderate titers (1: 1250 to <1: 31,250); the third, of 28 patients, to low titers (0 to <1: 1250). Three patterns of progression were observed concerning anti-ERT Ab titers; in the vast majority of patients (97%), titers either decreased or remained stable after 12 months of treatment, except in two patients, one of whom belonged to the group with the highest titers and the other to intermediate titers. Using the combined score of the MRC scale and the standing FVC, the authors compared treatment responses between the three Ab titer groups and found no significant differences between them, whether at baseline or after 3 years of treatment (*p* = 0.35 and *p* = 0.38, respectively). In one patient with high titers, the Ab had neutralizing effects on the enzyme, with a decline in FVC and strength. De Vries et al. (2017) [45] concluded, therefore, that there was a relationship between anti-alglucosidase alfa Ab titers and the development of AEs (statistical analysis not shown). Only 1/28 (4%) patients in the low-titer group had AEs, while 5/29 (17%) in the intermediate-titer group and 7/16 (44%) in the high-titer group experienced them.

**Table 5.** Antibody titers for patients with late-onset Pompe disease receiving enzyme replacement therapy, reduction in response to treatment, and incidence of AEs.

AE = adverse event. Ab = anti-alglucosidase alfa antibodies (measured by different methods). N/A = not available. N/E = not evaluated.

#### *3.7. Risk of Bias and Quality of Included Studies*

The risk of bias of the included NRSI is shown in Figure 8. The RCT included had some concerning issues, such as an imbalance in age at baseline and possible conflict of interest of the investigators. Most included articles showed a moderate to severe risk of bias, independently of the study design; only one showed a critical risk of bias [46].

#### *3.8. Certainty of Evidence by Outcomes*

All outcomes were assessed for certainty of evidence, with low certainty only for TOV. All other outcomes had very low certainty of evidence, mainly due to the uncontrolled observational design of the included studies, with data from secondary outcomes. A full analysis is available in the Supplementary Materials.

**Figure 8.** Risk of bias of included studies evaluated through the ROBIN-I tool.

#### **4. Discussion**

LOPD is a rare, serious disease with no specific treatment available other than ERT. Interpretation of the available evidence must always consider these facts. Given that only one double-blind RCT of GAA ERT for LOPD has been conducted [36], prospective observational trials were also evaluated in this review. The included studies all had small sample sizes (which is to be expected given the rarity of LOPD), as well as different ages (children, adolescents, and adults), stages, durations of disease burden prior to start of ERT, and phenotypic manifestations. Among the outcomes evaluated for LOPD, we showed a benefit for 6MWT, as also demonstrated in a previous meta-analysis [50]. Otherwise, this was the first meta-analysis to evaluate the effect of ERT on TOV and QOL. An improvement in functional capacity, measured through FVC, was not confirmed.

Two systematic reviews have already been published on the efficacy of alglucosidase alfa, neither of which assessed safety directly [14,50]: the first one in 2013, by Toscano et al. [14], and the second one in 2017, by Schoser et al. [50]. The latter included 22 papers (of which 10 were included in the present review), comprising case series and retrospective studies, with or without a control group, and evaluated 6MWT, FVC, and mortality by conducting a meta-analysis. The average age of patients included in the review was 46 years. [50] Toscano et al. included 21 papers (of which 14 were included in

the present review), also comprising case series and retrospective studies, with or without a control group. They evaluated, among other outcomes, 6MWT, FVC, the need for ventilatory support, and QOL, with most patients aged between 40 and 59 years (44%); any improvement was considered meaningful, without defined criteria for clinical relevance [14]. Our review included 22 papers only with prospective data, excluding case series, and the mean age of the included participants was 42.8 years (range, 7 to 76.3 years), with 32.5 months of follow-up.

The systematic review carried out by Toscano et al. [14] evaluated FVC for 124 treated patients and identified an improvement in 51.6%, stable disease in 13.7%, and decline in 34.7%. There was no correlation between the duration of treatment and improvement in lung capacity, with no description of comparison with a control group. Likewise, the meta-analysis carried out by Schoser et al. [50] demonstrated a beneficial effect of ERT on FVC in groups receiving alglucosidase alfa; their conclusion was based on an analysis using a fixed-effect model, which is not considered the best method of evaluating studies with heterogeneous populations. The results showed that untreated patients had a 2.3% decline in FVC% after 12 months and 6.2% after 4 years; treated patients had an initial increase in FVC% of 1.4% after 2 months, with a return to baseline FVC% and a slight decline in follow-up, with data not detailed. The difference in efficacy between control (a historical cohort without treatment) and treated patients varied from 4.5% after 1 year to 6% at 4 years, and therefore cannot be considered clinically significant. Our data, retrieved from 348 patients, do not confirm the findings of Schoser et al., suggesting that there is little effect of ERT on FVC.

Our results confirm a previously published meta-analysis concerning the effect of ERT on 6MWT (mean change 36.6 m (95% CI 10.72, 62.48)). Schoser et al. [50] demonstrated a beneficial effect of ERT on 6MWT in the groups receiving alglucosidase alfa at 12 months, with an average improvement 43 m greater than in controls, based on data from 171 patients undergoing treatment in comparison to a historical cohort without treatment. Toscano et al. [14] also included the 6MWT among their outcomes of interest and reported data from 122 patients, of which 77.9% improved, 8.2% stabilized, and 13.9% worsened. We found an increase of 36 m in this outcome, above the 26 m cutoff considered clinically relevant, and the improvement in walking distance described in the included articles ranged from 33 to 1000 m. The inclusion of the results from Bembi et al. (2010) [42] for this outcome highlights the issue of early intervention, as the greatest benefit of intervention was seen in the young group, which is of fundamental importance for the analysis of the available evidence, since additional benefits or a greater effect size may not have been found in other studies due to the inclusion of patients with established disease and very heterogeneous age at initiation of treatment.

Our study also indicates a beneficial effect of ERT on the physical component of QOL (mean change 1.96 (95% CI 0.33, 3.59)), and this improvement in endurance has positive aspects in making patients less dependent on caregivers. However, it bears stressing that generic QOL instruments were used, as there is no specific validated instrument for assessing the QOL of patients with PD. One systematic review without meta-analysis also evaluated QOL [14], and all included studies in this systematic review with *n* ≥ 5 were included in our search. In addition, 9 of the 21 included articles evaluated QOL, with 156 patients evaluated for this outcome using the SF-36 questionnaire. Qualitative synthesis of these studies showed that only 13/156 patients (8.3%) improved their QOL scores after treatment.

This is the first meta-analysis to show a trend toward a beneficial effect of ERT on TOV (mean change −2.64 h (95% CI −5.28, 0.00)), although previous systematic reviews have evaluated this outcome in different ways. In Schoser et al. [50], the percentage of patients dependent on ventilation varied between 14 and 100%, which is considered very high, due to the early age of the sample. They also reported that the number of patients requiring ventilation was maintained in patients treated with ERT, compared to a historical cohort that presented an increased proportion of patients using ventilation (data not provided by Schoser et al.)—with great heterogeneity between studies, however. Toscano et al. [14] evaluated data from 66 patients and demonstrated that ERT resulted in an improvement in the need for MV in 59.1% of patients, stabilization in 36.4% of patients, and worsening in only 4.5% of patients. Regarding the need for non-invasive ventilation, 64.1% improved, 32.1% stabilized, and 3.8% worsened (data not detailed). In our study, 265 patients were on any type of ventilatory support.

Concerning mortality, our study showed an incidence rate of 4.4/1000 events per person-year of patients with LOPD treated with ERT, which is considered very low. A previous systematic review with meta-analysis [50] was able to estimate that patients on ERT had a lower mortality rate compared to those not treated, using mortality data from included studies. It is important to note that the results of this study should be interpreted with caution, as the methodological steps followed by authors were not clearly mentioned.

Schoser et al. included six observational studies [31,32,43,46,51,52], of which only one [51] presented comparative data between a group on alglucosidase alfa ERT and untreated controls. According to this meta-analysis, the summary measure showed a 79% reduction in risk of death for those patients on alglucosidase alfa (HR = 0.21 (95% CI 0.11; 0.41)). However, it should be noted that the other studies, which were noncomparative case series, might have led to an overestimation of the effect. Although this study corrected the meta-analysis by covariables (meta-regression adjusted for age, sex, and severity of PD) and interpreted the sources of heterogeneity, the review had a serious risk of bias, according to the AMSTAR-2 tool. Gungor et al. (2013) [51] showed a 59% reduced risk of death for those patients on alglucosidase alfa compared to those who did not receive the therapy (HR = 0.41 (95% CI 0.19; 0.87)). This analysis was adjusted for age, sex, country of residence, and severity of PD. The other studies were case series, with no comparator group, reported a small number of deaths, and did not discuss whether these were related to treatment (absolute frequency in the five studies: 3 deaths among 151 patients). Gungor et al. (2013) [51] was the only study with a comparator group and cohort adjusted for potential confounders.

Although treatment-emergent or infusion-related AEs are common in ERT recipients, in most cases, these are mild and easily treatable. The development of IgG antibodies to alglucosidase alfa is also frequent; however, it does not seem to be related to the presence or absence of AEs or to the effect of treatment, although the measures used may not be sensitive enough to identify these. In IOPD, where the outcome is clearer, there is a direct correlation between patients with highly sustained and sustained intermediate Ab titers and clinical outcome. Therefore, more studies in LOPD are needed evaluating outcome measures that have the ability to capture small changes caused by ERT on them [53]. A systematic review conducted by Toscano et al. [14] described AEs as mostly mild to moderate in 303 patients undergoing treatment, with severe AEs reported in only four patients, all in studies included in the present article. They also reported the development of antibodies in 128 patients, three of whom had an anaphylactic reaction. It bears stressing that the evidence for safety is low, but although these studies have already reached a substantial length of follow-up, ERT is generally understood to be safe. No studies have demonstrated, for example, the effectiveness of physical therapy as the sole treatment strategy for these patients.

The included studies were highly heterogeneous, enrolling patients from different age groups and with different disease severities. Therefore, the findings of this review and meta-analysis should be interpreted with caution. In addition, most included studies were non-randomized studies of interventions, with low methodological quality, no comparison group, or comparison with a historical cohort. Another challenge is incompletely reported data, as publication bias was present. Usually, data from secondary outcomes were poorly reported, as studies were not designed to measure them. In most cases, however, conclusions were made without showing full data and results. Proper reporting of data is essential. One strength of the present study was that we included only prospective trials, in an attempt to avoid memory and selection bias. Prospective trials also have the advantage

of collecting data accordingly with the predefined outcomes of interest, which does not happen in retrospective trials.

In conclusion, alglucosidase alfa effectively increases the distance achieved in 6MWT, improves the physical component of QOL, and may decrease TOV in treated patients. Stabilization of functional capacity, measured through FVC, was not confirmed. The treatment is safe in the studied population, with generally mild adverse events. Further studies could evaluate the impact of the duration of follow-up in the included studies, taking into consideration that the efficacy of ERT may present some secondary decline after 3 to 5 years of treatment [54]. Moreover, the age at ERT initiation is an important aspect that should be addressed in future reviews.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2077-0 383/10/21/4828/s1, Table S1: Evaluation of the effect of enzyme replacement therapy on forced vital capacity in late-onset Pompe disease; Table S2: GRADEpro of forced vital capacity outcome; Table S3: Evaluation of the effect of enzyme replacement therapy on 6-min walking test in lateonset Pompe disease; Table S4: GRADEpro of 6-min walking test outcome; Table S5: Evaluation of the effect of enzyme replacement therapy on Walton & Gardner-Medwin scale scores in late-onset Pompe disease; Table S6: GRADEpro of Walton & Gardner-Medwin Scale outcome; Table S7: Evaluation of the effect of enzyme replacement therapy on upper limb strength in late-onset Pompe disease; Table S8: GRADEpro of upper limb strength outcome (assessed with QMFT and HHD); Table S9: Evaluation of the effect of enzyme replacement therapy on quality of life in late-onset Pompe disease; Table S10: GRADEpro of quality of life outcome (assessed with SF-36); Table S11: Evaluation of the effect of enzyme replacement therapy on time on ventilation in late-onset Pompe disease; Table S12: GRADEpro of time on ventilation; Table S13. Safety assessment of enzyme replacement therapy for patients with late-onset Pompe disease; Table S14: GRADEpro of safety outcomes; Table S15: GRADEpro of mortality.

**Author Contributions:** Conceptualization, A.D.D. and I.V.D.S.; Methodology, A.D.D., T.V.P., H.A.d.O.J. and I.V.D.S.; Software, T.V.P.; Formal Analysis, T.V.P.; Selection stage, A.P.P.J. and C.B.T.G.; Data extraction, A.D.D. and B.C.K.; Writing—Original Draft Preparation, A.D.D.; Writing—Review and Editing, A.P.P.J., P.S.K., J.C.L.J. and H.A.d.O.J.; Supervision, H.A.d.O.J.; Project Administration, I.V.D.S.; Funding Acquisition, I.V.D.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Hospital de Clínicas de Porto Alegre Event Incentive Fund (FIPE/HCPA), grant number (protocol code) 2019-0495, by the Pro-BIC HCPA/CNPq Undergraduate Research Program, by Casa dos Raros, Center for Comprehensive Care and Training in Rare Diseases, and by Casa Hunter. The funding body had no role in the design of the study, in the collection, analysis, and interpretation of data, or in writing the manuscript. T.V.P. is funded by the Chevening Scholarship Programme (Foreign and Commonwealth Office, UK).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Hospital de Clínicas de Porto Alegre (protocol code 2019-0495, date of approval 11 February 2020) and Plataforma Brasil (protocol code<PHONE_NUMBER>0005327, date of approval 11 February 2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets analyzed during the current study are available from the corresponding author on reasonable request.

**Conflicts of Interest:** P.S.K. has received research/grant support from Sanofi Genzyme, Valerion Therapeutics, Shire Pharmaceuticals, and Amicus Therapeutics. P.S.K. has received consulting fees and honoraria from Sanofi Genzyme, Shire Pharmaceuticals, Amicus Therapeutics, Vertex Pharmaceuticals, Maze Therapeutics, JCR Pharmaceutical and Asklepios BioPharmaceuticalBiopharmaceutical, Inc. (AskBio). P.S.K. is a member of the Pompe and Gaucher Disease Registry Advisory Board for Sanofi Genzyme., Amicus Therapeutics, and Baebies. P.S.K. has equity in Asklepios Biopharmaceutical, Inc. (AskBio), which is developing gene therapy for Pompe disease and Maze Therapeutics, which is developing small molecule in Pompe disease. All other authors declare that they have no competing interests.

#### **Abbreviations**

#### **References**

54. Harlaar, L.; Hogrel, J.Y.; Perniconi, B.; Kruijshaar, M.E.; Rizopoulos, D.; Taouagh, N.; Canal, A.; Brusse, E.; van Doorn, P.A.; van der Ploeg, A.T.; et al. Large variation in effects during 10 years of enzyme therapy in adults with Pompe disease. *Neurology* **2019**, *93*, e1756–e1767. [CrossRef]


*Article* **Natural Rubber-TiO2 Nanocomposite Film for Triboelectric Nanogenerator Application**

**Weeraya Bunriw 1, Viyada Harnchana 2,3,\* , Chalathorn Chanthad 4 and Van Ngoc Huynh 5**

	- 2800 Kongens Lyngby, Denmark<EMAIL_ADDRESS>
**Abstract:** In this research, natural rubber (NR)-TiO2 nanocomposites were developed for triboelectric nanogenerator (TENG) application to harvest mechanical energy into electrical energy. Rutile TiO2 nanoparticles were used as fillers in NR material to improve dielectric properties so as to enhance the energy conversion performance of the NR composite TENG. The effect of filler concentration on TENG performance of the NR-TiO2 composites was investigated. In addition, ball-milling method was employed to reduce the agglomeration of TiO2 nanoparticles in order to improve their dispersion in the NR film. It was found that the TENG performance was significantly enhanced due to the increased dielectric constant of the NR-TiO2 composite films fabricated from the ball-milled TiO2. The TENG, fabricated from the NR-TiO2 composite using 24 h ball-milled TiO2 at 0.5%wt, delivered the highest power density of 237 mW/m2, which was almost four times higher than that of pristine NR TENG. Furthermore, the applications of the fabricated NR-TiO2 TENG as a power source to operate portable electronics devices were also demonstrated.

**Keywords:** natural rubber; triboelectric nanogenerator; TiO2 nanoparticles; dielectric constant


**Author details**

Blaženka Kljaić Bukvić 1,2,3\*, Mario Blekić 1,2,3 and Marija Pečnjak1

1 Pediatric Department, General Hospital "Dr. Josip Benčević", Croatia

2 School of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia

3 Faculty of Dental Medicine and Health Care, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**11**

*Allergic March*

**References**

*DOI: http://dx.doi.org/10.5772/intechopen.85553*

[9] Paternoster L et al. Identification of atopic dermatitis subgroups in children from 2 longitudinal birth cohorts. The Journal of Allergy and Clinical Immunology. 2018;**141**(3):964-971

[10] Barker JN et al. Null mutations in the filaggrin gene (FLG) determine major susceptibility to early-onset atopic dermatitis that persists into adulthood. The Journal of Investigative Dermatology. 2007;**127**(3):564-567.

[11] Margolis DJ et al. Thymic stromal lymphopoietin variation, filaggrin loss of function, and the persistence of atopic dermatitis. JAMA Dermatology. 2014;**150**(3):254-259. DOI: 10.1001/

[12] Ashley SE et al. The skin barrier function gene SPINK5 is associated with challenge-proven IgE-mediated food allergy in infants. Allergy. 2017;**72**(9):1356-1364. DOI: 10.1111/

[13] Marenholz I, Esparza-Gordillo J, Lee YA. The genetics of the skin barrier in eczema and other allergic disorders. Current Opinion in Allergy and Clinical Immunology. 2015;**15**(5):426-434. DOI: 10.1097/aci.0000000000000194

[14] Cianferoni A, Spergel J. The importance of TSLP in allergic disease and its role as a potential therapeutic target. Expert Review of Clinical Immunology. 2014;**10**(11):1463-1474. DOI: 10.1586/1744666x.2014.967684

[15] Czarnowicki T, Krueger JG, Guttman-Yassky E. Skin barrier and immune dysregulation in atopic dermatitis: An evolving story with important clinical implications. The Journal of Allergy and Clinical Immunology. In Practice. 2014;**2**(4): 371-379. Quiz 380-1. DOI: 10.1016/j.

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[2] Nwaru BI et al. Prevalence of common food allergies in Europe: A systematic review and meta-analysis.

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[3] Prescott S, Allen KJ. Food allergy: Riding the second wave of the allergy epidemic. Pediatric Allergy and Immunology. 2011;**22**(2):155-160

[4] van der Hulst AE, Klip H, Brand PL. Risk of developing asthma in young children with atopic eczema: A systematic review. The Journal of Allergy and Clinical Immunology.

[5] Thomsen SF. Epidemiology and natural history of atopic diseases. European Clinical Respiratory Journal. 2015;**2**. DOI: 10.3402/ecrj.v2.24642

[6] Belgrave DC et al. Atopic dermatitis and respiratory allergy: What is the link. Current Dermatology Reports. 2015;**4**(4):221-227. DOI: 10.1007/

[7] Goksor E et al. The allergic march comprises the coexistence of related patterns of allergic disease not just the progressive development of one disease. Acta Paediatrica. 2016;**105**(12):1472-

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### *Allergic March DOI: http://dx.doi.org/10.5772/intechopen.85553*


**3. Case Study**

The stripper of the TEP simulator [22] is used as an example to test the feasibility of the proposed hybrid inverse problem approach to fault diagnosis. The fault set related to this stripper includes two types, that is, a step and a random type. This work chooses fault 7 and 8 as typical examples for these two types. The advantages and disadvantages of this approach are illustrated in comparison with the base approach with LSQ only.

#### *3.1. Solving the LSQ Inverse Problem with Di*ff*erent Initial Values*

LSQ is greatly affected by the initial values of iterated variables, so the first step is to discuss different setting methods for initial values of the LSQ algorithm. The following test is firstly based on the fault diagnosis process under fault 7. Fault 7 occurs at 8 h when the header pressure loss of the stripping stream entering the stripper bottom decreases abruptly. The fault diagnosis result using LSQ only is given in Figures 3 and 4, which has been published in our early work [16], henceforth referred to as the base case for comparison. In this base case, the overall running time and quantity of function evaluations (QFE) are 539 seconds and 1826, respectively. In the fault diagnosis algorithm, the boundary state at *t* = 0 is considered a normal state, this is, without anomaly. Therefore, the pressure loss coefficient is set as 1 uniformly in Figure 3. When *t* > 0, the fault diagnosis algorithm is activated to obtain the pressure loss coefficient in a timely manner based on the measurements of a plant.

**Figure 3.** Fault parameter in the base case of fault 7.

**Figure 4.** Function evaluation in the base case of fault 7.

(1) Set the initial value with the previous time point

The simplest value-setting method for the initial value is to directly use one from a previous time point. If there are only small changes in the fault parameter between two consecutive time points, the initial value given can thus be accepted. This is also the initial value-setting method adopted by the base case.

(2) Set the initial value with the linear fitting method

Figure 5 shows the diagnosis result with initial values given by the linear fitting method. It indicates that QFE decreases greatly and becomes stochastically stable before the fitted point number equals 30. After that, a rising function evaluation number curve is seen because of the growing time lag of the fitted line. For this reason, 5 or 8 is chosen as the candidate for the optimal fitting point number. Because more fitting points will definitely lead to a heavier computational load for the fitting operation, 5 is finally chosen as the optimal fitting point number. Despite this, QFE is only cut down by 2%, contributing little to the computational efficiency of the diagnosis algorithm.

**Figure 5.** Fault diagnosis of fault 7 with linear fitted initial values.

(3) Set the initial value with the parabolic fitting method

Following the linear fitting method presented above, the parabolic fitting method, the simplest nonlinear fitting method, is utilized here to predict the initial values. Figure 6 shows the diagnosis result with this method, revealing a larger QFE than the base case. This reflects the essentially linear change of the fault parameter between neighboring time points. Therefore, the nonlinear fitting method does not achieve the satisfactory goal of reducing QFE.

**Figure 6.** Fault diagnosis of fault 7 with parabola fitted initial values.

#### (4) Set the initial value with the grey model

Different from the above regression methods, grey system theory uses partial description information of a system to generate a grey sequence revealing the potential rule of data with the goal of system whitening [23]. It has advantages such as a small amount of data, fast operation, easy iteration, and high accuracy [24]. Figure 7 gives the diagnosis result with the grey model-predicted initial values. It shows that QFE decreases by 4.7% at most when adopting 15 as the number of sampling points. So far, the highest reduction ratio of function evaluations to the base case has been obtained by the grey model, which is, therefore, the best method for estimating initial values. At the same time, the limited improvement given by the grey model shows the need for further improvement of the diagnosis algorithm based on other factors.

**Figure 7.** Fault diagnosis of fault 7 with grey model-predicted initial values.

#### *3.2. Solving the LSQ Inverse Problem with Di*ff*erent Numbers of Iterations*

As one popular optimization algorithm, LSQ requires a large number of iterations to obtain accurate fault parameters at each sampling time, so its high computational cost is its main disadvantage. In fact, the aim of fault diagnosis is to find the abnormal trend of fault parameters in a timely manner during a given time interval. In this process, completely converged calculation at each time point is not necessary. Based on the idea of tracking approximation, the proposal of the present paper distributes the inner iterative computation into an outer integration progress to decrease the maximum number of iterations at each sampling point. Figure 8 shows the diagnosis result with different maximum numbers of iterations. It presents a great decrease of QFE when reducing the maximum number of iterations. In particular, when the iteration number equals 1, QFE decreases by 55% compared to the base case, being far greater than the value obtained by the grey model.

**Figure 8.** Fault diagnosis of fault 7 with different numbers of iterations.

The fault diagnosis result obtained by this strategy is shown in Figure 9, in contrast with that obtained by the base case. The exact coherence of fault parameters between these two cases evidences no loss of accuracy with this fast algorithm. Meanwhile, minor parameter fluctuation is observed due to the insufficient iterative computation of this algorithm.

**Figure 9.** Fault diagnosis of fault 7 with one iteration only.

#### *3.3. Hybrid Inverse Problem-Solving Strategy*

In the above sections, two kinds of improvements—increasing the prediction accuracy of initial values and decreasing the number of iterations—were conducted for the least squares algorithm. The computational results show that the latter has a significant effect on the fault diagnosis speed. Generally, these algorithms use the passive trial and error method to solve the inverse problem of fault diagnosis. Fault parameters are defined as input variables for the system model used by LSQ, different from their output variable role defined in the inverse problem. In the following, an alternative inverse problem model using a direct mapping of fault parameters from measurements will be considered to avoid the time-consuming model solving process.

In an information view of fault diagnosis, the inverse problem defined herein is a typical multiple input-multiple output (MIMO) system in which measurable/controllable variables and fault parameters constitute input and output parts, respectively. The linear MIMO model is given by the PLS method in this work owing to the small data change for both input and output variables in a short sampling interval. Furthermore, periodic correction for this linear model by LSQ is necessary to preserve its accuracy.

Figure 10 shows the comparison of fault diagnosis for the base case and the case using a hybrid strategy. It proves the feasibility and accuracy of this strategy, but indicates larger fluctuations of the fault parameter with the hybrid algorithm. Therefore, PLS is suitable for replacing LSQ, but its application should be controlled properly. Factors affecting the efficiency of this strategy will be discussed hereafter.

**Figure 10.** Fault diagnosis of fault 7 with the hybrid algorithm.

(1) Number of PLS components

PLS is something of a cross between multiple linear regression and principal component analysis. It constructs components as linear combinations of the original variables, while allowing for correlation between independent and dependent variables. The number of components is therefore of primary importance to the accuracy of the PLS model. Figure 11 depicts the percent of variance explained in the dependent variable as a function of the component number. A maximum of 16 components is assumed in Figure 11 because the independent variables consist of a total of 16 variables for the stripper in TEP. It can be seen that more than 95 percent variance was explained by the first three components, which were, accordingly, chosen as the principle components in the following PLS modeling process.

**Figure 11.** Number analysis of PLS components under fault 7.

(2) Sampling data set for PLS modeling

The training data sets for PLS modeling were composed of 12 measured variables, 4 manipulated variables, and 1 fault parameter. As shown in Figure 1, the fault parameter may be obtained from LSQ or PLS, so the PLS model can be built on LSQ-generated fault parameter sets (Vector I) or mixed sets (Vector II).

Figure 12a,b show the fault diagnosis results with Vector II as training data sets for PLS, whereas Figure 12c,d give results with Vector I as training data sets. The root mean square error (RMSE) of fault parameters between the base case and the proposed approach was also calculated and adhered to Figure 12a–d. As illustrated in Figure 1, a second PLS (PLS II) was performed with the aim of keeping the continuity of fault parameters when no abnormal signals were detected. Figure 12 shows the fault parameters obtained with (b) and without (a) in this second PLS based on Vector II. The worse result obtained with PLS than without PLS to predict the fault parameter in normal states evidences an adverse propagation effect of PLS prediction error on the PLS model itself. The fact that diagnosis results with Vector I as training data sets for PLS coincide exactly with the base case no matter whether they are with (d) or without (c) the second PLS further proves this conclusion. Consequently, PLS modeling should be conducted based on the fault parameters generated by the LSQ algorithm to preserve its accuracy.

Generally, the time-saving prediction of fault parameters with the PLS method may be equal to several sampling periods before each time-consuming LSQ in Figure 1. Although this scheme can cut down the running time of fault diagnosis greatly, the using frequency of PLS should be limited to an allowable range since the PLS accuracy strongly depends on new fault parameters generated by LSQ. In other words, it is crucial to correct the PLS model with LSQ-generated data after consecutive calls of PLS.

**Figure 12.** *Cont*.

**Figure 12.** Fault diagnosis under fault 7 (**a**) based on Vector II data, but without second PLS; (**b**) based on Vector II data and with second PLS; (**c**) based on Vector I data, but without second PLS; (**d**) based on Vector I data and with second PLS.

Figure 13 shows the effect of the correction interval on QFE, the running time, and RMSE, respectively. We can see from Figure 13a,b that QFE decreases, as does the running time of the fault diagnosis process, when increasing the correction interval. In particular, their decreasing magnitude becomes small when the correction interval exceeds 5. However, there appears to be a rapid growth of calculation error, as can be seen from Figure 13c. Although the effect of correction interval 4 or 5 on RMSE is not significant, both QFE and the running time will increase for the correction interval of 4 compared with 5. Therefore, 5 is the appropriate calling number of PLS in a correction interval. With this calling number, QFE decreases by 81.60% and the running speed increases about 1.7 times compared to the base case with this calling number.

Table 1 summarizes the approaches that can effectively reduce QFE in fault diagnosis. The best results obtained are indicated in boldface. It leads to the conclusion that the approach proposed in this paper evaluates the fault parameter markedly faster than the pure LSQ-based algorithm used in [16].

**Table 1.** Algorithm improvement concerning function evaluations.

The above feasibility test was implemented based on a step-type fault, with fault 7 as an example. Next, another type of fault, a random type with fault 8 as an example, will be tested with the aforementioned hybrid structure. In the case of fault 8, the composition of the feed stream (containing component A, B, and C only) changes randomly from the 8 h point. The essentially random sampling values form this fault are fed to the hybrid fault diagnosis algorithm as a preset input from an outside battery. It is satisfactory to discriminate the fault type from fault diagnosis results, with no need for a statistics analysis of random sampling values. Its fault diagnosis result with LSQ only is shown in Figures 14 and 15, also published in our early work [16]. The overall running time and QFE for this diagnosis process are 5103 seconds and 19,256, respectively. This case is referred to as the base case for fault 8 in the following discussions.

**Figure 13.** Fault diagnosis with different correction intervals of PLS under fault 7 with respect to (**a**) quantity of function evaluations (QFE); (**b**) the running time; (**c**) the root mean square error (RMSE).

**Figure 14.** Fault parameter in the base case of fault 8.

**Figure 15.** Function evaluation in the base case of fault 8.

Similar to Figure 11, Figure 16 shows the percent of variance explained by independent components. The first two components make more than 80% contributions and were thus selected as the components in PLS modeling under fault 8.

**Figure 16.** Number analysis of PLS components under fault 8.

Figure 17 shows the effect of the correction interval on QFE (a), the running time (b), and RMSE (c) under fault 8. We can see that QFE and the running time decrease, but RMSE increases, when increasing the correction interval. 5 is chosen as the optimal correction interval since the former two indices do not decrease significantly, while RMSE remains small under this choice. Besides, larger values of the former two indices than fault 7 are observed in Figure 17, indicating that a random-type fault consumes more time than a step-type fault due to its stochastic computing load.

**Figure 17.** Fault diagnosis with different correction intervals of PLS under fault 8 with respect to (**a**) QFE; (**b**) the running time; (**c**) RMSE.

With 5 as the correction interval, the diagnosis result of three compositions in the bottom feed of the stripper is shown in Figure 18. It indicates nearly the same composition trajectories for the hybrid approach and base case, and proves the feasibility and accuracy of our proposed approach. Finally,

**Figure 18.** Fault diagnosis with a hybrid scheme under fault 8 for the (**a**) composition of A; (**b**) composition of B; (**c**) composition of C.


• **Management of genital duplication**

Gender distribution shows a predilection of CDS in females with a complete but well-developed duplicated external and internal genital organ and preserved ovarian function. Almost always, corrective genital surgery in females is not necessary unless one side is hypoplastic and might cause menstrual flow obstruction or for cosmetic concerns, in which case, it can be removed [10]. If congenital uterine anomalies such as septate or subseptate uteri have a risk for reduced conception rate, increased risk of first trimester miscarriage (especially with septal implantation), preterm birth, and fetal malpresentation, patients with didelphys uterus do not appear to have reduced fertility and have less pregnancy complications, but might be at increased risk for preterm labor [17]. All our adult female patients carried pregnancies to term and in three out the four cases [3,18–20] presented for pregnancy related issues (genetic counseling, symptomatic ureteral compression or vaginal prolapse, and voiding dysfunction after multiple pregnancies). Cesarean section was performed in all cases. In cases with a history of multiple abdominal surgeries and depending on the complexity of procedures, the cesarean surgery should be assisted by a colorectal surgeon and/or urologist. Male patients present with complete or partial penile duplication and are more likely to be corrected for cosmetic reasons with removal of one of the abnormal shafts [13] or penoplasty [12]. There are no reports assessing fertility and sexual function into adult age in CDS male patients.


**6. OCT-angiography**

OCT-Angiography (OCT-A) enables the noninvasive visualization of 3D retinal capillary network. It correlates very well with fluorescein angiography (FA), and it is able to show even more capillaries in the pericentral macula than FA and to separate and individualize the superficial and deep capillary plexus.