Genomic Epidemiology of SARS-CoV-2 in Guangdong Province, China.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 infection and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, China's most populous province, during early 2020 resulted in 1,388 reported RNA-positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China, we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain because of low virus genetic variation early in the pandemic. Our results illustrate how the timing, size, and duration of putative local transmission chains were constrained by national travel restrictions and by the province's large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required, because the number of cases imported from other countries has increased.

Immunogenicity and safety of a recombinant COVID-19 vaccine (ZF2001) as heterologous booster after priming with inactivated vaccine in healthy children and adolescents aged 3-17 years: an open-labeled, single-arm clinical trial.

Considering that neutralizing antibody levels induced by two doses of the inactivated vaccine decreased over time and had fallen to low levels by 6 months, and homologous and heterologous booster immunization programs have been implemented in adults in China. The booster immunization of recombinant COVID-19 vaccine (ZF2001) after priming with inactivated vaccine in healthy children and adolescents has not been reported. We performed an open-labeled, single-arm clinical trial to evaluate the safety and immunogenicity of heterologous booster immunization with ZF2001 after priming with inactivated vaccine among 240 population aged 3-17 years in China. The primary outcome was immunogenicity, including geometric mean titers (GMTs), geometric mean ratios (GMRs) and seroconversion rates of SARS-CoV-2 neutralizing antibodies against prototype SARS-CoV-2 and Omicron BA.2 variant at 14 days after vaccination booster. On day 14 post-booster, a third dose booster of the ZF2001 provided a substantial increase in antibody responses in minors, and the overall occurrence rate of adverse reactions after heterologous vaccination was low and all adverse reactions were mild or moderate. The results showed that the ZF2001 heterologous booster had high immunogenicity and good safety profile in children and adolescents, and can elicit a certain level of neutralizing antibodies against Omicron.Trial registration NCT05895110 (Retrospectively registered, First posted in ClinicalTrials.gov date: 08/06/2023).

Viral hepatitis in China during 2002-2021: epidemiology and influence factors through a country-level modeling study.

BACKGROUND: Viral hepatitis imposes a heavy disease burden worldwide and is also one of the most serious public health problems in China. We aimed to describe the epidemiological characteristics of hepatitis in China and to investigate the influencing factors. METHODS: We first used the JoinPoint model to analyze the percentage change (APC) and average annual percentage change (AAPC) of hepatitis in Chinese provinces from 2002 to 2021. We then explored the influencing factors by using the time-series global principal component analysis (GPCA) and the panel fixed-effects model. RESULTS: The disease burden varied across different provinces from 2002 to 2021. The AAPC of the total HAV incidence decreased by 10.39% (95% CI: [-12.70%, -8.02%]) from 2002 to 2021. Yet the AAPC of HBV, HCV, and HEV increased by 1.50% (95% CI: [0.23%, 2.79%]), 13.99% (95% CI: [11.28%, 16.77%]), and 7.10% (95% CI: [0.90%, 13.69%]), respectively. The hotspots of HAV, HBV, HCV, and HEV moved from the west to the center, from the northwest to the southeast, from the northeast to the whole country, and from the northeast to the southeast, respectively. Different types of viral hepatitis infections were associated with hygiene, pollutant, and meteorological factors. Their roles in spatial-temporal incidence were expressed by panel regression functions. CONCLUSIONS: Viral hepatitis infection in China showed spatiotemporal heterogeneity. Interventions should be tailored to its epidemiological characteristics and determinants of viral hepatitis.

Induction of Broadly Cross-Reactive Antibody Responses to SARS-CoV-2 Variants by S1 Nanoparticle Vaccines.

Despite the rapid deployment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, the emergence of SARS-CoV-2 variants and reports of their immune evasion characteristics have led to an urgent need for novel vaccines that confer potent cross-protective immunity. In this study, we constructed three different SARS-CoV-2 spike S1-conjugated nanoparticle vaccine candidates that exhibited high structural homogeneity and stability. Notably, these vaccines elicited up to 50-times-higher neutralizing antibody titers than the S1 monomer in mice. Crucially, it was found that the S1-conjugated nanoparticle vaccine could elicit comparable levels of neutralizing antibodies against wild-type or emerging variant SARS-CoV-2, with cross-reactivity to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), the effect of which could be further enhanced using our designed nanoparticles. Our results indicate that the S1-conjugated nanoparticles are promising vaccine candidates with the potential to elicit potent and cross-reactive immunity against not only wild-type SARS-CoV-2, but also its variants of concern, variants of interest, and even other pathogenic betacoronaviruses. IMPORTANCE The emergence of SARS-CoV-2 variants led to an urgent demand for a broadly effective vaccine against the threat of variant infection. The spike protein S1-based nanoparticle designed in our study could elicit a comprehensive humoral response toward different SARS-CoV-2 variants of concern and variants of interest and will be helpful to combat COVID-19 globally.

Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus first identified in December 2019. Notable features that make SARS-CoV-2 distinct from most other previously identified betacoronaviruses include a receptor binding domain and a unique insertion of 12 nucleotides or 4 amino acids (PRRA) at the S1/S2 boundary. In this study, we identified two deletion variants of SARS-CoV-2 that either directly affect the polybasic cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). These deletions were verified by multiple sequencing methods. In vitro results showed that the deletion of NSPRRAR likely does not affect virus replication in Vero and Vero-E6 cells; however, the deletion of QTQTN may restrict late-phase viral replication. The deletion of QTQTN was detected in 3 of 68 clinical samples and 12 of 24 in vitro-isolated viruses, while the deletion of NSPRRAR was identified in 3 in vitro-isolated viruses. Our data indicate that (i) there may be distinct selection pressures on SARS-CoV-2 replication or infection in vitro and in vivo; (ii) an efficient mechanism for deleting this region from the viral genome may exist, given that the deletion variant is commonly detected after two rounds of cell passage; and (iii) the PRRA insertion, which is unique to SARS-CoV-2, is not fixed during virus replication in vitro These findings provide information to aid further investigation of SARS-CoV-2 infection mechanisms and a better understanding of the NSPRRAR deletion variant observed here.IMPORTANCE The spike protein determines the infectivity and host range of coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has two unique features in its spike protein, the receptor binding domain and an insertion of 12 nucleotides at the S1/S2 boundary resulting in a furin-like cleavage site. Here, we identified two deletion variants of SARS-CoV-2 that either directly affect the furin-like cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN), and we investigated these deletions in cell isolates and clinical samples. The absence of the polybasic cleavage site in SARS-CoV-2 did not affect virus replication in Vero or Vero-E6 cells. Our data indicate the PRRAR sequence and the flanking QTQTN sequence are not fixed in vitro; thus, there appears to be distinct selection pressures on SARS-CoV-2 sequences in vitro and in vivo Further investigation of the mechanism of generating these deletion variants and their infectivity in different animal models would improve our understanding of the origin and evolution of this virus.

Reduction of air pollutants and associated mortality during and after the COVID-19 lockdown in China: Impacts and implications.

Although strict lockdown measurements implemented during the COVID-19 pandemic have dramatically reduced the anthropogenic-based emissions, changes in air quality and its health impacts remain unclear in China. We comprehensively described air pollution during and after the lockdown periods in 2020 compared with 2018-2019, and estimated the mortality burden indicated by the number of deaths and years of life lost (YLL) related to the air pollution changes. The mean air quality index (AQI), PM10, PM2.5, NO2, SO2 and CO concentrations during the lockdown across China declined by 18.2 (21.2%), 27.0 µg/m3 (28.9%), 10.5 µg/m3 (18.3%), 8.4 µg/m3 (44.2%), 13.1 µg/m3 (38.8%), and 0.3 mg/m3 (27.3%) respectively, when compared to the same periods during 2018-2019. We observed an increase in O3 concentration during the lockdown by 5.5 µg/m3 (10.4%), and a slight decrease after the lockdown by 3.4 µg/m3 (4.4%). As a result, there were 51.3 (95%CI: 32.2, 70.1) thousand fewer premature deaths (16.2 thousand during and 35.1 thousand after the lockdown), and 1066.8 (95%CI: 668.7, 1456.8) thousand fewer YLLs (343.3 thousand during and 723.5 thousand after the lockdown) than these in 2018-2019. Our findings suggest that the COVID-19 lockdown has caused substantial decreases in air pollutants except for O3, and that substantial human health benefits can be achieved when strict control measures for air pollution are taken to reduce emissions from vehicles and industries. Stricter tailored policy solutions of air pollution are urgently needed in China and other countries, especially in well-developed industrial regions, such as upgrading industry structure and promoting green transportation.

Prolonged Persistence of SARS-CoV-2 RNA in Body Fluids.

We prospectively assessed 49 coronavirus disease cases in Guangdong, China, to estimate the frequency and duration of detectable severe acute respiratory syndrome coronavirus 2 RNA in human body fluids. The prolonged persistence of virus RNA in various body fluids may guide the clinical diagnosis and prevention of onward virus transmission.

Associations of maternal ozone exposures during pregnancy with maternal blood pressure and risk of hypertensive disorders of pregnancy: A birth cohort study in Guangzhou, China.

Although studies have assessed the associations of maternal exposure to ozone (O3) during pregnancy with blood pressure and the risk of hypertensive disorders of pregnancy (HDP), the results were inconsistent. Furthermore, no studies have been conducted in China where the ambient O3 concentration continuedly increased. The present study aimed to estimate the effects of maternal exposure to O3 during pregnancy on the HDP risk, systolic blood pressure (SBP), diastolic blood pressure (DBP) and pulse pressure (PP). All participants of pregnant women were selected from the prospective birth cohort study on Prenatal Environments and Offspring Health conducted in Guangzhou, China. A spatiotemporal land-use-regression model was used to estimate individual monthly air pollution exposure from three months before pregnancy to childbirth date. Information on HDP, SBP, DBP and PP was obtained from maternal medical records. A Logistic regression model and a mixed linear model were used to estimate the associations of maternal exposure to O3 with the risk of HDP and blood pressure (SBP, DBP and PP), respectively. We found significant associations of maternal exposure to O3 during the third (OR = 1.31, 95%CI: 1.07, 1.60) and the second month (OR = 1.25, 95%CI: 1.02, 1.51) before pregnancy with the risk of HDP. Observed significantly positive associations of O3 exposures with SBP, DBP and PP during the two months before pregnancy and during the early pregnancy. The peak effects of O3 exposure on SBP, DBP and PP were respectively observed during the second month of pregnancy (ß = 1.07  mmHg, 95%CI: 0.84, 1.31  mmHg), the first month before pregnancy (ß = 0.40  mmHg, 95%CI: 0.21, 0.50  mmHg) and the second month of pregnancy (ß = 0.78  mmHg, 95%CI: 0.59, 0.97  mmHg). Our results suggest that maternal exposure to O3 were positively associated with blood pressure and the risk of HDP, and the period from three months before pregnancy to the first trimester might be the critical exposure window.

Prevalence and antifungal susceptibility of Sporothrix species in Jiangxi, central China.

Sporotrichosis is a subcutaneous mycosis caused by traumatic inoculation of pathogenic Sporothrix species. Until recently, Sporothrix globosa was considered as the unique Chinese species causing this disorder. In the present study, 33 clinical Sporothrix strains isolated from Jiangxi, China, were classified and antifungal susceptibility for each strain was determined. Thirteen S. globosa strains and 20 S. schenckii strains were identified by morphology and by multilocus analysis using rDNA ITS, CAL, and EF1α (i.e., internal transcribed spacer, calmodulin and elongation factor-1α). In vitro antifungal susceptibility testing of yeast phases indicated that itraconazole, terbinafine, and posaconazole were most effective against both species, followed by amphotericin B and voriconazole, while fluconazole, 5-fluorocytosine had low efficacy with high MICs. Co-occurrence of S. schenckii and S. globosa in central China may indicate different routes of transmission in this area.

Transcriptional profiling of macrophages infected with Fonsecaea monophora.

Fonsecaea monophora is a member of dematiaceous fungi capable of causing chromoblastomycosis through traumatic injury. However, little is known about the pathogenesis and early interactions between F. monophora and host. The aim of this study was to explore the potential mechanism of macrophages against F. monophora, especially the role of melanin during the pathogenic process. We carried out RNA sequencing based on the Illumina system. It showed that according to melanin contents, different strains of F. monophora induced different transcriptional profilings in macrophages. Functional analyses suggested the biological functions of differentially expressed genes were closely related to immune response, and the melanin might affect the interactions by regulating the MAPK signalling pathway of macrophages. Our results provide insights into the pathogenesis of infection by F. monophora conidia.

Molecular epidemiology and in vitro antifungal susceptibility of Trichophyton schoenleinii, agent of tinea capitis favosa.

Trichophyton schoenleinii is an anthropophilic dermatophyte usually causing tinea favosa. Only few studies have provided data on molecular epidemiology and antifungal profiles of this fungus due to its limited prevalence after 1950s. Forty-nine strains from Asia (n = 27), Africa (n = 10), Europe (n = 10) and from unknown regions (n = 2) were analysed with amplified fragment length polymorphism fingerprinting (AFLP) to reveal intraspecific genetic diversity in this dataset. Amplified fragment length polymorphism fingerprinting genotyping revealed five clusters which did not correspond to geographic origins or clinical characteristics. Additionally, in vitro antifungal susceptibility to seven antifungals was provided for all strains. Terbinafine, ketoconazole, miconazole and itraconazole proved to be the most effective drugs, followed by griseofulvin. No correlation between genotypes and differences in antifungal susceptibility was observed. It is concluded that the AFLP groups are lineages within a single species.

New Molecular Markers Distinguishing Fonsecaea Agents of Chromoblastomycosis.

The species belonging to the genus Fonsecaea are the main causative agents of chromoblastomycosis. The invasive potential of Fonsecaea differs significantly among its various sibling species. Moreover, the lack of clarity on the virulence and availability of precise markers to distinguish and detect Fonsecaea species is attributed to the different ways of dissemination and pathogenicity. Therefore, the present study aimed to propose new molecular tools to differentiate between sibling species causing chromoblastomycosis. We used an infection model of chromoblastomycosis in BALB/c to study species-specific molecular markers for the in vivo detection of Fonsecaea species in biological samples. Specific primers based on the CBF5 gene were developed for Fonsecaea pedrosoi, Fonsecaea monophora, Fonsecaea nubica, and Fonsecaea pugnacius. In addition, a padlock probe was designed for F. pugnacius based on ITS sequences. We also assessed the specificity of Fonsecaea species using in silico, in vitro, and in vivo assays. The results showed that markers and probes could effectively discriminate the species in both clinical and environmental samples, enabling bioprospecting of agents of chromoblastomycosis, thereby elucidating the infection route of the disease.

Melanization of a meristematic mutant of Fonsecaea monophora increase the pathogenesis in a BALB/c mice infection model.

The role of melanin in agents of chromoblastomycosis in vivo still remains unclear. In this study, we addressed the question in a BALB/c mice infection model by using an albino mutant, which generated from a melanized meristematic mutant of F. monophora. The most severe clinical manifestations of infected mice were observed at 10-25 dpi (day post infection) and 7-15 dpi for melanized and albino strain, respectively. Histopathologic examination were similar in both groups at 15 dpi (acute infection phase), but not at 30 dpi (sustained infection phase) and 45 dpi (recovery phase). Sclerotic body could be revealed in melanized strain infection group compare with albino strain infection group at 45 dpi. We found significantly elevated of TH2 cytokines in melanized strain infected group at 15 dpi, while increased expression level of TH1 and TH17 cytokines in albino strain infection group at 15 and 30 dpi. In conclusion, the present data addressed our speculation that melanization of this meristematic mutant of F. monophora could increase the pathogenesis in vivo. The inhibition of TH1 and TH17, exacerbated TH2 ability of melanized F. monophora is the key to escape the host immune system at the initial recognition and persistence in BALB/c mice.

Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37-40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe-template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

Correction to: Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

The Editorial Office of Mycopathologia reports that several paragraphs of Najafzadeh et al. were transcribed with only minor edits from previously published material by Najafzadeh M.J.

Molecular identification of Histoplasma capsulatum using rolling circle amplification.

Histoplasmosis is a systemic fungal disease that occurs worldwide, causing symptomatic infection mostly in immunocompromised hosts. Etiological agent is the dimorphic fungus, Histoplasma capsulatum, which occurs in soil contaminated with bird or bat droppings. Major limitation in recognition of H. capsulatum infections is the low awareness, since other diseases may have similar symptomatology. The molecular methods have gained importance because of unambiguous diagnostic ability and efficiency. The aim of this study was to develop and evaluate a padlock probe in view of rolling circle amplification (RCA) detection method which targets ITS (Internal Transcribed Spacer) rDNA of H. capsulatum enabling rapid and specific detection of the fungus in clinical samples. Two padlock probes were designed and one of these (HcPL2) allowed specific amplification of H. capsulatum DNA while no cross-reactivity was observed with fungi used as negative controls. This method proved to be effective for H. capsulatum specific identification and demonstrated to be faster than the traditional method of microbiological identification.

Epidemiology of Superficial Fungal Infections in Guangdong, Southern China: A Retrospective Study from 2004 to 2014.

Superficial fungal infections are common worldwide; however, the distribution of pathogenic species varies among geographical areas and changes over time. This study aimed to determine the epidemiologic profile of superficial fungal infections during 2004-2014 in Guangzhou, Southern China. Data regarding the superficial mycoses from outpatients and inpatients in our hospital were recorded and analyzed. From the 3367 patients that were enrolled in the study, 3385 samples were collected from skin, hair and nail lesions. Of the 697 positive cultures, dermatophytes were the most prevalent isolates (84.36 %), followed by yeasts (14.92 %) and non-dermatophyte molds (0.72 %). Trichophyton rubrum (56.24 %) was the most common dermatophyte isolated from cases of tinea unguium (83.92 %), tinea pedis (71.19 %), tinea cruris (91.66 %), tinea corporis (91.81 %) and tinea manuum (65.00 %). Trichophyton mentagrophytes (13.35 %) and Microsporum canis (10.19 %) were the predominant species associated with cases of tinea faciei (54.55 %) and tinea capitis (54.13 %), respectively. Yeasts and molds were identified primarily from other cases of superficial fungal infections. In conclusion, when compared to previous studies in the same area, the epidemiology of superficial mycoses in Guangdong did not significantly change from 2004 to 2014. The prevalence of causative agents and the spectrum of superficial fungal infections, particularly tinea caused by dermatophyte infection, are similar to reports from several specific regions in China and Europe, whereas increasing incidences of Trichophyton mentagrophytes and Microsporum canis occurred in Guangdong, China.

Epidemiological changes in tinea capitis over the sixty years of economic growth in China.

BACKGROUND: Tinea capitis is a fungal infection of the scalp occurring commonly in children. Historical data indicate that clinical manifestations and the spectrum of etiologic agents vary greatly with geography, as well as socioeconomic affected populations. OBJECTIVE: To study the possible connection between socioeconomic status, the disease patterns and the variability of etiological agents. METHODS: We reviewed tinea capitis in China through literature since 1956. The disease pattern was correlated with economic and public health management protocols. Historical data on fungal identification were mostly obtained by morphology. The accuracy of these historical results was further confirmed by use of both morphological and ITS identification on a control set of 90 isolates collected recently from local hospital. RESULTS: Full agreement of the two identification methods implies that data from the literature were sufficiently reliable to allow comparison across reported cases. In sum, 88 papers involving 25 administrative provinces and municipalities with 38,962 clinical strains met the inclusion criteria of this review. Zoophilic species Microsporum canis is the most prevalent agent within large, modernized cities in China today accounting for over 80% of infections. In contrast, anthropophilic dermatophytes, particularly Trichophyton violaceum, are geographically endemic only in some southeastern and northwestern regions. CONCLUSION: Economic development and urbanization of cities favor a shift of etiological agents from anthroponoses to zoonoses in contemporary China. Pets are becoming the most likely sources of infection in modern lifestyles, replacing the earlier human-to-human transmission mode. However, the latter transmission mode is still prevalent in less developed areas lacking adequate social and public health facilities.

Development of an air-flow-assisted extractive electrospray ionization source for rapid analysis of phthalic acid esters in spirits.

RATIONALE: Although traditional analytical techniques like gas chromatography (GC) and GC/mass spectrometry (MS) offer satisfactory sensitivity and good reproducibility for the detection of phthalic acid esters (PAEs) in a variety of matrices, they involve laborious sample pretreatment, are time-consuming, and some are expensive and environmentally unfriendly. Furthermore, there are thousands of spirits on the market; therefore, rapid and high-throughout methods suitable for the consistent detection and quantification of PAEs in spirits are urgently required. METHODS: A new atmospheric pressure ionization method, named air-flow-assisted extractive electrospray ionization (EESI), has been developed. It is a variant on EESI and possesses the advantages of both EESI and air-flow-assisted ionization for direct analysis of samples without pretreatment. Combined with a quadrupole time-of-flight (QTOF) mass spectrometer, the method was used to directly analyze four PAEs, i.e., dipentyl phthalate, diethyl phthalate, benzyl butyl phthalate and didecyl phthalate, in spirits. RESULTS: The method exhibits excellent sensitivity, stability and convenience. Four different brands of spirits have been successfully analyzed. The total analysis time for one sample was within 1 min, and the limits of detection and limits of quantification of the samples are located in the range 0.011-0.035 and 0.038-0.087 µg g(-1), respectively. Very good linearities, with correlation coefficients of 0.9758-0.9990, are observed for the samples in the range of 0.035 to 10 µg g(-1). CONCLUSIONS: The results indicate that the air-flow-assisted EESI combined with tandem mass spectrometry is an effective method for rapid and direct determination of PAEs in spirits without sample pretreatment.

Three isothermal amplification techniques for rapid identification of Cladophialophora carrionii, an agent of human chromoblastomycosis.

In this study, we developed rapid and sensitive assays for the detection of Cladophialophora carrionii, a common agent of human chromoblastomycosis. The isothermal techniques evaluated were rolling-circle amplification (RCA), multiplex ligation-dependent probe amplification (MLPA), and loop-mediated isothermal amplification (LAMP). The probes for RCA and MLPA were designed with target sequences in the rDNA internal transcribed spacer gene (ITS) region, and LAMP primers were designed using the elongation factor 1α gene (EF1); these probes and primers specifically amplified DNA of isolates of the species. The three techniques were sufficiently specific and sensitive for discriminating target DNA of C. carrionii from that of related Cladophialophora species and other agents of chromoblastomycosis. RCA, MLPA, and LAMP are advantageous in their reliability and ease of operation compared to standard PCR and conventional methods.