Identification of an Oligostilbene, Vaticanol B, from Dryobalanops aromatica Leaves as an Antiviral Compound against the Hepatitis C Virus.

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Although current medications using direct-acting antivirals (DAAs) are highly effective and well-tolerated for treating patients with chronic HCV, high prices and the existence of DAA-resistant variants hamper treatment. There is thus a need for easily accessible antivirals with different mechanisms of action. During the screening of Indonesian medicinal plants for anti-HCV activity, we found that a crude extract of Dryobalanops aromatica leaves possessed strong antiviral activity against HCV. Bioassay-guided purification identified an oligostilbene, vaticanol B, as an active compound responsible for the anti-HCV activity. Vaticanol B inhibited HCV infection in a dose-dependent manner with 50% effective and cytotoxic concentrations of 3.6 and 559.5 µg/mL, respectively (Selectivity Index: 155.4). A time-of-addition study revealed that the infectivity of HCV virions was largely lost upon vaticanol B pretreatment. Also, the addition of vaticanol B following viral entry slightly but significantly suppressed HCV replication and HCV protein expression in HCV-infected and a subgenomic HCV replicon cells. Thus, the results clearly demonstrated that vaticanol B acted mainly on the viral entry step, while acting weakly on the post-entry step as well. Furthermore, co-treatment of the HCV NS5A inhibitor daclatasvir with vaticanol B increased the anti-HCV effect. Collectively, the present study has identified a plant-derived oligostilbene, vaticanol B, as a novel anti-HCV compound.

Consideration of the Cycle Threshold Values from Real-Time RT-PCR SARS-CoV-2 Interpretation for the Clinicians: Analysis of 339 Positive Cases from a Referral Laboratory in Jakarta, Indonesia.

BACKGROUND: real-time RT-PCR was recommended by WHO for COVID-19 diagnosis. The cycle threshold (Ct) values were expected to have an association with clinical manifestation. However, the diagnostic modalities such as quantitative molecular detection and virus isolation were not yet available for the routine test. This study has been conducted to analyze the relationship between the Ct values of qualitative rRT-PCR and the clinical manifestation and to describe the factors determining the result. METHODS: from March to April 2020, specimens were sent to our laboratory from different healthcare centers in Jakarta. The patient's characteristic and clinical manifestation were extracted from the specimen's epidemiology forms. The specimens extracted and tested using rRT-PCR, and the Ct value were collected. The data were analyzed using the appropriate statistic test. RESULTS: from 339 positive results, the mild to moderate case was 176 (52%) and the severe cases was 163 (48%). Female was dominant in the mild to moderate cases (58%), while the male was prevalent in the severe cases (60%). The median age for mild to moderate case was 35 years old and severe cases was 49 years old. Statistical analysis found relationship between both group with gender (p = 0.001) and age (p < 0.001), but not with the Ct value. CONCLUSION: many variables in specimen sampling and processing could affect the Ct value result. In addition, the disease's severity was depended with the host immune response, regardless the number of virus. There was suggested no significant difference between the Ct values of mild-moderate and severe COVID-19, and thus should not be loosely interpreted.

Universal health coverage in Indonesia: concept, progress, and challenges.

Indonesia is a rapidly growing middle-income country with 262 million inhabitants from more than 300 ethnic and 730 language groups spread over 17 744 islands, and presents unique challenges for health systems and universal health coverage (UHC). From 1960 to 2001, the centralised health system of Indonesia made gains as medical care infrastructure grew from virtually no primary health centres to 20 900 centres. Life expectancy improved from 48 to 69 years, infant mortality decreased from 76 deaths per 1000 livebirths to 23 per 1000, and the total fertility rate decreased from 5·61 to 2·11. However, gains across the country were starkly uneven with major health gaps, such as the stagnant maternal mortality of around 300 deaths per 100 000 livebirths, and minimal change in neonatal mortality. The centralised one size fits all approach did not address the complexity and diversity in population density and dispersion across islands, diets, diseases, local living styles, health beliefs, human development, and community participation. Decentralisation of governance to 354 districts in 2001, and currently 514 districts, further increased health system heterogeneity and exacerbated equity gaps. The novel UHC system introduced in 2014 focused on accommodating diversity with flexible and adaptive implementation features and quick evidence-driven decisions based on changing needs. The UHC system grew rapidly and covers 203 million people, the largest single-payer scheme in the world, and has improved health equity and service access. With early success, challenges have emerged, such as the so-called missing-middle group, a term used to designate the smaller number of people who have enrolled in UHC in wealth quintiles Q2-Q3 than in other quintiles, and the low UHC coverage of children from birth to age 4 years. Moreover, high costs for non-communicable diseases warrant new features for prevention and promotion of healthy lifestyles, and investment in a robust integrated digital health-information system for front-line health workers is crucial for impact and sustainability. This Review describes the innovative UHC initiative of Indonesia along with the future roadmap required to meet sustainable development goals by 2030.

Leptospirosis in Indonesia: diagnostic challenges associated with atypical clinical manifestations and limited laboratory capacity.

BACKGROUND: The burden of leptospirosis in Indonesia is poorly understood. Data from an observational study conducted from 2013 to 2016 in seven cities across Indonesia was used to estimate the incidence of leptospirosis and document its clinical manifestations in patients requiring hospitalization. METHODS: Specimens from patients hospitalized with acute fever were collected at enrollment, 14-28 days, and 3 months. Demographic and clinical information were collected during study visits and/or retrieved from medical records and double-entered into clinical report forms. After initially screening for dengue virus and other pathogens, specimens were tested at a central Reference Laboratory for anti-Leptospira IgM using commercial ELISA kits and for Leptospira DNA using an in-house quantitative real-time PCR assay. RESULTS: Of 1464 patients enrolled, 45 (3.1%) confirmed cases (by PCR and/or sero-coversion or four-fold increase of IgM) and 6 (0.4%) probable cases (by high titer IgM) of leptospirosis were identified by the Reference Laboratory. Disease incidence at sites ranged from 0 (0%) cases in Denpasar to 17 (8.9%) cases in Semarang. The median age of patients was 41.2 years (range of 5.3 to 85.0 years), and 67% of patients were male. Twenty-two patients (43.1%) were accurately diagnosed at sites, and 29 patients (56.9%) were clinically misdiagnosed as having another infection, most commonly dengue fever (11, 37.9%). Clinically, 20 patients (39.2%) did not present with hyperbilirubinemia or increased creatinine levels. Two patients (3.9%) died, both from respiratory failure. Fifteen patients (29.4%) clinically diagnosed with leptospirosis at sites were negative based on IgM ELISA and/or PCR at the Reference Laboratory. CONCLUSIONS: Leptospirosis remains an important cause of hospitalization in Indonesia. It can have diverse clinical presentations, making it difficult to differentiate from other common tropical infections. PCR combined with ELISA is a powerful alternative to the cumbersome gold-standard microscopic agglutination test, particularly in resource-limited settings.

Underdiagnoses of Rickettsia in patients hospitalized with acute fever in Indonesia: observational study results.

BACKGROUND: Reports of human rickettsial infection in Indonesia are limited. This study sought to characterize the epidemiology of human rickettsioses amongst patients hospitalized with fever at 8 tertiary hospitals in Indonesia. METHODS: Acute and convalescent blood from 975 hospitalized non-dengue patients was tested for Rickettsia IgM and IgG by ELISA. Specimens from cases with seroconversion or increasing IgM and/or IgG titers were tested for Rickettsia IgM and IgG by IFA and Rickettsia genomes using primers for Rickettsia (R.) sp, R. typhi, and Orientia tsutsugamushi. Testing was performed retrospectively on stored specimens; results did not inform patient management. RESULTS: R. typhi, R. rickettsii, and O. tsutsugamushi IgG antibodies were identified in 269/872 (30.8%), 36/634 (5.7%), and 19/504 (3.8%) of samples, respectively. For the 103/975 (10.6%) non-dengue patients diagnosed with acute rickettsial infection, presenting symptoms included nausea (72%), headache (69%), vomiting (43%), lethargy (33%), anorexia (32%), arthralgia (30%), myalgia (28%), chills (28%), epigastric pain (28%), and rash (17%). No acute rickettsioses cases were suspected during hospitalization. Discharge diagnoses included typhoid fever (44), dengue fever (20), respiratory infections (7), leptospirosis (6), unknown fever (6), sepsis (5), hepatobiliary infections (3), UTI (3), and others (9). Fatalities occurred in 7 (6.8%) patients, mostly with co-morbidities. CONCLUSIONS: Rickettsial infections are consistently misdiagnosed, often as leptospirosis, dengue, or Salmonella typhi infection. Clinicians should include rickettsioses in their differential diagnosis of fever to guide empiric management; laboratories should support evaluation for rickettsial etiologies; and public policy should be implemented to reduce burden of disease.

COVID-19 in Indonesia: Challenges and Multidisciplinary Perspectives for a Safe and Productive New Normal.

The COVID-19 pandemic has caused disruption in all aspects of life, and countries around the world have been combating this pandemic using multiple approaches. Success in one country does not guarantee a transferable approach to other countries with different contexts. This review describes the challenges of COVID-19 management in Indonesia as a populous, socially and culturally diverse, and archipelagic country. It aims to provide multidisciplinary perspectives for a safe, evidence-based, and productive new normal as well as a comprehensive and integrated actionable policy for COVID-19 control.

Southeast Asia Strategic Multilateral Dialogue on Biosecurity.

A strategic multilateral dialogue related to biosecurity risks in Southeast Asia, established in 2014, now includes participants from Singapore, Malaysia, Indonesia, Thailand, Philippines, and the United States. This dialogue is conducted at the nonministerial level, enabling participants to engage without the constraints of operating in their official capacities. Participants reflect on mechanisms to detect, mitigate, and respond to biosecurity risks and highlight biosecurity issues for national leadership. Participants have also identified factors to improve regional and global biosecurity, including improved engagement and collaboration across relevant ministries and agencies, sustainable funding for biosecurity programs, enhanced information sharing for communicable diseases, and increased engagement in international biosecurity forums.

INA-RESPOND: a multi-centre clinical research network in Indonesia.

Nationally representative observational and translational research is needed to address the public health challenges in Indonesia due to the geographic disparity, recently decentralized health system, and diverse infectious disease priorities. To accomplish this, the Indonesian Ministry of Health in collaboration with the US National Institute of Health has established INA-RESPOND (Indonesia Research Partnership on Infectious Disease) - a clinical research network comprising 9 referral hospitals, 7 medical faculties, and 2 research centres across Indonesia. The network provides a forum to conduct research at a national scale and to address scientific questions that would be difficult to address in smaller research settings. Further, it is currently conducting multi-centre research on the etiologies of fever, sepsis, and tuberculosis. There are opportunities to leverage existing network resources for other public health research needs. INA-RESPOND is an Indonesian-led network in a country with diverse population groups and public health needs which is poised to collaborate with researchers, universities, donors, and industry worldwide. This paper describes the network and its goals and values, as well as the management structure, process for collaboration, and future vision.

Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites, pheophorbide a and pyropheophorbide a, against hepatitis C virus.

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo-Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n-hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti-HCV activities with 50%-inhibitory concentrations (IC(50)) of 20.6, 6.1, and 6.6 µg/mL, respectively. Bioactivity-guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti-HCV compound present in the extracts (IC(50) = 0.3 µg/mL). It was also found that pyropheophorbide a possesses anti-HCV activity (IC(50) = 0.2 µg/mL). The 50%-cytotoxic concentrations (CC(50)) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 µg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti-HCV activities. Time-of-addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post-entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.

First Indonesian report of WGS-based MTBC L3 discovery.

OBJECTIVE: Recent spoligotyping results in the island nation of Indonesia had revealed the existence of Mycobacterium tuberculosis complex lineage 3 (MTBC L3) or Central Asian (CAS) strains. In this work, whole-genome sequencing (WGS) - based methods were used to search for the presence of MTBC L3. RESULTS: Two unrelated Indonesian L3 strains discovered by WGS-based SNP phylogenomics are presented here for the first time. Assemblies of their genomes yielded 96.95% (MTBC strain Mtb_S6970) and 98.35% (Mtb_S19106) of the known reference strain H37Rv. Their respective constructed genome coverages are 45.38 ± 12.95x and 63.13 ± 21.10x. The two L3 genomes have 4062 and 4121 genes, respectively, which are well within the number of genes predicted in MTBC strains. Instead of having three rRNA genes usually, Mtb_S6970 possesses four. These L3 isolates exhibit cross-class antibiotic susceptibility. FadD26, fadE24, fbpA, lprO, and panC, which are thought to be important in the pathophysiology of MTBC, were discovered to have 3-7 times more loci in L3 than L2 or L4. The penetration of L3 in the nation, despite its antibiotic sensitivity, is a concerning indicator of borderless global spread that may eventually be overcome by the phenotypes of acquired drug resistance.

Evaluation of SARS-CoV-2 quantification from oropharyngeal swabs, nasopharyngeal swabs, and naso-oropharyngeal swabs: A cross-sectional study.

The current naso-oropharyngeal swab for SARS-CoV-2 detection faces several problems, such as waste issues and its use for quantitative studies. This study aimed to evaluate the total RNA and viral loads from different upper respiratory tract swabs types and whether SARS-CoV-2 quantification can use the current internal control for normalization. This cross-sectional study collected positive specimens with single oropharyngeal or nasopharyngeal swabs and naso-oropharyngeal swabs. The samples were extracted, tested with qualitative RT‒PCR, and then tested with quantitative RT‒PCR. The RNA eluate was measured for the total RNA concentration. The total RNA concentration, viral load, and RNaseP Ct values were collected and analysed statistically. The positive results came from 41 oropharyngeal swabs, 34 nasopharyngeal swabs, and 36 naso-oropharyngeal swabs. The total RNA increased significantly from oropharyngeal swabs to nasopharyngeal swabs to naso-oropharyngeal swabs. Significant differences in RNaseP Ct values between groups and their correlations with total RNA were found. In addition, the increase in the total RNA and the RNaseP Ct values were unrelated to the viral load. The physical features in the naso-oropharyngeal area and the swabbing procedures could affect the total RNA but not the viral load. However, since the virus particles could present inside and outside human cells, the increase in collected human cells may not always be followed by the viral load increase. Normalization using the RNaseP Ct value became unnecessary due to the factors mentioned above. Therefore, a careful approach is needed in viral load studies of swab specimens.

Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis.

Background and Aim: Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis. Materials and Methods: Escherichia coli rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting. Results: PCR was able to amplify the LipL32 gene from E. coli rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic Leptospira. SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in anti-rLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic Leptospira serovar but not with E. coli or Staphylococcus aureus. Conclusion: IgG anti-rLipL32 antibody has high specificity and sensitivity against Leptospira pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis.

Clostridium perfringens sialidase interaction with Neu5Ac α-Gal sialic acid receptors by in-silico observation and its impact on monolayers cellular behavior structure.

Objective: This study aims to evaluate the effect of Clostridium perfringens sialidase treatment on monolayer cell behavior using computational screening and an in vitro approach to demonstrate interaction between enzyme-based drugs and ligands in host cells. Materials and Methods: The in silico study was carried out by molecular docking analysis used to predict the interactions between atoms that occur, followed by genetic characterization of sialidase from a wild isolate. Sialidase, which has undergone further production and purification processes exposed to chicken embryonic fibroblast cell culture, and observations-based structural morphology of cells compared between treated cells and normal cells without treatment. Results: Based on an in silico study, C. perfringens sialidase has an excellent binding affinity with Neu5Acα (2.3) Gal ligand receptor with Gibbs energy value (∆G)-7.35 kcal/mol and Ki value of 4.11 µM. Wild C. perfringens isolates in this study have 99.1%-100% similarity to the plc gene, NanH, and NanI genes, while NanJ shows 93.18% similarity compared to the reference isolate from GenBank. Sialidase at 750 and 150 mU may impact the viability, cell count, and cell behavior structure of fibroblast cells by significantly increasing the empty area and perimeter of chicken embryo fibroblast (CEF) cells, while at 30 mU sialidase shows no significant difference compared with mock control. Conclusion: Sialidase-derived C. perfringens has the capacity to compete with viral molecules for attachment to host sialic acid based on in silico analysis. However, sialidase treatment has an impact on monolayer cell fibroblasts given exposure to high doses.

Development of a multiassay algorithm (MAA) to identify recent HIV infection in newly diagnosed individuals in Indonesia.

Ongoing HIV transmission is a public health priority in Indonesia. We developed a new multiassay algorithm (MAA) to identify recent HIV infection. The MAA is a sequential decision tree based on multiple biomarkers, starting with CD4+ T cells >200/µL, followed by plasma viral load (pVL) > 1,000 copies/ml, avidity index (AI) < 0 · 7, and pol ambiguity <0 · 47%. Plasma from 140 HIV-infected adults from 19 hospitals across Indonesia (January 2018 - June 2020) was studied, consisting of a training set (N = 60) of longstanding infection (>12-month) and a test set (N = 80) of newly diagnosed (≤1-month) antiretroviral (ARV) drug naive individuals. Ten of eighty (12 · 5%) newly diagnosed individuals were classified as recent infections. Drug resistance mutations (DRMs) against reverse transcriptase inhibitors were identified in two individuals: one infected with HIV subtype C (K219Q, V179T) and the other with CRF01_AE (V179D). Ongoing HIV transmission, including infections with DRMs, is substantial in Indonesia.

Accelerating Detection of Variants During COVID-19 Surges by Diverse Technological and Public Health Partnerships: A Case Study From Indonesia.

Early detection of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) variants and use of data for public health action requires a coordinated, rapid, and high throughput approach to whole genome sequencing (WGS). Currently, WGS output from many low- and middle-income countries (LMIC) has lagged. By fostering diverse partnerships and multiple sequencing technologies, Indonesia accelerated SARS-CoV-2 WGS uploads to GISAID from 1,210 in April 2021 to 5,791 in August 2021, an increase from 11 submissions per day between January to May, to 43 per day between June to August. Turn-around-time from specimen collection to submission decreased from 77 to 5 days, allowing for timely public health decisions. These changes were enabled by establishment of the National Genomic Surveillance Consortium, coordination between public and private sector laboratories with WGS capability, and diversification of sequencing platform technologies. Here we present how diversification on multiple levels enabled a rapid and significant increase of national WGS performance, with potentially valuable lessons for other LMICs.

Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model.

Background and Aim: Clostridium toxins are widely used as medicinal agents. Many active metabolic enzymes, including sialidase (neuraminidase), hyaluronidase, and collagenase, contribute to the mechanism of action of these toxins. Sialidase from Clostridium perfringens recognizes and degrades sialic acid receptors in the host cell glycoprotein, glycolipid, and polysaccharide complexes. Sialic acid promotes the adhesion of various pathogens, including viruses, under pathological conditions. This study aimed to investigate the potential of C. perfringens sialidase protein to inhibit Newcastle disease virus (NDV) infection in ovo model. Materials and Methods: C. perfringens was characterized by molecular identification through polymerase chain reaction (PCR) and is cultured in a broth medium to produce sialidase. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was conducted to characterize the sialidase protein. In contrast, enzymatic activity and protein concentration were carried out using a neuraminidase assay kit and Bradford to obtain suitable active substances. Furthermore, embryonated chicken egg models were used to observe the toxicity of several sialidase doses. Then, the hemagglutination (HA) titer was obtained, and absolute quantitative reverse transcription-PCR assay was performed to measure the viral replication inhibitory activity of sialidase against NDV. Results: Each isolate had a specific sialidase gene and its product. The sialidase derived from C. perfringens could hydrolyze the sialic acid receptor Neu5Ac (2,6)-Gal higher than Neu5Ac (2,3)Gal in chicken erythrocytes, as observed by enzyme-linked lectin assay. A significant difference (p = 0.05) in the HA titer in the pre-challenge administration group at dosages of 375 mU, 187.5 mU, and 93.75 mU in the competitive inhibition experiment suggests that sialidase inhibits NDV reproduction. Quantification of infective viral copy confirmed the interference of viral replication in the pre-challenge administration group, with a significant difference (p = 0.05) at the treatment doses of 750 mU, 375 mU, and 46.87 mU. Conclusion: The potency of sialidase obtained from C. perfringens was shown in this study, given its ability to reduce the viral titer and copy number in allantoic fluids without adversely impacting the toxicity of the chicken embryo at different concentrations.

Case series in Indonesia: B.1.617.2 (delta) variant of SARS-CoV-2 infection after a second dose of vaccine.

BACKGROUND: The B.1.617.2 (delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in Maharashtra in late 2020 and has rapidly expanded across India and worldwide. It took only 2 mo for this variant to spread in Indonesia, making the country the new epicenter of the delta variant as of July 2021. Despite efforts made by accelerating massive rollouts of current vaccines to protect against infection, cases of fully-vaccinated people infected with the delta variant have been reported. AIM: To describe the demographic statistics and clinical presentation of the delta variant infection after the second dose of vaccine in Indonesia. METHODS: A retrospective, single-centre case series of the general consecutive population that worked or studied at Faculty of Medicine, Universitas Indonesia with confirmed Delta Variant Infection after a second dose of vaccine from 24 June and 25 June 2021. Cases were collected retrospectively based on a combination of author recall, reverse transcription-polymerase chain reaction (RT-PCR), and whole genome sequencing results from the Clinical Microbiology Laboratory, Faculty of Medicine, Universitas Indonesia. RESULTS: Between 24 June and 25 June 2021, 15 subjects were confirmed with the B.1.617.2 (delta) variant infection after a second dose of the vaccine. Fourteen subjects were vaccinated with CoronaVac (Sinovac) and one subject with ChAdOx1 nCoV-19 (Oxford-AstraZeneca). All of the subjects remained in home isolation, with fever being the most common symptom at the onset of illness (n = 10, 66.67%). The mean duration of symptoms was 7.73 d (± 5.444). The mean time that elapsed from the first positive swab to a negative RT-PCR test for SARS-CoV-2 was 17.93 d (± 6.3464). The median time that elapsed from the second dose of vaccine to the first positive swab was 87 d (interquartile range: 86-128). CONCLUSION: Although this case shows that after two doses of vaccine, subjects are still susceptible to the delta variant infection, currently available vaccines remain the most effective protection. They reduce clinical manifestations of COVID-19, decrease recovery time from the first positive swab to negative swab, and lower the probability of hospitalization and mortality rate compared to unvaccinated individuals.

Correction: An observational prospective cohort study of the epidemiology of hospitalized patients with acute febrile illness in Indonesia.

[This corrects the article DOI: 10.1371/journal.pntd.0007927.].

Evaluation of inhibitor activity of bacterial sialidase from Clostridium perfringens against Newcastle disease virus in the cell culture model using chicken embryo fibroblast.

Objective: The Newcastle disease virus (NDV) is an infectious disease that causes very high economic losses due to decreased livestock production and poultry deaths. The vaccine's ineffectiveness due to mutation of the genetic structure of the virus impacts obstacles in controlling the disease, especially in some endemic areas. This study aimed to provide an alternative treatment for NDV infection by observing the viral replication inhibitor activity of Clostridium perfringens sialidase in primary chicken embryo fibroblast (CEF) cells. Materials and Methods: The virus was adapted in CEF monolayer cells, then collected thrice using the freeze-thaw method and stored at -20°C for the next step in the challenge procedure. C. perfringens crude sialidase was obtained, but it was further purified via stepwise elution in ion exchange using Q Sepharose® Fast Flow and affinity chromatography with oxamic acid agarose. The purified sialidase was tested for its toxicity, ability to breakdown sialic acid, stopping viral replication, and how treated cells expressed their genes. Results: According to this study, purified C. perfringens sialidase at dosages of 187.5, 93.75, and 46.87 mU effectively hydrolyzes CEF cells' sialic acid and significantly inhibits viral replication on the treated cells. However, sialidase dosages of 375 and 750 mU affected the viability of monolayer CEF cells. Interestingly, downregulation of toll-like receptor (TLR)3 and TLR7 (p < 0.05) in the sialidase-treated group indicates viral endocytosis failure. Conclusions: By stopping endocytosis and viral replication in host cells, sialidase from C. perfringens can be used as an alternative preventive treatment for NDV infection.

A multicentre surveillance study on the characteristics, bacterial aetiologies and in vitro antibiotic susceptibilities in patients with acute exacerbations of chronic bronchitis.

BACKGROUND AND OBJECTIVE: Antimicrobial resistance is a global problem and the prevalence is high in many Asian countries. METHODS: A prospective observational study of the prevalence of bacterial pathogens and their antimicrobial susceptibilities in patients with acute exacerbations of chronic bronchitis (AECB) was conducted in Indonesia, Philippines, Korea, Thailand, Malaysia, Taiwan and Hong Kong from August 2006 to April 2008. The diagnosis of AECB was based on increased cough and worsening of two of following: dyspnoea, increased sputum volume or purulence. Patients who had taken antibiotics within 72 h of presentation were excluded. All bacterial strains were submitted to a central laboratory for re-identification and antimicrobial susceptibility testing to 16 antimicrobial agents according to Clinical and Laboratory Standards Institute. RESULTS: Four hundred and seven isolates were identified among 447 patients of AECB. The most frequent organisms isolated were Klebsiella pneumoniae and associated species (n = 91 + 17), Haemophilus influenzae (n = 71), Pseudomonas aeruginosa (n = 63), Streptococcus pneumoniae (n = 32), Acinetobacter baumannii (n = 22) and Moraxella catarrhalis (n = 21). According to Clinical and Laboratory Standards Institute susceptibility breakpoints, 85.7% and >90% of these pathogens were susceptible to levofloxacin and cefepime respectively. Other options with overall lower susceptibilities include imipenem, ceftazidime, ceftriaxone and amoxicillin/clavulanate. CONCLUSIONS: Gram-negative bacteria including Klebsiella spp., P. aeruginosa and Acinetobacter spp. constitute a large proportion of pathogens identified in patients with AECB in some Asian countries. Surveillance on the local prevalence and antibiotic resistance of these organisms is important in guiding appropriate choice of antimicrobials in the management of AECB.