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.

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.

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.

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.

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.

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.

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.].

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.

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.

Prevalence of HIV Infection and Resistance Mutations in Patients Hospitalized for Febrile Illness in Indonesia.

HIV prevalence in Indonesia is increasing, and only 64% of infected individuals know their status. In a prospective cohort of 1,453 hospitalized patients with unexplained fever, 46 (3.2%) had HIV, including 15 (1.1%) patients without a prior HIV diagnosis. Among 31 subjects previously known to have HIV, 21 (68%) had been receiving combination antiretroviral therapy (cART) at the time of enrollment. Of 39 HIV cases with HIV RNA levels ≥ 100 copies/mL, sequencing for genotype analysis and resistance testing was successful in 30 (77%) subjects. The most common HIV subtypes were AE (90%) and B (10%). Five (16.7%) subjects had resistance mutations to nucleoside and non-nucleoside reverse transcriptase inhibitors, and all of them were on cART. No evidence of transmitted drug resistance was found in newly diagnosed individuals. Hospital-based screening may be an efficient method to expand HIV testing and identify a significant number of new cases. Access to care, close monitoring, expansion of anti-retroviral options, and ensuring availability of CD4 determinations, viral load testing, and genotyping are crucial to control of the epidemic in Indonesia.

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.

Etiologies of severe acute respiratory infection (SARI) and misdiagnosis of influenza in Indonesia, 2013-2016.

BACKGROUND: Severe acute respiratory infection (SARI) accounts for a large burden of illness in Indonesia. However, epidemiology of SARI in tertiary hospitals in Indonesia is unknown. This study sought to assess the burden, clinical characteristics, and etiologies of SARI and concordance of clinical diagnosis with confirmed etiology. METHODS: Data and samples were collected from subjects presenting with SARI as part of the acute febrile Illness requiring hospitalization study (AFIRE). In tertiary hospitals, clinical diagnosis was ascertained from chart review. Samples were analyzed to determine the "true" etiology of SARI at hospitals and Indonesia Research Partnership on Infectious Diseases (INA-RESPOND) laboratory. Distribution and characteristics of SARI by true etiology and accuracy of clinical diagnosis were assessed. RESULTS: Four hundred and twenty of 1464 AFIRE subjects presented with SARI; etiology was identified in 242 (57.6%), including 121 (28.8%) viruses and bacteria associated with systemic infections, 70 (16.7%) respiratory bacteria and viruses other than influenza virus, and 51 (12.1%) influenza virus cases. None of these influenza patients were accurately diagnosed as having influenza during hospitalization. CONCLUSIONS: Influenza was misdiagnosed among all patients presenting with SARI to Indonesian tertiary hospitals in the AFIRE study. Diagnostic approaches and empiric management should be guided by known epidemiology. Public health strategies to address the high burden of influenza should include broad implementation of SARI screening, vaccination programs, clinician education and awareness campaigns, improved diagnostic capacity, and support for effective point-of-care tests.

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.

Chikungunya in Indonesia: Epidemiology and diagnostic challenges.

BACKGROUND: Chikungunya virus (CHIKV) is often overlooked as an etiology of fever in tropical and sub-tropical regions. Lack of diagnostic testing capacity in these areas combined with co-circulation of clinically similar pathogens such as dengue virus (DENV), hinders CHIKV diagnosis. To better address CHIKV in Indonesia, an improved understanding of epidemiology, clinical presentation, and diagnostic approaches is needed. METHODOLOGY/PRINCIPAL FINDINGS: Acutely hospitalized febrile patients ≥1-year-old were enrolled in a multi-site observational cohort study conducted in Indonesia from 2013 to 2016. Demographic and clinical data were collected at enrollment; blood specimens were collected at enrollment, once during days 14 to 28, and three months after enrollment. Plasma samples negative for DENV by serology and/or molecular assays were screened for evidence of acute CHIKV infection (ACI) by serology and molecular assays. To address the co-infection of DENV and CHIKV, DENV cases were selected randomly to be screened for evidence of ACI. ACI was confirmed in 40/1,089 (3.7%) screened subjects, all of whom were DENV negative. All 40 cases initially received other diagnoses, most commonly dengue fever, typhoid fever, and leptospirosis. ACI was found at five of the seven study cities, though evidence of prior CHIKV exposure was observed in 25.2% to 45.9% of subjects across sites. All subjects were assessed during hospitalization as mildly or moderately ill, consistent with the Asian genotype of CHIKV. Subjects with ACI had clinical presentations that overlapped with other common syndromes, atypical manifestations of disease, or persistent or false-positive IgM against Salmonella Typhi. Two of the 40 cases were possibly secondary ACI. CONCLUSIONS/SIGNIFICANCE: CHIKV remains an underdiagnosed acute febrile illness in Indonesia. Public health measures should support development of CHIKV diagnostic capacity. Improved access to point-of-care diagnostic tests and clinical training on presentations of ACI will facilitate appropriate case management such as avoiding unneccessary treatments or antibiotics, early response to control mosquito population and eventually reducing disease transmission.

Whole-genome sequencing analysis of multidrug-resistant Mycobacterium tuberculosis from Java, Indonesia.

Introduction. Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem globally, including in Indonesia. Whole-genome sequencing (WGS) analysis has rarely been used for the study of TB and MDR-TB in Indonesia.Aim. We evaluated the use of WGS for drug-susceptibility testing (DST) and to investigate the population structure of drug-resistant Mycobacterium tuberculosis in Java, Indonesia.Methodology. Thirty suspected MDR-TB isolates were subjected to MGIT 960 system (MGIT)-based DST and to WGS. Phylogenetic analysis was done using the WGS data. Results obtained using MGIT-based DST and WGS-based DST were compared.Results. Agreement between WGS and MGIT was 93.33 % for rifampicin, 83.33 % for isoniazid and 76.67 % for streptomycin but only 63.33 % for ethambutol. Moderate WGS-MGIT agreement was found for second-line drugs including amikacin, kanamycin and fluoroquinolone (73.33-76.67 %). MDR-TB was more common in isolates of the East Asian Lineage (63.3%). No evidence of clonal transmission of DR-TB was found among members of the tested population.Conclusion. Our study demonstrated the applicability of WGS for DST and molecular epidemiology of DR-TB in Java, Indonesia. We found no transmission of DR-TB in Indonesia.

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.

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.

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

BACKGROUND: The epidemiology of acute febrile illness, a common cause of hospitalization in Indonesia, has not been systematically studied. METHODOLOGY/PRINCIPAL FINDINGS: This prospective observational study enrolled febrile patients (temperature ≥38°C) aged ≥1 year from July 2013 until June 2016 at eight government referral teaching hospitals in seven provincial capitals in Indonesia. Patients were managed according to the hospital standard-of-care (SOC), and blood samples were drawn for molecular and serological assays. Clinical data, laboratory results, and specimens for additional tests were collected at enrollment, days 14-28, and at three months. Regular follow-up visits were then scheduled for every three months either until symptoms resolved or until one year. In total, this study included 1,486 adult and pediatric patients presenting with multi-organ (768, 51.7%), gastrointestinal (497, 33.0%), respiratory (114, 7.7%), constitutional (62, 4.2%), skin and soft-tissue (24, 1.6%), central nervous system (17, 1.1%), or genitourinary (4, 0.3%) manifestations. Microbiological diagnoses were found in 1,003/1,486 (67.5%) participants, of which 351/1,003 (35.0%) were not diagnosed during hospitalization using SOC diagnostic tests. Missed diagnoses included all cases caused by Rickettsia spp., chikungunya, influenza, and Seoul virus. The most common etiologic agents identified were dengue virus (467, 46.6%), Salmonella spp. (103, 10.3%), and Rickettsia spp. (103, 10.3%). The overall mortality was 89 (5.9%). CONCLUSIONS/SIGNIFICANCE: Febrile illness in Indonesia has various microbiologic etiologies and substantial overall mortality. Diagnostic limitations and lack of epidemiologic data resulted in potentially treatable, and at times fatal, diseases being missed.

Comparison of Commercial Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay for Diagnosis of Acute Rickettsia typhi Infections.

Murine typhus is a tropical disease caused by Rickettsia typhi and is endemic in resource-limited settings such as Southeast Asian countries. Early diagnosis of R. typhi infection facilitates appropriate management and reduces the risk of severe disease. However, molecular detection of R. typhi in blood is insensitive due to low rickettsemia. Furthermore, the gold standard of sero-diagnosis by immunofluorescence assay (IFA) is cumbersome, subjective, impractical, and unavailable in many endemic areas. In an attempt to identify a practical diagnostic approach that can be applied in Indonesia, we evaluated the performance of commercial R. typhi IgM and IgG enzyme-linked immunosorbent assay (ELISA) and IFA using paired plasma from previously studied R. typhi PCR-positive cases and controls with other known infections. Sensitivity and specificity of combined ELISA IgM and IgG anti-R. typhi using paired specimens were excellent (95.0% and 98.3%, respectively), comparable to combined IFA IgM and IgG (97.5% and 100%, respectively); sensitivity of ELISA IgM from acute specimens only was poor (45.0%), but specificity was excellent (98.3%). IFA IgM was more sensitive (77.5%), but less specific (89.7%) for single specimens.

Characterization and Synergistic Antimicrobial Evaluation of Lipopeptides from Bacillus amyloliquefaciens Isolated from Oil-Contaminated Soil.

Lipopeptides show great potential for biomedical application. Several lipopeptides exhibit narrow and broad-spectrum inhibition activities. The aim of the study is to characterize the lipopeptides produced by B. amyloliquefaciens strain MD4-12 and evaluate the synergistic antimicrobial activity in combination with a conventional antibiotic against Gram-negative bacteria. B. amyloliquefaciens strain MD4-12 was isolated from oil-contaminated soil. The isolate was cultivated in McKeen medium, and the lipopeptides were isolated by precipitation and extraction with methanol. Characterization of the lipopeptides by ESI-MS gave nine mass ion peaks with m/z 994-1072, resulted from protonating of the main ions in [M + H]+ and [M + Na]+ ion form. These mass ion peaks attributed to surfactin homologs. By tandem mass spectrometry, five variants of surfactin with the same amino acid sequence in peptide moiety could be revealed. The peptide moiety contains seven amino acids identified as Glu-Leu/Ile-Leu-Val-Asp-Leu-Leu/Ile while the fatty acid moiety comprises a different length of chain from C12 to C16. Surfactin showed antibacterial activity against various Gram-positive and Gram-negative bacteria. Combination surfactin with ampicillin showed a synergistic effect against P. aeruginosa ATCC 27853.