Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.

Rickettsia felis has recently emerged worldwide as a cause of human illness. Typically causing mild, undifferentiated fever, it has been implicated in several cases of non-fatal neurological disease in Mexico and Sweden. Its distribution and pathogenicity in Southeast Asia is poorly understood. We retroactively tested cerebrospinal fluid (CSF) or sera from 64 adult patients admitted to hospital in North Sulawesi, Indonesia with acute neurological disease. Rickettsia felis DNA was identified in the CSF of two fatal cases of meningoencephalitis using multi-locus sequence typing semi-nested PCR followed by Sanger sequencing. DNA from both cases had 100% sequence homologies to the R. felis reference strain URRWXCal2 for the 17-kDa and ompB genes, and 99.91% to gltA. The identification of R. felis in the CSF of two fatal cases of meningoencephalitis in Indonesia suggests the distribution and pathogenicity of this emerging vector-borne bacteria might be greater than generally recognized. Typically Rickettsia are susceptible to the tetracyclines and greater knowledge of R. felis endemicity in Indonesia should lead to better management of some acute neurological cases. Rickettsia felis is a flea-borne bacteria that has recently emerged worldwide as a cause of disease. Little is known about its distribution or details of transmission but typically it has been described as causing mild, self-limiting, febrile illness. It has, however, been incriminated in Mexico and Sweden of causing acute neurological disease. We report from North Sulawesi, Indonesia evidence that R. felis caused or contributed to the death of two adult men admitted to hospital with progressive meningoencephalitis. One case was a farmer and the other was an itinerant urban salesman. DNA unique to R. felis was identified in the patients’ cerebrospinal fluid using Sanger sequencing techniques. No other potential pathogens were found. These appear to be the first possible cases of fatal R. felis reported. Because rickettsial infections can usually be quickly, cheaply, and effectively treated with doxycycline and related tetracylines, it is important for clinicians in the region to consider using these antibiotics to treat suspect cases presumptively.

: Chikungunya virus (CHIKV) is recognized but rarely considered as a cause of central nervous system infection in endemic areas. A total of 244 patients with acute meningoencephalitis in Indonesia were retrospectively tested to identify whether any CHIKV infection was associated with neurological manifestations, especially in provinces known for CHIKV endemicity. Cerebrospinal fluid (CSF) and blood specimens were tested using CHIKV-specific real-time reverse transcription polymerase chain reaction and IgM ELISA, alongside a panel of neurotropic viruses. We report four cases of suspected or confirmed CHIKV-associated neurological disease, including CHIKV RNA detection in CSF of one patient and in acute serum of another, and CHIKV IgM in CSF of three patients and in serum of a fourth. In conclusion, CHIKV should be considered as a cause of neurologic disease in endemic areas and especially during outbreaks, in addition to the more common arboviral diseases such as dengue and Japanese encephalitis viruses.

Background: Dengue virus (DENV) is hyperendemic in Indonesia and of high public health concern globally. Without an effective antiviral therapy, an accurate diagnosis of the four viruses, DENV1-4, is crucial for patient management and outcome. We aim to assess the diagnostic accuracy and usefulness of a commercial DENV antigen rapid diagnostic test kit in point-of-care conditions. Design and methods: Six-hundred-forty-six patients with fever or history of fever, and without leukocytosis, were enrolled from Tabanan General Hospital, Bali in 2017–2018. All sera were tested for DENV NS1 antigen using the Standard Diagnostics Bioline NS1 Ag rapid test kit and Flavivirus genus specific RT-PCR. Positive specimens were further confirmed using DENV multiplex RT-PCR. Results: DENV was detected in 10.4% (67/646) of enrolled patients by molecular assays. Only 41 of the positives were found to be positive by the NS1 rapid test. Our findings indicate that the kit was the most sensitive in the first 2 days after disease onset with sensitivity > 75%, and declining at day 3 with a sensitivity of <60%. In addition, the kit was found to be the most sensitive to DENV-2 and least sensitive to DENV-4. Overall, the kit had a sensitivity of 59.7% [95% CI: 44.5–74.9] and specificity of 99.8% [95% CI: 99.5–100.0]. Conclusions: Our evaluation indicated that the rapid diagnostic test is useful for both surveillance and initial detection of DENV infection in community settings. However, it should be supplemented with additional diagnostic methods to ensure accurate confirmation for patient management.

Central nervous system involvement of dengue virus is increasingly reported from endemic areas. This study describes the clinical characteristics and laboratory features of a pediatric patient enrolled in a central nervous system illness study conducted in 2017–2018 to identify viral and bacterial etiologies in Indonesian children. Dengue diagnostics including molecular and serological testing were performed on an encephalitis patient who presented with both classical dengue and neurological clinical symptoms. Dengue virus serotype 1 RNA was detected in both cerebrospinal fluid and serum by serotype-specific reverse transcription polymerase chain reaction, and the E gene was successfully sequenced. Anti-dengue virus immunoglobulin M was detected in both admission and discharge sera, whereas anti-dengue virus immunoglobulin G was identified only in the discharge serum. This study describes the central nervous system complications in a case with dengue virus infection in West Java, Indonesia, and highlights the potential for dengue virus serotype 1, a serotype rarely associated with neurotropism, to cause encephalitis.

The role of microRiboNucleic Acids (miRNA), a small-non coding RNA has been associated with immune regulation in various viral infectionincluding dengue infection. The microRNA will bind a specific protein target in order to encourage an explosive expression of various cytokines, known as cytokines storm in Dengue infection.The objective of this study aimed to determine and evaluate themicroRNAs profile expression withinperipheral blood mononuclear cells having been infected with one of the dengue virus serotype.To obtained the PBMCs from a healthy donor, Ficoll density gradient centrifugation was used to isolate the PBMCs and then followed infecting it with a DENV-2 clinical isolate. Prior to PBMCs isolation, the virus has been propagated and having titration to get an optimal virus titer. We conducted the infection at the multiplication of infections 4 PFU/106 cells.MiRCURYLNATMExiqon was utilized on purpose to extract the RNA. Quantitative Real-Time PCR was applied in order for the miRNAs relative expression to be measured. The preliminary result reveals that miR-150, miR-146a, hsa-let-7e expression were increased 1.74 folds, 2 folds, and 1.49 foldsrespectively at 12 hours post-infection on PBMCs upon DENV-2 infection.The expression of microRNAswas discovered to behigher inPBMCsat the time of infection withDENV-2.ThemiRNAs expression in the uninfected PMBCs was lower than that of the miRNA. This high expression of miRNAsin dengue infection may proceedto dengue infection pathogenesis.