Dengue encephalitis: A rare manifestation of dengue fever.

Dengue fever, caused by the dengue virus transmitted by mosquitoes, usually manifests as flu-like symptoms and is a prevalent tropical illness. However, there are rare cases where the infection takes an unusual course, resulting in severe complications like dengue encephalitis. This case report delineates an occurrence of dengue encephalitis in a patient from Sri Lanka. This work provides insights into the clinical presentation, diagnostic difficulties, and treatment approaches linked to this uncommon manifestation of dengue fever.

Laboratory response to an infant with suspected measles vaccine associated fever and rash in Sri Lanka.

Background: Measles is a highly contagious illness. Sri Lanka (SL) has eliminated the measles in 2019. The country is at risk of importation of measles and there could be vaccine-associated measles like illnesses. Therefore, it is important to investigate patients with fever, rash to differentiate the wild-type from vaccine-type excluding other suspected pathogens to direct infection prevention and control strategies. The objective is to describe the laboratory investigation procedure in an immunocompetent child, developed fever, rash following measles containing vaccine in post-measles eliminated period, SL. Methods: This laboratory based investigation was carried out in National Measles Laboratory, SL. Blood and throat swab were received from a patient with fever, rash, cough and coryza developed at tenth day of receiving the measles containing live-attenuated vaccine. Samples were tested for measles, rubella, and other relevant pathogens according to the laboratory testing algorithm for an immunocompetent child with fever, rash and flu like symptoms. Results: Measles vaccine type A, Edmonston-strain virus was detected after sequencing in throat swab and measles IgM and IgG were positive at sixth-week of illness-onset. In addition, influenza A RNA was detected in throat swab at day-three with detectable parvoB19 IgM in blood sample received at sixth-week of post-onset symptoms. Conclusions: Measles like illness of this immunocompetent child who received measles containing vaccine could be due to measles vaccine-type A or influenza infection. In a measles eliminated, resource-limited setting in SL, there should be a well-defined, testing algorithm to exclude prevalent possible pathogens according to epidemiological and clinical information.

The first laboratory-confirmed neonatal Mpox infection in Sri Lanka.

In 2022-2023, a global outbreak of Mpox was reported especially in nonendemic countries. We report the first laboratory-confirmed neonatal case of Mpox infection complicated by bronchopneumonia in Sri Lanka.

Dengue Virus and Yellow Fever Virus Detection Using Reverse Transcription-Insulated Isothermal PCR and Comparison with Real-Time RT-PCR.

Real-time reverse transcriptase PCR (rRT-PCR) is the most accurate method for the detection of dengue virus (DENV) and yellow fever virus (YFV) in acute illness. However, performing rRT-PCR is not feasible for many laboratories in regions of endemicity. The current study compared new reverse transcription-insulated isothermal PCRs (the POCKIT DENV and YFV reagent sets) with laboratory-developed rRT-PCRs for both viruses using clinical samples and viral strains from different endemic regions. Sensitivity and specificity of the POCKIT DENV Reagent Set were 87.2% (68/78 samples) and 98.2% of samples (54/55), respectively. The YFV reagent set demonstrated sensitive detection of YFV RNA from six viral strains down to an estimated concentration of 2.5 log10 copies/mL and proved to be specific for YFV. Although the POCKIT assays require RNA extraction, they may provide accurate and less-complex options for molecular testing in laboratory settings where rRT-PCR is not practical.

The laboratory investigation of a measles outbreak in the eve of its elimination in Sri Lanka.

BACKGROUND: Measles is highly contagious and cause significant morbidity and mortality. Sri-Lanka has the goal to eliminate endogenous measles by 2020 in par with WHO. OBJECTIVE: To describe laboratory confirmation and genotype distribution of measles cases during the outbreak occurred from mid-March to May 2019, Sri-Lanka STUDY DESIGN: This retrospective study was conducted at National Measles Reference Laboratory (NMRL), Sri-Lanka. All samples received were tested according to the testing flow chart at NMRL with WHO recommended kits. Blood samples were tested for anti-measles IgM and IgG with IgG avidity for IgG positives. Samples within 5days post-onset rash were tested with measles real-time RT-PCR. Products of genotyping PCR were sent to Regional Reference Laboratory, Thailand for sequencing. Subsequent phylogenetic analysis was done at NMRL. Data were analyzed by descriptive statistics. RESULTS: A total of 182 blood and 46 throat/nasopharyngeal swabs were received from 195 suspected cases and 37(19 %) were positive for measles by anti-measles IgM, rRT-PCR or both. Majority was females, with mean age of 20 years. Cases represented three main geographical areas; Western-35 %, Central-32 % and Southern-27 %. High avidity IgG was detected in 27/37(73 %). Sequencing data of six cases (4 from Western and 2 from Central province) revealed genotype D8. CONCLUSION: Nineteen percent of the suspected patients were measles positive with 73 % having re-infections. Majority were 22 years or over. Measles genotype was D8 in two provinces, suggesting the spread of virus within the country. Laboratory confirmation with measles PCR; IgG avidity and sequencing/genetic analysis is critical in the verge of measles elimination.

Rotavirus infection among hospitalized children under five years of age with acute watery diarrhea in Sri Lanka.

BACKGROUND: Rotavirus is the leading cause of acute watery diarrhoea among children and is vaccine preventable. The aim of this hospital-based sentinel surveillance was to study the prevalence, demographic and clinical characteristics of rotavirus infections and to describe rotavirus genotype distribution patterns among children under five years of age hospitalized for acute watery diarrhea during the period of 2009-2016. METHODS: Prospective, sentinel hospital-based surveillance was conducted in Lady Ridgeway Hospital (LRH) from 2009 to 2016. Stool samples of children admitted with acute watery diarrhea were tested by rotavirus antigen detection 'ProSpecT' Enzyme Immunoassay (EIA) at Department of Virology, Medical Research Institute, Colombo. Specimens that tested positive for rotavirus were further analyzed at the Regional Reference Laboratory (RRL) participating in the World Health Organization (WHO)-coordinated Global Rotavirus Surveillance Network (GRSN) to determine the genotype of strains by reverse-transcriptase polymerase chain reaction. RESULTS: Of the 6090 children with diarrhea admitted, 1801 (29.5%) had stools taken and tested. In years with at least 11 months of data (2010 and 2013) rotavirus was detected in 36.5% (228/624) of specimens. Genotype G1P[8] was the most common genotype detected throughout the surveillance period (30.1%; 123/408) with G2P [8], G9P[8] and G3P[8] also detected. CONCLUSIONS: Rotavirus is a common cause of pediatric diarrhea hospitalizations in Sri Lanka. National introduction of rotavirus vaccine could reduce the burden of pediatric diarrhea.

Characterization of Dengue Virus Infections Among Febrile Children Clinically Diagnosed With a Non-Dengue Illness, Managua, Nicaragua.

Background: We sought to characterize dengue virus (DENV) infections among febrile children enrolled in a pediatric cohort study who were clinically diagnosed with a non-dengue illness ("C cases"). Methods: DENV infections were detected and viral load quantitated by real-time reverse transcription-polymerase chain reaction in C cases presenting between January 2007 and January 2013. Results: One hundred forty-one of 2892 C cases (4.88%) tested positive for DENV. Of all febrile cases in the study, DENV-positive C cases accounted for an estimated 52.0% of patients with DENV viremia at presentation. Compared with previously detected, symptomatic dengue cases, DENV-positive C cases were significantly less likely to develop long-lasting humoral immune responses to DENV, as measured in healthy annual serum samples (79.7% vs 47.8%; P < .001). Humoral immunity was associated with viral load at presentation: 40 of 43 patients (93.0%) with a viral load ≥7.0 log10 copies/mL serum developed the expected rise in anti-DENV antibodies in annual samples versus 13 of 68 (19.1%) patients with a viral load below this level (P < .001). Conclusions: Antibody responses to DENV-positive C cases differ from responses to classic symptomatic dengue. These findings have important implications for DENV transmission modeling, immunology, and epidemiologic surveillance.

Diagnosis of Zika virus infection on a nanotechnology platform.

We developed a multiplexed assay on a plasmonic-gold platform for measuring IgG and IgA antibodies and IgG avidity against both Zika virus (ZIKV) and dengue virus (DENV) infections. In contrast to IgM cross-reactivity, IgG and IgA antibodies against ZIKV nonstructural protein 1 (NS1) antigen were specific to ZIKV infection, and IgG avidity revealed recent ZIKV infection and past DENV-2 infection in patients in dengue-endemic regions. This assay could enable specific diagnosis of ZIKV infection over other flaviviral infections.

Homotypic Dengue Virus Reinfections in Nicaraguan Children.

BACKGROUND: Infection with any of the 4 related dengue virus serotypes (DENV-1-4) is thought to result in lifelong immunity to homotypic reinfection (ie, reinfection with the same serotype). METHODS: Archived serum samples collected as part of an ongoing pediatric dengue cohort study in Nicaragua were tested for DENV by real-time reverse transcription polymerase chain reaction. Samples were collected from 2892 children who presented with an acute febrile illness clinically attributed to a non-DENV cause (hereafter, "C cases"). Test results were added to a database of previously identified symptomatic dengue cases in the cohort to identify repeat infections. RESULTS: Four patients with homotypic DENV reinfections were identified and confirmed among 29 repeat DENV infections (13.8%) with serotype confirmation. Homotypic reinfections with DENV-1, DENV-2, and DENV-3 occurred 325-621 days after the initial infection. Each patient experienced 1 symptomatic dengue case and 1 DENV-positive C case, and 2 patients presented with symptomatic dengue during their second infection. These DENV-positive C cases did not elicit long-lived humoral immune responses, despite viremia levels of up to 6.44 log10 copies per mL of serum. CONCLUSIONS: We describe the first set of virologically confirmed homotypic DENV reinfections. Such cases challenge the current understanding of DENV immunity and have important implications for modeling DENV transmission.

Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection.

BACKGROUND: Dengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection. OBJECTIVES: In the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV-CHIKV rRT-PCR). STUDY DESIGN: From an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV-CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua. RESULTS: The pan-DENV-CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10copies/µL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/µL. The pan-DENV-CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples). CONCLUSIONS: The single-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.

Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis.

BACKGROUND: Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. METHODOLOGY/PRINCIPAL FINDINGS: For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. CONCLUSIONS/SIGNIFICANCE: The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

Commutability of the Epstein-Barr virus WHO international standard across two quantitative PCR methods.

The commutability of international reference standards is critical for ensuring quantitative agreement across different viral load assays. Here, we demonstrate the commutability of the Epstein-Barr virus (EBV) WHO international standard for the BamHI-W and artus EBV assays.

Multiplex nucleic acid amplification test for diagnosis of dengue fever, malaria, and leptospirosis.

Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.

Comparison of the FDA-approved CDC DENV-1-4 real-time reverse transcription-PCR with a laboratory-developed assay for dengue virus detection and serotyping.

Dengue virus (DENV) is the agent of the most common vector-borne disease worldwide. Using 199 clinical samples collected from Nicaragua and Sri Lanka, a laboratory-developed DENV multiplex real-time reverse transcription-PCR (rRT-PCR) proved more clinically sensitive than the FDA-approved CDC assay for DENV serotypes 1 to 4 when measured against a composite reference standard, with sensitivities of 97.4% versus 87.1%, respectively.

Development of an internally controlled real-time reverse transcriptase PCR assay for pan-dengue virus detection and comparison of four molecular dengue virus detection assays.

A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic acid amplification tests (NAATs). However, reports describing a direct comparison of different NAATs have been limited. In this study, we report the design of an internally controlled real-time reverse transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay, a commercially produced, internally controlled DENV rRT-PCR (the Altona assay), a widely used heminested RT-PCR, and a serotype-specific multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 1.0 to 7.0 log10 cDNA equivalents/µl and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/µl, depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved to be more clinically sensitive than either the Altona or heminested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (P ≤ 0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day 5 of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed molecular diagnosis of DENV infection can be provided.

Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses.

BACKGROUND: Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction. METHODOLOGY/PRINCIPAL FINDINGS: An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log10 cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested. CONCLUSIONS/SIGNIFICANCE: This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.