MERS-CoV seroconversion amongst Malaysian Hajj pilgrims returning from the Middle East, 2016-2018: results from the MERCURIAL multiyear prospective cohort study.

Prospective cohort study to investigate the potential exposure to the Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) following Hajj pilgrims is still very limited. Here, we report the antibody seroconversion study results obtained from successive three years cohort studies (2016-2018) involving the Malaysian Hajj pilgrims returning from the Middle East. A cohort study of Hajj pilgrims from Malaysia enrolled 2,863 participants from 2016-2018, all of whom consented to provide paired blood samples for both pre- and post-Hajj travel to the Middle East. ELISAs and micro-neutralization assays were performed to detect the presence of MERS-CoV IgG antibodies. Sociodemographic data, symptoms experienced during Hajj, and history of exposure to camels or camel products were recorded using structured pre- and post-Hajj questionnaires. A 4-fold increase in anti-MERS-CoV IgG between paired pre-Hajj and post-Hajj serum samples in twelve participants was observed. None of the twelve ELISA-positive sera had detectable levels of virus-neutralizing antibodies. All reportedly had mild symptoms of respiratory symptoms at a certain point during the pilgrimage, implying mild or asymptomatic infections. No association between post-Hajj serum positivity and a history of exposure to camels or camel products was obtained. Findings from the study suggest that serologic conversion to MERS-CoV occurred in at least 0.6% of the Hajj pilgrims returning from the Middle East. Since all the seroconvertants had mild to no symptoms during the sampling period, it highlights the likelihood of occurrence of only low infectivity spillover infections among the Hajj pilgrims.

Effectiveness of Dengue Awareness Calendar on Indigenous Population: Impact on Knowledge, Belief and Practice.

BACKGROUND: Dengue is prevalent among the indigenous community due to impoverished living conditions near the forest fringe areas and lack of health awareness. The study aims to determine the effect of a dengue awareness calendar on knowledge, belief, and practices (KBP) among the indigenous population. METHOD: A cross-sectional study was conducted in nine selected indigenous villages in Selangor, Malaysia. A dengue awareness calendar was distributed to the indigenous communities after pre-intervention. The KBP scores were compared between the pre-and post-intervention. RESULT: A total of 609 paired responses were obtained. Knowledge, perceived severity, cues to action, self-efficacy, and prevention practices were significantly improved after the intervention (p > 0.00). Participants with primary (Odds Ratio (OR) 2.627; 95% CI 1.338-5.160) and secondary level education (OR 2.263; 95% Cl 1.126-4.550) reported a high increment in practices score. High increments in dengue knowledge scores (OR 2.190; 95% CI 1.521-3.757, p < 0.00) were significantly more likely to report a high increment in practices score. Housewives (OR 0.535; 95% Cl 0.289-0.950), perceived severity (OR 0.349; 95% CI 0.184-0.662), and perceived susceptibility (OR 0.474; 95% CI 0.286-0.785) were significantly less likely to report an increment in prevention practices score. CONCLUSION: Findings inferred that the dengue awareness calendar significantly improved knowledge and practices. Our findings revealed the effectiveness of the dengue awareness calendar in dengue prevention among indigenous communities.

Epidemiological serosurvey of chikungunya fever post outbreak at Tanjung Sepat, Malaysia.

INTRODUCTION: Chikungunya fever is a mosquito-borne viral disease that usually presents with prominent arthralgia. An outbreak of chikungunya fever was reported in Tanjung Sepat, Malaysia in 2019. The outbreak was limited in size with a low number of cases being reported. The present study sought to determine the possible variables that could have affected the transmission of the infection. METHODOLOGY: A cross-sectional study involving 149 healthy adult volunteers from Tanjung Sepat was performed soon after the outbreak had subsided. All the participants donated blood samples and completed the questionnaires. Laboratory detection of anti-CHIKV IgM and IgG antibodies was performed using enzyme-linked immunoassays (ELISA). Risk factors associated with chikungunya seropositivity were determined using logistic regression. RESULTS: The majority (72.5%, n = 108) of the study participants tested positive for CHIKV antibodies. Only 8.3% (n = 9) of the participants out of all the seropositive volunteers had an asymptomatic infection. Participants who resided with a febrile (p < 0.05, Exp(B) = 2.2, confidence interval [CI] 1.3-3.6) or a CHIKV-diagnosed person (p < 0.05, Exp(B) = 2.1, CI 1.2-3.6) in the same household were found likely to be tested positive for CHIKV antibodies. CONCLUSIONS: Findings from the study support that asymptomatic CHIKV infections and indoor transmission occurred during the outbreak. Hence, widespread community testing and indoor use of mosquito repellent are among the possible measures that can be implemented to reduce CHIKV transmission during an outbreak.

Group IV Getah Virus in Culex Mosquitoes, Malaysia.

A new Getah virus (GETV) strain, B254, was isolated from Culex fuscocephalus mosquitoes captured at Mount Ophir, Malaysia, in 2012. Phylogenetic analyses revealed that GETV B254 is distinct from the old Malaysia GETV MM2021 strain but closely related to group IV GETV from Russia (LEIV16275Mag), China (YN12031), and Thailand (GETV/SW/Thailand/2017).

Emergence of B.1.524(G) SARS-CoV-2 in Malaysia during the third COVID-19 epidemic wave.

The COVID-19 pandemic first emerged in Malaysia in Jan 2020. As of 12th Sept 2021, 1,979,698 COVID-19 cases that occurred over three major epidemic waves were confirmed. The virus contributing to the three epidemic waves has not been well-studied. We sequenced the genome of 22 SARS-CoV-2 strains detected in Malaysia during the second and the ongoing third wave of the COVID-19 epidemic. Detailed phylogenetic and genetic variation analyses of the SARS-CoV-2 isolate genomes were performed using these newly determined sequences and all other available sequences. Results from the analyses suggested multiple independent introductions of SARS-CoV-2 into Malaysia. A new B.1.524(G) lineage with S-D614G mutation was detected in Sabah, East Malaysia and Selangor, Peninsular Malaysia on 7th October 2020 and 14th October 2020, respectively. This new B.1.524(G) group was not the direct descendant of any of the previously detected lineages. The new B.1.524(G) carried a set of genetic variations, including A701V (position variant frequency = 0.0007) in Spike protein and a novel G114T mutation at the 5'UTR. The biological importance of the specific mutations remained unknown. The sequential appearance of the mutations, however, suggests that the spread of the new B.1.524(G) lineages likely begun in Sabah and then spread to Selangor. The findings presented here support the importance of SARS-CoV-2 full genome sequencing as a tool to establish an epidemiological link between cases or clusters of COVID-19 worldwide.

Detecting dengue outbreaks in Malaysia using geospatial techniques.

Dengue is a complex disease with an increasing number of infections worldwide. This study aimed to analyse spatiotemporal dengue outbreaks using geospatial techniques and examine the effects of the weather on dengue outbreaks in the Klang Valley area, Kuala Lumpur, Malaysia. Daily weather variables including rainfall, temperature (maximum and minimum) and wind speed were acquired together with the daily reported dengue cases data from 2001 to 2011 and converted into geospatial format to identify whether there was a specific pattern of the dengue outbreaks. The association between these variables and dengue outbreaks was assessed using Spearman's correlation. The result showed that dengue outbreaks consistently occurred in the study area during a 11-year study period. And that the strongest outbreaks frequently occurred in two high-rise apartment buildings located in Kuala Lumpur City centre. The results also show significant negative correlations between maximum temperature and minimum temperature on dengue outbreaks around the study area as well as in the area of the high-rise apartment buildings in Kuala Lumpur City centre.

Multiyear prospective cohort study to evaluate the risk potential of MERS-CoV infection among Malaysian Hajj pilgrims (MERCURIAL): a study protocol.

INTRODUCTION: Middle East respiratory syndrome (MERS) is a viral respiratory infection caused by the MERS-CoV. MERS was first reported in the Kingdom of Saudi Arabia in 2012. Every year, the Hajj pilgrimage to Mecca attracts more than two million pilgrims from 184 countries, making it one of the largest annual religious mass gatherings (MGs) worldwide. MGs in confined areas with a high number of pilgrims' movements worldwide continues to elicit significant global public health concerns. MERCURIAL was designed by adopting a seroconversion surveillance approach to provide multiyear evidence of MG-associated MERS-CoV seroconversion among the Malaysian Hajj pilgrims. METHODS AND ANALYSIS: MERCURIAL is an ongoing multiyear prospective cohort study. Every year, for the next 5 years, a cohort of 1000 Hajj pilgrims was enrolled beginning in the 2016 Hajj pilgrimage season. Pre-Hajj and post-Hajj serum samples were obtained and serologically analysed for evidence of MERS-CoV seroconversion. Sociodemographic data, underlying medical conditions, symptoms experienced during Hajj pilgrimage, and exposure to camel and untreated camel products were recorded using structured pre-Hajj and post-Hajj questionnaires. The possible risk factors associated with the seroconversion data were analysed using univariate and multivariate logistic regression. The primary outcome of this study is to better enhance our understanding of the potential threat of MERS-CoV spreading through MG beyond the Middle East. ETHICS AND DISSEMINATION: This study has obtained ethical approval from the Medical Research and Ethics Committee (MREC), Ministry of Health Malaysia. Results from the study will be submitted for publication in peer-reviewed journals and presented in conferences and scientific meetings. TRIAL REGISTRATION NUMBER: NMRR-15-1640-25391.

Detection and confirmation of dengue pre- and postintroduction of dengue NS1-antigen test at the University Malaya Medical Centre: An observational study.

Early diagnosis of dengue is important to ensure proper management of patients and effective implementation of control measures. The present study was undertaken to determine the outcome of the implementation of dengue NS1-antigen (Ag) rapid diagnostic test (RDT) in the confirmation of dengue at the first patient hospital visit at the University Malaya Medical Centre. A total of 1036 and 1097 sera from the year 2008 and 2015 were used, representing samples from before and after dengue NS1-Ag RDT was implemented as routine diagnostic at the hospital. Results showed that similar dengue confirmation percentage (56%) was made in 2008 and 2015, regardless of the main laboratory diagnostic method used. Confirmation of dengue, however, increased to 68% and 73% when dengue NS1-Ag test or dengue immunoglobulin M-capture enzyme-linked immunosorbent assay was used as the second test for the 2008 and 2015 samples, respectively. Detection of dengue virus (DENV) using multiplex reverse transcription-polymerase chain reaction (RT-PCR) showed that DENV-1 was the highest in circulation in 2008 and that both DENV-1 and DENV-2 were dominant in 2015. In summary, the present study demonstrated that the introduction and use of the dengue NS1-Ag RDT did not change or compromise confirmation of dengue, highlighting the advantage of using the method. With the reducing cost of molecular detection tools, DENV detection using RT-PCR remains a viable option for further confirmation of dengue in hospital settings.

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages.

BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required. METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay. RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001). CONCLUSION: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Possible Factors Influencing the Seroprevalence of Dengue among Residents of the Forest Fringe Areas of Peninsular Malaysia.

Dengue is an endemic mosquito-borne viral disease prevalent in many urban areas of the tropic, especially the Southeast Asia. Its presence among the indigenous population of Peninsular Malaysia (Orang Asli), however, has not been well described. The present study was performed to investigate the seroprevalence of dengue among the Orang Asli (OA) residing at the forest fringe areas of Peninsular Malaysia and determine the factors that could affect the transmission of dengue among the OA. Eight OA communities consisting of 491 individuals were recruited. From the study, at least 17% of the recruited study participants were positive for dengue IgG, indicating past exposure to dengue. Analysis on the demographic and socioeconomic variables suggested that high seroprevalence of dengue was significantly associated with those above 13 years old and a low household income of less than MYR500 (USD150). It was also associated with the vast presence of residential areas and the presence of a lake. Remote sensing analysis showed that higher land surface temperatures and lower land elevations also contributed to higher dengue seroprevalence. The present study suggested that both demographic and geographical factors contributed to the increasing risk of contracting dengue among the OA living at the forest fringe areas of Peninsular Malaysia. The OA, hence, remained vulnerable to dengue.

Dengue fever among febrile patients in Taiz City, Yemen during the 2016 war: Clinical manifestations, risk factors, and patients knowledge, attitudes, and practices toward the disease.

The current war in Yemen has displaced millions of people from their homes into living in cramped shelters where the healthcare is limited. The breakdown of Yemen's healthcare and sanitation systems has facilitated the spread of infectious diseases including mosquito-borne diseases. The present study aimed to describe the prevalence of dengue virus (DENV) infection among the febrile patients of the Taiz governorate, Yemen as well as their knowledge, attitude and preventive practices (KAPs) regarding dengue fever (DF), and to investigate the factors associated with dengue preventive practices during the war. A total of 384 clinically dengue-suspected patients who sought health care in Taiz, Yemen during the period from July 2016 until October 2016 were recruited for the study. Serum samples were obtained and screened for the presence of DENV RNA and anti-DENV antibodies by reverse transcription-recombinase polymerase amplification (RT-RPA) and dengue IgM/IgG-capture ELISA, respectively. KAP questionnaires were obtained from all participants too. In the study, dengue was laboratory confirmed in approximately 49.3% (189/384) of the clinically suspected dengue patients. In general, 67.1% of the patients had low knowledge scores regarding DF. Low scores for knowledge about DF was significantly associated with those in the age groups of ≤20 years and 21-30 years, illiterates and patients with non-skilled jobs or jobless. The most common preventive practices reported by participants were covering stored water (78.6%) and putting a screen on the house's windows (65.3%). A low proportion of participants (6.7%) had 51-100% of good DF preventive practices. Low scores of positive attitudes toward DF was identified as a risk factor. The study participants showed poor knowledge about DF and their ways of dealing with the various aspects of DF prevention was quite limited, hence, preventive measures against the disease were less likely to be undertaken. Findings from the study highlight the peril of dengue in Taiz, Yemen, which is now comparable to that of endemic regions. The ongoing civil war with disruption in regular health services compounded by the low knowledge about DF as well as the limited DF preventive practices could result in entrenchment of dengue in Yemen.

Dengue Outbreak during Ongoing Civil War, Taiz, Yemen.

We identified dengue in ≈51% of patients given a clinical diagnosis of suspected dengue in Taiz, Yemen, during 2016. The cosmopolitan genotype of dengue virus type 2 was most common; viruses appeared to have originated in Saudi Arabia. Damage to public health infrastructure during the ongoing civil war might enable dengue to become endemic to Yemen.

Seroprevalence of Borrelia burgdorferi among the indigenous people (Orang Asli) of Peninsular Malaysia.

INTRODUCTION: Lyme disease has been well-described in the North America and European countries. However, information is still very limited in the developing countries including Malaysia. The Orang Asli (OA), the indigenous people of Peninsular Malaysia reside mostly in the forest and forest fringe areas abundant with the vector for Lyme disease. Here, we described the seroprevalence of Borellia burgdorferi (B. burgdorferi) among the OA and demographic variables that could be associated with seroprevalence. METHODOLOGY: A total of 16 OA villages distributed across 8 states in Peninsular Malaysia participated in this study. Sera obtained from 904 OA volunteers were screened for anti-B. burgdorferi IgG antibodies. ELISA results obtained and demographic information collected were analysed to identify possible variables associated with seroprevalence. RESULTS: A total of 73 (8.1%) OA tested positive for anti-B. burgdorferi IgG antibodies. Among all the variables examined, village of residence (p = 0.045) was the only significant predictor for seropositivity. High (> 10.0%) prevalence was associated with three OA villages. Those living in one particular village were 1.65 times more likely to be seropositive as compared to other OA villages. Age, gender, marital status, household size, level of education, monthly household income and occupation were not significant predictors for seropositivity. CONCLUSION: Results of the present study support earlier findings that B. burgdorferi infection among Malaysians is currently under-recognized. Further studies will be needed at these locations to confirm the presence of Lyme disease among these populations.

A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus.

Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.

Emergence of the Asian lineage dengue virus type 3 genotype III in Malaysia.

BACKGROUND: Dengue virus type 3 genotype III (DENV3/III) is associated with increased number of severe infections when it emerged in the Americas and Asia. We had previously demonstrated that the DENV3/III was introduced into Malaysia in the late 2000s. We investigated the genetic diversity of DENV3/III strains recovered from Malaysia and examined their phylogenetic relationships against other DENV3/III strains isolated globally. RESULTS: Phylogenetic analysis revealed at least four distinct DENV3/III lineages. Two of the lineages (DENV3/III-B and DENV3/III-C) are current actively circulating whereas the DENV3/III-A and DENV3/III-D were no longer recovered since the 1980s. Selection pressure analysis revealed strong evidence of positive selection on a number of amino acid sites in PrM, E, NS1, NS2a, NS2b, NS3, NS4a, and NS5. The Malaysian DENV3/III isolates recovered in the 1980s (MY.59538/1987) clustered into DENV3/III-B, which was the lineage with cosmopolitan distribution consisting of strains actively circulating in the Americas, Africa, and Asia. The Malaysian isolates recovered after the 2000s clustered within DENV3/III-C. This DENV3/III-C lineage displayed a more restricted geographical distribution and consisted of isolates recovered from Asia, denoted as the Asian lineage. Amino acid variation sites in NS5 (NS5-553I/M, NS5-629 T, and NS5-820E) differentiated the DENV3/III-C from other DENV3 viruses. The codon 629 of NS5 was identified as a positively selected site. While the NS5-698R was identified as unique to the genome of DENV3/III-C3. Phylogeographic results suggested that the recent Malaysian DENV3/III-C was likely to have been introduced from Singapore in 2008 and became endemic. From Malaysia, the virus subsequently spread into Taiwan and Thailand in the early part of the 2010s and later reintroduced into Singapore in 2013. CONCLUSIONS: Distinct clustering of the Malaysian old and new DENV3/III isolates suggests that the currently circulating DENV3/III in Malaysia did not descend directly from the strains recovered during the 1980s. Phylogenetic analyses and common genetic traits in the genome of the strains and those from the neighboring countries suggest that the Malaysian DENV3/III is likely to have been introduced from the neighboring regions. Malaysia, however, serves as one of the sources of the recent regional spread of DENV3/III-C3 within the Asia region.

Seroprevalence of Q Fever Among the Indigenous People (Orang Asli) of Peninsular Malaysia.

Q fever is a disease caused by Coxiella burnetii. It is a disease of public health concern in many parts of the world. In this study, we described the seroprevalence of Q fever among selected populations of Orang Asli (OA), indigenous people, many of whom live within the forest fringe areas of Peninsular Malaysia. Serum samples were obtained from 887 OA participants from selected villages. Samples were analyzed for the presence of IgG antibodies reactive against C. burnetii by enzyme-linked immunosorbent assay. Statistical methods were used to identify possible associations between seropositivity for C. burnetii and a number of demographic variables obtained from the questionnaires. In total, 9.6% (n = 85/887) of the serum samples were reactive to C. burnetii. Statistical results suggest that elderly male OA residing in OA village, Bukit Payung, were most likely to be tested seropositive for C. burnetii. This study suggests that OA are at a significant risk of contracting C. burnetii infection, and both demographic and geographic factors are important contributors to this risk. Further prospective studies are needed to establish the true burden of C. burnetii infection within the indigenous population as well as within Peninsular Malaysia as a whole.

Disruption of predicted dengue virus type 3 major outbreak cycle coincided with switching of the dominant circulating virus genotype.

Dengue is hyperendemic in most of Southeast Asia. In this region, all four dengue virus serotypes are persistently present. Major dengue outbreak cycle occurs in a cyclical pattern involving the different dengue virus serotypes. In Malaysia, since the 1980s, the major outbreak cycles have involved dengue virus type 3 (DENV3), dengue virus type 1 (DENV1) and dengue virus type 2 (DENV2), occurring in that order (DENV3/DENV1/DENV2). Only limited information on the DENV3 cycles, however, have been described. In the current study, we examined the major outbreak cycle involving DENV3 using data from 1985 to 2016. We examined the genetic diversity of DENV3 isolates obtained during the period when DENV3 was the dominant serotype and during the inter-dominant transmission period. Results obtained suggest that the typical DENV3/DENV1/DENV2 cyclical outbreak cycle in Malaysia has recently been disrupted. The last recorded major outbreak cycle involving DENV3 occurred in 2002, and the expected major outbreak cycle involving DENV3 in 2006-2012 did not materialize. DENV genome analyses revealed that DENV3 genotype II (DENV3/II) was the predominant DENV3 genotype (67%-100%) recovered between 1987 and 2002. DENV3 genotype I (DENV3/I) emerged in 2002 followed by the introduction of DENV3 genotype III (DENV3/III) in 2008. These newly emerged DENV3 genotypes replaced DENV3/II, but there was no major upsurge of DENV3 cases that accompanied the emergence of these viruses. DENV3 remained in the background of DENV1 and DENV2 until now. Virus genome sequence analysis suggested that intrinsic differences within the different dengue virus genotypes could have influenced the transmission efficiency of DENV3. Further studies and continuous monitoring of the virus are needed for better understanding of the DENV transmission dynamics in hyperendemic regions.

Penicillin-Susceptible, Oxidase-Negative, Nonhemolytic, Nonmotile Bacillus megaterium in Disguise of Bacillus anthracis.

Bacillus anthracis is a bacterial pathogen of major concern. The spores of this bacteria can survive harsh environmental conditions for extended periods and are well recognized as a potential bioterror weapon with significant implications. Accurate and timely identification of this Bacillus species in the diagnostic laboratory is essential for disease and public health management. Biosafety Level 3 measures and ciprofloxacin treatment were instituted when B. anthracis was suspected from a patient with gangrenous foot. 16S rDNA sequencing was performed to accurately identify the suspected bacterium, due to the superiority of this method to accurately identify clinically isolated bacteria. B. megaterium was identified as the causative agent and the organism was subsequently treated as a Biosafety Level 2 pathogen.

The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen.

Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.

Dengue death with evidence of hemophagocytic syndrome and dengue virus infection in the bone marrow.

INTRODUCTION: HPS is a potentially life-threatening histiocytic disorder that has been described in various viral infections including dengue. Its involvement in severe and fatal dengue is probably more common but is presently under recognized. CASE DESCRIPTION: A 38-year-old female was admitted after 5 days of fever. She was deeply jaundiced, leukopenic and thrombocytopenic. Marked elevation of transaminases, hyperbilirubinemia and hypoalbuminemia were observed. She had deranged INR values and prolonged aPTT accompanied with hypofibrinogenemia. She also had splenomegaly. She was positive for dengue IgM. Five days later she became polyuric and CT brain image showed gross generalized cerebral edema. Her conditions deteriorated by day 9, became confused with GCS of 9/15. Her BMAT showed minimal histiocytes. Her serum ferritin level peaked at 13,670.00 µg/mL and her sCD163 and sCD25 values were markedly elevated at 4750.00 ng/mL and 4191.00 pg/mL, respectively. She succumbed to the disease on day 10 and examination of her tissues showed the presence of dengue virus genome in the bone marrow. DISCUSSION AND EVALUATION: It is described here, a case of fatal dengue with clinical features of HPS. Though BMAT results did not show the presence of macrophage hemophagocytosis, other laboratory features were consistent with HPS especially marked elevation of ferritin, sCD163 and sCD25. Detection of dengue virus in the patient's bone marrow, fifteen days after the onset of fever was also consistent with the suggestion that the HPS is associated with dengue virus infection. CONCLUSIONS: The findings highlight HPS as a possible complication leading to severe dengue and revealed persistent dengue virus infection of the bone marrow. Detection of HPS markers; ferritin, sCD163 and sCD25, therefore, should be considered for early recognition of HPS-associated dengue.