Diagnostic performances of four commercially available assays for the identification of SARS-CoV-2, influenza type A/B virus and RSV.

We evaluated the diagnostic performance of 4 commercially NAAT for detecting SARS-CoV-2 RNA, Influenza type A/B virus and RSV. Included tests were the Allplex™ SARS-CoV-2 fast PCR Assay (RNA extraction-free), Allplex™ RV Master Assay, Allplex™ SARS-CoV-2 fast MDx Assay (LAMP) and Aptima™ SARS-CoV-2/Flu Assay (RT-TMA). The assays' performance characteristics were determined using nasopharyngeal swabs from 270 patients with suspected SARS-CoV-2 infection. A total of 215 SARS-CoV-2 positive, 55 negative nasopharyngeal swabs and 19 bacteria strains were included. The sensitivities and specificities for detecting SARS-CoV-2, Influenza type A virus and RSV ranged between 81.8% and 100% with extremely good agreements (κ ≥ 86.8 %). The Aptima™ SARS-CoV-2/Flu Assay introduced a new result parameter, that is, TTime. Here, we showed that TTime may be used as a surrogate for Ct-value. We concluded that all assays assessed in this study can be used for routine detection of SARS-CoV-2, Influenza type A virus and RSV.

Epidemiology of Mycoplasma genitalium and Trichomonas vaginalis in the primary health care setting in the Netherlands.

The aim of this paper is to describe the prevalence of Mycoplasma genitalium and Trichomonas vaginalis in patients who visited general practitioners in the Netherlands. Additionally, we describe the prevalence of M. genitalium resistance to azithromycin and moxifloxacin. We used data from 7,411 consecutive female patients who were screened for Chlamydia trachomatis, Neisseria gonorrhoeae, M. genitalium, and T. vaginalis and data from 5,732 consecutive male patients screened for C. trachomatis, N. gonorrhoeae, and M. genitalium. The prevalence of M. genitalium and T. vaginalis in female patients was 6.7% (95% CI: 6.2 to 7.4) and 1.9% (95%CI: 1.6 to 2.2%), respectively. M. genitalium prevalence in male patients was 3.7% (3.3 to 4.3). M. genitalium co-occurred with C. trachomatis in 1.4% (0.3 to 0.6%) of female and in 0.7% (0.5 to 0.9) of male patients. Macrolide resistance gene mutations and fluoroquinolone resistance gene mutations were detected in 73.8% and 9.9%, respectively. We concluded that M.genitalium is relatively infrequently found in a large general practitioner population in the Netherlands. It can co-occur with C. trachomatis, and is often resistant to azithromycin. Therefore, when treating sexually transmitted infections, these prevalence and resistance data should be taken into account.

Rapid detection of dermatophytes using the Seegene Novaplex™ dermatophyte assay.

OBJECTIVES: We assessed the performance of the Seegene Novaplex™ Dermatophyte Assay for diagnosis of dermatophytosis. METHODS: Sixty-one clinical samples from skin, nail, hair and culture were selected based on RT-PCR according to Wisselink et al. Of these samples, 26 samples were negative and 35 samples were positive with 39 dermatophytes strains. Emerging fungal strains harbouring terbinafine resistance (i.e. T. indotineae and T. mentagrophytes) were included. RESULTS: The specificities of the Novaplex™ Dermatophyte Assay ranged between 94.3% and 97.9%. The sensitivities for the detection of T. rubrum complex, T. mentagrophytes/T. interdigitale species complex and C. albicans were 94.1% (95% CI: 71.3-99.9), 78.6% (95% CI: 49.2-95.3) and 100% (95% CI: 69.2-100), respectively, with Cohen's kappa of at least 72.9%. CONCLUSIONS: The Seegene Novaplex™ Dermatophyte Assay can be used for reliable screening of dermatophytes, including emerging strains in a routine laboratory setting.

Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens.

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

An analysis of the predictive value of the HIV Ag/Ab screening assay within the performance characteristics of the DiaSorin LIAISON XL for the detection of blood-borne viruses.

BACKGROUND: Correct identification of blood borne viral infections, such as hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is crucial in detection and follow up of infection in patients. OBJECTIVES: We evaluated the diagnostic performance of the DiaSorin LIAISON XL (LIAISON XL) for screening of HBV, HCV and HIV infection. In addition, we investigated the variability of the signal-to-cuttoff ratio (S/CO) of the LIAISON XL HIV Ag/Ab assay and it's predictive value in subsequent confirmation of HIV-1 infection. STUDY DESIGN: We analyzed 16,497 blood samples on which HBV, HCV and HIV screening was performed. We defined A) archived samples previously tested with an arbitrary result in the Abbott ARCHITECT i2000SR system; B) prospectively collected samples which were simultaneously tested on the LIAISON XL and ARCHITECT i2000SR; C) prospectively collected serum samples for HIV testing which were tested solely on the LIAISON XL. RESULTS: The agreements of HBV-, HCV-, and HIV markers between the two compared systems are remarkably high. Among the samples which were prospectively tested for HIV Ab/Ag on the LIASON XL, 229 (1.6%) were reactive of which 141 (61.6%) could be confirmed. Increasing the signal-to-cutoff value to 4 could increase the positive predictive value (PPV) to 88.1% without decreasing sensitivity. CONCLUSIONS: The LIAISON XL system proved to be an excellent system for diagnosing HBV, HCV, and HIV. Our data for the first time showed that increasing the HIV S/CO ratio was safe and increased the PPV for confirmed HIV infection in the tested population.

Spatiotemporal dynamics of dengue epidemics, southern Vietnam.

An improved understanding of heterogeneities in dengue virus transmission might provide insights into biological and ecologic drivers and facilitate predictions of the magnitude, timing, and location of future dengue epidemics. To investigate dengue dynamics in urban Ho Chi Minh City and neighboring rural provinces in Vietnam, we analyzed a 10-year monthly time series of dengue surveillance data from southern Vietnam. The per capita incidence of dengue was lower in Ho Chi Minh City than in most rural provinces; annual epidemics occurred 1-3 months later in Ho Chi Minh City than elsewhere. The timing and the magnitude of annual epidemics were significantly more correlated in nearby districts than in remote districts, suggesting that local biological and ecologic drivers operate at a scale of 50-100 km. Dengue incidence during the dry season accounted for 63% of variability in epidemic magnitude. These findings can aid the targeting of vector-control interventions and the planning for dengue vaccine implementation.

High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections.

Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples (n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia.

Quantifying the emergence of dengue in Hanoi, Vietnam: 1998-2009.

BACKGROUND: An estimated 2.4 billion people live in areas at risk of dengue transmission, therefore the factors determining the establishment of endemic dengue in areas where transmission suitability is marginal is of considerable importance. Hanoi, Vietnam is such an area, and following a large dengue outbreak in 2009, we set out to determine if dengue is emerging in Hanoi. METHODS AND PRINCIPAL FINDINGS: We undertook a temporal and spatial analysis of 25,983 dengue cases notified in Hanoi between 1998 and 2009. Age standardized incidence rates, standardized age of infection, and Standardized Morbidity Ratios (SMR) were calculated. A quasi-Poisson regression model was used to determine if dengue incidence was increasing over time. Wavelet analysis was used to explore the periodicity of dengue transmission and the association with climate variables. After excluding the two major outbreak years of 1998 and 2009 and correcting for changes in population age structure, we identified a significant annual increase in the incidence of dengue cases over the period 1999-2008 (incidence rate ratio  =  1.38, 95% confidence interval  =  1.20-1.58, p value  =  0.002). The age of notified dengue cases in Hanoi is high, with a median age of 23 years (mean 26.3 years). After adjusting for changes in population age structure, there was no statistically significant change in the median or mean age of dengue cases over the period studied. Districts in the central, highly urban, area of Hanoi have the highest incidence of dengue (SMR>3). CONCLUSIONS: Hanoi is a low dengue transmission setting where dengue incidence has been increasing year on year since 1999. This trend needs to be confirmed with serological surveys, followed by studies to determine the underlying drivers of this emergence. Such studies can provide insights into the biological, demographic, and environmental changes associated with vulnerability to the establishment of endemic dengue.

The role of climate variability and change in the transmission dynamics and geographic distribution of dengue.

The mounting evidence for anthropogenic changes in global climate raises many pressing questions about the potential effects on biological systems, and in particular the transmission of infectious diseases. Vector-borne diseases, such as dengue, may be particularly sensitive to both periodic fluctuations and sustained changes in global and local climates, because vector biology and viral replication are temperature- and moisture-dependent. This paper reviews the current state of knowledge on the associations between climate variability, climate change and dengue transmission, and the tools being used to quantify these associations. The underlying causes of dengue's recent global expansion are multifactorial and poorly understood, but climatic factors should be considered within the context of the sociodemographic, economic and immunological determinants that have contributed to dengue's spread. These factors may mediate the direct effects of climate on dengue and many may operate at a very local level. Translating theoretical models of dengue transmission based on historical data into predictive models that can inform public health interventions is a critical next step and efforts should be focused on developing and refining models at smaller spatial scales to characterize the relationships between both climatic and non-climatic factors and dengue risk.

Age-specificity of clinical dengue during primary and secondary infections.

BACKGROUND: This study aims to estimate the age-specific risks of clinical dengue attack (i.e., the risk of symptomatic dengue among the total number of dengue virus (DENV) infections) during primary and secondary infections. METHODS: We analyzed two pieces of epidemiological information in Binh Thuan province, southern Vietnam, i.e., age-specific seroprevalence and a community-wide longitudinal study of clinical dengue attack. The latter data set stratified febrile patients with DENV infection by age as well as infection parity. A simple modeling approach was employed to estimate the age-specific risks of clinical dengue attack during primary and secondary infections. RESULTS: Using the seroprevalence data, the force of infection was estimated to be 11.7% (95% confidence intervals (CI): 10.8-12.7) per year. Median age (and the 25-75 percentiles) of dengue fever patients during primary and secondary infections were 12 (9-20) and 20 (14-31) years, respectively. The estimated age-specific risk of clinical dengue increases as a function of age for both primary and secondary infections; the estimated proportion of symptomatic patients among the total number of infected individuals was estimated to be <7% for those aged <10 years for both primary and secondary infections, but increased as patients become older, reaching to 8-11% by the age of 20 years. CONCLUSIONS/SIGNIFICANCE: For both primary and secondary infections, higher age at DENV infection was shown to result in higher risk of clinical attack. Age as an important modulator of clinical dengue explains recent increase in dengue notifications in ageing countries in Southeast Asia, and moreover, poses a paradoxical problem of an increase in adult patients resulting from a decline in the force of infection, which may be caused by various factors including time-dependent variations in epidemiological, ecological and demographic dynamics.

Internally controlled, generic real-time PCR for quantification and multiplex real-time PCR with serotype-specific probes for serotyping of dengue virus infections.

Dengue has become a global public health problem and a sensitive diagnostic test for early phase detection can be life saving. An internally controlled, generic real-time PCR was developed and validated by testing serial dilutions of a DENV positive control RNA in the presence of a fixed amount of IC with results showing a good linearity (R(2) = 0.9967) and a LOD of at least 1.95 × 10(4) copies/mL. Application of the generic PCR on 136 patient samples revealed a sensitivity of 95.8% and specificity of 100%. A newly developed multiplex real-time PCR with serotype-specific probes allowed the serotyping of DENV for 80 out of 92 (87%) generic real-time PCR positive patients. Combined these real-time PCRs offer a convenient diagnostic tool for the sensitive and specific quantification of DENV in clinical specimens with the possibility for serotyping.

Dengue dynamics in Binh Thuan province, southern Vietnam: periodicity, synchronicity and climate variability.

BACKGROUND: Dengue is a major global public health problem with increasing incidence and geographic spread. The epidemiology is complex with long inter-epidemic intervals and endemic with seasonal fluctuations. This study was initiated to investigate dengue transmission dynamics in Binh Thuan province, southern Vietnam. METHODOLOGY: Wavelet analyses were performed on time series of monthly notified dengue cases from January 1994 to June 2009 (i) to detect and quantify dengue periodicity, (ii) to describe synchrony patterns in both time and space, (iii) to investigate the spatio-temporal waves and (iv) to associate the relationship between dengue incidence and El Niño-Southern Oscillation (ENSO) indices in Binh Thuan province, southern Vietnam. PRINCIPAL FINDINGS: We demonstrate a continuous annual mode of oscillation and a multi-annual cycle of around 2-3-years was solely observed from 1996-2001. Synchrony in time and between districts was detected for both the annual and 2-3-year cycle. Phase differences used to describe the spatio-temporal patterns suggested that the seasonal wave of infection was either synchronous among all districts or moving away from Phan Thiet district. The 2-3-year periodic wave was moving towards, rather than away from Phan Thiet district. A strong non-stationary association between ENSO indices and climate variables with dengue incidence in the 2-3-year periodic band was found. CONCLUSIONS: A multi-annual mode of oscillation was observed and these 2-3-year waves of infection probably started outside Binh Thuan province. Associations with climatic variables were observed with dengue incidence. Here, we have provided insight in dengue population transmission dynamics over the past 14.5 years. Further studies on an extensive time series dataset are needed to test the hypothesis that epidemics emanate from larger cities in southern Vietnam.

Clinical, epidemiological and virological features of Dengue virus infections in Vietnamese patients presenting to primary care facilities with acute undifferentiated fever.

OBJECTIVES: To explore clinical and virological characteristics and describe the epidemiology of dengue in patients who presented with acute undifferentiated fever (AUF) at primary health centers (PHC) in Binh Thuan Province, Vietnam. METHODS: A prospective observational study was conducted from 2001 to 2006 to study the aetiology in AUF patients. Demographic and clinical information was obtained, and dengue polymerase chain reaction (RT-PCR) and serology were performed on a random selection of patients. RESULTS: Three hundred fifty-one serologically confirmed dengue patients including 68 primary and 283 secondary infections were included in this study. In 25% (86/351) dengue virus (DENV) was detected by RT-PCR among which 32 DENV-1, 16 DENV-2, 1 DENV-3 and 37 DENV-4 were identified. The predominant dengue serotype varied by year with seasonal fluctuation: DENV-4 in 2001-2002, DENV-1 and DENV-2 from 2003 to 2006. Primary dengue was more common in children. Higher viraemia levels (P=0.010) were found in primary infections compared to secondary infections. DENV-1 infected patients had higher viraemia levels than DENV-2 (P=0.003) and DENV-4 (P<0.001) infected patients. Clinical symptoms were often seen in adults. Few differences in clinical symptoms were found between primary and secondary infection and no significant differences in clinical symptoms between the serotypes were observed. CONCLUSIONS: Our data provide insight in the epidemiology, clinical profile and virological features of mild symptomatic dengue patients who presented to PHC with AUF in Vietnam.

Detection of dengue nonstructural 1 (NS1) protein in Vietnamese patients with fever.

Diagnosing dengue in febrile patients is challenging. Of a total of 459 patients with acute undifferentiated fever, randomly selected from 12 primary health facilities and 1 clinic of the provincial malaria station in southern Vietnam, dengue-specific antibody (Ab) and NS1Ag enzyme-linked immunosorbent assay (ELISA) (Platelia, Bio-Rad Laboratories, Hercules, CA 94547, US) were performed on acute (t0) and convalescent (t3 weeks) sera. Polymerase chain reaction (PCR) was used for confirmation. Based on a composite of the NS1Ag-ELISA, Ab-ELISA, and PCR results, 54 (12%) patients had acute dengue. Positive and negative predictive values were 65% and 98% for the Ab-based diagnosis and 91% and 92% for NS1Ag, respectively. The agreement between Ab- and NS1Ag-based diagnosis was poor (kappa value, 0.2). Two patients without dengue had detectable NS1Ag on t0 and t3, 1 just above the cutoff value and 1 with very high values. For 5 dengue patients, NS1Ag was still detectable at very high levels at t3. Dengue NS1Ag can be used for early diagnosis of dengue; infrequent false-positive results need further clarification.

Seroepidemiology and serological follow-up of anti-leptospiral IgG in children in Southern Vietnam.

A follow-up study was conducted with 23 months interval to investigate the seroepidemiology and persistence of Leptospira IgG antibodies among healthy children in Binh Thuan province, Southern Vietnam. Sera from 262 children (7-13 years of age) were collected and analysed with a commercially available enzyme-linked immunosorbent assay (ELISA) for Leptospira IgG. Seroconversion was observed in 10.4% (22 of 211, 95% CI: 5.6-26.7) of the children, of whom 18 (8.5%) had probably and four (1.9%) had certainly been exposed to Leptospira. Based on the reduction of sero-negatives of 1.9% among children who have been certainly exposed, the annual seroconversion rate, a measure of the incidence rate of Leptospira infections, corresponds to 0.99% (95% CI: 0.39-2.52). In 61% (31 of 51, 95% CI: 47.1-73.0) of the children with past-infection, Leptospira IgG antibodies remain detectable after 2 years. Data from this study indicate that IgG antibody responses against Leptospira may persist at least for 2 years in children without manifestations of leptospirosis. Results of study uncover the true incidence of leptospirosis infection, the dynamics of waxing and waning antibody concentrations and points at a larger burden of clinically non-significant Leptospira infections in Southern Vietnam. This also indicates background reactivity for serological testing and thus serological result of a single serum sample must be carefully interpreted.

Incidence of primary dengue virus infections in Southern Vietnamese children and reactivity against other flaviviruses.

OBJECTIVE: To study the incidence of asymptomatic primary dengue infections among children and reactivity against other flaviviruses. METHODS: A total of 216 children, who had no dengue-specific IgG antibodies during a serosurvey in 2003 were re-examined 23 months later to determine if seroconversion had occurred. Dengue-specific IgG was demonstrated with enzyme-linked immunosorbent assay (ELISA) and reactivity patterns against other flaviviruses were assessed by using immunofluorescence assay (IFA). RESULTS: Sixty-six children had seroconverted for dengue virus-specific IgG; the true annual incidence of primary dengue was thus 17.3% (95% CI: 13.8-21.4). Japanese Encephalitis virus (JEV)-specific IgG antibodies were detected by IFA among three (4.6%) samples that showed seroconversion in the dengue ELISA, because of cross-reactivity. CONCLUSION: Our findings highlight the high incidence of dengue among Vietnamese children; JEV infections are rare. The true annual incidence of dengue can be estimated with a single cross-sectional seroprevalence survey.

Evaluation of two rapid immunochromatographic assays for diagnosis of dengue among Vietnamese febrile patients.

Results from two dengue rapid tests, the PanBio Duo cassette and the SD Bioline strip test, were compared to those of enzyme-linked immunosorbent assays (Focus Diagnostics) from sera of 200 Vietnamese febrile patients. The PanBio assay was superior, with sensitivity and specificity values for acute-phase serum samples of 54% and 70% (immunoglobulin M) and 70% and 88% (immunoglobulin G), respectively.

Seroepidemiology of leptospirosis in southern Vietnamese children.

OBJECTIVE: To estimate the seroprevalence of human leptospirosis in southern Vietnam. METHODS: All pupils (n=961) of two primary schools in two communes in southern Vietnam were screened for the presence of serum Leptospira immunoglobulin (Ig)G. Leptospira IgM was tested in 92 randomly selected samples. IgM and IgG were tested with a commercially available enzyme-linked immunosorbent assay (ELISA). RESULTS: Leptospira IgG was found in 123 (12.8%, 95% CI: 10.8-15.1) children with a male:female ratio of 1.5:1. At the age of 7 years the prevalence was 11%. Log-linear binary regression of the IgG seroprevalence by age showed a mean seroconversion rate of 1.5% (95% CI: 1.23-1.75) per year. Five (5.4%, 95% CI: 2.3-12.1) samples were definitely positive for IgM. Presence of serum Leptospira IgG antibodies was significantly associated with swimming in rivers (RR: 1.745, 95% CI: 1.117-2.724) and wading through water (RR 1.793, 95% CI: 1.181-2.722). CONCLUSION: Our data emphasize the importance of leptospirosis among children in Vietnam, despite the absence of severe disease, and stress the need for adequate and cheap diagnostics.

Seroprevalence of dengue antibodies, annual incidence and risk factors among children in southern Vietnam.

Dengue is highly endemic in southern Vietnam and all four serotypes of dengue virus have already been identified. To determine the age-specific prevalence of dengue and associated risk factors, we conducted a serological study at two primary schools and assessed risk factors by analysing children's questionnaires and household surveys. Sera were collected from 961 primary schoolchildren in Binh Thuan Province and tested for the presence of dengue virus serum antibodies using an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA). The antibody prevalence of the total population was 65.7% (n=631) which increased from 53.0 to 88.2% with age. The annual incidence of a first dengue infection, estimated by binary regression of the seroprevalence by age, was 11.7%. Interestingly, the prevalence of dengue IgG antibodies was significantly higher in children who confirmed using a pit latrine (RR 1.467, 95% CI: 1.245-1.730) and whose domestic environment contained discarded cans (RR 1.238, 95% CI: 1.042-1.470) and pigs (RR 1.228, 95% CI: 1.002-1.504). The epidemiology of dengue in southern Vietnam is stable with a constantly high annual incidence of first infections. Transmission occurs mainly peri-domestically, which has important public health implications.