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"Blacksell, Stuart D"
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Commercial Dengue Rapid Diagnostic Tests for Point-of-Care Application : Recent Evaluations and Future Needs?
2012
Dengue fever, dengue haemorrhagic fever, and dengue shock syndrome (DF/DHF/DSS) are tropical diseases that cause significant humanitarian and economic hardship. It is estimated that more than 2.5 billion people are at risk of infection and more than 100 countries have endemic dengue virus transmission. Laboratory tests are essential to provide an accurate diagnosis of dengue virus infection so that appropriate treatment and patient management may be administered. In many dengue endemic settings, laboratory diagnostic resources are limited and simple rapid diagnostic tests (RDTs) provide opportunities for point-of-care diagnosis. This paper addresses current issues relating to the application of commercial dengue RDTs for the diagnosis of acute dengue virus infection, recent diagnostic evaluations, and identifies future needs.
Journal Article
Risk mapping using serologic surveillance for selected One Health and transboundary diseases in Cambodian goats
2023
In Cambodia, goat production and meat consumption are customary among Muslim communities. Recently, goat meat has gained popularity among Cambodians. Goat farmers use a traditional management system, including grazing, requiring minimal labour. The close proximity between humans and animals could increase the risk of zoonotic disease transmission. A serological survey was undertaken to estimate the prevalence of some priority zoonoses and high-impact animal diseases in the Cambodian goat population. A total of 540 samples were collected from goats in six provinces and analysed with commercially available enzyme-linked immunosorbent assays for Brucella species, Q fever (C oxiella burnetii ), Foot and Mouth Disease virus non-structural protein (FMDV NSP) and Peste des Petits Ruminants virus (PPRV). True seroprevalences with a 95% Confidence Interval (CI), taking into account imperfect tests, risk factors and odds ratios (ORs), were calculated to better understand the disease distribution and epidemiology. Independent variables used in statistical modellings included sex, body condition score, age, vaccination history, province and commune, while dependent variables were ELISA test results. The overall true prevalence of antibodies to Brucella spp ., C . burnetii , FMDV and PPRV, were 0.1% (95% CI 0.0, 1.0), 7.2% (95% CI 5.3, 9.7), 57.7% (95% CI 53.1, 62.3) and 0.0% (95% CI 0.0, 0.0), respectively. There was no identified risk factor for brucellosis and PPR. The two risk factors for C . burnetii seropositivity were sex (p-value = 0.0005) and commune (p-value <0.0001). However, only the OR of C . burnetii seropositive female goat was significant at 9.7 (95% CI 2.7, 35.5) times higher than male. The risk factors of FMD NSP seropositivity were age (p-value = 0.001) and commune (p-value <0.0001). Only the age ’more than two-year-old’ group with a significant OR of 6.2 (95% CI 2.1, 18.4) using the ’up to one-year-old’ group as the reference. In summary, Brucella spp . seroprevalence was low, while no evidence of PPRV antibodies was detected in the goat populations. C . burnetii seroprevalence in female goats was significantly higher than for males, and there were significant differences in C . burnetii seroprevalence between communes. The overall FMDV NSP seroprevalence was high, especially in older animals. Vaccination should be advocated to protect animals from FMDV and improve productivity. As the impacts of these zoonoses on human and animal health were still unknown, further investigation of these zoonotic diseases’ epidemiology is recommended.
Journal Article
Scrub typhus point-of-care testing: A systematic review and meta-analysis
by
Mukaka, Mavuto
,
Saraswati, Kartika
,
Blacksell, Stuart D.
in
Accuracy
,
Agglutination tests
,
Antigens
2018
Diagnosing scrub typhus clinically is difficult, hence laboratory tests play a very important role in diagnosis. As performing sophisticated laboratory tests in resource-limited settings is not feasible, accurate point-of-care testing (POCT) for scrub typhus diagnosis would be invaluable for patient diagnosis and management. Here we summarise the existing evidence on the accuracy of scrub typhus POCTs to inform clinical practitioners in resource-limited settings of their diagnostic value.
Studies on POCTs which can be feasibly deployed in primary health care or outpatient settings were included. Thirty-one studies were identified through PubMed and manual searches of reference lists. The quality of the studies was assessed with the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2). About half (n = 14/31) of the included studies were of moderate quality. Meta-analysis showed the pooled sensitivity and specificity of commercially available immunochromatographic tests (ICTs) were 66.0% (95% CI 0.37-0.86) and 92.0% (95% CI 0.83-0.97), respectively. There was a significant and high degree of heterogeneity between the studies (I2 value = 97.48%, 95% CI 96.71-98.24 for sensitivity and I2 value = 98.17%, 95% CI 97.67-98.67 for specificity). Significant heterogeneity was observed for total number of samples between studies (p = 0.01), study design (whether using case-control design or not, p = 0.01), blinding during index test interpretation (p = 0.02), and QUADAS-2 score (p = 0.01).
There was significant heterogeneity between the scrub typhus POCT diagnostic accuracy studies examined. Overall, the commercially available scrub typhus ICTs demonstrated better performance when 'ruling in' the diagnosis. There is a need for standardised methods and reporting of diagnostic accuracy to decrease between-study heterogeneity and increase comparability among study results, as well as development of an affordable and accurate antigen-based POCT to tackle the inherent weaknesses associated with serological testing.
Journal Article
Diagnostic accuracy of the WHO clinical definitions for dengue and implications for surveillance: A systematic review and meta-analysis
by
Mukaka, Mavuto
,
Loganathan, Shanghavie
,
Blacksell, Stuart D.
in
Biology and life sciences
,
Care and treatment
,
Dengue
2021
Dengue is the world's most common mosquito-borne virus but remains diagnostically challenging due to its nonspecific presentation. Access to laboratory confirmation is limited and thus most reported figures are based on clinical diagnosis alone, the accuracy of which is uncertain. This systematic review assesses the diagnostic accuracy of the traditional (1997) and revised (2009) WHO clinical case definitions for dengue fever, the basis for most national guidelines.
PubMed, EMBASE, Scopus, OpenGrey, and the annual Dengue Bulletin were searched for studies assessing the diagnostic accuracy of the unmodified clinical criteria. Two reviewers (NR/SL) independently assessed eligibility, extracted data, and evaluated risk of bias using a modified QUADAS-2. Additional records were found by citation network analysis. A meta-analysis was done using a bivariate mixed-effects regression model. Studies that modified criteria were analysed separately. This systematic review protocol was registered on PROSPERO (CRD42020165998). We identified 11 and 12 datasets assessing the 1997 and 2009 definition, respectively, and 6 using modified criteria. Sensitivity was 93% (95% CI: 77-98) and 93% (95% CI: 86-96) for the 1997 and 2009 definitions, respectively. Specificity was 29% (95% CI: 8-65) and 31% (95% CI: 18-48) for the 1997 and 2009 definitions, respectively. Diagnostic performance suffered at the extremes of age. No modification significantly improved accuracy.
Diagnostic accuracy of clinical criteria is poor, with significant implications for surveillance and public health responses for dengue control. As the basis for most reported figures, this has relevance to policymakers planning resource allocation and researchers modelling transmission, particularly during COVID-19.
Journal Article
Optimization of heat inactivation protocols for Orientia and Rickettsia species
2025
Heat treatment, or thermal disinfection, is one of the simplest and most widely used methods for microbial inactivation. Proper heat inactivation protocols are essential to ensure the safe transportation and handling of infectious materials, particularly for organisms in risk group 3, such as
Rickettsia
and
Orientia
. In this study, we examined the inactivation of four bacterial species—
Orientia tsutsugamushi
,
Rickettsia typhi
,
Rickettsia conorii
, and
Rickettsia honei
—at temperatures of 56 °C, 80 °C, and 90 °C for durations of 5, 15, 30, and 60 min. Observations were made at 0, 1, 3, 7, 10, and 14 days post-infection (dpi) to assess bacterial infectivity by monitoring bacterial DNA copies in newly infected cells. Our results indicate that 56 °C for 5 min was the minimum temperature and time required to inactivate
O. tsutsugamushi
,
R. typhi
,
R. conorii
, and
R. honei
.
O. tsutsugamushi
exhibited a higher reduction factor at 56 °C compared to
R. typhi
,
R. conorii
, and
R. honei
. Additionally, a strong inverse correlation between incubation time and log10 reduction factor was observed for
O. tsutsugamushi
and
R. typhi
, underscoring the importance of both time and temperature in effective heat treatment. However, no such correlation was observed for
R. conorii
and
R. honei
. These findings highlight the variable responses of bacteria to heat, emphasizing the need for pathogen-specific approaches in inactivation protocols. Optimizing heat treatment strategies based on these insights is critical for enhancing biosafety and ensuring effective pathogen eradication.
Journal Article
The validity of diagnostic cut-offs for commercial and in-house scrub typhus IgM and IgG ELISAs: A review of the evidence
by
Saraswati, Kartika
,
Phanichkrivalkosil, Meghna
,
Blacksell, Stuart D.
in
Accuracy
,
Antibodies
,
Antibodies, Bacterial - blood
2019
Scrub typhus is a neglected tropical disease that causes acute febrile illness. Diagnosis is made based upon serology, or detection of the causative agent-Orientia tsutsugamushi-using PCR or in vitro isolation. The enzyme-linked immunosorbent assay (ELISA) is an objective and reproducible means of detecting IgM or IgG antibodies. However, lack of standardization in ELISA methodology, as well as in the choice of reference test with which the ELISA is compared, calls into question the validity of cut-offs used in diagnostic accuracy studies and observational studies.
A PubMed search and manual screening of reference lists identified 46 studies that used ELISA antibody cut-offs to diagnose scrub typhus patients, 22 of which were diagnostic accuracy studies. Overall, 22 studies (47.8%) provided little to no explanation as to how the ELISA cut-off was derived, and 7 studies (15.2%) did not even state the cut-off used. Variation was seen locally in reference standards used, in terms of both the diagnostic test and cut-off titer. Furthermore, with the exception of studies using ELISAs manufactured by InBios, there was no standardization of the selection of antigenic strains. As a result, no consensus was found for determining a cut-off, ELISA methodology, or for a single value diagnostic cut-off.
We have concluded that there is a lack of consensus in the determination of a cut-off. We recommend interpreting the results from these studies with caution. Further studies will need to be performed at each geographic location to determine region-specific cut-offs, taking into consideration background antibody levels to discriminate true disease from healthy individuals.
Journal Article
RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection
by
Naksith, Piyaporn
,
Anuracpreeda, Panat
,
Sutipatanasomboon, Arpaporn
in
631/1647/1888
,
631/61/32
,
Anaplasma marginale
2024
Anaplasma marginale
infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to
A. marginale
infection in bovines targeting the
msp4
gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/μl of
Anaplasma
using
msp4
marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines.
Journal Article
Distribution and Ecological Drivers of Spotted Fever Group Rickettsia in Asia
by
Robinson, Matthew T
,
Blacksell, Stuart D
,
Satjanadumrong, Jaruwan
in
Arthropods
,
Fever
,
Land use
2019
Spotted fever group and related rickettsia (SFGR) are a neglected group of pathogens that belong to the genus Rickettsia. SFGR are zoonotic and are transmitted by arthropod vectors, primarily ticks, fleas and mites to accidental hosts. These emerging and re-emerging infections are widely distributed throughout the world. Land-use change and increasing human–wildlife conflict compound the risk of SFGR infection to local people in endemic areas and travelers to these regions. In this article, we discuss the rickettsial organisms causing spotted fever and related diseases, their arthropod vectors in Asia and the impact of land-use change on their spread.
Journal Article
Causes of acute undifferentiated fever and the utility of biomarkers in Chiangrai, northern Thailand
by
Laongnualpanich, Achara
,
Wuthiekanun, Vanaporn
,
Wangrangsimakul, Tri
in
Adults
,
Alkaline phosphatase
,
Antibiotics
2018
Tropical infectious diseases like dengue, scrub typhus, murine typhus, leptospirosis, and enteric fever continue to contribute substantially to the febrile disease burden throughout Southeast Asia while malaria is declining. Recently, there has been increasing focus on biomarkers (i.e. C-reactive protein (CRP) and procalcitonin) in delineating bacterial from viral infections.
A prospective observational study was performed to investigate the causes of acute undifferentiated fever (AUF) in adults admitted to Chiangrai Prachanukroh hospital, northern Thailand, which included an evaluation of CRP and procalcitonin as diagnostic tools. In total, 200 patients with AUF were recruited. Scrub typhus was the leading bacterial cause of AUF (45/200, 22.5%) followed by leptospirosis (15/200, 7.5%) and murine typhus (7/200, 3.5%), while dengue was the leading viral cause (23/200, 11.5%). Bloodstream infections contributed to 7/200 (3.5%) of the study cohort. There were 9 deaths during this study (4.5%): 3 cases of scrub typhus, 2 with septicaemia (Talaromyces marneffei and Haemophilus influenzae), and 4 of unknown aetiologies. Rickettsioses, leptospirosis and culture-attributed bacterial infections, received a combination of 3rd generation cephalosporin plus a rickettsia-active drug in 53%, 73% and 67% of cases, respectively. Low CRP and white blood count were significant predictors of a viral infection (mainly dengue) while the presence of an eschar and elevated aspartate aminotransferase and alkaline phosphatase were important predictors of scrub typhus.
Scrub typhus and dengue are the leading causes of AUF in Chiangrai, Thailand. Eschar, white blood count and CRP were beneficial in differentiating between bacterial and viral infections in this study. CRP outperformed procalcitonin although cut-offs for positivity require further assessment. The study provides evidence that accurate, pathogen-specific rapid diagnostic tests coupled with biomarker point-of-care tests such as CRP can inform the correct use of antibiotics and improve antimicrobial stewardship in this setting.
Journal Article
Diagnostic heterogeneity in scrub typhus serology: A scoping review of IFA thresholds and regional standardisation needs (2005–2024)
by
Wongsantichon, Jantana
,
Le, Khanh Kim
,
Saraswati, Kartika
in
Antibodies, Bacterial - blood
,
Fluorescent Antibody Technique, Indirect - methods
,
Fluorescent Antibody Technique, Indirect - standards
2025
The indirect immunofluorescence assay (IFA) remains the most widely used reference method for diagnosing scrub typhus. However, inconsistent cut-off thresholds and strain selections across studies hinder standardisation and complicate cross-regional comparisons. This scoping review examines diagnostic heterogeneity in IFA-based scrub typhus serology and assesses the need for region-specific standardisation.
We conducted a systematic search of peer-reviewed literature published between January 2005 and May 2024 across PubMed, Scopus, and Web of Science databases. Studies were included if they employed IFA for diagnosing or conducting seroepidemiological investigations of scrub typhus and reported specific IgM or IgG titre thresholds. Data were extracted regarding IFA methodology, antigen strains used, titre cut-offs for positivity, sample populations, and geographic settings. The studies were mapped and synthesised to identify trends, methodological diversity, and regional variation in IFA practices.
A total of 84 studies met the inclusion criteria, covering 16 countries across Asia-Pacific and South Asia. The diagnostic cut-off titres for IgM ranged widely from 1:10 to 1:25,600, with considerable variability both within and between countries. Many studies lacked a clearly stated rationale for threshold selection or did not reference region-specific validation. Antigen panels were often limited to prototype strains (e.g., Karp, Gilliam, Kato), with few incorporating locally circulating genotypes. Seroprevalence estimates were significantly influenced by the selected cut-off and antigen composition. Only a minority of studies employed standardised or validated thresholds aligned with regional disease endemicity.
This review underscores significant heterogeneity in IFA cut-offs and strain selection in scrub typhus serology, highlighting the urgent need for regionally validated diagnostic standards. Greater harmonisation of IFA protocols, including rational cut-off determination and inclusion of locally relevant strains, is crucial for improving diagnostic accuracy and informing surveillance and public health strategies.
Journal Article