Wanitda Watthanaworawit

A prospective evaluation of diagnostic methodologies for the acute diagnosis of dengue virus infection on the Thailand-Myanmar border

Summary Clinically useful diagnostic tests of dengue virus infection are lacking. We prospectively evaluated the performance of real-time reverse transcriptase (rRT)-PCR, NS-1 antigen and IgM antibody tests to confirm dengue virus infection in acute blood specimens from 162 patients presenting with undifferentiated febrile illness compatible with dengue infection. rRT-PCR was the most sensitive test (89%) and potentially could be used as a single test for confirmation of dengue infection. NS-1 antigen and IgM antibody were not sufficiently sensitive to be used as a single confirmatory test with sensitivities of 54% and 17% respectively. The specificities of rRT-PCR, NS-1 antigen and IgM antibody tests were 96%, 100% and 88% respectively. Combining NS-1 and rRT-PCR or the combination of all three tests resulted in the highest sensitivity (93%) but specificities dropped to 96% and 83% respectively. We conclude that at least the combination of two tests, either agent detection (rRT-PCR) or antigen detection (NS-1) plus IgM antibody detection should be used for laboratory confirmation of dengue infection.

A Prospective Evaluation of Real-Time PCR Assays for the Detection of Orientia tsutsugamushi and Rickettsia spp. for Early Diagnosis of Rickettsial Infections during the Acute Phase of Undifferentiated Febrile Illness

One hundred and eighty febrile patients were analyzed in a prospective evaluation of Orientia tsutsugamushi and Rickettsia spp. real-time polymerase chain reaction (PCR) assays for early diagnosis of rickettsial infections. By paired serology, 3.9% (7 of 180) and 6.1% (11 of 180) of patients were confirmed to have acute scrub or murine typhus, respectively. The PCR assays for the detection of O. tsutsugamushi and Rickettsia spp. had high specificity (99.4% [95% confidence interval (CI): 96.8–100] and 100% [95% CI: 97.8–100], respectively). The PCR results were also compared with immunoglobulin M (IgM) immunofluorescence assay (IFA) on acute sera. For O. tsutsugamushi, PCR sensitivity was twice that of acute specimen IgM IFA (28.6% versus 14.3%; McNemars P = 0.3). For Rickettsia spp., PCR was four times as sensitive as acute specimen IgM IFA (36.4% versus 9.1%; P = 0.08), although this was not statistically significant. Whole blood and buffy coat, but not serum, were acceptable specimens for these PCRs. Further evaluation of these assays in a larger prospective study is warranted.

An integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens.

Tropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83.1% and a specificity of 100% for P. falciparum; a sensitivity of 91.3% and a specificity of 99.3% for P. vivax; a positive 90.0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85.0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens.

Pregnancy Outcome in Relation to Treatment of Murine Typhus and Scrub Typhus Infection: A Fever Cohort and a Case Series Analysis

Background There is a paucity of published reports on pregnancy outcome following scrub and murine typhus despite these infections being leading causes of undifferentiated fever in Asia. This study aimed to relate pregnancy outcome with treatment of typhus. Methodology/Principal Findings Data were analyzed from: i) pregnant women with a diagnosis of scrub and/or murine typhus from a fever cohort studies; ii) case series of published studies in PubMed using the search terms “scrub typhus” (ST), “murine typhus” (MT), “Orientia tsutsugamushi”, “Rickettsia tsutsugamushi”, “Rickettsia typhi”, “rickettsiae”, “typhus”, or “rickettsiosis”; and “pregnancy”, until February 2014 and iii) an unpublished case series. Fever clearance time (FCT) and pregnancy outcome (miscarriage and delivery) were compared to treatment. Poor neonatal outcome was a composite measure for pregnancies sustained to 28 weeks or more of gestation ending in stillbirth, preterm birth, or delivery of a growth restricted or low birth weight newborn. Results There were 26 women in the fever cohort. MT and ST were clinically indistinguishable apart from two ST patients with eschars. FCTs (median [range] hours) were 25 [16–42] for azithromycin (n = 5), 34 [20–53] for antimalarials (n = 5) and 92 [6–260] for other antibiotics/supportive therapy (n = 16). There were 36.4% (8/22) with a poor neonatal outcome. In 18 years, 97 pregnancies were collated, 82 with known outcomes, including two maternal deaths. Proportions of miscarriage 17.3% (14/81) and poor neonatal outcomes 41.8% (28/67) were high, increasing with longer FCTs (p = 0.050, linear trend). Use of azithromycin was not significantly associated with improved neonatal outcomes (p = 0.610) Conclusion The published ST and MT world literature amounts to less than 100 pregnancies due to under recognition and under diagnosis. Evidence supporting the most commonly used treatment, azithromycin, is weak. Collaborative, prospective clinical trials in pregnant women are urgently required to reduce the burden of adverse maternal and newborn outcomes and to determine the safety and efficacy of antimicrobial treatment.

High Rates of Pneumonia in Children under Two Years of Age in a South East Asian Refugee Population

Background There are an estimated 150 million episodes of childhood pneumonia per year, with 11–20 million hospital admissions and 1.575 million deaths. Refugee children are particularly vulnerable, with poorly defined pneumonia epidemiology. Methods We followed a birth cohort of 955 refugee infants, born over a one-year period, until two years of age. Clinical and radiographic pneumonia were diagnosed according to WHO criteria. Detailed characteristics were collected to determine risk factors for clinical, radiological and multiple episodes of pneumonia. Investigations were taken during a pneumonia episode to help determine or to infer an aetiological diagnosis. Findings The incidence of clinical pneumonia was 0.73 (95% CI 0.70–0.75) episodes per child year (/CY) and of radiological primary endpoint pneumonia (PEP) was 0.22/CY (95% CI 0.20–0.24). The incidence of pneumonia without severe signs was 0.50/CY (95% CI 0.48–0.53), severe pneumonia 0.15/CY (95% CI 0.13–0.17) and very severe pneumonia 0.06/CY (0.05–0.07). Virus was detected, from a nasopharyngeal aspirate, in 61.3% of episodes. A reduced volume of living space per person (IRR 0.99, 95% CI 0.99–1.0, p = 0.003) and young maternal age (IRR 1.59, 95% CI 1.12–2.27, p = 0.01) were risk factors for developing pneumonia. The risk of a child having >1 episode of pneumonia was increased by having a shorter distance to the next house (IRR 0.86, 95% CI 0.74–1.00, p = 0.04). Infants were at risk of having an episode of PEP if there was a shorter distance from stove to bed (IRR 0.89, 95% CI 0.80–0.99, p = 0.03). Raised CRP and neutrophil values were associated with PEP. Conclusions There was a high incidence of pneumonia in young children in this SE Asian refugee population. Viral infections were important, however CXR and non-specific marker findings suggested that bacteria may be involved in up to a third of cases.

Underrecognized arthropod-borne and zoonotic pathogens in northern and northwestern Thailand: serological evidence and opportunities for awareness.

Abstract Although scrub typhus and murine typhus are well-described tropical rickettsial illnesses, especially in Southeast Asia, only limited evidence is available for rickettsia-like pathogens contributing to the burden of undifferentiated febrile illness. Using commercially available kits, this study measured immunoglobulin G (IgG) antibody seroprevalence for Coxiella burnetii, Ehrlichia chaffeensis, Bartonella henselae, Anaplasma phagocytophilum, and spotted fever group rickettsiae (SFGR) in 375 patients enrolled in undifferentiated febrile illness studies at Chiangrai (northern Thailand) and Mae Sot (Thai–Myanmar border). Ehrlichia and SFGR were the most common causes of IgG seropositivity. A distinct relationship between age and seropositivity was found in Chiangrai with acquisition of IgG titers against Ehrlichia, Bartonella, Anaplasma, and SFGR in young adulthood, suggesting cumulative exposure to these pathogens. At Mae Sot, high early IgG titers against Ehrlichia and SFGR were common, whereas Anaplasma and Bartonella IgG titers increased at 50–60 years. Q fever associated with low IgG positivity at both study sites, with significantly higher prevalence at 30 years of age in Chiangrai. These data suggest that other rickettsial illnesses could contribute to the burden of febrile illness in Thailand and possibly adjacent regions. Improved diagnostics and better understanding of antibody longevity and cross-reactivity will improve identification and management of these easily treatable infectious diseases.

A High Burden of Respiratory Syncytial Virus Associated Pneumonia in Children Less than Two Years of Age in a South East Asian Refugee Population

Background Pneumonia is a major cause of childhood mortality and morbidity approximately 1.6 million deaths and 150 million episodes occur annually in children <5 years. Respiratory syncytial virus (RSV) may be responsible for up to 25% of cases and 12% of deaths making it an important potential vaccine target, although data from South East Asia is scarce. Methods We followed a birth cohort of Burmese refugee children, born over a one year period, for two years. Pneumonia episodes were diagnosed using WHO criteria. A chest radiograph, nasopharyngeal aspirate and non-specific markers of infection were taken during each episode. Results The incidence of RSV-associated pneumonia was 0.24 (95% CI 0.22–0.26) episodes per child year. All children with pneumonia received antibiotic treatment, following WHO guidelines. The highest incidence was in the 2–12 month age group. The commonest diagnosis in a child with RSV-associated pneumonia was non-severe pneumonia (239/362∶66.0%), however the incidence of RSV-associated severe or very severe pneumonia was 0.08 (95% CI 0.01–0.10) episodes per child year. Birth in the wet season increased the risk of severe disease in children who had their first episode of RSV-associated pneumonia aged 2–11 months (OR 28.7, 95% CI 6.6–125.0, p<0.001). RSV episodes were highly seasonal being responsible for 80.0% of all the pneumonia episodes occurring each October and November over the study period. Conclusions There was a high incidence of RSV associated pneumonia in this refugee population. Interventions to prevent RSV infection have the potential to reduce the incidence of clinically diagnosed pneumonia and hence unnecessary antibiotic usage in this population.

Influenza in refugees on the Thailand-Myanmar border, May-October 2009.

TOC Summary: Influenza viruses can be identified in up to 22% of patients who have acute respiratory infections.

A longitudinal study of the infant nasopharyngeal microbiota: The effects of age, illness and antibiotic use in a cohort of South East Asian children.

A longitudinal study was undertaken in infants living in the Maela refugee camp on the Thailand-Myanmar border between 2007 and 2010. Nasopharyngeal swabs were collected monthly, from birth to 24 months of age, with additional swabs taken if the infant was diagnosed with pneumonia according to WHO clinical criteria. At the time of collection, swabs were cultured for Streptococcus pneumoniae and multiple serotype carriage was assessed. The bacterial 16S rRNA gene profiles of 544 swabs from 21 infants were analysed to see how the microbiota changes with age, respiratory infection, antibiotic consumption and pneumococcal acquisition. The nasopharyngeal microbiota is a somewhat homogenous community compared to that of other body sites. In this cohort it is dominated by five taxa: Moraxella, Streptococcus, Haemophilus, Corynebacterium and an uncharacterized Flavobacteriaceae taxon of 93% nucleotide similarity to Ornithobacterium. Infant age correlates with certain changes in the microbiota across the cohort: Staphylococcus and Corynebacterium are associated with the first few months of life while Moraxella and the uncharacterised Flavobacteriaceae increase in proportional abundance with age. Respiratory illness and antibiotic use often coincide with an unpredictable perturbation of the microbiota that differs from infant to infant and in different illness episodes. The previously described interaction between Dolosigranulum and Streptococcus was observed in these data. Monthly sampling demonstrates that the nasopharyngeal microbiota is in flux throughout the first two years of life, and that in this refugee camp population the pool of potential bacterial colonisers may be limited.

Diagnostic Accuracy Assessment of Immunochromatographic Tests for the Rapid Detection of Antibodies Against Orientia tsutsugamushi Using Paired Acute and Convalescent Specimens

We assessed the diagnostic accuracy of two immunochromatographic tests (ICTs), the Access Bio CareStart Scrub Typhus test (Somerset, NJ) (IgM), and the SD BIOLINE Tsutsugamushi test (Kyonggi-do, Republic of Korea) (IgG, IgM, or IgA) compared with indirect immunofluorescence assay (IFA) and real-time PCR results as reference tests using 86 paired acute and convalescent specimens from febrile patients. The sensitivity and specificity of the CareStart test were 23.3% (95% confidence interval [CI]: 11.8–38.6) and 81.4% (95% CI: 66.6–91.6), respectively, for acute specimens and 32.6% (95% CI: 19.1–48.5) and 79.1% (95% CI: 64.0–90.0), respectively, for convalescent specimens. For the SD BIOLINE test, sensitivity and specificity were 20.9% (95% CI: 10.0–36.0) and 74.4% (95% CI: 58.8–86.5), respectively, for acute specimens and 76.7% (95% CI: 61.4–88.2) and 76.7% (95% CI: 61.4–88.2), respectively, for convalescent specimens. The poor sensitivity obtained for both ICTs during this study when performed on acute specimens highlights the difficulties in prompt diagnosis of scrub typhus.