Case series of three malaria patients from Thailand infected with the simian parasite, Plasmodium cynomolgi.

BACKGROUND: While human cases of Plasmodium knowlesi are now regularly recognized in Southeast Asia, infections with other simian malaria species, such as Plasmodium cynomolgi, are still rare. There has been a handful of clinical cases described, all from Malaysia, and retrospective studies of archived blood samples in Thailand and Cambodia have discovered the presence P. cynomolgi in isolates using polymerase chain reaction (PCR) assays. CASE PRESENTATION: In Thailand, an ongoing malaria surveillance study enrolled two patients from Yala Province diagnosed with Plasmodium vivax by blood smear, but who were subsequently found to be negative by PCR. Expanded PCR testing of these isolates detected mono-infection with P. cynomolgi, the first time this has been reported in Thailand. Upon re-testing of 60 isolates collected from Yala, one other case was identified, a co-infection of P. cynomolgi and P. vivax. The clinical course for all three was relatively mild, with symptoms commonly seen in malaria: fever, chills and headaches. All infections were cured with a course of chloroquine and primaquine. CONCLUSION: In malaria-endemic areas with macaque populations, cases of simian malaria in humans are being reported at an increasing rate, although still comprise a very small percentage of total cases. Plasmodium cynomolgi and P. vivax are challenging to distinguish by blood smear; therefore, PCR can be employed when infections are suspected or as part of systematic malaria surveillance. As Thai MoPH policy schedules regular follow-up visits after each malaria infection, identifying those with P. cynomolgi will allow for monitoring of treatment efficacy, although at this time P. cynomolgi appears to have an uncomplicated clinical course and good response to commonly used anti-malarials.

Retrospective Survey of Borrelia spp. From Rodents and Ticks in Thailand.

Borrelia is a genus of spirochetal bacteria with several species known to cause disease in humans. The distribution of Borrelia has rarely been studied in Thailand. In this study, a retrospective survey of Borrelia was conducted in ticks and wild rodents to better characterize the prevalence, diversity, and distribution of Borrelia across Thailand. Several pools of DNA from tick samples were positive for Borrelia spp. (36/258, 13.9%). Borrelia theileri/B. lonestari was found in 17 tick samples (16 pools of Haemaphysalis bandicota and 1 pool of Rhipicephalus sp.), and Borrelia yangtzensis was found in 8 tick samples (2 pools of H. bandicota and 6 pools of Ixodes granulatus). Borrelia spp. were detected at low prevalence levels in rodent tissue samples (24/2001, 1.2%), with 19 identified as B. theileri or B. lonestari and 5 identified as B. miyamotoi. Several geographic and species-specific infection trends were apparent, with Ixodes ticks infected with B. yangtzensis and Haemaphysalis and Rhipicephalus ticks infected with both B. yangtzensis and B. theileri/B. lonestari. Notably, B. yangtzensis showed a similar geographic distribution to B. miyamotoi, which was identified in new areas of Thailand in this study. The flagellin gene sequence from B. miyamotoi was more similar to European (99.3-99.9%) than Japanese (96.9-97.6%) genotypes. This study greatly expands the knowledge of Borrelia in Thailand and identified several Borrelia species for the first time. It also found several ticks and rodents infected with the pathogen that were not previously known to carry Borrelia.

Molecular Detection of Mutations in the Propeller Domain of Kelch 13 and pfmdr1 Copy Number Variation in Plasmodium falciparum Isolates from Thailand Collected from 2002 to 2007.

We determined the prevalence of Kelch 13 mutations and pfmdr1 copy number in samples collected from the Thailand-Myanmar border, the Thailand-Cambodia border, and southern Thailand from 2002 to 2007. C580Y was the most prevalent in Trat (Thailand-Cambodia border) and Ranong (Thailand-Myanmar border) at 42% (24/57) and 13% (6/48), respectively. Less predominant mutations were also identified including R539T (7%, 4/57) and Y493H (2%, 1/57) in Trat, P574L (6%, 3/48) and P553L (2%, 1/48) in Ranong, and N537I and D452E (7%, 1/15) in Sangkhlaburi (Thailand-Myanmar border). Samples from Mae sot (33%, 11/33) harbored the highest percentage of multiple pfmdr1 copies, followed by Trat (18%, 10/57), Chiang Dao in 2003 (13%, 4/30), Phang Nga (5%, 2/44), and Chiang Dao in 2002 (4%, 1/26). This retrospective study provides geographic diversity of K13 and pfmdr1 copies and the emergence of these molecular markers in Thailand, an important background information for future surveillance in the region.

Dengue viremia kinetics in asymptomatic and symptomatic infection.

BACKGROUND: Dengue infection is a global health threat. While symptomatic cases contribute to morbidity and mortality, the majority of infected people are asymptomatic but serve as an important reservoir. However, the kinetics of viremia in asymptomatic infections remains unknown. METHODS: We enrolled 279 hospital-based symptomatic index cases and quantified dengue virus (DENV) RNA at enrollment and at the day of defervescence. To identify asymptomatic cases, 175 household members of index cases were monitored for clinical symptoms during follow-up, and blood was taken twice weekly to test for and quantify DENV RNA until cleared. RESULTS: We detected DENV in thirteen asymptomatic household members (7.43%). Their DENV serotypes were primarily the same as those of their family index cases. The median peak DENV viremia in asymptomatic subjects was lower than that of symptomatic individuals during the febrile phase, and the viral decay rate was slower in asymptomatic infections. CONCLUSIONS: DENV level and kinetics in asymptomatic individuals differed significantly from those of symptomatic cases. Despite the lower viremia, the slower decay rate in asymptomatic infections could lead to their prolonging the infectious reservoir. The improvement of transmission control to prevent such long-lived asymptomatic infections from transmitting the DENV is needed.

Metagenomic Approach to Characterizing Disease Epidemiology in a Disease-Endemic Environment in Northern Thailand.

In this study, we used a metagenomic approach to analyze bacterial communities from diverse populations (humans, animals, and vectors) to investigate the role of these microorganisms as causative agents of disease in human and animal populations. Wild rodents and ectoparasites were collected from 2014 to 2018 in Nan province, Thailand where scrub typhus is highly endemic. Samples from undifferentiated febrile illness (UFI) patients were obtained from a local hospital. A total of 200 UFI patient samples were obtained and 309 rodents and 420 pools of ectoparasites were collected from rodents (n = 285) and domestic animals (n = 135). The bacterial 16S rRNA gene was amplified and sequenced with the Illumina. Real-time PCR and Sanger sequencing were used to confirm the next-generation sequencing (NGS) results and to characterize pathogen species. Several pathogens were detected by NGS in all populations studied and the most common pathogens identified included Bartonella spp., Rickettsia spp., Leptospira spp., and Orientia tsutsugamushi. Interestingly, Anaplasma spp. was detected in patient, rodent and tick populations, although they were not previously known to cause human disease from this region. Candidatus Neoehrlichia, Neorickettsia spp., Borrelia spp., and Ehrlichia spp. were detected in rodents and their associated ectoparasites. The same O. tsutsugamushi genotypes were shared among UFI patients, rodents, and chiggers in a single district indicating that the chiggers found on rodents were also likely responsible for transmitting to people. Serological testing using immunofluorescence assays in UFI samples showed high prevalence (IgM/IgG) of Rickettsia and Orientia pathogens, most notably among samples collected during September-November. Additionally, a higher number of seropositive samples belonged to patients in the working age population (20-60 years old). The results presented in this study demonstrate that the increased risk of human infection or exposure to chiggers and their associated pathogen (O. tsutsugamushi) resulted in part from two important factors; working age group and seasons for rice cultivation and harvesting. Evidence of pathogen exposure was shown to occur as there was seropositivity (IgG) in UFI patients for bartonellosis as well as for anaplasmosis. Using a metagenomic approach, this study demonstrated the circulation and transmission of several pathogens in the environment, some of which are known causative agents of illness in human populations.

Detection of ß-lactoglobulin in human breast-milk 7 days after cow milk ingestion.

BACKGROUND: ß-lactoglobulin (BLG), a major allergen in cow milk (CM) can be detected in human breast-milk (BM) and is associated with exacerbation of symptoms in breastfed infants with cow milk protein allergy (CMPA). Currently, it is not known how long lactating mothers who consume dairy products need to withhold breastfeeding. OBJECTIVE: To elucidate the kinetics of BLG in BM after maternal ingestion of a single dose of CM. METHODS: Nineteen lactating mothers, four of whom had infants with CMPA, were instructed to avoid CM for 7 days before ingesting a single dose of CM and to continue to withhold CM thereafter throughout the study period. BLG was measured by ELISA in BM from 15 mothers of healthy infants before and at 3, 6 and 24 h, and 3 and 7 days after CM ingestion. Four pairs of mothers and CMPA infants were enrolled for BM challenge after the mothers had ingested CM. RESULTS: After CM ingestion, the level of BLG in BM increased significantly from 0.58 ng/ml (0.58 g/L) (IQR 0.38-0.88) to a peak level of 1.23 ng/ml (IQR 1.03-2.29), p < 0.001. The BLG level on day 3 (1.15 ng/ml, IQR 0.89-1.45) and day 7 (1.08 ng/ml (IQR 0.86-1.25) after CM ingestion was significantly higher than baseline (p = 0.01 and p = 0.001, respectively). BLG was detected in all BM samples from the four mothers of CMPA infants after CM ingestion, and the level was not different from that in the mothers of the 15 healthy infants. Three of the four CMPA infants developed symptoms such as maculopapular rash and hypersecretion in the airways after BM challenge. CONCLUSIONS: BLG can be detected in BM up to 7 days after CM ingestion. Lactating mothers should suspend breastfeeding to CMPA infants more than 7 days after CM ingestion.

A Field-Deployable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of the Chikungunya Virus.

BACKGROUND: Chikungunya virus (CHIKV) is a mosquito-borne virus currently transmitted in about 60 countries. CHIKV causes acute flu-like symptoms and in many cases prolonged musculoskeletal and joint pain. Detection of the infection is mostly done using RT-RCR or ELISA, which are not suitable for point-of-care diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of the CHIKV was developed. The assay sensitivity, specificity, and cross-reactivity were tested. CHIKV RT-RPA assay detected down to 80 genome copies/reaction in a maximum of 15 minutes. It successfully identified 18 isolates representing the three CHIKV genotypes. No cross-reactivity was detected to other alphaviruses and arboviruses except O'nyong'nyong virus, which could be differentiated by a modified RPA primer pair. Seventy-eight samples were screened both by RT-RPA and real-time RT-PCR. The diagnostic sensitivity and specificity of the CHIKV RT-RPA assay were determined at 100%. CONCLUSIONS/SIGNIFICANCE: The developed RT-RPA assay represents a promising method for the molecular detection of CHIKV at point of need.

Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

BACKGROUND: Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. METHODOLOGY/PRINCIPAL FINDINGS: Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. CONCLUSIONS/SIGNIFICANCE: During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.