Prevalence of asymptomatic Plasmodium infections with sub-microscopic parasite densities in the northwestern border of Thailand: a potential threat to malaria elimination.

BACKGROUND: Asymptomatic infections with sub-microscopic Plasmodium serve as a silent reservoir of disease, critical to sustaining a low level of remanent malaria in the population. These infections must be effectively identified and targeted for elimination. The sensitivity of light microscopy, the traditional method used for diagnosing Plasmodium infections, is frequently insufficient for detecting asymptomatic infections due to the low density of parasitaemia. The objective of this study was to explore the current prevalence of asymptomatic sub-microscopic Plasmodium carriages to evaluate the parasite reservoir amongst residents from 7 hamlets in Tak Province in northwestern Thailand using a highly sensitive molecular method. METHODS: Malaria infection was screened in a real-world setting from 3650 finger-prick blood specimens collected in a mass cross-sectional survey using light microscopy and loop-mediated isothermal amplification (LAMP). LAMP results were later confirmed in a laboratory setting in Bangkok using nested PCR, restriction enzyme digestion and DNA sequencing. The association of malaria infection with demographic factors was explored. RESULTS: Parasite prevalence was 0.27% (10/3650) as determined by microscopy. Sub-microscopic infection prevalence was 2.33% (85/3650) by LAMP. Of these, 30.6% (26/85) were infected with Plasmodium falciparum, 52.9% (45/85) with Plasmodium vivax, 2.4% (2/85) with Plasmodium malariae, 4.7% (4/85) with mixed P. falciparum and P. vivax, and 9.4% (8/85) had parasite densities too low for species identification. Asymptomatic carriages (T < 37.5 °C) accounted for 95% (76/80) of all sub-microscopic cases with the highest prevalence occurring in the subjects 31-45 years of age (p ≤ 0.035). Participants working on plantations or as merchants had an increased infection risk. Evaluation by microscopy identified 10.53% (10/95) of all Plasmodium infected participants. CONCLUSION: Participants carrying asymptomatic Plasmodium infections with sub-microscopic parasite densities are considerable in this area. These findings provide the true disease burden and risk factors in this region. This information helps to direct policy makers towards better schemes and delivery of targeted interventions. Moreover, this is the first study to use LAMP in mass screening for sub-clinical and sub-microscopic infections in a field setting in Thailand. LAMP proves to be a sensitive and field-deployable assay suitable for national malaria control screening campaigns.

Loop-mediated isothermal amplification assay for rapid diagnosis of malaria infections in an area of endemicity in Thailand.

The loop-mediated isothermal amplification (LAMP) method, developed by our group for diagnosis of four human malaria parasites, was evaluated on a large scale at a remote clinic in Thailand where malaria is endemic. A total of 899 febrile patients were analyzed in this study. LAMP was first evaluated in 219 patients, and the result was compared to those of two histidine-rich protein (HRP)-2 rapid diagnostic tests (RDTs) and microscopy as a gold standard. LAMP DNA extraction was conducted by a simple boiling method, and the test results were assessed visually. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 95.7%, 100%, 100%, and 98%, respectively, for LAMP and 98.6%, 98%, 95.8%, and 99.3%, respectively, for RDTs. Since RDT-positive results were based on one out of two RDTs, the sensitivity of RDTs was slightly higher than that of LAMP. However, LAMP tended to be more specific than RDTs. LAMP next was evaluated in 680 patients, and the result was compared to that of microscopy as a gold standard. Sensitivity, specificity, PPV, NPV, and diagnostic accuracy of LAMP were 88.9%, 96.9%, 92.2%, 95.5%, and 94.6%, respectively. Nested PCR was used to confirm the discrepant results. Malaria LAMP in a remote clinic in Thailand achieved an acceptable result, indicating that LAMP malaria diagnosis is feasible in a field setting with limited technical resources. Additionally, the rapid boiling method for extracting DNA from dried blood spots proved to be simple, fast, and suitable for use in the field.

End-point rapid detection of total and pathogenic Vibrio parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) in raw seafood using a colorimetric loop-mediated isothermal amplification-xylenol orange technique.

Background: Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis in humans worldwide. To ensure seafood safety and to minimize the occurrence of seafood-borne diseases, early detection of total V. parahaemolyticus (pathogenic and non-pathogenic strains) and pathogenic V. parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) is required. This study further improved a loop-mediated isothermal amplification (LAMP) assay using xylenol orange (XO), a pH sensitive dye, to transform conventional LAMP into a one-step colorimetric assay giving visible results to the naked eye. LAMP-XO targeted rpoD for species specificity and tdh, trh1, and trh2 for pathogenic strains. Multiple hybrid inner primers (MHP) of LAMP primers for rpoD detection to complement the main primer set previously reported were designed by our group to maximize sensitivity and speed. Methods: Following the standard LAMP protocol, LAMP reaction temperature for rpoD, tdh, trh1, and trh2 detection was first determined using a turbidimeter. The acquired optimal temperature was subjected to optimize six parameters including dNTP mix, betaine, MgSO4, Bst 2.0 WarmStart DNA polymerase, reaction time and XO dye. The last parameter was done using a heat block. The color change of the LAMP-XO result from purple (negative) to yellow (positive) was monitored visually. The detection limits (DLs) of LAMP-XO using a 10-fold serial dilution of gDNA and spiked seafood samples were determined and compared with standard LAMP, PCR, and quantitative PCR (qPCR) assays. Subsequently, the LAMP-XO assay was validated with 102 raw seafood samples and the results were compared with PCR and qPCR assays. Results: Under optimal conditions (65 °C for 75 min), rpoD-LAMP-XO and tdh-LAMP-XO showed detection sensitivity at 102 copies of gDNA/reaction, or 10 folds greater than trh1-LAMP-XO and trh2-LAMP-XO. This level of sensitivity was similar to that of standard LAMP, comparable to that of the gold standard qPCR, and 10-100 times higher than that of PCR. In spiked samples, rpoD-LAMP-XO, tdh-LAMP-XO, and trh2-LAMP-XO could detect V. parahaemolyticus at 1 CFU/2.5 g spiked shrimp. Of 102 seafood samples, LAMP-XO was significantly more sensitive than PCR (P < 0.05) for tdh and trh2 detection and not significantly different from qPCR for all genes determined. The reliability of tdh-LAMP-XO and trh2-LAMP-XO to detect pathogenic V. parahaemolyticus was at 94.4% and 100%, respectively. Conclusions: To detect total and pathogenic V. parahaemolyticus, at least rpoD-LAMP-XO and trh2-LAMP-XO should be used, as both showed 100% sensitivity, specificity, and accuracy. With short turnaround time, ease, and reliability, LAMP-XO serves as a better alternative to PCR and qPCR for routine detection of V. parahaemolyticus in seafood. The concept of using a one-step LAMP-XO and MHP-LAMP to enhance efficiency of diagnostic performance of LAMP-based assays can be generally applied for detecting any gene of interest.

Enhancement of loop mediated isothermal amplification's sensitivity and speed by multiple inner primers for more efficient identification of Vibrio parahaemolyticus.

The modified loop-mediated isothermal amplification (LAMP), called multiple hybrid, inner primers (MHP)-LAMP, was developed to enhance the efficiency of the existing LAMP-based assay for Vibrio parahaemolyticus detection. The method was built on a conventional LAMP assay by employing 2 newly designed extra sets of primers to increase the initial binding sites of core primers on the V. parahaemolyticus's rpoD gene from 8 to 12. With this strategy, the assay detection sensitivity was increased by 10 folds, with the detection limit (DL) approaching 100 copies of purified target genomic DNA (gDNA) as analyzed by real-time turbidity measurement and gel electrophoresis. The MHP also accelerated the rate of DNA amplification by 30%, rendering the assay faster. The MHP-LAMP assay did not cross- react with other pathogens, indicating that it was highly specific for V. parahaemolyticus detection. Whilst V. parahaemolyticus was used as a study model herein, our idea of using MHP to maximize assay sensitivity and speed is considered as a universal strategy that can be applied to enhance efficiency of LAMP-based assays for detecting any DNA and RNA of interest. •The strategy of using multiple hybrid, inner primers (MHP) to enhance LAMP assay's efficiency was demonstrated with success.•The MHP enhanced the sensitivity and speed of the existing LAMP assay, designed to detect V. parahaemolyticus, by 10 times and 30%, respectively.•The proposed strategy can be applied to boost up any other LAMP-based assay's diagnostic performance.

Incidence, genetic diversity, and antimicrobial resistance profiles of Vibrio parahaemolyticus in seafood in Bangkok and eastern Thailand.

Background: Emergence of Vibrio parahaemolyticus pandemic strain O3:K6 was first documented in 1996. Since then it has been accounted for large outbreaks of diarrhea globally. In Thailand, prior studies on pandemic and non-pandemic V. parahaemolyticus had mostly been done in the south. The incidence and molecular characterization of pandemic and non-pandemic strains in other parts of Thailand have not been fully characterized. This study examined the incidence of V. parahaemolyticus in seafood samples purchased in Bangkok and collected in eastern Thailand and characterized V. parahaemolyticus isolates. Potential virulence genes, VPaI-7, T3SS2, and biofilm were examined. Antimicrobial resistance (AMR) profiles and AMR genes (ARGs) were determined. Methods: V. parahaemolyticus was isolated from 190 marketed and farmed seafood samples by a culture method and confirmed by polymerase chain reaction (PCR). The incidence of pandemic and non-pandemic V. parahaemolyticus and VPaI-7, T3SS2, and biofilm genes was examined by PCR. AMR profiles were verified by a broth microdilution technique. The presence of ARGs was verified by genome analysis. V. parahaemolyticus characterization was done by multilocus sequence typing (MLST). A phylogenomic tree was built from nucleotide sequences by UBCG2.0 and RAxML softwares. Results: All 50 V. parahaemolyticus isolates including 21 pathogenic and 29 non-pathogenic strains from 190 samples had the toxRS/old sequence, indicating non-pandemic strains. All isolates had biofilm genes (VP0950, VP0952, and VP0962). None carried T3SS2 genes (VP1346 and VP1367), while VPaI-7 gene (VP1321) was seen in two isolates. Antimicrobial susceptibility profiles obtained from 36 V. parahaemolyticus isolates revealed high frequency of resistance to colistin (100%, 36/36) and ampicillin (83%, 30/36), but susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (100%, 36/36). Multidrug resistance (MDR) was seen in 11 isolates (31%, 11/36). Genome analysis revealed ARGs including blaCARB (100%, 36/36), tet(34) (83%, 30/36), tet(35) (42%, 15/36), qnrC (6%, 2/36), dfrA6 (3%, 1/36), and blaCTX-M-55 (3%, 1/36). Phylogenomic and MLST analyses classified 36 V. parahaemolyticus isolates into 5 clades, with 12 known and 13 novel sequence types (STs), suggesting high genetic variation among the isolates. Conclusions: Although none V. parahaemolyticus strains isolated from seafood samples purchased in Bangkok and collected in eastern Thailand were pandemic strains, around one third of isolates were MDR V. parahaemolyticus strains. The presence of resistance genes of the first-line antibiotics for V. parahaemolyticus infection raises a major concern for clinical treatment outcome since these resistance genes could be highly expressed under suitable circumstances.

Single-Strand Conformation Polymorphism Fingerprint Method for Dictyostelids.

Dictyostelid social amoebae are a highly diverse group of eukaryotic soil microbes that are valuable resources for biological research. Genetic diversity study of these organisms solely relies on molecular phylogenetics of the SSU rDNA gene, which is not ideal for large-scale genetic diversity study. Here, we designed a set of PCR-single-strand conformation polymorphism (SSCP) primers and optimized the SSCP fingerprint method for the screening of dictyostelids. The optimized SSCP condition required gel purification of the SSCP amplicons followed by electrophoresis using a 9% polyacrylamide gel under 4°C. We also tested the optimized SSCP procedure with 73 Thai isolates of dictyostelid that had the SSU rDNA gene sequences published. The SSCP fingerprint patterns were related to the genus-level taxonomy of dictyostelids, but the fingerprint dendrogram did not reflect the deep phylogeny. This method is rapid, cost-effective, and suitable for large-scale sample screening as compared with the phylogenetic analysis of the SSU rDNA gene sequences.

Validation of PfSNP-LAMP-Lateral Flow Dipstick for Detection of Single Nucleotide Polymorphism Associated with Pyrimethamine Resistance in Plasmodium falciparum.

The loop-mediated isothermal amplification coupled with lateral flow dipstick (PfSNP-LAMP-LFD) was recently developed to detect single nucleotide polymorphism (AAT → ATT), corresponding to substitution of asparagine to isoleucine at amino acid position 51 in the P. falciparumdhfr-ts gene associated with antifolate resistance. In this present study, the PfSNP-LAMP-LFD was validated on 128 clinical malaria samples of broad ranged parasite densities (10 to 87,634 parasites per microliter of blood). The results showed 100% accuracy for the detection of single nucleotide polymorphism for N51I mutation. Indeed, the high prevalence of N51I in the Pfdhfr-ts gene detected in the clinical samples is in line with reports of widespread antifolate resistant P. falciparum in Thailand. The relationship between enzyme choice and reaction time was observed to have an effect on PfSNP-LAMP-LFD specificity; however, the method yielded consistent results once the conditions have been optimized. The results demonstrate that PfSNP-LAMP-LFD is a simple method with sufficient sensitivity and specificity to be deployed in routine surveillance of antifolate resistance molecular marker and inform antimalarial management policy.

Identification and characterization of protein 14-3-3 in carcinogenic liver fluke Opisthorchis viverrini.

Protein 14-3-3s are abundant phospho-serine/threonine binding proteins, which are highly conserved among eukaryotes. Members of this protein family mediate metabolism and signal transduction networks through binding to hundreds of other protein partners. Protein 14-3-3s have been studied in other species of parasitic helminthes, but little is known about this protein in the carcinogenic liver fluke Opisthorchis viverrini. In this study, we identified and characterized protein 14-3-3s of O. viverrini. Seven protein 14-3-3 encoded sequences were retrieved from the O. viverrini genome database. Multiple alignment and phylogenetic analysis were performed. Two isoforms (protein 14-3-3 zeta and protein 14-3-3 epsilon) that have been previously found in the excretory-secretory (ES) products of O. viverrini were produced as recombinant protein in E. coli and the proteins were then used to immunize mice to obtain specific antibodies. Western blot analysis showed that both proteins were detected in all obtainable developmental stages of O. viverrini and the ES products. Immunolocalization revealed that both isoforms were expressed throughout tissues and organs except the gut epithelium. The highest expression was observed in testes especially in developing spermatocytes, suggesting their role in spermatogenesis. Prominent expression was also detected on tegumental surface of the parasite and on epical surface of bile duct epithelium indicates their additional role in host-parasite interaction. These findings indicate that protein 14-3-3s play important role in the life cycle of the parasite and might be involved in the pathogenesis of O. viverrini infection.

Infectivity of symptomatic and asymptomatic Plasmodium vivax infections to a Southeast Asian vector, Anopheles dirus.

Plasmodium vivax is now the predominant species causing malarial infection and disease in most non-African areas, but little is known about its transmission efficiency from human to mosquitoes. Because the majority of Plasmodium infections in endemic areas are low density and asymptomatic, it is important to evaluate how well these infections transmit. Using membrane feeding apparatus, Anopheles dirus were fed with blood samples from 94 individuals who had natural P. vivax infections with parasitemias spanning four orders of magnitude. We found that the mosquito infection rate was positively correlated with blood parasitemia and that infection began to rise when parasitemia was >10parasites/µl. Below this threshold, mosquito infection is rare and associated with very few oocysts. These findings provide useful information for assessing the human reservoir of transmission and for establishing diagnostic sensitivity required to identify individuals who are most infective to mosquitoes.

Evaluation of loop-mediated isothermal amplification (LAMP) for malaria diagnosis in a field setting.

We used the loop-mediated isothermal amplification (LAMP) method developed by our group for malaria diagnosis with genus-specific and species-specific primers for the four human malaria parasites at a field clinic in comparison with standard microscopy. Among 110 blood samples collected from the malaria clinic in Thailand, LAMP detected 59 of 60 samples positive by microscopy (sensitivity = 98.3%) and none of the 50 microscopy-negative samples (specificity = 100%). Negative predictive value (NPV) and positive predictive value (PPV) of LAMP were 98% and 100%, respectively. These results indicate that LAMP is an effective tool for malaria diagnosis at a field clinic in a field setting.

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for clinical detection of Plasmodium falciparum gametocytes.

Plasmodium falciparum gametocytes are usually present in peripheral blood at a very low level, thus requiring a sensitive assay detection method. In this study, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed for clinical detection of P. falciparum gametocytes. Transcripts of Pfs16 of sexually committed ring and Pfs25 of mature gametocytes were detected by RT-LAMP in 82 clinical blood samples using nested RT-PCR as a gold standard. RT-LAMP demonstrated a detection limit of 1 parasitized red blood cell (RBC)/500microl of blood for both Pfs16 and Pfs25 transcripts. For Pfs16 transcript, RT-LAMP detected all 30 samples positive by nested RT-PCR (100% sensitivity) and 1 in 52 samples negative by nested RT-PCR (98.1% specificity). For Pfs25 transcript, RT-LAMP detected all 15 samples positive by nested RT-PCR (100% sensitivity) and none of 67 samples negative by nested RT-PCR (100% specificity). Negative predictive value (NPV) and positive predictive value (PPV) of RT-LAMP for detection of Pfs16 transcript were 100% and 96.8%, respectively, and 100% for both when employing Pfs25 transcript. Detection rate of Pfs16 and Pfs25 transcripts by RT-LAMP in microscopically gametocyte-negative samples was 91.7% and 29.2%, respectively. Compared with nested RT-PCR, RT-LAMP had a higher sensitivity but similar specificity, with the advantage of a shorter assay time. As RT-LAMP requires very basic instruments and the results can be obtained by visual inspection, this technique provides a simple and reliable tool for epidemiological studies of malaria transmission and in gametocyte-targeted control programmes.