Evaluation of Kato-Katz and multiplex quantitative polymerase chain reaction performance for clinical helminth infections in Thailand using a latent class analysis

Using an appropriate diagnostic tool is essential to soil-transmitted helminth control and elimination efforts. Kato-Katz (KK) is the most commonly used diagnostic, but recently other tools, such as real-time quantitative polymerase chain reaction (multiplex qPCR), are starting to be employed more. Here, we evaluated the performance of these two diagnostic tools for five helminth species in Thailand. In the absence of a gold standard, diagnostic performance can be evaluated using latent class analysis. Our results suggest that in moderate to high prevalence settings above 2% multiplex qPCR could be more sensitive than KK, this was particularly apparent for Opisthorchis viverrini in the northeastern provinces. However, for low prevalence, both diagnostics suffered from low sensitivity. Specificity of both diagnostics was estimated to be high (above 70%) across all settings. For some specific helminth infection such as O. viverrini, multiplex qPCR is still a preferable choice of diagnostic test. KK performed equally well in detecting Ascaris lumbricoides and Taeniasis (Taenia spp.) when the prevalence is moderate to high (above 2%). Neither test performed well when the prevalence of infection is low (below 2%), and certainly in the case for hookworm and Trichuris trichiura. Combination of two or more diagnostic tests can improve the performance although the cost would be high. Development of new methods for helminth surveillance at the pre-elimination phase is therefore very important. This article is part of the theme issue 'Challenges and opportunities in the fight against neglected tropical diseases: a decade from the London Declaration on NTDs'

The effect of mimicking febrile temperature and drug stress on malarial development

<p>Abstract</p> <p>Background</p> <p>Malaria remains one of the most important tropical diseases of human with 1–2 million deaths annually especially caused by <it>P. falciparum</it>. During malarial life cycle, they exposed to many environmentally stresses including wide temperature fluctuation and pharmacological active molecules. These trigger malarial evolutionarily adaptive responses. The effect of febrile temperature on malarial growth, development and drug susceptibility by mimicking patient in treatment failure before and after drug uptake was examined.</p> <p>Methods</p> <p>Sensitivities of <it>P. falciparum </it>to antimalarial drug (chloroquine, mefloquine, quinine and artesunate) were investigated based on the incorporation of [³H] hypoxanthine into parasite nucleic acids or radioisotopic technique. The number of parasites was examined under microscope following Giemsa staining and the parasite development at the end of each phase was counted and comparison of parasite number was made. The proteome was separated, blotted and hybridized with anti-Hsp70s primary antibody. The hybridized proteins were separately digested with trypsin and identified by MALDI-TOF peptide mass fingerprint.</p> <p>Results</p> <p>The results show that febrile temperature is capable of markedly inhibiting the growth of field isolate <it>P. falciparum </it>but not to K1 and 3D7 standard strains. K1 and 3D7 grown under heat shock developed greater and the reinfection rate was increased up to 2-folds when compared to that of non-heat shock group. The IC₅₀value of K1 toward chloroquine, mefloquine and quinine under heat shock was higher than that of K1 under non-heat shock which is opposite to that of 3D7. Heat shock caused death in field isolated parasite. It was also found that the febrile temperature coped with chloroquine uptake had no effect to the development, drug sensitivity and the parasite number of K1 strain. In the opposite way, heat shock and chloroquine shows extremely effect toward 3D7 and field isolate PF91 as shown by higher number of dead parasites compared to that of control group. After culture under high temperature with artesunate, the total parasite number of all strains including K1, 3D7 and PF91 was extremely decreased and the parasite was not found at the end. Additionally, the expression of <it>pf</it>Hsp70s was found in all strains and conditions as shown in 120 kDa hybridized band. However, the proteome extracted from K1 grown under heat shock with chloroquine, anti-<it>pf</it>Hsp70 interacted with additional three bands identified by MALDI-TOF as elongation factor-1α (83 kDa), pf<it>Hsp</it>86 (60 kDa) and phosphoethanolamine <it>N</it>-methyltransferase (43 kDa).</p> <p>Conclusion</p> <p>In conclusion, febrile temperature was capable of markedly inhibiting the growth of field isolate <it>P. falciparum </it>while the development, reinfection rate and drug (chloroquine, mefloquine and quinine) resistant level of standard strain K1 was enhanced. However, the febrile temperature coped with chloroquine had no effect to the development, drug sensitivity and the parasite number of K1 strain. In the opposite way, heat shock and chloroquine showed extremely effect toward 3D7 and field isolate PF91 as shown by some died parasites. Heat shock protein 70 (<it>pf</it>HSP70) of strain K1 under heat shock with chloroquine might involved in many pathways in order to sustain the parasite.</p

Real-Time PCR Detection and Phylogenetic Relationships of \u3ci\u3eNeorickettsia\u3c/i\u3e In Digeneans From Egypt, Phillipines, Thailand, Vietnam and the United States

Neorickettsia (Rickettsiales, Anaplasmataceae) is a genus of obligate intracellular bacterial endosymbionts of digeneans (Platyhelminthes, Digenea). Some Neorickettsia are able to invade cells of the digenean\u27s vertebrate host and are known to cause diseases of domestic animals, wildlife, and humans. In this study we report the results of screening digenean samples for Neorickettsia collected from bats in Egypt and Mindoro Island, Philippines, snails and fishes from Thailand, and fishes from Vietnam and the USA. Neorickettsia were detected using a real-time PCR protocol targeting a 152bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1853bp long region of the GroESL operon and a 1371bp long region of 16S rRNA. Eight unique genotypes of Neorickettsia were obtained from digenean samples. Neorickettsia sp. 8 obtained from Lecithodendrium sp. from Egypt; Neorickettsia sp. 9 and 10 obtained from two species of Paralecithodendrium from Mindoro, Philippines; Neorickettsia sp. 11 from Lecithodendrium sp. and Neorickettsia sp. 4 (previously identified from Saccocoelioides lizae, from China) from Thailand; Neorickettsia sp. 12 from Dicrogaster sp. Florida, USA; Neorickettsia sp. 13 and SF agent from Vietnam. Sequence comparison and phylogenetic analysis demonstrated that the forms, provisionally named Neorickettsia sp. 8-13, represent new genotypes. We have for the first time detected Neorickettsia in a digenean from Egypt (and the African continent as a whole), the Philippines, Thailand and Vietnam based on PCR and sequencing evidence. Our findings suggest that further surveys from the African continent, SE Asia, and island countries are likely to reveal new Neorickettsia lineages as well as new digenean host associations

Taeniasis and Other Helminthic Infections in the Northern and Northeastern Border Provinces of Thailand

Abstract icroscopic stool examinations to diagnose taeniasis and other helminthic infections were performed in three provinces: Nan in the north, and Ubon Ratchathani and Khon Kaen in the northeast. In Nan, lowland communities and hill-tribe communities were treated separately. By Kato thick-smear technique, the results indicated similar prevalence (13-15%) of helminthic infections in all three provinces. In Nan, higher infection rates were found among lowland (34.9%) than hilltribe inhabitants (13.0%). The most significant contributors to the high infection rate were minute intestinal flukes (29.4%) in lowland Nan, whereas in the hill-tribe community, prevalence was very low (0.5%). Infections in the two northeastern provinces were mainly caused by Opisthorchis liver flukes (6-7%). Hookworm infection rates were 4-6% in all study areas. Taenia eggs were found in 2% of both Nan groups, 3.7% in Ubon Ratchathani and 0.9% in Khon Kaen. Other helminths found included Ascaris lumbricoides (4%), Enterobius vermicularis (0.5%) in Nan hill-tribe communities, Strongyloides stercoralis (about 1-2%), Trichuris trichiura in both northeastern provinces (&lt; 1%), and A. lumbricoides in Ubon Ratchathani (&lt; 1%). Thirteen cases positive for Taenia eggs were treated with 2 g niclosamide, and five bowel movements were observed immediately following cathartic administration. Eleven of these 13 cases had Taenia segments in their stool. Long-chain strobilae were commonly expelled in the first two bowel movements. The head portion, or scolex, was released in five cases during any of the five bowel movements. One case expelled two separate long-chain strobilae with two scolices. All Taenia worms found were identified morphologically as T. saginata, either by scolex or gravid proglottids

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure

Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein

Immunoproteomics to identify species-specific antigens in Neospora caninum recognised by infected bovine sera

Bovine neosporosis is a disease of concern due to its global distribution and significant economic impact through massive losses in the dairy and meat industries. To date, there is no effective chemotherapeutic drug or vaccine to prevent neosporosis. Control of this disease is therefore dependent on efficient detection tests that may affect treatment management strategies. This study was conducted to identify the specific immunoreactive proteins of Neospora caninum tachyzoites recognised by sera from cattle infected with N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and B. bigemina, and by sera from uninfected cattle using two-DE dimensional gel electrophoresis (2-DE) combined with immunoblot and mass spectrometry (LC-MS/MS). Among 70 protein spots that reacted with all infected sera, 20 specific antigenic spots corresponding to 14 different antigenic proteins were recognised by N. caninum-positive sera. Of these immunoreactive antigens, proteins involved in cell proliferation and invasion process were highly immunogenic, including HSP90-like protein, putative microneme 4 (MIC4), actin, elongation factor 1-alpha and armadillo/beta-catenin-like repeat-containing protein. Interestingly, we discovered an unnamed protein product, rhoptry protein (ROP1), possessing strong immunoreactivity against N. caninum but with no data on function available. Moreover, we identified cross-reactive antigens among these apicomplexan parasites, especially N. caninum, T. gondii and C. parvum. Neospora caninum-specific immunodominant proteins were identified for immunodiagnosis and vaccine development. The cross-reactive antigens could be evaluated as potential common vaccine candidates or drug targets to control the diseases caused by these apicomplexan protozoan parasites

Helminth-Derived Immunomodulatory Molecules

Infection of man with parasitic helminths leads to potent activation and modulation of the host immune response. This modulation of immunity by helminth infections may have bystander effects in altering, either suppressing or exacerbating, unrelated inflammatory processes. Various ongoing clinical trials are testing the therapeutic application of helminth infection of patients with inflammatory diseases, including inflammatory bowel disease and allergic disorders. Rather than the use of five helminth infection, with the potential for side effects, an alternative approach is to identify the immune modulatory molecules (IM) produced by helminths that can alter immune functions. In this review, we will focus on characterized helminth-derived IMs that may have potential to be developed as novel therapeutics for inflammatory diseases

Maoberry (Antidesma bunius) ameliorates oxidative stress and inflammation in cardiac tissues of rats fed a high-fat diet

Abstract Backgound Chronic fat-rich diets consumption is increased risk associated with cardiovascular diseases (CVD). Prevention or reduction the progression of cardiac tissue deterioration could benefit in CVD. This study aimed to examine the effects of maoberry (Antidesma bunius), a antioxidant-rich tropical fruit, supplementation on oxidative stress and inflammation in cardiac tissues of rats fed a high-fat diet (HFD). Methods The male rats orally received HFD with maoberry extract doses of 0.38, 0.76 or 1.52 g/kg or simvastatin (10 mg/kg) for 12 weeks. At the end of the experimental period, the rats were fasted, euthanized and harvested for the hearts. Results Significantly reduced oxidative stress (malondialdehyde levels) and enhanced antioxidant capacity (ferric-reducing activities) in cardiac tissues of the rats were found. Maoberry extract remarkably ameliorated the expressions of genes involved with pro-inflammatory such as the tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and endothelial nitric oxide synthase (eNOS). Conclusions Our findings suggest that maoberry extract has remarkable effects on preventing progression of cardiac tissue deterioration at least through lowering oxidative stress and inflammation

A Mitochondria-Penetrating Peptide Exerts Potent Anti-Plasmodium Activity and Localizes at Parasites&rsquo; Mitochondria

Mitochondria are considered a novel drug target as they play a key role in energy production and programmed cell death of eukaryotic cells. The mitochondria of malaria parasites differ from those of their vertebrate hosts, contributing to the drug selectivity and the development of antimalarial drugs. (Fxr)3, a mitochondria-penetrating peptide or MPP, entered malaria-infected red cells without disrupting the membrane and subsequently killed the blood stage of P. falciparum parasites. The effects were more potent on the late stages than on the younger stages. Confocal microscopy showed that the (Fxr)3 intensely localized at the parasite mitochondria. (Fxr)3 highly affected both the lab-strain, chloroquine-resistant K1, and freshly isolated malaria parasites. (Fxr)3 (1 ng/mL to 10 &mu;g/mL) was rarely toxic towards various mammalian cells, i.e., mouse fibroblasts (L929), human leukocytes and erythrocytes. At a thousand times higher concentration (100 &mu;g/mL) than that of the antimalarial activity, cytotoxicity and hemolytic activity of (Fxr)3 were observed. Compared with the known antimalarial drug, atovaquone, (Fxr)3 exhibited more rapid killing activity. This is the first report on antimalarial activity of (Fxr)3, showing localization at parasites&rsquo; mitochondria