Comparative Study of DNA Extraction Methods for the PCR Detection of Intestinal Parasites in Human Stool Samples.

Stool samples typically contain PCR inhibitors; however, helminths are difficult to lyse and can cause false-negative PCR results. We assessed the effective methods for extracting DNA from different kinds of intestinal parasites. We compared the most common DNA extraction methods from stool samples, including the phenol-chloroform technique with or without a bead-beating step (P and PB), a QIAamp Fast DNA Stool Mini Kit (Q), and a QIAamp PowerFecal Pro DNA Kit (QB). Genomic DNA was extracted from 85 stool samples collected from patients infected with Blastocystis sp., Ascaris lumbricoides, Trichuris trichiura, hookworm, and Strongyloides stercoralis. DNA quantity and DNA quality were evaluated via spectrophotometry, and DNA integrity was assessed by PCR. We found that P and PB provided higher DNA yields (~4 times) than when using Q and QB. However, P showed the lowest detection rate of PCR (8.2%), wherein only S. stercoralis (7 out of 20 samples) was detected. QB showed the highest detection rate of PCR (61.2%). After plasmid spikes, only 5 samples by QB were negative while 60 samples by P were still negative. Remarkably, QB could extract DNA from all the groups of parasites that we tested. These results indicate that QB is the most effective DNA extraction method for the diagnosis and monitoring of intestinal parasites via PCR.

Bacillus thuringiensis Cry5B is Active against Strongyloides stercoralis in vitro.

Strongyloidiasis, caused by Strongyloides stercoralis infection, is an important neglected tropical disease that causes significant public health problems in the tropics and subtropics. The disease can persist in hosts for decades and may be life-threatening because of hyperinfection and dissemination. Ivermectin (mostly) and albendazole are the most common anthelmintics used for treatment. Albendazole is suboptimal for this parasite, and although ivermectin is quite effective in immunocompromised patients, a multiple-course regimen is required. Furthermore, reliance on a single drug class for treating intestinal nematodes is a recipe for future failure. Therefore, it is important to discover new anthelmintics to treat or prevent human strongyloidiasis. One promising candidate is the Bacillus thuringiensis crystal protein Cry5B. Cry5B is highly potent against parasitic nematodes, for example, hookworms and Ascaris suum. Here, we investigated the potential of Cry5B against S. stercoralis. Multiple stages of S. stercoralis, including the first larval stage (L1s), infective stage (iL3s), free-living adult stage, and parasitic female stage, were all susceptible to Cry5B as indicated by impairment of motility and decreased viability in vitro. In summary, Cry5B demonstrated strong potential as an effective anthelmintic for treatment and transmission control of human strongyloidiasis, justifying further experiments to investigate in vivo therapeutic efficacy.

Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths.

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin-ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

The Genetic Polymorphisms of 24 Base Pair Duplication and Point G102S of Human Chitotriosidase to Bancroftian Filariasis at the Thai⁻Myanmar Border.

Lymphatic filariasis, caused by lymphatic filarial parasites, Wuchereria bancrofti, and Brugia malayi, causes significant morbidity and disability to 120 million people in the tropics and subtropics. Chitin has an important role for embryogenesis in adult worms and is a component of microfilaria sheath. Human chitotriosidase (CHIT1) is a chitin-degrading enzyme which provides a protective role against chitin-containing pathogens. Here, we determined the association of CHIT1 polymorphisms with susceptibility to bancroftian filariasis (BF) in 88 individuals at the Thai⁻Myanmar border. Two common polymorphisms of CHIT1, contributing inactive CHIT protein, including 24 base pair (24 bp) duplication in exon 10, and p. G102S in exon 4 were genotyped by allele-specific Polymerase Chain Reaction (PCR) and PCR sequencing, respectively. Unexpectedly, genotype frequencies of 24 bp duplication insertion homozygous (INS/INS) were significantly higher in endemic normal (EN) (40.0%) than BF patients (31.4%). In contrast, genotype frequencies of p. G102S homozygous (A/A) in BF patients (21.6%) was higher than in EN (19.0%) without statistical difference. Mutant allele frequencies of 24 bp duplication were 0.6125 (98/160) and p. G102S were 0.392 (69/176). Genotype and allele frequencies of CHIT1, 24 bp duplication, and p. G102S, showed no association with BF patients.

A Simple Genotyping Method for Rapid Differentiation of Blastocystis Subtypes and Subtype Distribution of Blastocystis spp. in Thailand.

Blastocystis spp. is one of the most common protozoa of humans and animals worldwide. The genetic diversity of Blastocystis spp. might be associated with a wide range of symptoms. However, the prevalence of each subtype is different in each country. Until now, there is no standard method for subtyping of Blastocystis spp. We developed a sequential restriction fragment length polymorphism (RFLP) analysis for the rapid differentiation of human Blastocystis subtypes. A large-scale study was also conducted to determine the subtype distribution of Blastocystis spp. in Thailand. Stool samples were collected from 1025 school-age students in four regions of Thailand. Blastocystis infections were identified by direct smear, formalin ethyl-acetate concentration technique (FECT), Boeck and Drbohlav's Locke-Egg-Serum (LES) medium culture, and polymerase chain reaction (PCR) of small-subunit ribosomal DNA (SSU rDNA). Subtypes of Blastocystis spp. were determined by RFLP. Phylogenetic tree of partial SSU rDNA sequences of Blastocystis spp. was constructed using the Maximum Likelihood (ML) method. Out of 1025 students, 416 (40.6%) were positive for Blastocystis spp. Using two steps of RFLP reactions, we could determine subtype one⁻three among these students. Subtype 3 was the most common subtype (58.72%) in Thai students, followed by subtype 1 (31.2%), and subtype 2 (10.1%). Blastocystis subtype 3 was the most prevalent in all regions of Thailand. The subtype distribution of Blastocystis spp. in Thailand was different from other countries.

The Experimental Infections of the Human Isolate of Strongyloides Stercoralis in a Rodent Model (The Mongolian Gerbil, Meriones Unguiculatus).

Strongyloidiasis is life-threatening disease which is mainly caused by Strongyloides stercoralis infection. Autoinfection of the parasite results in long-lasting infection and fatal conditions, hyperinfection and dissemination (primarily in immunosuppressed hosts). However, mechanisms of autoinfection and biology remain largely unknown. Rodent models including mice and rats are not susceptible to the human isolate of S. stercoralis. Variations in susceptibility of the human isolate of S. stercoralis are found in dogs. S. ratti and S. venezuelensis infections in rats are an alternative model without the ability to cause autoinfection. The absence of appropriate model for the human isolate of strongyloidiasis hampers a better understanding of human strongyloidiasis. We demonstrated the maintenance of the human isolate of the S. stercoralis life cycle in the Mongolian gerbil (Meriones unguiculatus). The human isolate of S. stercoralis caused a patent infection in immunosuppressed gerbils, more than 18 months. The mean number of recovery adult parasitic worms were 120 ± 23 (1.2% of the initial dose) and L1s were 12,500 ± 7,500 after day 28 post-inoculation (p.i.). The prepatent period was 9⁻14 days. Mild diarrhoea was found in gerbils carrying a high number of adult parasitic worms. Our findings provided a promising model for studying biology and searching new alternative drugs against the parasites. Further studies about the hyperinfection and dissemination would be performed.

Larvicidal Activity of Synthesized Silver Nanoparticles from Curcuma zedoaria Essential Oil against Culex quinquefasciatus.

Culex quinquefasciatus is the major vector of the bancroftian filarial parasite which causes human lymphatic filariasis and St. Louis encephalitis. The simple way to stop the transmission is to control the vector by using synthetic chemicals. However, herbal essential oils have biological properties, such as a larvicidal effect and are ecofriendly to use. In this study, we investigated the larvicidal activity of Curcuma zedoaria essential oil (ZEO) and biosynthesized silver nanoparticles using this essential oil (ZEO-AgNPs). The larvicidal activity against both insecticide-susceptible and -resistant strains of Cx. quinquefasciatus larvae of ZEO were investigated and compared with ZEO-AgNPs. The ZEO-AgNPs showed the utmost toxicity against both strains of Cx. quinquefasciatus. After 24 h of exposure, LC50 and LC99 of ZEO against susceptible strain were 36.32 and 85.11 ppm, respectively. While LC50 and LC99 of ZEO against the resistant strain were 37.29 and 76.79 ppm, respectively. Whereas ZEO-AgNPs offered complete larval mortality within 24 h of exposure, LC50 and LC99 of ZEO-AgNPs against the susceptible strain, were 0.57 and 8.54 ppm, respectively. For the resistant strain, LC50 and LC99 values were 0.64 and 8.88 ppm, respectively. The potency in killing Cx. quinquefasciatus and stability of ZEO-AgNPs have made this product a good candidate for the development of novel natural larvicides.

A Cross-Sectional Study on Intestinal Parasitic Infections in Children in Suburban Public Primary Schools, Saraburi, the Central Region of Thailand.

Intestinal parasitic infection rate among school-aged children in Thailand has been decreasing. However, certain intestinal parasites remain problematic in some regions. This cross-sectional study was conducted between February and September 2016 in three suburban government primary schools (KK, BR, and HK), Saraburi, Thailand. Stool was collected from 263 asymptomatic subjects (4-15 years old), using simple direct smear, formalin-ether concentration, Boeck and Drbohlav's Locke-Egg-Serum (LES) medium culture, and agar plate culture. A self-administered questionnaire was used to collect data about lifestyle and socioeconomic status. The overall rate of intestinal parasites was 22.1% (15.6% single infection and 6.5% multiple infections). The helminths involving the digestive system found were Strongyloides stercoralis (1.5%) and Opisthorchis viverrini (0.4%). For protozoan infection, the major cause was Blastocystis hominis (17.5%). The other protozoa included Endolimax nana (4.6%), Entamoeba coli (3.4%), Entamoeba histolytica/Entamoeba dispar (1.1%), and Giardia intestinalis (0.8%). The sensitivity for the detection of B. hominis increased with the LES culture technique. The infection rate of each organism was not significantly different among the three schools except for B. hominis which showed the highest prevalence in the HK school (P = 0.001). This was correlated with the questionnaire results in which the HK school showed the highest risk of drinking contaminated water (P = 0.004). The present study emphasized the persistent problems of protozoan infections among suburban school-aged children. Lifestyle was still an important factor for intestinal parasitic infections among suburban school-aged Thai children in this study. Health education as well as routine surveillance was necessary to control the infections.

PREVALENCE OF INTESTINAL PROTOZOAN INFECTIONS AMONG CHILDREN IN THAILAND: A LARGE-SCALE SCREENING AND COMPARATIVE STUDY OF THREE STANDARD DETECTION METHODS.

A significant impact of intestinal parasitic infections on public health has mostly been neglected. Parasitic infections are one of risk factors for malnutrition in children. In this study, a large-scale screening of intestinal parasitic infections among children in 16 schools in 6 regions of Thailand was performed. In addition, we compared sensitivity of methods currently employed for detection of intestinal parasitic infection. Fecal samples collected from 1,909 students were examined for intestinal parasites by simple smear, formalin-ethyl acetate concentration (FECT), and Locke-egg-serum (LES) medium culture methods. Seven hundred and thirteen samples were infected with at least one intestinal parasite. The highest prevalence (82.8%) was found in Kanchanaburi Province, western Thailand. Blastocystis spp was the most common (32.8%) parasite, followed by Giardia duodenalis (4.2%), Ascaris lumbricoides (3.6%), hookworms (1.6%), Entamoeba histolytica (0.7%), Trichuris trichiura (0.5%), Enterobius vermicularis (0.5%), Strongyloides stercoralis (0.4%), minute intestinal flukes (0.2%), and Taenia spp (0.1%). Mixed parasitic infections were found in 121 students. In a comparative study, we found that FECT was more sensitive (74.0%) than simple smear (55.0%) method for detecting helminths. However, sensitivity of these two methods is not significantly different for protozoan detection (31.2% by simple smear and 33.5% by FECT). LES culture technique was the most sensitive method (77.5%) for detecting Blastocystis spp. Our results indicate a high prevalence of intestinal parasite infection among Thai students. More sensitive methods should be developed for a large-scale screening of intestinal protozoan infection.

Susceptibility of Wolbachia, an endosymbiont of Brugia malayi microfilariae, to doxycycline determined by quantitative PCR assay.

Lymphatic filariasis, caused by filarial nematodes, is a mosquito-borne disease that affects over 120 million people in the tropics and subtropics. The disease is caused mainly by Wuchereria bancrofti and Brugia malayi. Fertile adult female worms release offsprings (microfilariae) into the host blood circulatory system. Transmission-blocking agents as well as antimicrobial agents have been used to reduce microfilarial density in human and animal reservoir hosts. Doxycycline and rifampicin have an effect on the obligate intracellular gram-negative bacteria, Wolbachia, which appears to exert an influence on filarial nematode embryonic and larval development, adult female fertility, and filarial survival. We investigated the effects of doxycycline, rifampicin and ciprofloxacin on B. malayi microfilarial motility, expressed as minimum effective concentration (MEC), and on Wolbachia proliferation using quantitative PCR, expressed as the concentration of the drug to inhibit bacteria growth by 50% (IC50). MEC of doxycycline was 128 and 32 microg/ml at 12 and 52 hours, respectively, but rifampicin and ciprofloxacin were ineffective (MEC >256 microg/ml). IC50 of doxycycline was 32 and 2 microg/ml at 12 and 52 hours, but this for rifampicin (8 microg/ml) and ciprofloxacin (32 microg/ml) were obtained only after 52 hour treatment. Thus, MEC and IC50 assay methods used in this study could be applied to screen other agents targeting filariae and their endosymbiont bacteria.

Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation.

Molecular cloning and functional characterization of P-III snake venom metalloproteinases (SVMPs) will give us deeper insights in the pathogenesis of viper bites. This may lead to novel therapy for venom-induced local tissue damages, the complication refractory to current antivenom. The aim of this study was to elucidate the in vitro activities of a new SVMP from the green pit viper (GPV) using recombinant DNA technology. We report, here, a new cDNA clone from GPV (Cryptelytrops albolabris) venom glands encoding 614 amino acid residues P-III SVMP, termed albocollagenase. The conceptually translated protein comprised a signal peptide and prodomain, followed by a metalloproteinase domain containing a zinc-binding motifs, HEXGHXXGXXH-CIM and 9 cysteine residues. The disintegrin-like and cysteine-rich domains possessed 24 cysteines and a DCD (Asp-Cys-Asp) motif. The albocollagenase deduced amino acid sequence alignments showed approximately 70% identity with other P-III SVMPs. Notably, the prodomain was highly conserved, while the metalloproteinase, disintegrin-like and cysteine-rich domains contained several differences. Albocollagenase without the signal peptide and prodomain was expressed in Pichia pastoris with an N-terminal six-histidine tag. After affinity purification from the supernatant of methanol-induced media, SDS-PAGE and Western blot analysis in both reducing and non-reducing conditions showed a protein band of approximately 62 kDa. The recombinant albocollagenase could digest human type IV collagen from human placenta basement membrane within 1 min. After 10-min incubation, it also inhibited collagen-induced platelet aggregation with 50% inhibitory concentration (IC50) of 70 nM. This is the first report of the active recombinant SVMP enzymes expressed in P. pastoris. The results suggest the significant roles of P-III SVMP in local and systemic pathology of envenomated patients. Inhibitors of this SVMP will be investigated in further studies to find a better treatment for viper bites.

A single dose of doxycycline in combination with diethylcarbamazine for treatment of bancroftian filariasis.

Standard treatment of lymphatic filariasis with diethylcarbamazine (DEC) is associated with systemic adverse reactions, thought to be due to the release of microfilariae material and Wolbachia endosymbiotic bacteria into the blood. Combination treatments with doxycycline for 3-8 weeks are more effective than standard treatment. However, long-term use of antibiotics may contribute to drug resistance and are not practical for use in remote areas. We assessed whether a single dose of doxycycline combined with the standard DEC regimen would reduce the incidence and severity of adverse reactions and increase the efficacy of standard treatment. Forty-four subjects from Tak Province were recruited into the randomized double-blind clinical trial study: 25 received DEC (300 mg) combined with a placebo, and 19 received DEC (300 mg) combined with doxycycline (200 mg). The incidences of adverse reactions to standard treatment were lower in the doxycycline group (45.5%) than in the placebo group (58.8%). Severe reactions occurred only in the placebo group (3 of 25 subjects). The severity of adverse reactions was significantly lower in the doxycycline group (mean score 0.45) than in the placebo group (mean score 1.17). The levels of IL-6 and Wolbachia DNA in the plasma were significantly lower in the doxycycline group. The filarial antigen levels were significantly lower in the doxycycline group at months 6 after treatment.

Association between Toll-like receptor 2 (TLR2) polymorphisms and asymptomatic bancroftian filariasis.

Lymphatic filariasis is mainly caused by the filarial nematodes Wuchereria bancrofti and Brugia malayi. Wolbachia, intracellular symbiotic bacteria in filarial parasite, is known to induce immune response predominantly through Toll-like receptor 2 (TLR2). This study was performed to investigate the association between polymorphisms of the TLR2 gene and susceptibility to asymptomatic bancroftian filariasis. A total of 142 unrelated asymptomatic bancroftian filariasis patients and 151 endemic normal controls in Tak province, Thailand were recruited into this study. The -196 to -173 deletion (del) polymorphism in the 5' untranslated region was investigated by allele-specific polymerase chain reaction. Two single nucleotide polymorphisms, +597 T>C and +1350 T>C, in exon 3 were identified by polymerase chain reaction-restriction fragment length polymorphism analysis. Furthermore, we analyzed the functional difference between the TLR2 -196 to -173 del and wild-type (wt) alleles using the luciferase reporter assay. All three polymorphisms were associated with a higher risk of asymptomatic bancroftian filariasis and were in strong linkage disequilibrium with each other. The TLR2 haplotype -196 to -173del/+597C/+1350C was strongly associated with an increased risk of asymptomatic bancroftian filariasis. The TLR2 -196 to -173 del allele had a significantly lower transcriptional activity than wt allele. The results of our study indicate that TLR2 -196 to -173 del, +597 T>C and +1350 T>C polymorphisms are associated with asymptomatic bancroftian filariasis in Thailand. Our functional study also supports this finding with respect to differential TLR2 gene expression by -196 to -173 del polymorphism.

Mosquito distribution and Japanese encephalitis virus infection in the immigration bird (Asian open-billed stork) nested area in Pathum Thani province, central Thailand.

Japanese encephalitis virus infection is a mosquito-borne emerging or re-emerging infectious disease in several countries. The ecology of this virus in nature includes amplifying avian or mammal hosts and mosquito vectors. Infected immigration birds from epidemic areas may play important roles in the outbreak of the disease. The prevalence is high during the raining season in Thailand and human cases have been reported from several provinces including Bangkok suburbs. This study was conducted to investigate the mosquito distribution and Japanese encephalitis virus infection in the immigration bird (Asian open-billed stork) nested area, Pathum Thani province, central Thailand. Mosquitoes were collected by using CO(2)-baited Centers for disease control and prevention (CDC) light traps, and dry ice was used as a source of CO(2) to attract mosquitoes from March 2008 to January 2009. Eight traps were operated from 4 p.m. until 7 a.m. on each study day. There were seven genera collected: Aedes, Anopheles, Armigeres, Coquillettidia, Culex, Mansonia, and Uranotaenia. Culex tritaeniorhynchus was the most collected species in each month, except November, in which Culex gelidus was the most collected species. Sixty pools of C. gelidus and of C. tritaeniorhynchus, each of which had 50 mosquitoes, were tested for Japanese encephalitis virus infection by using reverse transcription polymerase chain reactions; however, none of them was infected with the virus.

DNA-based diagnosis of lymphatic filariasis.

Lymphatic filariasis (LF) is still a major public health problem. The disease is ranked by the World Health Organization (WHO) as the second leading cause of permanent and long-term disability, and has been targeted for elimination by 2020. Effective diagnosis LF is required for treatment of infected individuals, for epidemiological assessment and for monitoring of the control program. Conventional diagnosis of LF depends on detection of microfilariae (Mf) in blood specimens, which has low sensitivity and specificity. Detection of specific circulating filarial antigens is regarded by WHO as the 'gold standard' for diagnosis of LF. However, the limitations of the antigen tests are cost and inconsistent availability. Although anti-filarial IgG4 antibody levels are associated with active LF infections, however, cross-reactivity with other filarial parasites is common. Not as sensitive as antigen tests, DNA-based techniques have been developed to diagnose and differentiate filarial parasites in humans, animal reservoir hosts, and mosquito vectors. These include DNA hybridization, polymerase chain reaction (PCR) amplification using specific primers (eg Ssp I repeat, pWb12 repeat, pWb-35 repeat, and LDR repeat for Wuchereria bancrofti and Hha I repeat, glutathione peroxidase gene, mitochondrial DNA for Brugia malayi), and universal primers, multiplex-PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), PCR-enzyme linked immunosorbent assay (PCR-ELISA), as well as quantitative PCR. Furthermore, because bancroftian filariasis is endemic on the Thai-Myanmar border, the potential now exists for a re-emergence of bancroftian filariasis in Thailand, and random amplified polymorphic DNA (RAPD) analysis has proved effective to differentiate Thai and Myanmar strains of W. bancrofti.

Screening for intestinal parasitic infections among Myanmar migrant workers in Thai food industry: a high-risk transmission.

The impact of intestinal parasitic infections on public health has been neglected. Millions of Myanmar natives have migrated to work in Thailand. We performed a study of intestinal parasitic infections in Myanmar-migrants working in the Thai food industry. A total of 338 Myanmar migrant workers in a food plant at Samut Sakhon Province, Thailand, were recruited for this study. 284 (84%) returned requested stool samples. Samples were examined for intestinal parasites by means of simple smear, formalin-ether concentration, Locke-Egg-Serum medium, and Harada-Mori culture methods. We found parasites in 177 (62.3%) migrants (29 of 46 males; 148 of 238 females). The majority (89.3%) were infected with parasites transmitted by fecal-oral route, including Blastocystis hominis (41.5%), Trichuris trichiura (22.2%), Giardia lamblia (14.1%), and Ascaris lumbricoides (1.8%). Mixed infections were common (40.7%). The highest prevalence (73.3%) was found among migrants from Kohsong city, Myanmar. This high parasite infection rate in Myanmar migrant workers is an obvious public health hazard.

Distribution of mosquito (Diptera: Culicidae) species and Wolbachia (Rickettsiales: Rickettsiaceae) infections during the bird immigration season in Pathumthani province, central Thailand.

Mosquito distribution in the immigration bird-nested area, Pathumthani province, was investigated from August to December in 2006. Mosquitoes were collected by using CO2-baited Centers for Disease Control light traps in which dry ice was used as a source of CO2 to attract mosquitoes. Six traps were operated from 4 p.m. until 7 a.m. on each study day. Four genera, which were Anopheles, Armigeres, Culex, and Mansonia with 14 species of mosquitoes were collected. Culex gelidus (13.94-59.41%) and Culex tritaeniorhynchus (32.87-70.30%) were most collected species in this area for every month. Other two species with moderate distribution in this area were Anopheles barbirostris (0.76-3.30%) and Mansonia uniformis (1.55-11.36%). Polymerase Chain Reactions were performed for testing Wolbachia infection in Cx. gelidus and Cx. tritaeniorhynchus only. Fifty-four percent (15/28 pools) of Cx. gelidus and none (0/20 pools) of Cx. tritaeniorhynchus were positive for Wolbachia infection. Wolbachia infection in other mosquito species collected in this and other areas need to be investigated to understand species and geographic variation of Wolbachia infection in mosquitoes in nature.

FlindersTechnology Associates (FTA) filter paper-based DNA extraction with polymerase chain reaction (PCR) for detection of Pneumocystis jirovecii from respiratory specimens of immunocompromised patients.

We evaluated the diagnostic value of Flinders Technology Associates (FTA) filter paper together with polymerase chain reaction (PCR) for detection of Pneumocystis jirovecii (carinii) from induced sputum (IS) and bronchoalveolar lavage fluid (BALF) samples. The study involved 162 patients with clinical diagnosis of pneumocystis pneumonia (PcP) of human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients and other immunocompromised patients. P. jirovecii cysts or trophozoites were detected in IS and BALF by cytological method. The mitochondrial 5S ribosomal ribonucleic acid (rRNA) gene of P. jirovecii was amplified from these samples by using FTA filters together with a one-step PCR method (FTA-PCR). With the FTA-PCR method, the sensitivity and specificity of the test compared to microscopic examination were 67% and 90% for IS, while they were 67% and 91% for BALF, respectively. The sensitivity and specificity of the FTA-PCR test was also comparable to PCR with the conventional deoxyribonucleic acid (DNA) extraction method. We concluded that FTA-PCR is useful to detect P. jirovecii in noninvasive IS.

Random amplified polymorphic DNA (RAPD) for differentiation between Thai and Myanmar strains of Wuchereria bancrofti.

BACKGROUND: Lymphatic filariasis (LF) is a mosquito-borne disease caused by mosquito-transmitted filarial nematodes, including Wuchereria bancrofti and Brugia malayi. The Lymphatic Filariasis Elimination Program in Thailand has reduced the prevalence of nocturnally subperiodic W. bancrofti (Thai strain), mainly transmitted by the Ochlerotatus (Aedes) niveus group in Thailand to 0.57/100,000 population. However, it is estimated that more than one million Myanmar migrants with high prevalence of bancroftian filariasis have settled in the large urban cities of Thailand. These infected migrants carry the nocturnally periodic W. bancrofti (Myanmar strain) which has Culex quinquefasciatus as the main mosquito vector. Although transmissions of the Myanmar strain of W. bancrofti by the Thai Cx. quinquefasciatus has never been reported, previous study showed that Cx. quinquefasciatus could nurture the Myanmar strain of W. bancrofti to the infective stage. Thus, the potential now exists for a re-emergence of bancroftian filariasis in Thailand. The present study was undertaken in an attempt to differentiate between the Thai and Myanmar strains of W. bancrofti. METHODS: The microfilarial periodicity of Thai and the Myanmar strains of W. bancrofti were determined. Comparative morphology and morphometry of microfilariae and a study of random amplified polymorphic DNA (RAPD) was performed. The Nei's genetic distance was calculated, and a phylogenetic tree was constructed using the Unweighted Pair Group Method with Arithmetic mean (UPGMA). RESULTS: The Thai strain of W. bancrofti was nocturnally subperiodic, and the Myanmar strain of W. bancrofti was nocturnally periodic. The body length, cephalic space length, and cephalic space width of the Thai strain of W. bancrofti were significantly larger than those of the Myanmar strain of W. bancrofti (p < 0.05). However, an overlapping mean of these parameters made it impractical for field application. RAPD-PCR profiles showed specific bands characteristic for the Myanmar strain of W. bancrofti. The phylogenetic tree indicated two genetically distinct clusters of the Thai and Myanmar strains of W. bancrofti. DISCUSSION: This study was the first report on the genetic polymorphism of the Thai and Myanmar strains of W. bancrofti. Differentiation between the Thai and Myanmar strains of W. bancrofti could not rely on morphological criteria alone. However, RAPD profiles revealed a significant diversity between the two strains. The RAPD-PCR technique was suitable for differentiating Thai and Myanmar strains of W. bancrofti. The RAPD marker could be used for epidemiological assessment of the Myanmar strains of W. bancrofti in Thailand.

Proinflammatory cytokine gene expression by murine macrophages in response to Brugia malayi Wolbachia surface protein.

Wolbachia, an endosymbiotic bacterium found in most species of filarial parasites, is thought to play a significant role in inducing innate inflammatory responses in lymphatic filariasis patients. However, the Wolbachia-derived molecules that are recognized by the innate immune system have not yet been identified. In this study, we exposed the murine macrophage cell line RAW 264.7 to a recombinant form of the major Wolbachia surface protein (rWSP) to determine if WSP is capable of innately inducing cytokine transcription. Interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF) mRNAs were all upregulated by the rWSP stimulation in a dose-dependant manner. TNF transcription peaked at 3 hours, whereas IL-1beta and IL-6 transcription peaked at 6 hours post-rWSP exposure. The levels of innate cytokine expression induced by a high-dose (9.0 microg/mL) rWSP in the RAW 264.7 cells were comparable to the levels induced by 0.1 microg/mL E. coli-derived lipopolysaccharides. Pretreatment of the rWSP with proteinase-K drastically reduced IL-1beta, IL-6, and TNF transcription. However, the proinflammatory response was not inhibited by polymyxin B treatment. These results strongly suggest that the major Wolbachia surface protein molecule WSP is an important inducer of innate immune responses during filarial infections.