Real-Time Polymerase Chain Reaction for Detection of Strongyloides stercoralis in Stool

The use of real-time polymerase chain reaction (PCR) for detection of Strongyloides stercoralis in stool has recently been described. We compared five DNA extraction methods by using normal human stool spiked with Strongyloides ratti and tested by using a real-time PCR. The PowerSoil kit was found to be the best technique in terms of sensitivity and ease of use. The PCR detected DNA extracted from one spiked S. ratti larva diluted 10⁻². The PowerSoil kit was then used to extract DNA from 160 human survey samples. All culture positive specimens with a high and moderate larval load were identified by real-time PCR, but only 15% of specimens with low larval load were positive. Specificity was greater than 99%. The combination of the PowerSoil kit and real-time PCR reliably detected high to moderate larval numbers of S. stercoralis in stools but was less sensitive when the larval load was low.     

Detection of Leishmania siamensis DNA in Saliva by Polymerase Chain Reaction

Polymerase chain reaction was used to detect Leishmania siamensis DNA from clinical samples collected from six leishmaniasis patients during 2011–2012. The samples used in this study came from bone marrow, blood, buffy coat, saliva, urine, and tissue biopsy specimens. Saliva was a good source for L. siamensis DNA by polymerase chain reaction. L. siamensis DNA was also found in saliva of an asymptomatic case-patient. Levels of L. siamensis DNA in saliva decreased until being undetectable after treatment. These levels could be used as a marker to evaluate efficacy of the treatment. A larger study is needed to evaluate this method as a screening and survey tool to study the silent background of Leishmania infection among the at-risk population.Leishmaniasis is a neglected tropical diseases caused by an obligate intracellular protozoa belonging to the genus Leishmania. The disease is transmitted to vertebrate hosts by infected female sand flies taking a blood meal.¹ Leishmaniasis presents in three clinical forms; visceral, cutaneous, and mucocutaneous.² Clinical presentation of leishmaniasis depends on the species of Leishmania and the immunity of the host.³Detection and species identification of the parasites is essential for prognostic and therapeutic reasons and surveys.⁴ Several laboratory techniques have been used for diagnosis of Leishmania infection. They are microscopy, culture, immunologic techniques (enzyme-linked immunosorbent assay, direct agglutination test, and recombinant protein K39 dipstick test), and molecular techniques (polymerase chain reaction [PCR] and quantitative PCR).^5–7 In comparisons of microscopic examination, culture, and PCR in detecting Leishmania parasites, PCR has shown to have a significantly higher sensitivity than culture and microscopic examination (97%, 78%, and 76%^8–10 sensitivity, respectively). New cases of leishmaniasis caused by L. siamensis, a novel species of Leishmania, have been documented in patients in Thailand^11–17 and Myanmar (unpublished data). The infection was described in immunocompromised patients, mostly persons infected with human immunodeficiency virus (HIV).^11–17 Clinical presentations of these patients have included visceral,^11–13 diffuse cutaneous,¹⁵ and overlapping diffuse cutaneous and visceral forms.¹⁴With low prevalence of leishmaniasis in Thailand and Myanmar, screening tests for leishmaniasis such as enzyme-linked immunosorbent assay, direct agglutination test, and recombinant protein K39 dipstick test are not readily available. Moreover, sensitivity and specificity of these serologic tests for detection of L. siamensis infection have never been fully documented. Diagnosis of L. siamensis infection relies on microscopic examination, culture, and detection of parasite DNA by PCR.^11–15 Although microscopic examination and culture of Leishmania parasites have high specificity, they require experience and have low sensitivity. The PCR is commonly used to diagnose leishmaniasis caused by L. siamensis. Bone marrow, blood, buffy coat, tissue, saliva, and urine have been successfully used for detection of L. siamensis DNA by PCR.¹⁴Saliva has shown to be a good source for L. siamensis DNA.¹⁴ There are also reports of using saliva to identify other Leishmania species.^18–20 Collection of saliva is noninvasive and convenient for field studies. We describe a PCR to amplify the internal transcribed spacer1 (ITS1) gene of L. siamensis from six infected patients and compare it with specimens collected from patients and different clinical presentations. Details of clinical presentations and management of some patients enrolled in this study have been described.¹⁴Bone marrow, blood or saliva was smeared on glass slides, and fixed with absolute methanol (Sigma-Aldrich, St. Louis, MO) for one minute. The slides were then stained with Giemsa (Sigma-Aldrich) in phosphate buffer, pH 7.2. Tissue biopsy specimens were stained with hematoxylin and eosin. Stained slides were then examined under a light microscope (Olympus, Tokyo, Japan).Schneider''s insect medium (Sigma-Aldrich) containing 20% fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Sigma-Aldrich) was used for culturing Leishmania parasites. One hundred microliters of bone marrow, blood, or saliva was inoculated into 5 mL of culture media in a 25-cm³ flask and incubated at 25 ± 2°C. Cultures were inspected for parasites every 24 hours by using an inverted microscope (Olympus).Two hundred microliters of blood or bone marrow and 50 μL of buffy coat were used for DNA extraction by using a blood DNA extraction kit (Invisorb^® Spin Blood Mini Kit (STRATEC Molecular, Berlin, Germany). Thirty milliliters of urine or 1 mL of saliva were centrifuged for 5 minutes at 5,000 × g, and the pellets were collected and used for further DNA extraction. Tissue specimens, urine pellets, and saliva pellets were used for DNA extraction by tissue DNA extraction (Invisorb^® Spin Tissue Mini Kit) according to the manufacturer''s instructions. Extracted DNA was eluted in 80 μL of elution buffer. Quantity and quality of the extracted DNA was determined by using a Nanodrop 2000c Apparatus (Thermo Scientific, Singapore). Extracted DNA samples were kept at −80°C for long-term storage. Blood, saliva, and urine were collected from three healthy uninfected persons and used for the PCR as negative controls.The PCR was performed in a final volume of 25 μL containing approximately 100 ng of extracted DNA, 10 μM of each primer, 25 mM of MgCl₂, 2 mM of dNTPs, and 1 unit of Taq DNA polymerase (Fermentas, Pittsburgh, PA). The primers were designed to anneal specifically to the ITS1 regions of ribosomal RNA (rRNA) of Leishmania parasites described by Spanakos and others:²¹ (LeF: 5′-TCC GCC CGA AAG TTC ACC GAT A-3′ and LeR: 5′-CCA AGT CAT CCA TCG CGA CAC G-3′). The PCRs were performed in a PCR Mastercycler^® Pro (Eppendorf, Hamburg, Germany) with conditions as follows: denaturation at 94°C for 4 minutes; followed by 40 cycles at 94°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. Aliquots of the PCR amplicons were analyzed by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and visualized with Quantity One Quantification Analysis Software version 4.5.2 (Gel Doc EQ System; Bio-Rad, Hercules, CA). The extracted DNA samples from an uninfected persons and a no DNA template (double-distilled water) were used as negative controls in every PCR. Reactions in which either or both negative controls contained bands were discarded.Confirmatory testing for PCR was performed by using another set of primers to amplify the partial small subunit (SSU) rRNA genes of the Leishmania parasite. The primer sequences (R221: 5′-GGT TCC TTT CCT GAT TTA CG-3′ and R332: 5′-GGC CGG TAA AGG CCG AAT AG-3′) and PCR conditions were described by Van Eys and others.²²The PCR amplicons were ligated into the pGEM-T Easy Vector (Promega, Madison, WI). Ligation reactions mixture was composed of 5 μL of 2× Rapid ligation buffer, 3 μL of PCR products, 1 μL pGEM-T Easy Vector, and 1 μL double-distilled water. The ligated vectors were transformed into DH5α competent cells and chimeric plasmids were screened by blue–white colony selection system. The suspected positive colonies were cultured and used for further plasmid DNA extraction by using the Invisorb^® Spin Plasmid Mini Kit following the manufacturer''s instructions. Purified plasmids were sequenced by 1st BASE DNA sequencing services (1st Base Laboratories, Kuala Lumpur, Malaysia) by using universal forward T7 primer. Nucleotide sequences were analyzed by using BioEdit Sequence Alignment Editor Version 7.0.9.0 (www.mbio.ncsu.edu/bioedit/bioedit.html), and consensus sequences were compared with available sequence data in a GenBank by BLAST search (www.ncbi.nlm.gov/BLAST).The study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (IRB no. 385/55).Patient 1 was a 46-year-old Thai man who was a rubber planter from southern Thailand. He has been given a diagnosis with HIV infection in 2003 and received boosted lopinavir and lamivudine. His CD4+ T cell count was 175 cells/mm³, and serum virus levels were < 40 copies/mL. He also had Evans syndrome (an autoimmune disorder with destruction of erythrocytes, platelets, and leukocytes), a left knee ulcer, and hepatosplenomegaly. A bone marrow study showed Leishmania amastigotes within macrophages. Bone marrow, blood, discharge from the ulcer, saliva, and urine were cultured and DNA was extracted for PCR.He improved after two weeks of intravenous amphotericin B deoxycholate (1 mg/kg/day), followed by 400 mg of itraconazole/day. A recurrence after two months of itraconazole therapy was re-treated with three weeks of intravenous liposomal amphotericin B (3 mg/kg/day), followed by 400 mg of itraconazole per day. Blood and saliva were collected for PCR three months after re-treatment and were negative. Details of this patient were reported by Chusri and others.¹⁴Patient 2 was 30-year-old pet store owner from southern Thailand who had been given a diagnosis of HIV infection in 1999. The patient received tenofovir, lamivudine, and nevirapine. His CD4+ T-cell count was 111 cells/mm³ and viral RNA was not detectable (< 40 copies/mL). He had multiple papules and plaques with ulceration and discharges. He also had anemia, thrombocytopenia, and hepatosplenomegaly. Bone marrow, papule, and ulcer biopsy specimens showed Leishmania amastigotes within macrophages. Bone marrow, blood, tissue biopsy specimens, and saliva and urine samples were used for culture and PCR. The patient received intravenous amphotericin B deoxycholate (1 mg/kg/day) for two weeks, followed by 400 mg of itraconazole per day. Blood and saliva were collected three months later and were negative for Leishmania and no recurrence was observed. This patient was reported by Chusri and others.¹⁴Patient 3 was a 60-year-old man with diabetes who lived in Yangon, Myanmar and had not previously traveled abroad. He showed development of fever, multiple infiltrative skin lesions, and oral ulcers. A skin biopsy specimen indicated Sweet''s syndrome or acute febrile neutrophilic dermatosis, which is a skin disease with fever and painful skin lesions that is commonly present on arms, neck, face, and back. He was treated with systemic corticosteroids for two months without improvement. He came to Thailand for further evaluation. Multiple erythematous, shiny, infiltrative plaques; nodules on face, trunk, and extremities; and oral ulcers and white patches on the buccal mucosa were observed. Lymph nodes, liver, and spleen were not palpable. Complete blood counts, blood urea nitrogen levels, creatinine levels, and liver function test results were within normal limits. Results for antinuclear antibodies, antibodies against HIV, and C-reactive protein levels were unremarkable.A new skin biopsy specimen of a trunk nodule showed diffuse histiocytic infiltrate and multinucleated giant cells in the upper and deep dermis. Many round-to-oval small organisms were present in histiocytes and fibrous stroma. They stained positive with Periodic Acid–Schiff (PAS) stain. Numerous small yeast-liked organisms were present in histiocytes and in the stroma. Some of these organisms were large and contained small basophilic dots in cytoplasm near nuclei stained positively with PAS stain but not with Gomori''s methenamine silver stain and Giemsa. This finding suggested the presence of Leishmania parasites. Blood samples, tissue biopsy specimens, saliva and urine samples were used for culture and were negative. PCR testing of the ITS1 region of the rRNA gene in blood, skin biopsy specimen, urine, and saliva, and DNA sequencing identified L. siamensis. The patient was treated with intravenous amphotericin B for 40 days (total dose = 2.1 grams). After regression of the cutaneous lesions, he was discharged. When seen again two months later, he had gained weight but a few skin lesions were still present. He returned home and was lost to follow-up.Patient 4 was the 22-year-old daughter of patient 3; she came to Thailand with her father. She was healthy and lived with her father in Yangon. Results of her physical examination were unremarkable. Saliva and urine samples were collected and used for culture; these samples were negative for L. siamensis. PCR testing of the ITS1 region of the rRNA gene in saliva DNA, followed by DNA sequencing, identified L. siamensis. Whole blood and buffy coat were then collected for Leishmania detection by culture, staining with Giemsa, and PCR. Only PCR identified L. siamensis in buffy coat. We planned to follow-up the patient for detection of Leishmania parasites without treatment of leishmaniasis but the patient was lost to follow-up.Patient 5 was a 45-year-old Thai man living in Chiang Rai, Thailand. He has been given a diagnosis of HIV infection in 2005. He had a CD4+ T cell count of < 50 cells/mm³. He was treated with stavudine, lamivudine, and nevirapine. In 2007, He showed development of lumpy skin lesions that were first not investigated. He was later hospitalized with fever, oral candidiasis, pancytopenia, pancreatitis, type 2 diabetes mellitus, epistaxis, perianal abscess, urinary retention, and abnormal liver function test results. Skin biopsy specimens from lesions present for more than five years showed epidermal hyperplasia, diffuse fibrosis, dilated blood vessels, and mild perivascular lymphohistiocytic infiltrates in the dermis. There were a few clumps of small parasites in histiocytes and extracellularly between collagen fibers in the upper dermis. They stained with Giemsa. Blood, tissue biopsy specimen, and saliva and urine samples were used for culture but were negative. A PCR of ITS1 region of the rRNA gene in blood, tissue biopsy specimen, and saliva DNA, followed by DNA sequencing, confirmed the presence of L. siamensis. The patient was then confirmed as having cutaneous leishmaniasis. He was severely debilitated and died of systemic bacterial infection without treatment of leishmaniasis.Patient 6 was a 34-year-old Burmese man from Yangon who was seropositive for HIV for six years. He was treated with truvada, legalon, isoniazid, rifampicin, ethambutal, and pyrazinamide. Five months later, he was hospitalized with high fever and diarrhea. He was empirically treated with clarithromycin and moxifloxacin. He showed development of multiple, umbilicated, erythematous papules on his neck, arms, and chest wall. Skin biopsy specimens showed a moderately dense superficial and deep perivascular and interstitial histiocytic infiltrate. Many small yeast-like organisms were present in the cytoplasm of histiocytes and between collagen fibers. They stained with PAS and Giemsa, leading to diagnosis of cutaneous leishmaniasis. Blood, tissue, saliva, and urine samples were cultured. A PCR of the ITS1 region of the rRNA gene in blood, skin biopsy specimen, and saliva DNA, followed by DNA sequencing, identified L. siamensis. The patient was treated with liposomal amphotericin B and anti-tuberculosis therapy was continued. Clinical recovery resulted within one month.Specimens were obtained from patients with different clinical presentations of leishmaniasis (

An alarming rate of drug-resistant tuberculosis at Ngwelezane Hospital in northern KwaZulu Natal, South Africa.

OBJECTIVE: To obtain a profile of tuberculosis (TB) cases caused by Mycobacterium tuberculosis in northern KwaZulu Natal (KZN) for 2001. METHOD: The demographic and drug resistance characteristics were determined. A total of 251 smear and culture confirmed cases was obtained and the resistance of three major anti-tuberculosis drugs was tested using the proportion method. RESULTS: The patients were mainly from Ngwelezane (57.8%), Stanger (23.5%) and Manguzi (18.7%); 59.0% were male; 54.2% of the isolates were obtained from new TB patients. The mean age of the TB patients was 29.6 years, with the maximum notification age range at 25-34 years. A high prevalence of drug-resistant and multiresistant TB was observed in this region. At least 55% of previously treated and 19% of new cases from all areas were resistant to at least one of the drugs tested. New patients from Ngwelezane and Manguzi area had a high prevalence of any rifampicin resistance (11.0%) and ethambutol resistance (3.9%), respectively. CONCLUSION: Our results indicate that the TB epidemic in northern KZN, especially in Ngwelezane, is severe. An infrastructure to educate stakeholders about the need to ensure consistency in treatment is needed.     

High Prevalence of Schistosoma japonicum Infection in Water Buffaloes in the Philippines Assessed by Real-Time Polymerase Chain Reaction

Difficulty in controlling human Schistosoma japonicum infection is partly attributed to the presence of non-human definitive hosts. Water buffaloes are a major reservoir for transmission of S. japonicum to humans in China. However, in the Philippines, reports based on microscopic examination of buffalo stool identified a low prevalence of S. japonicum, and mathematical models using these data concluded that water buffaloes are not a major reservoir for transmission of S. japonicum to humans. We collected stool from 81 buffaloes in Macanip, Leyte, the Philippines, and assayed for S. japonicum infection by the Danish Bilharziasis Laboratory technique, the Kato-Katz technique, miracidia hatching, and a highly validated real-time polymerase chain reaction. The prevalence defined by each assay was 3.7%, 3.7%, 0%, and 51.5% respectively. Our results demonstrate that microscopic-based techniques dramatically underestimate the prevalence of S. japonicum infection in water buffaloes in the Philippines and warrant reexamination of the role of bovines in transmission of S. japonicum to humans in the Philippines.     

套式PCR用于恙虫病的早期诊断及恙螨幼虫体内立克次体的检测

从恙虫病立克次体（Ｒｔ）Ｋａｒｐ株的群特异性抗原基因序列设计两对引物建立套式ＰＣＲ检测现场鼠体采集的恙螨幼虫体内及恙虫病现症病人外周血单核细胞内的ＲｔＤＮＡ。除一份已用过药病人标本外，其余９份均可检见８８ｂｐＤＮＡ扩增产物；检测结果表明：现场鼠体采集的地里纤恙螨（Ｌｅｐｔｏｔｒｏｍｂｉｄｉｕｍｄｅｌｉｅｎｓｅ）幼虫的Ｒｔ携带率是１３．３％，证实套式ＰＣＲ可用于急性期恙虫病的诊断和媒介恙螨流行病学的调查。     

用PCR技术检测大鼠弓形虫DNA

目的 探讨弓形虫病大鼠外周血弓形虫DNA检测的意义。方法 自行设计一对引物,用聚合酶链反应技术扩增弓形虫P30基因的一段保守序列。结果 设计的这对引物对健康人、大鼠、小鼠外周血白细胞以及阴道毛滴虫、溶组织内阿米巴均不能扩增,表明具有特异性。反应体系经35个循环扩增,可检测到2条弓形虫DNA,表明具有较高的敏感性。结论 PCR法对大鼠弓形虫感染可做出早期诊断。     

Detection of Legionella Species in Clinical Samples: Comparison of Polymerase Chain Reaction and Urinary Antigen Detection Kits

Abstract Background: Recently, two excellent methods have been used for the diagnosis of Legionnaires’ disease: urinary antigen detection and PCR. The purpose of the present study is to analyze and evaluate the sensitivity and specificity of three different urinary antigen detection kits as well as PCR. Materials and Methods: A total of 148 samples were collected from 33 patients between 1993 and 2004. These consisted of 73 urine samples obtained from 33 patients, 57 serum samples provided by 29 patients, and 18 respiratory tract specimens from 13 patients. Three commercially available kits were used to detect urinary antigen. For the 5S PCR reaction, primers L5SL2 and L5SR84 were used. Results: Positive results were shown in all patients’ urine (representing 79.5% of total samples) using the Binax EIA kit, in 93.9% patients (representing 75.3% samples) using the Binax NOW immunochromatographic kit, and in 90.9% (representing 72.6% samples) using the Biotest EIA kit. Urine samples from 12.1% patients (representing 6.8% of total samples), serum samples from 41.4% patients (representing 35.1% of total samples), and respiratory samples from 84.6% patients (representing 88.9% of total samples) showed positive results with PCR. Conclusion: In testing urine of legionellosis patients, it was suggested that three kits were all valuable tools for diagnosis of legionellosis. Since over one-third of patients’ serum samples and most respiratory specimens showed positive results with PCR, the addition of PCR for testing of these samples might be useful, particularly in cases of culture negative and serum antibody negative patients.     

新疆妇女宫颈脱落细胞HPV16E6基因的PCR及荧光定量PCR检测

目的:了解新疆妇女人乳头瘤病毒16型(13PV16)的感染状况。方法:用PCR及荧光定量PCR(FQ—PCR)方法检测妇科门诊普通患宫颈脱落细胞及分泌物中人乳头瘤病毒16型E6基因(HPV16E6),以普通门诊患宫颈脱落细胞及分泌物DNA作为样本,以人乳头瘤病毒(HPV16E6)作为扩增的靶基因,同时以人β—actin基因片段作为细胞内参照,借助于两对引物,两个特异的荧光探针,用荧光定量PCR(FQ—PCR)方法对这两个片段进行扩增,得到单位细胞HPV16E6的相对含量;同时进行定性PCR检测。结果:宫颈脱落细胞标本159例中,β—actin阳性154例;HPV16E6阳性共12例,阳性率为7．8％。PCR与FQ—PCR结果基本一致,但FQ—PCR更敏感。结论:建立的FQ—PCR检测宫颈脱落细胞内：HPV16E6基因的方法,能反映单位细胞内病毒的复制情况,可用于性传播感染(STI)及宫颈癌的筛查。     

致病性钩端螺旋体微量DNA的聚合酶链反应(PCR)

本研究首次通过对黄疸出血群钩端螺旋体 DNA 的分子克隆的反复筛选,获得了两个重组DNA片段,其中一个具有广泛针对各群致病性钩体的特性,另一个仅对黄疸出血群钩体起杂交反应。经 DNA 序列分析,分别合成两对 PCR 寡核苷酸引物——Primer B1.2和 Primer B3.4。本研究证明 PCR 是一种灵敏度极高,针对性很强的 DNA 检测技术,可用于钩体病早期诊断和流行病学分类鉴定。     

Detection of Onchocerca volvulus in Skin Snips by Microscopy and Real-Time Polymerase Chain Reaction: Implications for Monitoring and Evaluation Activities

Microscopic evaluation of skin biopsies is the monitoring and evaluation (M and E) method currently used by multiple onchocerciasis elimination programs in Africa. However, as repeated mass drug administration suppresses microfilarial loads, the sensitivity and programmatic utility of skin snip microscopy is expected to decrease. Using a pan-filarial real-time polymerase chain reaction with melt curve analysis (qPCR-MCA), we evaluated 1) the use of a single-step molecular assay for detecting and identifying Onchocerca volvulus microfilariae in residual skin snips and 2) the sensitivity of skin snip microscopy relative to qPCR-MCA. Skin snips were collected and examined with routine microscopy in hyperendemic regions of Uganda and Ethiopia (N = 500 each) and “residual” skin snips (tissue remaining after induced microfilarial emergence) were tested with qPCR-MCA. qPCR-MCA detected Onchocerca DNA in 223 residual snips: 139 of 147 microscopy(+) and 84 among microscopy(−) snips, suggesting overall sensitivity of microscopy was 62.3% (139/223) relative to qPCR-MCA (75.6% in Uganda and 28.6% in Ethiopia). These findings demonstrate the insufficient sensitivity of skin snip microscopy for reliable programmatic monitoring. Molecular tools such as qPCR-MCA can augment sensitivity and provide diagnostic confirmation of skin biopsies and will be useful for evaluation or validation of new onchocerciasis M and E tools.     

Detection of Hb H Disease Genotypes Common in Northern Thailand by Quantitative Real-Time Polymerase Chain Reaction and High Resolution Melting Analyses

We used quantitative real-time polymerase chain reaction (qPCR) and high resolution melting (HRM) analyses for the detection of the common α-thalassemia (α-thal) genotypes in 40 northern Thai Hb H (β4) patients. The α⁰-thal [– –^SEA (Southeast Asian) deletion] was detected by a multiplex gap real-time PCR. To determine the α⁺-thal, three primer pairs were designed. The A-primer pair was used to amplify the 3' terminal DNA sequences of the HBA2 gene, and the B-primer pair amplified the 5' flanking region of the HBA1 gene. The C-primer pair amplified the 3' terminal DNA sequences of the HBA1 gene and was used as an internal control. The –α^4.2 (leftward) and –α^3.7 (rightward) deletions were determined by monitoring the absence of PCR product(s). The Hb H patients who had a negative PCR result for the A-primer pair but positive for the B- and C-primer pairs carried the –α^4.2 deletion, while the –α^3.7 deletion carriers were negative for the A- and B-primer pairs. In the case of Hb H with Hb Constant Spring (Hb CS, α142, Term→Gln; HBA2: c.427T>C), all primer pairs were positive, HRM analysis of the PCR product of the A-primer pair was introduced to analyze the Hb CS gene. It can distinguish clearly between normal and Hb CS genotypes. The applied methods for the determination of the α⁰- and α⁺-thal genotypes revealed the results to be as accurate as conventional gap-PCR and the direct DNA sequencing methods but resulted in a much simpler and faster procedure.     

Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers'' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.     

地里纤恙螨红、白体色体内恙虫病立克次体的聚合酶链反应技术检测及其传病作用

本文应用聚合酶链反应技术（ＰＣＲ）检测了红、白体色地里纤恙螨亲代成虫及子代幼虫体内恙虫病立克次体ＤＮＡ的动态变化,结果表明：恙虫病立克次体能在红、白体色恙螨体内生长繁殖,且至少持续２７０天,并可以经卵传递给子代。叮咬试验证明两体色恙螨幼虫均具有叮咬和传病能力。     

Molecular Evidence of Bartonella Infection in Domestic Dogs from Algeria,North Africa,by Polymerase Chain Reaction (PCR)

Bartonella species are being recognized as important bacterial human and canine pathogens, and are associated with multiple arthropod vectors. Bartonella DNA extracted from blood samples was obtained from domestic dogs in Algiers, Algeria. Polymerase chain reaction (PCR) and DNA sequence analyses of the ftsZ gene and the 16S-23S intergenic spacer region (ITS) were performed. Three Bartonella species: Bartonella vinsonii subsp. berkhoffii, Bartonella clarridgeiae, and Bartonells elizabethae were detected infecting Algerian dogs. To our knowledge, this study is the first report of detection by PCR amplification of Bartonella in dogs in North Africa.     

Natural Leishmania Infection of Lutzomyia auraensis in Madre de Dios, Peru, Detected by a Fluorescence Resonance Energy Transfer-Based Real-Time Polymerase Chain Reaction

Abstract. Leishmania species of the Viannia subgenus are responsible for most cases of New World tegumentary leishmaniasis. However, little is known about the vectors involved in disease transmission in the Amazon regions of Peru. We used a novel real-time polymerase chain reaction (PCR) to assess Leishmania infections in phlebotomines collected in rural areas of Madre de Dios, Peru. A total of 1,299 non-blood fed female sand flies from 33 species were captured by using miniature CDC light traps. Lutzomyia auraensis was the most abundant species (63%) in this area. Seven of 164 pools were positive by PCR for Leishmania by kinetoplast DNA. The real-time PCR identified four Lu. auraensis pools as positive for L. (Viannia) lainsoni and L. (V.) braziliensis. The minimum infection prevalence for Lu. auraensis was estimated to be 0.6% (95% confidence interval = 0.20-1.42%). Further studies are needed to assess the importance of Lu. auraensis in the transmission of New World tegumentary leishmaniasis in hyperendemic areas of Peru.     

Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe.