Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C_T) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10⁴ colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.     

Giardia intestinalis: DNA extraction approaches to improve PCR results

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.     

Absence of lower genital tract lesions among women of reproductive age infected with Schistosoma mansoni: A cross-sectional study using a colposcope in Western Kenya

BackgroundFemale genital schistosomiasis (FGS) constitutes four different lesions known to be caused by Schistosoma haematobium ova deposited in the genital tract. Schistosoma mansoni ova may also be found in the genital tract. However, it is not known if S. mansoni causes lower genital tract lesions characteristic of FGS.MethodologyThis study was conducted in 8 villages along the shores of Lake Victoria, western Kenya. Stool and urine samples, collected from women of reproductive age on three consecutive days, were analysed for S. mansoni and S. haematobium infection. S. mansoni positive and S. haematobium negative willing participants, aged 18–50 years were invited to answer a questionnaire (demographics, symptoms), undergo a gynaecological examination and cytology specimen collection by an FGS expert.Principal findingsGynaecologic investigations were conducted in 147 S. mansoni-positive women who had a mean infection intensity of 253.3 epg (95% CI: 194.8–311.9 epg). Nearly 90% of them used Lake Victoria as their main water source. None were found to have cervicovaginal grainy sandy patches or rubbery papules. Homogenous yellow patches were found in 12/147 (8.2%) women. Women with homogenous yellow patches were significantly older (47 years) than the rest (34 years, p = 0.001). No association was found between intensity of S. mansoni infection and homogenous yellow patches (p = 0.70) or abnormal blood vessels (p = 0.14). S. mansoni infection intensity was not associated with genital itch, bloody or malodorous vaginal discharge.ConclusionS. mansoni infection was neither associated with lower genital tract lesions nor symptoms typically found in women with FGS.     

Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (101 cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 102 spores and 75 % for 101 spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.     

Development of Procedures for Direct Extraction of Cryptosporidium DNA from Water Concentrates and for Relief of PCR Inhibitors

Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/μl or 25 ng of T4 gene 32 protein/μl to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.     

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

Background

Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis.

Methodology/Principal Findings

A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%).

Conclusions/Significance

We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas.     

Emergent canine visceral leishmaniasis in Argentina: Comparative diagnostics and relevance to proliferation of human disease

BackgroundVisceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL.Methodology/Principal findingsSampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis.Conclusions/SignificanceSeroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.     

Evaluation of QIAamp DNA Stool Mini Kit for ecological studies of gut microbiota

Cell lysis efficiency and the quality of DNA extracts from complex bacterial ecosystems are two major concerns in molecular ecological studies of gut microbiota. In this study, we use PCR-denaturing gradient gel electrophoresis (DGGE) DNA profiling, random cloning and sequence analysis of 16S rRNA genes to compare the QIAamp DNA Stool Mini Kit with the bead beating technique in the preparation of DNA extracts from gut microbiota of pigs. We also developed a washing procedure that can release more than 93% of bacterial cells attached to the gut mucosa. Both the QIAamp kit and bead beating method lysed approximately 95% of bacterial cells. PCR-DGGE DNA profiles of ileal and cecal microbiota from both digesta and mucosa that were generated from the DNA extracts using the two methods were nearly identical. Random cloning and sequence analysis also demonstrated the high quality of DNA extracts using the two methods. Two random clone sets of 16S rRNA genes generated from the DNA extracts had a similar degree of bacterial diversity. Different preparations of DNA extract from a single sample using the QIAamp kit consistently produced similar PCR-DGGE DNA profiles with similarity indexes higher than 99%. Our data suggest the appropriateness of the QIAamp DNA Stool Mini Kit for the studies of gut microbial ecology and the effectiveness of the QIAamp kit in processing multiple samples for cell lysis and DNA extraction.     

Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

Background

Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.

Methodology/Principal Findings

We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.

Conclusions

M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10⁵ cells g⁻¹ using Griffiths and 4.25×10⁶ cells g⁻¹ using FastDNA® Spin kit.     

Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors

Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/mul or 25 ng of T4 gene 32 protein/mul to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.     

沼气池污泥微生物总DNA提取方法的比较

沼气发酵系统是一个复杂的生态系统,其污泥微生物超过99%是不可培养的。为了优化沼气池纤维素的转化效率、沼气的产率和开展污泥微生物多样性研究,本研究采用化学裂解法、溶菌酶裂解法和QIAampDNA Stool Mini Kit提取了沼气池污泥样品中微生物的总DNA,对三种方法的DNA得率、纯度、大片段提取效果以及是否含有PCR反应抑制剂进行了研究,最后对16S rRNA基因V3区的扩增产物进行了PCR变性梯度凝胶电泳(PCR-DGGE)分析。与化学裂解法和QIAamp DNA Stool Mini Kit法相比,溶菌酶裂解法得到的DNA量大、片段长、片段分布广、PCR扩增效率高;同时PCR-DGGE图谱显示,溶菌酶裂解法可更好地展示沼气池污泥中微生物的多样性。该结果为进一步提高沼气池中纤维素的转化效率和沼气生产优势菌种的质和量打下了一定的前期基础。     

Comparison of DNA extraction methods for human gut microbial community profiling

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome.     

Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum

BackgroundCurrent methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime.ObjectivesThus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR).MethodsThree DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added.ResultsThe DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood &; Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA.ConclusionsIn our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis.     

体重差异双胎新生儿 粪便样本DNA提取的优化及分析

摘要：目的 优化新生儿粪便样本DNA提取方法,提取及分析体重差异双胎新生儿粪便样本DNA。方法 从7种DNA提取试剂盒方法中选择对成人粪便样本DNA提取效果最佳的QIAamp DNA Stool Mini Kit法为基准方法,通过钢珠打断前处理、DNA吸附柱收集全部裂解液上清和洗脱液重复洗脱的优化,建立了用于新生儿粪便样本DNA的提取方法。结果 该优化方法用于婴儿（出生1个月）粪便样本,结果显示DNA提取浓度平均提高了2.4倍。用于48对双胞胎新生儿出生第1天和第3天粪便样本DNA的提取,经酶标仪及PCR扩增检测,结果显示出生第1天粪便样本DNA提取率为32%,出生3天提取率达83%。RT-PCR显示新生儿第1天到第3天肠道微生物量呈现增长趋势。结论 优化的QIAamp DNA Stool Mini Kit法适用于新生儿粪便样本DNA的快速提取,为后续扩增子高通量测序和研究体重差异双胎新生儿肠道菌群构成规律奠定基础。     

Diagnostic Performance of Schistosoma Real-Time PCR in Urine Samples from Kenyan Children Infected with Schistosoma haematobium: Day-to-day Variation and Follow-up after Praziquantel Treatment

Background

In an effort to enhance accuracy of diagnosis of Schistosoma haematobium, this study explores day-to-day variability and diagnostic performance of real-time PCR for detection and quantification of Schistosoma DNA compared to other diagnostic tools in an endemic area before and after treatment.

Methodology

Previously collected urine samples (N = 390) from 114 preselected proven parasitological and/or clinical S. haematobium positive Kenyan schoolchildren were analyzed by a Schistosoma internal transcribed spacer-based real-time PCR after 14 years of storage. Pre-treatment day-to-day fluctuations of PCR and microscopy over three consecutive days were measured for 24 children using intra-class correlation coefficient. A combined ‘gold standard’ (PCR and/or microscopy positive) was used to measure sensitivity and negative predictive value (NPV) of several diagnostic tools at baseline, two and 18 months post-treatment with praziquantel.

Principal Findings

All 24 repeatedly tested children were PCR-positive over three days with little daily variation in median Ct-values, while 83.3% were found to be egg-positive for S. haematobium at day 1 and 75.0% at day 2 and 3 pre-treatment, signifying daily fluctuations in microscopy diagnosis. Of all 114 preselected schoolchildren, repeated microscopic measurements were required to detect 96.5% versus 100% of positive pre-treatment cases by single PCR. At two months post-treatment, microscopy and PCR detected 22.8% versus 69.3% positive children, respectively. Based on the ‘gold standard’, PCR showed high sensitivity (>92%) as compared to >31% sensitivity for microscopy, both pre- and post-treatment.

Conclusions/Significance

Detection and quantification of Schistosoma DNA in urine by real-time PCR was shown to be a powerful and specific diagnostic tool for detection of S. haematobium infections, with less day-to-day variation and higher sensitivity compared to microscopy. The superior performance of PCR before, and two and 18 months post-treatment provides a compelling argument for PCR as an accurate and reproducible tool for monitoring treatment efficacy.     

A comparison of five methods for extraction of bacterial DNA from human faecal samples

The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type.     

Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis

OBJECTIVE: The aim of this study was to compare and evaluate three methods of DNA extraction for the amplification of Chlamydia trachomatis in uterine cervical samples collected in PreservCyt solution. ThinPrep is the trade name for the slide preparation. METHODS: Thirty-eight samples collected in LCx buffer medium, which were identified as C. trachomatis infected by ligase chain reaction (LCR), were selected for this study. DNA from the PreservCyt samples was extracted by three methods: (i) QIAamp kit, (ii) boiling in Tris-EDTA buffer with Chelex purification, and (iii) Proteinase K digestion with Chelex purification. Sample DNA was tested for the presence of C. trachomatis by PCR using cryptic plasmid research (CTP) primers and major outer membrane protein research momp gene (MOMP) primers. Real-time (LightCycler) PCR for relative C. trachomatis quantification following DNA extraction was performed using primers (Hsp 60) for the 60 kDa heat-shock protein hsp60 gene. RESULTS: Amplification using CTP primers was the most successful with each of the extraction protocols. Boiling in buffer was the least successful extraction method. QIAamp was the best extraction method, yielding the most positives with both the CTP and MOMP primers. Proteinase K-Chelex extraction gave similar sensitivity to QIAamp extraction with CTP primers but lower for MOMP primers. CONCLUSIONS: The DNA extraction method must be carefully selected to ensure that larger PCR amplicons can be successfully produced by PCR and to ensure high sensitivity of detection of C. trachomatis. In this study it was found that the QIAamp extraction method followed by PCR with the CTP primers was the most successful for amplification of C. trachomatis DNA.     

A New Mouse Model for Female Genital Schistosomiasis

Background

Over 112 million people worldwide are infected with Schistosoma haematobium, one of the most prevalent schistosome species affecting humans. Female genital schistosomiasis (FGS) occurs when S. haematobium eggs are deposited into the female reproductive tract by adult worms, which can lead to pelvic pain, vaginal bleeding, genital disfigurement and infertility. Recent evidence suggests co-infection with S. haematobium increases the risks of contracting sexually transmitted diseases such as HIV. The associated mechanisms remain unclear due to the lack of a tractable animal model. We sought to create a mouse model conducive to the study of immune modulation and genitourinary changes that occur with FGS.

Methods

To model FGS in mice, we injected S. haematobium eggs into the posterior vaginal walls of 30 female BALB/c mice. A control group of 20 female BALB/c mice were injected with uninfected LVG hamster tissue extract. Histology, flow cytometry and serum cytokine levels were assessed at 2, 4, 6, and 8 weeks post egg injection. Voiding studies were performed at 1 week post egg injection.

Results

Vaginal wall injection with S. haematobium eggs resulted in synchronous vaginal granuloma development within 2 weeks post-egg injection that persisted for at least 6 additional weeks. Flow cytometric analysis of vaginal granulomata revealed infiltration by CD4⁺ T cells with variable expression of the HIV co-receptors CXCR4 and CCR5. Granulomata also contained CD11b⁺F4/80⁺ cells (macrophages and eosinophils) as well as CXCR4⁺MerTK⁺ macrophages. Strikingly, vaginal wall-injected mice featured significant urinary frequency despite the posterior vagina being anatomically distant from the bladder. This may represent a previously unrecognized overactive bladder response to deposition of schistosome eggs in the vagina.

Conclusion

We have established a new mouse model that could potentially enable novel studies of genital schistosomiasis in females. Ongoing studies will further explore the mechanisms by which HIV target cells may be drawn into FGS-associated vaginal granulomata.     

Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil

Background

We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively.

Methodology/Principal Findings

Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups.

Conclusions

The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.     

Eosinophil Granule Proteins ECP and EPX as Markers for a Potential Early-Stage Inflammatory Lesion in Female Genital Schistosomiasis (FGS)

Background

Genital granulomas induced by Schistosoma haematobium eggs can manifest as different lesion types visible by colposcopy; rubbery papules (RP), homogenous sandy patches (HSP) and grainy sandy patches (GSP). Pronounced tissue eosinophilia is a candidate marker for active S. haematobium pathology, as viable schistosome egg granulomas often are eosinophil rich. Here it was investigated whether eosinophil granule proteins ECP (eosinophil cationic protein) and EPX (eosinophil protein-X) in urine and genital lavage can be used as markers for active FGS lesions.

Methods

Uro-genital samples from 118 Malagasy women were analysed for ECP and EPX by standard sandwich avidin/biotin amplified ELISA.

Principal findings

The women with RP lesions had significantly higher levels of ECP and EPX in both lavage and urine. Furthermore, women with RP lesions were significantly younger than those with GSP. This could indicate that RP lesions might be more recently established and thus represent an earlier inflammatory lesion stage.

Conclusion

ECP in genital lavage might be a future tool aiding the identification of FGS pathology at a stage where reversibility remains a possibility following praziquantel treatment.