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.     

Molecular detection of Histoplasma capsulatum indoors: A public health approach

BackgroundHistoplasmosis, caused by the dimorphic fungus Histoplasma capsulatum, represents an important public health problem, especially in urban environments where bats and humans cohabit indoors.AimsTo detect the presence of H. capsulatum indoors, using samples of bat droppings collected in roost sites inside houses.MethodsA Real-Time TaqMan PCR assay targeting the ITS1 region of the ribosomal DNA of H. capsulatum was carried out.ResultsFifty-nine sampling points in the municipality of São Paulo were inspected, all of them located at inhabited places. H. capsulatum was isolated from nine samples.ConclusionsThe rapid identification and monitoring of sites where the fungus is present may contribute to make a more reliable database of H. capsulatum distribution.     

Fast protocol for the production of Histoplasma capsulatum antigens for antibody detection in the immunodiagnosis of histoplasmosis

Background

Current methods for the production of Histoplasma capsulatum antigens are problematic in terms of standardization, specificity, stability, repeatability and reproducibility.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.     

Validation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica serovars

In Europe, alternative methods for the detection of food-borne pathogens can be used instead of the standard ISO/CEN reference protocol, if validated according to the protocol outlined in ISO 16140, 2003. In this study, the performance of two novel methods for the detection of Salmonella sp. using real-time PCR technology in tandem with an adapted two-step enrichment protocol were assessed and validated against a reference culture method, ISO 6579, 2004. The DNA and RNA real-time PCR assays amplified a 270 bp region of the hilA gene of Salmonella enterica serovars, and incorporated an internal amplification control (IAC) which was co-amplified with the hilA gene to monitor potential PCR inhibitors and ensure successful amplification. The inclusivity and exclusivity of the hilA primer set was examined for both the DNA and RNA methods and detected the 30 S. enterica serovars but not the 30 non-salmonellae strains. The inoculation of meat carcass swabs with five different S. enterica serovars at five different inocula, indicated both PCR methods were able to detect between 1 and 10 CFU per carcass swab. The real-time DNA PCR assay performed as well as the traditional cultural method in detecting Salmonella sp. in artificially contaminated salad, chocolate, fish and cheese samples. The relative accuracy, relative sensitivity and relative specificity of the DNA PCR real-time method were determined to be 98.5, 98.1 and 100%, respectively. The DNA method was further validated in a collaborative inter-laboratory trial according to ISO 16140, 2003. The validated methods provide an accurate means for the rapid detection and tracking of S. enterica serovars giving equivalent results to the standard method within three days, thus providing an alternative testing method to the reference microbiological method. The real-time PCR methodology not only offers significant time-saving advantages compared to traditional methods, it can also be applied to a wide range of samples types.     

Rapid Identification of Histoplasma capsulatum Directly from Cultures by Multiplex PCR

The multiplex PCR developed from a suspension of the yeast fungi correctly identified fifty-one clinical of H. capsulatum var. capsulatum strains isolated from clinical samples and soil specimens. The multiplex PCR was developed by combining two pairs of primers, one of them was specific to the H. capsulatum and the other one, universal for fungi, turned out to be specific to H. capsulatum, regardless of the fungus isolate studied. Primers designed to amplify a region of about 390-bp (Hc I–Hc II) and a region of approximately 600-bp (ITS1–ITS4) were used to identify a yeast isolated as H. capsulatum when both regions could be amplified. Absolute agreement (100?% sensitivity) could be shown between this assay and the cultures of H. capsulatum according to their morphological characteristics. Failure to amplify the target DNA sequence by PCR with primers Hc I–Hc II in the presence of the ITS1–ITS4 amplicon in isolates of P. brasiliensis, Cryptococcus neoformans, Trichosporon spp, Candida glabrata, C. albicans, C. tropicalis, C. parapsilosis, C. krusei, or Penicillium marneffei was an unequivocal sign of the high specificity of this assay. The assay specificity was also found to be 100?%. Incipient yeast forms obtained from clinical samples were identified as H. capsulatum by the PCR assay described before the morphological characteristics were registered shortening the time of diagnosis.     

Multiplex real-time PCR detection of P. falciparum, P. vivax and P. malariae in human blood samples

Two duplex real-time PCR assays were developed to diagnose three human parasites: Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. TaqMan duplex real-time PCR was evaluated in 263 blood samples of suspected malaria patients by comparing results against those obtained with microscopy and nested PCR. Compared with nested PCR, duplex real-time PCR assays showed 100% sensitivity and specificity. Duplex real-time PCR detected all mixtures of P. falciparum and P. vivax DNA, except at threshold detection limits for both parasites in which P. vivax was not amplified. Threshold detection limits of real-time PCR were 3.1, 0.3 and 0.8 parasites per microlitre of blood for P. falciparum, P. vivax and P. malariae, respectively. Duplex real-time PCR allows the detection of malarial cases, including mixed species infection, it simplifies analysis and reduces cost. Thus, this protocol may prove invaluable for use in the diagnosis of human infection, trial treatments and epidemiologic studies in which high-throughput analyses are often required.     

Specific and sensitive detection of Ralstonia solanacearum in soil with quantitative, real-time PCR assays

Aims:  The aim of this study was to develop a sensitive and an effective method suitable for large-scale detection and quantification of Ralstonia solanacearum in soil.
Methods and Results:  Based on the specific sequence of R. solanacearum strain G1000, the primer pair R.sol1-R.sol2 and the TaqMan probe Rs-pro were designed, and specific and sensitive PCR detection methods were successfully established. The detection limit was 100 fg μl⁻¹ DNA in conventional PCR and 1·2 fg μl⁻¹ in real-time PCR. By combining real-time PCR with the modified protocols to extract DNA from soil, it was possible to achieve real-time detection of R. solanacearum in soil, and the degree of sensitivity was 100 fg μl⁻¹. To detect inhibition in soil samples, an exogenous internal positive control (IPC) was included preventing false negative results, and IPC was successfully amplified from all samples tested. The methodology developed was used to detect the presence of R. solanacearum in tobacco fields in China.
Conclusions:  The real-time PCR combined with the protocol to extract DNA from soil led to the development of a specific, sensitive and rapid detection method for R. solanacearum in soil.
Significance and Impact of the Study:  The real-time PCR improves the detection sensitivity and specificity and provides an important tool for routine detection of R. solanacearum in soil samples and for epidemiological and ecological studies.     

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne’s disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold’s egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne’s) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.     

Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Validation in a Multicenter Collaborative Trial

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters (Campylobacter jejuni, C. coli, and C. lari) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.     

An unusual presentation of disseminated histoplasmosis in a non-HIV patient from Vietnam

BackgroundHistoplasmosis is a systemic infection caused by the dimorphic fungus Histoplasma capsulatum, naturally found in nitrogen-rich soil, whose main transmission route is the inhalation of conidia. Up to 95% of histoplasmosis cases are asymptomatic or transient, and the remaining 5% of cases have pathological manifestations in the lungs, bone marrow, liver, spleen, intestine, mucous membranes, and rarely on the skin. This mycosis has been reported from many endemic areas, mainly in immunosuppressed patients, such as HIV-positive patients, and its disseminated form is rarely reported.Case reportHistoplama capsulatum was isolated and identified by means of microscopy, culture characteristics and nested PCR from the cutaneous lesions of a non-HIV patient from Vietnam. The patient improved significantly with systemic itraconazole treatment.ConclusionsDisseminated histoplasmosis with cutaneous involvement in non-HIV patients is an extremely unusual presentation.     

Comparison between micro-hematocrit centrifugation technique and polymerase chain reaction (PCR) to detect Trypanosoma evansi in experimentally inoculated goats

Natural Trypanosoma evansi infection in the Canary Islands has only been diagnosed in the camel population, but dissemination of the disease in other hosts has not been excluded. To evaluate the role of the goats in the dissemination of the disease, 8 goats were inoculated and examined during 6 months using a polymerase chain reaction (PCR) with a primer targeting a repetitive region specific for Trypanozoon subgenus used to amplify a 227 bp fragment from the genomic DNA. PCR was able to detect parasitemia in all tested samples; therefore it was considered as gold standard test in this study. The results were compared with those obtained using the micro-hematocrit centrifugation technique showing a sensitivity of 92.7%, specificity of 100%, positive predictive value of 1 and negative predictive value of 0.87. Both techniques seem to be adequate to detect T. evansi from infected goats.     

Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp

A real-time PCR method was developed to detect monodon baculovirus (MBV) in penaeid shrimp. A pair of MBV primers to amplify a 135 bp DNA fragment and a TaqMan probe were developed. The primers and TaqMan probe were specific for MBV and did not cross react with Hepatopancreatic parvovirus (HPV), White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic virus (IHHNV) and specific pathogen free (SPF) shrimp DNA. A plasmid (pMBV) containing the target MBV sequence was constructed and used for determination of the sensitivity of the real-time PCR. This real-time PCR assay had a detection limit of one plasmid MBV DNA copy. Most significantly, this real-time PCR method can detect MBV positive samples from different geographic locations in the University of Arizona collection, including Thailand and Indonesia collected over a 13-year period.     

Brote de histoplasmosis en la provincia de Neuquén,Patagonia Argentina

BackgroundIn Argentina, there are no reports of autochthonous cases of histoplasmosis in the southern regions of the country.AimTo report a histoplasmosis outbreak in Zapala town, Province of Neuquén, Patagonia Argentina.MethodsWe evaluated the clinical and epidemiological characteristics of 5 patients involved in the outbreak. Environmental studies were conducted to determine the source of infection. The genetic profile of Histoplasma capsulatum strains isolated from the index case (IC) were compared with clinical isolates from Argentinean patients not related to the outbreak, using RAPD-PCR with primers 1281-1283.ResultsThe patients were residents of Zapala, and had not visited other geographical areas before. All patients had an influenza-like syndrome, and X-ray revealed disseminated micronodular images throughout the lung parenchyma. The IC needed specific antifungal therapy; the remaining 4 patients had mild symptoms, and did not require therapy. All of them had a good clinical outcome. Strains of H. capsulatum isolated from blood culture and lung biopsy of the IC showed a genetic profile different from other strains analyzed. The presence of the fungus in the environment was demonstrated by the detection of anti-Histoplasma antibodies in BALB/c mice inoculated with soil obtained in a culvert where workers had dug up earth after a landslide.ConclusionsThis outbreak suggests the histoplasmosis endemic area is under the 38° S parallel. Patients from Neuquén, Patagonia Argentina, with compatible symptoms of histoplasmosis should be tested, regardless of their travel or exposure history.     

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer''s protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit''s protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.     

Optimal swab processing recovery method for detection of bioterrorism-related Francisella tularensis by real-time PCR

Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5′nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations.     

Development of a new probe for specific and sensitive detection of 'Candidatus Phytoplasma mali' in inoculated apple trees

A new TaqMan minor groove binding (MGB) probe and new PCR conditions were designed for quick, specific and sensitive detection of ‘Candidatus Phytoplasma mali’. The new probe can distinguish a single mismatch between ‘Ca. P. mali’ and ‘Candidatus Phytoplasma prunorum’, this constituting an improvement over a previously published method. In our study, the relative position of the mismatch in the MGB probe influenced greatly the specificity of detection. Our new real‐time PCR protocol was able to detect one plasmid copy in water and 100 copies in healthy plant DNA extracts. The sensitivity of this new real‐time PCR method, three other real‐time PCR protocols and a conventional PCR with fU5/rU3 primers was compared. Periwinkles and MM106 rootstocks were grafted with infected material and surveyed over time by symptom observation, conventional PCR and real‐time PCR. Phytoplasma infection was detected by symptom observation in all periwinkles within 4 months and in 75% of the MM106 rootstocks by the end of 7 months. Conventional PCR detected phytoplasma infection in all periwinkles within 4 months and in all MM106 rootstocks within 7 months. Best results were obtained by our real‐time PCR, which detected phytoplasma infection in all grafted plants within 3 months after inoculation.     

PCR/16sRNA联合核苷酸测序法检测化脓性脑膜炎病原菌的临床价值

目的:探讨PCR/16sRNA联合核苷酸测序法在化脓性脑膜炎病原菌检测中的临床诊断价值。方法:选择2016年4月至2017年2月上海儿童医学中心临床考虑中枢感染的43例化脓性脑膜炎患儿的脑脊液标本,所有患儿标本同时进行培养,并行PCR/16sRNA联合核苷酸测序法检测,记录检测结果,并统计检测方法的灵敏度和特异度,以脑脊液培养检测结果为金标准,对比脑脊液培养和PCR/16sRNA联合核苷酸测序法的灵敏度和特异度。结果:脑脊液培养的灵敏度为21.7%,特异度为100.0%;PCR/16sRNA联合核苷酸测序的灵敏度为69.6%,特异度为95.0%;两者的灵敏度比较差异具统计学意义(P0.05),而两者特异性比较差异无统计学意义(P0.05)。PCR/16sRNA联合核苷酸测序可检出脑脊液培养阴性的病原体。结论:PCR/16sRNA联合核苷酸测序具有较高的灵敏度,可检出脑脊液培养阴性的病原体,且受抗菌药物影响小,可为临床早期提供化脓性脑膜炎的病原学依据,降低致死率及致残率。     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

Primers targeting mitochondrial genes of avian haemosporidians: PCR detection and differential DNA amplification of parasites belonging to different genera

Haemosporida is a diverse group of vector-borne parasitic protozoa, ubiquitous in terrestrial vertebrates worldwide. The renewed interest in their diversity has been driven by the extensive use of molecular methods targeting mitochondrial genes. Unfortunately, most studies target a 478?bp fragment of the cytochrome b (cytb) gene, which often cannot be used to separate lineages from different genera found in mixed infections that are common in wildlife. In this investigation, an alignment constructed with 114 mitochondrial genome sequences belonging to four genera (Leucocytozoon, Haemoproteus, Plasmodium and Hepatocystis) was used to design two different sets of primers targeting the cytb gene as well as the other two mitochondrial DNA genes: cytochrome c oxidase subunit 1 and cytochrome c oxidase subunit 3. The design of each pair of primers required consideration of different criteria, including a set for detection and another for differential amplification of DNA from parasites belonging to different avian haemosporidians. All pairs of primers were tested in three laboratories to assess their sensitivity and specificity under diverse practices and across isolates from different genera including single and natural mixed infections as well as experimental mixed infections. Overall, these primers exhibited high sensitivity regardless of the differences in laboratory practices, parasite species, and parasitemias. Furthermore, those primers designed to separate parasite genera showed high specificity, as confirmed by sequencing. In the case of cytb, a nested multiplex (single tube PCR) test was designed and successfully tested to differentially detect lineages of Plasmodium and Haemoproteus parasites by yielding amplicons with different sizes detectable in a standard agarose gel. To our knowledge, the designed assay is the first test for detection and differentiation of species belonging to these two genera in a single PCR. The experiments across laboratories provided recommendations that can be of use to those researchers seeking to standardise these or other primers to the specific needs of their field investigations.