Enteroaggregative Escherichia coli quantification in children stool samples using quantitative PCR

Enteroaggregative Escherichia coli (EAEC) is a common cause of infectious diarrhea, especially in children living in poor‐resource countries. In this article, we present a SYBR Green‐based real‐time polymerase chain reaction (qPCR) method for quantitative detection of EAEC in DNA directly extracted from human stool samples. To test the proposed qPCR system, we examined specificity, sensitivity, repeatability, and also the degree of DNA extraction efficiency using EAEC strain 042 spiked into EAEC‐free stool sample. The specificity of this assay was proved using six strains of EAEC, seven strains of other E. coli types, and one strain of Shigella. The detection limit of qPCR was 67 CFU/reaction. In naturally infected stool samples, we found EAEC in quantities varying from 6.7 × 10⁵ to 2 × 10⁹ CFU/g of feces. We could not detect any reduction after stool DNA extraction for the amounts of 10⁷ and 10⁶ CFU/mL of spiked EAEC. This qPCR assay is simple, rapid, reproducible, sensitive, specific, and allows rapid EAEC quantification to be used in a variety of further EAEC studies. This new quantitative method provides a relatively simple means to quantify EAEC, which will likely be key to understanding the pathophysiology and impact of EAEC infection.     

Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR

Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial strains. Twenty-two of the strains represented all of the presently available 13 phenotypic variants of G. anatis originating from different geographical locations. Nine strains represented each of the additional six Gallibacterium species and 21 strains represented other poultry associated bacterial species of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae. Regarding specificity none of non-G. anatis strains tested positive with the proposed assay. To test and compare the qPCR method’s ability to detect G. anatis from field samples, the sensitivity was compared to a previously published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10⁸ colony forming units (CFU)/ml to as low as 10⁰?CFU/ml copies. The proposed qPCR method is thus highly specific, sensitive and reproducible. In conclusion, we have developed a qPCR method that allows species-specific identification of G. anatis.     

Rapid Identification and Typing of Staphylococcus aureus by PCR-Restriction Fragment Length Polymorphism Analysis of the aroA Gene

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 × 10² CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.     

Evaluation of an internally controlled real-time polymerase chain reaction assay targeting the groEL gene for the detection of Bartonella spp. DNA in patients with suspected cat-scratch disease

Bartonella (B.) henselae is the causative agent of cat-scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. This study reports the development and evaluation of an internally controlled real-time polymerase chain reaction targeting the groEL gene for detection of Bartonella spp. DNA was extracted using the MagNA Pure system. The lower detection limit was 10–100 fg DNA and the in vitro sensitivity of the assay was not affected by duplexing with an internal control PCR. The real-time PCR assay detected DNA from all five B. henselae strains tested, and from B. birtlesii, B. vinsonii subsp. vinsonii, B. vinsonii subsp. arupensis and B. doshiae. The assay generated negative results with a selection of other bacteria, including several Mycobacterium spp., Streptococcus pyogenes and Staphylococcus aureus. Results of real-time PCR in clinical samples were compared with those of a conventional 16S rDNA-based PCR assay. During the period described in the Material and methods section, real-time PCR and conventional 16S PCR were performed on 73 clinical samples. Of these samples, 29 (40%) were found to give positive results and 44 (60%) gave negative results, both by real-time PCR and by conventional PCR, with a 100% agreement between the two tests. The PCR developed in this study is a rapid, sensitive, and simple method for the detection of Bartonella spp. in CSD and is suitable for implementation in the diagnostic laboratory.     

A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources

An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10⁸ cfu/ml into a suspension of Ped-2E9 (10⁶/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test, Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 μm filter.     

Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection

Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert^®, and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert^® assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert^® assay. The predicted median for probit = 0.9 was 54 (44–76) CFU/ml for M. hyorhinis and 16 (13–23) CFU/ml for M. hominis by the nested PCR, but 431 (346–593) CFU/ml and 105 (87–142) CFU/ml, respectively, with MycoAlert^®. Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Pentaplex PCR Assay for Detection of Hemorrhagic Bacteria from Stool Samples

Diarrheal diseases cause illness and death among children younger than 10 years in developing countries. Conventional testing for the detection of hemorrhagic bacteria takes 2 to 5 days to yield complete information on the organism and its antibiotic sensitivity pattern. Hence, in the present study, we developed a molecular-based diagnostic assay that identifies common hemorrhagic bacteria in stool samples. A set of specific primers were designed for the detection of Salmonella spp., Shigella spp., enterohemorrhagic Escherichia coli (EHEC), and Campylobacter spp., suitable for use in a one-tube PCR assay. The assay in the present study simultaneously detected five genes, namely, ompC for the Salmonella genus, virA for the Shigella genus, eaeA for EHEC, 16S rRNA for the Campylobacter genus, and hemA for an internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. Validation with 20 Gram-negative and 17 Gram-positive strains yielded 100% specificity. The limit of detection of the multiplex PCR assay was 1 × 10³ CFU at the bacterial cell level and 100 pg at the genomic DNA level. Further evaluation of the multiplex PCR with 223 bacterium-spiked stool specimens revealed 100% sensitivity and specificity. We conclude that the developed multiplex PCR assay was rapid, giving results within 4 h, which is essential for the identification of hemorrhagic bacteria, and it might be useful as an additional diagnostic tool whenever time is important in the diagnosis of hemorrhagic bacteria that cause diarrhea. In addition, the presence of an internal control in the multiplex PCR assay is important for excluding false-negative cases.     

Development of an immunochromatographic strip for the rapid detection of Pseudomonas syringae pv. maculicola in broccoli and radish seeds

A portable immunochromatographic (IC) strip based on gold-labelled monoclonal antibodies was developed for the rapid detection of Pseudomonas syringae pv. maculicola, a plant pathogen. The IC strip detected 10⁵ colony-forming units CFU/ml P. syringae pv. maculicola in pure culture within 10 min and had good specificity. Compared with sandwich enzyme-linked immunosorbent assay, the strip was equally sensitive; however, it is portable and time-efficient. On broccoli and radish seeds, the IC strips had a sensitivity of 5 × 10⁵ CFU/ml and 10⁶ CFU/ml, respectively. The results revealed that the IC strip could have potential applications for P. syringae pv. maculicola detection in plant seeds.     

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit M^Grade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.     

Triplex PCR‐based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk

Although many strains of Bacillaceae are considered nonpathogenic, Bacillus cereus is recognized worldwide as a bacterial pathogen in a variety of foods. The ability of B. cereus to cause gastroenteritis following ingestion of contaminated food is due to the production of enterotoxins. The ubiquity of this genus makes it a persistent problem for quality assurance in food processing environments. The primary objective of this study was to develop and apply a multiplex real‐time PCR‐based assay for rapid and sensitive detection of enterotoxigenic B. cereus. Template DNA was separately extracted from tryptic soy broth (TSB)‐grown and 2.5% Nonfat Dry Milk (NFDM)‐grown B. cereus using a commercial system. Three enterotoxin gene fragments (hblC, nheA, and hblA) were simultaneously amplified in real‐time followed by melting curve analysis to confirm amplicon identity. Resolution of melting curves (characteristic T_m) was achieved for each amplicon (hblC = 74.5 °C; nheA = 78 °C; and hblA = 85.5 °C in TSB and 84 °C in NFDM) with an assay sensitivities of 10¹ CFU/ml for both TSB and NFDM‐grown B. cereus compared to 10⁴ CFU/ml in either matrix using gel electrophoresis. The results demonstrate the potential sensitivity of real‐time bacterial detection methods in a heterogenous food matrix using real‐time PCR. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Prevalence,identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia

Objectives

We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates.

Material and methods

Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus.

Results

The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases.

Conclusion

We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources.     

Microbiological survey of a South African poultry processing plant

Bacterial populations associated with poultry processing were determined on neck skin samples, equipment surfaces and environmental samples by replicate surveys. Aerobic plate counts, Enterobacteriaceae counts and Pseudomonas counts were performed by standard procedures and the prevalence of Listeria, presumptive Salmonella and Staphylococcus aureus determined. Statistically significant (P < 0.05) increases in counts of all types of bacteria were obtained on product samples as a result of processing. Although bacterial counts on neck skin samples decreased by 0.3 to 0.4 log CFU g^?1 after spray washing of carcasses, subsequent spinchilling and packaging of whole carcasses resulted in 0.7 to 1.2 log CFU g^?1 increases. Bacterial numbers on equipment surfaces, however, decreased significantly from the ?dirty”? to the ?clean”? areas of the abattoir. Transport cages, ?rubber fingers”?, defeathering curtains, shackles and conveyor belts repeatedly showed aerobic plate counts in excess of 5.0 log CFU 25 cm^?2. Aerobic plate counts of scald tank and spinchiller water were 2 log CFU ml^?1 higher than those of potable water samples. Bacterial numbers of the air in the ?dirty”? area were higher than those of the ?clean”? area. Listeria, presumptive Salmonella and Staphylococcus aureus were isolated from 27.6, 51.7 and 24.1% of all product samples, respectively, and Listeria and Staphylococcus aureus were also isolated from selected equipment surfaces.     

MRSA screening: can one swab be used for both culture and rapid testing? An evaluation of chromogenic culture and subsequent Hain GenoQuick PCR amplification/detection

The use of a single swab for both MRSA culture and for rapid testing by PCR was evaluated, using the Hain GenoQuick (GQM) methicillin resistant Staphylococcus aureus (MRSA) assay for the rapid detection of MRSA, as a single swab would be the preferred option for routine diagnostic testing. GQM detected current prevalent Irish MRSA strains incorporating all known SSCmec types, including Panton–Valentine leukocidin-positive strains. Using the GQM method, all methicillin-resistant coagulase-negative staphylococci tested were confirmed to be negative, although three of seven gentamicin-resistant methicillin-sensitive Staphylococcus aureus strains tested were identified as MRSA. The theoretical ex-vivo limit of detection of the assay was 704 CFU per GQM assay reaction (1.7 × 10⁴ CFU/mL) when MRSA suspensions were used for DNA extraction, or 1.4 × 10³ CFU/swab (1.4 × 10⁴ CFU/mL) using MRSA absorbed onto Copan screening swabs. Swab processing on chromogenic agar prior to PCR resulted in some inhibition of the PCR reaction, increasing the limit of detection of the assay by a factor of four. Based on 540 single swab screening specimens (nasal and groin) processed first for culture assay, then by GQM, the specificity and positive predictive value were both 100%, the negative predictive value was 92%, and the sensitivity was 57. Culture followed by PCR from a single specimen is not optimal for the rapid detection of MRSA. Further laboratory validation of the GQM assay is required to determine the true diagnostic sensitivity and value of this kit in routine microbiology laboratories, modifying the protocol for single specimens, or using two specimens.     

A chelating-bond breaking and re-linking technique for rapid re-immobilization of immune micro-sensors

With high sensitivity and specificity to antigen, immune micro-sensors can be used in rapid detection of pathogenic microbial. This study proposes and develops a method for rapidly regeneration of antibody on a resonant micro-cantilever sensor. A nitrilotriacetic acid (NTA) derivative is synthesized with cystine and bromoacetic acid, then added with 2-mercaptoethanol to prepare a mixed self-assembled monolayer (SAM) on Au (111) surface of the cantilever. Ni²⁺ ions are thereafter chelated on the mixed SAM to form a breakable and re-linkable chelating-bond layer. Repeatable cycles of antibody immobilization and erasing are experimentally validated with a detectable marker of synthesized biotinylated poly peptides harboring six histidine residues (named as His-Bio). Two distinguished pathogenic microbial, Escherichia. coli O157:H7 and Bacillus Anthracis, are detected with the rapidly regenerated sensor. The E. coli O157:H7 sensor exhibits a three-time repeated detection to the 10³ CFU/ml concentration microbial. Then, an E. coli O157:H7 sensor is eluted with Tris–HCl (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH = 3.0) and rapidly reconstructed into a B. Anthracis sensor by changing the re-immobilized antibody. The cantilever sensor no longer responses to E. coli O157:H7 even in a high concentration of 10⁷ CFU/ml. In contrast, the sensor is experimentally confirmed being resoluble to low concentration B. Anthracis at 10³ spores/ml level. The proposed fast regeneration method is promising in repeatedly or multi-target detection applications of micro/nano immune-sensors, e.g. the resonant micro-cantilevers.     

A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory

A multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 10(2)-10(3) DEC CFU/PCR in the presence of 10(6) non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 10(6) CFU/mL stool sample.     

Rapid identification of bacteria by PCR-single-strand conformation polymorphism.

A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.     

聚合酶链式反应快速检测E型肉毒神经毒素基因

目的Ｅ型肉毒中毒是人类肉毒中毒的主要型别之一，本试验旨在建立用于Ｅ型肉毒梭菌鉴定的ＰＣＲ方法。方法用人工合成的寡核苷酸引物扩增Ｅ型肉毒神经毒素基因的一段３６８ｂｐ的ＤＮＡ片段，快速检测Ｅ型肉毒神经毒素基因，对梭状芽胞杆菌属的４０株保藏菌株及３３份土壤标本进行了鉴定，用Ｅ型肉毒梭菌ＣＭＣＣ（Ｂ）６４５０１对其灵敏度进行了检查。结果从所有Ｅ型神经毒素原性菌株或标本均能扩增出目的片段，且能用特定的限制性内切酶切成相应的片段。其它菌株均未能扩增出目的片段。灵敏度试验可从３０个菌体扩增出目的片段。结论此方法用于Ｅ型神经毒素原性梭菌的鉴定具有较高的灵敏度及特异性。     

Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays

Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 10¹ to 2.9 × 10⁴ CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.     

A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii,Histoplasma capsulatum,and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r² of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log₁₀ copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.     

Development of a single tube multiplex real-time PCR to detect the most clinically relevant Mucormycetes species

Mucormycetes infections are very difficult to treat and a delay in diagnosis could be fatal for the outcome of the patient. A molecular diagnostic technique based on Real Time PCR was developed for the simultaneous detection of Rhizopus oryzae, Rhizopus microsporus and the genus Mucor spp. in both culture and clinical samples. The methodology used was Molecular beacon species-specific probes with an internal control. This multiplex real-time PCR (MRT-PCR) was tested in 22 cultured strains and 12 clinical samples from patients suffering from a proven mucormycosis. Results showed 100% specificity and a detection limit of 1 fg of DNA per microlitre of sample. The sensitivity was 100% for clinical cultured strains and for clinical samples containing species detected by the PCR assay. Other mucormycetes species were not detected in clinical samples. This technique can be useful for clinical diagnosis and further studies are warranted.