Evaluation of a rapid antigen detection test for <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> in urine sediment for diagnosis of gonococcal urethritis in males

We evaluated a rapid antigen detection method with an immunochromatographic assay for Neisseria gonorrhoeae (NOW Gonorrhea Test) by using urine samples from patients with urethritis to diagnose gonococcal infection in males. Among 58 male patients who underwent urethral swab culture, 34 cases (58.6%) were found to have N. gonorrhoeae infection. The sensitivity and specificity of the NOW Test compared with the results of standard culture were 94.1% (32/34) and 95.8% (23/24), respectively. The predictive values of positive NOW and negative NOW were 96.9% (32/33) and 92.0% (23/25), respectively. The detection limit of this assay was determined as 5 × 10⁴cfu/ml using N. gonorrhoeae suspension as an antigen. In contrast to standard cultures, gonococcal antigens in specimens were still detectable by this method up to 45h of storage at either room temperature or 4°C. Considering the rapidity and ease of this method, our results suggest that the NOW method might be a useful and reliable diagnostic screening tool for gonococcal urethritis.     

Binding of Complement Factor H to Loop 5 of Porin Protein 1A: A Molecular Mechanism of Serum Resistance of Nonsialylated Neisseria gonorrhoeae

Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N. gonorrhoeae.     

Genotyping of Helicobacter pylori strains from gastric biopsies by multiplex polymerase chain reaction. How advantageous is it?

Recent application of multiplex polymerase chain reaction (PCR) for genotyping Helicobacter pylori direct from biopsies revealed variable results (detection of amplicons from DNA extracted by boiling biopsies, variable size amplicons and deletions, uniform intensity of amplicon bands). We aimed to look at how applicable the technique is for determining cagA and vacA genotypes and to correlate the results with the severity of the disease. H. pylori strains from 52 patients (35 duodenal ulcers [DUs], 7 gastric ulcers [GUs], 10 gastritis) were included. Three antral biopsies were obtained for Campylobacter-like organism (CLO) and PCR. Primers for cagA, vacA s1s2, and m1m2 alleles were used. No PCR amplicons were detected from boiling biopsies; thus, DNA was extracted by QIAamp kit. H. pylori was positive in 84.6% of the patients (85.7% DU, 100% GU, and 70% gastritis). The cagA gene was detected in 86.6% DU, 71.4% GU, and 57.0% gastritis patients. The vacA allelic distribution among cagA-positive strains was 80.7% s1m1 in DU and 60.0% in GU patients, whereas 75.0% of gastritis had s1m2. No variability in the amplicon sizes was found, and the intensity of the amplicon bands was not uniform. A deleted band of approximately 420 bp below the m1 band was detected in strains from 2 DU and 1 GU patients. Although the multiplex PCR is a rapid and an effective tool for detecting several genes in a single-step system, one has to adjust for optimization of the technique when genotyping H. pylori direct from biopsies. A significant association was found between the cagA-positive vacA-s1m1 genotype and peptic ulcers.     

Evaluation of the cobas 4800 CT/NG Test for detecting Chlamydia trachomatis and Neisseria gonorrhoeae DNA in urogenital swabs and urine specimens

We have evaluated 696 samples (488 swabs and 208 urine specimens) with the cobas 4800 (c4800) CT/NG Test for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA in swab and urine specimens. c4800 results were compared with those obtained from COBAS AMPLICOR (CAM) CT/NG Test. Discordant results were reanalyzed with the MultiNA system and compared with clinical data. For C. trachomatis detection by both methods, we obtained 93.8%, 100%, 100%, and 99.1% for sensitivity, specificity, and positive and negative predictive values, respectively. For urine specimens analyzed in c4800, our results were 96.6%, 100%, 100%, and 99.4%, respectively. For N. gonorrhoeae detection, swab results were:88.0%, 100%, 100%, and 99.4%. For urine specimen, results obtained were 100%, 100%, 100%, and 100%. Reanalyses were all concordant between both methods. c4800 results were comparable with those obtained with the CAM system. We had an excellent correlation between swab and urine specimens analyzed by c4800.     

Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens

We evaluated a new multiplex polymerase chain reaction (mPCR), “STDFinder assay”, a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing.     

Single dose of cefodizime completely eradicated multidrug-resistant strain of Neisseria gonorrhoeae in urethritis and uterine cervicitis

Cefodizime (CDZM) has strong antimicrobial activity to Neisseria gonorrhoeae in vitro. However, multidrug-resistant N. gonorrhoea emerged and has been increasing in Japan. To know the effectiveness of CDZM on gonococcal urethritis and uterine cervicitis even in the era of multidrug-resistant N. gonorrhoeae, a clinical trial of single-dose therapy of CDZM for gonococcal urethritis and uterine cervicitis was conducted. N. gonorrhoeae was eradicated from 100% of patients with gonococcal urethritis and uterine cervicitis by a single dose of CDZM. In conclusion, CDZM is one of most suitable drugs for the treatment of gonococcal genital infection in the era of multidrug-resistant N. gonorrhoeae.     

Evaluation of Seeplex® STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections

Traditionally, the diagnosis of bacterial sexually transmitted infection (STI) has been dependent on the isolation of the causative pathogens by culturing endocervical or urethral swab specimens on selective media. While such procedures typically provide excellent diagnostic accuracy, they are often time-consuming and expensive. A multiplex polymerase chain reaction (PCR) assay, based on a semi-automated detection system, was evaluated for the detection of six STI causative organisms. The Seeplex^® STD6 ACE (auto-capillary electrophoresis) Detection assay employed six pairs of dual priming oligonucleotide (DPO?) primers specifically targeted to unique genes of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis. A total of 739 specimens (304 cervical swabs and 435 urine samples) collected for 4 months were tested, and results were compared to those obtained with a combined monoplex PCR. The concordance between the multiplex PCR and monoplex PCR assay was 100% for both sensitivity and specificity. We also tested for the presence of two pathogenic bacteria (C. trachomatis and N. gonorrhoeae) and compared the results obtained with the multiplex PCR and BD ProbeTec duplex strand displacement amplification (SDA). The results of the multiplex PCR and duplex SDA were 99.7% concordant for C. trachomatis and 100% concordant for N. gonorrhoeae. The multiplex PCR assay using the Seeplex^® STD6 ACE Detection kit proved to be a novel cost-effective and fast diagnostic tool with high sensitivity and specificity for the simultaneous detection of six STI pathogens.     

Development of a single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. directly in clinical samples

This study describes the development and evaluation of a multiplex single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. used as target species-specific or genus-specific genes. The assay enables the detection of 5 to 50 pg of bacterial DNA. The sensitivity of the assay was evaluated as 100% for P. aeruginosa, S. aureus, and Streptococcus spp., and 94.3% for H. influenzae; the specificity was 100% for all 4 microorganisms (positive predictive value, 100%; negative predictive value, 98.2%). The assay permits rapid and accurate detection of these 4 microorganisms in a wide range of clinical samples such as whole blood, cerebrospinal, ear, pleural and ophthalmic fluids, as well as bronchoalveolar lavage and bronchial secretions.     

Pharyngeal <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> detection in oral-throat wash specimens of male patients with urethritis

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in the pharynx has been highlighted in the prevention of the unexpected spread of sexually transmitted diseases. We tried to clarify the detection rate of Neisseria gonorrhoeae in the pharynx and the clinical relevance of oral-throat wash specimens to detect the organism in heterosexual men with gonococcal and nongonococcal urethritis. In our cohort of 79 male patients with urethritis, oral throat wash specimens were collected after they had gargled with normal saline for approximately 30 to 60 s. Positive pharyngeal N. gonorrhoeae was defined as a positive result on the strand displacement amplification test for the specimen from the oral-throat wash. N. gonorrhoeae was detected in the oral-throat wash specimens of 13 (31.7%) of the 41 male patients with gonococcal urethritis. Oral-throat wash with a nucleic acid amplification test can detect pharyngeal N. gonorrhoeae easily and efficiently.     

Simple,Rapid, and Inexpensive Detection of Neisseria gonorrhoeae Resistance Mechanisms Using Heat-Denatured Isolates and SYBR Green-Based Real-Time PCR

Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials. There is now growing concern that without the availability of appropriate public health strategies to combat this problem, gonorrhea could become untreatable. For this reason, surveillance for gonococcal antimicrobial resistance must be optimal both in terms of obtaining a representative sample of gonococcal isolates and in terms of having the appropriate tools to identify resistance. To aid with this surveillance, molecular tools are increasingly being used. In the present study, we investigated the use of a simple heat denaturation protocol for isolate DNA preparation combined with SYBR green-based real-time PCR for the identification of mutations associated with N. gonorrhoeae antimicrobial resistance. A total of 109 clinical gonococcal isolates were tested by high-resolution melting (HRM) curve analysis for chromosomal mutations associated with gonococcal resistance to beta-lactam antibiotics: a penA 345A insertion, ponA L421P, mtrR G45D, substitutions at positions 120 and 121 in porB1b, and an adenine deletion in the mtrR promoter. An allele-specific PCR assay was also investigated for its ability to detect the adenine deletion in the mtrR promoter. The results were compared to those obtained by DNA sequencing. Our HRM assays provided the accurate discrimination of heat-treated isolates in which the sequence types differed in GC content, including isolates with the penA 345A insertion and the ponA L421P and mtrR G45D mutations. The allele-specific PCR assay accurately identified isolates with the adenine deletion in the mtrR promoter. Heat-denatured DNA combined with SYBR green-based real-time PCR offers a simple, rapid, and inexpensive means of detecting gonococcal resistance mechanisms. These methods may have broader application in the detection of polymorphisms associated with phenotypes of interest.Neisseria gonorrhoeae is the etiologic agent of the sexually transmitted disease gonorrhea, causing an estimated 60 million new infections annually (7). In females, gonorrhea is a major cause of pelvic inflammatory disease and may lead to tubal infertility, ectopic pregnancy, and chronic pelvic pain, whereas in males it primarily causes urethritis (2, 3, 7). The control of gonorrhea is achieved through the provision of effective antimicrobial therapy to eradicate the infection, reduce transmission, and prevent the development of complications. However, N. gonorrhoeae has managed to develop resistance to multiple classes of antimicrobials, including penicillins and tetracyclines, and quinolones have been withdrawn from use in many parts of the world because of widespread resistance. Even the more recently available macrolides now have limited utility, leaving extended-spectrum cephalosporins (both oral and injectable) and spectinomycin (when it is available) as the mainstays of treatment. Without appropriate public health strategies to combat this problem, there is a real threat that gonorrhea will become untreatable (18). The Centers for Disease Control and Prevention has recently expressed fears regarding the ability to control gonorrhea, the need to explore alternate treatment options as a response to the increasing cephalosporin resistance in N. gonorrhoeae, and the requirement for enhanced monitoring of gonococcal resistance globally (4, 30).The volatile nature of antimicrobial resistance in gonococci means that surveillance for resistance for public health purposes must be optimal both in terms of obtaining a sufficiently large and representative sample of gonococcal isolates and in terms of using the appropriate tools to identify resistance (19). The increasing use of nucleic acid amplification assays in industrialized countries and the widespread application of syndromic management principles in less developed countries have increasingly restricted the availability of gonococcal isolates for the phenotypic detection of resistance rates. Logistical problems with gonococcal storage and transport and the intrinsic fragility of N. gonorrhoeae also have adverse effects on the availability of viable isolate (21, 31).Molecular tools are being used to provide an understanding of the genetic basis of resistance as well as to supplement phenotypic antimicrobial susceptibility testing (9, 15, 16, 20, 22, 23, 24). These tools have not yet advanced to a stage where they can be used in place of phenotypic testing, mainly because the genetic determinants of resistance to most antibiotics are not yet fully known. Nevertheless, for well-characterized resistance mechanisms, molecular tools offer an accurate and objective means of detection and systems for the detection of pivotal resistance determinants for public health purposes when viable isolates are not required have been described (22, 23).An ongoing limitation of molecular assays is that they can be prohibitively expensive. The advent of real-time PCR technology has, for the most part, alleviated the need for costly DNA sequencing. However, real-time PCR assays targeting gonococcal resistance mechanisms have to date predominantly combined initial DNA extraction with probe-based real-time PCR. Both of these factors can impinge on the cost-effectiveness of the use of real-time PCR; commercial DNA extraction methods can be expensive, and the use of fluorophore-labeled probes can involve considerable initial setup costs. This is particularly the case when multiple mutations are being investigated, as different probe sets specific for each mutation need to be purchased. Furthermore, we have found that the accuracy of probe-based real-time PCR can be undermined by unexpected variations in the sequences of the probe targets (28, 29). This is particularly relevant to N. gonorrhoeae, given that the species comprises numerous subtypes that exhibit considerable sequence diversity (25, 27) as well as because some of the genes implicated in conferring resistance, including porB1b, are highly variable.In the study described here, we sought to develop a simple and cost-effective means of identifying mutations associated with gonococcal resistance for potential applications in screening for antimicrobial resistance for public health purposes. We did this using two approaches: first, we sought to use a simple heat denaturation protocol rather than DNA extraction for isolate DNA preparation to decrease costs and the preparation time. This also allows the examination of nonviable organisms. Second, we used SYBR green-based real-time PCR, thereby removing the need for the initial purchase of expensive fluorophore-labeled probes and allowing the use of only a single primer pair. Briefly, the study was conducted by developing high-resolution melting (HRM) curve analysis assays targeting five key chromosomal mutations associated with gonococcal resistance to beta-lactam antibiotics, including an Asp-345A codon insertion in the penA gene, which encodes penicillin binding protein 2 (PBP 2) (5, 6); the L421P substitution in the ponA gene, which encodes PBP 1 (13); a G45D substitution in the encoding region of the mtrR gene (32); an adenine deletion in the mtrR promoter region (32); and various substitutions in amino acids 120 and 121 of the porB1b protein (8, 12). An allele-specific PCR assay was also investigated for its ability to detect the adenine deletion in the mtrR promoter.     

Detection of Helicobacter pylori by enzyme-linked immunosorbent assay of thermophilic helicase-dependent isothermal DNA amplification

An enzyme-linked immunosorbent assay (ELISA) of thermophilic helicase-dependent isothermal DNA amplification (tHDA) was developed for detection of Helicobacter pylori. The primers targeting ureC were used for the amplification of bacterial DNA by the isothermal digoxigenin (DIG)-labeling tHDA process, resulting in the accumulation of DIG-labeled DNA amplicons. The amplicons were denatured using heat and then hybridized with a specific biotinylated DNA probe, which was noncovalently immobilized on streptavidin-coated microtiter plate. The hybrids were colorimetrically detected by the addition of an anti-DIG antibody HRP conjugate and 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) substrate solution. Results obtained from the gastric biopsy samples showed 90% and 95.7% of sensitivity and specificity, respectively, in comparison with culture results, and 96.6% and 96.8% of sensitivity and specificity, respectively, in comparison with those of the histologic studies. This assay significantly reduces the time needed for the identification of H. pylori and has the potential to facilitate early detection of this gastrointestinal pathogen.     

16S ribosomal assay for early diagnosis of gonococcal meningitis with negative CSF culture: A case report

Gonococcal meningitis is an exceedingly rare infectious disease, and if not diagnosed and treated in time, it can be severe. We present a case of gonococcal meningitis occurring in a 31-year old healthy woman. She was admitted with fever and persistent headache without urogenital symptoms. Blood cultures were positive and identified as N.gonorrhoeae, but CSF and cervical secretions cultures were both negative. Further testing confirmed the presence of N.gonorrhoeae by 16S ribosomal gene amplification and sequencing in all samples. These results suggest that the case may be a disseminated infection caused by untreated gonorrhea. Our case also shows that nucleic acid detection plays an important role in the rapid and precise diagnosis of gonococcal meningitis and in finding the origin of the pathogen.     

Detection of Mycobacterium avium complex DNA directly in clinical respiratory specimens: opportunities for improved turn-around time and cost savings

We developed, evaluated, and implemented a Taqman multiplex real-time polymerase chain reaction (PCR) assay for the detection of Mycobacterium avium complex (MAC), targeting the 16S-23S rRNA internal transcribed spacer, which we have combined with an existing Mycobacterium tuberculosis complex assay for use directly in clinical respiratory specimens. Evaluation of the performance of this assay for MAC detection included 464 clinical respiratory specimens tested prospectively. This real-time PCR assay was found overall to have a sensitivity of 71.1%, a specificity of 99.5%, a positive predictive value of 98.0%, and a negative predictive value of 90.2% for MAC. The assay provides results prior to the availability of cultured material and identification, most within 24 h of specimen receipt, and may reduce the need to culture MAC-PCR–positive specimens when susceptibility testing is not requested. Additionally, we have found significant cost savings of approximately $21.00 per specimen and staff time reductions of 3.75 h per specimen with implementation of this assay.     

Multiplex TaqMan real-time PCR platform for detection of Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone

A multiplex TaqMan real-time PCR platform was developed in this study for combined detection of opa and/or porA genes (identification of N. gonorrhoeae) and the key mutations (Ala501Val/Thr/Pro, and/or Gly545Ser) in penicillin-binding protein 2 (PBP2) associated with decreased susceptibility to extended-spectrum cephalosporins (ESCs). Firstly, the specificities of the TaqMan probes/primers for the multiplex TaqMan real time PCR platform were confirmed by Basic Local Alignment Search Tool (BLAST) analysis. Then the multiplex PCR platform was performed on 77 isolates with decreased susceptibility to ceftriaxone (CRO) and 100 isolates with full susceptibility to CRO under universal optimized reaction conditions. As a result, based on cultivation-based matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and antimicrobial susceptibility testing in vitro, the multiplex platform had a sensitivity of 100% and a specificity of 95.0% for identifying cultured isolates of Neisseria gonorrhoeae (N. gonorrhoeae, NG, GC) with decreased susceptibility to CRO. When directly screening N. gonorrhoeae with decreased susceptibility to CRO from clinical urogenital swabs, the multiplex platform offered a sensitivity of 96.1% and a specificity of 95.0%. Therefore, on the basis of sample culture and antimicrobial susceptibility testing in vitro, the multiplex TaqMan real time PCR platform has been proven to be a sensitivity of 100% and a specificity of 95.0% useful tool for screening cultured isolates of N. gonorrhoeae with decreased susceptibility to CRO, which can be finished within 2 days.     

Rapid detection of vancomycin-resistant enterococci from rectal swabs by the Cepheid Xpert vanA/vanB assay

The detection of vancomycin-resistant enterococci using a novel commercial multiplex real-time polymerase chain reaction assay (Xpert™ vanA/vanB, Cepheid, Sunnyvale, CA) was evaluated on 804 rectal swab specimens. Compared to enriched culture, sensitivity and negative predictive value of this method were 100%. Many false-positive results were recovered (sensitivity, 85.4%; positive predictive value, 8.7%), especially for the vanB gene.     

Detection of Helicobacter pylori in dental plaque using a DNA biosensor for noninvasive diagnosis

Noninvasive diagnosis of Helicobacter pylori (H. pylori) infection is very attractive. This study investigated the single strand DNA (ssDNA) acquisition method from H. pylori in dental plaque, and the integration of our previously developed 43-mer H. pylori DNA biosensor with the obtained target ssDNA (tDNA). Dental plaque samples were collected from 34 patients/volunteers, whose gastric H. pylori infection statuses were tested with the ¹³C urea breath test (UBT). The samples were treated with colony polymerase chain reaction (PCR) to obtain double strand DNA (dsDNA) of 104 basepairs (bp) long. A blocker ssDNA was designed and used in thermal treatment of the dsDNA to release the 104-mer tDNA, which contains the 43-mer DNA sequence in the middle. PCR primers were designed, and the tDNA releasing and detection conditions with the biosensor were optimized. The limit of detection with the biosensor was 12 fM dsDNA. The dental plaque detection results correlated quite well with the UBT results, with a sensitivity of 100%, and specificity of 97%. These results indicate that the residence of H. pylori in dental plaque is highly associated with gastric H. pylori infection, and detection of dental plaque samples with our DNA biosensor is promisingly applicable in noninvasive diagnosis of H. pylori infection.

H. pylori in dental plaque was detected with a DNA biosensor with results correlating well with the ¹³C urea breath test.     

Usefulness and influence of age of a novel Rapid HpStAR™ stool antigen for the diagnosis of Helicobacter pylori infection in children

A novel rapid monoclonal enzyme immunoassay stool antigen for Helicobacter pylori detection (Rapid HpStAR™) was evaluated in 16 infected and 92 noninfected children. The overall sensitivity, specificity, and positive and negative predictive values were 87.5%, 97.8%, 87.5%, and 97.8%, respectively, with an accuracy of 96.2%. The negative predictive value was good in all age groups, reaching 100% in younger children.     

Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis

A real-time polymerase chain reaction (PCR) assay was evaluated in comparison with the combination of conventional methods (microscopic examination and antigen detection assay) during the period 2006 to 2008 on 771 fecal samples belonging to 386 patients to assess its usefulness for an accurate laboratory diagnosis of giardiasis. The real-time PCR assay detected Giardia intestinalis DNA in 195 samples (106 patients), including 26 samples (21 patients) negative by the conventional assays. Among the 21 patients, in 8 cases, giardiasis was previously diagnosed also by conventional methods in additional samples of the same patients, whereas in 13, it would have been undiagnosed if real-time PCR assay was not used. The real-time PCR assay demonstrated a detection limit of 2 cysts per reaction and 100% specificity and sensitivity compared to conventional methods. A genotype analysis targeting the β-giardin gene allowed to identify 53 samples (23 patients) containing genotype A and 59 samples (45 patients) containing genotype B.     

Simplified protocol for DNA extraction and amplification of 2 molecular markers to detect and type Giardia duodenalis

We evaluated the ability of 3 kits: QIAmp® DNA stool mini kit (Qiagen, Hilden, Germany), PureLink PCR Purification®, and PureLink™ Genomic DNA® (Invitrogen, Carlsbad, CA, USA) for DNA extraction, and of 2 molecular markers (heat shock protein [HSP] and β-giardin genes) for detection and genotyping of Giardia duodenalis stool samples. The detection and typing limits of the markers were determined by the DNA concentration of trophozoites and cysts and were tested in 26 clinical samples. Of the 3 kits tested, the PureLink PCR Purification gave the best results when tested with clinical samples with low, intermediate, and high numbers of cysts. The DNA extracted from trophozoites and cysts was diluted successively in 1:2 ratios until it was no longer possible to observe the amplified product in polyacrylamide gel. Similarly, a suspension of cysts was diluted until no cysts were observed, and then the DNA was extracted. The amount of DNA of trophozoites and cysts for the typing of the parasite was smaller for the HSP marker than for β-giardin. Combined use of both markers allowed us to detect DNA of Giardia in parasitologically positive samples in a higher percentage (75%) than the results obtained for each marker and in 1 parasitologically negative sample, indicating that this combination increased the potential to accurately detect and genotype this parasite. We also concluded that the HSP marker has a higher limit of detection and typing than the β-giardin marker and that the DNA extraction method tested for G. duodenalis is simpler and more efficient than those that are currently in use and can be applied on a large scale.