Polymerase Chain Reaction DNA Sequencing Research Articles

Epidemiological study of antimicrobial-resistant bacteria in healthy free-ranging bantengs (Bos javanicus) and domestic cattle.

Antimicrobial-resistant microorganisms (ARMs) have been increasing among wild animals. Interactions occurring at the interface between wildlife, humans, and livestock can lead to the transmission of ARMs. Thus, the prevalence of ARMs in wild and domestic animals should be determined to address and prevent this issue. This study aimed to determine the resistance patterns of cefotaxime (CTX)-resistant Escherichia coli and identify the presence of extended-spectrum beta-lactamase (ESBL) genes in ESBL-producing E. coli among a population of wild banteng (Bos javanicus) and domestic cattle kept on farms located close to the Lam Pao non-hunting area, Kalasin province, Thailand. Forty-five fecal samples were taken from wild bantengs inhabiting the Lam Pao non-hunting area in Thailand, alongside 15 samples from domestic cattle. Bacterial culture, triple sugar iron, and motile indole lysine tests were conducted to identify E. coli. A polymerase chain reaction (PCR) was conducted for specific confirmation. MacConkey agar supplemented with 2 μg/mL of CTX was used to identify CTX-resistant E. coli, which would be used to identify ESBL production based on a double-disk synergy test. Extended-spectrum beta-lactamase-producing samples were subjected to disk diffusion tests to determine resistant patterns, and the sizes of PCR bands and DNA sequencing were used to differentiate ESBL gene types. All samples tested positive for E. coli. Forty-five isolates from 15 banteng samples and three isolates from one domestic cattle sample displayed CTX-resistant and ESBL-producing traits. The banteng and domestic cattle populations exhibited nine and three distinct resistant patterns, respectively. The PCR results indicated that the banteng isolates harbored the following genes: Cefotaxime-M1 (n = 38), CTX-M9 (n = 5), and the SHV group (n = 2). All three isolates from the domestic cattle sample contained the CTX-M1 gene. Classification of ESBL genes based on the DNA sequences of the banteng isolates showed the characteristics of CTX-M15 (n = 20), CTX-M55 (n = 6), CTX-M14 (n = 5), and CTX-M79 (n = 1). The three domestic cattle isolates exhibited the characteristics of CTX-M15, CTX-M55, and CTX-M79. Despite no previous antibiotic applications, approximately one-third of the banteng samples displayed CTX resistance, indicating ARM contamination within the ecosystem. The similarity in ESBL genes between the banteng and domestic cattle populations suggests potential gene transmissions between these animal groups. However, the initial source of ARMs remains unclear, as the banteng population exhibited more ESBL genes than the domestic cattle, suggesting the possibility of multiple ARM sources. These findings raise concerns because the banteng population inhabits an area that is an important source of freshwater and nourishes the entire north-east region of Thailand and other South-east Asian countries, including Laos, Cambodia, and Southern Vietnam.

Evaluation of the Performance of a Multiplex Real-Time PCR Assay for the Identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii Simultaneously from Sputum in Multicenter.

PurposeTo evaluate the performance of a multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii from sputum.Patients and MethodsSputum samples (n=537) from patients with suspected invasive fungal infection (IFI) were collected from four centers; they were tested by both multiplex real-time PCR assay and DNA sequencing. DNA sequencing was considered as the reference method, and the performance of the multiplex real-time assay was evaluated by determining the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The interference experiment, repeatability, reproducibility, and stability of the multiplex real-time PCR assay were also evaluated.ResultsThe detection performance of the multiplex real-time assay, compared with that of DNA sequencing, for the three pathogens was as follows: sensitivity, specificity, PPV, and NPV for Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii were 99.40%, 98.64%, 97.09%, and 99.73%; 100%, 99.59%, 96.36%, and 100.00%; and 99.28%, 98.50%, 95.80%, and 99.75%, respectively. The consistency of the two methods was almost perfect: the kappa value was between 0.97 and 0.98. The minimum detection limit of the multiplex real-time assay for each of the three pathogens was 1250 copies/mL. Interference experiment showed that blood and normally used antifungal drugs had no effect on the results. No cross-reactivity was detected for any bacteria or fungi. In 40 patients, mixed infections by Aspergillus and/or Cryptococcus neoformans and/or Pneumocystis jirovecii were detected by the multiplex real-time assay. Among these patients, those with acquired immune deficiency syndrome (AIDS) ranked first, with Aspergillus and Pneumocystis mixed infection accounting for 75%.ConclusionThe multiplex real-time PCR assay is fast, sensitive, and specific and has good clinical application prospects.

Characterization of Sequence Types and Mechanisms of Resistance to Tigecycline Among Acinetobacter baumannii Isolated from Children.

The present study aimed to investigate the mechanisms of resistance to tigecycline and to determine sequence types of Acinetobacter baumannii isolates recovered from children, using the Multilocus Sequence Typing (MLST). A total of 74 A. baumannii isolates were recovered from patients at one of the children's hospital in Tehran, Iran. Antimicrobial susceptibility testing of the isolates was performed for different classes of antibiotics and minimum inhibitory concentrations of colistin and tigecycline were determined using broth microdilution method and E-test strips, respectively. The presence of ISAba1, AbaR, tet(39), and tetX and the expressions of adeB, adeG, and adeJ efflux pump genes were measured using Polymerase Chain Reaction (PCR) and quantitative real-time PCR (RT-PCR), respectively. The diversity of mutations across the regulatory genes of RND efflux pumps (adeRS, adeL, and adeN) and trm gene were determined using their PCR amplification and DNA sequencing in tigecycline-resistant isolates. In addition, STs of tigecycline-resistant isolates were determined using MLST method. Three A. baumannii isolates were resistant to tigecycline. Several amino acid substitutions were identified in AdeRS, AdeN, and Trm but no alteration was found in AdeL. Nevertheless, adeB, adeG, and adeJ overexpression were observed in 1, 2, and 1 isolates, respectively. The tigecycline-resistant isolates belonged to ST1720 and ST2285. This is the first study reporting on ST2285 in A. baumannii populations. Among 74 isolates, two tigecycline susceptible isolates carried tet(39) gene but no tetX gene was detected. We concluded that mutations in regulatory genes of RND efflux pumps and the trm gene may play some important role in A. baumannii resistance to tigecycline.

Long-read Amplicon Sequencing of the CYP21A2 in 48 Thai Patients With Steroid 21-Hydroxylase Deficiency

Congenital adrenal hyperplasia is most commonly caused by 21-hydroxylase deficiency (21-OHD), an autosomal recessive disorder resulting from biallelic pathogenic variants (PVs) in CYP21A2. With a highly homologous pseudogene and various types of single nucleotide and complex structural variants, identification of PVs in CYP21A2 has been challenging. To leverage long-read next-generation sequencing combined with locus-specific polymerase chain reaction (PCR) to detect PVs in CYP21A2 and to determine its diagnostic yield in patients with 21-OHD. Forty-eight Thai patients with 21-OHD comprising 38 sporadic cases and 5 pairs of siblings were enrolled. Two previously described locus-specific PCR methods were performed. Amplicons were subject to long-read sequencing. Ninety-six PVs in CYP21A2 in the 48 patients were successfully identified. The combined techniques were able to detect 26 structural chimeric variants (27%; 26/96) in 22 patients with 18 having monoallelic and 4 having biallelic chimeras. The remaining PVs were pseudogene-derived mutations (63%; 60/96), entire gene deletions (2%; 2/96), missense variants (3%; 3/96), a splice-site variant (2%; 2/96), frameshift variants (2%; 2/96), and a nonsense variant (1%; 1/96). Notably, a splice-site variant, IVS7 + 1G > T, which was identified in a pair of siblings, has not previously been reported. Our approach exploiting locus-specific PCR and long-read DNA sequencing has a 100% diagnostic yield for our cohort of 48 patients with 21-OHD.

Human serum albumin variants in China: a molecular epidemiological investigation and literature review.

BackgroundBisalbuminemia is a hereditary and/or acquired abnormality characterized by a double albumin (ALB) band on serum protein electrophoresis. However, there have been no epidemiological investigations of ALB variants in Chinese populations.MethodsThis retrospective study examined 71,963 unrelated subjects from five provinces in southern China. ALB variants were screened by cellulose acetate electrophoresis at pH 8.6 and ALB mutations were confirmed by polymerase chain reaction-DNA sequencing.ResultsThe average incidence of inherited bisalbuminemia in the southern Chinese population was 0.0264% (19/71,963). Thirteen cases showed slow and six showed fast genetic variants on cellulose acetate electrophoresis. Four kinds of ALB variants were identified: proalbumin Lille (p.Arg23His), ALB Castel di Sangro (p.Lys560Glu), ALB Fukuoka-1 (p.Asp587Asn), and a novel ALB Wuxi (p.Lys562Glu). The gene frequency of ALB variants in the Wuxi region (0.126%, 13/10,297) was significantly higher than in other regions in southern China, and 90.9% (10/11) of cases of proalbumin Lille were also found in the Wuxi region.ConclusionsThis study provides the first report of the detailed prevalence and molecular characterization of ALB variants in southern China. Compared with other areas of China, Wuxi had a different pattern of ALB variants and a high prevalence of proalbumin Lille.

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii.

Cronobacter sakazakii is a foodborne pathogen that can cause a rare, septicemia, life-threatening meningitis, and necrotizing enterocolitis in infants. In general, standard methods for pathogen detection rely on culture, plating, colony counting and polymerase chain reaction DNA-sequencing for identification, which are time, equipment and skill demanding. Recently, nanoparticle- and surface-based immunoassays have increasingly been explored for pathogen detection. We investigate the functionalization of gold nanoparticles optimized for irreversible and specific binding to C. sakazakii and their use for spectroscopic detection of the pathogen. We demonstrate how 40-nm gold nanoparticles grafted with a poly(ethylene glycol) brush and functionalized with polyclonal antibodies raised against C. sakazakii can be used to specifically target C. sakazakii. The strong extinction peak of the Au nanoparticle plasmon polariton resonance in the optical range is used as a label for detection of the pathogens. Individual binding of the nanoparticles to the C. sakazakii surface is also verified by transmission electron microscopy. We show that a high degree of surface functionalization with anti-C. sakazakii optimizes the detection and leads to a detection limit as low as 10 CFU/mL within 2 h using a simple cuvette-based UV-Vis spectrometric readout that has great potential for further optimization.

Construction of srtA-deletion mutant of Streptococcus mutans by an in-frame deletion system

To construct srtA-gene deletion mutant of Streptococcus mutans (S. mutans) UA159 with IFDC2 cassette through overlapping polymerase chain reaction (PCR) and allelic homologous recombination. First, the upstream and downstream fragments surrounding the srtA and IFDC2 cassette were PCR amplified and ligated through overlapping PCR. The resulting amplicon was transformed into UA159, and positive transformants were selected on BHI plates containing erythromycin. Second, upstream and downstream fragments of srtA with overlap regions were generated by PCR and were overlapped to create upΔ-down amplicon. Then, the upΔ-down amplicon was transformed into the aforementioned positive transformants and selected on BHI plates containing p-Cl-Phe. The PCR analysis and DNA sequencing results indicated that the coding region of the srtA was completely deleted, and the upstream and downstream regions flanking the srtA were ligated seamlessly. The markerless srtA-deletion mutant of S. mutans was constructed successfully, which laid a foundation for further study of its biological function and influence on the biofilm formation of S. mutans.

Investigation of serotype distribution and resistance genes profile in group B Streptococcus isolated from pregnant women: a Chinese multicenter cohort study.

We surveyed the group B Streptococcus (GBS) strains isolated from four teaching hospitals during 1-year period to investigate the current serotypes and antimicrobial resistance status of these strains. A total of 231 non-duplicate colonizing GBS isolates were collected from pregnant women. Antimicrobial susceptibility of these isolates was tested by the disk diffusion method. Serotype was performed by a multiplex polymerase chain reaction (PCR) method. Analysis of the resistance mechanisms was performed by PCR amplification and DNA sequencing. Seven serotypes (Ia, Ib, II, III, V, VI, and VIII) were identified, and the prevalence ranged from 0.9 to 35.9%. All isolates were susceptible to the penicillin, ceftriaxone, and vancomycin. The resistance of all the isolates to erythromycin, clindamycin, and levofloxacin was 61.5, 51.9, and 35.5%, respectively. The erythromycin resistance was mainly associated with the genes ermB and ermB-mef(A/E) (69.8%). The most predominant phenotype was cMLSB (77.5%). Five gene panels, including gyrA, parC, parE, gyrA-parC, and gyrA-parC-parE, were detected. The most predominant genotype was gyrA-parC-parE triple mutation (69.5%). The S81L in gyrA gene, S79Y mutation in parC gene, and H225Y mutation in parE gene were discovered. The isolates with serotype III, V, and Ia were the most important clone concerning the prevalence and resistance.

Molecular Identification of Q Fever in Patients with a Suspected Diagnosis of Dengue in Brazil in 2013–2014

Q fever is an important cause of undifferentiated fever that is rarely recognized or reported in Brazil. The objective of this study was to look for the presence of Coxiella burnetii during a dengue fever outbreak in the municipality of Itaboraí, Rio de Janeiro, Brazil, where this bacterium had previously infected humans and domesticated animals. Blood samples from clinically suspected dengue fever patients were tested by polymerase chain reaction (PCR) for C. burnetii; the DNA was detected in nine (3.3%) of 272 patients. One was coinfected with dengue virus, which was also detected in another 166 (61.3%) patients. The nucleotide sequence of PCR amplification and DNA sequencing of the IS1111 transposase elements in the genome of C. burnetii exhibited 99% identity with the sequence in GenBank. The detection of C. burnetii in patients suspected of dengue fever indicates that awareness and knowledge of Q fever should be strengthened and that this bacterium is present in Brazil. Finally, because a negative molecular result does not completely rule out the diagnosis of Q fever and the serological assay based on seroconversion was not available, the actual number of this zoonosis is likely to be much higher than that reported in this study.

Rapid identification of apolipoprotein E genotypes by high-resolution melting analysis in Chinese Han and African Fang populations.

Apolipoprotein E (APOE) gene polymorphism can affect APOE gene transcription, serum lipid levels and repair of tissue damage, which could place individuals at serious risk of cardiovascular disease or certain infectious diseases. Recently, high-resolution melting (HRM) analysis was reported to be a simple, inexpensive, accurate and sensitive method for the genotyping or/and scanning of rare mutations. For this reason, an HRM analysis was used in the present study for APOE genotyping in the Southern Chinese Han and African Fang populations. A total of 100 healthy Southern Chinese Han and 175 healthy African Fang individuals attended the study. Polymerase chain reaction-DNA sequencing was used as a reference method for the genotyping of these samples. The six APOE genotypes could all be rapidly and efficiently identified by HRM analysis, and 100% concordance was found between the HRM analysis and the reference method. The allele frequencies of APOE in the Southern Chinese Han population were 7.0, 87.5 and 5.5% for ɛ2, ɛ3 and ɛ4, respectively. In the African Fang population, the allele frequencies of APOE were 24.3, 65.7 and 10.0% for ɛ2, ɛ3 and ɛ4, respectively. A statistically significant difference was found between the allele frequencies between the populations (P<0.05). In conclusion, the present study revealed the molecular characterization of APOE gene polymorphism in the Han population from the Chaozhou region of Southern China and the Fang population from Equatorial Guinea. The findings of the study indicated that HRM analysis could be used as an accurate and sensitive method for the rapid screening and identification of APOE genotypes in prospective clinical and population genetic analyses.

Molecular detection of Neospora caninum from naturally infected dogs in Lorestan province, West of Iran

Neospora caninum is a coccidian protozoan that causes abortion in dairy and beef cattle and neurological disorders in dogs and horses. To identify N. caninum oocysts in the dog feces the molecular approaches are known as sensitive methods that specifically detect the oocysts. In present study, a polymerase chain reaction (PCR) targeting N. caninum specific Nc5 genomic fragment was performed to identify the N. caninum DNA in the feces of naturally infected dogs of Lorestan province, West Iran. Fecal samples of dogs living in small dairy farms were collected. The samples were homogenized in 2.5% Potassium dichromate (K2Cr2O7) and stored at 4 °C. Genomic DNA was extracted from the feces using CTAB protocol. PCR assay and DNA sequencing were performed with specific primers. DNA amplification of the Nc5 formed a 340bp fragment for the N. caninum specimens; however, the fragment was 99% identical to the homologous sequences from Neospora caninum isolates. Totally, 9 positive samples of N. caninum were detected by PCR from 428 fecal specimens.

Bartonella quintana in Cimex hemipterus, Rwanda

The common bed bug species Cimex lectularius, which has a worldwide distribution, and C. hemipterus, which has tropical climate distribution, are hematophagous arthropods.1 The bugs are attracted to the human host by warmth and carbon dioxide, and bed bug infestations are rapidly increasing worldwide. Moreover, bed bugs are likely to be more problematic in the future because of travel, immigration, and insecticide resistance. Reactions to bites vary, and some patients present only asymptomatic macules, whereas others develop indurated, bulbous lesions that resemble erythema multiforme.2 Although no case of bed bug-borne infection has been reported yet, bed bug infestations can have an adverse effect on health and quality of life in the general population, particularly among homeless persons living in shelters.1,3 The transmission of more than 45 human diseases has been attributed to bed bugs; however, there is little evidence that they are vectors of communicable disease.1 The objective of this study was to analyze with molecular assays a large number of bed bugs from Rwanda, where epidemiological and clinical studies on zoonosis are scarce. Bed bugs were collected from two prisons in Rwanda (Miyove and Muhanga prisons) (Figure 1) from May to January of 2011 and taken to a local laboratory. Bed bugs were preserved in sterile conditions at room temperature and then sent to our reference center in Marseille to test for lice-borne infections. Before DNA isolation, each bug was rinsed two times in sterile water for 15 minutes. Overall, 100 bed bugs were tested. DNA was extracted from each bug using a QIAamp tissue kit (Qiagen, Hilden, Germany) and then used as template in a real-time polymerase chain reaction (PCR) assay targeting a portion of the Bartonella 16S–23S intergenic spacer region (ITS) as described previously.4 Two specific B. quintana genes (fabF3 and yopP) encode for 3-oxoacyl-[acyl-carrier-protein] synthase and a hypothetical intracellular effector, respectively.5 All three sets were positive by real-time PCR for one bed bug. Amplicons were then purified using the QIAquick Spin PCR Purification Kit (Qiagen, Courtaboeuf, France) and sequenced on an ABI 3100 Automated Sequencer (Perking Elmer, Courtaboeuf, France) using the dRhodamine Terminator Cycle-Sequencing Ready Reaction Kit (PE Applied Biosystems, Les Ulis, France) according to the manufacturer's instructions. Sequences obtained after sequencing of the ITS gene shared 100% similarity to the corresponding ITS fragment of the genome of B. quintana (Toulouse strain; GenBank accession number {type:entrez-nucleotide,attrs:{text:BX897700,term_id:49239191,term_text:BX897700}}BX897700). B. quintana-positive bugs were further studied by PCR amplification and DNA sequencing of the mitochondrial (16S DNA) gene as previous described.6 Sequences obtained shared 99.7% similarity to the corresponding 16S DNA fragment of the genome of C. hemipterus (GenBank accession number {type:entrez-nucleotide,attrs:{text:GU985560.1,term_id:294999274,term_text:GU985560.1}}GU985560.1). Moreover, B. quintana-positive bugs were triturated in brain–heart medium before inoculation onto Columbia 5% sheep blood agar plates (BioMerieux, Marcy l'Etoile, France). Plates were placed in polyethylene bags and incubated at 37°C in 5% CO2 (Genbag CO2 system; BioMerieux). The agar plates were examined weekly. After 1 month, no evidence of growth was observed. Figure 1. The two areas in Rwanda where the bed bugs were collected. In this study, we identified the presence of B. quintana in C. hemipterus collected from a prison in Rwanda. B. quintana and Rickettsia prowazekii have been previous identified in body lice collected from a similar jail in Rwanda,7 and recently, B. quintana was found in head and body lice in Ethiopia.8 To avoid the potential of misinterpretation of the PCR data caused by laboratory contamination, three different genes were assessed, and all of them were successfully detected. However, we were not able to cultivate B. quintana from this bug. This result suggests that living microorganisms may be dead before testing or that only DNA but no living organisms were present in the bug. C. hemipterus might be contaminated when they feed on the blood of prisoners infected with B. quintana. Unfortunately, a human blood sample was not collected at the time of sampling the bugs. There are few data to support bed bugs as vectors for transmission of human disease agents, and older studies consist of only logical and not evidence-based postulates.9 Recently, Delaunay and others1 found no published evidence for completion of all the necessary steps leading to the conclusion that bed bugs transmit a pathogen. It is possible that the bed bug might only play the role of vector in pathogen transmission, and consequently, it may be involved in human disease in special circumstances not yet discovered.1 Nonetheless, the role of the bed bugs in the maintenance and transmission of B. quintana remains to be determined, and it is currently being tested in our laboratory.

Oral Candida carriage among individuals chewing betel-quid with and without tobacco

The aim was to assess oral Candida carriage among individuals chewing betel-quid (BQ) with and without tobacco. A retrospective and comparative study of oral Candida carriage among individuals chewing BQ with and without tobacco. Oral yeast samples were collected from 103 BQ-chewers (52 chewing BQ with tobacco and 51 chewing BQ without tobacco) and 100 non-chewers. Candida strains were cultured in Sabouraud dextrose agar and identified using the API 32-C System and polymerase chain reaction-DNA sequencing. Tongue lesions were clinically identified and numbers of missing teeth were recorded. Unstimulated whole salivary flow rate was recorded. Oral Candida species were isolated from 72.7% BQ-chewers (73.1% in individuals chewing BQ with tobacco and 72.4% in individuals chewing BQ without tobacco) and 61% non-chewers. Chewing BQ (with or without tobacco) does not influence oral Candida carriage.

Development of Sequence Based Molecular Diagnostic Test to Evaluate MDR and XDR in &lt;i&gt;M.&lt;/i&gt; &lt;i&gt;t&lt;/i&gt;&lt;i&gt;uberculosis&lt;/i&gt; Patients from Western India

Globally, Tuberculosis is the most distress disease which remains as a major challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. The increasing knowledge in molecular biology techniques has improved our understanding of the molecular mechanisms of drug resistance for the major first and second-line anti- tubercular drugs. Thus, rapid and accurate diagnosis of Tuberculosis would improve patient care and limit its transmission. This study is aimed to identify mutations in drug resistance genes of first and second line drugs against Mycobacterium tuberculosis by using polymerase chain reaction (PCR) and DNA sequencing methods. This study involves sputum samples of 20 patients diagnosed to have pulmonary tuberculosis. The genomic DNA from clinical isolates was isolated using simple boiling method. PCR amplification and DNA sequencing were performed for katG, inhA, ndh, kasA, pncA, embB, rrs, rpsL, rpoB, gyrA, gyrB and thyA genes. Sequence analysis was done using Bioedit, a bioinformatics tool. The isolates of Mycobacterium tuberculosis from western part of India were analyzed by DNA sequencing revealed total 29 mutations in first line drugs. Out of them, 5(25%) in rpoB at codon 516 and 531, 6(30%) in katG at codon 315, 8(40%) in rpsL at codon 43 & 88, 3(15%) in embB at codon 306 and 7(35%) in pncA at codon 195.No mutations in inhA, ndh, kasA and rrs genes were observed in our study. In the second line drugs, 2(10%) at codon 90 & 100% mutations at codon 95 in gyrA gene, and no mutation in gyrB and thyA genes were observed.

Rare case of post-cataract-surgery Prevotella endophthalmitis diagnosed by polymerase chain reaction DNA sequencing

We report the presentation, diagnosis, and management of endophthalmitis caused by the opportunistic Prevotella species. The case was referred to us following uneventful phacoemulsification and intraocular lens implantation. Accurate identification of this rare cause of endophthalmitis was made using bacterial polymerase chain reaction DNA sequencing. Subsequent prompt modification of antibacterial therapy allowed resolution of signs and symptoms and significant visual recovery. To our knowledge, this is the first reported case of diagnosis and management of post-cataract-surgery endophthalmitis caused by the opportunistic Prevotella species.

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

Clinical and molecular genetic study of 12 Italian families with autosomal recessive Stargardt disease

Stargardt disease was diagnosed in 12 patients from 12 families using complete ophthalmologic examination, fundus photography, fundus autofluorescence, and spectral-domain optical coherence tomography. DNA was extracted for polymerase chain reaction (PCR) and direct DNA sequencing (ABCA4 gene). Genetic counseling and eye examination were offered to 16 additional family members. Various patterns of presentation were observed in patients with clinical diagnoses of Stargardt disease. The genetic study identified 2 mutations in 75% of families (9/12); a second mutation could not be found in the remaining 25% of families (3/12). The most frequent mutation was G1961E, found in 17% of families (2/12). This finding is similar to that of a previous analysis report of an Italian patient series. Four new mutations were also identified: Tyr1858Asp, Leu1195fsX1196, p.Tyr850Cys, and p.Thr959Ala. Our results suggest that PCR and direct DNA sequencing are the most appropriate techniques for the analysis of the ABCA4 gene. However, this method requires supplementation with specific PCR analysis to diagnose large deletions. The study of the families identified healthy carriers and affected subjects in presymptomatic stages and was also useful for evaluating the risk of transmission to progeny. Combined ophthalmologic and genetic evaluation enabled better clinical management of these families.

Viral load, genomic integration frequency of human papillomavirus 16 in cervical cancer and precancerous lesions

To investigate the association between viral load, genomic integration frequency of HPV 16 and cervical carcinogenesis and assess the probability that HPV 16 integration frequency may be utilized as a marker for cervical cancer. Forty cases of HPV16 single infection were selected from 337 cervical scrape samples by PCR (polymerase chain reaction) amplification and DNA sequencing. The copy numbers of E2, E6 and β-actin were quantified to evaluate the viral load and integration status by real-time PCR. (1) The median number of adjusted viral load of normal group, LSIL (low-grade squamous intraepithelial lesion) group, HSIL (high-grade squamous intraepithelial lesion) group and squamous cervical cancer group were 0.11, 18.55, 44.63 and 7.6 copies per cell respectively. The viral load was higher in LSIL-HSIL group than that in normal group while lower in squamous cervical cancer group than that in HSIL group. (2) The median number of E2/E6 was 0.93 in normal group, 0.84 in precancerous group (LSIL-HSIL) and 0.64 in squamous cervical cancer group respectively. It showed a descending trend with the progression of cervical disease. (3) With the disease development, the proportion of episomal form decreased (normal group 4/5, LSIL group 4/6, HSIL group 10/16, cervical squamous cancer group 5/13) whereas that of integrated form (mixed and totally integrated) increased (normal group 1/5, LSIL group 2/6, HSIL group 6/16, cervical squamous cancer group 8/13). Both totally integrated cases were cervical squamous cancer. (1) HPV 16 viral load may not be an ideal marker to predict cervical carcinogenesis. (2) Due to a positive correlation between HPV16 genomic integration frequency and cervical neoplastic progression, HPV 16 integration frequency may be a potential marker of early diagnosis for cervical lesion progression.

Molecular Detection of Autosomal-Dominant Feline Polycystic Kidney Disease by Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction

Feline autosomal-dominant polycystic kidney disease (ADPKD), with its characteristic growth of fluid-filled cysts of different sizes, is the most prevalent inherited genetic disease of cats. The point mutation (C-->A transversion) in exon 29 of the PKD1 gene is known to contribute to ADPKD development and can thus serve as a target for the molecular genetic diagnosis of ADPKD. To this end, a simple amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) was designed with 3 primers: 2 forward primers specifically targeting either the mutant or normal allele, and 1 universal reverse primer for amplification of both alleles. The new method was tested on the DNA from 35 feline blood samples, which included 15 mutant cats and 20 wild type cats. As verified by direct DNA sequencing, both sensitivity and specificity of this tri-primer ARMS PCR were 100%. As the multiplex ARMS PCR test can be performed in a single PCR reaction without other post-PCR procedures, it is a simple and accurate method for molecular studies of feline ADPKD.

Diagnosis and phylogenetic analysis of Orf virus from goats in China: a case report

BackgroundOrf virus (ORFV) is the etiological agent of contagious pustular dermatitis and is the prototype of the genus Parapoxvirus (PPV). It causes a severe exanthematous dermatitis that afflicts domestic and wild small ruminants.Case presentationIn the present study, an outbreak of proliferative dermatitis in farmed goats. The presence of ORFV in tissue scrapings from the lips was confirmed by B2L gene polymerase chain reaction (PCR) amplification. The molecular characterization of the ORFV was performed using PCR amplification, DNA sequencing and phylogenetic analysis of the B2L gene.ConclusionThe results of this investigation indicated that the outbreak was caused by infection with an ORFV that was closely related genetically to Nantou (DQ934351), which was isolated from the Tai wan province of China and Hoping (EU935106), which originated from South Korea in 2008. This is the first report of the phylogenetic analysis of ORFV from goats in China.