Evaluating a semi-nested PCR to support histopathology reports of fungal rhinosinusitis in formalin-fixed paraffin-embedded tissue samples.

BACKGROUND: Fungal rhinosinusitis (FRS) encompasses a various spectrum of diseases. Histopathology is the "reference method" for diagnosing FRS, but it cannot determine the genus and species. Moreover, in more than 50% of the histopathologically proven cases, the culture elicited no reliable results. This study was an attempt to evaluate the diagnostic efficiency of semi-nested polymerase chain reaction (PCR) from formalin-fixed paraffin-embedded (FFPE) functional endoscopic sinus surgery (FESS) in FRS patients. METHODS: One hundred ten specimens were subjected to DNA extraction and histopathology examination. The amplification of the ß-globin gene by conventional PCR was used to confirm the quality of extracted DNA. The semi-nested PCR was performed using ITS1, ITS2, and ITS4 primers during two steps. Sequencing the internal transcribed spacer region (ITS1-5.8S-ITS2) to identify causative agents was performed on PCR products. RESULTS: Sixty-four out of 110 samples were positive by histopathology evidence, of which 56 samples (87.5%) were positive by PCR. Out of 46 negative samples by histopathological methods, five samples (10.9%) yielded positive results by PCR. Sensitivity, specificity, positive predictive value, and negative predictive value of the semi-nested PCR method were reported 87.5%, 89.2%, 92.7%, and 85.2%, respectively. The kappa factor between PCR and histopathological methods was 0.76, indicating substantial agreements between these two tests. CONCLUSION: Due to the acceptable sensitivity and specificity of the present method, it might be used to diagnose fungal sinusitis infections along with microscopic techniques. This method is recommended to confirm the diagnose of suspected fungal sinusitis with negative histopathology results.

Accompanying a semi-nested PCR assay to support histopathology findings of fungal keratitis in formalin-fixed paraffin-embedded corneal samples.

BACKGROUND: Fungal species are responsible for 40%-50% of all microbial keratitis cases. Due to the low amount of extracted DNA in ocular Formalin-fixed Paraffin-embedded (FFPE) samples, selecting a reliable molecular method is a substantial issue in this field. METHODS: Sixty-six samples were collected via the penetrating keratoplasty (PK) technique. Histopathology assays were performed using hematoxylin-eosin (H&E) and periodic acid Schiff (PAS) staining methods. The ITS1/ITS4 and ITS1/ITS2 primer pairs were used in a semi-nested polymerase chain reaction (PCR) to target the universal internal transcribed spacer (ITS) region. Some PCR results were validated through sequencing. RESULTS: Fungal DNA was detected in 44 of 66 samples (66.7%), and histopathology was positive for 41 of 66 samples (62.1%). Of 41 histopathologically proven fungal-positive cases, 39 were PCR-positive (95%). Moreover, of 44 PCR-positive samples, 39 (88.6%) were histopathology-positive, and 5 (11.3%) were histopathology-negative. Totally in 39 cases (59%), both histopathology and PCR yielded positive results. The Kappa agreement rate between the two diagnostic methods, including histopathology and PCR, was 0.77. Sensitivity, specificity, positive predictive value, and false predictive value were reported as 88.64%, 90.9%, 95.12%, and 80%, respectively. CONCLUSION: As we reached the acceptable Kappa agreement rate, we concluded that applying the semi-nested PCR assay is a promising method for supporting the evidence by histopathology. Finally, we suggest targeting more specific gene regions using primer pairs that amplify smaller amplicon sizes and surveying novel molecular methods such as NGS to achieve higher sensitivity and Kappa agreement rates.

Molecular characterization of non-polio enteroviruses isolated from acute flaccid paralysis patients in Uganda.

Enteroviruses (EVs) are RNA viruses that can cause many clinical syndromes including acute flaccid paralysis (AFP). Within the global polio laboratory network, EVs are categorized either as polioviruses or non-polio enteroviruses (NPEVs). Specific NPEVs have been described in polio-like residual paralytic events in AFP patients. Retrospective analysis of 112 NPEV isolates from AFP patients was performed and thirty one NPEV types were identified of which 91% were Enterovirus B and 9% were Enterovirus A species. The NPEVs were distributed across the country with most patients in the eastern region (41/89; 46.1%). The highest proportion of patients were children less than 5 years (77/89; 86.5%) and male patients were more common (54/89; 60.7%). Echovirus 11 (11/89; 12.4%) was frequently observed and phylogenetic analysis of these sequences revealed high diversity. Coxsackievirus B5 (CV-B5), CV-B6, E21, and EV-B69 were only seen in patients with residual paralysis. Analyses of the EV-A71 sequence indicated a unique genogroup.

ABL1 tyrosine kinase domain mutations in chronic myeloid leukemia treatment resistance.

The development of mutations in the BCR-ABL1 fusion gene transcript causes resistance to tyrosine kinase inhibitors (TKIs) based therapy in chronic myeloid leukemia (CML). Thereby, screening for BCR-ABL1 mutations is advised especially in patients undergoing poor response to treatment. In the current study the authors investigated 43 patients with CML that failed or had suboptimal response to TKIs treatment. Blood samples were collected from patients that were treated with TKIs. The analysis of genetic mutations was performed using a semi-nested PCR assay, followed by Sanger sequencing. The analysis revealed 15 mutations (32.55%): 14 point mutations and an exon 7 deletion. In roughly 30% of cases, mutations in the BCR-ABL1 fusion gene are common causes for treatment resistance.

PCR detection of Ehrlichia ruminantium and Babesia bigemina in cattle from Kwara State, Nigeria: unexpected absence of infection.

Ticks and tick-borne diseases (TBDs) continue to pose an insidious and ever-present threat to livestock and livelihoods across the globe. Two of the most significant TBDs of cattle in Africa are heartwater and babesioisis, caused by Ehrlichia ruminantium and Babesia bigemina respectively. Both pathogens are endemic in Nigeria. However, to date, little data has been published regarding the number of cattle infected. In this study, blood samples were collected from cattle of the Kwara State, north-central Nigeria. Probe-based quantitative PCR (qPCR) and semi-nested PCR were used to investigate the presence of both pathogens, respectively. Our study found all samples (n = 157) to be surprisingly negative for both B. bigemina and E. ruminantium. These results contribute new information on the current burden of these two pathogens in Kwara State and may be helpful in informing more effective targeting of control strategies in Nigeria.

An Alternative Method for Extracting Plasmodium DNA from EDTA Whole Blood for Malaria Diagnosis.

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris-EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/µl for P. falciparum and 35.2 parasites/µl for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/µl for P. falciparum and 1.4 parasites/µl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

Human Rhinoviruses A9, A49, B14 and Echovirus 3, 9 among the patients with acute respiratory infection.

  Background: Acute respiratory infection result in high mortality and morbidity worldwide. There are several viral factors that originate respiratory diseases among them Enteroviruses(EVs) and Human Rhinoviruses(HRVs) can be mentioned. HRVs and EVs belong to Picornaviridae family and they have been recently classified under Enteroviruses. The pattern of respiratory infections generating organisms varies according to geographical locations. Therefore, it seems necessary to organize an appropriate plan to manage common viral diseases exclusively about Rhinoviruses and Enteroviruses. PATIENT AND METHODS: A total of 100 samples were collected from patients with acute respiratory infections (ARIs) who were hospitalized in Ahvaz city hospitals during December 2012 to November 2013 (one year longitude). Semi-Nested PCR was done on samples for detection of HRVs and EVs using region gene of VP4/VP2. Phylogenetic and molecular evolutionary analyses performed with MEGA version 5 software find out the sequence homology among the detected HRV and EV serotype. RESULTS: The results of this study revealed that from of 100 cases of ARIs 19 patients (19%) were HRV positive and 3 (3%) patients positive for EVs. Most positive cases of HRVs were observed in the autumn season while 3 positive cases of EVs were equally found in spring, summer and autumn. Phylogenetic analyses showed that the HRV strains were HRV-A9, HRV-A49, HRV-B14 and EV strains were Echo3 and 9. CONCLUSION: The results of this study revealed that high prevalence of 19% HRVs, HRV-A9, HRV-A49, HRV-B14 serotypes and low frequency of 3% Echo Viruses, Echo3 and Echo 9 serotypes have been detected in patients with ARI.

Molecular diagnosis of Acanthamoeba keratitis: evaluation in rat model and application in suspected human cases.

Acanthamoeba keratitis (AK) is a progressive corneal infection that demands rapid and sensitive techniques for diagnosis to avoid risk of visual impairment. We evaluated two DNA extraction techniques and a semi-nested-PCR (snPCR) targeting the 18S rRNA gene to detect Acanthamoeba cysts and trophozoites. The most effective protocol was evaluated in samples of corneal scrapings and biopsies from an AK rat model and applied to diagnosis of human cases of AK. DNA extraction performed with a commercial kit based on DNA binding to magnetic beads was more efficient than a method based on alkaline lysis, allowing the detection of one trophozoite and one cyst of Acanthamoeba in samples prepared from cultures. This technique and sn-PCR were applied in corneal scrapings of rats experimentally infected with Acanthamoeba (n = 6), resulting in 100% of positivity, against 16.7% (n = 6) of positive identification in culture method using non-nutrient agar (NNA) with Escherichia coli. Corneal biopsies from rats were also tested (n = 6) and resulted in positivity in all samples in both molecular and culture methods. Eight out of ten presumptive human cases of Acanthamoeba keratitis were also confirmed by sn-PCR of scrapping samples, while the culture method was positive in only four cases. We discuss that animal model of AK can be an efficient tool to validate diagnostic methods and conclude that DNA extraction with the kit and snPCR protocol described here is an effective alternative for diagnosis of AK.

Molecular identification and phylogenetic analysis of Wuchereria bancrofti from human blood samples in Egypt.

Lymphatic filariasis (LF) is a serious vector-borne health problem, and Wuchereria bancrofti (W.b) is the major cause of LF worldwide and is focally endemic in Egypt. Identification of filarial infection using traditional morphologic and immunological criteria can be difficult and lead to misdiagnosis. The aim of the present study was molecular detection of W.b in residents in endemic areas in Egypt, sequence variance analysis, and phylogenetic analysis of W.b DNA. Collected blood samples from residents in filariasis endemic areas in five governorates were subjected to semi-nested PCR targeting repeated DNA sequence, for detection of W.b DNA. PCR products were sequenced; subsequently, a phylogenetic analysis of the obtained sequences was performed. Out of 300 blood samples, W.b DNA was identified in 48 (16%). Sequencing analysis confirmed PCR results identifying only W.b species. Sequence alignment and phylogenetic analysis indicated genetically distinct clusters of W.b among the study population. Study results demonstrated that the semi-nested PCR proved to be an effective diagnostic tool for accurate and rapid detection of W.b infections in nano-epidemics and is applicable for samples collected in the daytime as well as the night time. PCR products sequencing and phylogenitic analysis revealed three different nucleotide sequences variants. Further genetic studies of W.b in Egypt and other endemic areas are needed to distinguish related strains and the various ecological as well as drug effects exerted on them to support W.b elimination.

Molecular diagnosis of Toxoplasma gondii infection in Libya.

BACKGROUND: Toxoplasma gondii infections are prevalent in humans and animals throughout Libya. Current diagnosis is based on detection of Toxoplasma-specific IgM and IgG. In this study, we established and optimized a diagnostic PCR assay for molecular diagnosis of T. gondii in Libya. METHODS: From January to December, 2010, 177 blood and serum samples were collected from suspected patients. This includes: 140 women who have had spontaneous abortions, 26 HIV-positive patients, nine patients with leukemia and lymphoma, and two infants with ocular infection. Samples were screened for anti-Toxoplasma IgG and IgM antibodies before DNA extraction. The surface antigen gene 2 (SAG2) was targeted in a semi-nested PCR to amplify a 999 bp and a 614 bp fragment in the first and the second run respectively. RESULTS: A total of 54/140 (38.5 %) women who have had spontaneous abortions, 23/26 (88 %) HIV patients, 6/9 (66.6 %) of the leukaemia and lymphoma patients, and one child with ocular infection were seropositive for anti-Toxoplasma IgG and/or IgM. Genomic DNA was extracted from 38 selected seropositive samples. The PCR was sensitive enough to detect DNA concentration of 12 ng/µL. PCR analysis was performed for 38 selected seropositive patients (16 women who have had spontaneous abortions, 15 positive HIV patients, six leukaemia patients and one child with ocular infection). Our designed primers were successfully amplified in 22/38 (57.9 %) samples; 5/12 (35.7 %) from serum and 17/26 (65.8 %) from whole blood samples. All PCR positive samples were IgG-positive except two samples which were IgM and IgG & IgM-positive serum samples respectively. The semi-nested PCR confirmed five more samples. These included two leukaemia and two HIV-positive whole blood samples and one serum sample from an aborted woman. CONCLUSION: The ability of PCR to diagnose active toxoplasmosis is needed in immunocompromised patients and congenital toxoplasmosis cases, especially when serological techniques fail. For the first time in Libya, we established and optimized semi-nested PCR of SAG2 gene. The developed PCR method was able to detect as little as 12 ng/µL of T. gondii DNA and was useful to diagnose the diseases in women who have had spontaneous abortions, HIV-positive patients, patients with leukemia and lymphoma, and infants with ocular infection.

Novel Semi-Nested Real-Time PCR Assay Leveraging Extendable Blocking Probes for Improved SHOX2 Methylation Analysis in Lung Cancer.

Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. SHOX2 gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting SHOX2 methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated SHOX2 DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated SHOX2 DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting SHOX2 methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings.

Hepatitis B virus reverse transcriptase mutations in treatment Naïve chronic hepatitis B patients.

Mutations in the reverse transcriptase (RT) region of the hepatitis B virus (HBV) genome lead to decreased susceptibility to nucleos(t)ide analogs approved for treatment of HBV infection. The aim of this study was to detect and analyze pre-existing HBV RT mutations in treatment naïve patients with chronic hepatitis B. Seventy one chronic HBV treatment naïve patients were enrolled from January 2009 to June 2011. HBV RT sequence analysis was done by using direct bidirectional sequencing of semi-nested PCR products. HBV genotypes were determined by multiplex PCR. Genotype D was found in 64 patients (90.1%) followed by genotype C and A which were present in 5 (7.0%) and 2 (2.8%) patients respectively. The results of the RT sequence analysis showed mutations in 34 (47.9%) patients. The rtH248N mutation was the most common mutation, accounting for 47.1% patients. Other common mutations included rtD263E/S, rtM129L, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D, which were present in 26.5% (9/34), 29.4% (10/34), 20.6% (7/34), 20.6% (7/34), 20.6% (7/34), 17.6% (6/34), 14.7% (5/34), 14.7% (5/34), 11.8% (4/34), 11.8% (4/34) and 11.8% (4/34) patients respectively. The known primary drug resistance mutations were found in 3 (8.8%) patients. The present study shows the presence of RT amino acid substitutions in treatment-naïve patients with chronic hepatitis B, which may decrease susceptibility to available oral antiviral drugs. On the basis of the finding of this study, genotypic testing is recommended before the start of therapy in naïve patients, so that suitable antiviral drugs can be prescribed.

Development and evaluation of a semi-nested PCR technique for amplification and determination of different surface gene variation patterns in patients with chronic HBV infection: development of an amplification method for HBV S gene.

Aim: Development of an amplification method for further investigation of HBV S gene variation patterns. Background: Pre-S/S variants in patients with chronic HBV infection may contribute to the progression of liver damage and Hepatocellular carcinoma (HCC). Methods: This study was performed on ten patients with chronic HBV infection. Viral DNA was extracted from patient's plasma, primer design was performed, and a semi-nested PCR method was set up to amplify the pre-S/S region of HBV genome. Subsequently, sequencing was performed to analyze the variants of this region. Results: In the current study, the semi-nested PCR method was successfully set up, and types of variation in the studied samples were investigated. Conclusion: Pre-S/S variants should be routinely determined in HBV carriers to help identify individuals who may be at a high risk of less favorable liver disease progression. This study showed that the technique could accurately amplify the pre-S/S region, and the product can be successfully used for variation detection by direct sequencing.

The impact of increasing non-albicans Candida trends on diagnostics in immunocompromised patients.

Invasive candidiasis (IC) represents a growing concern worldwide, with a considerable increase in non-albicans Candida (NAC) species. The study's primary goal was to determine if species identification by semi-nested PCR (sn-PCR) with primers for the five most prevalent Candida species is sufficient to deal with the current trends of Candida infections in cancer patients. Over one year, Candida isolates were collected from samples of patients with hematological and solid organ tumors in a single center. Species of Candida were identified by chromagar and multiplex sn-PCR using specific primers for Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, and the Candida parapsilosis complex. Most Candida infection episodes are caused by NAC species (70.5% of 105 isolates). Rare species (14 isolates) accounted for 13.3% of isolates and were not identified by sn-PCR using the five most common Candida species primers. More than half of these rare species caused candidemia in cancer patients (57.1%; p = 0.011). The risk factor for candidiasis was recent surgeries (p = 0.020) in adults and chemotherapy in pediatric patients (p = 0.006). Prolonged hospitalization and genitourinary tract cancer were significantly associated with invasive infections (p = 0.005 and 0.049, respectively). Recent surgery was a significant risk factor associated with C. parapsilosis and C. glabrata infections (P = 0.038 and 0.003, respectively), while C. tropicalis was significantly more common in patients with hematological malignancies (P = 0.012). Techniques with a broader identification spectrum than the major five Candida species are crucial for the optimal management of cancer patients.

Molecular Evaluation of t(14;18)(bcl-2/IgH) Translocation in Follicular Lymphoma at Diagnosis Using Paraffin-Embedded Tissue Sections.

OBJECTIVE: Follicular lymphoma (FL) is one of the most common lymphomas, and is characterized by t(14;18)(q32;q21) in more than 80% of patients. The aim of this study was to determine the rate of t(14;18) positivity based onthe detection of mbr or mcr in paraffin-embedded tissue samples. MATERIAL AND METHODS: The study included 32 paraffin-embedded tissue samples collected from 32 consecutive FL patients that were diagnosed and followed-up at our hospital between 1999 and 2006. The MBR breakpoint wasidentified based on real-time PCR using a LightCycler v.2.0 t(14;18) Quantification Kit (MBR), multiplex PCR, and seminestedPCR. To identify the mcr breakpoint, real-time PCR was performed using specific primers and the FastStart DNAMaster SYBR Green I Kit. To detect t(14;18) via fluorescence in situ hybridization (FISH) nuclei from paraffin-embeddedtissue sections were extracted and used together with LSI IgH (immunoglobulin heavy chain) (spectrum green)/bcl-2(B-cell leukemia-lymphoma 2) (spectrum orange) probes. RESULTS: The DNA and nuclei isolation success rate for B5 formalin-fixed, paraffin-embedded tissue sections (n = 12)was 42% and 33%, respectively, versus 95% and 60%, respectively, for 20 tissue sections fixed in formalin only. In all,24 paraffin-embedded tissue sections were analyzed and mbr positivity was observed in the DNA of 82.14% via seminested PCR, in 53.57% via multiplex PCR, and in 28.57% via real-time PCR. We did not detect mcr rearrangementin any of the samples. In all, 15 of 16 patients (93.75%) whose nuclei were successfully isolated were observed to bet(14;18) positive via the FISH method. CONCLUSION: Semi-nested PCR and FISH facilitated the genetic characterization of FL tumors. As such, FISH and PCR complement each other and are both essential for detecting t(14;18) translocation.

Successful treatment of severe form of bovine tropical theileriosis in dairy cattle and genotyping of Theileria annulata isolates of Tamil Nadu, India.

Bovine tropical theileriosis (BTT) is a tick-borne protozoan disease of cattle and responsible for major economic losses to the dairy farmers in India. This report describes diagnosis, genotyping and successful treatment of heavy infection of Theileria annulata in an organized dairy farm at Kattupakkam, Chennai. Four cross bred cows of 2 to 5 years of age showed clinical signs i.e., anorexia, salivation and panting. Clinical examination revealed pyrexia (40.0 °C to 40.1 °C), pale mucus membranes, enlarged prescapular lymph nodes and haemoglobinuria. The peripheral blood smear examination of infected cows revealed presence of piroplasm within the RBCs indicating high parasitemia. Haematology results suggested that decreased levels of Hb, RBC, WBC and PCV in the infected cows when compared with normal reference values. There were increased serum ALT and AST values and reduced serum total protein, albumin, calcium and phosphorous values in the infected cows. Semi-nested PCR using T. annulata specific oligonucleotide primers amplified 199 bp of the partial T. annulata 18S rRNA gene. Presence of four satellite markers TS6, TS8, TS9, and TS12 in the Theileria annulata isolates 1 and 2 indicating that the isolates were the same haplotype and suggested the infection in the farm was due to a single haplotype of T. annulata parasite. Based on the clinical signs, microscopic examination of blood smear and molecular diagnosis, the condition was diagnosed as tropical theileriosis. Infected cows were successfully treated with a single deep intramuscular injection of buparvaquone (Zubion®, INTAS pharmaceuticals LTD, Ahmedabad, India) along with supportive medication.

Molecular Detection of Gurltia paralysans by Semi-Nested PCR in Cerebrospinal Fluid and Serum Samples from Domestic Cats (Felis catus).

Gurltia paralysans is an angio-neurotropic metastrongyloid nematode that infects domestic and wild cats, invading the veins of the subarachnoid space of the spinal cord and mainly causing progressive paralysis of the pelvic limbs. The definitive diagnosis of feline gurltiosis can only be achieved by post-mortem examination that reveals the presence of the nematode in the spinal cord vein vasculature. An early diagnosis with conclusive results is required since laboratory and imaging findings are not sufficient. Therefore, the purpose of this study was to detect the presence of G. paralysans, via semi-nested PCR, in samples of cerebrospinal fluid (CSF) and the sera of domestic cats naturally infected with the parasite. A total of 12 cats with a diagnosis suggestive of feline gurltiosis were selected, and they underwent a complete neurological and imaging examination. DNA samples were analysed by semi-nested PCR, with universal (AaGp28Sa1/AaGp28Ss1) and specific (Gp28Sa3/Aa28Ss2) primers, for G. paralysans (G. paralysans 18S rRNA gene, partial sequence; ITS 1, 5.8S rRNA gene, and ITS 2, complete sequence; and 28S rRNA gene, partial sequence) and Aelurostrongylus abstrusus, obtaining amplifications of 356 and 300 bp, which indicated the presence or absence of nematode DNA, respectively. The presence of G. paralysans was detected in the CSF of four out of nine cats, and the sera of seven out of seven cats. In the sera analysis of five out of seven cats, a mixed infection with A. abstrusus was found, despite no alterations of the respiratory tract being observed during the necropsies. It is proposed that serum samples could be more effective than CSF in detecting the parasite by PCR analysis. Sequencing analysis showed high percentages of identity with G. paralysans, which indicated the feasibility of detection and the sensitivity/specificity of the method used, suggesting the implementation of semi-nested PCR as a routine diagnostic test for early and timely detection of feline gurltiosis.

Molecular Diagnosis of Mycoplasma Species Infection in Camels Using Semi-nested PCR.

BACKGROUND AND OBJECTIVE: Bacteriological isolation and identification of Mycoplasma species is difficult and time-consuming, therefore, molecular identification of Mycoplasma using PCR targeting specific genes is considered a specific and sensitive method for identification. The aim of current study was to isolate, characterize Mycoplasma infection in dromedary camels in Saudi Arabia. MATERIALS AND METHODS: Nasal swabs were randomly collected from 93 camels and tested for Mycoplasma by sequencing of their 16S rRNA genes using universal primers. RESULTS: The 93 samples, 24 were positive for Mycoplasma. However, no positive results were obtained using species-specific primers for Mycoplasma arginine, M. bovis or M. mycoides subsp. mycoides, thus, 16S rDNA sequencing methods and semi-nested PCR were employed. Sequences were matched to those in GenBank and phylogenetic analysis was performed. Mycoplasma edwardii (77-84% similarity with Mycoplasma edwardii ATCC 23462) and one isolate of Mycoplasma yeastsii (100% similarity with M. yeastsii GM274B) were identified. Further, some Mycoplasma species were identified as previously uncultured. The incidence of Mycoplasma infection in camels in Taif city, Saudi Arabia, was approximately 26%. CONCLUSION: This study provides insights into the accuracy and efficiency of PCR and universal primers for the detection and identification of Mycoplasma, thereby circumventing conventional culturing methods that require several days to complete and exhibit low accuracy.

Screening for BKV-DNAEMIA after renal transplantation in a resource limited setting.

Costs may hinder the implementation of BK polyomavirus (BKV)-DNAemia screening in resource-limited kidney transplant (KT) centers. We analyzed data from two studies to assess the performance and potential cost saving of a dual-step screening strategy based on the use of a preliminary qualitative semi-nested PCR (snPCR) assay followed by BKV-DNAemia quantification after KT. In the preliminary study, in which 130 samples from 33 KT recipients were screened for BKV-DNAemia, the estimated positive and negative predictive values of snPCR, as compared to quantitative PCR (qPCR), were 88% and 99%, respectively. In the second study, which included 84 KT recipients, BKV-DNAemia was detected by snPCR in 28/472 (5.9%) samples and confirmed by qPCR in 26 samples of 21 (25%) subjects. No graft loss occurred among KT recipients who developed BKV-DNAemia. Cost analyses suggested that this strategy might be a cost saving alternative for BKV-DNAemia screening for some resource-limited settings.

Identification of novel microsatellite markers flanking GJB2 gene in order to use in preimplantation genetic diagnosis of hearing loss: A comparison of whole-genome amplification and semi-nested PCR.

Hearing loss is the most prevalent sensorineural disorder which can be caused by genetic factors in more than half of the cases. GJB2 mutations with the frequency of 18.7% are the most common cause of autosomal recessive non-syndromic hearing loss (ARNSHL) in the Iranian population. The aim of the current study was to genotype 100 healthy individuals for eight microsatellite markers flanking the GJB2 gene, and to study markers on ten blastomeres using semi-nested PCR and Whole-genome amplification (WGA). All microsatellite markers within 1 Mb flanking the GJB2 gene were identified. From the identified markers, four with potentially high heterozygosity values were selected. The heterozygosity indices of four newly discovered markers and four previously reported markers were calculated. The markers and the GJB2 gene were also validated on single lymphocytes and blastomeres. Totally, 77 alleles were observed in eight loci. D13S046 showed the highest polymorphism and D13S141 showed the lowest. The observed heterozygosities of all markers, except D13S141, were higher than 50%. All single cells were genotyped successfully by the two techniques. Our findings indicate a high degree of polymorphism of the selected markers. Due to the high rate of successful amplification of markers in all ten blastomeres and the low level of allelic drop out (ADO), a combination of these eight microsatellite markers in conjunction with direct mutation detection is suggested for performing preimplantation genetic diagnosis (PGD) of hearing loss due to GJB2 mutations.