Specific Nested Polymerase Chain Reaction Research Articles

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis.

In recent years, the “milky disease” caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/μL) and internal transcribed spacer (ITS; 6.74 × 105 copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

Diagnostics of Pulmonary Aspergillosis in Critically Ill Hematological and Non-Hematological Patients in the Intensive Care Unit By Combined Use of Galactomannan, 1-3-Beta-D-Glucan, Aspergillus Specific PCR and Conventional Culture in Concurrent Blood and Bronchoalveolar Lavage Samples — Results of a Multicenter Prospective Pilot Study

Invasive pulmonary aspergillosis (IPA) is a life-threatening infectious complication mainly affecting immunocompromised hematological patients. Therapeutic success requires early and precise diagnostics. Few data are available on the performance of current diagnostic tests in patients admitted to the intensive care unit (ICU) as the majority of diagnostic studies is conducted in hematological patients and results cannot readily be transferred to patients in the ICU setting.In a prospective multicenter study, we evaluated concurrent bronchoalveolar lavage (BAL) samples and concurrent blood samples from 44 patients admitted to the ICU who required mechanical ventilation and therefore underwent a diagnostic work-up including chest CT scan and BAL (NCT01695499). The subsequent diagnostic work-up comprised of conventional methods for direct detection of Aspergillus spp. as well as biomarkers such as galactomannan (GM) and 1-3-beta-D-glucan (BDG) and an Aspergillus specific nested polymerase chain reaction (PCR).14 patients (32%) suffered from a hematological/hematooncological disease, of which 7 had received allogeneic stem cell transplantation. 6 patients (14%) had a solid tumor requiring chemotherapy. The remaining 24 patients (54%) had no hematological or oncological disease. 11 patients were neutropenic with ANC <1.0/nl (10 belonged to the hematooncological group). According to AspICU classification (Blot et al., Am J Respir Crit Care Med 2012) 9 patients (20%) had putative IPA (3 patients with hematological malignancy). Other invasive fungal diseases (IFD) besides IPA were observed: positive growth of Rhizopus microsporus in BAL was seen in one patient. Furthermore, two cases of disseminated candidiasis with positive growth in blood culture (C.albicans, C.krusei) and one case with positive growth in ascites (C.albicans). Additionally, two cases of Pneumocystis jirovecii pneumonia (PcP) were diagnosed.Diagnostic performance of each single test and of test combinations was analyzed. GM and PCR showed superior performance in BAL (sensitivity/specificity of 56%/94% (GM) and 44%/94% (PCR) compared to 33%/97% and 11%/94% in serum). Despite a better sensitivity of 89%, BDG showed a specificity of only 31% in BAL and 26% in serum, reducing the diagnostic odds ratio (DOR) to 3.7 and 2.8, respectively. For hematological patients the mean GM optical density index (ODI) in BAL was 0.97 and in serum 0.58 compared to 0.59 and 0.16 in non-hematological patients. The strength of agreement between GM in BAL and GM in serum as calculated by Cohen's kappa intertest coherence analysis was 0.62 [0.29-0.95], which is considered to be good. BDG levels and performance of the PCR assay did not differ between hematological and non-hematological patients. The combined use of biomarkers in BAL showed a diagnostic performance of sensitivity/specificity/PPV/NPV and DOR of 89%/31%/25%/92% and 3.67. Testing of GM and BDG solely in serum resulted in a combined test performance of 89%/26%/ 24%/ 90% and 2.77. GM (BAL) combined with BDG (serum) showed high joint sensitivity of 100% with specificity of 25% (DOR 6.81). Using PCR instead of GM dropped sensitivity to 89% (specificity 23%, DOR 2.37). Diagnostic performance of combined GM and PCR testing in BAL showed sensitivity/specificity/PPV/NPV and DOR 67%/89%/60%/91% and 15.0. Cohen's kappa for PCR (BAL) vs. GM (BAL) was 0.49 [0.145 to 0.840], which is considered to be moderate. If a diagnostic algorithm defines BDG (serum) as an initial test that is, in case of a positive result, followed by a confirming test combination from BAL with strong or even exclusive specificity for Aspergillus spp. (GM, PCR and culture) a diagnostic performance is achieved with sensitivity/specificity/positive predictive value/negative predictive value of 88%/97%/88%/97% and DOR >100.This study intended as a pilot study underscores the concept of combined testing in serum and BAL for optimized detection of IPA and provides valuable data for the emerging cohort of critically ill patients, both hematological and non-hematological. Further studies with hematological ICU patients in larger cohorts are planned. DisclosuresBoch:Pfizer research grant (WS 2274964): Research Funding. Buchheidt:Pfizer research grant (WS 2274964): Research Funding.

Identification of tick-borne pathogens by metagenomic next-generation sequencing in Dermacentor nuttalli and Ixodes persulcatus in Inner Mongolia, China

BackgroundHard ticks act as arthropod vectors in the transmission of human and animal pathogens and are widely distributed in northern China. The aim of this study is to screen the important tick-borne pathogens (TBPs) carried by hard ticks in Inner Mongolia using metagenomic next-generation sequencing (mNGS) and to estimate the risk of human infection imposed by tick bites.MethodsThe adult Dermacentor nuttalli (n = 203) and Ixodes persulcatus (n = 36) ticks feeding on cattle were collected. The pooled DNA samples prepared from these ticks were sequenced as the templates for mNGS to survey the presence of TBPs at the genus level. Individual tick DNA samples were detected by genus--specific or group-specific nested polymerase chain reaction (PCR) of these TBPs and combined with DNA sequencing assay to confirm the results of mNGS.ResultsR. raoultii (45.32%, 92/203), Candidatus R. tarasevichiae (5.42%, 11/203), Anaplasma sp. Mongolia (26.60%, 54/203), Coxiella-like endosymbiont (CLE) (53.69%, 109/203), and Babesia venatorum (7.88%, 16/203) were detected in D. nuttalli, while R. raoultii (30.56%, 11/36), Anaplasma sp. Mongolia (27.80%, 10/36), and CLE (27.80%, 10/36) were detected in I. persulcatus. The double- and triple-pathogen/endosymbiont co-infections were detected in 40.39% of D. nuttalli and 13.89% of I. persulcatus, respectively. The dual co-infection with R. raoultii and CLE (14.29%, 29/203) and triple co-infection with R. raoultii, Anaplasma sp. Mongolia, and CLE (13.79%, 28/203) were most frequent in D. nuttalli.ConclusionsThis study provides insight into the microbial diversity of D. nuttalli and I. persulcatus in Inner Mongolia, China, reporting for the first time that Candidatus R. tarasevichiae had been found in D. nuttalli in China, and for the first time in the world that Anaplasma sp. Mongolia has been detected in I. persulcatus. This study proves that various vertically transmitted pathogens co-inhabit D. nuttalli and I. persulcatus, and indicates that cattle in Inner Mongolia are exposed to several TBPs.Graphical

Epidemiological study on Anaplasma phagocytophilum in cattle: Molecular prevalence and risk factors assessment in different ecological zones in Iran

Anaplasma phagocytophilum is a tick-borne pathogen affecting humans and domestic animals worldwide. This study aimed to determine the molecular epidemiology and its associated risk factors of A. phagocytophilum infection in cattle in four ecological zones of Iran. A multi-stage stratified random sampling method was utilized during 2017−2018. A total of 1851 blood samples from 320 cattle farms were collected and examined using specific nested polymerase chain reaction (nPCR) based on the 16S rRNA gene. The overall prevalence of A. phagocytophilum was 15.5% (286/1851) by using nPCR. All four zones were A. phagocytophilum positive, the presence of A. phagocytophilum DNA was detected in eight out of nine tested provinces. Univariable analysis of risk factors indicated that climate, altitude, longitude, latitude, season, farm-type, feeding method, hygiene of the farm, tick infestation, use of acaricides by the farmer, distance from other farms, contact with wild animals, race, sex, and milk yield were significant determinants (P < 0.05) for A. phagocytophilum infection. The multivariable analysis determined that longitude, latitude, season, feeding method, and hygiene of the farm remained as significant risk factors for A. phagocytophilum infection (P < 0.05). Specific (SaTScan) cluster analysis identified two high risks and four low risks statistically significant clusters for A. phagocytophilum infection amongst the study areas (P < 0.001). Phylogenetic analysis indicated that A. phagocytophilum 16S rRNA isolates were 96–99% identical to sequences deposited in the GenBank. To the best of our knowledge, this is the first comprehensive molecular study on the epidemiology and risk factors analysis of A. phagocytophilum infection in cattle in different climatic zones of Iran. Further investigations are necessary to be performed regarding the tick vectors, reservoir animals, and the zoonotic potential of the A. phagocytophilum in the endemic region of Iran.

Iberian Lynx Lynx pardinus Temminck, 1827 (Mammalia: Carnivora: Felidae) in central Spain: trophic niche of an isolated population

Understanding predator-prey relationships is fundamental to develop effective conservation plans. Between 2015 and 2018, we combed 21 transects, each 7km long, searching for Iberian Lynx Lynx pardinus scat within the province of Madrid in central Spain. In order to minimise inherent subjectivity of visual identification as much as possible, we performed a double specific nested polymerase chain reaction (PCR) followed by a primer extension assay addressed to two Iberian Lynx diagnostic single nucleotide polymorphisms. Forty-six scat samples were positively identified as belonging to Iberian Lynx through genetic analysis. From these, we extracted remains of consumed prey, which we determined to the lowest possible taxonomic level, mainly through hair identification. Identified prey was divided into four types: lagomorphs, small mammals, birds, and ungulates. The species’ diet composition was described based on the frequency of occurrence (FO) of each prey and niche breadth, and also compared with prior knowledge of the species using four prior studies as a comparative reference through the calculation of the niche overlap value. The FO of lagomorphs (39%) was the lowest, while the FO of small mammals (54%) was the highest recorded to date. The niche breath (0.36) was higher than recorded in prior studies, but still showing the specialist character of the Iberian Lynx. Niche overlap was low (C = 0.49), showing differences in trophic niche between the population in our study area and the one studied in southern Spain. This indicates that the Iberian Lynx is adept at switching its main prey, an ability that has previously been firmly rejected. It is, however, capable of adapting to alternative prey more often than recorded to date, which could be a behavioural response to the patchy distribution of European Rabbit Oryctolagus cuniculus in the study area.

Aspergillus specific nested PCR from the site of infection is superior to testing concurrent blood samples in immunocompromised patients with suspected invasive aspergillosis.

Invasive aspergillosis (IA) is a severe complication in immunocompromised patients. Early diagnosis is crucial to decrease its high mortality, yet the diagnostic gold standard (histopathology and culture) is time-consuming and cannot offer early confirmation of IA. Detection of IA by polymerase chain reaction (PCR) shows promising potential. Various studies have analysed its diagnostic performance in different clinical settings, especially addressing optimal specimen selection. However, direct comparison of different types of specimens in individual patients though essential, is rarely reported. We systematically assessed the diagnostic performance of an Aspergillus-specific nested PCR by investigating specimens from the site of infection and comparing it with concurrent blood samples in individual patients (pts) with IA. In a retrospective multicenter analysis PCR was performed on clinical specimens (n=138) of immunocompromised high-risk pts (n=133) from the site of infection together with concurrent blood samples. 38 pts were classified as proven/probable, 67 as possible and 28 as no IA according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions. A considerably superior performance of PCR from the site of infection was observed particularly in pts during antifungal prophylaxis (AFP)/antifungal therapy (AFT). Besides a specificity of 85%, sensitivity varied markedly in BAL (64%), CSF (100%), tissue samples (67%) as opposed to concurrent blood samples (8%). Our results further emphasise the need for investigating clinical samples from the site of infection in case of suspected IA to further establish or rule out the diagnosis.

Molecular Epidemiology and Risk Factors Assessment of Anaplasma spp. on Dairy Cattle in Southwest of Iran

DOI : 10.26650/actavet.2019.18014 The present study was carried out to determine Anaplasma species and potential risk factors associated with molecular prevalence of Anaplasma spp. among dairy cattle in southwest of Iran. A total of 88 samples out of 200 generated an expected amplicon of 866 bp from Anaplasma marginale msp4 gene. Six samples that were identified as A. marginale gave also positive results for A. phagocytophilum 16S rRNA gene with specific nested polymerase chain reaction (nPCR). The multivariate analysis of risk factors revealed that the cattle of mountain regions were significantly (p=0.0001) at higher risk as compared to the plain regions. Cattle <1 year age and the latitude 32-33°C were significantly at lower risk (p<0.01). The cattle with low milk yield were significantly (p=0.002) at lower risk. Low hygienic farms were significantly (p=0.011) at higher risk as compared to good and normal hygienic farms. Distance from other farms (<1Km) was another important risk factor which showed significant association with the occurrence of Anaplasma infection (p=0.021). The results of this study can be used in strategic planning for prevention and control of bovine anaplasmosis in dairy cattle in the southwest of Iran.

FIRST MOLECULAR DETECTION OF ANAPLASMA PHAGOCYTOPHILUM IN DROMEDARIES ( CAMELUS DROMEDARIUS).

Anaplasma phagocytophilum infects a wide variety of wild and domestic animals and causes an emerging zoonotic tick-borne disease. There are no available data regarding the presence of A. phagocytophilum in camels ( Camelus dromedarius). Therefore, the objective of this study was to investigate the prevalence of A. pagocytophilum in Iranian camels. Whole blood of 207 camels from five geographical regions of Iran was tested for A. phagocytophilum using polymerase chain reaction (PCR), nested PCR, and specific nested PCR based on 16S rRNA. The overall prevalence of infection in tested animals was 34.2% (71/207). Sex was not identified as a risk factor for A. phagocytophilum infection, but analysis revealed significant differences in age and region. In conclusion, Iranian camels can be potential reservoirs for A. phagocytophilum, and Iran must be considered an enzootic area for this infection as indicated by the high subclinical infection rate in camels.

Hepatitis B virus (HBV) genome detection and genotyping in virally suppressed patients using nested polymerase chain reaction-based Sanger sequencing

Hepatitis B virus (HBV) genotypes have important clinical implications. Current genotyping methods are less sensitive in patients with ultra-low HBV viral load. We report a highly sensitive and specific nested polymerase chain reaction (PCR) assay for genotyping patient HBV. Total DNA derived from plasma of 14 (HBsAg+ and/or HBsAg-) HBcAb+ patients was used for HBV-specific nested PCRs targeting the preC/C, X/BCP/preC, and surface regions. All patients were treated with long-term nucleos(t)ide analogues (NAs), and 12/14 have undetectable viremia (clinical PCR: sensitivity >10 IU/mL). Surface amplicons were sequenced, aligned with reference genomes, and used in phylogenetic tree construction to determine genotype. HBV DNA was detected in 14/14, including 3 occult (HBsAg-/HBcAb+) cases. Genotypes identified were 6/14 B, 6/14 C, and 2/14 D. This assay in virologically suppressed patients may be useful for future studies requiring genotype prior to assessment of immunomodulatory and/or direct acting anti-viral therapeutics in patients on potent NAs.

Molecular investigation of Lawsonia intracellularis in diarrheic and healthy captive ostriches (Struthio camelus) in Iran

Lawsonia intracellularis is the causative agent of proliferative enteropathy (PE) in pigs, a disease of economic importance worldwide. The present study aimed to investigate the occurrence of L. intracellularis in ostriches using a sensitive and specific nested polymerase chain reaction (nested PCR). A total number of 112 fecal samples (64 healthy, 48 diarrheic) were collected from 11 ostrich farms located in four provinces in Iran (Semnan, Tehran, Gilan, Yazd). The results showed the presence of L. intracellularis in three diarrheic and four apparently healthy birds. The frequency of positive results was the same in both groups (6.25% healthy vs. 6.25% diarrheic). Although the results of this study did not reveal any relationship between this organism and diarrhea (P > 0.05), the molecular detection of L. intracellularis on a farm where birds were suffering from the typical clinical symptoms of PE highlights the need for evaluation of PE in ratites.

Detection of invasive pulmonary aspergillosis in critically ill patients by combined use of conventional culture, galactomannan, 1-3-beta-D-glucan and Aspergillus specific nested polymerase chain reaction in a prospective pilot study

Invasive pulmonary aspergillosis (IPA) is an emerging and life-threatening infectious disease in patients admitted to the intensive care unit (ICU). Most diagnostic studies are conducted in hematological patients and results cannot readily be transferred to ICU patients lacking classical host factors.In a multicenter, prospective clinical trial including 44 ICU patients, hematological (n = 14) and non-hematological patients (n = 30), concurrent serum and bronchoalveolar lavage (BAL) samples were analyzed by conventional culture, galactomannan (GM), 1-3-beta-D-glucan (BDG) as well as an Aspergillus specific nested polymerase chain reaction (PCR). Nine patients (20%) had putative IPA according to AspICU classification.GM and PCR showed superior performance in BAL with sensitivity/specificity of 56%/94% and 44%/94% compared to 33%/97% and 11%/94% in serum. Despite better sensitivity of 89%, BDG showed poor specificity of only 31% (BAL) and 26% (serum). Combination of GM and PCR (BAL) with BDG (serum) resulted in 100% sensitivity, but also reduced specificity to 23%. Whereas mean GM levels were significantly higher in hematological patients BDG and PCR did not differ between hematological and non-hematological patients.Under present clinical conditions test combinations integrating both BAL and blood samples are advantageous. BDG might best serve as possible indicator for ruling out IPA. Trial registrationClinicalTrials.gov Identifier: NCT01695499. First posted: September 28, 2012, last update posted: May 8, 2017.

Long-Distance Spread of Verticillium dahliae Through Rivers and Irrigation Systems.

Verticillium dahliae Kleb. is a soilborne pathogen causing Verticillium wilt disease on several hosts. The pathogen survival structure (i.e., microsclerotia) can be efficiently spread by different dispersal methods. In the present study, the medium to long dispersal spread of the pathogen through rivers and irrigation canals was investigated. Samples of sediments (n = 29) were gathered from eight Lebanese rivers and three regional irrigation canals, in addition to samples of soil particles and plant residues (n = 14) from irrigation filters in commercial orchards. Specific conventional and real-time nested polymerase chain reaction assays detected the pathogen in six rivers-Al Kabir, Al Bared, Litani, Al Awali, Ostwan, and Litani South-and in all sampled canals-Ostwan, Al Bared, and Litani Canal 900. Starting DNA quantities ranged from 0.2 pg to 21.318 ng and an inoculum density, determined by a traditional plating method, varied between nondetectable and 0.2 microsclerotia/g. Viable V. dahliae microsclerotia were also found in residues collected from mesh-type irrigation filters of five commercial orchards. This study confirms that water is an important inoculum source of V. dahliae, being involved in the efficient spread of microsclerotia in Lebanese agricultural areas.

Nested polymerase chain reaction targeting 16S rRNA gene in diagnosis of acute bacterial meningitis

CONTEXT: Diagnosing acute bacterial meningitis (ABM) among children presenting to tertiary health care settings is often difficult because of prior administration of antimicrobials. AIMS: The present study was an attempt to diagnose cases of ABM in children with the help of nested polymerase chain reaction (PCR). SETTINGS AND DESIGN: It is a prospective observational study, in which a total of 84 clinically suspected cases and cerbrospinal fluid (CSF) biochemical parameters suggestive of ABM were included in the study. METHODS AND MATERIAL: CSF samples were subjected to Gram staining, bacterial culture and biochemical identification tests as well as panbacterial nested PCR targeting 16S rRNA gene sequence. Subsequently, a nested multiplex PCR for detection of the three fastidious organisms, viz. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae, was performed. STATISTICAL ANALYSIS USED: The sensitivity, specificity, PPV, NPV, LR+, and LR− of the tests were calculated. RESULTS: The sensitivity of Gram stain, bacterial culture, and nested PCR targeting 16SrRNA were observed to be 16.21%, 9.45%, and 97.29% respectively. Further, the NPV and LR− the PCR were found to be 83.33 and 0.02 respectively. Species specific nested multiplex PCR was able to detect S. pneumoniae (n = 7), N. meningitidis (n = 2) and H. influenzae (n = 1). CONCLUSIONS: The results indicates that nested PCR targeting 16S rRNA gene may be used in diagnosis of ABM. Further, nested multiplex PCR targeting the three important fastidious bacterial pathogens in ABM cases has showed their presence in our region.

Viruses in the environment - presence and diversity of bacteriophage and enteric virus populations in the Umhlangane River, Durban, South Africa.

Due to the continued persistence of waterborne viral-associated infections, the presence of enteric viruses is a concern. Notwithstanding the health implications, viral diversity and abundance is an indicator of water quality declination in the environment. The aim of this study was to evaluate the presence of viruses (bacteriophage and enteric viruses) in a highly polluted, anthropogenic-influenced river system over a 6-month period at five sampling points. Cytopathic-based tissue culture assays revealed that the isolated viruses were infectious when tested on Hep-G2, HEK293 and Vero cells. While transmission electron microscopy (TEM) revealed that the majority of the viruses were bacteriophages, a number of presumptive enteric virus families were visualized, some of which include Picornaviridae, Adenoviridae, Polyomaviridae and Reoviridae. Finally, primer specific nested polymerase chain reaction (nested-PCR)/reverse transcription-polymerase chain reaction (RT-PCR) coupled with BLAST analysis identified human adenovirus, polyomavirus and hepatitis A and C virus genomes in river water samples. Taken together, the complexity of both bacteriophage and enteric virus populations in the river has potential health implications. Finally, a systematic integrated risk assessment and management plan to identify and minimize sources of faecal contamination is the most effective way of ensuring water safety and should be established in all future guidelines.

Development of nested polymerase chain reaction-based diagnosis of duck enteritis virus and detection of DNA polymerase gene from non-descriptive duck breeds of West Bengal, India.

Aim:The study was undertaken to detect the clinical signs, postmortem lesions of embryonated duck plague (DP) infected eggs, and histopathological changes of chorioallantoic membrane (CAM) in non-descriptive ducks of West Bengal with special reference to standardize nested polymerase chain reaction (PCR).Materials and Methods:After postmortem of suspected carcasses, samples were collected for virus isolation and identification through specific pathogen free (Khaki Campbell) embryonated duck eggs. PCR was also done as confirmatory test after doing postmortem of duck embryos. DP specific nested PCR was standardized for better confirmation of the disease. Sensitivity of nested primers was also tested for DP virus.Results:Gross, postmortem and histopathological changes were prominent in dead embryos. First set of primer was able to detect 602 bp fragments of DNA polymerase gene of duck enteritis virus from infected CAM. Subsequently, a DP specific nested PCR which was very much sensitive for very small amount of viral genome was successfully standardized. After NCBI blast nucleotide sequence of nested PCR product (Accession No. HG425076) showed homology with the sequences data available in GenBank.Conclusion:The study concludes that PCR assay is very much helpful to diagnose DP disease and developed nested PCR is a double confirmatory diagnostic tool for DP.

HBV Genotyping and Phylogenetic Analysis through Multiplex PCR Using Type Specific Primer Pairs in Pakistani Population

Background: Hepatitis B virus causing acute and chronic hepatocellular carcinoma relics a serious health issue affecting more than 350 million people worldwide. Geographically, nine genotypes of HBV have been identified so far. Four open reading frames named (ORF P, S, F and X) renders it to specifically targets hepatocyte cells. Additionally, reverse transcription process present in the life cycle of HBV caused four order higher mutation rates as compared to adenovirus. The structural and functional differences between HBV genotypes are the mainstay to severity, complications, treatment and possibly vaccination against the virus. Hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, but the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis and medical therapies. In Pakistan, there is no reported data available on molecular evolutionary analysis of HBV. A study was, therefore, much needed to evaluate the spectra of dominant strains prevalent here. Objectives: This study was aimed to figure out the prevalence of HBV genotypes among population in Pakistan. In this research, we focused to find out the new strains and sub genotypes of HBV. Pakistan is one of the HBV endemic countries but there is no data on subgenotypes and their recombination or the phylogenetic relatedness of the virus endemic in the country. The present study was carried out in this perspective. This study will help future researcher to study some other aspects of HBV related strains in Pakistan and also about its medical therapies. Place and duration of study: The present study was carried out at Polymerase Chain Reaction (PCR) lab, Nuclear Medicine, Oncology and Radiotherapy Institute [NORI], Islamabad, and the samples were collected between September 2012 to February 2014. Study design: A total of 450 Hepatitis B surface antigen HBsAg and HBV DNA positive samples were collected in the first step during the period of September 2012 to February 2014 and sent to Nuclear Medicine Oncology and Radiotherapy Institute [NORI], for further analysis. All the samples were genotyped by type specific nested PCR primer pair method for 8 HBV genotypes from A through H. The patients were randomly selected irrespective of their age and gender and a written consent (parental consent in case of less than 18 years of age) was obtained. The study was approved by ethical review committee. In our research, we have used the type specific primer technique to find out the dominant strain in Pakistani populations and furthermore its phylogenetic analysis with reference sequences retrieved from NCBI databases. Results: Our results showed a direct relationship between viral load and identified genotypes and pinpointed that genotype D is the most pre-dominant genotype and prevailing in high proportions among all genotypes in Pakistani population. Mix HBV genotype infections of genotype A with B & C, D with B and C constitute about 17 percent of all the samples. Subgenotypes of different HBV strains have been identified on the basis of differences in the complete nucleotide sequences. Positive PCR results were repeated twice for confirmation. Furthermore, phylogenetic analysis revealed a homology between our reported sequences and reference sequences. The phylogenetic method showed that genotypes/sub-genotypes varies in the geographical areas and correlate strongly with ethnicity. Conclusion: Genotype D was identified and come out to be the most dominant genotype in Pakistani community showing sub-genotypes of D1 & D3 in our study. Genotype A and D are present as co-infection with each other and contributed as the second prevailing genotypic group. Additionally, phylogenetic analysis revealed that genotypes varies in the geographical areas and correlate strongly with reference sequences from NCBI.

Aspergillus Specific PCR and Galactomannan of Bronchoalveolar Lavage Are Superior to Concomitant Same-Day Testing of Concurrent Blood Samples in Immunocompromised Hematological Patients with Suspected Invasive Aspergillosis

BackgroundInvasive pulmonary aspergillosis (IPA) is a frequent complication in hematologic patients (pts) and proper diagnosis is difficult as culture based methods are time consuming and show only modest sensitivity, especially under the current practice of early mold-active antifungal therapy (AFT) or prophylaxis. Analyzing specimens representing the specific site of infection, i.e. bronchoalveolar lavage (BAL), is supposed to be superior compared to testing blood samples. However, that has not yet been shown in direct comparison in individual patients within a 24h time window.To further elucidate this issue the diagnostic performance of an established Aspergillus specific nested Polymerase Chain Reaction (PCR) assay and of Galactomannan antigen testing (GM ELISA) in BAL was evaluated and compared to results obtained from concurrent blood samples of immunocompromised patients, most of all with hematological malignancies.Methods:We retrospectively identified 125 immunocompromised pts suspected to harbor IPA in whom BAL (n=92) and concurrent serum samples (within 24 hours) had been analyzed with Aspergillus PCR. Of these, 30/125 pts (24%) had proven/probable IPA according to 2008 EORTC/MSG criteria while the remaining 95 were classified as possible (n=65) or no IPA (n=30). Furthermore we also identified pts with same-day BAL and serum samples that had been analyzed with GM ELISA. Among those were another 26 pts that could be classified as proven/ probable IPA.ResultsPCR positivity in samples directly from the site of infection (BAL) was observed for 19/30 samples yielding cumulative sensitivity/specificity values of 64%/100%. PCR performance of single corresponding blood samples of these pts was significantly inferior with only 3/33 patients with proven/probable IPA resulting in a sensitivity/specificity of 8%/87% (p<0.005) GM ELISA positivity in BAL was observed for 22/26 pts with sensitivity of 85%. Identical to PCR testing, GM ELISA was significantly more sensitive for detection of IPA when performed in BAL compared to same-day serum samples (p<0.005).ConclusionsIn this retrospective, direct comparison of clinical specimens we observed a significantly superior performance of an Aspergillus PCR as well as GM ELISA assay directly from the site of infection compared to concurrent blood sample testing for pts with suspected IPA during AFT.Thus, PCR analyses or GM ELISA for early diagnosis of IA should be preferably directed at specimens such as BAL when IPA is suspected. DisclosuresBertz:GILEAD Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Buchheidt:Astellas: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Gilead: Consultancy, Honoraria.

Retrospective study of central nervous system lesions and association with Parelaphostrongylus species by histology and specific nested polymerase chain reaction in domestic camelids and wild ungulates.

Formalin-fixed, paraffin-embedded tissues from elk (Cervus elaphus), goats, and camelids with case histories and lesions suggestive of Parelaphostrongylus tenuis were examined by histology to characterize lesions that could aid in definitively diagnosing P. tenuis infection. Additionally, sections of paraffin-embedded tissue were used in a nested polymerase chain reaction (nPCR) using Parelaphostrongylus-specific primers to determine how PCR results corresponded with histological findings. Histological changes in brain and spinal cord consisted of linear tracks of hemorrhage; tracks or perivascular accumulations of hemosiderin-laden macrophages; acute foci of axonal degeneration and/or linear glial scars; and perivascular, parenchymal, or meningeal accumulations of eosinophils and/or lymphocytes and plasma cells. Of the 43 samples with histologic lesions consistent with neural larval migrans, 19 were PCR positive; however, only 8 were confirmed Parelaphostrongylus by DNA sequencing. Additionally, 1 goat was identified with a protostrongylid that had a 97% identity to both Parelaphostrongylus odocoilei and a protostrongylid nematode from pampas deer (Ozotoceros bezoarticus celer) from Argentina. None of the histologic lesions individually or in combination correlated statistically to positive molecular tests for the nematode. The results indicate that it is possible to extract Parelaphostrongylus DNA from formalin-fixed, paraffin-embedded tissue, but extended fixation presumably can cause DNA crosslinking. Nested PCR provides another diagnostic tool to identify the cause of neurologic disease in camelids and elk with histologic lesions consistent with neural larval migrans. Furthermore, potential novel protostrongylid DNA was detected from a goat with lesions consistent with P. tenuis infection, suggesting that other neurotropic Parelaphostrongylus species may occur locally.

Pneumocystis jirovecii colonisation in HIV‐positive and HIV‐negative subjects in Cameroon

To determine the prevalence of Pneumocystis pneumonia (PCP), a major opportunistic infection in AIDS patients in Europe and the USA, in Cameroon. Induced sputum samples from 237 patients without pulmonary symptoms (126 HIV-positive and 111 HIV-negative outpatients) treated at a regional hospital in Cameroon were examined for the prevalence of Pneumocystis jirovecii by specific nested polymerase chain reaction (nPCR) and staining methods. CD4 counts and the history of antiretroviral therapy of the subjects were obtained through the ESOPE database system. Seventy-five of 237 study participants (31.6%) were colonised with Pneumocystis, but none showed active PCP. The Pneumocystis colonisation rate in HIV-positive subjects was more than double that of HIV-negative subjects (42.9% vs. 18.9%, P<0.001). In the HIV-positive group, the colonisation rate corresponds to the reduction in the CD4 lymphocyte counts. Subjects with CD4 counts >500cells/μl were colonised at a rate of 20.0%, subjects with CD4 counts between 200 and 500cells/μl of 42.5%, and subjects with CD4 counts <200cells/μl of 57.1%. Colonisation with Pneumocystis in Cameroon seems to be comparable to rates found in Western Europe. Prophylactic and therapeutic measures against Pneumocystis should be taken into account in HIV care in western Africa.

Fungal polymerase chain reaction testing in equine ulcerative keratitis

To assess the diagnostic utility of fungal polymerase chain reaction (PCR) in forty-three horses with naturally acquired corneal ulcers presenting to a private practice. Routine evaluation of cytologic, histologic, and microbiologic samples was performed. Two PCR approaches were compared - generic and specific fungal nested PCR followed by sequencing and quantitative PCR (qPCR). PCRs were applied to pure control fungal cultures, corneal tissue from ulcerated eyes and in a subset of 9 horses, to swabs from contralateral normal eyes. The expected fungus was identified by nested PCR and qPCR in all control fungal cultures. In all fungal culture-positive affected eyes (10/43), one or more fungi were identified by nested PCR and 4/10 were positive by qPCR. In 6/10 animals, the same fungus was identified by nested PCR and culture. Of these 6, only three were positive by qPCR. Fungal agents were identified by morphology in 8/10 horses. Diagnosis of fungal keratitis was reserved for only those cases in which the same fungus could be identified by PCR, culture, and morphology (5 horses). In 33/43 culture-negative affected eyes and in 6/9 unaffected eyes, one or more fungi were identified by nested PCR in 26 samples and by qPCR in 2 samples. Apart from Aspergillus spp, similar fungi were identified in affected and control eyes. Most eyes harbored mixed bacterial and fungal agents. Nested PCR results confirmed all cytologically positive cases of fungal keratitis. Nested PCR identified a greater spectrum of agents than either culture or qPCR.