Diagnostic Polymerase Chain Reaction Assay Research Articles

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

A conserved active site PenA β-lactamase Ambler motif specific for Burkholderia pseudomallei/B. mallei is likely responsible for intrinsic amoxicillin-clavulanic acid sensitivity and facilitates a simple diagnostic PCR assay for melioidosis

Burkholderia pseudomallei is a soil- and water-dwelling Gram-negative bacterium that causes melioidosis in humans and animals. Amoxicillin-clavulanic acid (AMC) susceptibility has been hailed as an integral part of the screening algorithm for identification of B. pseudomallei, but the molecular basis for the inherent AMC susceptibility of this bacterium remains undefined. This study showed that B. pseudomallei (and the closely-related B. mallei) wild-type strains are the only Burkholderia spp. that contain a 70STSK73 PenA Ambler motif. This motif was present in >99.5% of 1820 analysed B. pseudomallei strains and 100% of 83 analysed B. mallei strains, and is proposed as the likely cause for their inherent AMC sensitivity. The authors developed a polymerase chain reaction (PCR) assay that specifically amplifies the penA70ST(S/F)K73-containing region from B. pseudomallei and B. mallei, but not from the remaining B. pseudomallei complex species or the 70STFK73 region from the closely-related penB of B. cepacia complex species. The abundance and purity of the 193-bp PCR fragment from putative B. pseudomallei isolates from clinical and environmental samples is likely sufficient for reliable confirmation of the presence of B. pseudomallei. The PCR assay is designed to be especially suited for use in resource-constrained areas. While not further explored in this study, the assay may allow diagnosis of putative B. mallei in culture isolates from animal and human samples.

Genetic Variation in Rhipicephalus sanguineus s.l. Ticks across Arizona.

Rhipicephalus sanguineus s.l. (Latreille, 1806), the brown dog tick, is the most widely distributed tick species in the world. The two dominant lineages, a temperate group and a tropical group, are recognized as important disease vectors for both dogs and humans. The temperate and tropical lineages overlap in range in some regions of the world, including the southwestern United States, where recent outbreaks of Rocky Mountain spotted fever are linked to R. sanguineus s.l. While it is unclear to what extent they may differ in their capacity to transmit pathogens, finer-scale resolution of temperate and tropical lineage distribution may provide insight into the ecology of these two tick groups and the epidemiology of R. sanguineus s.l.-vectored diseases. Using diagnostic polymerase chain reaction assays, we examined the geospatial trends in R. sanguineus s.l. lineages throughout Arizona. We found the temperate and tropical lineages were well delineated, with some overlap in the eastern part of the state. In one county, tropical and temperate ticks were collected on the same dog host, demonstrating that the two lineages are living in sympatry in some instances and may co-feed on the same host.

First Report of Pectobacterium polaris Causing Soft Rot and Black Leg of Potato in Russia.

Blackleg and soft rot of potato (Solanum tuberosum) were monitored in the Central European part of Russia within a period of 2012- 2019. Symptoms included decay of tubers, blackening of stem vascular bundles, and partial yellowing of leaves. The disease causes serious potato yield losses in the field and storage. Pectobacterium parmentieri, P. brasiliense, P. versatile (syn. Ca. Pectobacterium maceratum), P. carotovorum, P. atrosepticum, Dickeya dianthicola, and D. solani are considered as main causal agents of soft rot and blackleg disease in Russia (Voronina et al. 2019, Ngoc Ha et al., 2019, Shirshikov et al. 2018, Kornev et al. 2012). Potato plant samples collected in commercial fields in routine plant health assay were used for bacteria isolation on crystal violet pectate agar (CVP) (Helias et al. 2012) as described previously (Voronina et al. 2019). Bacterial colonies producing pitting on CVP were re-isolated and purified on nutrient broth yeast extract medium. DNA of bacterial isolates was extracted, and polymerase chain reaction (PCR) amplifications were performed using gapA primers (Cigna et al. 2017) followed by sequencing. DNA sequence alignment showed that the isolates F099, F100, F106, F109, and F118 were identical (deposited as part of NCBI Ref.Seq. for F109 NZ_RRYS01000004.1, locus KHDHEBDM_RS06360) and grouped together with the type strain Pectobacterium polaris NIBIO1006T (CP017481), a new species described as a potato pathogen (Dees et al. 2017). These strains were negative in diagnostic PCR assays using specific primers Y45/Y46 for the detection of P. atrosepticum, Br1f and L1r for P. brasiliense (Duarte et al. 2004), and ADE1/ADE2 for Dickeya sp. (Nassar et al. 1996). To further validate the identification, strain F109 of P. polaris was selected for genome sequencing. The genome of P. polaris strain F109, (NCBI Reference Sequence NZ_RRYS00000000.1) reveals >99% sequence similarity with type strain P. polaris IPO_1606 (GenBank accession GCA_902143345.1). The strain F109 was deposited to All-Russian Collection of Microorganisms under number VKM V-3420. Thus, the characterization of five isolates provided evidence that a previously unreported pathogen was present in the surveyed fields. The isolates were uniform in genetic and physiological properties; they were gram negative, facultative anaerobes with pectinolytic activity, negative for oxidase, urease, indole production, gelatin liquefaction. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, xylose, and trehalose, and were tolerant to 5% NaCl, unable to utilize malonate and citrate. All the isolates grew at 37°C. All isolates caused soft rot symptoms on 10 inoculated potato tubers. They produced typical black leg rot symptoms in young potato plants inoculated with 107 CFU/ml of the pathogen by stem injection and incubated at 25°C for 48 h. The bacteria were re-isolated successfully from symptomatic potato and pathogen confirmed by gapA sequencing to complete Koch's postulates. To our knowledge, this is the first report of blackleg and soft rot caused by P. polaris on potato in the Russian Federation. According to the data of commercial diagnostic laboratory "PhytoEngineering" (Moscow region), P. polaris occurred in 5% potato seed stocks harvested in 2017-2019 in the Moscow region. This finding may indicate that new Pectobacterium strains have adapted to a diverse environment, which is consistent with widespread distribution of commercial seed potatoes. The author(s) declare no conflict of interest. Funding: This work was supported by Russian Science Foundation grant #16-16-00073.

Vaginal microbiota and human papillomavirus: a systematic review

Accumulating evidence indicates the potential correlation between the vaginal microbioma and the acquisition and persistence of human papillomavirus (HPV) infection. This study aims to demonstrate the potential relationship through a systematic review of the current literature. A search was conducted on the following medical databases: PubMed and Scopus. Nineteen studies met the inclusion criteria and were incorporated in the present review. A total of 12.204 patients and their demographic characteristics were studied. Commercially available DNA tests and polymerase chain reaction (PCR) were used for the detection of different HPV subtypes, while the identification of the microbiomes was performed through specific diagnostic methods and PCR assay. The most frequently encountered species were classified based on their protective or detrimental impact on the progression of HPV infection. The beneficial role of some types of Lactobacillus (Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus crispatus) is generally supported. On the other hand, high microbial diversity and specific microorganisms such as Sneathia, Anaerococcus tetradius, Peptostreptococcus, Fusobacterium and Gardnerella vaginalis were found to be implicated with higher frequency and severity of disease, potentially resulting in pre-cancerous and cancerous cervical lesions.The role of vaginal microbiota appears to play an as yet not fully understood role in the susceptibility to HPV infection and its natural history.

Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named as 2019-nCoV) is responsible for the recent COVID-19 pandemic and polymerase chain reaction (PCR) is the current standard method for its diagnosis from patient samples. This study conducted a reassessment of published diagnostic PCR assays, including those recommended by the World Health Organization (WHO), through the evaluation of mismatches with publicly available viral sequences. An exhaustive evaluation of the sequence variability within the primer/probe target regions of the viral genome was performed using more than 17 000 viral sequences from around the world. The analysis showed the presence of mutations/mismatches in primer/probe binding regions of 7 assays out of 27 assays studied. A comprehensive bioinformatics approach for in silico inclusivity evaluation of PCR diagnostic assays of SARS-CoV-2 was validated using freely available software programs that can be applied to any diagnostic assay of choice. These findings provide potentially important information for clinicians, laboratory professionals and policy-makers.

Meningitis and epididymitis caused by Toscana virus infection imported to Switzerland diagnosed by metagenomic sequencing: a case report

BackgroundWe report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia (Italy) 10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland.Case presentationThe patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days.ConclusionsThis case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis.

Eimeria spp. in captive-reared corncrakes (Crex crex): results of a GeneScan assay consistent with high prevalence of infection and extra-intestinal life stages

ABSTRACTEimeria crecis and Eimeria nenei have been detected in association with enteric disease (“coccidiosis”) in the corncrake (Crex crex: Family Rallidae, Order Gruiformes). Both parasite species are common in apparently healthy free-living corncrakes, but captive-bred juvenile birds reared for reintroduction appeared particularly susceptible to clinical coccidiosis. We investigated the occurrence and relative pathogenicity of these Eimeria species in this juvenile corncrake population and developed a diagnostic species-specific polymerase chain reaction (PCR) for their identification. PCR amplification and sequencing of 18S rDNA were performed on genomic DNA extracted from samples of corncrake intestine, liver and spleen. Sequences generated were used to design a GeneScan diagnostic PCR assay targeting a species-specific TTA indel located within the 18S rDNA – the results suggested this assay was more sensitive than the 18S rDNA/amplicon sequencing approach. Eimeria sp. DNA (consistent with Eimeria sp. infection) was detected at a high prevalence and E. crecis was the predominant species. Each Eimeria species was detected in cases with and without histological evidence of coccidiosis: parasite detection was not statistically associated with disease. In addition to intestinal tissue, liver and spleen samples were positive for Eimeria sp. DNA. Its detection in tissues other than intestine is unusual and a novel finding in corncrakes, although extra-intestinal infection occurs with closely related Eimeria species in cranes (Family Gruidae, Order Gruiformes). Eimeria sp. infection of corncrakes appears typically to be chronic, and to exhibit extra-intestinal spread: as in cranes, these characteristics may be adaptations to the host’s migratory nature.

Diagnostics for Yaws Eradication: Insights From Direct Next-Generation Sequencing of Cutaneous Strains of Treponema pallidum.

Yaws-like chronic ulcers can be caused by Treponema pallidum subspecies pertenue, Haemophilus ducreyi, or other, still-undefined bacteria. To permit accurate evaluation of yaws elimination efforts, programmatic use of molecular diagnostics is required. The accuracy and sensitivity of current tools remain unclear because our understanding of T. pallidum diversity is limited by the low number of sequenced genomes. We tested samples from patients with suspected yaws collected in the Solomon Islands and Ghana. All samples were from patients whose lesions had previously tested negative using the Centers for Disease Control and Prevention (CDC) diagnostic assay in widespread use. However, some of these patients had positive serological assays for yaws on blood. We used direct whole-genome sequencing to identify T. pallidum subsp pertenue strains missed by the current assay. From 45 Solomon Islands and 27 Ghanaian samples, 11 were positive for T. pallidum DNA using the species-wide quantitative polymerase chain reaction (PCR) assay, from which we obtained 6 previously undetected T. pallidum subsp pertenue whole-genome sequences. These show that Solomon Islands sequences represent distinct T. pallidum subsp pertenue clades. These isolates were invisible to the CDC diagnostic PCR assay, due to sequence variation in the primer binding site. Our data double the number of published T. pallidum subsp pertenue genomes. We show that Solomon Islands strains are undetectable by the PCR used in many studies and by health ministries. This assay is therefore not adequate for the eradication program. Next-generation genome sequence data are essential for these efforts.

Novel genotype of Tritrichomonas foetus from cattle in Southern Africa.

Bovine trichomonosis caused by Tritrichomonas foetus is a significant reproductive disease of cattle. Preputial samples were collected using sheath washing technique in bulls in Namibia. Thirty-six trichomonad cultures were characterized using the TaqMan-probe commercial real-time polymerase chain reaction (PCR) diagnostic assay (VetMAX™-Gold Trich Detection Kit) and CYBR real-time PCR assay based on TFR3/4 primers. Diagnostic real-time PCRs and DNA sequencing of the internal transcribed region confirmed presence of T. foetus in 35 out of 36 samples. Multilocus genotyping using cysteine proteases (CP1, CP2, CP4, CP5, CP6, CP7, CP8, CP9) and malate dehydrogenase (MDH1) gene sequences demonstrate that the T. foetus in Namibia are genetically distinct from those characterized elsewhere. We report the discovery of a novel genotype of T. foetus in Namibian cattle, distinct from other T. foetus genotypes in Europe, South and North America and Australia. We suggest recognition of a 'Southern African' genotype of T. foetus. Identification of the new genotype of T. foetus demonstrates the need for wider global sampling to fully understand the diversity and origin of T. foetus causing disease in cattle or cats.

Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR).

Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.

The role of Streptococcus pneumoniae in community-acquired pneumonia among adults in Europe: a meta-analysis

The primary objective of this meta-analysis was to estimate the prevalence of adult community-acquired pneumonia (CAP) caused by Streptococcus pneumoniae in Europe, adjusted for possible independent covariates. Two reviewers conducted a systematic literature search using PubMed on English-language articles that involved human subjects with CAP during the period from January 1990 to November 2011 across European countries. A mixed-effects meta-regression model was developed and populated with 24,410 patients obtained from 77 articles that met the inclusion criteria. The model showed that the observed prevalence of S. pneumoniae in CAP significantly varies between European regions, even after adjusting for explanatory covariates, including patient characteristics, diagnostic tests, antibiotic resistance, and health-care setting. The probability of detecting S. pneumoniae was substantially higher in studies that performed more frequently a diagnostic polymerase chain reaction assay compared to all the other diagnostic tests included. Furthermore, S. pneumoniae was more likely to be confirmed as the cause of a CAP in studies with intensive care unit patients as compared to those with hospital- or community-treated patients. This study provides estimates of the average observed prevalence of S. pneumoniae, which could be used for projecting the health and economic benefits of pneumococcal immunization.

A diagnostic polymerase chain reaction assay for Zika virus

Zika virus (ZIKV) is a mosquito-borne flavivirus. Infection results in a dengue-like illness with fever, headache, malaise, and a maculopapular rash. Nearly all cases are mild and self-limiting but in 2007, a large outbreak of ZIKV was reported from the island of Yap (in Micronesia, northwest of Indonesia). Singapore is already endemic for dengue, and its impact on public health and economic burden is significant. Other dengue-like infections (e.g., Chikungunya virus) are present. Yet only 10% of reported dengue cases have laboratory confirmation. The identification and control of other dengue-like, mosquito-transmitted infections is thus important for the health of Singapore's population, as well as its economy. Given that ZIKV shares the same Aedes mosquito vector with both dengue and Chikungunya, it is possible that this virus is present in Singapore and causing some of the mild dengue-like illness. A specific and sensitive one-step, reverse transcription polymerase chain reaction (RT-PCR) with an internal control (IC) was designed and tested on 88 archived samples of dengue-negative, Chikungunya-negative sera from patients presenting to our hospital with a dengue-like illness, to determine the presence of ZIKV in Singapore. The assay was specific for detection of ZIKV and displayed a lower limit of detection (LoD) of 140 copies viral RNA/reaction when tested on synthetic RNA standards prepared using pooled negative patient plasma. Of the 88 samples tested, none were positive for ZIKV RNA, however, the vast majority of these were from patients admitted to hospital and further study may be warranted in community-based environments.

Molecular characterization of the 2011 Hong Kong scarlet fever outbreak.

A scarlet fever outbreak occurred in Hong Kong in 2011. The majority of cases resulted in the isolation of Streptococcus pyogenes emm12 with multiple antibiotic resistances. Phylogenetic analysis of 22 emm12 scarlet fever outbreak isolates, 7 temporally and geographically matched emm12 non-scarlet fever isolates, and 18 emm12 strains isolated during 2005-2010 indicated the outbreak was multiclonal. Genome sequencing of 2 nonclonal scarlet fever isolates (HKU16 and HKU30), coupled with diagnostic polymerase chain reaction assays, identified 2 mobile genetic elements distributed across the major lineages: a 64.9-kb integrative and conjugative element encoding tetracycline and macrolide resistance and a 46.4-kb prophage encoding superantigens SSA and SpeC and the DNase Spd1. Phenotypic comparison of HKU16 and HKU30 with the S. pyogenes M1T1 strain 5448 revealed that HKU16 displays increased adherence to HEp-2 human epithelial cells, whereas HKU16, HKU30, and 5448 exhibit equivalent resistance to neutrophils and virulence in a humanized plasminogen murine model. However, in contrast to M1T1, the virulence of HKU16 and HKU30 was not associated with covRS mutation. The multiclonal nature of the emm12 scarlet fever isolates suggests that factors such as mobile genetic elements, environmental factors, and host immune status may have contributed to the 2011 scarlet fever outbreak.

Phylogenetic relationships of Mongolian Babesia bovis isolates based on the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c genes

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4–26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.

Rapid diagnosis of the invasive species, Frankliniella occidentalis (Pergande): a species‐specific COI marker

AbstractThe western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is an invasive species and currently occurs in only a few areas in China. An easy, accurate and developmental‐stage independent method to identify F. occidentalis would be a valuable tool to facilitate pest management decision making and, more importantly, to provide an early warning so actions can be taken to prevent its introduction into non‐infested areas. Morphological identification of thrips adults and, to a lesser extent, of second‐stage larvae is the main method currently available to identify F. occidentalis. Molecular identification, however, can be easily carried out by a non‐thrips‐specialist with a little training. In this study, DNA sequence data [within the mitochondrial cytochrome oxidase I gene (COI)] and polymerase chain reaction (PCR) were utilized to develop a molecular diagnostic marker for F. occidentalis. A primer set and PCR cycling parameters were designed for the amplification of a single marker fragment (340 bp) of F. occidentalis COI mtDNA. Specificity tests performed on 28 thrips species, efficacy tests performed on five immature developmental stages as well as on male and female adults and tests on primer sensitivity all demonstrated the diagnostic utility of this marker. Furthermore, the primer set was tested on seventeen F. occidentalis populations from different countries and invaded areas in China and proved to be applicable for all geographic populations. It was used successfully to clarify the distribution of F. occidentalis in the Beijing metro area. These results suggested that this diagnostic PCR assay provides a quick, simple and reliable molecular technique for the identification of F. occidentalis.

The allelic state at the major semi-dwarfing genes in a panel of Turkish bread wheat cultivars and landraces

Dwarfing genes play an important role in improving yield and adaptability of wheat cultivars in most production environments. Understanding the allelic distribution at dwarfing loci is very important for any wheat-breeding programmes. In this study, we reported the allelic constitution at microsatellite locus Xgwm261 and the two major height-reducing genes Rht-B1 and Rht-D1 among a set of 56 bread wheat cultivars and nine landraces, based on diagnostic polymerase chain reaction assays. With respect to Rht-B1, 37% of the accessions carried the dwarfing allele Rht-B1b, while at Rht-D1, only one accession carried the dwarfing allele Rht-D1b. The allelic state at Rht8 was assayed indirectly by genotyping for the linked microsatellite locus Xgwm261. About 26% of the accessions carried the 192 bp allele (linked with Rht8 gene in some cases), whereas 35 and 12% genotypes carried 165 and 174 bp allele at the microsatellite locus Xgwm261. Cultivars released from 1980 onwards increasingly carried either Rht-B1b or Rht8. This information should allow for a more rational use of this collection for the purpose of wheat improvement in Turkey.

Low Prevalence of Hepatitis B and Hepatitis C Infection in Western India

© 2011, INASL 11 Conclusion: The prevalence of positive serological markers for HBV and HCV was as per the national prevalence. Conflict of Interest: None Oral Plenary Session Detection of Diagnosis Escape Variants of Hepatitis B Virus-impact on Viral Load P Balkumar Reddy*, RM Mukherjee*, J Aparna*, PN Rao**, S Mitnala*, R Gupta**, DN Reddy** *Asian Health Care Foundation, **Asian Institute of Gastroenterology, Somajiguda, Hyderabad, India Background/Objective: Hepatitis B virus (HBV) DNA in serum is measured by real time polymerase chain reaction (PCR) assay as viral load—a marker in guided antiviral therapy. Being quasispecies in nature, HBV produce replication variants, which whether escape detection by the commercially used diagnostic PCR assays and thus alter the burden of viral load has not been elucidated. We evaluated an in-house nested PCR assay to detect diagnosis escape variants (DEV) of HBV undetected by commercially available real time PCR assay. Methods: Sera of HBsAg +ve subjects (n = 178) were screened for HBV DNA by a commercially available real time TaqMan based PCR and an in-house nested PCR assay followed by direct sequencing and densitometric quantification of amplicons to obtain viral load. Serum ALT, hepatitis B virus e antigen (HBeAg) and antibody (anti-HBe) status were evaluated by standard biochemical and ELISA procedures. Result: The in-house assay detected 80% of real time PCR positive samples of which, 79.7% had single band (858 bp) and 20.3% showed mixed type (858 bp ± 192 bp). Among 70 real time PCR negative subjects, 5.7% were positive by the in-house assay having 192 bp amplicon containing A1762T and G1764A mutations with variable viral load. Conclusion: Viral load estimated on 858 bp product only showed conformity with real time assay while inclusion of 192 bp product increased the total viral load which might have significance in determining rapid or sustained virological response (RVR/SVR) in treated subjects. Detection of DEV, particularly in real time PCR negative subjects, warrant customization of the in-house assay to real time format for better diagnosis and management of HBV infection. Conflict of Interest: None Low Prevalence of Hepatitis B and Hepatitis C Infection in Western India D Amarapurkar*, N Patel** *Bombay Hospital and Medical Research Centre, Mumbai, Maharashtra, India **Jivandeep Hospital, Anand, Gujarat, India Introduction: Prevalence rates of hepatitis B viral infection (HBV) in India vary from 3% to 12%, which falls in intermediate prevalence zone. Prevalence of hepatitis C viral infection (HCV) in India varies from 0.3% to 4% among blood donor population. Previous data from western India suggests HBV prevalence of 1.6–5.9% and HCV prevalence of 0.2–15.9%. Most of these data are almost 10 years old, and most are based on select population. We aimed to study HBV and HCV recent prevalence from multiple sources. Methods: Data on prevalence of HBV and HCV was collected prospectively from multiple sources like: general population, blood donor population, hospital attending population, and liver clinic population. Results: HBV prevalence was as follows: general population data: 166/17581 (0.94%); blood donor data: 2039/172316 (1.18%); hospital attending population: 133/13177 (1%); liver clinic data: 2637/12251 (21.5%). HCV prevalence was as follows: general population: 1/2388 (0.04%); blood donor data: 666/171306 (0.38%); hospital attending population: 12/2597 (0.46%); liver clinic data: 916/12251 (7.4%). Conclusion: In Western India, there is very low prevalence of HBV and HCV; making it a low prevalence area. Conflict of Interest: None 03_JCEH-Abstract.indd 11 3/18/2011 11:13:03 AM

Detection of Diagnosis Escape Variants of Hepatitis B Virus-impact on Viral Load

© 2011, INASL 11 Conclusion: The prevalence of positive serological markers for HBV and HCV was as per the national prevalence. Conflict of Interest: None Oral Plenary Session Detection of Diagnosis Escape Variants of Hepatitis B Virus-impact on Viral Load P Balkumar Reddy*, RM Mukherjee*, J Aparna*, PN Rao**, S Mitnala*, R Gupta**, DN Reddy** *Asian Health Care Foundation, **Asian Institute of Gastroenterology, Somajiguda, Hyderabad, India Background/Objective: Hepatitis B virus (HBV) DNA in serum is measured by real time polymerase chain reaction (PCR) assay as viral load—a marker in guided antiviral therapy. Being quasispecies in nature, HBV produce replication variants, which whether escape detection by the commercially used diagnostic PCR assays and thus alter the burden of viral load has not been elucidated. We evaluated an in-house nested PCR assay to detect diagnosis escape variants (DEV) of HBV undetected by commercially available real time PCR assay. Methods: Sera of HBsAg +ve subjects (n = 178) were screened for HBV DNA by a commercially available real time TaqMan based PCR and an in-house nested PCR assay followed by direct sequencing and densitometric quantification of amplicons to obtain viral load. Serum ALT, hepatitis B virus e antigen (HBeAg) and antibody (anti-HBe) status were evaluated by standard biochemical and ELISA procedures. Result: The in-house assay detected 80% of real time PCR positive samples of which, 79.7% had single band (858 bp) and 20.3% showed mixed type (858 bp ± 192 bp). Among 70 real time PCR negative subjects, 5.7% were positive by the in-house assay having 192 bp amplicon containing A1762T and G1764A mutations with variable viral load. Conclusion: Viral load estimated on 858 bp product only showed conformity with real time assay while inclusion of 192 bp product increased the total viral load which might have significance in determining rapid or sustained virological response (RVR/SVR) in treated subjects. Detection of DEV, particularly in real time PCR negative subjects, warrant customization of the in-house assay to real time format for better diagnosis and management of HBV infection. Conflict of Interest: None Low Prevalence of Hepatitis B and Hepatitis C Infection in Western India D Amarapurkar*, N Patel** *Bombay Hospital and Medical Research Centre, Mumbai, Maharashtra, India **Jivandeep Hospital, Anand, Gujarat, India Introduction: Prevalence rates of hepatitis B viral infection (HBV) in India vary from 3% to 12%, which falls in intermediate prevalence zone. Prevalence of hepatitis C viral infection (HCV) in India varies from 0.3% to 4% among blood donor population. Previous data from western India suggests HBV prevalence of 1.6–5.9% and HCV prevalence of 0.2–15.9%. Most of these data are almost 10 years old, and most are based on select population. We aimed to study HBV and HCV recent prevalence from multiple sources. Methods: Data on prevalence of HBV and HCV was collected prospectively from multiple sources like: general population, blood donor population, hospital attending population, and liver clinic population. Results: HBV prevalence was as follows: general population data: 166/17581 (0.94%); blood donor data: 2039/172316 (1.18%); hospital attending population: 133/13177 (1%); liver clinic data: 2637/12251 (21.5%). HCV prevalence was as follows: general population: 1/2388 (0.04%); blood donor data: 666/171306 (0.38%); hospital attending population: 12/2597 (0.46%); liver clinic data: 916/12251 (7.4%). Conclusion: In Western India, there is very low prevalence of HBV and HCV; making it a low prevalence area. Conflict of Interest: None 03_JCEH-Abstract.indd 11 3/18/2011 11:13:03 AM

Development of a Ribosomal DNA ITS2 Marker for the Identification of the Thrips,Scirtothrips dorsalis

The thrips Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) is an invasive pest that poses a significant economical threat to U.S. agriculture and trade. In this study, DNA sequence data and polymerase chain reaction (PCR) were utilized to develop a molecular diagnostic marker for S. dorsalis. The DNA sequence variation from the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA (rDNA) was analyzed from various thrips species, including S. dorsalis. A primer set and polymerase chain reaction cycling parameters were designed for the amplification of a single marker fragment of S. dorsalis ITS2 rDNA. Specificity tests performed on ten thrips species, efficacy tests performed on fifteen S. dorsalis populations, and tests on primer sensitivity and robustness all demonstrated the diagnostic utility of this marker. This diagnostic PCR assay provides a quick, simple, and reliable molecular technique to be used in the identification of S. dorsalis.