RNA Polymerase Chain Reaction Research Articles

Multiplex polymerase chain reaction test for the diagnosis of acute viral hepatitis A

Background/AimsThe early diagnosis of acute hepatitis A (AHA) is hindered because serum IgM against hepatitis A virus (HAV) can yield false-negative results during the window period. This study evaluated the diagnostic accuracy of a polymerase chain reaction (PCR) kit for HAV RNA for the diagnosis of AHA.MethodsSamples were collected from 136 patients with acute severe hepatitis at their admission to Asan Medical Center between June 2010 and July 2010. Samples were analyzed for serum IgM anti-HAV using an immunoassay test and for qualitative HAV RNA using the Magicplex HepaTrio PCR test kit. The diagnostic accuracies of these methods were tested on the basis of clinical and laboratory diagnoses of AHA.ResultsThe concordance rate and kappa value between IgM anti-HAV and HAV RNA PCR were 88.2% and 0.707, respectively. For the diagnosis of AHA, the sensitivity and specificity of IgM anti-HAV were 90.7% and 100%, respectively, when an "equivocal" result was regarded as positive; and 79.1% and 100%, respectively, when an "equivocal" result was regarded as negative. The sensitivity and specificity of HAV RNA PCR were 81.4% and 100%, respectively. All four patients with negative IgM anti-HAV and positive HAV RNA PCR results and all four patients with equivocal IgM anti-HAV RNA and positive HAV RNA PCR results were eventually diagnosed with AHA.ConclusionsThe qualitative HAV RNA PCR test has an equivalent diagnostic accuracy for AHA compared to IgM anti-HAV and may be more sensitive during the window period.

Detection of Acute HIV Infection: We Can’t Close the Window

Acute human immunodeficiency virus (HIV) infection poses a dilemma for diagnosis, clinical management, and public health. It has been variously defined as the transient symptomatic illness associated with high-titer viral replication [1], the period from initial infection to complete seroconversion [2], and the phase between the appearance of detectable HIV RNA and detectable antibodies to HIV [3]. In the context of therapeutic trials, primary HIV infection includes the acute infection interval and the first 6 months after seroconversion, during which viral set point is established [4, 5]. ‘‘Detecting acute infection’’ has also been used synonymously with ‘‘closing the window,’’ the period during which tests for HIV are negative in persons who are infected. In this issue of the journal, Rosenberg et al [6] evaluated how well the Determine HIV-1/2 Ag/Ab Combo rapid test (Combo RT) compared with an algorithm of parallel antibody tests followed by RNA polymerase chain reaction. The Combo RT, designed for use at the point of care with finger-stick whole blood to detect HIV antibody and HIV antigen (and distinguish the two) in 20 minutes, did not fare very well in this comparison. The Combo RT is a lateral flow test (ie, an immunoassay that incorporates all necessary reagents into a single test strip). The test result is read visually, without instrumentation, after the specimen flows across the strip. Lateral flow tests have revolutionized HIV testing because they are easy to perform, require minimal training, have long-term stability, require no dedicated instrumentation, and can use whole blood specimens. In the United States, lateral flow HIV tests are used extensively in nonclinical settings. However, high hopes for point-of-care rapid tests able to identify acute infection, predicated on detecting p24 antigen, may be unrealistic. Recently developed fourthgeneration HIV antigen-antibody (Ag/Ab) combination assays, designed for use in the laboratory, detect both p24 antigen and antibodies against HIV but do not distinguish between the two. They exhibit an analytic sensitivity for p24 antigen of 11–18 pg/mL [7], equivalent to approximately 30 000–50 000 copies/mL of HIV RNA [8]. In contrast, lateral flow assays using colloidal gold have a lower limit of detection in the range of 1 ng/mL [9], 100-fold higher than the p24 antigen concentration usually present in plasma [8]. With the ultrasensitive procedure that includes heat dissociation of p24 antigen–antibody immune complexes, signal amplification, and instrumentation, the limit of detection for p24 (without additional pretreatment of the specimen to disrupt viral particles) can be $250 000 RNA copies/mL [10]. The findings of Rosenberg et al substantiate this: ultrasensitive methods were used to test 7 of the 8 acute specimens and failed to detect p24 antigen in 2 that had RNA concentrations of 45 000 and $750 000 copies/mL. In contrast, the ability of the Combo RT to establish the presence of antibodies to HIV was excellent. The Combo RT was positive for antibody in 162 (99.4%) of the 163 persons with established infection and in 2 of the 8 patients with acute HIV infection who were negative on other antibody tests. This latter finding suggests that the Combo RT might be able to identify many of the earliest infections. As shown in Table 1, antibody tests differ in their sensitivity to detect HIV during seroconversion [11]. Most lateral flow rapid tests in current use (and also Western blot analysis) are based on second-generation immunoassay principles that detect only immunoglobulin (Ig) G–class antibodies and become positive, on average, 20–25 days after RNA appears [12]. The antibody component of the Combo RT detects both IgMand IgGclass antibodies. Several studies suggest Received and accepted 9 November 2011; electronically published 29 December 2011. Correspondence: Bernard M. Branson, MD, CDC, 1600 Clifton Rd, MS D-21, Atlanta, GA 30333 (bbranson@cdc.gov). The Journal of Infectious Diseases 2012;205:521–4 Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2011. DOI: 10.1093/infdis/jir793

Molecular Detection of Microorganisms in Distal Airways of Patients Undergoing Lung Cancer Surgery

Whereas proximal airways of patients undergoing lung cancer surgery are known to present specific microbiota incriminated in the occurrence of postoperative respiratory complications, little attention has been paid to distal airways and lung parenchyma considered to be free from bacteria. We have hypothesized that molecular culture-independent techniques applied to distal airways should allow identification of uncultured bacteria, virus, or emerging pathogens and predict the occurrence of postoperative respiratory complications. Microbiological assessments were obtained from the distal airways of resected lung specimens from a prospective cohort of patients undergoing major lung resections for cancer. Microorganisms were detected using real-time polymerase chain reaction (PCR) assays targeting the bacterial 16s ribosomal RNA gene and Herpesviridae, cytomegalovirus (CMV), and herpesvirus simplex. All postoperative microbiological assessments were compared with the PCR results. In all, 240 samples from 87 patients were investigated. Colonizing agents were exclusively Herpesviridae (CMV, n=13, and herpesvirus simplex, n=1). All 16s ribosomal RNA PCR remained negative. Thirteen patients (15%) had a positive CMV PCR (positive-PCR group), whereas the remaining 74 patients constituted the negative-PCR group. Postoperative pneumonia occurred in 24% of the negative-PCR group and in 69% of the positive-PCR group (p=0.003). Upon stepwise logistic regression, performance status, percent of predicted diffusion lung capacity for carbon monoxide, and positive PCR were the risk factors of postoperative respiratory complications. The CMV PCR had a positive predictive value of 0.70 in prediction of respiratory complications. When tested by molecular techniques, lung parenchyma and distal airways are free of bacteria, but CMV was found in a high proportion of the samples. Molecular CMV detection in distal airways should be seen as a reliable marker to identify patients at risk for postoperative respiratory complications.

HIV diagnostics in infants vertically exposed and receiving anti-retroviral treatment

In this multicenter, prospective, cohort study of neonates born to mothers infected with HIV, the value of performance of testing for both HIV-1 DNA and HIV-1 RNA was compared in infant aged ≤ 6 months. The novelty of this investigation is that the 1567 studied infants exposed to HIV received postnatal prophylaxis (and were not breastfed). The DNA and RNA tests performed similarly as diagnostic tests. Although both HIV-1 RNA and HIV-1 DNA polymerase chain reaction (PCR) tests had specificity of 100% for infants infected with HIV (except in first few postnatal days), sensitivity of tests rose to 100% only at 3 months of age (after discontinuation of 4-6 week course of anti-retroviral prophylaxis). At 1 month of age, 11% of children infected with HIV had negative DNA and RNA PCR test results. Our expert reviewers suggested that these data make the current World Health Organization's goal of diagnosing HIV-1 infections using tests with at least 95% sensitivity for all infants exposed to HIV by 4-6 weeks of age nonfeasible given the current standard of peri- and postnatal management and current limitations of viral detection methodology. Article page 60▶ Performance of HIV-1 DNA or HIV-1 RNA Tests for Early Diagnosis of Perinatal HIV-1 Infection during Anti-Retroviral ProphylaxisThe Journal of PediatricsVol. 160Issue 1PreviewTo compare performance of testing for human immunodeficiency virus (HIV)-1 DNA and HIV-1 RNA for diagnosis of HIV-1 infection in infants receiving preventive antiretroviral therapy. Full-Text PDF

Index of Suspicion in The Nursery

A 4-day-old male infant presents in late November to the pediatrics clinic with decreased activity and poor feeding. The mother reports that he had been feeding and acting well until 1 day before presentation. The mother states that he has not had any rhinorrhea, nasal congestion, cough, vomiting, diarrhea, rash, or any atypical movements that could be seizures. She had not taken his temperature, and she states that he has not felt hot. The pediatrician becomes immediately concerned as she recognizes a lethargic infant in respiratory distress. A code is called, and the resuscitation is begun. The infant was a 3,083-g product of a 38+1 week gestation to a 28-year-old G2P1 woman. The pregnancy was only complicated by a positive group B streptococcus screen at 35 weeks' gestation and a history of herpes simplex virus in the past, but no lesions were noted before or during delivery. On presentation to labor and delivery, the fetus was noted to be tachycardic and the mother had mild abdominal tenderness, leading to initiation of ampicillin and gentamicin 16 hours before delivery. The duration of rupture of membranes was 4 hours. The infant was born via spontaneous vaginal delivery and was vigorous, with Apgar scores of 9 and 9 at 1 and 5 minutes. Because the mother was neither febrile nor tachycardic, it was decided that she did not have chorioamnionitis, so the infant received no further laboratory evaluation or antibiotics. The mother and infant were discharged from the hospital after 2 uneventful days. In the clinic, the 4-day-old infant has initial vital signs that included a temperature of 98.9°F, a heart rate of 170 beats per minute, a respiratory rate of 71 breaths per minute, an oxygen saturation (SaO2) of 80% on room air, and blood pressure 84/35 mm …

Determination of HIV status of infants born to HIV-infected mothers: A review of the diagnostic methods with special focus on the applicability of p24 antigen testing in developing countries

In 2009, 2.5 million children under the age of 15 y were living with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS); 370,000 were diagnosed with HIV and 260,000 died due to AIDS. More than 90% of the children infected with HIV live in sub-Saharan Africa. Most children infected with HIV contract the infection in utero, during delivery, or via breast milk. This review outlines the current diagnostic methods to determine the HIV status of infants born to HIV-infected mothers. The HIV DNA and RNA polymerase chain reaction (PCR) tests are highly accurate and are recommended as the first-choice diagnostic methods. However, they are expensive and require complex laboratory procedures. Consequently, a search for less costly and complicated methods has led to the testing of p24 antigen analyses as an alternative to the gold-standard PCR tests, with encouraging results. The p24 antigen Perkin Elmer assay currently most often used has a sensitivity of 98.8% and a specificity of 100% (infants 6 weeks of age). Larger-scale studies should be performed in resource-limited settings to confirm these findings.

Expression and activity of metalloproteinases in the digital laminae of horses with starch gruel-induced laminitis

Expression and activity of metalloproteinases in the digital laminae of horses with starch gruel-induced laminitis

Efficiency of diversion of the first aliquot of blood and prestorage leukoreduction for preventing bacterial contamination in red blood cell concentrates assessed using a rapid polymerase chain reaction-based bacterial detection system

Sepsis caused by the bacterial contamination of blood products is a major infection risk associated with blood transfusion. Diversion of the initial 25 mL of blood and prestorage leukoreduction were implemented in Japan in 2007 for all donated blood products. We assessed the efficacy of these new collection procedures in preventing bacterial contamination of red blood cell (RBC) concentrates. Broad-range 16S ribosomal RNA polymerase chain reaction was used to determine bacterial contamination in segment samples of RBCs before and after implementation of the new collection procedures. To evaluate whether these new procedures reduced bacterial contamination, we compared bacterial contamination rates of blood samples from diversion pouches with those of segment samples from the same donor's RBCs. The rate of bacterial contamination of RBCs before implementation of the new collection procedures was 1.27%. Most of the isolated bacteria were Staphylococcus epidermidis or Propionibacterium acnes. After implementation, this rate was significantly reduced to 0.10%. Of the 233 whole blood samples obtained from the Mie Red Cross Blood Center, 1.72% of blood samples from diversion pouches were contaminated, but no bacterial contamination was detected in segment samples from the same donor's RBCs after prestorage leukoreduction. The new collection procedure significantly reduced bacterial contamination of RBC concentrates.

Public Health Implications of Rapid Hepatitis C Screening With an Oral Swab for Community-Based Organizations Serving High-Risk Populations

Between April and September of 2009 we evaluated the accuracy of the OraQuick HCV rapid antibody test and assessed its feasibility for use by community-based organizations (CBOs) serving populations at high risk for HCV in New York City. We compared the results of screening by OraQuick (oral swab) and enzyme immunoassay (EIA; blood draw). We performed ribonucleic acid polymerase chain reaction testing for discordant results. We also assessed research staff perceptions through a survey and focus group. Overall, 97.5% of OraQuick and EIA results matched. Testing of discordant samples indicated that the rapid test was more likely than the EIA to provide a correct diagnosis. Research staff preferred the rapid test and identified challenges that would be overcome with its use. CBOs could benefit from increased testing capacity, and clients might benefit from more rapid access to education, counseling, and referrals. OraQuick's accuracy is comparable to the EIA. The oral swab rapid test could help HCV screening programs reach individuals unaware of their status and expand testing into nonclinical settings such as mobile units.

The Impact of Illicit Drug Use on Spontaneous Hepatitis C Clearance: Experience from a Large Cohort Population Study

Background and AimsAcute hepatitis C infection usually ends in chronic infection, while in a minority of patients it is spontaneously cleared. The current population-based study is performed on a large cohort in Golestan province of Iran to examine the demographic correlates of Spontaneous Hepatitis C Clearance.MethodsSerum samples used in this study had been stored in biorepository of Golestan Cohort Study. These samples were evaluated for anti hepatitis C Virus by third generation Enzyme-linked immunosorbent assay (ELISA). Subjects who tested positive were then invited and tested by Recombinant Immunoblot Assay (RIBA) and Ribonucleic Acid Polymerase Chain Reaction test (PCR). If tested positive for RIBA, subjects were recalled and the two tests were re-done after 6 months. Those subjects who again tested positive for RIBA but negative for PCR were marked as cases of spontaneous clearance.Results49,338 serum samples were evaluated. The prevalence of Chronic Hepatitis C Virus (CHCV) infection based on PCR results was 0.31%. Among those who had acquired hepatitis C, the rate of SC was 38%. In multivariate analysis, illicit drug use both Injecting Use (OR = 3.271, 95% CI: 1.784–6.000, p-value<0.001) and Non-Injecting Use (OR = 1.901, 95% CI: 1.068–3.386, p-value = 0.029) were significant correlates of CHCV infection versus SC.ConclusionsIllicit drug use whether intravenous or non-intravenous is the only significant correlate of CHCV, for which several underlying mechanisms can be postulated including repeated contacts with hepatitis C antigen.

Performance of HIV-1 DNA or HIV-1 RNA Tests for Early Diagnosis of Perinatal HIV-1 Infection during Anti-Retroviral Prophylaxis

To compare performance of testing for human immunodeficiency virus (HIV)-1 DNA and HIV-1 RNA for diagnosis of HIV-1 infection in infants receiving preventive antiretroviral therapy. This substudy of the French multicenter prospective cohort of neonates born to HIV-infected mothers, included 1567 infants tested for HIV with polymerase chain reaction (PCR) in a single laboratory, receiving post-natal prophylaxis, not breastfed, and having simultaneous HIV-1 DNA and RNA results before 45 days. The performance of PCR was assessed in reference to the 6-month HIV-1 RNA result. Specificity of both HIV-1 RNA and HIV-1 DNA PCR was 100% at all ages (except 99.8% for DNA at birth); sensitivity was 58% (RNA) and 55% (DNA) at birth, and 89% at 1 month, 100% at 3 months for both, and 100% at 6 months (DNA). Concordance between HIV-1 DNA and RNA results was 0.78 and 0.81 (Kappa) at birth and 1 month and 100% at 3 and 6 months. Type of maternal and neonatal prophylaxis had no effect on sensitivity, but influenced viral load. The performances of testing for HIV-1 DNA and RNA were similar with 100% sensitivity at 3 months. At 1 month during prophylaxis, 11% of infected children had negative PCR results.

Biotechnological approaches to determine the impact of viruses in the energy crop plant Jatropha curcas.

BackgroundGeminiviruses infect a wide range of plant species including Jatropha and cassava both belonging to family Euphorbiaceae. Cassava is traditionally an important food crop in Sub - Saharan countries, while Jatropha is considered as valuable biofuel plant with great perspectives in the future.ResultsA total of 127 Jatropha samples from Ethiopia and Kenya and 124 cassava samples from Kenya were tested by Enzyme-Linked Immunosorbent Assay (ELISA) for RNA viruses and polymerase chain reaction for geminiviruses. Jatropha samples from 4 different districts in Kenya and Ethiopia (analyzed by ELISA) were negative for all three RNA viruses tested: Cassava brown streak virus (CBSV), Cassava common mosaic virus, Cucumber mosaic virus, Three cassava samples from Busia district (Kenya) contained CBSV. Efforts to develop diagnostic approaches allowing reliable pathogen detection in Jatropha, involved the amplification and sequencing of the entire DNA A molecules of 40 Kenyan isolates belonging to African cassava mosaic virus (ACMV) and East African cassava mosaic virus - Uganda. This information enabled the design of novel primers to address different questions: a) primers amplifying longer sequences led to a phylogenetic tree of isolates, allowing some predictions on the evolutionary aspects of Begomoviruses in Jatrophia; b) primers amplifying shorter sequences represent a reliable diagnostic tool. This is the first report of the two Begomoviruses in J. curcas. Two cassava samples were co - infected with cassava mosaic geminivirus and CBSV. A Defective DNA A of ACMV was found for the first time in Jatropha.ConclusionCassava geminiviruses occurring in Jatropha might be spread wider than anticipated. If not taken care of, this virus infection might negatively impact large scale plantations for biofuel production. Being hosts for similar pathogens, the planting vicinity of the two crop plants needs to be handled carefully.

Dengue Virus Seroprevalence Among Febrile Patients in Bamako, Mali: Results of a 2006 Surveillance Study

Dengue viruses (DENV) are endemic in over 100 countries worldwide, and annually 50 to 100 million people are infected by one of the four DENV serotypes, whereas over 2.5 billion people are at risk for infection. West African countries lack the surveillance to determine the true incidence of dengue; hence, this disease is likely significantly underestimated. In Mali, ?14 million people are potentially at risk of acquiring a dengue infection. A serosurvey for DENV was conducted on 95 human serum samples obtained from the Institute National de Recherche en Sante Publique in 2006. DENV-specific IgM and IgG enzyme-linked immunosorbent assays were performed on all samples, and a subset was tested using the plaque-reduction neutralization test against the DENV and yellow fever virus (YFV). Samples collected during the acute infection (0-5 days postonset of symptoms) were tested for dengue NS1 antigen and reverse-transcriptase polymerase chain reaction for Flaviviruses, Alphaviruses, and Bunyaviruses RNA. A total of 87 (93%) of samples were positive for anti-DENV IgG antibodies. Of a subset of 13 IgG positive samples, 2 samples neutralized monotypically against DENV-1 and -2, whereas 3 others neutralized broadly against YFV and multiple DENV. Although no polymerase chain reaction positives were found, DENV NS1 was detected in 1 of the 20 acute samples tested. Of the 93 human serum samples tested, the dengue prevalence based on dengue IgG enzyme-linked immunosorbent assay results was 93%. Three DENV specific positive samples and two YFV positives were identified by plaque-reduction neutralization test. Finally, one sample tested positive for dengue NS1, thus suggestive of an acute infection within 14 days of obtaining the sample from the patient. Based on these serological data from this study, YFV and DENV appear to be co-circulating in Mali.

Nephrotic Syndrome and UnrecognizedPlasmodium malariaeInfection in a US Navy Sailor 14 Years After Departing Nigeria

A 34-year-old Nigerian man presented with nephrotic syndrome. Renal biopsy revealed chronic membranous glomerulopathy with focal segmental sclerosis. Blood Giemsa smear contained rare Plasmodium sp. trophozoites and small subunit ribosomal RNA polymerase chain reaction amplification confirmed the presence of Plasmodium malariae. This case highlights the importance of obtaining even remote travel histories from ill immigrants and considering occult quartan malaria in patients from endemic locations with nephrotic syndrome.

LEUNIG_HOMOLOG and LEUNIG Regulate Seed Mucilage Extrusion in ArabidopsisF

LEUNIG (LUG) and LEUNIG_HOMOLOG (LUH) encode two closely related Arabidopsis proteins, belonging to the Gro/TLE family of transcriptional co-repressors. These two genes were previously shown to exhibit partially overlapping functions in embryo and flower development. In this report, the role of both LUH and LUG on seed mucilage extrusion was examined. Seed mucilage extrusion occurs after the seeds are imbibed, serving as functional aid in seed hydration, germination, and dispersal. While luh-1 mutants exhibited strong defects in seed mucilage extrusion, lug-3 mutants exhibited a minor phenotype in mucilage extrusion. Further characterization indicates that luh-1 does not exhibit any obvious defect in seed epidermal cell differentiation, mucilage synthesis, or mucilage deposition, suggesting a specific role of LUH in mucilage extrusion. This seed mucilage phenotype of luh-1 is identical to that of mucilage modified 2 (mum2) mutants. MUM2 encodes a β-galactosidase required for the modification of the mucilage. Quantitative reverse transcription polymerase chain reaction of RNA extracted from siliques detected a slight decrease of MUM2 mRNA in the luh-1 mutant compared to the wild type. Together, LUH and possibly LUG may specifically regulate mucilage extrusion by promoting the expression of genes required for mucilage maturation.

T helper (Th)1, Th2, and Th17 interleukin pathways in infertile patients with minimal/mild endometriosis

In the present study, interleukin (IL)-10, IL-12, IL-17, and IL-23 levels were measured in serum and peritoneal fluid of women with minimal or mild endometriosis and compared with levels in controls without endometriosis. Higher IL-23 levels were encountered in the peritoneal fluid of women with endometriosis, suggesting a possible role of this cytokine in these women's infertility.

Preliminary Results of Ozone Therapy as a Possible Treatment for Patients with Chronic Hepatitis C

Medical ozone is more bactericidal, fungicidal, and virucidal than any other natural substance. Some studies proved that ozone infused into donated blood samples can kill viruses 100% of the time. Ozone, because of its special biologic properties, has theoretical and practical attributes to make it a potent hepatitis C virus (HCV) inactivator, which suggests an important role in the therapy for hepatitis C. The study aim is to evaluate the role of ozone therapy in decreasing HCV ribonucleic acid (HCV RNA) load and its effect on the liver enzymes among patients with chronic hepatitis C. This study included 52 patients with chronic hepatitis C (positive polymerase chain reaction [PCR] for HCV RNA and raised serum alanine transaminase [ALT] for more than 6 months). All patients were subjected to meticulous history taking and clinical examination. Complete blood count, liver function tests, and abdominal ultrasonography were requested for all patients. The ozone group included 40 patients who received major autohemotherapy, minor autohemotherapy, and rectal ozone insufflation. The other 12 patients (conventional group) received silymarin and/or multivitamins. There were significant improvements of most of the presenting symptoms of the patients in the ozone group in comparison to the conventional group. ALT and aspartate transaminase (AST) levels normalized in 57.5% and 60% in the ozone group, respectively, in comparison to 16.7% and 8% in the conventional group, respectively. Polymerase chain reaction (PCR) for HCV RNA was negative among 25% and 44.4% after 30 and 60 sessions of ozone therapy, respectively, in comparison to 8% among the conventional group. Ozone therapy significantly improves the clinical symptoms associated with chronic hepatitis C and is associated with normalized ALT and AST levels among a significant number of patients. Ozone therapy is associated with disappearance of HCV RNA from the serum (-ve PCR for HCV RNA) in 25%-45% of patients with chronic hepatitis C.

MPL Mutation Profile in JAK2 Mutation-negative Patients With Myeloproliferative Disorders

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Comparison of RNA Extraction and Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Detection of Porcine Reproductive and Respiratory Syndrome Virus in Porcine Oral Fluid Specimens

The objective of the current study was to evaluate various RNA extraction and polymerase chain reaction (PCR) protocols for the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine oral fluids. Extraction protocols were selected based on ease of use and compatibility with high-throughput, automated systems. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited by unknown factors in the oral fluid matrix and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.

Detection and subtype identification of Blastocystis isolates from wastewater samples in the Philippines

To provide further evidence of waterborne transmission of Blastocystis, a total of 31 wastewater treatment plants from geographically distinct locations across the Philippines were sampled for influent and effluent sewage samples. In vitro cultivation was the method of choice to increase sensitivity of detection. Blastocystis cysts were detected in 15% (9/62) of the samples using in vitro culture. Moreover, influent and effluent samples were 23% (7/31) and 7% (2/31) positive for the parasite, respectively. The presence of viable cysts in treated samples may be an indication of the inefficiency of the treatment process in preventing Blastocystis from entering the environment. Polymerase chain reaction and sequencing of the full-length small subunit ribosomal RNA (SSU rRNA) genes of the nine wastewater isolates were performed. The SSU rRNA gene sequences of the isolates showed very high similarity (98 to 99%) to homologous sequences of Blastocystis described previously. The phylogenetic tree constructed showed that the wastewater isolates clustered with each other with good bootstrap support and belonged to two subtypes (ST) - ST1 and ST2. This is the first report of subtyping Blastocystis isolates from wastewater samples and gives further emphasis to the remarkable genetic diversity of the parasite.