Positive Polymerase Chain Reaction Results Research Articles

Aportaciones de las técnicas moleculares en la fase crónica de la Enfermedad de Chagas en ausencia de tratamiento

IntroductionThe chronic phase of Chagas disease (CD) is characterised by a low and intermittent parasitaemia. The Polymerase Chain Reaction (PCR) presents a variable sensitivity in this stage limiting its use as a diagnostic tool. Despite this, the use of PCR in untreated patients can provide information on the parasite behaviour and its presence in peripheral blood. MethodsA timely real-time PCR determination was performed on a cohort of 495 untreated chronic CD patients. Also, a subcohort of 29 patients was followed-up by serial real-time PCR during a period from 8 to 12 months in which they could not have access to the treatment due to lack of supply. ResultsThe positive percentage of real-time PCR in our series was 42%. Nevertheless, real-time PCR positive results were significantly higher in patients with five years or less of residence in Spain (P=.041). The detection of DNA was not related to the existence of cardiac and/or gastrointestinal abnormalities.In the follow-up subgroup, real-time PCR was consistently positive in 13.8% of patients, consistently negative in 31%, and intermittent in 55.2%. ConclusionsThe different real-time PCR results regarding the time of residence suggests the possible relationship of external factors in the parasite presence in peripheral blood. On the other hand, specific host factors may be involved in the behaviour of parasitaemia over time.

Seasonality of Acute Retinal Necrosis.

PurposeTo study the seasonal variability in the occurrence of acute retinal necrosis (ARN) in a series of polymerase chain reaction (PCR)-positive patients.Methods Consecutive patients clinically diagnosed with ARN and a positive PCR result of aqueous humor during a seven-year period were studied retrospectively. Patients' demographics, causative viral agent(s), and the date of disease onset were extracted from medical records.ResultsTwenty eyes of 20 patients were enrolled; the mean age at presentation was 39.6 14.4 (range, 6–62) years. Nine patients were female. The most common causative agent was varicella-zoster virus in 16 patients (80%), followed by herpes simplex virus in two patients (10%). The disease onset was in winter in 10 patients (50%), and the highest incidence was in February (five patients, 25%). The cumulative occurrence of ARN was significantly higher in the first half of the year (winter and spring) compared to the second half of the year (summer and fall) (P = 0.030). In general, seasons with a high incidence of ARN were preceded by cold seasons.Conclusion In our series, we observed seasonal variability in the incidence of ARN, with the highest incidence during winter and spring. However, further epidemiologic studies in different geographical areas are required to elucidate the true seasonal nature of ARN.

Identification of Dengue Serotypes using a Single Serum Specimen Algorithm in a Tertiary Care Hospital, Alappuzha, Kerala, India

Introduction: The geographic location of Alappuzha, a district in the South Indian state of Kerala, the distinct weather conditions and frequent natural calamities present a unique ecology that contributes to the prevalence of vector-borne diseases like dengue. Early dengue virus infection can be detected by using a combination of tests on a single serum specimen. Aim: To identify the dengue virus serotypes among hospitalised patients in a South Indian teaching hospital in Alappuzha, Kerala, India. Materials and Methods: Patient samples that tested positive for dengue non-structural protein-1 (NS1) antigen by ELISA were further evaluated for dengue virus RNA by real-time, multiplex reverse transcriptase Polymerase Chain Reaction (PCR) and the serotype was determined. Anonymised patient data was collected using a questionnaire as a data collection tool. The data was analysed for statistical significance. Results: Among 422 non-duplicate patient serum samples received in the Department of Microbiology, in the year 2019, 30 were positive for dengue NS1 antigen by ELISA. Dengue viral RNA was detected in 50% of the samples (15/30). DENV-3 serotype was the most prevalent (nine) followed by DENV-1 (five) and DENV-2 (one). Common presentations of the patients were fever, headache, and myalgia. No statistically significant association was found between a PCR positive result and the presence of warning signs and thrombocytopenia. Conclusion: DENV-3 was the most common serotype in the study population. Early dengue virus infection is associated with varied symptoms.

Polymerase chain reaction for the diagnosis of herpesvirus infections in dermatology : Analysis of clinical data.

SummaryBackgroundRapid identification of human herpesviruses from lesion swabs is necessary for timely initiation of antiviral treatment, especially with infections involving neonates and immunocompromised individuals. The aim of the study was to investigate the results of an in-house polymerase chain reaction (PCR) test for herpesviruses in patients with symptoms suggestive for a herpesvirus infection.Patients and methodsIn this single center retrospective study the results of 3677 lesion swab specimens tested for human herpes simplex virus 1 and 2 (HSV 1 and 2) and varicella zoster virus (VZV) were analyzed in the context of data sheets giving details of the suspected diagnosis, medical history as well as the demographic data of the patients. The PCR procedures for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpes virus 8 (HHV-8) were applied on special occasions.ResultsOf the samples 3369 (91.6%) were swabs and a minority were tissue or blood samples. Of the 3015 samples tested for HSV‑1, HSV‑2 and VZV concomitantly, 52.3% were positive for at least one of these viruses. Clinically distinct conditions, such as herpes zoster and varicella had a high rate of positive PCR results, ranging from 81% to 88%, respectively. Among HSV‑2 positive samples, 23.7% derived from human immunodeficiency virus (HIV) positive patients, in contrast to the 10.8% originating from immunocompetent patients, the difference being statistically significant (p < 0.002). The HSV‑2 was detected more often in women than in men.ConclusionDistinct clinical diagnoses have a high correlation rate with positive PCR results. A significantly higher number of HSV‑2 positive results were found in HIV positive patients and in women.

How to Interpret a Positive Campylobacter PCR Result Using the BD MAXTM System in the Absence of Positive Culture?

The aim of this study was to evaluate, using two independent polymerase chain reaction (PCR) formats, the results of Campylobacter detection by the BD MAXTM Enteric Bacterial Panel PCR (Becton Dickinson, Le Pont de Claix, France) in the absence of positive culture. A total of 77 samples found positive for Campylobacter on BD MAXTM but negative by culture were studied. Upon reception, one in-house real-time-PCR for Campylobacter sp. and a PCR with the RIDAGENE Bacterial Stool Panel (r-biopharm, Darmstadt, Germany) were performed. The data obtained using these two PCR formats were evaluated with respect to the cycle threshold (Ct) and fluorescence intensity values (FI) obtained on BD MAXTM. Ct and FI values were also obtained for 80 positive Campylobacter cases by culture. Among the 77 samples, 33 were positive with the two PCRs, and 37 remained negative. For the 33 double-positive PCRs samples, the Ct values obtained on BD MAXTM were lower than 30 in 93.9%, and FI > 2000 for 97% of cases. For the 37 double-negative PCRs samples, the Ct values obtained on BD MAXTM were <30 in only 18.9%, however FI were >2000 for 40.5% of cases. Positive culture cases had Ct values < 30 in 96.2% and FI > 2000 in 98.8%. We showed that the Ct values obtained on BD MAXTM can help to interpret the results. Almost 96% of the Campylobacter sp. cases detected by culture or with the two reference PCRs positive showed a Ct value on BD MAXTM, meaning that stools detected as positive with BD MAXTM and having a Ct > 30 may be false positives.

Clinical Factors Associated With Congenital Cytomegalovirus Infection: A Cohort Study of Pregnant Women and Newborns.

The aim of this prospective cohort study was to determine clinical factors associated with the occurrence of congenital cytomegalovirus infection (cCMV) in pregnant women. Between March 2009 and November 2017, newborns born at a primary maternity hospital received polymerase chain reaction (PCR) analyses for CMV DNA in their urine with informed consent of the mothers at a low risk. Clinical data, including age, gravidity, parity, body mass index, occupation, maternal fever/flulike symptoms, pregnancy complications, gestational weeks at delivery, birth weight, and automated auditory brainstem response, were collected. Logistic regression analyses were performed to determine clinical factors associated with cCMV. cCMV was diagnosed by positive PCR results of neonatal urine in 9 of 4125 pregnancies. Univariate and multivariable analyses revealed that the presence of fever/flulike symptoms (odds ratio [OR], 17.9; 95% confidence interval [CI], 3.7-86.7; P < .001) and threatened miscarriage/premature labor in the second trimester (OR, 6.0; 95% CI, 1.6-22.8; P < .01) were independent clinical factors associated with cCMV. Maternal fever/flulike symptoms or threatened miscarriage/premature labor in the second trimester had 100% sensitivity, 53.2% specificity, and a maximum Youden index of .85. This cohort study for the first time demonstrated that these clinical factors of pregnant women and newborns were associated with the occurrence of cCMV. This is useful information for targeted screening to assess risks of cCMV in low-risk mothers, irrespective of primary or nonprimary CMV infection.

Mycobacterium avium Subspecies paratuberculosis DNA and Antibodies in Dairy Goat Colostrum and Milk.

Mycobacterium avium subspecies paratuberculosis (MAP) is endemic in the Dutch dairy goat population causing economic loss, and negatively influencing welfare. Moreover, there are concerns about a potential zoonotic risk. Therefore the industry’s objectives are to decrease MAP prevalence, limit economic losses as well as reduce the concentration of MAP in (bulk) milk. To diminish within-farm spread of infection, vaccination, age dependent group housing with separation of newborns from adults, as well as rearing on artificial or treated colostrum and milk replacers are implemented. However, the importance of MAP contaminated colostrum and milk as a route of infection in dairy goat herds is unknown. Therefore the aim of this study was to detect the presence of MAP DNA in colostrum and milk from dairy goats in infected herds. A convenience sample of 120 colostrum samples and 202 milk samples from MAP infected dairy goat herds were tested by IS900 real-time Polymerase Chain Reaction (PCR) for MAP DNA. Furthermore, 22 colostrum samples and 27 post mortem milk samples of goats with clinical signs consistent with paratuberculosis from known infected herds were tested. The majority of samples were from goats vaccinated against MAP. Positive or doubtful PCR results were obtained in none of the 120 and two of the 22 colostrum samples, and in eight of the 202 and four of the 27 milk samples Negative PCR results were obtained in the remaining 140 (99%) colostrum samples and 217 (95%) milk samples.

Monitoring diagnosis, retention in care and viral load suppression in children testing HIV polymerase chain reaction-positive in two districts in South Africa.

Retention in care is associated with improved virological control and survival among HIV-infected children. However, retention of children in HIV care remains a challenge. To describe, using routine laboratory HIV test data, the retention-in-care and virological outcomes of HIV-infected children aged &lt;18 months in two districts in South Africa. HIV polymerase chain reaction (PCR)-positive results of children from uMkhanyakude and Tshwane districts in KwaZulu-Natal and Gauteng provinces, respectively, tested between April 2015 and May 2016, were extracted from the National Health Laboratory Service's Corporate Data Warehouse (CDW). HIV-related tests (PCR, viral load (VL), CD4+) were documented longitudinally for each child for ≥13 months after the first positive PCR result by manually searching demographics within the CDW, supplemented by an automated patient-linking algorithm. Test sets were linked if two or more demographics (surname, name, date of birth, folder number) matched exactly. Programmatic indicators assessed included age at first positive PCR test, presumed confirmatory test rates, retention in care, and VL suppression at 6 and 12 months. Ninety-four and 304 children tested HIV PCR-positive in uMkhanyakude and Tshwane, respectively. The median age at diagnosis was 3.6 months (interquartile range (IQR) 1.4 - 7.1) for uMkhanyakude and 2.3 months (IQR 0.1 - 6.7) for Tshwane. In uMkhanyakude, confirmed in utero infections accounted for 18.1% of transmissions (n=17), compared with 29.6% (n=90) in Tshwane. Presumed confirmatory test rates following an initial positive PCR result were 77.7% and 71.7% for uMkhanyakude and Tshwane, respectively. Within 6 months of starting antiretroviral therapy, 43 children (58.9%) were lost to follow-up in uMkhanyakude compared with 160 (73.4%) in Tshwane. Of those retained in care at 6 months with a VL measurement, 15 (60.0%) from uMkhanyakude had a VL &lt;1 000 copies/mL, compared with 24 (48.0%) in Tshwane. For both districts, a third of all HIV PCR-positive children were retained in care at the end of follow-up, with 29 (30.9%) in uMkhanyakude and 99 (32.5%) in Tshwane. Of these, 12 (41.4%) had a VL &lt;1 000 copies/mL in uMkhanyakude compared with 28 (28.3%) in Tshwane. We demonstrate the value of routine laboratory data in monitoring diagnosis, retention and VL suppression in HIV-infected children. This approach is scalable, can be reported near real-time, is relatively inexpensive to implement, and provides a tool for improving paediatric HIV services until clinical databases can assume this role.

Scabies polymerase chain reaction with standardized dry swab sampling: an easy tool for cluster diagnosis of human scabies.

Expert visualization of Sarcoptes scabiei remains essential for diagnosing human scabies, but access to said experts can be difficult. Polymerase chain reaction (PCR) is a specific tool for the detection and confirmation of S. scabiei but has poor sensitivity. To evaluate PCR as a diagnostic method for scabies using nonexpert-dependent standardized sampling. The dry swab was systematically rubbed across the front of both wrists, the eight interdigital spaces and on any suspected scabies lesions in all patients referred for scabies. A new PCR-based diagnostic test was run on the samples. All patients underwent clinical and dermoscopic examination. Scabies diagnosis was confirmed when dermoscopic examination was positive or the patient had typical clinical signs of scabies. Of 183 suspected cases of scabies, 164 patients were sampled, 87 had confirmed scabies (dermoscopy positive n = 87, typical clinical signs n = 1) and 77 did not. Of the 87 patients with proved scabies, 33 patients had positive scabies PCR, resulting in a 37·9% [95% confidence interval (CI) 28·4-48·4%] sensitivity and a 61·7% (95% CI 52·4-72·7%) negative predictive value. None of the 77 patients ruled out for scabies had a positive PCR result. This method is nontraumatic, repeatable and non-expert-dependent. It shows sensitivity similar to previous studies involving expert skin scraping. However, this method facilitates the multiplication of sampling, which increased the sensitivity for cluster scabies diagnosis. This method may be suitable as a first-line diagnosis tool where a large cluster scabies outbreak is suspected. What's already known about this topic? Scabies diagnosis requires expertise. Scabies polymerase chain reaction (PCR) is specific but has poor sensitivity. Poor sensitivity is the consequence of the low efficiency of sampling methods. What does this study add? This PCR-based diagnostic method based on nontraumatic standardized skin sampling is not expert-dependent and is reproducible. This diagnostic method may be relevant as a non-expert sentinel diagnosis tool in large clusters where a scabies outbreak is suspected.

Serodiagnosis of Human Bocavirus 1 Infection among Hospitalised Children with Lower Respiratory Tract Infection in Latvia

Abstract Since its discovery in 2005, human bocavirus 1 (HBoV1) has globally been one of the most common respiratory viruses. It is currently accepted that HBoV1 is a pathogen, causing upper and lower respiratory tract infections (LRTIs) in children. However, due to the prolonged HBoV1 DNA shedding from the upper airways and the subsequent high rate of co-detections with other respiratory viruses, the interpretation of positive polymerase chain reaction results is challenging. The aim of this study was to identify acute HBoV1 infections by the presence of HBoV1-specific IgM and IgG measured by competition enzyme immunoassay, to elucidate the induction of Th1/Th2 cytokines, and to describe the clinical characteristics associated with acute HBoV1 infection in hospitalised children less than five years of age with LRTI. HBoV1 IgM was detected in 19/102 (18.6%) and IgG in 66/102 (64.7%) patients. HBoV1 IgM was most frequently found in patients aged 13 to 24 months. Pneumonia and acute wheezing were the most common clinical diagnoses among HBoV1 IgM positive patients. The seroprevalence of HBoV1-specific IgG increased with age, reaching 85% by the age of five years. INF-γ, IL-4, IL-5, and IL-10 were observed to be higher in patients with acute HBoV1 infection.

Use of Routine Complete Blood Count Results to Rule Out Anaplasmosis Without the Need for Specific Diagnostic Testing.

Anaplasmosis presents with fever, headache, and laboratory abnormalities including leukopenia and thrombocytopenia. Polymerase chain reaction (PCR) is the preferred diagnostic but is overutilized. We determined if routine laboratory tests could exclude anaplasmosis, improving PCR utilization. Anaplasma PCR results from a 3-year period, with associated complete blood count (CBC) and liver function test results, were retrospectively reviewed. PCR rejection criteria, based on white blood cell (WBC) and platelet (PLT) counts, were developed and prospectively applied in a mock stewardship program. If rejection criteria were met, a committee mock-refused PCR unless the patient was clinically unstable or immunocompromised. WBC and PLT counts were the most actionable routine tests for excluding anaplasmosis. Retrospective review demonstrated that rejection criteria of WBC ≥11 000 cells/µL or PLT ≥300 000 cells/µL would have led to PCR refusal in 428 of 1685 true-negative cases (25%) and 3 of 66 true-positive cases (5%) involving clinically unstable or immunocompromised patients. In the prospective phase, 155 of 663 PCR requests (23%) met rejection criteria and were reviewed by committee, which endorsed refusal in 110 of 155 cases (71%) and approval in 45 (29%), based on clinical criteria. PCR was negative in all 45 committee-approved cases. Only 1 of 110 mock-refused requests yielded a positive PCR result; this patient was already receiving doxycycline at the time of testing. A CBC-based stewardship algorithm would reduce unnecessary Anaplasma PCR testing, without missing active cases. Although the prospectively evaluated screening approach involved medical record review, this was unnecessary to prevent errors and could be replaced by a rejection comment specifying clinical situations that might warrant overriding the algorithm.

The yield of monitoring adenovirus in pediatric hematopoietic stem cell transplant patients

Human adenovirus (HAdV) is recognized as a serious pathogen after allogeneic hematopoietic stem cell transplantation (HSCT), causing morbidity and mortality. Currently, there is no universal agreement regarding routine HAdV surveillance after HSCT. We assessed the impact of HAdV weekly monitoring by polymerase chain reaction (PCR) on HAdV viremia rates and the risk factors that influence survival. Three-hundred and fifty-six pediatric allogeneic HSCT were done between 2007 and 2015. Until July 2011, HAdV testing was performed based on clinical suspicion (cohort 1, n = 175) and from August 2011, weekly blood-HAdV monitoring was done (cohort 2, n = 181) until day +100. Twenty-three patients (4 [2.3%] from cohort 1 and 19 [10.5%] from cohort 2, p = .001) were found with HAdV viremia and seven of them died. Both cohorts had a similar incidence of HAdV-associated mortality (3/175; 1.7% in cohort 1 and 4/181; 2.2% in cohort 2). Respiratory failure was the cause of death in all patients. Clinical symptoms appeared prior to or within 5 days of HAdV detection in cohort 2. In summary, weekly monitoring was associated with higher detection of HAdV. The study could not assess survival benefit due to small numbers of HAdV-positive cases. In many instances, symptoms occurred with the development of positive HAdV blood PCR results and hence, symptomatology could have triggered the test. Future studies are needed to provide data that help establishing a uniform approach for regular monitoring of HAdV post-transplant.

English

Bovine tuberculosis causes huge economic losses to the dairy industry in terms of poor growth, reduce milk production potential of infected animals, emaciation and poor quality of carcass. Bovine tuberculosis due to Mycobacterium bovis is a major threat to public health. Therefore, this study was aimed at to know the frequency of Mycobacterium bovis infection in cattle and buffaloes. A total of 265 animals, including adult cattle (132) and buffaloes (133) of different age and sex were screened for tuberculosis infection with tuberculin test. Equal numbers of milk and nasal swab samples were collected from tuberculin reactor and non-reactor cattle and buffaloes for the isolation and identification of the causative organism. The infectious agent was identified and confirmed through procedures such as culture characteristics, ZN (Ziehl-Neelsen) staining, and PCR (Polymerase Chain Reaction) was also performed. The study revealed an overall 10.56% (95% C.I. 7.28 to 14.71) prevalence of bovine tuberculosis, the frequency of infectious agent was higher in buffaloes (11.04%) than cattle (9.76%). TB (Tuberculosis) was present in 100% herds. Multivariate logistic regression analysis with the backward elimination procedure revealed that, age (OR=1.564), body weight (OR=1.008) and status (OR=5.72) showed a significant association with the occurrence of tuberculosis. Nasal samples yielded more positive PCR results than milk samples. By considering PCR as a gold standard, ZN was more sensitive and PPD was a more specific test. Among all tests, PCR proved most accurate and fast test for the confirmation of bovine tuberculosis

КОМПЛЕКСНОЕ ИССЛЕДОВАНИЕ МОКРОТЫ ВПЕРВЫЕ ВЫЯВЛЕННЫХ БОЛЬНЫХ ТУБЕРКУЛЁЗОМ ЛЁГКИХ С ИСПОЛЬЗОВАНИЕМ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ И МИКРОБИОЛОГИЧЕСКОГО КОНТРОЛЯ ЖИЗНЕСПОСОБНОСТИ МИКОБАКТЕРИЙ ТУБЕРКУЛЁЗА

Objective: The use of polymerase chain reaction (PCR) in a comprehensive study of sputum in newly diagnosed patients with pulmonary tuberculosis (PTB) in the control of mycobacterium tuberculosis (MTB) viability by the microbiological method. Methods: The object of the study – 59 newly diagnosed patients with PTB in admission for treatment and 28 patients with PTB two months after the start of anti-tuberculosis chemotherapy. The study material is the sputum of PTB patients. Identification of the dead and persistent MTB was noted with the positive result of PCR (PCR+) and the absence of growth of MTB on the dense of nutritional environment of Levenstein-Jensen. The DNASorb-B and Litekh sets were used for DNA extraction of Mycobacterium tuberculosis complex. For amplification, Politub and AmpliSens MBT kits were used. Detection of amplification products was carried out with electrophoresis in 1.7% agarose gel in the presence of bromide ethidium Results: Before treatment, PCR was detected in 52 (88.1%) of the 59 PTB patients examined, 34 (57.6%) tested positive for inoculation (culture+). 25 (42.4%) of patients, lack of growth of MTB out of 59 PTB patients. After 2 months of chemotherapy, PCR+ was detected in 23 (82.1%) out of 28 patients with PTB, culture+ – in 13 (46.4%), no growth of MTB was revealed in 15 (53.6%). Conclusion: Before treatment, PCR was detected in 52 (88.1%) of the 59 PTB patients examined, 34 (57.6%) tested positive for inoculation (culture+). 25 (42.4%) of patients, lack of growth of MTB out of 59 PTB patients. After 2 months of chemotherapy, PCR+ was detected in 23 (82.1%) out of 28 patients with PTB, culture+ – in 13 (46.4%), no growth of MTB was revealed in 15 (53.6%). Keywords: Pulmonary tuberculosis, polymerase chain reaction, microbiological method, mycobacterium tuberculosis viability, chemotherapy.

Clinical Utility of Molecular Diagnosis of Blood Stream Infections in Allogeneic Hematopoietic Stem Cell Transplantation Recipients with Hematologic Malignancies

Blood stream infections (BSIs) are a serious problem in patients with hematologic malignancies receiving allogeneic hematopoietic stem cell transplantation (ASCT). We evaluated the clinical utility of molecular diagnosis for the management of BSIs in such patients. We prospectively performed a polymerase chain reaction (PCR) analysis of microbial DNA in blood samples from 10 consecutive patients with hematological malignancies at least once a week for one month after ASCT. In total, 51 and 54 samples were analyzed by bacterial and fungal PCR assays, respectively. Bacteria were detected in 24 samples from 8 patients by PCR, but in only 2 samples from one patient by blood culture. Notably, the bacteria detected in at least half of the 24 samples were considered to have originated from the oral cavity. Fungi were detected in 5 samples from 3 patients by PCR, but not by blood culture. Most cases with positive PCR results were manageable with empirical antimicrobial therapy without disclosure of DNA data. Our DNA analyses did not directly contribute to management of BSIs, but did provide valuable microbiological evidence for the patients. Additionally, oral management appears to require a critical re-evaluation to reduce the occurrence of BSIs in ASCT recipients.

Detection of single-nucleotide polymorphism Gap junction protein Beta-2 genes in deaf schoolchildren of javanese population in Surabaya, Indonesia

Background: Genetic factors account for about 50%–75% responsible for hearing loss. The existence of single-nucleotide polymorphism (SNP) as genetic factors that affect hereditary hearing loss. The widely studied SNP was the gap junction protein beta-2 (GJB2) gene encoding the gap junction beta-2 protein (connexin26) that found in cochlea and required to convert sound waves into electrical nerve impulse. This study was aimed to detect the SNP GJB2 gene of hereditary hearing loss patients from Javanese population in Surabaya, Indonesia. Methods: The design of this study was a cross-sectional, analytic observational. The participant was taken randomly among the students from a deaf School in Surabaya. The questionnaire was completed by the parents of the deaf children. Blood sampling was taken from venous peripheral blood. DNA was extracted and amplified on GJB2 gene area by polymerase chain reaction (PCR). The positive results of PCR were processed further for sequencing. The sequencing results were analyzed to detect the GJB2 gene SNP with reference sequence/rs-80338939. Results: A total of 22 children participated in this study; all were profound sensorineural hearing loss (SNHL). The hereditary hearing loss was obtained with fewer in five children (22.73%), who had a history of hearing loss in their family. It was compared to 17 children (77.27%) who had no family history of hearing loss. It was found that the nucleotide variation in nucleotide number 8473 of GJB2 gene as much as 3 (13.64%) out of 22 children in hereditary hearing loss patients in Deaf School Type B Surabaya. Conclusions: This study did not found any SNP GJB2 gene (rs-80338939) of hereditary hearing loss patients from the Javanese population in Surabaya, Indonesia. There was the nucleotide substitution G to A in nucleotide number 8473 of GJB2 gene, which indicated the change of amino acid code genetic code table (valine) to amino acid code genetic code table (isoleucine). It may as the cause of SNHL.

First Report of East African Cassava Mosaic Virus-Uganda Infecting the Nile Tulip Tree in Western Kenya

Whitefly-transmitted cassava mosaic begomoviruses (CMBs; family Geminiviridae; genus Begomovirus) are viruses with bipartite genomes (components referred to as DNA-A and DNA-B) containing a conserved (between the two components) noncoding sequence known as the common region (CR) required for DNA replication and transcription (Fondong 2013). CMBs cause cassava mosaic disease (CMD), a major limitation to cassava production in sub-Saharan Africa. In Kenya, CMD is associated with the begomoviruses East African cassava mosaic virus (EACMV), African cassava mosaic virus (ACMV), East African cassava mosaic Kenya virus, and East African cassava mosaic Zanzibar virus, as well as several recombinant viruses, such as the virulent EACMV-Uganda (EACMV-UG) that so far occurs only in western Kenya (Nyongesa Were et al. 2016). Within the CMB-cassava pathosystem, various noncassava plant species may play an epidemiological role as virus reservoirs for the whitefly vector Bemisia tabaci (Alabi et al. 2008). In May 2017, leaf samples were collected from three plants exhibiting virus-like symptoms including mosaic, mottling, twisted leaves, and reduction in leaf surface area as well as from two nonsymptomatic plants belonging to the family Bignoniaceae growing in three cassava fields in western Kenya. Total genomic DNA was extracted from leaf samples using the ZR Plant/Seed DNA MiniPrep kit as per the manufacturer’s instructions (Zymo Research Corp., U.S.A.). The identity of the plant species was confirmed as Markhamia lutea by phenotypic characteristics and by polymerase chain reaction (PCR) amplification, Sanger sequencing, and BLASTn analysis of the chloroplast rubisco large-chain gene using the primer pair RbcLa-F (5′-ATGTCACCACAAACAGAGACTAAAGC-3′)/RbcLa-R (5′-GTAAAATCAAGTCCACCRCG-3′). Samples were screened by PCR with the diagnostic primer pairs JSP001/JSP002 for ACMV, JSP001/JSP003 for EACMV (Fondong et al. 2000), and UV AL1-F1/ACMV CP-R3 for EACMV-UG (Zhou et al. 1997). Samples from nonsymptomatic plants did not yield amplification products, confirming absence of viral infection. A 1,650-bp fragment of the expected size was amplified from one symptomatic sample with the primer pair UV AL1-F1/ACMV CP-R3, suggesting the presence of EACMV-UG. To confirm the positive PCR result, the genome of the virus was enriched by rolling-circle amplification (RCA) using a TempliPhi 100 RCA Kit (GE Healthcare, U.S.A.). The library was prepared using the NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, U.S.A.) according to the manufacturer’s instructions and sequenced using an Illumina MiSeq platform with the MiSeq Reagent Kit version 3 (600 cycles) (Illumina, U.S.A.). Following de novo sequence assembly, 344 and 89 paired-end reads aligned to generate two contigs identified by BLASTn search as the DNA-A and DNA-B of a bipartite virus. The nucleotide sequences were deposited in GenBank as accession numbers MF974568 and MH379641. The 2,798-nucleotide (nt) DNA-A and 2,774-nt DNA-B had an approximately 160-nt EACMV-like CR with 53%-nt sequence identity. DNA-A shared 99% nucleotide sequence identity with the DNA-A of an EACMV-UG isolate (AJ717522) from western Kenya, whereas the DNA-B was closely related to another EACMV-UG isolate (AJ704962) from western Kenya at 97% nucleotide sequence identity. To the best of our knowledge, this is the first report of occurrence of EACMV-UG in a species of the family Bignoniaceae. Further investigations are required to determine the epidemiological significance of M. lutea in the CMB-cassava pathosystem.

Detection of Occult Hepatitis B Virus Among Iranian HCV-Infected Patients with Hemophilia Treated with Direct-Acting Antiviral Agents

Background: One of the challenges to treat hepatitis C virus (HCV) infection is the activation of hepatitis B virus (HBV) that occurs during treatment of HCV infection with direct-acting antivirals (DAAs) in some patients. The detection of serum or liver HBV DNA in the absence of serum HBsAg (HBV surface antigen) is described as occult HBV infection (OBI). Objectives: The current study aimed at determining the prevalence of OBI in Iranian DAA-naive HCV-infected patients with hemophilia. Methods: The current study was conducted on 100 patients with hemophilia receiving DAAs. The sera obtained from these patients were tested for the presence of HBsAg. Then, the presence of the HBV DNA was detected in peripheral blood mononuclear cell (PBMC) and also plasma samples using nested polymerase chain reaction (PCR). Results: Among the 100 study subjects, 81 (81%) were male and 19 (19%) female. The mean age of the patients was 37 ± 10.50 years. All patients had previously received HBV vaccine. In the current study, HBV DNA was observed in 1% of plasma and in 3% of PBMC samples. In addition, none of the patients who had positive result for HBV detection test previously had markers of HBV infection (anti-HBc (HB core antigen) antibodies, anti-HBs antigens, and positive result of DNA PCR) and all had negative results for HCV RNA after treatment. Conclusions: Generally, the prevalence of OBI was low, but however, HBsAg negativity was not sufficient to completely exclude the presence of HBV DNA. Thus, the serological markers of HBV infection should be confirmed by molecular tests for the presence of possible occult infection.

Clinical Results and Utility of Herpesviruses Multiplex Polymerase Chain Reaction: Assessment of Aqueous Humor Samples From Patients With Corneal Endotheliitis and High Intraocular Pressure.

The main purpose of this study was to evaluate herpesvirus infection in patients with corneal endotheliitis and high intraocular pressure (IOP) using multiplex polymerase chain reaction (PCR) in aqueous humor samples. This was a retrospective, observational study of immunocompetent patients living in South Korea. Eligible subjects had typical corneal endotheliitis with an IOP≥21 mm Hg or required antiglaucoma medication. Multiplex PCR was performed using aqueous humor samples obtained at first visit to detect the DNA of 6 herpesviruses. Forty-two eyes from 42 patients with >6 months' follow-up were analyzed. Of these, 16 were herpesvirus-positive: 3 herpes simplex virus 1, 3 varicella-zoster virus, 9 cytomegalovirus, and 1 Epstein-Barr virus. Eyes with coin-shaped or fine keratic precipitates (kps), high IOP, and a low baseline endothelial cell count were more likely to show a positive result on multiplex PCR. Univariate analysis showed that male sex (P=0.014), a previous history of uveitic glaucoma (P=0.048), and the presence of fine kps (P=0.031) were significantly associated with a positive PCR result. On multivariate analysis, male sex (P=0.010) and a previous history of uveitic glaucoma (P=0.031) showed a significant positive association. Cytomegalovirus was the most commonly detected herpesvirus in patients with corneal endotheliitis and high IOP. A positive PCR result was seen more frequently in male individuals and patients with fine kps or a history of uveitic glaucoma.

Molecular Identification of Leishmania spp. DNA from Archived Giemsa-Stained Slides of Patients from Salta, Argentina.

Cutaneous leishmaniasis is endemic in Salta province, which belongs to the northwest of Argentina. Leishmania spp. DNA from Giemsa-stained slides of up to 12 years in storage of patients from Salta was characterized through polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). One hundred smears positive for microscopy, classified in a semiquantitative scale for amastigote density, were analyzed. Also, Leishmanin skin test (LST) results were included. DNA extraction was carried out applying lysis buffer with proteinase K, and then DNA was amplified with ribosomal internal transcribed spacer 1 primers. PCR products were digested with HaeIII enzyme. All PCR-positive smears (74/100) belonged to Viannia subgenus. A statistically significant, directly proportional relationship between semiquantitative microscopy and PCR results was detected. All patients had LST-positive results (induration ≥ 5 mm), and the smears of those with smaller induration (LST < 19 mm) gave a higher proportion of positive PCR results. This study determined that smear age did not affect PCR positivity, which allows retrospective analyzes and suggests smears might be useful for molecular complementary diagnosis. Because Leishmania (Viannia) braziliensis is the main circulating species in the study area, determining Viannia subgenus in all analyzed samples confirms previous findings. PCR positivity showed statistically significant differences according to semiquantitative microscopy, highlighting the importance of parasite burden in the diagnostic sensitivity of the method. Considering that smears of patients with smaller LST induration were more positive in PCR, a negative smear from patients with positive LST response, but < 19 mm, could actually represent a false-negative result.