Multiplex Nested PCR Research Articles

Pattern and Prevalence of Hepatitis B virus genotypes in Among Discordant Partners Enugu, Southeastern Nigeria

Hepatitis B virus (HBV) continues to cause significant global health problems despite the presence of a potential vaccine and other clinical approaches. This study was designed to investigate the pattern and spread of HBV genotypes among discordant and concordant partners of hepatitis B virus infection in Enugu State, Nigeria. The study involved a cross-sectional study consisting of 150 subjects (120 discordant and concordant partners of hepatitis B, and 30 controls). HBsAg was screened using a rapid ELISA diagnostic strip and re-screened to reaffirm the result using Ichroma Fluorescence Immunoassay (FIA) analyser. Multiplex nested polymerase chain reaction using type specific primers was used to identify the HBV genotypes. Statistical analysis was performed using Graph Pad Prism. HBV genotype E (75%), A (10%) and a mixed genotype of A and E (mixed infection (15%) were gotten with genotype E being the most predominant. This study has established the high prevalence of genotype E as the most predominant HBV genotype in circulation in Enugu, southeastern Nigeria.

HIGH RISK HPV DETECTION IN PRECURSOR LESIONS OF CERVIX USING LBC SAMPLES.

Background: Liquid-based cytology (LBC) was developed as an alternative to conventional cytology screening to improve specimen adequacy and sensitivity in detecting cervical abnormalities. High risk HPV (HR HPV) types 16 and 18 are responsible for about 70% of all cervical cancer cases worldwide. Objectives: This study aimed (1) to compare the diagnostic efciency of liquid based cytology LBC and conventional tests in detecting cervical dysplasia and (2) detection of high risk HR HPV types 16 and 18 in cervical precursor lesions using liquid based cytology samples. Methods: A prospective comparative study was conducted from the year August 2014 – February 2019 at Basavatarakam Indoamerican Cancer Hospital and Research Institute, Hyderbad.A total of 200 women undergoing screening for cervical cancer were included in the study. HR-HPV 16 & 18 detection was done by Nested Multiplex Polymerase Chain Reaction (NMPCR). Results: The area under the ROC curve for conventional Pap smear was 0.725 and LBC was 0.974, showing that the diagnostic accuracy of LBC method in diagnosing precursor lesions and carcinoma of cervix is better than the conventional Pap smear method. HR-HPV 16/18 were positive in 43/102 (42.2%) of cases. HPV 16 alone was positive in 32/43(74.4%), HPV 18 alone in 6/43 (13.9%) and both HPV-16 and HPV-18 were positive in 5/43 (11.6%) of the total HR-HPV positive cases. Conclusion: The detection rate of epithelial abnormalities and infections in LBC preparation was better than CPS. LBC samples can be used for HR-HPV genotype detection to explore new paradigms of screening strategies.

High Prevalence of Asymptomatic Malarial Anemia and Association with Early Conversion from Asymptomatic to Symptomatic Infection in a Plasmodium falciparum Hyperendemic Setting in Cameroon.

Asymptomatic malarial parasitemia is highly prevalent in Plasmodium falciparum endemic areas and often associated with increased prevalence of mild to moderate anemia. The aim of this study was to assess the prevalence of anemia during asymptomatic malaria parasitemia and its interplay with persistent infection in highly exposed individuals. A household-based longitudinal survey was undertaken in a malaria hyperendemic area in Cameroon using multiplex nested polymerase chain reaction to detect plasmodial infections. Residents with P. falciparum asymptomatic parasitemia were monitored over a 3-week period with the aid of structured questionnaires and weekly measurements of axillary temperatures. Of the 353 individuals included (median age: 26 years, range 2-86 years, male/female sex ratio 0.9), 328 (92.9%) were positive for malaria parasitemia of whom 266 (81.1%) were asymptomatic carriers. The prevalence of anemia in the study population was 38.6%, of which 69.2% were asymptomatic. Multivariate analyses identified high parasitemia (> 327 parasites/µL) and female gender as associated risk factors of asymptomatic malarial anemia in the population. Furthermore, risk analyses revealed female gender and anemia at the time of enrolment as key predictors of early development of febrile illness (< 3 weeks post enrolment) among the asymptomatic individuals. Together, the data reveal an extremely high prevalence of asymptomatic malaria parasitemia and anemia in the study area, unveiling for the first time the association of asymptomatic malarial anemia with early clinical conversion from asymptomatic to symptomatic infection. Furthermore, these findings underscore the negative impact of asymptomatic malaria parasitemia on individual health, necessitating the development of appropriate control and preventive measures.

The detection and phylogenetic analysis of equine herpesviruses 1, 4 and 5 identified in nasal swab samples of asymptomatic horses from Serbia and Bosnia and Herzegovina.

Nasal swabs originating from 112 apparently clinically healthy and unvaccinated horses of different age, breed and from diverse rearing conditions from Serbia and Bosnia and Herzegovina were examined for the presence of equine herpesviruses 1, 4 and 5 using multiplex nested PCR (Mn‑PCR) and virus isolation. The detected viruses were subsequently characterised by gB gene nucleotide sequencing and their phylogenetic analysis was performed. The infections with EHV‑1, EHV‑4, and EHV‑5 in the examined horse populations are apparently chronic, subclinical and persistent, whilst the shedding of EHV‑1 and EHV‑5 was confirmed by their successful isolation. A connection was established between the finding of EHVs and rearing conditions since horses kept together in stables were positive for at least one EHV in contrast to animals held free grazing or individually. EHV‑5 was found most often in younger horses, however descending in frequency in animals up to 10 years of age. The phylogenetic analysis showed that the identified EHV strains group mostly with Turkish and German strains of respective viruses. A certain degree of genetic heterogeneity was determined regarding the identified EHV‑5 strains in contrast to EHV‑1 and EHV‑4.

E6-E7 based nested multiplex PCR assay for genital HPV detection and simultaneous typing of 15 high and low-risk HPV types

PurposeDue to a wide range of Human Papillomavirus (HPV) types associated with genital cancers; HPV genotyping remains important for the introduction of an appropriate vaccine, disease diagnosis, follow-up and epidemiological surveys. Currently, available molecular genotyping assays are not only expensive but also requires dedicated and expensive equipment which is not feasible in the majority of low-and-middle-socioeconomic countries. The purpose of the study was to develop and evaluated a cost-effective nested-multiplex polymerase chain reaction (NM-PCR) assay for HPV genotyping. MethodsHPV-DNA containing plasmids and cervical scrapings from histologically confirmed cervical cancer cases were used to evaluate the NM-PCR. In the first round PCR, a set of consensus primers were used to amplify 38 mucosal HPV types. HPV Type-specific primers were used in the second-round polymerase chain reaction (PCR) to amplify 15 HPV types in three multiplex cocktails. The assay sensitivity was determined with the control panel containing one to 1010genome equivalents (GE). DNA sequencing was done to confirm the PCR results. ResultsThe assay was able to amplify all HPV types and detected as few as 50GE per reaction. A total of 23 endo-cervical samples obtained from healthy, HPV negative subjects and 52 histologically confirmed cervical scrapings were processed for HPV genotyping by NM-PCR. HPV DNA was detected in all histologically confirmed samples. DNA sequencing results showed complete concordance with PCR results. ConclusionsThe designed nested PCR based assay had good concordance with clinical histology and sequencing results and appears to be a promising tool for HPV genotyping especially in resource-constrained settings.

Feasibility of a High-Volume Filter Sampler for Detecting SARS-CoV-2 RNA in COVID-19 Patient Rooms.

Aerosolization of SARS-CoV-2 by COVID-19 patients can put healthcare workers and susceptible individuals at risk of infection. Air sampling for SARS-CoV-2 has been conducted in healthcare settings, but methods vary widely and there is need for improvement. The objective of this study was to evaluate the feasibility of using a high-volume filter sampler, BioCapture z720, to detect SARS-CoV-2 in COVID-19 patient rooms in a medical intensive care unit, a dedicated COVID-19 ward, and at nurses’ stations. In some locations, the BioSpot-VIVAS, known for high efficiency in the collection of virus-containing bioaerosols, was also operated. The samples were processed for SARS-CoV-2 RNA with multi-plex nested polymerase chain reaction. One of 28 samples collected with the high-volume filter sampler was positive for SARS-CoV-2; all 6 samples collected with BioSpot-VIVAS were negative for SARS-CoV-2. The high-volume filter sampler was more portable and less intrusive in patient rooms than the BioSpot-VIVAS, but limits of detection remain unknown for this device. This study will inform future work to evaluate the reliability of these types of instruments and inform best practices for their use in healthcare environments for SARS-CoV-2 air sampling.

A new highly sensitive single-tube nested real-time PCR assay: Clinical utility in perinatal HIV-1 diagnosis

Real time PCR is one of the major tools for molecular diagnosis, however not always reaches the required sensitivity, especially in detecting early infectious disease. To overcome this problem, nested PCR is commonly performed, since it is highly sensitive, but it is time-consuming, prone to cross-contamination and difficult to standardize. Therefore, we developed a sensitive and specific single-tube nested real-time PCR (STN-real-time PCR) assay and evaluated its clinical utility on early infant HIV-1 diagnosis (EID). The STN-real-time PCR enables the simultaneous amplification of four HIV-1 specific amplicons by the use of an internal and external pair of primers targeting ltr/gag region, and another one corresponding to human albumin as an internal control. Thermocycling had different annealing temperatures to favor the sequential use of each pair of primers, and included an initial touchdown step to broaden specificity and increase sensitivity. Finally, HIV-1 was detected by melting curve analysis. A total of 234 samples collected retrospectively and prospectively from HIV-1 exposed infants aged <18 months were used to evaluate the performance of the assay and compare it with a routine diagnostic nested-multiplex PCR. The developed assay had a limit of detection of 3 copies of HIV-1 DNA per reaction and had a sensitivity of 31 % more than routine diagnostic nested-multiplex PCR when testing samples near delivery. In conclusion, we developed a new assay by turning a conventional nested-PCR into a faster, more sensitive and feasible STN-real-time PCR assay for EID and potentially useful for detection of pathogens with variable genomes and present in low copy numbers.

Investigation of LAMP Technique in Diagnosis Type of Plasmodium Species in Anopheles Mosquitoes :A Fast and Practical Technique to Detect Malaria Pathogens in the Field.

Malaria is one of the main parasitic diseases and a major health issue in some countries. This study aims to determine the rate and type of infections of Anopheles mosquitoes with malaria parasites using the molecular LAMP method in the Southeastern Iran. In this study, 400 Anopheles mosquitoes were collected by the Zahedan Medical Insecticide Center in Nikshahr City, a high-risk area of malaria transmission in Sistan-Baluchestan Province. The mosquitoes were caught manually (by hand) in domestic (humans and animals), natural, and artificial outdoor places (Shelter pits). After DNA extraction, the LAMP method was used, which was compared with Multiplex Nested-PCR as a standard method. Out of 400 samples collected from Nikshahr City, 6 samples (1.5%) were infected with Plasmodium vivax. No Plasmodium falciparum or a mix (Plasmodium vivax and Plasmodium falciparum) was detected in this study. The results of this study indicate that in places with transmission of both species, i.e. Plasmodium vivax and Plasmodium falciparum, detection of malaria parasites by the LAMP method could be very useful in spotting infections in the field. Thus, molecular epidemiological studies could be conducted annually to monitor malaria in endemic regions. The results of this research show that contamination with mosquito malaria vectors is increasing in Nikshahr City, and it seems that more studies will be required to eliminate malaria until 2026.

Cytomegalovirus Glycoprotein Polymorphisms and Increasing Viral Load in Non-Transplant Patients with Hematological Malignancies Undergoing Chemotherapy: A Prospective Observational Study.

IntroductionCytomegalovirus (CMV) predisposes to several clinical complications and is a major cause of morbidity and mortality in immunocompromised patients, including patients with hematological malignancies (HM). The present study was carried out to determine the distribution of CMV glycoprotein B, N, and O (gB, gN, and gO) genotypes and their potential effect on its viral load and on clinical outcomes in a cohort of Tunisian non-hematopoietic stem cell transplant (HSCT) patients with HM undergoing chemotherapy.MethodsCMV viral load was evaluated by real-time quantitative PCR. The gB, gN, and gO genotypes of the CMV strains were analyzed by multiplex nested PCR and sequencing.ResultsThis prospective study involved 60 clinical isolates obtained from 60 non-HSCT patients with HM undergoing chemotherapy. Mixed CMV gB, gN, and gO genotypes were the predominant glycoprotein genotypes in 31%, 41.4%, and 46.4% of patients, respectively. Mixed gB genotypes were associated with higher initial levels of CMV load (p = 0.001), increased rate of fever (0.025), and co-infection with other herpesviruses (HHVs) (p = 0.024) more frequently than in single gB genotype. Mixed gN genotypes were more associated with severe lymphopenia (ALC < 500/µL) (p = 0.01) and increased risk of death (p = 0.042) than single gN genotype. Single gO2b genotype had also a more unfavorable outcome (p = 0.009) than the other single gO genotype. Mixed gO genotypes were associated with female gender (p = 0.015), acute leukemia disease (p = 0.036), initial high level of CMV viral load (at least 1000 copies/mL) (p = 0.029), skin rash (p = 0.01) more frequently than in single gO genotype. The gO1a/gN3b linkage was associated with an increased initial viral load (p = 0.012).ConclusionInfection with mixed CMV genotypes was common and multiple gB, gN, and gO genotypes were associated with clinical manifestation and higher viral load.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40121-021-00457-z.

Molecular identification of Entamoeba histolytica, E. dispar, and E. moshkovskii in children with diarrhea from Maracaibo, Venezuela

ResumenIntroducción. Las amebas no patógenas Entamoeba dispar, Entamoeba moshkovskii y Entamoeba bangladeshi son morfológicamente idénticas a Entamoeba histolytica, parásito responsable de la amebiasis, por lo cual se necesitan técnicas moleculares para diferenciarlas.Objetivo. Determinar la frecuencia de las diferentes especies de Entamoeba mediante reacción en cadena de la polimerasa (Polymerase Chain Reaction, PCR) en muestras fecales de niños menores de cinco años con diarrea, provenientes de Maracaibo (Venezuela).Materiales y métodos. Se recolectó una muestra fecal por individuo en 75 niños con diarrea (grupo de casos) y en 25 niños sin diarrea (grupo control). Las heces se evaluaron mediante examen microscópico, método de concentración de formól-éter y PCR múltiple anidada en una sola ronda para identificar E. histolytica, E. dispar y E. moshkovskii. Además, se hizo una encuesta en la que se recopilaron los datos demográficos, signos, manifestaciones clínicas y estrato socioeconómico de los niños.Resultados. El 48 % de los participantes (38 del grupo de casos y 10 del grupo de control) tenían enteroparásitos. Solo en las muestras de cuatro de los niños, se encontraron quistes del complejo Entamoeba (tres en el grupo de casos y uno en el de control). Mediante PCR se amplificaron nueve muestras (9 %) para la detección de las amebas estudiadas. En el grupo de casos se registraron tres (28,13 %) de E. histolytica, cuatro (30,50 %) de E. dispar y una (9,37 %) de E. moshkovskii, en tanto que solo una (25 %) muestra amplificó para E. dispar en el grupo de control.Conclusión. En general, predominó E. dispar; sin embargo, todos los infectados con E. histolytica se detectaron en el grupo de niños con diarrea y se detectó el primer caso de E. moshkovskii en la región.

Identification of Candida Species Circulating in Dakar using Multiplex Nested PCR

Background: In most developing countries, laboratory identification of Fungi belonging to the genus Candida is often problematic. The germ test tube known as Blastese test, which is the most used has the limitation that it only differentiates the C. albicans complex from other Candida complexes. Not only it is important to know the involvement of other species in the occurrence of fungal infections, but also the difference in sensitivity to the azoles used in the treatment of these fungal infections makes it necessary to identify the Fungi at the species level. The objective of this study was to identify by multiplex nested PCR the Candida species isolated in the laboratory of Parasitology and Mycology of Aristide Le Dantec Hospital, Senegal. Methodology: Candida isolates were obtained from patients who were admitted to the laboratory of Parasitology and Mycology with suspected fungal infections. After identification by conventional methods, isolates were preserved in a storage medium and kept at -20°C. Species identification was performed by nested PCR. Results: A total of 42 yeast isolates were collected after culture on Sabouraud medium, among which 10 (24%) from vaginal swabs and 32 (76%) from patients suffering from superficial mycoses (skin and nails). C. albicans represented 68% and nonCandida albicans (NAC) species 32%. The distribution of the NAC species was as follows: C. parapsilosis (group I and II) 17% (8), C. tropicalis 8.5% (4), C. kefyr 4.25% (2) and C. lusitaniae 2.15% (1). Mixed infection with two species represented 17.39% of the isolates. Conclusion: This study shows that other Candida species are common in Dakar, although their identification is not routinely performed in most laboratories. Thus, it is necessary to improve the technical facilities in these diagnostic laboratories.

Circulation profile of respiratory viruses in symptomatic and asymptomatic children from Midwest Brazil.

Acute respiratory infection (ARI) is a major cause of morbidity and mortality worldwide. Most of these infections are caused by viruses. Infections pose as important triggers of acute episodes of chronic respiratory diseases (CRD). This study sought to evaluate the frequency and circulation profile of respiratory viruses among ARI symptomatic patients and completely asymptomatic children in Midwest Brazil. The study enrolled symptomatic children with and without ARI symptoms. During 1 year, 225 nasal respiratory samples were obtained from patients aged 4–14 years old. The samples were screened by multiplex nested-PCR for 16 common respiratory viruses. From 225 samples, 42 had at least one virus detected. Samples from four different patients had multiple viruses detected. The viral detection rate in symptomatic (20.1%) and asymptomatic patients (14.8%) showed no significant difference. The most frequent viruses detected were rhinovirus (28.6%), FLUA (11.9%), adenovirus (11.9%), human bocavirus (HBoV) (11.9%), and respiratory syncytial virus (RSV) antigenic group A (9.5%). Monthly detection rate was higher during the rainy season. RSVs were detected during the months with higher rainfall indexes and higher air humidity, while FLU and HBoV were detected during the winter months. The obtained results reinforce the importance of viral pathogens in pediatric population, emphasizing similar viral occurrence in symptomatic and asymptomatic children.Electronic supplementary materialThe online version of this article (10.1007/s42770-020-00368-0) contains supplementary material, which is available to authorized users.

Detection of human herpes viruses 1-5 in miscarriage: A case-control study.

BackgroundMiscarriage is the spontaneous pregnancy loss before 24 wk of gestation. The incidence rate of miscarriage over the past few decades has shown steady or even growing trends. Viral intrauterine infections are one of the probable etiological causes of miscarriage. Previous evidence have shown that human herpes viruses (HHVs) could be considered as the potential reasons for intrauterine infections and adverse pregnancy outcomes.ObjectiveThis case-control study aimed to detect HHV1-5 DNAs in placental tissues and assess their association with miscarriage during the first 24 wk of pregnancy in spontaneous and therapeutic abortions.Materials and MethodsPlacental tissues from 83 women with spontaneous abortions during the first and the second trimesters of pregnancy and 81 women with therapeutic abortion during the same gestational age were collected. The DNA extraction was performed by the phenol/chloroform method. A part of the DNA polymerase gene of HHVs was amplified with multiplex nested-polymerase chain reaction. The polymerase chain reaction products were subjected to sequencing.ResultsThe results showed the presence of human cytomegalovirus genome in the placenta of both spontaneous (8.4%) and therapeutic (4.9%) abortions. No statistically significant differences were found between these two groups. The other investigated viruses were not detected here.Conclusion In conclusion, like some other studies, no correlation was detected between the HHVs placental infections and the increased risk of spontaneous abortions. In order to find the actual role of HHVs infections in miscarriage, further investigations should be performed on a larger sample size in different areas.

Molecular Pattern of Anti-malarial Drug Resistance of Plasmodium falciparum in Bangladeshi Troops Working in Endemic Areas of Bangladesh and Africa

Members of Bangladesh Armed Forces work in two different malaria endemic area, Chittagong Hill Tracts (CHT) in Bangladesh and Sub-Saharan countries in Africa. This under-recognized group remained unexplored for long in respect to drug resistant falciparum malaria they usually suffer from. In this study, a total of 252 ‘dried blood samples on filter paper’ were collected between November 2014 and February 2016, from Plasmodium falciparum positive Bangladeshi troops working in Chittagong Hill Tracts (CHT), Bangladesh and five Sub Saharan African Countries namely, Central African Republic (CAR), Democratic Republic of Congo (DRC), Liberia, Mali and Ivory Coast. After DNA extraction from all these samples (94 from Bangladesh and 138 from African countries), plasmodium species was confirmed by a nested PCR following standard protocol with minor modifications. Thereafter, a multiplex nested PCR followed by restriction fragment length polymorphism (RFLP) method was employed to investigate the presence of chloroquine resistance marker ‘K76T mutation’ in P. falciparum chloroquine resistance transporters (pfcrt) gene and lumifantrine and mefloquine resistance marker ‘N86Y mutation’ in P. falciparum multidrug resistance1 (pfmdr1) gene. The P. falciparum DNA was confirmed in 35 (37.23%) Bangladeshi and 45 (28.48%) African samples. The ‘pfcrt (K76T) mutation’ that confers resistance to chloroquine, was detected in 93.10% Bangladeshi and 29.27% African samples. The ‘pfmdr1 (N86Y) mutation’ that confers resistance to lumifantrine and mefloquine, was detected in 20.69% Bangladeshi and only 2.44% African samples. The higher prevalence of chloroquine resistance of P. falciparum in Bangladesh than in African countries revealed that possible withdrawal of chloroquine from endemic areas and also periodic molecular survey to monitor pf resistance to chloroquine, mefloquine, lumefantrine and artemisinin among these troops working in both endemic areas.&#x0D; Bangladesh J Microbiol, Volume 37 Number 1 June 2020, pp 1-6

Molecular Detection of Occult Hepatitis B virus in plasma and urine of renal transplant patients in Khartoum state Sudan

ObjectiveThe aim of this study was to determine the OBI in plasma and urine samples from renal transplant patients using Multiplex Nested PCR. MethodA total of 100 samples (plasma and urine) were collected from renal transplant patients admitted to the renal transplant center in Khartoum north, Sudan in 2019. For each sample, HBsAg, HBeAg and anti HBcAg were detected using Enzyme linked Immune sorbent assay (ELISA). The viral DNA was then extracted using viral DNA extraction kit and were then tested for HBV DNA by using multiplex nested PCR. Statistical analysis was done using statistical package of social science (IBM SPSS version 20.0) considering a P value ≤ 0.05 as a level of significance. ResultsHBsAg were not detected in al patient but, HBeAg were 14 (14%) and anti HBcAg were 36 (36%)were detect by using ELISA. A total 18 (18%) and 3 out of 100 were found positive in plasma and urine samples, respectively. Regarding the virus genotypes, D, E and mixed D/E genotypes were detected in all positive samples. Females were significantly (P value=0.013) higher detectable with HBV than males in plasma samples ConclusionOBI incidence in renal transplant patients is high in Sudan. The multiplex nested PCR had identified OBI with a high rate supporting the efficiency of using molecular techniques in detecting of HBV. This will lead to an appropriate diagnosis and minimizing the risk to be infected by HBV.

Clinical Significance of Common Gene Mutations in 53 Patients with Acute Myeloid Leukemia Harboring 11q23/MLL Rearrangements

To investigate the clinical significance of AML patients with 11q23/MLL rearrangement, and to evaluate the effect of those mutations on the AML patients. 53 cases involving translocations of chromosome 11q23 were identified by chromosome banding analysis. MLL rearrangements were detected by fluorescence in situ hybridization and/or multiplex nested PCR. The samples were screened for mutations in the candidate genes FLT3-ITD, FLT3-TKD, TET2, N-RAS, ASXLI, EZH2, DNMT3, C-Kit, NPM1, WT1, CEBPA by using genomic DNA-PCR and deep-sequencing. 21/53 MLL-rearranged AML cases showed at least one additional chromosomal aberrations. The most common additional aberration was +8. Gene mutations were observed in 23 cases (43.4%) and most cases showed singal mutation. N-RAS mutation was more frequent (8 cases, 15.1%), followed by WT1 mutation in 4 cases (7.5%), FLT3-ITD mutation in 3 cases, ASXL1 mutation in 2 cases, DNMT3A mutation in 2 cases, EZH2 mutation in 1 case, c-Kit17 mutation in 1 case, FLT3-TKD mutation in 1 case, and FLT3-ITD and TKD mutation coexistent in 1 case. No mutation was detected in CEBPA, NPM1, C-KIT8, TET2. Median OS for gene mutated patients was 8.5 months and 13 months for no mutated patients. Median OS for patients who received hematopoietic stem cell transplantation (HSCT) was 22.5 months and 7.5 months for patients who olny received chemotherapy. A relatively high mutation frequency is observed in AML patients with 11q23/MLL rearrangements and most cases shows single mutation. The RAS signaling pathway alterations are most common. Gene mutation does not affect the OS of these patients, who show poor prognosis. A significantly higher Hb at initial diagnosis in FLT3 mutated patients is significantly higher than that in FLT3 wild-type cases. Patients who underwent HSCT show a better prognosis than those only received chemotherapy.

Simultaneous Detection and Direct Identification of Phytoplasmas in the Aster Yellows (16SrI), Clover Proliferation (16SrVI) and Stolbur (16SrXII) Groups Using a Multiplex Nested PCR Assay in Potato Plants

Phytoplasmas in the aster yellows (16SrI), clover proliferation (16SrVI) and stolbur (16SrXII) groups have a wide host range with common hosts. In the current study, specific primers based on the 16-23S rRNA spacer region were designed and optimised for the detection and identification of phytoplasmas in these groups in a multiplex nested PCR assay. Fifty-two phytoplasma symptomatic potato plants were tested, and clover proliferation (16SrVI) and stolbur (16SrXII) phytoplasmas detected coinfecting 16 potato plants with single infection of these phytoplasmas in 5 and 21 samples, respectively. ‘Candidatus P. asteris’ (16SrI) was not detected. The specificity of the primers was proven in both single and multiplex PCR assays. Detection of multiple infections can be useful in assisting epidemiological studies and in deciding appropriate disease management strategies.

Prevalent Latent Human Cytomegalovirus Genotype b2 in Biopsy Samples of Gastric Cancer

Aim: Genotyping and prevalence of human cytomegalovirus (HCMV) in gastrointestinal tract remains unclear. Objective: Characterization of HCMV and its viral load in samples of patients with gastric cancer (GC) in northeast Iran. Methods: A total of 21 biopsies were collected from patients with GC. HCMV was detected and genotyped using multiplex nested PCR. Quantitative real-time PCR has been used to determine the viral load. Staining was performed to detect viral inclusions. Results: 76.19% of the samples had HCMV genotypic b2 with viral load &lt;0.1 IU/μl. 66.67% of patients were positive for H. pylori. Conclusion: Multiplex nested-PCR method can detect CMV populations with low viral loads in GC biopsies. For the first time, it was determined that the genotype b2 of HCMV is prevalent in the gastrointestinal tract.

Self-Collected Specimens Revealed a Higher Vaccine- and Non-Vaccine-Type Human Papillomavirus Prevalences in a Cross-Sectional Study in Akuse

Background Population-specific epidemiologic data on human Papillomavirus infection, which are limited in most of the SubSaharan African countries, are necessary for effective cervical cancer prevention. This study aimed to generate population-specific data on human Papillomavirus infections, and determine which of these, self-collected and provider-collected specimens, gives a higher estimate of the prevalence of human Papillomaviruses, including vaccine and non-vaccine-type human Papillomavirus. Methods In this cross-sectional study, following a questionnaire-based collection of epidemiological data, self-, and provider-collected specimens, obtained from women 15−65 years of age, were analysed for human Papillomavirus types by a nested-multiplex polymerase chain reaction, and for cervical lesions by Pap testing. HPV data were categorised according to risk type and vaccine types for further analysis. Results The difference between the overall human Papillomavirus infection prevalences obtained with the self-collected specimens, 43.1% (95% CI of 38.0–51.0%) and that with the provider-collected samples, 23.3% (95% CI of 19.0–31.0%) were significant (p ≤ 0.001). The prevalence of quadrivalent vaccine-type human Papillomaviruses was 12.3% with self-collected specimens, but 6.0% with provider-collected specimens. For the nonavalent vaccine-types, the prevalences were 26.6% and 16.7% respectively. There were multiple infections involving both vaccine-preventable and nonvaccine preventable high-risk human Papillomavirus genotypes. Conclusion The Akuse subdistrict can, therefore, be said to have a high burden of human Papillomavirus infections, which included nonvaccine types, as detected with both self-collected and provider-collected specimens. These imply that self-collection is to be given a higher consideration as a means for a population-based high-risk human Papillomavirus infections burdens assessment/screening. Additionally, even with a successful implementation of the HPV vaccination, if introduced in Ghana, there is still the need to continue with the screening of women.

Genotypes of glycoprotein B gene among the Indian symptomatic neonates with congenital CMV infection

BackgroundCytomegalovirus [CMV] is a causative agent of congenital infection worldwide and often leads to neurological deficits and hearing loss in newborns. Infants born with symptomatic congenital Cytomegalovirus infection [cCMV] are at significant high risk for developing adverse long-term outcomes. In this study, we look into the sequence variability of surface glycoprotein B [gB] encoding region in newborns with symptomatic CMV infection for the first time in Eastern region of India.Methods576 suspected newborns from seropositive mothers were subjected to the study and ELISA was used to confirm CMV infection. Different genotypes and their subtypes were determined using multiplex nested-PCR. Viral load of different glycoprotein B [gB] genotypes was measured using RT-PCR. Sequencing and phylogenetic analysis was then performed using Bayesian interference.ResultsThe overall frequency of cCMV infection was 18.4%, where 16.0% neonates were symptomatic. Among the different gB genotypes, gB1 had the highest frequency [23.5%] and gB4 showed the lowest occurrence [5.8%]. 23.5% of symptomatic neonates had mixed genotypes of gB, probably indicating matrenal reinfection with CMV strains in Indian population. Significant genotypic clades [gB1-gB2-gB3-gB5] were grouped closely based on gene sequences, but the gB4 sequence was in the outlier region of the phylogenetic tree indicating the genetic polymorphism.ConclusionThis is the first study on cCMV genotyping and its phylogenetic analysis from Eastern Indian neonatal population. The study holds importance in the assessment of cCMV seroprevalence in global perspective. gB protein can be used as a potential therapeutic target against CMV infection.