Hemi-nested Polymerase Chain Reaction Research Articles

Phylogenetic analysis of small ruminant lentiviruses in Mongolian sheep supports an ancient east-west split for the genotype A.

The ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) are small ruminant lentiviruses (SRLVs) with striking genetic and structural similarities. The presence of SRLV in Mongolian sheep and goats was serologically demonstrated more than a decade ago; however, the viral genotype remains unknown. In total, 329 blood samples were collected from two sheep breeds (i.e., Khalkha and Sumber) in Tov, Govisumber, Arkhangay, Dornogovi, Zavkhan, and Sukhbaatar provinces, Mongolia. Serological and phylogenetic analyses were performed regardless of any apparent clinical signs, although most of the animals appeared healthy. All sheep in three of the six provinces were seronegative, whereas the seroprevalence in the Tov, Govisumber, and Zavkhan provinces averaged 7.9%. Genomic DNA from seropositive animals was tested using hemi-nested polymerase chain reaction, and sub-genomic SRLV sequences were determined from nine samples. Mongolian SRLV sequences clustered within the divergent subtype A22, which was previously found only in Fertile Crescent regions, including Lebanon, Jordan, and Iran, where the first sheep-domestication (Ovis aries) occurred. According to the phylogenetic analysis, genotype A has two ancestors from the ancient Fertile Crescent: (1) Turkish strains and (2) Iranian, Jordanian, and Lebanese strains. The first ancestor spread westward, whereas the second spread eastward, ultimately reaching Mongolia.

A Genotyping Method for Detecting Foreign Buffalo Material in Mozzarella di Bufala Campana Cheese Using Allele-Specific- and Single-Tube Heminested-Polymerase Chain Reaction.

Mozzarella di Bufala Campana (MdBC) cheese is a Protected Designation of Origin (PDO) product that is important for the economy and cultural heritage of the Campania region. Food fraud can undermine consumers' trust in this dairy product and harm the livelihood of local producers. The current methods for detecting adulteration in MdBC cheese due to the use of buffalo material from foreign countries could exhibit limitations associated with the required use of expensive equipment, time-consuming procedures, and specialized personnel. To address these limits here, we propose a rapid, reliable, and cost-effective genotyping method that can detect foreign buffalo milk in a counterpart from the PDO area and in MdBC cheese, ensuring the quality and authenticity of the latter dairy product. This method is based on dedicated allele-specific and single-tube heminested polymerase chain reaction procedures. By using allele-specific primers that are designed to detect the nucleotide g.472G>C mutation of the CSN1S1Bbt allele, we distinguished an amplicon of 330 bp in the amplification product of DNA when extracted from milk and cheese, which is specific to the material originating from foreign countries. By spiking foreign milk samples with known amounts of the counterpart from the PDO area, the sensitivity of this assay was determined to be 0.01% v/v foreign to PDO milk. Based on a rough estimate of its simplicity, reliability, and cost, this method could be a valuable tool for identifying adulterated buffalo PDO dairy products.

Ultrasonographic features of malignant catarrhal fever induced corneal oedema in cattle

The study was aimed to record the ocular ultrasonographic changes in the confirmed clinical cases of MCF. The cases were confirmed using hemi-nested polymerase chain reaction assay. Ocular ultrasonography was performed using a 10–18 MHz linear transducer (My Lab 40 Vet). Clinical signs observed were corneal oedema, lacrimation, photophobia, corneal oedema and hyperaemia of conjunctiva. In two animals, mild corneal oedema was observed while in four animals each, moderate and severe corneal oedema was recorded. In all the animals, corneal oedema initially appeared at the limbus with subsequent involvement of the entire cornea with varying degree of severity. Hemi-nested polymerase chain reaction was used for confirming the diagnosis. Ocular ultrasonographic examination revealed significant increase in the thickness of the cornea, iris and ciliary body. Anterior uveitis, characterized by thickened iris, exhibiting crust and trough formation covering the major part of the anterior surface of the lens was recorded in nine animals. There was no recovery in any of the case and the animals died within one month of the diagnosis of the MCF. Significant ultrasonographic changes were evident in eyes of animals affected with the malignant catarrhal fever. Ultrasonography may therefore be used for monitoring the ocular changes especially in the anterior segment of the opaque eyes.

Molecular detection and characterization of ovine herpesvirus-2 using heminested PCR in Pakistan.

BackgroundMalignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo, and pigs caused by the gamma-herpesviruses alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2).ObjectivesThis study aimed to determine the prevalence of OvHV-2 in sheep, goats, cattle, and buffalo in Rawalpindi and Islamabad, Pakistan, by applying molecular and phylogenetic methods.MethodsBlood samples were aspirated from sheep (n = 54), goat (n = 50), cattle (n = 46) and buffalo (n= 50) at a slaughterhouse and several farms. The samples were subjected to heminested polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the OvHV-2 POL gene and the OvHV-2 ORF75 tegument protein gene.ResultsThe highest percentage of MCF positive samples was in sheep (13%), whereas goat, cattle, and buffalo had lower positive percentages, 11%, 9%, and 6.5%, respectively. Four OvHV-2-positive PCR products obtained from sheep samples were sequenced. The sequences obtained were submitted to the NCBI GenBank database (MK852173 for the POL gene; MK840962, MK852171, and MK852172 for the ORF75 tegument protein gene). Phylogenetic analysis revealed a close similarity of study sequences with those of worldwide samples.ConclusionsThis study is the first cross-sectional study on the prevalence and molecular detection of OvHV-2 in apparently healthy cattle and buffalo that could be carrying OvHV-2 acquired from OvHV-2-positive sheep and goats. The results indicate that OvHV-2 is circulating in Pakistan. Further studies are needed to characterize OvHV-2 and elucidate further its prevalence.

Lineage analysis of human papillomavirus types 31 and 45 in cervical samples of Iranian women.

Knowing the regional lineages/sublineages of human papillomavirus 31 (HPV31) and 45 would be of great importance for further evolutionary, epidemiological, and biological analysis. In this regard, to characterize more common lineages and sublineages of HPV 31 and 45, the sequence variations of E6 gene were investigated in normal, premalignant, and malignant samples collected from the cervix in Iran. In total, 54 HPV 31- and 24 HPV 45-positive samples were analyzed by hemi-nested polymerase chain reaction (PCR) and nested-PCR, respectively. All PCR products were subjected to direct sequencing analysis. The results indicated that all three lineages A, B, and C were detected in HPV 31-positive samples; among which HPV 31 lineage A was dominant as it was found in 66.7% of all samples. HPV 31 lineages B and C were identified in 5.5% and 27.8% of samples, respectively. In HPV 45-infected samples, lineage B comprised of 62.5% of all samples and the remaining 37.5% belonged to lineage A. In conclusion, our findings showed that lineage A of HPV 31 was predominant in Iran. Lineage B of HPV 45 was also dominant among Iranian women. However, further studies with larger sample size should be addressed to estimate the pathogenicity risk of HPV 31 or HPV 45 lineages/sublineages in the development of cervical cancer among Iranian women.

A novel flavivirus strain detected in phlebotomine sandflies in Bosnia and Herzegovina.

Aim Phlebotominae sandflies are primary vectors of phleboviruses, causing the sandfly fever disease. The aim of this study was to detect and report the presence of flaviviruses in Phlebotominae sandflies captured in Bosnia and Herzegovina. Methods After a microscopic and morphometric analysis, the final identification of collected Phlebotomus specimens was confirmed by PCR, using a hemi-nested polymerase chain reaction on extracted and reversely transcribed RNA. Results We obtained a 155 nt long fragment of the viral non-structural protein 5 (NS5) gene (GenBank accession no. MN090154). The acquired nucleotide sequence, provisionally named as Drežnica, showed a maximum of 70-80% identity in 70-88% (110-137 nucleotides) of the query coverage with several Anopheles, Sabethes, Calbertado and Culex flaviviruses. Maximum likelihood phylogenetic analysis showed that the new flavivirus Drežnica clusters together with the flavivirus isolated from Culiseta annulata mosquitos. Conclusion We report the presence of flavivirus in Phlebotominae sandflies, captured in Drežnica, Herzegovina for the first time. The next phase of research will be directed towards virus cultivation, obtaining a longer or complete virus sequence and clarifying the medical and epidemiological importance of the Drežnica virus.

The Impact of Rotavirus Vaccines on Genotype Diversity: A Comprehensive Analysis of 2 Decades of Australian Surveillance Data.

Introduction of rotavirus vaccines into national immunization programs (NIPs) could result in strain selection due to vaccine-induced selective pressure. This study describes the distribution and diversity of rotavirus genotypes before and after rotavirus vaccine introduction into the Australian NIP. State-based vaccine selection facilitated a unique comparison of diversity in RotaTeq and Rotarix vaccine states. From 1995 to 2015, the Australian Rotavirus Surveillance Program conducted genotypic analysis on 13051 rotavirus-positive samples from children <5 years of age, hospitalized with acute gastroenteritis. Rotavirus G and P genotypes were determined using serological and heminested multiplex reverse-transcription polymerase chain reaction assays. G1P[8] was the dominant genotype nationally in the prevaccine era (1995-2006). Following vaccine introduction (2007-2015), greater genotype diversity was observed with fluctuating genotype dominance. Genotype distribution varied based on the vaccine implemented, with G12P[8] dominant in states using RotaTeq, and equine-like G3P[8] and G2P[4] dominant in states and territories using Rotarix. The increased diversity and differences in genotype dominance observed in states using RotaTeq (G12P[8]), and in states and territories using Rotarix (equine-like G3P[8] and G2P[4]), suggest that these vaccines exert different immunological pressures that influence the diversity of rotavirus strains circulating in Australia.

Rotavirus prevalence and genotype distribution in children with acute gastroenteritis in Adana province

Rotavirus is the most common agent of acute gastroenteritis in children under five years of age and still a major cause of mortality and morbidity mainly in devoloping countries. The aim of this study was to determine the circulating rotavirus genotypes in children less than five years of age with acute gastroenteritis in Adana province, before the introduction of the rotavirus vaccine into the national immunization programme. Fecal specimens were collected from children enrolled between December 2012 and November 2016 and rotavirus antigen was searched by a rapid immunochromatographic test method. The antigen positive samples were G and Pgenotyped by hemi-nested type-specific multiplex reverse-transcriptase polymerase chain reaction (RT-PCR). Rotavirus antigen positive fecal specimens from 201 patients of 107 (53.3%) boys and 94 (46.8%) girls were included in this study. Rotavirus infection was most frequently detected in patients with gastroenteritis between the ages of 12 and 23 months. It has been determined that 45% of rotavirus gastroenteritis was determined in children under two years of age. Rotavirus gastroenteritis was more frequent between September and April, but the highest incidence was detected in January. Of 201 rotavirus antigen-positive stool specimens, 167 were PCR positive. Five common G (G1, G2, G3, G4 and G9) and two common P genotypes (P[8] and P[4]) were determined as 91.6% and 96.4% of the isolates, respectively. The most prevalent G and P genotype combination was G1P8 with a frequency of 29.9%, followed by 21% G9P8 About 78% of rotavirus isolates were classified into five common G-P combination genotypes, including G1P8, G9P8 G3P8, G2P4, and G2P8. During the study period, there were significant changes in the distribution of rotavirus genotype. The prevalence of G9P8 genotype sharply decreased from 40% to 8.1% and the prevalence of G1P8 genotype increased from 21.3% to 48.6%. Although no G3P8 isolates were detected in the first two years of the study, this genotype was detected in 18.5% and 13.5% of the samples in 2015 and 2016, respectively. There were no relationship between the age onset of the disease, sex, dehydration development risk and rotavirus genotype. The results from this study indicated that the predominant circulating strains of rotavirus determined in clinical specimens were G1P8 and G9P8. Significant changes were observed in the prevalence of the most common rotavirus isolates over the years. Rotavirus surveillance is important to determine the circulating strains of rotavirus to assess vaccine effectiveness, to monitor vaccine failures, and to detect potential emerging strains.

Differential Expression of MicroRNAs in Uterine Cervical Cancer and Its Implications in Carcinogenesis; An Integrative Approach.

Cervical cancer is the second most common cancer in women in developing countries, including India. Recently, microRNAs (miRNAs) are gaining importance in cancer biology because of their involvement in various cellular processes. The present study aimed to profile miRNA expression pattern in cervical cancer, identify their target genes, and understand their role in carcinogenesis. Human papillomavirus (HPV) infection statuses in samples were assessed by heminested polymerase chain reaction followed by direct DNA sequencing. Next-generation sequencing and miRNA microarray were used for miRNA profiling in cervical cancer cell lines and tissue samples, respectively. MicroRNA signature was validated by quantitative real-time PCR, and biological significance was elucidated using various in silico analyses. Cervical cancer tissues samples were mostly infected by HPV type 16 (93%). MicroRNA profiling showed that the pattern of miRNA expression differed with respect to HPV positivity in cervical cancer cell lines. However, target and pathway analyses indicated identical involvement of these significantly deregulated miRNAs in HPV-positive cervical cancer cell lines irrespective of type of HPV infected. Microarray profiling identified a set of miRNAs that are differentially deregulated in cervical cancer tissue samples which were validated using quantitative real-time PCR. In silico analyses revealed that the signature miRNAs are mainly involved in PI3K-Akt and mTOR pathways. The study identified that high-risk HPV induces similar carcinogenic mechanism irrespective of HPV type. The miRNA signature of cervical cancer and their target genes were also elucidated, thereby providing a better insight into the molecular mechanism underlying cervical cancer development.

Clinical, laboratory and epidemiological aspects of HPV infection in a low-income population from South Bahia, Brazil.

The aim of this study was to determine the prevalence and risk factors for human papillomavirus (HPV) infection in the Southern region of the State of Bahia, evaluating the performance of alternative complementary methods for cervical lesion detection. Cervical samples from women who attended healthcare units were collected and diagnosed by visual inspection, cervical cytology and nested polymerase chain reaction (PCR). Moreover, hemi-nested PCR was performed to detect different HPV genotypes. The prevalence of HPV infection was 47·7%, with genotype 16 detected in most cases. Infection was associated with dyspareunia and bleeding (P < 0·001, odds ratio (OR) 5·6, 95% confidence interval (CI) 2·815-11·14) and hormonal contraceptive use (P = 0·007, OR 2·33, 95% CI 1·25-4·34). There was a positive correlation between positive PCR and positive visual inspection, cervical cytology and symptoms reported. Furthermore, visual inspection was twice as specific, and had a greater positive predictive value than cytology. We showed a high prevalence of HPV infection in Southern Bahia, with HPV 16 being the most common type, and visual inspection being most effective at detecting HPV lesions, corroborating the suggestion that it can be applied in routine gynecologic examinations for low-income populations.

High level of B19 strain detection in Brazilian cattle semen.

Semen contaminated with microorganisms can disseminate serious diseases including brucellosis. The objectives of this study were to detect Brucella-specific antibodies and Brucella abortus DNA in samples of blood and fresh semen from 100 animals older than 20months. The samples were collected on farms and in semen collection and processing centers (CCPS). The serum samples were evaluated by Rose Bengal test (RBT). B. abortus DNA was detected by a polymerase chain reaction (PCR) using BAB and IS771 primers. The difference between the vaccine field strain was identified using ery-1, ery-2, and ery-3 primers, using the hemi-nested PCR method. No anti-B. abortus antibodies were detected in the serum samples. Out of the total semen samples, 68% (68/100) presented amplifications of the B. abortus genes. All (68/68) were identified as B19 strain of Brucella abortus vaccine. It was concluded that even bulls that are seronegative for brucellosis can eliminate the bacteria in the semen. The presence in the DNA of the B19 vaccine strain should be investigated for a better understanding of the epidemiological importance of this strain in these animals.

The eIF3a Arg803Lys genetic polymorphism is associated with susceptibility to and chemoradiotherapy efficacy in cervical carcinoma

We aimed to explore the correlations between eukaryotic translation initiation factor 3, subunit A (eIF3a) polymorphisms and susceptibility to and chemoradiotherapy efficacy in cervical carcinoma. Between August 2007 and August 2011, 176 patients with cervical carcinoma were enrolled as the case group, and 180 healthy individuals were selected as the control group. eIF3a Arg803Lys C>T genotypes were detected by hemi-nested polymerase chain reaction restriction fragment length polymorphism. All patients received chemoradiotherapy and were evaluated for efficacy. Compared with carriers of the CC genotype, carriers of the T genotype of the eIF3a Arg803Lys C>T polymorphism had a higher risk of cervical carcinoma. The eIF3a Arg803Lys C>T polymorphism was associated with tumor size, differentiation degree, Federation of Gynecology and Obstetrics (FIGO) stage, and lymph node metastasis (LNM). The overall response rate of the case group was 69.32% (122/176). The response rate of CC genotype carriers was higher compared to patients with the CT+TT genotypes. Binary-logistic regression analysis showed that tumor size, FIGO stage, LNM, and the eIF3a Arg803Lys C>T polymorphism were influencing factors for chemoradiotherapy efficacy. Univariate analysis revealed that age, eIF3a Arg803Lys C>T polymorphism, differentiation degree, FIGO stage, and LNM were prognostic factors of cervical carcinoma, and multivariate analysis showed that age ≥ 60 years, higher FIGO stage, and LNM, as well as the CT and TT genotypes of the eIF3a Arg803Lys C>T polymorphism, were risk factors related to the prognosis of cervical carcinoma. The eIF3a Arg803Lys C>T polymorphism is connected with a higher susceptibility to cervical carcinoma and may affect chemoradiotherapy efficacy in and prognosis of cervical carcinoma.

The new frontier of diagnostics: Molecular assays and their role in infection prevention and control.

Recent advances in technology over the last decade have propelled the microbiology laboratory into a pivotal role in infection prevention and control. The rapid adaptation of molecular technologies to the field of clinical microbiology now greatly influences infectious disease management and significantly impacts infection control practices. This review discusses recent developments in molecular techniques in the diagnosis of infectious diseases. It describes the basic concepts of molecular assays, discusses their advantages and limitations, and characterizes currently available commercial assays with respect to cost, interpretive requirements, and clinical utility.

Detection of rabies in camel, goat and cattle in Sudan using Fluorescent antibody test (FAT) and hemi nested Polymerase Chain Reaction (hnRT-PCR)

Objective: The objective of this study was to identify rabies virus in camels and other animals in Sudan. Materials and methods: Four camel samples were collected from Garraht Elzawia, Kab-kabia and North Darfur areas in Sudan. The samples were collected based on clinical signs. In addition, two camel samples were obtained from Khartoum and Tambool, one goat sample was collected from El-Fashir, and one cattle sample was obtained from Atbara. The samples were transported to the Veterinary Research Institute (VRI) at Khartoum, Sudan for further studies. The samples were subjected for nested and hemi nested RT-PCR (hnRT-PCR) along with the gold standard Fluorescent antibody test (FAT) to diagnose rabies. Results: Out of eight samples, seven were found to be positive by both FAT and RT-PCR methods. The remaining one sample was positive by FAT but negative by hnRT-PCR indicating the suitablity of hnRT-PCR along with FAT for accurate diagnosis of rabies in animals. Conclusion: The study concluded that FAT and RT-PCR are useful tools for research and diagnosis of rabies.

Cross-sectional study indicates nearly a quarter of sheep population in Karnataka state of India is infected with ovine herpesvirus 2.

In a cross-sectional study, prevalence of ovine herpesvirus 2 (family: Herpesviridae, subfamily: Gammaherpesvirinae, genus Macavirus and species: Ovine herpesvirus 2) infection was estimated in sheep population of Karnataka state in India. Based on the three stage cluster sampling method, whole blood samples (356) of sheep were collected from 11 sheep-dense districts of the state. The samples were tested for presence of OvHV-2 genome by recommended hemi-nested polymerase chain reaction (PCR) test. The true prevalence of OvHV-2 infection in sheep population of Karnataka was 24.44%. Of the 11 district surveyed, highest true prevalence of 42.42% (CI 25.56-59.29) was found in Raichur followed by Tumkur (39.02%, CI 24.09-53.96). Inverse distance weighted interpolation of prevalence indicated that OvHV-2 prevalence within a given district is not uniform and there are areas of varied prevalence. The nucleotide sequence of the 422bp DNA fragment, amplified in PCR, matched 99% with OvHV-2 reference sequence and other sequences reported from India. Grouping of OvHV-2 sequences obtained from Karnataka with those from Andhra Pradesh, Tamil Nadu and Jammu and Kashmir in the neighbour joining tree indicated a close relationship among the OvHV-2s circulating in India. This is the first study in the country where systematic screening of sheep population of a state for the presence of OvHV-2 infection has been carried out, which indicated a widespread prevalence calling for an urgent need for policy measures to prevent economic losses due to the disease in susceptible cattle and buffalo species.

Towards single cell ad hoc protocols: First report of a healthy birth after preimplantation genetic diagnosis for methylmalonic acidemia

IntroductionMethylmalonic acidemia is an autosomal recessive disease, caused in 60% of cases by mutations in the MUT gene. The lack of l-methylmalonyl-CoA mutase leads to the accumulation of toxic compounds due to a metabolic block, leading to progressive encephalopathy and hyperammonemia that can end in death. Carrier couples are asymptomatic, and this disorder is not always included in the neonatal screening panel in Spain. Different reproductive options were explained to a couple with a previous affected son, and preimplantation genetic diagnosis was chosen. Both of them were carriers in heterozygosis of the same mutation c.671_678dup (p.V227fs) in exon 3 of the MUT gene. Material and methodsFifteen short tandem repeat markers closely linked to the MUT gene were tested, and the mutation c.671_678dup was detected by performing multiplex fluorescent heminested polymerase chain reaction. ResultsEight short tandem repeat markers were informative or partially informative, and the closest downstream and upstream to the MUT gene were selected in order to establish the disease-bearing haplotype. Nine blastomeres from nine embryos were analyzed, and one single embryo was transferred, resulting in a healthy birth. DiscussionCustomized direct preimplantation genetic diagnosis is a valid reproductive alternative to avoid transmission of methylmalonic acidemia to offspring. The ad hoc protocol designed for this couple allowed the birth of a healthy boy.

Comparison of immunochromatographic diagnostic test with Hheminested Reverse transcriptase polymerase chain reaction for detection of rabies virus from brain samples of various species.

Aim:Detection of rabies is a cause of serious concern in developing countries, where dearth of highly equipped laboratories and trained personnel to handle sophisticated investigations is felt. The availability of a diagnostic kit, which can be used in the field, is essential for diagnosis and control programs as well as for epidemiological surveillance of the prevalence of the disease. This study was planned to evaluate anigen rabies Ag test kit for its efficacy to be used for rapid diagnosis of rabies under field conditions. The test results were compared with hemi-nested reverse transcriptase polymerase chain reaction and with a gold standard fluorescent antibody test.Materials and Methods:A total of 34 brain samples from different rabies suspected animals including dogs, buffaloes, cow, horse, and cat were examined in this study.Results:Sensitivity of the kit was found to be 91.66%, specificity 100%, and accuracy was 94.11%.Conclusion:The study implies that the immunochromatographic diagnostic test kit may be employed for diagnosis of rabies in field conditions.

Refractory giardiasis: a molecular appraisal from a tertiary care centre in India.

The intestinal flagellate Giardia lamblia includes many genetically distinct assemblages, of which assemblage A and B, predominantly infect humans. Nitroimidazoles derivatives (metronidazole and tinidazole) and nitazoxanide are some of the therapeutic agents for treatment of giardiasis. Nevertheless, some individuals with giardiasis are non-responsive to standard therapy. The present study highlights cases of refractory giardiasis and attempts to elucidate if genetic heterogeneity in the parasite is associated with treatment failure. Three stool samples were obtained on three consecutive days from 4000 patients with diarrhoea and were microscopically examined for the detection of trophozoites, and/or cysts, using both normal saline and Lugol's iodine. A hemi-nested polymerase chain reaction (PCR) assay using triose phosphate isomerase (tpi) as the target gene was performed to determine the assemblages. Sequencing of the PCR products of the patients showing failure to treatment of giardiasis was also performed. Two per cent (82/4000) of the total patients were microscopically positive for Giardia lamblia in the stool samples. All these patients were treated with metronidazole/tinidazole as per the standard regimens. However, eight patients showed treatment failure to giardiasis as stool examinations were repeatedly positive even after treatment with multiple courses of anti-giardial therapy. Genetic characterisation of all eight Giardia isolates showed that they belonged to Assemblage B and had homogeneous sequences. These patients were either treated with extended regimens or with combination therapy of anti-giardials. In our experience, combination of two or more drugs for a longer duration is the treatment modality to treat refractory giardiasis.

Rotavirus G and P Types Circulating in the Eastern Region of Kenya

The World Health Organization has recommended that rotavirus (RV) vaccines be included in all national immunization programs as part of a strategy to control RV-associated diarrheal diseases. Hospital-based surveillance of RV infection is therefore crucial in monitoring the impact pre- and post-vaccine introduction and also to document changes in genotype distribution. This study sought to determine the RV genotypes circulating in the eastern region of Kenya before introduction of the RV vaccine. During September 2009 to August 2011, 500 stool samples were collected from children <5 years of age admitted for acute diarrhea in hospitals in the eastern region of Kenya and analyzed for the presence of group A RV using an enzyme immunoassay. G and P genotypes were determined using hemi-nested reverse transcriptase polymerase chain reaction. One hundred and eighty nine out of 500 (38%) samples analyzed were positive for rotavirus. The following G types were detected: G9 (50.9%), G1 (26.8%), G8 (12.1%), G12 (3.1%), G2 (0.6%), mixed G (1.3%) and 5.1% were G nontypeable. P types detected included: P[8] (63.7%), P[4] (12.1%), P[6] (4.5%), mixed P (7.6%) and 12.1% were P nontypeable. The most dominant strain was G9P[8] (35%), followed by G1P[8] (26.8%), G8P[4] (9.6%), G12P[6] (2.5%), G9P[6] (1.9%), G9P[4] (1.3%), G8P[8] (1.3%), and G2P[4] (0.6%). The present study demonstrates the recurring changing genotypes of RV circulating in Kenya, with genotypes G9, G1 and G8 being the dominant strains circulating in the eastern region of Kenya between 2009 and 2011. Additionally, G12 genotype was detected for the first time in Kenya.

Evaluation of efficiency of hemi-nested PCR assay for the detection of ‘Candidatus Liberibacter’ infecting citrus

The present study reports the development and evaluation of a hemi-nested polymerase chain reaction (hnPCR) assay for the efficient detection of ‘Candidatus Liberibacter’ infecting citrus plants. A set of new primers was designed by alignment of nucleotide sequences of the β-operon (rplKAJL-rpoBC) ribosomal protein genes from all the known ‘Candi-datus Liberibacter asiaticus’ isolates reported in Genbank. Hemi-nested PCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from hemi nested-PCR reactions confirmed the specificity of new primer pairs for ‘Candidatus Liberibacter asiaticus’. The reliability and sensitivity of hnPCR was evaluated by comparing it to real-time PCR.