Nested Polymerase Chain Reaction Technique Research Articles

Leishmania in skin of Rattus rattus from the urban area in the Corrientes city, Argentina

Objective: the objective of the present work was the detection of Leishmania sp. in Rattus rattus tail base skin through polymerase chain reaction (PCR) techniques. Material and Method: We analyzed 45 Rattus rattus skin samples from the urban area of the city of Corrientes in Argentina. The Leishmania spp detection was performed by nested PCR technique. Results: Leishmania DNA was detected in 22 samples out of 45 processed (49%) in the first round of amplification, and in 14 samples (31%) in the second round (31%). Conclusions: These results contribute to increase the existing information in our region on the possible relationship between Leishmania and Rattus rattus, considering the high prevalence found in skin added to the total absence of lesions. Other aspects should be further studied to establish the role of these animals in the epidemiological chain of the disease in an urban area endemic to leishmaniasis in other animal species.

Genetic variability and mutation of Epstein‒Barr virus (EBV)-encoded LMP-1 and BHRF-1 genes in EBV-infected patients: identification of precise targets for development of personalized EBV vaccines.

The critical Epstein‒Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) and BamHI fragment H rightward open reading frame 1 (BHRF-1) genes affect EBV-mediated malignant transformation and virus replication during EBV infection. Therefore, these two genes are considered ideal targets for EBV vaccine development. However, gene mutations in LMP-1 and BHRF-1 in different cohorts may affect the biological functions of EBV, which would seriously hinder development of personalized vaccines for EBV. In the present study, by performing nested polymerase chain reaction (nested PCR) and DNA sequence techniques, we analyzed the nucleotide variability and phylogeny of LMP-1 containing a 30bp deletion region (del-LMP-1) and BHRF-1 in EBV-infected patients (N = 382) and healthy persons receiving physical examination (N = 98; defined as the control group) in Yunnan Province, China. Three BHRF-1 subtypes were identified in this study: 79V88V, 79L88L, and 79V88L, with mutation frequencies of 58.59%, 24.24%, and 17.17%, respectively. Compared with the control group, the distribution of BHRF-1 subtypes of the three groups showed no significant difference, suggesting that BHRF-1 is highly conserved in EBV-related samples. In addition, a short fragment of del-LMP-1 was found in 133 cases, and the nucleotide variation rate was 87.50% (133/152). For del-LMP-1, a significant distribution in three groups was detected, as characterized by a high mutation rate. In conclusion, our study illustrates gene variability and mutations of EBV-encoded del-LMP-1 and BHRF-1 in clinical samples. Highly mutated LMP-1 might be associated with various types of EBV-related diseases, indicating that BHRF-1 combined with LMP-1 may be used as an ideal target for development of EBV personalized vaccines.

Occurrence of Cryptosporidium spp. in Traditional Milk and Dairy Products Supplied in Yazd City, Central Iran

Background: Cryptosporidium is one of the most important agents of food-borne diseases with gastrointestinal symptoms such as diarrhea in either livestock or humans. This protozoon can be transmitted to human through consuming contaminated raw milk and dairy products. The present study aimed to detect Cryptosporidium spp. in traditional raw cow milk, cream, and cheese consumed in Yazd city, Central Iran.
 Methods: Two hundred traditional (unpasteurized) milk and dairy samples were collected from five different regions of the studied area, including 100 traditional cow milk, 50 cow cheese, and 50 cow cream. DNA extraction was performed. Then, molecular detection was performed using the nested Polymerase Chain Reaction technique. Statistical analysis was performed using SPSS version 23.0.
 Results: The findings of this study showed that 11% samples were contaminated with Cryptosporidium spp., including 4.0% (4/100) milk, 18.0% (9/50) cream, and 18.0% (9/50) cheese (p<0.05). Besides, parasite contamination was 8.8, 12.5, 12.8, 8.3, and 12.8% in the Center, North, East, South, and West of the studied region, respectively (p>0.05).
 Conclusion: Due to the occurrence of Cryptosporidium spp. in unpasteurized dairy samples in Central Iran, developing and designing control and prevention programs is necessary against this parasite.

Molecular study of epstein Barr virus among patients with malignant diseases in al Najaf and Diwaniyah governorates

The present study is an effort to study the Epstein-Barr virus and it is relationship to the different types of cancers and their relationship to the virus and to the demographic distribution of the patients. our study started from Sep 2019 to Dec 2020 , blood samples were collected (2 ml of venous blood) were drawing by disposable syringe under a septic conditions. From patients with age grouped more 13 years old , represented by 47 specimens that subjected to molecular examination in Nested PCR technique detecting EBV LMP-1 in patients' blood as a main route. The study has included patients with different types of cancers from different areas. 47 specimens submitted to molecular examination, polymerase chain reaction (PCR) and nested PCR. Where EBV LMP-1 detected in 20 positive Specimens. All the virus isolates were sent to NCBI-BLAST to confirm the variation of the isolates. The study summarize that there is a strong relationship between EBV and some cancer diseases and show no significant difference according to age or gender or hereditary.

Seroprevalence and Genetic Characterization of Toxoplasma gondii among Children with Neurodevelopmental Disorders in Egypt.

Toxoplasma gondii is a parasite with a special predilection for the central nervous system. Toxoplasmosis's contribution to the triggering of many neurodevelopmental disorders was established. This study aimed to detect the seroprevalence and genotypes of T. gondii strains in children with neurodevelopmental disorders. The study included 180 children with neurodevelopmental disorders and 180 children in the control group. Assessment of seropositivity of Toxoplasma IgM and IgG antibodies in patients and controls was carried out. Genetic characterization of T. gondii was obtained by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) technique targeting dense granule gene (GRA6). Our results showed that the overall seroprevalence of T. gondii antibodies in the patient and controls was 35.6% and 11.7%, respectively. Nested PCR showed positivity in 11.1% of the patient group for T. gondii DNA. T. gondii seropositivity rate was significantly high in patients with hydrocephalus and also in patients with epilepsy. Positive nested PCR was significantly high in children with hydrocephalus only. Genotyping using nested PCR-RFLP showed genotype I (80%) followed by atypical strains (20%) with no association with any specific clinical presentation. In conclusion, among toxoplasmosis-positive children with neurodevelopmental disorders, analysis of T. gondii GRA6 locus revealed the predominance of type I genotype followed by atypical strains.

First Molecular Study and Sequencing of Cryptosporidium spp. Isolated from Human, Cattle and Sheep in Wasit Province, Iraq

The current study aimed to investigate Cryptosporidium spp. that infect humans and some animal species as well as to compare among various isolates obtained. A total of 96 human stool samples and 96 animal fecal samples (48 for cattle and 48 for sheep) were collected and examined by using Nested PCR technique followed by DNA sequencing analysis through phylogenetic tree to know the parasite species. The whole infection rate in human was 36.4% (35/96), while in cattle was 22.9% (11/48) and in sheep was 14.5% (7/48) by nested polymerase chain reaction technique. The findings of DNA sequencing based on 18s rRNA gene (654 bp) showed the presence of three Cryptosporidium spp. which namely C. hominis, C. parvum, and C. bovis, all of them have identity 99 %, Cryptosporidium parvum was the most widespread species being detected in 60% (9/15), followed by C. bovis 33.33% (5/15), and C. hominis 6.66% (1/15). This is the first report revealed the molecular sequencing among human, cattle and sheep in Wasit province, Iraq and the role of livestock (cattle, sheep) in zoonotic transmission of the cryptosporidiosis.

Molecular detection and identification of Babesia bovis and Trypanosoma spp. in one-humped camel (Camelus dromedarius) breeds in Egypt.

Background and Aim:Camels are a unique source of milk and meat, which helps recover from several diseases that affect humans worldwide. In Egypt, one of the great obstacles for this industry is tick-borne diseases. This study aimed to characterize blood parasite infections, such as Babesia (B.) bovis and Trypanosoma (T.) spp. in one-humped camel (Camelus dromedarius) (n=142) breeds in Halayeb and Shalateen, Egypt, through phylogenetic analysis.Materials and Methods:The prevalence of B. bovis and Trypanosoma spp. was identified in camels using polymerase chain reaction (PCR) assays targeting the Rhoptry-Associated Protein-1 and internal transcribed spacer 1 genes, respectively. A nested PCR technique was conducted to detect B. bovis. At the same time, KIN multispecies PCR assay was employed to diagnose and classify trypanosome DNA in camels.Results:B. bovis was detected in 4/142 camels with an infection rate of 2.81%. Sequencing and phylogenetic analyses revealed that the strain of B. bovis isolated from this population was closely related to strains isolated from Argentine, the United States, and Brazil. Moreover, Trypanosoma evansi was detected in 8/142 camels with an infection rate of 5.63%. Sequencing and phylogenetic analyses revealed that this isolated strain T. evansi was closely related to Trypanosoma theileri detected from cattle in Brazil.Conclusion:The obtained data indicated the existence of B. bovis and T. evansi in camels from two provinces of Egypt. The obtained findings have economic significance and reflect the importance of implementing effective prevention and control methods across Egypt to reduce the incidence of B. bovis and T. evansi in camels.

Evaluating Plasmodium misdiagnosis using microscopy compared with nested polymerase chain reaction technique in Rivers State, Nigeria

Evaluating Plasmodium misdiagnosis using microscopy compared with nested polymerase chain reaction technique in Rivers State, Nigeria

Detection of B1 Gene in Toxoplasma Gondii and the Role of Interleukin 2 in Abortive Women Infected by this Parasite

The study included 80 blood and serum samples of aborted women with Toxoplasma gondii infectionand healthy, uninfected and non-aborted women in the control group. For the period from August-2018to June-2019, in collaboration with the Falluja teaching hospital for women and children and the Ramaditeaching hospital for women and children, the Enzyme-Linked Immunosorbent Assay (ELISA), also to theToxoplasma gondii (B1) gene, the Nested polymerase chain reaction technique are used. The results of thestudy showed by that Elisa Toxoplasma gondii, IgG infection (50%), IgM infection (12.5%), and IgG & IgMInfection (12.5%), were mean (108.16±47.02) (99.02±31.47) (96.45±20.68) respectively compared with thecontrol group (25%) (0.94±0.93) and Interleukin-2 shown in IgG group (48.66±12.01) and control group(14.28±6.84) decreased compared to IgM (81.19±9.66), and IgG & IgM (80.71±9.18) group. Nonetheless,using nested PCR, the B1 gene showed just (12.5%) of all study samples in IgM and IgG&IgM alone andall IgG negativly

Performance of a Multiplex Nested Polymerase Chain Reaction in Detecting 7 Pathogens Containing DNA in Their Genomes Associated With Congenital Infections.

Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests. To develop a multiplex nested polymerase chain reaction (PCR) technique for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection. Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications. Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P < .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings. The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.

Identification of Amelogenin Gene on Burnt Teeth Samples through Nested Polymerase Chain Reaction Amplification for Sex Identification

Sex determination is one of the basic components in victim identification. This study aims to ascertain the sex of an individual from burnt teeth samples exposed at different temperature and time through nested polymerase chain reaction (PCR) on the amelogenin (AMEL) sex marker, to calculate the specificity and sensitivity, and to compare with previous relevant studies. A total of 17 teeth samples was subjected to burning at different temperatures ranging from 100°C to 500°C, at 2 to 10 minutes. The whole tooth was used for deoxyribonucleic acid (DNA) extraction by phenol-chloroform method. All samples were quantified for DNA concentration and then analyzed with nested PCR using two pairs of AMEL primer and results of sex typing were recorded. Out of 17 samples, genomic DNA extracted from 6 samples have concentrations ranging from 27.3 – 130.6 ng/µL. Nested PCR could amplify 16 samples for AMEL gene. Sex typing using AMEL gene showed 76.47% accuracy. Sensitivity of AMEL primer was increased from 6.67% to 63.64% using nested PCR technique; specificity of both external and internal primer was reported at 100%. Nested PCR of AMEL gene proved to be a suitable method for unequivocal determination of sex from degraded DNA samples. DOI : http://dx.doi.org/10.17576/JSKM-2019-1701-12

Clinical and laboratory features of PCR-confirmed periocular tuberculosis in China

Experts lack knowledge of periocular tuberculosis (TB) in China. Nested polymerase chain reaction (PCR) shows advantages in diagnosis of extrapulmonary TB. Our study aims to explore the clinical and laboratory features of PCR-confirmed periocular TB. We retrospectively reviewed medical records of presumptive periocular TB and performed nested PCR test to confirm diagnosis. Nine cases were recruited. Clinical symptoms were chronic and insidious. Eight cases achieved favorable visual acuity, while one underwent enucleation due to fungal-TB panophthalmitis. Sensitivity of caseous necrosis, acid-fast bacilli (AFB) staining and interferon γ release assay (T-SPOT) test are 33.3%, 44.4% and 85.7% respectively. Low lymphocyte percentage (P=0.019) and high monocyte-lymphocyte ratio (P=0.042) positively correlate with AFB staining. Male gender (P=0.048) and Langhans giant cell (P=0.048) positively correlate with caseous necrosis. To conclude, traditional TB ancillary tests are not as sensitive as nested PCR technique. Several factors facilitate diagnosis including male gender, decreased lymphocytes, and typical Langhans giant cells.

Risk factors of a viral nervous necrosis disease in grouper (Epinephelus spp.) cultured in Bintan district, Indonesia.

Aim:The aim of the study was to determine the prevalence and risk factors of viral nervous necrosis (VNN) disease in tiger grouper cultured in the floating net cage in Bintan district, Indonesia.Materials and Methods:Sampling used multiple stages with ponds as interest units. The selection conducted by systematic random sampling from the entire net cage in Bintan district. The fish samples were selected based on the appearance of clinical signs of infected fish. The risk factors investigated in this study included net cage, technical, and sample fish information, culture, water quality, and feed management. A total of 195 fishes pooled to the 39 net cages and tested using the nested polymerase chain reaction technique to determine the VNN status. The brain and eye processed for histopathology.Results:The prevalence rate of VNN on the net cage was 38% (15/39). The risk factors affecting VNN using bivariate analysis was cleaning the net (χ2=9.80; p=0.002), replacement of net cage (χ2=5.20; p=0.0226), and floating net cage technicians knowledge (χ2=4.13; p=0.042). The variables of positive risk factors affecting VNN by multivariate analysis were the juvenile source and the level of mid-weather changes (technician experience and dissolved oxygen [DO]). Seven variables associated to the VNN outbreak have detected. The positive multiplier factors were the source of juveniles, mid weather changes, technician experience, and the DO, while the negative factors were salinity, mixed feed, and the low weather changes. Histopathologically, the grouper fish showed the brain, eye, and muscle vacuolization and kidney necrosis.Conclusion:It proves that the Bintan waters contaminated by VNN had a prevalence rate of 38% from the total sample based on bivariate method, net cleaning, net replacement, and knowledge on the VNN outbreak. Natural infection of VNN in grouper leads to vacuolization of the brain, the eye, and muscles nearby the eye as well the kidney necrosis.

Oral shedding of HSV-1 and EBV and oral manifestations in paediatric chronic kidney disease patients and renal transplant recipients

Objective: Previous research demonstrated that salivary shedding of HSV-1 and EBV occurs often in adult renal transplant recipients, but there is a lack of studies on the presence of them in the saliva of paediatric population. Therefore, the objective of this study is to describe oral characteristics and to compare the shedding profile of HSV-1 and EBV in the saliva of children with renal transplant to that of chronic kidney disease patients and controls.Methods: This is a cross-sectional study involving 100 children, being 25 renal transplant recipients, 25 chronic kidney disease patients and 50 healthy children. Demographic and oral clinical characteristics were assessed. Saliva samples were collected and submitted to screening for EBV and HSV-1 by using nested polymerase chain reaction technique. Fisher’s exact, Pearson’s chi-square and Kruskal–Wallis tests were used for statistical analysis at a significance level of 5%.Results: Oral shedding of HSV-1 (28%) and EBV (60%) were significantly higher in renal transplant recipients compared to the other groups. Single vesicles in the oral mucosa were statistically associated with the presence of HSV-1 (p = .035). In children with chronic kidney disease, there was a higher prevalence of pale oral mucosa (32%) and enamel hypoplasia (40%) compared to paediatric renal transplant recipients and controls. Dental calculus (36%), candidiasis (8%), drug-induced gingival overgrowth (16%), mouth blisters (8%), xerostomia (12%) and salivary gland enlargement (20%) were more common in paediatric renal transplant recipients.Conclusions: Therefore, it can be concluded that salivary shedding of HSV-1 and EBV in paediatric patients was more often found in renal transplant recipients than in the renal failure and control children. Transplanted recipients showed more oral manifestations than renal failure and control children did.

Nested polymerase chain reaction technique for the detection of Gpc3 and Afp mRNA in liver cancer micrometastases.

The incidence of liver cancer has gradually risen to a high level in China, and tumor metastasis occurs via multiple pathways. Alpha fetal protein (AFP) is the main biomarker of liver cancer micrometastases. A recent study showed that glypican-3 (GPC3), which is abundant in hepatoma cells, has promising specificity and could be used to determine the presence of malignant cells. The nested polymerase chain reaction (PCR) technique is superior in experimental sensitivity. Using rat models of liver cancer in the current study, we utilized nested PCR to detect Gpc3 and Afp mRNA to determine their relationship with liver cancer micrometastases. The aim was to provide an experimental basis for clinical examination. We randomly assigned male Sprague-Dawley rats to sham and experimental groups. The experimental group constituted a liver cancer model induced by diethylnitrosamine, whereas the sham group was administered with an equivalent volume of normal saline. Gpc3 and Afp mRNA was detected using nested PCR. Analysis was performed to determine statistical significance. Compared with the sham group, the rates of occurrence of Gpc3 and Afp mRNA were significantly higher in the experimental group (P < 0.05). Compared with the total positive ratio of hepatoma cells examined by joint detection, the rates of occurrence of Gpc3 and Afp mRNA increased significantly in the four subgroups of the experimental group (P < 0.05). The use of nested PCR significantly improved sensitivity for the detection of Gpc3 and Afp mRNA in liver cancer micrometastases.

Molecular and Serological Assessment of Chronic Parvovirus B19 among Chronic Hemolytic Anemia Children

Background: Parvovirus B19 infections can suppress erythropoiesis and induce acute erythroblastopenia which is often referred to as transient aplastic crisis with hematological disorders especially chronic hemolytic anemia. Objectives: to detect the presence of parvovirus B19 (B19) DNA together with its IgG antibodies in the sera of children with chronic hemolytic anemia and in apparently healthy children in Menoufia University Hospitals and to detect the prevalence of chronic parvovirus B19 infection through detection of PCR among positive anti-parvo virus B19 IgG in different types of anemia. Methodology: The study was conducted on 60 children with chronic hemolytic anemia (40 children with chronic hemolytic anemia without a history of aplastic crisis and 20 children with chronic hemolytic anemia with a history of aplastic crisis) and 20 age and sex-matched apparently healthy children. All patients were subjected to full history taking, clinical examination and laboratory investigations. The presence of B19 IgG levels were measured by using anti-parvovirus B19 ELISA kits (EUROIMMUN), as well as detection of its DNA by nested-polymerase chain reaction technique. Results: Anti-parvovirus B19 IgG antibodies were detected in 62.5% and 100% of patients in group I and group II respectively. Only 20% in control group had a detectable level of anti-parvovirus B19 IgG. There were significant positive correlations between age of patients, frequency of transfusion, and transfusion index in both groups and the level of anti-parvovirus B19 IgG. Seven B19 IgG seropositive cases (14.3%) had B19 DNA. Although78% of children with f thalassemia major had a detectable level of anti- parvovirus B19 virus IgG antibodies, only 15.4% of them had B19 DNA. Children with sickle cell anemia were presented only in group II with aplastic crisis by percentage of 25%, where the prevalence of anti-B19 IgG antibodies among them was 100% but only one child had both anti-B19 IgG antibodies and B 19 DNA (20%).Patients who had anti- parvo virus B19 IgG and those who had both B19 DNA anti-parvo virus B19 IgG cases had a higher transfusion index compared to negative cases. Conclusion: All children with hematological disorders must be screened for B19. Direct detection of DNA by PCR needs to be performed along with serology in these children. Genotyping and quantification of the virus can be more useful for diagnosis and staging of infection.

Genotypic analysis of Giardia duodenalis in children at Egypt

Giardia duodenalis is among the most common and frequent intestinal protozoan infecting Egyptian children. The present study aims to identify the genotypic features of G. duodenalis among children fecal samples complaining of diarrhea at Beni-Suef Governorate, Egypt, and to study the association between Giardia assemblages and clinical presentation of the disease among this category. One hundred thirty diarrheic stool samples were subjected to direct stool examination, and positive samples for Giardia were subjected to copro-DNA extraction and amplification targeting the triose phosphate isomerase (tpi) gene using nested polymerase chain reaction (nPCR) technique. Then amplified DNA products were purified and sequenced. Out of 36 microscopically positive samples for Giardia, 28 samples were successfully purified and sequenced; subassemblages AII, BIII, and BIV were detected (10.7, 14,3, and 17,8 %, respectively), and it was difficult to subgroup 16 samples that belong to assemblage B (57 %). Children below the age of 6 are significantly 16 times at risk of infection with assemblage B than assemblage A (p value = .001). Flatulence and presence of fat particles on microscopic examination were significantly associated with infection (p value = 0.001, 0.027, respectively). However, assemblage B was associated with variations of symptomology than A. The present study focuses on giardiasis among Beni-Suef community. Infection is due to both assemblages: assemblage B is more prevalent (89.3 %) than A (10.7 %) and assemblage AI was not recorded. More studies are needed to identify source of infection.

Lack of strong association of Chlamydia pneumoniae and atherosclerosis in a Jordanian population.

The correlation of Chlamydia pneumoniae to coronary artery disease (CAD) in Jordan was investigated in this study. Totals of 361 atherosclerotic patients and 392 apparently healthy controls of both sexes were enrolled. C. pneumoniae-specific IgG antibodies were measured by the microimmunofluorescence assay (MIF). The presence of the bacterial DNA in the blood by polymerase chain reaction (PCR) was also tested. The overall IgG seroprevalence, estimated at a titer of 1/16, was insignificantly higher in patients (75.9%) than in controls (71.7%). About 59.3% of patients demonstrated seropositivity at titers ≤ 1/256, which are suggestive of chronic or presumed past infection, whereas 54.1% of controls were seropositive at these titers (p > 0.05). Analysis of gender-specific seroprevalences revealed no obvious relation between C. pneumoniae and atherosclerosis in males (78.9% and 77.9% in atherosclerotic and control males, respectively; p > 0.05). However, a significantly elevated seropositivity was detected in atherosclerotic females (71.7%) compared with control females (64.2%). On the other hand, the PCR-based detection of C. pneumoniae DNA failed to correlate the bacterium to atherosclerosis. We were unable to show a strong association between C. pneumoniae and CAD, potentially because of the presence of high seroprevalence of C. pneumoniae antibodies and the unreliability of the whole blood-based nested PCR technique used.

Asymptomatic falciparum malaria and genetic polymorphisms of Pfcrt K76T and Pfmdr1 N86Y among almajirai in northeast Nigeria.

Malaria remains a public health challenge, especially in sub-Saharan Africa where asymptomatic malaria is not uncommon. In the present study, the prevalence of asymptomatic falciparum malaria was investigated in almajirai, and the genetic polymorphisms of chloroquine (CQ) resistance biomarkers were assessed. A total of 440 apparently healthy almajirai between 3 and 12 years of age were randomly enrolled in Maiduguri, northeast Nigeria, between July and December 2010. Parasitemia and gametocytemia were assessed by light microscopy, and polymorphisms of Pfcrt K76T and Pfmdr1 N86Y were detected by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. The mean age of the subjects was 8.3 ± 4.5 years, with subjects ≤ 5 years accounting for 10.7% (47/440) of the population. Prevalence of asymptomatic falciparum parasitemia and gametocytemia were 12.7% (56/440) and 8.6% (38/440), respectively. Geometric mean parasite density (GMPD) was 240 (160-630) parasites/µL, while geometric mean gametocyte density (GMGD) was 53 (24-96) gametocytes/µL. The GMPD was higher among subjects ≤ 5 years of age (p = 0.027). Pfcrt 76T was detected in 5.4% (3/56) of the isolates, and no isolates harbored Pfmdr1 86Y mutant. The study reveals asymptomatic falciparum malaria in almajirai and low levels of Pfcrt 76T and Pfmdr1 86Y alleles. These findings could hinder malaria control measures, and hence almajirai should be incorporated into malaria control programs.

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39–31.27% for rpoB-PCR, 36.44–60.83% for IS6110- PCR, 75.29–92.93% for nested-rpoB PCR, and 87.98–99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15–100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results.