Sensitive Nested Polymerase Chain Reaction Research Articles

CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood

BackgroundToxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity.MethodsThis study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection.ResultsThe results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi).ConclusionsThis study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field.Graphical abstract

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis.

In recent years, the “milky disease” caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/μL) and internal transcribed spacer (ITS; 6.74 × 105 copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

Highly sensitive nested polymerase chain reaction to improve the detection of Leishmania species in clinical specimens.

Cutaneous leishmaniasis (CL) is one of the most neglected tropical diseases and an important health problem in many countries. It is an endemic disease in most regions of Iraq, while being non-endemic in the Kurdistan Region. The techniques frequently used for detection of CL are not very sensitive. Therefore, this study aimed to identify a sensitive method for diagnosis of CL in clinical samples. The present study was performed in December 2019 to December 2020 in Kalar General Hospital. Clinical samples were collected from 85 suspected CL cases. Sixty-four (75.29%), 71 (83.53%) and 84 (98.82%) cases were detected as positive for CL by microscopy, PCR, and nested PCR, respectively. Of the 84 nested PCR-confirmed CL patients, 46 (54.8%) were female and 38 (45.2%) were male. The most predominate rate of infection was in the 30-39-year age group (29.76%) and the lowest was in the ≥ 60-year group (3.57%). Forty (47.62%) patients had a single lesion. The statistical analysis showed significant differences between age groups and between the number of lesions. The sensitivities of microscopy, conventional PCR, and nested PCR were 80.77%, 86.6% and 100%, respectively, while all three methods showed 100% specificity. Furthermore, PCR-ITS1 followed by a simple restriction fragment length polymorphism (RFLP) analysis using HaeIII endonuclease indicated that Leishmania major was responsible for all CL infections in the study area.

Detection of Brucella Abortus Using Nested-PCR Molecular Method

Common detection methods of brucellosis are always accompanied by technical problems in infection diagnosis. In this study, the nested PCR technique was used for the detection of Brucella abortus, and it was compared with other methods. This case-control study was done in Kurdistan. Blood samples were collected from 50 patients with clinical symptoms and 50 healthy people with no clinical symptoms. Serologic and culture tests were done for all the samples. DNA was extracted using a Promega kit. Molecular detection was done by nested PCR. In the end, the results of phenotypical method and nested PCR were compared. The results of culture and serology tests indicate the limited capabilities of these methods to detect Brucella species. According to the results among all samples, only 14 (28%) were cultured. Rose Bengal test sensitivity was 100% but its specificity 26.09%. Also, Wright's test (tube agglutination) sensitivity was also 85% and nested PCR sensitivity was 46.15%. The sensitivity of Rose Bengal, Wright's and nested PCR tests were 100%, 85%, and 46.15%, respectively, for this sampling.

Direct Epstein-Barr Virus (EBV) Typing on Peripheral Blood Mononuclear Cells: No Association Between EBV Type 2 Infection or Superinfection and the Development of Acquired Immunodeficiency Syndrome–Related Non-Hodgkin's Lymphoma

AbstractIn the literature, a correlation has been suggested between the occurrence of acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (NHL) and Epstein-Barr virus (EBV) type 2 infection. To further investigate a possible role for EBV type 2 infection in the development of AIDS-NHL, we developed a sensitive and type-specific nested polymerase chain reaction (PCR) assay and analyzed EBV types directly on peripheral blood mononuclear cells (PBMC) in three subgroups of human immunodeficiency virus (HIV)-1 infected individuals: 30 AIDS-NHL patients, 42 individuals progressing to AIDS without lymphoma (PROG), either developing opportunistic infections (AIDS-OI) or Kaposi's sarcoma (AIDS-KS), and 18 long-term asymptomatic individuals (LTA). Furthermore, EBV type analysis was performed on PBMC samples obtained from AIDS-NHL patients in the course of HIV-1 infection. The results showed that: (1) direct analysis of PBMC is superior to analysis of B-lymphoblastoid cell lines (B-LCL) grown from the same PBMC samples; (2) in HIV-1 infected individuals, there is a high prevalence of EBV type 2 infection (50% in LTA, 62% in progressors, and 53% in AIDS-NHL) and superinfection with both type 1 and 2 (24% in LTA, 40% in progressors, and 47% in AIDS-NHL); (3) EBV type 2 (super)infection is not associated with an increased risk for development of AIDS-NHL; (4) type 2 infection can be found early in HIV-1 infection, and neither type 2 infection nor superinfection correlates with a failing immune system.

Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina.

Bovine babesiosis is a tick-transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick-hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species-specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C-terminal region of rhoptry-associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa-stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis.

Hepatitis B virus (HBV) genome detection and genotyping in virally suppressed patients using nested polymerase chain reaction-based Sanger sequencing

Hepatitis B virus (HBV) genotypes have important clinical implications. Current genotyping methods are less sensitive in patients with ultra-low HBV viral load. We report a highly sensitive and specific nested polymerase chain reaction (PCR) assay for genotyping patient HBV. Total DNA derived from plasma of 14 (HBsAg+ and/or HBsAg-) HBcAb+ patients was used for HBV-specific nested PCRs targeting the preC/C, X/BCP/preC, and surface regions. All patients were treated with long-term nucleos(t)ide analogues (NAs), and 12/14 have undetectable viremia (clinical PCR: sensitivity >10 IU/mL). Surface amplicons were sequenced, aligned with reference genomes, and used in phylogenetic tree construction to determine genotype. HBV DNA was detected in 14/14, including 3 occult (HBsAg-/HBcAb+) cases. Genotypes identified were 6/14 B, 6/14 C, and 2/14 D. This assay in virologically suppressed patients may be useful for future studies requiring genotype prior to assessment of immunomodulatory and/or direct acting anti-viral therapeutics in patients on potent NAs.

Molecular investigation of Lawsonia intracellularis in diarrheic and healthy captive ostriches (Struthio camelus) in Iran

Lawsonia intracellularis is the causative agent of proliferative enteropathy (PE) in pigs, a disease of economic importance worldwide. The present study aimed to investigate the occurrence of L. intracellularis in ostriches using a sensitive and specific nested polymerase chain reaction (nested PCR). A total number of 112 fecal samples (64 healthy, 48 diarrheic) were collected from 11 ostrich farms located in four provinces in Iran (Semnan, Tehran, Gilan, Yazd). The results showed the presence of L. intracellularis in three diarrheic and four apparently healthy birds. The frequency of positive results was the same in both groups (6.25% healthy vs. 6.25% diarrheic). Although the results of this study did not reveal any relationship between this organism and diarrhea (P > 0.05), the molecular detection of L. intracellularis on a farm where birds were suffering from the typical clinical symptoms of PE highlights the need for evaluation of PE in ratites.

Sensitivity and Specificity of Nested PCR for Diagnosing Malaria: Cases in Several Areas of Indonesia

Indonesia is still included in high endemic area of malaria infection. Early detection as well as appropriate and quick treatment is needed to be able to prevent and treat malaria in Indonesia. Laboratory examination using a microscopic method is still used as the gold standard to diagnose malaria cases. However, the morphology similarity of some Plasmodium species and the number of parasites that can be seen under microscopy causes malaria diagnosis become difficult if only relying on microscopy diagnostic method. The purpose of this study is to analyze the sensitivity and specificity of nested PCR compared to microscopic examination in diagnosing malaria cases. A cross-sectional study has been carried out in some areas of Indonesia and the microscopic analysis as well as nest PCR was done in Laboratory of Parasitology and Laboratory of Central Biomedical Faculty of Medicine, Universitas Brawijaya, Malang East Java Indonesia. A total of 149 blood samples from patients with clinical symptoms of malaria had been obtained from Sumatra, Sulawesi and East Java during December 2011 to December 2013. From 149 sample, 81.9% samples were diagnosed malaria positive by microscopy examination, whereas the PCR results showed that 90.6% of samples were positive. Nested PCR sensitivity is 97.5%, and microscopy 88.2%. Nested PCR specificity is 40.7%, whereas microscopy 78.5%. PPV and NPV for nested PCR are 88,2% and 78.5% respectively, and for microscopy are 97.5% and 40.7% respectively. Nested PCR has a higher sensitivity than microscopy in diagnosing malaria and is able to detect mixed infection better than microscopic examination. However, it is statistically less specific than microscopy examination.

Effective Detection of Porcine Cytomegalovirus Using Non-Invasively Taken Samples from Piglets.

Shortage of human organs forced the development of xenotransplantation using cells, tissues, and organs from pigs. Xenotransplantation may be associated with the transmission of porcine zoonotic microorganisms, among them the porcine cytomegalovirus (PCMV). To prevent virus transmission, pigs have to be screened using sensitive methods. In order to perform regular follow-ups and further breeding of the animals, samples for testing should be collected by low-invasive or non-invasive methods. Sera, ear biopsies, as well as oral and anal swabs were collected from ten 10-day-old Aachen minipigs (AaMP) and tested for PCMV using sensitive nested polymerase chain reaction (PCR) as well as uniplex and duplex real-time PCR. Porcine cytomegalovirus DNA was detected most frequently in oral and anal swabs. Comparison of duplex and uniplex real-time PCR systems for PCMV detection demonstrated a lower sensitivity of duplex real-time PCR when the copy numbers of the target genes were low (less 200). Therefore, to increase the efficacy of PCMV detection in piglets, early testing of oral and anal swabs by uniplex real-time PCR is recommended.

Avian Malaria is Absent in Juvenile Colonial Herons (Ardeidae) but notCulex pipiensMosquitoes in the Camargue, Southern France

Apicomplexan blood parasites Plasmodium and Haemoproteus (together termed “Avian malaria”) and Leucocytozoon are widespread, diverse vector-transmitted blood parasites of birds, and conditions associated with colonial nesting in herons (Ardeidae) and other waterbirds appear perfect for their transmission. Despite studies in other locations reporting high prevalence of parasites in juvenile herons, juvenile Little Egrets (Egretta garzetta) previously tested in the Camargue, Southern France, had a total absence of malaria parasites. This study tested the hypotheses that this absence was due to insufficient sensitivity of the tests of infection; an absence of infective vectors; or testing birds too early in their lives. Blood was sampled from juveniles of four species shortly before fledging: Little Egret (n = 40), Cattle Egret (Bubulcus ibis; n = 40), Black-crowned Night-Heron (Nycticorax nycticorax, n = 40), and Squacco Heron (Ardeola ralloides; n = 40). Sensitive nested-Polymerase Chain Reaction was used to test for the presence of parasites in both birds and host-seeking female mosquitoes captured around the colonies. No malaria infection was found of in any of the heron species. Four different lineages of Plasmodium were detected in pooled samples of female Culex pipiens mosquitoes, including two in potentially infective mosquitoes. These results confirm that the absence of malaria parasites previously demonstrated in Little Egret is not due to methodological limitations. Although the prevalence of infection in mosquitoes was low, conditions within the colonies were suitable for transmission of Plasmodium. These colonial heron species may have evolved strategies for resisting malaria infection through physiological or behavioral mechanisms.

Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis

Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming. A new highly sensitive nucleic acid amplification test, combined nested and real-time polymerase chain reaction (PCR) in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantage of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the insertion sequence (IS) 6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS6110 real-time PCR and IS6110 one-tube nested real-time PCR were 100 fg/μL and 1 fg/μL of M. tuberculosis DNA, respectively. AdvanSure TB/non-tuberculous mycobacteria (NTM) real-time PCR, IS6110 real-time PCR, and two-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and NTM isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation.

Occult hepatitis B virus infection in liver transplant patients in a Brazilian referral center

Estimates of occult hepatitis B virus (HBV) infection prevalence varies among different studies depending on the prevalence of HBV infection in the study population and on the sensitivity of the assay used to detect HBV DNA. We investigated the prevalence of occult HBV infection in cirrhotic patients undergoing liver transplantation in a Brazilian referral center. Frozen liver samples from 68 adults were analyzed using a nested polymerase chain reaction assay for HBV DNA. The specificity of the amplified HBV sequences was confirmed by direct sequencing of the amplicons. The patient population comprised 49 (72.1%) males and 19 (27.9%) females with a median age of 53 years (range=18-67 years). Occult HBV infection was diagnosed in three (4.4%) patients. The etiologies of the underlying chronic liver disease in these cases were alcohol abuse, HBV infection, and cryptogenic cirrhosis. Two of the patients with cryptic HBV infection also presented hepatocellular carcinoma. Markers of previous HBV infection were available in two patients with occult HBV infection and were negative in both. In conclusion, using a sensitive nested polymerase chain reaction assay to detect HBV DNA in frozen liver tissue, we found a low prevalence of occult HBV infection in cirrhotic patients undergoing liver transplant, probably due to the low prevalence of HBV infection in our population.

Sensitivity of nested-PCR for plasmodium detection in pooled whole blood samples and its usefulness to blood donor screening in endemic areas

Transfusion-transmitted malaria is a severe disease with high fatality rate. Most Brazilian blood banks in the Amazon region perform malaria screening using microscopic examination (thick smears). Since low parasite concentrations are expected in asymptomatic blood donors a high sensitivity test should be used for donor screening. This study determined the sensitivity of a nested-PCR for plasmodium detection in pooled samples. We performed a one-stage criterion validation study with 21 positive samples pooled with samples from ten negative volunteer until three different concentrations were reached (0.33; 0.25; 0.20 parasites/μL - p/μL). Nested PCR was performed as described by Snounou et al. (1993). Sensitivities (and confidence intervals) were determined by stratum of final parasite concentration on the pooled samples. All samples with parasitemia values of 0.33 and 0.25 p/μL had 100% sensitivity (95%CI=86.3-100). One negative result was obtained from a sample with 0.20 p/μL sensitivity=95.2% (95%CI=76.2-99.9). Compared to parasitemia detectable under ideal conditions of thick smear, this nested-PCR in pooled sample was able to detect 40 times more parasites per microliter. Nested-PCR in pooled samples should be considered as a high sensitive alternative to thick smear for donor screening in blood banks at endemic regions. Local authorities need to assess cost:benefit advantages of this method compared to alternatives.

Vector Sequences Are Not Detected in Tumor Tissue from Research Subjects with Ornithine Transcarbamylase Deficiency Who Previously Received Adenovirus Gene Transfer

A 66-year-old woman heterozygous for a mutation in the ornithine transcarbamylase gene (Otc) participated in a phase I gene therapy trial for OTC deficiency. She received an adenovirus (Ad) vector expressing the functional OTC gene by intraportal perfusion. Fourteen years later she developed and subsequently died of hepatocellular carcinoma. A second subject, a 45-year-old woman, enrolled in the same trial presented with colon cancer 15 years later. We sought to investigate a possible association between the development of a tumor and prior adenoviral gene transfer in these two subjects. We developed and validated a sensitive nested polymerase chain reaction assay for recovering recombinant Ad sequences from host tissues. Using this method, we could not detect any Ad vector DNA in either tumor or normal tissue from the two patients. Our results are informative in ruling out the possibility that the adenoviral vector might have contributed to the development of cancer in those two subjects.

Detection of oncogenic human papillomavirus genotypes on spermatozoa from male partners of infertile couples

To evaluate the prevalence of human papillomavirus (HPV) sperm infection and its correlation with sperm parameters in patients who attended a fertility clinic. Cross-sectional clinical study. University-affiliated reproductive medicine clinic. A total of 308 male partners of couples undergoing in vitro fertilization techniques. Specimens of semen were collected from all patients. Sperm parameters were evaluated according to the World Health Organization manual. The presence of HPV DNA was researched by the combined use of two HPV assays and a highly sensitive nested polymerase chain reaction assay followed by HPV genotyping. To examine whether HPV was associated with the sperm, in situ hybridization (ISH) analysis was performed. Results of HPV investigation were compared with sperm parameters and ISH analysis. Twenty-four out of 308 semen samples (7.8%) were HPV DNA positive, but HPV infection did not seem to affect semen quality. Moreover, ISH revealed a clear HPV localization at the equatorial region of sperm head in infected samples. Oncogenic HPV genotypes were detected on spermatozoa from asymptomatic subjects, but a role of the infection in male infertility was not demonstrated.

Hepatitis B virus quasispecies in hepatic and extrahepatic viral reservoirs in liver transplant recipients on prophylactic therapy

The characterization of hepatitis B virus (HBV) quasispecies in different compartments in liver transplant (LT) recipients may be helpful in optimizing prophylaxis regimens. The aims of this study were to evaluate liver, peripheral blood mononuclear cells (PBMC), and plasma samples for HBV and to compare the quasispecies in hepatic and extrahepatic sites in LT recipients on long-term prophylaxis. For 12 patients followed for up to 15 years post-LT, liver, plasma, and PBMC samples [all HBV DNA-negative according to conventional polymerase chain reaction (PCR) assays] were evaluated for HBV DNA by a sensitive nested PCR method [covalently closed circular DNA (cccDNA) for liver and PBMC samples] and by the sequencing and phylogenetic analysis of polymerase quasispecies. For the 10 patients on prophylaxis with no clinical recurrence (median time post-LT = 15.5 months, range = 12-96 months), liver samples were HBV DNA-reactive in 9 of 10 cases, plasma samples were HBV DNA-reactive in 3 of 10 cases, and PBMC samples were HBV DNA-reactive in 2 of 7 cases (including 1 case with HBV cccDNA in PBMCs). The sequence analysis showed that all HBV clones had a wild-type (WT) sequence in the liver and PBMCs. In 2 patients with early HBV recurrence post-LT who were treated with nucleosides only, HBV DNA was detected in serum, PBMC, and liver samples, and HBV cccDNA was found in liver samples. An HBV lamivudine-resistant variant with an M204I mutation was identified in liver (70% and 18% of the clones) and plasma samples (100% of the clones), but a WT sequence was found in 70% and 100% of the PBMC clones. In conclusion, despite prophylaxis and the absence of HBV DNA in serum according to conventional assays, HBV is detectable in the serum, liver, and PBMCs of almost all patients, and this supports the use of continued anti-HBV therapy in this group. Antiviral drug-resistant variants are more frequent in the liver versus PBMCs, but both compartments are potential sources of reinfection.

Molecular Detection of Murine Herpesvirus 68 in Ticks Feeding on Free-living Reptiles

The MHV-68 (designed as Murid herpesvirus 4 (MuHV 4) strain 68) isolated from two rodents, Myodes glareolus and Apodemus flavicollis, is considered as a natural pathogen of free-living murid rodents. Recently, the detection of MHV antibodies in the blood of animals living in the same biotope as MHV-infected mice has suggested that ticks may have a role in the transmission of this pathogen. Ixodes ricinus is one the most abundant tick species in Europe known to transmit multiple pathogens causing human and animal diseases. In this study, nymphs and larvae feeding on 116 individuals of a temperate lizard species-the green lizard Lacerta viridis captured in the Slovak Karst National Park, were examined for MHV-68. The specific sequence of virion glycoprotein 150 was amplified in DNA individually isolated from I. ricinus ticks using single-copy sensitive nested polymerase chain reaction. MHV-68 was detected in ten of 649 nymphs and in five of 150 larvae, respectively. We found that 9.6% of green lizards fed at least one MHV-68-infected immature tick. Occurrence of MHV-68 within all ticks tested was 1.8%. This study is first to show that immature I. ricinus ticks feeding on free-living lizards in a Central European region could be infected with gammaherpesvirus (MHV-68), naturally infecting free-living murid rodents. Our results provide evidence supporting the hypothesis that ticks may play a mediating role in circulation of MHV-68 in nature.

Detection of bovine herpesvirus 1sequences in yaks (Bos grunniens) with keratoconjunctivitis, using a highly sensitive nested polymerase chain reaction

Thirty-seven yaks (Bos grunniens) with keratoconjunctivitis and 22 healthy yaks were used to investigate the role of bovine herpesvirus 1 (BoHV-1) in keratoconjunctivitis in yaks. Nucleic acid sequences of BoHV-1 glycoproteins B and E were detected in conjunctival swabs from all yaks with keratoconjunctivitis using a nested polymerase chain reaction (PCR). In 21 yaks, BoHV-1 sequences were detected along with Moraxella bovis (M. bovis) and Neisseria spp. The amplified BoHV-1 sequences were identical, and no nucleotide variation was observed when compared with a BoHV-1 reference strain using single-strand conformation polymorphism analysis of the amplified DNA sequences. Interestingly, BoHV-1 sequences could not be detected in samples from healthy yaks. However, conjunctival swabs from two healthy yaks (9.09%) yielded M. bovis and Neisseria spp. Samples from 35 yaks with keratoconjunctivitis showed positive reactions in an avidin biotin enzyme-linked immunosorbent assay for BoHV-1 antibodies; all the healthy yaks were seronegative. This is the first report of a possible association of BoHV-1 with keratoconjunctivitis in yaks.

Absence of detectable xenotropic murine leukemia virus-related virus in plasma or peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected blood donors or individuals in Africa.

Since the identification of xenotropic murine leukemia virus-related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV-1)-infected individuals to gain evidence of XMRV infection in this geographic region. A total of 199 plasma samples, 19 PBMNC samples, and 50 culture supernatants from PBMNCs of blood donors from Cameroon found to be infected with HIV-1 and HIV-1 patients from Uganda were screened for XMRV infection using a sensitive nested polymerase chain reaction (PCR) or reverse transcription (RT)-PCR assay. Using highly sensitive nested PCR or RT-PCR and real-time PCR assays capable of detecting at least 10 copies of XMRV plasmid DNA per reaction, none of the 268 samples tested were found to be XMRV DNA or RNA positive. Our results failed to demonstrate the presence of XMRV infection in African blood donors or individuals infected with HIV-1. More studies are needed to understand the prevalence, epidemiology, and geographic distribution of XMRV infection worldwide.