Nested Polymerase Chain Reaction Protocol Research Articles

Preliminary study on buffy coat smear and molecular detection of microfilaria in domestic chickens (Gallus gallus domesticus) raised in Southern Thailand

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_ R2-OnchoCOI_ R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required. Keywords: Buffy coat smear, Chickens, Cytochrome c oxidase I, Microfilaria, Nested-polymerase chain reaction.

Nested PCR-Based Rapid Detection of Phytoplasma Leaf Wilt Disease of Coconut in Sri Lanka and Systemic Movement of the Pathogen

Phytoplasmas are associated with many plant diseases. In palms, lethal bronzing disease, Texas Phoenix palm decline, and coconut lethal yellowing decline are some of them. In Sri Lanka, coconut leaf wilt decline has been reported in the Weligama area of the Southern province, and the disease is called Weligama coconut leaf wilt disease (WCLWD). Unlike other phytoplasma diseases of palms, WCLWD shows slow disease progress. Pathogen detection entirely relies on nested polymerase chain reaction (PCR). However, inconsistencies in pathogen detection have been experienced, i.e., symptomatic plants often produce negative results. The objectives of this study were to reconsider the choice of primers and to determine the best sampling tissue types for consistent detection of the pathogen. Among the six universal primer combinations tested, P1/Tint nested with fU5/rU3 produced consistent results. BLASTn searches of the sequences showed 99-100% similarity to sugarcane white leaf disease (SWL) or grassy shoot (SGS) disease-causing phytoplasma. The optimized nested PCR protocol was successful, with the minimum success rating of 88% and 100% specificity. Midribs of milky white bud leaf samples were the best tissue type for rapid detection. Systemic movement of the pathogen and a tentative latent period were also reported. The findings are helpful in the early detection of the disease.

Myrrh Oil in Vitro Inhibitory Growth on Bovine and Equine Piroplasm Parasites and Babesia microti of Mice.

The present experimental study was conducted for the assessment of the efficacy of in vitro inhibition of myrrh oil on the propagation of Babesia bovis, B. divergens, B. bigemina, Theileria equi, and B. caballi and in vivo efficacy on B. microti in mice through fluorescence assay based on SYBR green I. The culture of B. divergens B. bovis and was used to evaluate the in vitro possible interaction between myrrh oil and other commercial compound, such as pyronaridine tetraphosphate (PYR), diminazene aceturate (DA), or luteolin. Nested-polymerase chain reaction protocol using primers of the small-subunit rRNA of B. microti was employed to detect any remnants of DNA for studied parasitic species either in blood or tissues. Results elucidated that; Myrrh oil significantly inhibit the growth at 1% of parasitic blood level for all bovine and equine piroplasm under the study. Parasitic regrowth was inhibited subsequently by viability test at 2 µg/mL for B. bigemina and B. bovis, and there was a significant improvement in the in vitro growth inhibition by myrrh oil when combined with DA, PYR, and luteolin. At the same time; mice treated with a combination of myrrh oil/DA showed a higher inhibition in emitted fluorescence signals than the group that challenged with 25 mg/kg of diminazene aceturate at 10 and 12 days post-infection. In conclusion, this study has recommended the myrrh oil to treat animal piroplasmosis, especially in combination with low doses of DA.

Multiplex nested polymerase chain reaction targeting multiple genes for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens.

Objectives:The objective of this study was to design and evaluate a novel multiplex nested polymerase chain reaction (PCR) protocol for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens obtained from symptomatic patients clinically suspected of sexually transmitted infections (STIs), targeting two different genes each for these pathogens.Materials and Methods:A total of 116 genitourinary specimens were collected from men (n = 12) and women (n = 104). Direct microscopy, culture isolation, and antimicrobial susceptibility testing for N. gonorrhoeae were performed. Multiplex nested PCR was performed on clinical samples using novel designed primers targeting porA pseudogene and opa gene of N. gonorrhoeae and momp gene and cryptic plasmid of C. trachomatis simultaneously. DNA sequence analysis of nested PCR amplicons for each of four gene targets was carried out for the validation of in-house designed primers and PCR protocol.Results:A total of 51.72% (60/116) patients were detected to have either of the two STIs. About 35.35% (41/116) of patients were positive for C. trachomatis and 33.62% (39/116) for N. gonorrhoeae by employing multiplex nested PCR. Coinfection with N. gonorrhoeae and C. trachomatis was detected in 17.24% (20/116) patients. 31.5% endocervical swabs (n = 54), 64.4% speculum-assisted high vaginal swabs (n = 45), and 80% self-collected vaginal swabs (n = 5) were detected positive for either of two STIs.Conclusions:The multiplex nested PCR protocol designed and employed in the present study may be used in the diagnosis and management of both symptomatic as well as asymptomatic cases of N. gonorrhoeae and C. trachomatis, particularly among high-risk groups.

Molecular detection of DHFR gene polymorphisms in Pneumocystis jirovecii isolates from Indian patients.

Pneumocystis pneumonia (PCP) is an opportunistic life-threatening infection, especially for immunocompromised individuals. A trimethoprim-sulfamethoxazole (TMP-SMX) combination is commonly used for the treatment of PCP, targeting both dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes. Several studies have already shown that polymorphisms in the DHPS gene are associated with drug resistance. The present study analyzed DHFR gene polymorphisms in Pneumocystis jirovecii recovered from clinical samples from patients admitted to a tertiary care health center in New Delhi, India. Detection of P. jirovecii was performed using Gomori methenamine silver staining (GMS) and nested polymerase chain reaction (PCR) assay targeting the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) gene. The DHFR gene was amplified using nested PCR protocol and was sequenced for detection of polymorphisms. Of 180 clinical samples, only 4% (7/180) were positive by GMS staining, and 10% (18/180) were positive by mt LSU rRNA PCR assay. Of these 18 positive samples, only 77% (14/18) were amplified by the DHFR gene PCR assay. A total of 16 nucleotide substitutions were observed in 42% (6/14) samples targeted for the DHFR gene, of which 8 nucleotide substitutions were synonymous and the rest were non-synonymous. The DHFR gene mutations found in this study may possibly indicate an association of process likely to contribute to therapeutic failure or an evolutionary process, and warrant continuous monitoring.

Genetic polymorphisms in the glutamate-rich protein of Plasmodium falciparum field isolates from a malaria-endemic area of Brazil

The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.

Genetic polymorphisms in the glutamate-rich protein of Plasmodium falciparum field isolates from a malaria-endemic area of Brazil

The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.

Detection and Characterization ofGiardia duodenalisandCryptosporidiumspp. on Swine Farms in Ontario, Canada

As part of the C-EnterNet surveillance program of the Public Health Agency of Canada, 122 pooled swine manure samples from 10 farms in Ontario, Canada were collected and tested for Giardia and Cryptosporidium. Giardia duodenalis cysts and Cryptosporidium spp. oocysts were detected using immunofluorescence microscopy. Nested-polymerase chain reaction protocols were performed to amplify the small subunit rRNA gene and the β-giardin gene for G. duodenalis, and the small subunit rRNA gene and the heat shock protein-70 gene for Cryptosporidium spp. The DNA amplicons were sequenced to determine genotypes and species. A mixed multivariable method was used to compare the presence of Giardia and Cryptosporidium in different stages of production. Both Giardia cysts and Cryptosporidium oocysts were present on all tested farms, with 50.8% of the samples positive for G. duodenalis and 44.3% positive for Cryptosporidium spp. by microscopy, and 66.4% and 55.7%, respectively, positive by polymerase chain reaction (PCR). No significant agreement was observed between microscopy and PCR method to detect Giardia and Cryptosporidium (p<0.05). The prevalence of Giardia in manure pits and finisher pigs did not differ (p>0.05), however, it was less frequent (odds ratio, OR=0.21 [0.07, 0.63]) among sows. Cryptosporidium was more likely (OR=3.6 [1.3, 9.9]) to be detected in manure pits and weaners (OR=3.3 [1.1, 10.0]) compared to finisher pigs, and it was less frequent (OR=0.06 [0.007, 0.55]) in sows than in finishers (p<0.05). DNA sequencing demonstrated that 92.1% of the Giardia isolates were Assemblage B and 7.9% were Assemblage E. The most prevalent Cryptosporidium were Cryptosporidium parvum (55.4%), and Cryptosporidium sp. pig genotype II (37.5%). These findings indicate that the occurrence of zoonotic isolates of G. duodenalis and Cryptosporidium is very high on swine farms in southern Ontario, and that there is a potential for transmission between swine and humans by means of cyst and oocyst contaminated water or foods.

Dihydropteroate synthase (DHPS) gene mutation study in HIV-Infected Indian patients with Pneumocystis jirovecii pneumonia

Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations' (55th and 57th codon) association with prior sulfa prophylaxis failure has been reported from both developed and developing countries. We conducted a prospective study to determine the prevalence of P. jirovecii DHPS mutations from 2006 to 2009 on P. jirovecii isolates obtained from HIV-infected patients with a clinical diagnosis of Pneumocystis carinii pneumonia (PCP) admitted to our tertiary care reference health center in New Delhi, India. Detection of P. jirovecii cysts was performed by direct fluorescent antibody (DFA) staining and by Grocott's-Gomori methenamine silver staining (GMS). DNA detection was performed by polymerase chain reaction (PCR) using primers for the major surface glycoprotein (MSG) gene. P. jirovecii DHPS gene was amplified by nested PCR protocol and sequenced for detecting mutations at the 55th and 57th codons. Out of 147 HIV-positive patients with suspected Pneumocystis pneumonia (PCP), 16 (10.8%) PCP positive cases were detected. Of 16 cases, nine (56.2%) were positive by DFA staining, four (25%) were positive by Grocott's-Gomori methenamine silver staining, and all 16 were positive by MSG PCR. DHPS mutations at the 55th and 57th codons were observed in 6.2% of HIV patients studied, which was relatively low compared to reports from developed nations. Prevalence of Pneumocystis jirovecii DHPS mutations associated with cotrimoxazole treatment failure may be low in the Indian subpopulation of HIV-positive patients and warrants larger studies to elucidate the true picture of Pneumocystis jirovecii sulfa drug resistance in India.

Are the Proposed env Mutations Actually Associated With Resistance to Maraviroc?

Are the Proposed env Mutations Actually Associated With Resistance to Maraviroc?

Single Cell Analysis of Mutations in the APC Gene

Introduction: Mutation analysis of inherited monogenic diseases is an important aspect of preimplantation genetic diagnosis. Familial adenomatous polyposis of the colon is an autosomal dominant inherited disorder caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). A characteristic of this severe disease is the development of thousands of polyps in the colon which untreated evolve into malignant colon carcinomas. Here we present a novel approach for the establishment of mutation detection in the APC gene in single cells applicable for preimplantation genetic diagnosis. Methods: Single fixed lymphocytes were isolated via laser microdissection and transferred to reaction centers of planar chemically structured glass slides via a recently developed horizontal low-pressure single particle adsorbing transfer system (SPATS). A multiplex nested polymerase chain reaction protocol in a 1-μl reaction volume, followed by sequencing and fragment length analysis was applied in order to detect mutations in the APC gene. Results: Reliable isolation and transfer of single lymphocytes was demonstrated. High amplification efficiency and low allelic drop out (ADO) rates for polymorphic microsatellite markers and mutation specific amplification products of various mutations in the APC gene were obtained from fixed single cells. Conclusions: This novel nanotechnological downscaling approach enables a reliable validation of genetic testing using diploid single lymphocytes, and will open a wide range of single cell diagnostics.

Comparison between Different Polymerase Chain Reaction Methods for the Diagnosis of Ehrlichia canis Infection

The aim of this study was to compare different polymerase chain reaction (PCR) methods for the detection of Ehrlichia canis in blood samples and to relate these results to clinical findings and serology to E. canis using the indirect fluorescent antibody (IFA) test. Nine seropositive and nine seronegative dogs were included in this study. DNA was extracted once and used in one simple PCR and five nested PCR protocols previously described. In selected dogs (three seropositive and one seronegative) blood samples were aseptically collected in order to attempt the isolation of E. canis in the DH82 cell line. Results show that nested PCR protocols seem to be more sensitive than the simple PCR. Considering only nested PCR protocols, 33% of the IFA-positive samples were PCR positive using the five different protocols. The rest of the IFA-positive samples were PCR positive or negative depending on the protocol used. Clinical signs and laboratory findings compatible with canine monocytic ehrlichiosis (CME) were found in 67% of dogs positive by the IFA test. All samples positive by both techniques (IFA test and PCR) were from dogs suffering from clinical CME. IFA-negative samples were PCR negative, except 22% that were PCR positive when using only one of the nested PCR protocols. Isolation of the agent was exclusively achieved in the only case in which the IFA test and all the PCR protocols were also positive.

A Cautionary Note on the Use of Nested PCR for Parasite Screening—An Example From Avian Blood Parasites

The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp. However, in certain cases when a bird was infected with avian malaria, we obtained a slightly longer PCR product during the detection of Leucocytozoon spp. Our data imply that these "false" Leucocytozoon fragments are the consequences of strong amplification of certain malaria lineages in the first PCR, which can also be detected after the second PCR amplification that is specific to Leucocytozoon spp. parasites. Because these "false" Leucocytozoon fragments are slightly longer than the normal Leucocytozoon fragments, we suggest the use of well-separating agarose gels, several positive controls, and molecular standards to facilitate their separation. If one obtains a fragment that differs in length from the one expected for Leucocytozoon spp., sequencing is essential. More generally, in order to limit this type of problem with nested PCR protocols, we suggest that the first and the second primer pair be chosen so that they have different annealing temperatures.

Preimplantation genetic diagnosis for fragile X syndrome using multiplex nested PCR

Fragile X syndrome is caused by a dynamic mutation in the FMR1 gene. Normal individuals have <55 CGG repeats in the 5′ untranslated region, premutation carriers have 55–200 repeats and a full mutation has >200 repeats. Female carriers are at risk of having affected offspring. A multiplex nested polymerase chain reaction protocol is described for preimplantation genetic diagnosis (PGD) of fragile X syndrome with simultaneous amplification of the CGG-repeat region, the Sry gene and several flanking polymorphic markers. The amplification efficiency was ≥96% for all loci. The allele dropout rate in heterozygotic females was 9% for the FMR1 CGG-repeat region and 5–10% for the polymorphic markers. Amplification failure for Sry occurred in 5% of single leukocytes isolated from males. PGD was performed in six patients who underwent 15 cycles. Results were confirmed in all cases by amniocentesis or chorionic villous sampling. Five clinical pregnancies were obtained (31% per cycle), four of which resulted in a normal delivery and one miscarried. This technique is associated with high efficiency and accuracy and may be used in carriers of full mutations and unstable high-order premutations.

Human immunodeficiency virus‐1 subtypes and antiretroviral drug resistance profiles among drug‐naïve Brazilian blood donors

Human immunodeficiency virus-1 (HIV-1) genetic variability and its implication on the development of new reagents were investigated by correlating time of infection and resistance to antiretroviral drugs. Seventy-four plasma samples from Brazilian drug-naïve blood donors were assayed to further develop a panel of biologic reagents to be used in serology and molecular tests. After viral RNA extraction, cDNA was generated and used in nested polymerase chain reaction protocols with primers for the ENV (C2-V3 and gp41), protease (PR), and reverse transcriptase (RT) of HIV-1. Genomic sequencing was conducted to define HIV-1 subtypes and drug resistance mutations. Subtype B was found in 83.8 percent of all samples, subtype F in 2.7 percent, and BF mosaics in 11 percent of the cases. B and F different genetic profiles were evidenced: B(PR)B(RT)B(gp120)B(gp41), F(PR)F(RT)F(gp120)F(gp41), F(PR)F(RT)B(gp120)B(gp41), F(PR)B(RT)B(gp120)B(gp41), and B(PR)B(RT)F(gp120)B(gp41). A single sample (1.4%) was characterized as a BC mosaic (B(PR)C(RT)U(gp120)C(gp41)) and an AGH mosaic (AG(PR)G(RT)H(gp120)H(gp41)) was first observed in the country. Antiretroviral resistance to nucleoside RT inhibitor was observed in one sample (1.35%) showing M41L and T215S mutations. Nonnucleoside RT inhibitor and major PR inhibitor resistance mutations were not observed. Previously unseen patterns of resistance to T20 were found among HIV-1-infected drug-naïve individuals in Brazil. Recent infections were characterized in 21.6 percent of the blood donor samples included in this study. The availability of characterized plasma samples and HIV isolates will allow the development of biologic reagents necessary to implement quality control programs and to develop, validate, and evaluate the performance of diagnostic kits, considering all HIV-1-circulating variants in the country.

Development and evaluation of a qualitative reverse-transcriptase nested polymerase chain reaction protocol for same-day viral validation of human immunodeficiency virus type 1 ribonucleic acid in processed semen

To develop a method for same-day validation of processed semen in the setting of assisted reproductive techniques (ART) with patients who are seropositive for human immunodeficiency virus, type 1 (HIV-1). Laboratory experiments. University hospital. Volunteers who are HIV-1 seronegative and seropositive. Evaluation of the sensitivity of a reverse-transcriptase (RT)-nested polymerase chain reaction (PCR) in HIV-1 RNA-positive blood plasma, in artificially infected blood plasma and semen, and in 85 semen samples of 29 HIV-1-seropositive volunteers. Semen was submitted to gradient separation, followed by swim-up. Qualitative detection of HIV-1 RNA in blood plasma and in different parts of semen preparation by using RT-nested PCR, PCR inhibition control by dilution of samples, and an internal control. The detection limit of our PCR was 20 HIV-1 RNA copies per milliliter. Among seropositive patients, RNA was detected in 25% of fresh semen, 36.5% of seminal plasma, 27.5% of gradient supernatants, and 7.1% of final preparations before the migration-sedimentation stage. Positive final preparations were observed in patients who had blood viral loads of >/=20,000 HIV-1 RNA copies per milliliter. Inhibition was present in 17.6% of seminal plasma and in 20% gradient supernatants and in 2 final preparations among 69 tested. Among 25 preparations tested after the migration-sedimentation stage, 2 were positive (1 patient; 70,000 HIV-1 RNA copies per milliliter). The RT-nested PCR detects low viral load and allows the validation of semen preparations of HIV-1-seropositive patients for ART on the day of sampling. For this purpose, the validation is performed on spermatozoa that are obtained after gradient separation before swim-up. Inhibition of the PCR must be controlled by using an internal control that is well-designed to explore the detection limit of the method.

EVIDENCE FOR CRYPTIC SPECIATION OF LEUCOCYTOZOON SPP. (HAEMOSPORIDA, LEUCOCYTOZOIDAE) IN DIURNAL RAPTORS

Species of Leucocytozoon (Haemosporida, Leucocytozoidae) traditionally have been described based on morphological characters of their blood stages and host cells, with limited information on their avian host specificity. Based on the current taxonomy, Leucocytozoon toddi is the sole valid species of leucocytozoids parasitizing falconiform birds. Using a nested polymerase chain reaction protocol, we determined the prevalence of Leucocytozoon infection in 5 species of diurnal raptors from California. Of 591 birds tested, 177 (29.9%) were infected with Leucocytozoon toddi. Subsequent phylogenetic analysis of the cytochrome b gene revealed that distinct haplotypes are present in hawks of these genera. Haplotypes present in Buteo spp. are not found in Accipiter spp., and there is a 10.9% sequence divergence between the 2 lineage clades. In addition, Leucocytozoon sp. from Accipiter spp. from Europe group more closely with parasites found in Accipiter spp. from California than the same California Accipiter species do with their sympatric Buteo spp. Similarly, a Leucocytozoon haplotype from a Common Buzzard (Buteo buteo) from Kazakhstan forms a monophyletic lineage with a parasite from B. jamaicensis from California. These results suggest that Leucocytozoon toddi is most likely a group of cryptic species, with 1 species infecting Buteo spp. and 1 or more species, or subspecies, infecting Accipiter spp.

Preimplantation genetic diagnosis of skin fragility-ectodermal dysplasia syndrome

Skin fragility-ectodermal dysplasia syndrome is an autosomal recessive disorder caused by loss-of-function mutations in the desmosomal protein, plakophilin 1. Clinically, there may be considerable morbidity from extensive skin erosions and painful fissures on the palms and soles. In the absence of any specific treatment, prenatal diagnosis is an option for couples at reproductive risk of recurrence. In 2000, we developed and applied a single cell nested polymerase chain reaction protocol to test one couple for compound heterozygous plakophilin 1 gene mutations by preimplantation genetic diagnosis (PGD). Although pregnancy was established, an unrelated trisomy 22 led to a spontaneous abortion. However, eight embryos of known genetic status were cryopreserved at that stage, and we planned to undertake subsequent frozen embryo replacement cycles that might lead to the birth of an unaffected child in this family. Embryo cryopreservation was carried out in June 2000 using standard protocols in a three-step freezing procedure. Four embryos were thawed in March 2003, one of which was viable and was used in a frozen embryo replacement cycle, but pregnancy did not occur. The remaining four embryos were thawed in February 2004, two of which were viable (both carriers of the paternal mutation) and these were used in a second frozen embryo replacement cycle, and a singleton pregnancy was established. The child's plakophilin 1 genotype was assessed by direct nucleotide sequencing across the site of both potential mutations. Following two frozen embryo replacement cycles, and almost 4 years after the initial embryo biopsy and mutation analysis, a pregnancy was achieved that progressed to term with the birth of a healthy baby girl. Nucleotide sequencing of cord blood DNA, taken immediately after delivery, showed that the child was a heterozygous carrier of the paternal mutation but not of the maternal mutation. This case demonstrates the value of embryo cryopreservation, which can increase the number of embryo replacement procedures and hence the cumulative pregnancy rate per retrieval cycle. Moreover, this is the first report of successful full-term pregnancy and birth of a healthy baby following exclusion of a severe genodermatosis by PGD. The successful outcome of PGD in this case illustrates what is technically possible for couples at risk of recurrence of a severe inherited skin disease.

Occurrence of two newly named oral treponemes -Treponema parvum and Treponema putidum- in primary endodontic infections

Recent evidence from molecular genetic studies has revealed that oral Treponema species are involved in infections of endodontic origin. This study assessed the occurrence of two newly named oral treponemes - Treponema parvum and Treponema putidum - in primary endodontic infections using a culture-independent identification technique. Genomic DNA was isolated directly from clinical samples, and a 16S rRNA gene-based nested polymerase chain reaction (PCR) assay was used to determine the presence of T. parvum and T. putidum. Species-specific primer pairs were developed by aligning closely related 16S rRNA gene sequences. The specificity for each primer pair was validated by running PCR against a panel of oral bacteria and by sequence analysis of PCR products from positive clinical samples. T. parvum was detected in 52% of the root canals associated with chronic apical periodontitis, in 20% of the cases diagnosed as acute apical periodontitis, and in no abscessed case. In general, T. parvum was detected in 26% of the samples from primary endodontic infections. T. putidum was found in only one case of acute apical periodontitis (2% of the total number of cases investigated). The devised nested PCR protocol was able to identify both T. parvum and T. putidum directly in clinical samples and demonstrated that these two treponemes can take part in endodontic infections.

Absence of simian virus 40 DNA sequences in primary central nervous system lymphoma in HIV-negative patients.

Simian virus 40 (SV40) is known to induce primary brain tumors and lymphomas in animal models. Recently, it was also associated with the pathogenesis of human non-Hodgkin's lymphomas. In the present study, we investigated primary central nervous system lymphomas (PCNSL), a defined subgroup of diffuse large B-cell lymphoma confined to the central nervous system, for the presence of SV40 DNA. Frozen tissue samples of 23 PCNSL derived from human immunodeficiency virus-negative patients were analyzed by two different, fully nested polymerase chain reaction protocols. SV40 DNA sequences could not be detected in any of these samples. Thus, SV40 can be added to the list of viruses that have already been excluded as pathogenetically relevant cofactors in PCNSL.