Genetic diversity of Pneumocystis carinii f. sp. hominis based on variations in nucleotide sequences of internal transcribed spacers of rRNA genes

A variety of genes have been used to type Pneumocystis carinii. In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type Pneumocystis carinii f. sp. hominis DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple P. carinii strains. The genetic polymorphism observed demonstrates the degree of diversity of Pneumocystis strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.     

Internal transcribed spacer regions of rRNA genes of Pneumocystis carinii from monkeys

Analysis of sequence variations among isolates of Pneumocystis carinii f. sp. macacae from 14 Indian rhesus monkeys (Macaca mulatta) at the internal transcribed spacer (ITS) regions of the nuclear rRNA gene was undertaken. Like those from P. carinii f. sp. hominis, the ITS sequences from various P. carinii f. sp. macacae isolates were not identical. Two major types of sequences were found. One type of sequence was shared by 13 isolates. These 13 sequences were homologous but not identical. Variations were found at 13 of the 180 positions in the ITS1 region and 28 of the 221 positions in the ITS2 region. These sequence variations were not random but exhibited definite patterns when the sequences were aligned. According to this sequence variation, ITS1 sequences were classified into three types and ITS2 sequences were classified into five types. The remaining specimen had ITS1 and ITS2 sequences substantially different from the others. Although some specimens had the same ITS1 or ITS2 sequence, all 14 samples exhibited a unique whole ITS sequence (ITS1 plus ITS2). The 5.8S rRNA gene sequences were also analyzed, and only two types of sequences that differ by only one base were found. Unlike P. carinii f. sp. hominis infections in humans, none of the monkey lung specimens examined in this study were found to be infected by more than one type of P. carinii f. sp. macacae. These results offer insights into the genetic differences between P. carinii organisms which infect distinct species.     

PCR for capsular typing of Haemophilus influenzae.

A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine.     

汉坦病毒R36株的基因分型研究

采用汉坦病毒Ｍ片段特异性核苷酸分型引物，对Ｒ３６等７株汉坦病毒进行反转录和聚合酶链式反应扩增鉴定，结果Ｒ３６株仅能被Ⅰ型分型引物扩增出特异性片段，而不能被Ⅱ型引物扩增；ＰＣＲ产物的序列分析结果表明，Ｒ３６与ＨＴＮ型病毒７６－１１８株的同源性为７８．４％，与ＳＥＯ型病毒Ｒ２２株的同源性为６８．１％。文中对Ｒ３６与汉坦病毒其它毒株的核苷酸同源性进行了配对比较。该项研究为从核苷酸水平对Ｒ３６株的分型，提供了科学依据。     

Diversity of host species and strains of Pneumocystis carinii is based on rRNA sequences.

We have amplified by PCR Pneumocystis carinii cytoplasmic small-subunit rRNA (variously referred to as 16S-like or 18S-like rRNA) genes from DNA extracted from bronchoalveolar lavage and induced sputum specimens from patients positive for P. carinii and from infected ferret lung tissue. The amplification products were cloned into pUC18, and individual clones were sequenced. Comparison of the determined sequences with each other and with published rat and partial human P.carinii small-subunit rRNA gene sequences reveals that, although all P. carinii small-subunit rRNAs are closely related (approximately 96% identity), small-subunit rRNA genes isolated from different host species (human, rat, and ferret) exhibit distinctive patterns of sequence variation. Two types of sequences were isolated from the infected ferret lung tissue, one as a predominant species and the other as a minor species. There was 96% identity between the two types. In situ hybridization of the infected ferret lung tissue with oligonucleotide probes specific for each type revealed that there were two distinct strains of P. carinii present in the ferret lung tissue. Unlike the ferret P. carinii isolates, the small-subunit rRNA gene sequences from different human P. carinii isolates have greater than 99% identity and are distinct from all rat and ferret sequences so far inspected or reported in the literature. Southern blot hybridization analysis of PCR amplification products from several additional bronchoalveolar lavage or induced sputum specimens from P. carinii-infected patients, using a 32P-labeled oligonucleotide probe specific for human P. carinii, also suggests that all of the human P. carinii isolates are identical. These findings indicate that human P. carinii isolates may represent a distinct species of P. carinii distinguishable from rat and ferret P. carinii on the basis of characterization of small-subunit rRNA gene sequences.     

Pulmonary colonisation with Pneumocystis carinii in an immunosuppressed HIV-negative patient: detection and typing of the fungus by PCR

Mostly Pneumocystis carinii isolates from patients with acute pneumocystosis (PCP) have been typed until now. This report describes the typing of P. carinii organisms obtained from an HIV-negative patient without PCP. The patient underwent a broncho-alveolar lavage (BAL) to investigate an abnormal chest X-ray. He was diagnosed with sarcoidosis. However, a low level of P. carinii organisms undetectable by microscopy was detected in BAL fluid by two subsequent nested PCR assays: one assay amplifying a portion of the mitochondrial large subunit RNA gene and a second one amplifying the internal transcribed spacers (ITS) 1 and ITS 2 of the nuclear rRNA operon. This low level of the fungus did not reflect acute PCP. Indeed, the clinical outcome was improvement despite the absence of specific treatment. The patient was considered to be only colonised by the fungus. Analysis of sequences of ITS PCR products led to identification of genotype Gg. This information constitutes the first data concerning P. carinii ITS genotype from a patient without acute PCP and HIV. This type has been described previously in AIDS patients diagnosed with PCP. These results show that PCR and ITS genotyping could represent efficient tools for the further investigation of the role played by HIV-negative patients with pulmonary colonisation in the human reservoir of P. carinii.     

Study of internal transcribed spacer and mitochondrial large-subunit genes of Pneumocystis carinii hominis isolated by repeated bronchoalveolar lavage from human immunodeficiency virus-infected patients during one or several episodes of pneumonia.

The objective of this study was to type, analyze, and compare Pneumocystis carinii hominis strains obtained from different samples during a given or recurrent episodes of P. carinii pneumonia (PCP) for epidemiologic purposes. We studied 36 bronchoalveolar lavage (BAL) or induced sputum (IS) samples from 16 human immunodeficiency virus-infected patients with one or several episodes of PCP. PCR amplification and direct sequencing were performed on the two internal transcribed spacers (ITS1 and ITS2) of P. carinii hominis rRNA genes by using DNA extracted from BAL or IS samples, and the sequences were compared to the mitochondrial large-subunit (mt LSU) gene sequence determined in a previous study in our laboratory. The studies of the mt LSU and ITS sequences showed that some patients (n = 10) were infected with the same strains of P. carinii hominis during a given episode of PCP. In one patient infected with strains with identical sequences in several episodes, the recurrence could have been due to reactivation of organisms not eliminated by treatment during the first episode or to de novo infection by an identical strain. In five patients infected with strains with different sequences in each episode, recurrence was due to de novo infection. Sequence analysis of these two P. carinii hominis gene regions showed that de novo infection can occur in AIDS patients with recurrent PCP.     

Genotyping of Mycoplasma pneumoniae clinical isolates reveals eight P1 subtypes within two genomic groups

Three methods for genotyping of Mycoplasma pneumoniae clinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the M. pneumoniae cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of M. pneumoniae genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the M. pneumoniae isolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct M. pneumoniae genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.     

M protein gene typing of Streptococcus pyogenes by nonradioactively labeled oligonucleotide probes.

A new approach for the typing of Streptococcus pyogenes is described. Oligonucleotide probes of 30 nucleotides in length were derived from currently known sequences of the N-terminal regions of M protein genes (emm genes). The oligonucleotides were labeled with digoxigenin-dUTP and hybridized to dot-blotted genomic DNA from 116 group A streptococcal strains of serotypes M-1, M-2, M-3, M-5, M-6, M-12, M-18, M-19, M-24, and M-49. Hybridization reactions were visualized with a chemiluminescent substrate. In comparison with conventional serological typing of expressed M proteins, the binding of the probes to the corresponding emm genes exhibited 100% sensitivity and specificity. The results emphasize the high degree of type-specific conservation of the N-terminal regions of emm genes from reference strains and epidemiologically unrelated U.S. and European clinical isolates. The existence of two distinct genetic subgroups among eight investigated M-49 strains was unequivocally shown by hybridization assays and further confirmed by nucleotide sequence data obtained from four selected M-49 strains. Because oligonucleotide probes are relatively easy to prepare, easy to handle, and known to give consistent interlaboratory results, the "oligotyping" technique appears to offer potential advantages over conventional serological typing methods.     

Genotyping and coalescent phylogenetic analysis of Pneumocystis jiroveci from South Africa

Sequence analysis of Pneumocystis jiroveci internal transcribed spacer (ITS) regions has become an important epidemiological tool. The objectives of the present study were to investigate sequence variations in the ITS1-5.8S ribosomal DNA (rDNA)-ITS2 regions; determine the P. jiroveci genotypes present in Cape Town, South Africa; and resolve the lineage evolution of the types by use of the coalescent theory. ITS regions were amplified from samples collected from 19 patients. PCR products were cloned, and four to five clones were sequenced from each specimen. Statistical parsimony was applied for coalescence-based network genotype analysis. The most prevalent type was Eg (14 of 19 patients, 33 of 83 clones), followed by Gg (4 of 19 patients, 7 of 83 clones), Eu (3 of 19 patients, 5 of 83 clones), and Gh (2 of 19 patients, 2 of 83 clones). Four new combinations (Eo, Je, Ge, and No), 11 new ITS1 sequences, and 13 new ITS2 sequences were identified. A new ITS2 type was detected in three patients and was designated type u. Coinfection appeared to be common, with 15 of 19 patients harboring more than one type and with up to six types per specimen. The resultant parsimony network identified Eg as the most probable ancestral haplotype and supported the occurrence of recombinational events within the population studied. Although the 5.8S rDNA region revealed only 13 clones containing one to two nucleotide polymorphisms, it may assist in defining types. Coalescent theory proposed that Eg is an ancestral type from which microevolutionary subtypes radiate.     

Identification of dermatophytes by an oligonucleotide array

Species of dermatophytes are classified into three anamorphic (asexual) genera, Epidermophyton, Microsporum, and Trichophyton. Conventional methods used to identify dermatophytes are often lengthy and may be inconclusive because of atypical microscopic or colony morphology. Based on the internal transcribed spacer 1 (ITS-1) and ITS-2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 17 dermatophyte species. The method consisted of PCR amplification of the ITS regions using universal primers, followed by hybridization of the digoxigenin-labeled PCR products to an array of oligonucleotides (17- to 30-mers) immobilized on a nylon membrane. Of 198 dermatophyte strains and 90 nontarget strains tested, the sensitivity and specificity of the array were 99.5% and 97.8%, respectively. The only strain not identified (Microsporum audouinii LMA 597) was found to have a nucleotide insertion at the ITS-2 region where the probe was designed. Two nontarget strains, Microsporum equinum LMA 40396666 and Trichophyton gourvilii var. intermedium CBS 170.65, were misidentified as Microsporum canis and Trichophyton soudanense, respectively. Sequence analysis of the ITS regions revealed that the two misidentified strains displayed high sequence homology with the probes designed for M. canis and T. soudanense, respectively. The present method can be used as a reliable alternative to conventional identification methods and can be completed with isolated colonies within 24 h.     

Comparison of Different DNA Fingerprinting Techniques for Molecular Typing of Bartonella henselae Isolates

Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.     

Rapid DNA diagnosis of herpes simplex virus serotypes.

The presence of nucleotide sequences specific for each of herpes simplex virus (HSV) serotypes was demonstrated. These sequences were applied for dot DNA-DNA hybridization and for PCR for rapid DNA diagnosis of HSV infections. These sequences were found by molecular cloning of HSV-DNA fragments after digestion of DNA by KpnI enzyme. The type 1-specific sequence was found around the 5' end of BamHI B-fragment in the L region of type 1 DNA (corresponds to alpha gene 27, promoter-regulatory region) and the type 2-specific sequence was around the junction region of the L and S of type 2 DNA (corresponds to a' sequence). Both simple dot blot hybridization and PCR of HSV DNA's, employing these type-specific nucleotide sequences, were proven to be much more useful than immunofluorescence in terms of type-specific diagnosis of HSV infections.     

A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases

The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing.     

Detection and typing of 46 genital human papillomaviruses by the L1F/L1R primer system based multiplex PCR and hybridization

There are several genital HPV DNA detection systems described, however most of them present different disadvantages regarding the number of amplified and detected types, sensitivity, type specificity. A new PCR and hybridization based detection method was developed for sensitive and balanced amplification and specific typing of HPV DNA from clinical samples. The technique amplifies and detects 46 HPV types: 2a, 3, 6, 7, 10, 11, 13, 16, 18, 26, 27, 28, 29, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44/55, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 66, 67, 68, 70, 72, 73(MM9), 74, 82(MM4), 84(MM8), 89, 90, 91. Key elements of the L1F/L1R PCR and hybridization system are: the special selection of the amplified region, a novel and optimized amplification primer set, circumspectly designed general and type-specific oligonucleotide probes. Detection following a multiplex PCR is based on solid phase hybridization in microtiter plate format using general and type-specific probes at medium stringency, which makes the detection robust in case of small sequence variations. The assay is highly reproducible and suitable for automation. The method was compared to Hybrid Capture II test, and after clarifying conflicting results, the comparison showed an excellent agreement (96.2%).     

Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array

The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies.     

Oropharyngeal samples for genotyping and monitoring response to treatment in AIDS patients with Pneumocystis carinii pneumonia.

A nested PCR, amplifying a portion of the gene encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) of Pneumocystis carinii sp. f. hominis was applied to oropharyngeal samples obtained on repeated occasions from 12 HIV-infected patients with P. carinii pneumonia (PCP) to monitor response to anti-P. carinii treatment. Genotyping of P. carinii sp. f. hominis was also performed on paired samples of oropharyngeal and broncho-alveolar lavage samples before the start of treatment, and on oropharyngeal samples during the course of treatment, by analysis of sequence variation at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon. When a simple dilutional method was used, a reduction in the amount of amplification product was observed in samples from all patients during the course of treatment. In eight of the 12 patients, a single ITS sequence type was found in the oropharyngeal samples and also in the paired broncho-alveolar lavage sample. A mixed infection was identified in the samples from three patients. In eight patients, the ITS sequence types identified in the oropharyngeal sample were the same as in the broncho-alveolar lavage sample. Nested PCR amplifying the mt LSU rRNA on oropharyngeal samples provides a non-invasive method of monitoring response to treatment of PCP. ITS sequence typing of P. carinii sp. f. hominis from oropharyngeal samples appears to be a reliable alternative to broncho-alveolar lavage samples and provides a non-invasive tool for further epidemiological studies.     

Characterization of the HLA-A polymorphism by locus-specific polymerase chain reaction amplification and oligonucleotide hybridization

This report describes a PCR-based typing protocol for the HLA-A polymorphism. Locus-specific primers selectively amplified HLA-A sequences from exon 1 to exon 3 in a single PCR that avoided co-amplification of other classical and nonclassical class I genes. The allelic variation in exons 2 and 3 of the HLA-A gene was examined with a set of 44 oligonucleotide probes. According to the recognized HLA-A sequences the protocol is potentially able to distinguish all known HLA-A alleles with unique nucleotide sequences in this gene region. The related HLA-A genotypes can also be identified in both homozygous and heterozygous individuals. Thus the protocol provides the highest resolution for HLA-A typing. The PCR-SSO typing technique is accurate, reliable, and particularly suitable for a large number of samples. The DNA typing results from 42 Tenth IHWS B-cell lines are compatible with the serologic and IEF definitions. Sixty-six unrelated donors from a northern Chinese population were also tested, with 16 HLA-A alleles detected. Four subtypes of HLA-A2 were found in this population. The distribution of HLA-A subtypes in the population indicated that 40% of donor-recipient pairs thought to be matched for HLA-A by serology would be mismatched. Two novel HLA-A alleles were identified by unusual oligonucleotide hybridization patterns.     

Detection and direct typing of herpes simplex virus by polymerase chain reaction

A method for the detection and direct typing of herpes simplex virus (HSV) by the polymerase chain reaction (PCR) technique has been developed. One common upstream primer and two type-specific downstream primers were prepared to amplify DNA from the HSV type 1 and type 2 DNA polymerase gene. Using these three primers simultaneously in the PCR reaction mixtures, both types of HSV DNA were amplified to produce products of different sizes. By direct gel analysis, the products of standard HSV type 1 and type 2 strains had the predictive sizes of 469 and 391 base pairs, respectively, and the difference in molecular mass enabled us to type the HSV strain. A total of 24 strains (type 1; 16 and type 2; 8 strains) were examined by PCR, and the results were consistent with those determined by immunofluorescence using type-specific monoclonal antibodies. No specific amplification was observed using other herpes virus or human genomic DNAs. The PCR method was then applied to clinical specimens. Of 15 samples obtained from oral lesions of children with herpetic gingivostomatitis, all (100%) were HSV positive by PCR, compared with 13 (86.7%) using standard cell culture methods. Three specimens from vulvar lesions of women with genital herpes were positive using both PCR and cell cultures. There was complete agreement in the typing of HSV strains using the PCR method or virus isolation. On the basis of these results, it is suggested that DNA amplification and typing by PCR is particularly useful for material from which virus isolation might be difficult.