Genetic diversity of glucose phosphate isomerase from Entamoeba histolytica

To investigate the molecular basis of zymodeme analysis in the enteric protozoan parasite Entamoeba histolytica, genes encoding glucose phosphate isomerase (GPI) were isolated from four representative E. histolytica strains belonging to zymodeme II, II-, XIV, or XIX. Two alleles were obtained from each strain; six alleles with eight polymorphic nucleotide positions were identified among the four strains. Two of these eight polymorphic nucleotides resulted in non-conserved amino acid substitutions. Three GPI isoenzymes with distinct predicted isoelectric points were identified, which agrees well with the observed electrophoretic patterns of GPI from these strains. Amino acid comparisons of GPI from E. histolytica and other organisms revealed that all amino acid residues implicated for substrate binding and catalysis were conserved. Biochemical characterization of recombinant E. histolytica GPI confirmed that it possessed kinetic parameters similar to GPI from other organisms. The electrophoretic mobility of three GPI isoenzymes was examined by starch gel electrophoresis. Thus, we have established the molecular basis of the classical isoenzymes patterns that have been used for grouping E. histolytica isolates and for differentiation of E. histolytica from non-pathogenic Entamoeba dispar.     

Cytopathologic and genetic diagnosis of pulmonary amebiasis: a case report

BACKGROUND: Amebiasis is a parasitic infection with Entamoeba histolytica. Pulmonary amebiasis is rare since the infection is commonly manifested as amebic colitis or liver abscess. Most pleuropulmonary amebiasis is seen in patients with amebic liver abscesses. A pulmonary amebic lesion without either a liver abscess or amebic colitis is extremely rare. Thus, reported cases of sputum cytologic diagnosis of a pulmonary amebic lesion from a patient without a liver abscess are also very rare. CASE: A 53-year-old man presented with a dry cough and mild fever. Chest radiography revealed an abnormal solitary mass lesion in the right upper lung field. The clinical diagnosis was a bacterial lung abscess. Sputum cytologic examination demonstrated many trophozoites of E. histolytica. Following sputum cytodiagnosis, serologic tests revealed a slightly high but almost normal titer of IgG antibodies to E. histolytica, indicating the possible presence of the pathogen. Polymerase chain reaction (PCR) using E. histolytica-specific primers for DNA extracted from the sputum sample revealed specific DNA product. CONCLUSION: Pulmonary amebiasis without either a liver abscess or amebic colitis must be distinguished from bacterial abscesses and neoplastic disease. A sputum cytologic examination combined with PCR for DNA extracted from a sputum sample is a good approach to the diagnosis of a pulmonary amebic abscess.     

Diagnosis of nocardiosis by polymerase chain reaction: an experimental study in mice

In this study, we have established a sensitive semi-nested polymerase chain reaction (PCR) for detection of Nocardia in clinical specimens by first amplifying a 422 bp DNA fragment from the groEL gene, followed by a second amplification of 342 bp DNA by targeting sequences internal to the first amplicon. The semi-nested PCR was evaluated in a murine model of nocardiosis for detection of Nocardia in blood and visceral organs. Healthy BALB/c mice were intravenously infected with 0.2 ml suspension of 2.9 × 10⁵/ml cfu of Nocardia asteroides and N. farcinica. Viable counts and semi-nested PCR were performed post-infection with samples of blood as well as lung, liver, spleen, kidney and brain at 5 minutes, 3 h, and then every 24 h for 3 days. Of the 20 blood samples tested, 15 (75%) were Nocardia positive by culture and 19 (95%) were positive by semi-nested PCR. Likewise, in case of N. asteroides infection, 46% organ samples were positive by culture and 58% by semi-nested PCR. The positivity of organ samples was higher with N. farcinica, 60% by culture and 72% by PCR, which may be attributed to its increased virulence as compared to N. asteroides. These results demonstrate that semi-nested PCR is a rapid and sensitive method for detection of Nocardia in blood and different visceral organs. The diagnostic application of this method provides an additional advantage over culture techniques, as PCR can also detect L-forms of Nocardia in clinical specimens, which otherwise fail to grow on routine isolation medium.     

Identification of two putative ATP-cassette genes in Encephalitozoon intestinalis.

Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis. It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica. The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene. We report here two complete sequences EiABC1 and EiABC2, encoding different ATP-binding cassette genes from E. intestinalis, including a P-gp.     

Detection of Streptococcus pneumoniae DNA by using polymerase chain reaction and microwell hybridization with Europium-labelled probes

The present paper describes a novel modification of polymerase chain reaction (PCR) for the detection of Streptococcus pneumoniae DNA in clinical specimens. PCR was based on the detection of a 209-base pair segment of the S. pneumoniae pneumolysin gene. For the demonstration of the amplification product, microwell hybridization with a Europium-labelled oligonucleotide probe complementary to a biotinylated strand of the PCR product was performed, and the presence of the PCR product was monitored by time-resolved fluorescence (TRF) of the Europium chelate. The sensitivity of the assay for purified S. pneumoniae DNA was 50 fg DNA corresponding to 20 genome equivalents of S. pneumoniae DNA. The efficiency of the hybridization step was monitored by using known amounts of synthetic target oligonucleotides as standards. Sensitivity of 3×10⁸ molecules per individual reaction well was achieved with a 30-min attachment time and a 3-h hybridization time.

Detection of PCR-amplified products by the microwell hybridization technique and TRF was compared to agarose gel electrophoresis in 50 middle ear fluid samples obtained from children with acute otitis media. The agarose gel and TRF detection methods identified all culture-positive samples, but both were also positive for 55% of the culture-negative samples. The results suggest that the detection of amplified PCR products by microwell hybridization using Europium-labelled oligonucleotides is a reliable method for the demonstration of the pneumolysin gene fragment. Furthermore, the method is suitable for automation and, thus, for testing high numbers of samples. The clinical significance of the PCR findings remains to be studied.     

An IC-PCR method for detection of Cryptosporidium and Giardia in natural surface waters in Finland

We developed an immunocapture-based polymerase chain reaction (PCR) assay for simultaneous detection of Cryptosporidium parvum oocysts and Giardia intestinalis cysts in surface water. Using primer pairs Cry9/Cry15 and LaxA/LaxB for Cryptosporidium and Gdh1/Gdh4 for Giardia, the sensitivity of the entire detection procedure (dealing with concentration, separation, DNA purification and PCR amplification) was at the level of 50–100 oocysts and cysts. Of 54 surface water samples, 4 were positive for Cryptosporidium and 1 for Giardia. Cryptosporidium and Giardia were detected for the first time in surface water in Finland.     

An interlaboratory comparison for the detection of Mycoplasma pneumoniae in respiratory samples by the polymerase chain reaction

A panel of 78 respiratory samples collected from 43 patients was analyzed in three different Centers for the presence of Mycoplasma pneumoniae DNA by polymerase chain reaction (PCR). One Center collected the samples and extracted the DNA by two different methods. DNA extracted according to the first method was amplified using primers targetting the 16 S rRNA gene. DNA extracted according to the second method was amplified using the same primers in a semi-nested format and was sent to the two other Centers. The latter Centers both used the same primers targetting the P1 gene but with a different detection format. Thirty-nine samples (50%) from 19 patients were positive by at least two PCR assays. None of the laboratories were free of false positive or false negative PCR results. Calculated specificities of the individual PCR assays ranged from 97.4% to 87.2% and sensitivities ranged from 97.4% to 89.2%. Complement fixation was done on sera of 33 patients. The calculated specificity and sensitivity of serology was 100% and 58.8%, respectively. Several aspects concerning false positive and false negative results with PCR are discussed.     

Detection of Pneumocystis carinii DNA by polymerase chain reaction compared to direct microscopy and immunofluorescence.

The polymerase chain reaction (PCR) using published primers and probes has been compared to conventional stains and immunofluorescence for diagnosis of Pneumocystis carinii. We have screened 71 bronchoalveolar lavage (BAL) fluids from HIV-immunosuppressed patients. Of 34 samples negative by conventional stains and immunofluorescence, only one was positive by PCR. Thirty of 35 samples positive by conventional stains and immunofluorescence were also positive by PCR. One BAL sample, negative by conventional stains but positive by immunofluorescence, was negative by PCR. These data are discussed in relation to clinical and therapeutic conditions of the patients.     

Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (SPR)

A method for the rapid detection of verotoxin-producing Escherichia coli O157:H7 in stools was evaluated. Strains possessing Shiga toxin-2 (stx-2) genes were isolated from stool samples and amplified using oligonucleotide primers. Stools spiked with cultured E. coli O157:H7 (strain 298 or strain 1646) were detected to be polymerase chain reaction (PCR) positive at 10(2) cfu per 0.1 g of stool. Stool samples from patients and healthy carriers showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E. coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10 of 10 stool samples). These PCR products could be detected using a BIAcore 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe. In this report we use this method for the rapid detection of DNA from significant pathogenic organisms.     

Detection of the protozoan Neospora caninum Using in situ Polymerase Chain Reaction

A new method to detect the protozoan Neospora caninum using indirect in situ polymerase chain reaction (PCR) is described. In situ PCR combines the advantages of the extraordinarily high sensitivity and specificity of PCR and the in situ representation of immunohistochemical methods. We describe an indirect in situ PCR, whereby the amplified products were detected using a primed in situ (PRINS) reaction with hapten-labeled nucleotides and visualized using fluorochrome-labeled antibodies. This technique was carried out in both infected cell cultures and formalin fixed, paraffin embedded tissues. Clear signals were obtained in the N. caninum positive samples using in situ PCR, whereas control slides with Toxoplasma gondii infected tissues always yielded negative results.     

Toward a rational design of selective multi-trypanosomatid inhibitors: a computational docking study

Compound V7, a benzothiazole which was recently found as selective inhibitor of trypanosomal TIMs, was docked into TIMs from Trypanosoma cruzi, Trypanosoma brucei, Entamoeba histolytica, Plasmodium falciparum, yeast, and human. Structural analyses revealed the importance of the accessibility to the two aromatic clusters located at the dimer’s interface for the selective inhibition of trypanosomal TIMs. Thus, it was found that different accessibilities of the protein interface of TIMs plays an important role in the inhibitory activity of benzothiazoles. These findings will contribute to the rational development and improvement of benzothiazoles to be used as multi-trypanosomatid inhibitors.     

A rapid DNA isolation procedure for the identification of Campylobacter jejuni by the polymerase chain reaction

We have developed an efficient process for rapidly isolating campylobacter DNA using mechanical disruption combined with the guanidine-based reagent DNAzol. Template DNA was isolated by this method from cultures of Campylobacter jejuni resistant to lysis by boiling or enzymes and identified following polymerase chain reaction (PCR) amplification using primers specific for the hippuricase gene. Direct detection of campylobacters in poultry-processing samples by PCR is demonstrated in chicken carcass rinses spiked with lysis-resistant C. jejuni. Our results indicate that this method of DNA isolation may be ideal for direct PCR detection of pathogenic bacteria in complex samples of widely varied origin, especially when the target organisms are difficult to lyse by other means.     

A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area

The aim of this study was to evaluate the efficacy of a polymerase chain reaction (PCR)-based method to detect Schistosoma mansoni DNA in stool samples from individuals living in a low-endemicity area in Brazil. Of the 125 initial stool samples, 80 were ELISA reactive and eggs were identified in 19 of the samples by parasitological examination. For the PCR evaluations, 56 stool samples were selected and divided into five groups. Groups I-IV were scored negative for S. mansoni eggs by parasitological examination. Groups I and II were ELISA reactive, whereas Groups III and IV were ELISA nonreactive. Groups II and III were positive for other intestinal parasites. PCR testing scored eight samples as positive from these four groups. Group V represented the S. mansoni -positive group and it included ELISA-reactive samples that were scored positive for S. mansoni by one or more parasitological examinations (6/19 were positive by Kato-Katz method, 9/17 by saline gradient and 10/13 by Helmintex®). PCR scored 13 of these 19 samples as positive for S. mansoni . We conclude that while none of these methods yielded 100% sensitivity, a combination of techniques should be effective for improving the detection of S. mansoni infection in low-endemicity areas.     

Detection of Helicobacter ganmani-like 16S rDNA in pediatric liver tissue

BACKGROUND: To determine the presence of Helicobacter species in the liver biopsy specimens from children with various chronic liver diseases as data in adult literature suggests a possible role of these bacteria in their pathogenesis. MATERIALS AND METHODS: Paraffin sections of 61 liver biopsies of pediatric patients with miscellaneous diseases and autopsy liver tissue from 10 control subjects with no evidence of preexisting liver disease were examined for the presence of Helicobacter species by a genus-specific seminested polymerase chain reaction (PCR) assay. PCR-products of positive samples were further characterized by denaturing gradient gel electrophoresis (DGGE) and DNA-sequence analysis. Based on those results, a seminested PCR assay for H. ganmani was developed and applied to the samples. RESULTS: On analysis, 40/61 patient samples were positive in the genus-specific Helicobacter PCR and 4/10 from the control group. The nucleotide sequences of 16S rDNA fragments were 99-100% similar to mainly Helicobacter sp. 'liver' and H. ganmani. PCR-products similar to H. canis and H. bilis were also found. The 16S rDNAs of control specimens showed similarity to Helicobacter sp. 'liver'. In the H. ganmani-specific PCR analysis 19 patients, but none of the controls, were positive. CONCLUSIONS: Amplified Helicobacter 16S rDNAs were related to Helicobacter sp. 'liver' or H. ganmani in liver biopsy specimens of pediatric patients. The possible significance of Helicobacter species in pediatric liver diseases needs to be evaluated further in prospective studies.     

Opisthorchis viverrini: detection by polymerase chain reaction (PCR) in human stool samples

A polymerase chain reaction (PCR) assay was evaluated for detection of Opisthorchis viverrini eggs in the stool specimens of light and heavily infected individuals in Khon Kaen province of Thailand. A total of 75 fecal specimens were analyzed by PCR following DNA extraction. All the microscopically positive samples were positive by PCR, while 23 of 30 (76.6%) microscopically negative samples were also PCR positive. The sensitivity of the assay was 5 eggs/g of stool. This method is potentially useful in the diagnosis of human opisthorchiasis in endemic areas for treatment and in epidemiological investigations.     

Increased sensitivity of malaria detection by nested polymerase chain reaction using simple sampling and DNA extraction

Malaria remains a disease of underdeveloped and remote regions of the world. The application of polymerase chain reaction (PCR) technology to malaria epidemiology has the potential for increasing our knowledge and understanding of this disease. In order to study malaria in all geographical locations it is important that specimen collection and DNA extraction for PCR be kept simple. Here we report a method for extracting DNA from dried blood spots on filter paper which is capable of detecting one Plasmodium falciparum and two Plasmodium vivax parasites/μl of whole blood by nested PCR without compromising the simplicity of specimen collection or DNA extraction.     

Flying foxes as carriers of pathogenic Leptospira species

Recent serologic studies have identified flying foxes (Pteropus spp.) as carriers of leptospirosis; however, little is known about the role of flying foxes as carriers of pathogenic Leptospira spp. To determine if Australian Pteropus spp. are carriers of pathogenic Leptospira spp., TaqMan real-time polymerase chain reaction (PCR) was used to detect leptospiral DNA in kidney and urine specimens from four species of flying fox, including the spectacled flying fox (Pteropus conspicillatus), black flying fox (Pteropus alecto), grey-headed flying fox (Pteropus poliocephalus), and little red flying fox (Pteropus scapulatus). Of the 173 kidney samples tested, 19 (11%) were positive for leptospiral DNA. Positive individuals were detected in all four species; significant differences in prevalence were not detected between species, between species within the same geographic area, or between geographically separated samples from the same species. Of the 46 urine samples tested, 18 (39%) tested positive by PCR, confirming that flying foxes shed leptospires into the environment. The detection of leptospiral DNA in the kidneys and urine of flying foxes suggests that flying foxes are carriers of pathogenic Leptospira spp. No evidence collected in the present study, however, suggests that flying foxes pose a significant risk of leptospirosis to the wider community or that humans who are in regular, close contact with flying foxes are at risk for leptospirosis.     

病毒分离和PCR-RFLP法对急性结膜炎标本中腺病毒感染及其型别的分析

目的分析2003年下半年收集的哈尔滨医科大学第一临床医学院急性结膜炎标本中的腺病毒(Adenovirus,AdV)感染情况及其型别。方法采集59例临床确诊为急性结膜炎患者的结膜拭子标本,在观察和分析接种细胞的病变(CPE)情况的基础上,采用聚合酶链式反应-限制性片段长度多态性分析(PCR—RFLP)法,特异性检测CPE阳性细胞中腺病毒核酸的存在情况,并对PCR阳性扩增产物进行病毒型别的分析。结果70．7％(41／59)的急性结膜炎标本接种于培养细胞后可以引起明显的CPE;其中,53．6％(22／41)的CPE阳性标本可以扩增出腺病毒核酸,RFLP分析证实68．18％(15／22)的腺病毒感染为Ad3型,31．82％(7／22)的腺病毒感染为Ad7。结论2003年下半年哈尔滨市急性结膜炎主要以腺病毒3、7型感染为主。提示结合病毒分离培养和PCR—RFLP方法,可以广泛应用于急性结膜炎相关的病毒感染及其型别的进一步分析与研究。     

人细小病毒B19与自然流产关系的研究

无菌留取 5 4例自然流产妇女和 43例妊娠无异常孕妇血清 ,用聚合酶链反应 (PolymeraseChainReaction ,PCR)检测的人细小病毒B19(HumanParvovirusB19,B19)DNA ,在自然流产组中人细小病毒B19DNA有 15例阳性 ,阳性率为 2 7.78%。正常对照组中 ,人细小病毒B19DNA有 2例为阳性 ,阳性率为 4.65 % ,用x2 检验 ,x2 =8.86,P <0 .0 1,两组有非常显著性差异。由此总结 ,人细小病毒B19感染可能是导致自然流产的原因之一     

Species-specific and cross-reactive antigens of Entamoeba

Specific and cross-reactive antigens were defined in four species of Entamoeba: invadens, moshkovskii, Laredo and histolytica strains HM1, HM3, HM38 and HK9. Among these species extensive common reactivities were observed by immunoblot. Eight E. histolytica antigenic markers were revealed after blocking common specificities with antigens of other Entamoeba species. A monoclonal antibody (mAb) defined two protein markers of E. histolytica, M, 29 and 25 kDa. The four strains of E. histolytica, which varied in virulence as determined by the development of liver abscesses in hamsters, showed the same antigenic patterns with the mAb and with polyclonal antibodies.