Development of a ceuE-based multiplex polymerase chain reaction (PCR) assay for direct detection and differentiation of Campylobacter jejuni and Campylobacter coli in Thailand.

A novel ceuE-based multiplex PCR system was developed as an efficient diagnostics test to detect and differentiate C. jejuni and C. coli. There is no cross reactivity between C. jejuni and C. coli. In addition, the assay does not produce a positive signal from other enteric bacteria including Salmonella, Shigella and Escherichia coli strains. Campylobacter detection sensitivity was determined to be equivalent to previously reported PCR for other enteric bacteria. We also noticed that silicon dioxide extraction can improve Campylobacter detection sensitivity from infected stool samples. It was demonstrated that the PCR assay developed in this study had a much better Campylobacter detection rate than the traditional culturing method (77% versus 56%). However, we also identified small numbers of culture positive stools (8%, or 16 out of 202 samples) that did not yield PCR positive results for Campylobacter. These PCR negative/culture positive stools were proven to be inhibitory to PCR amplification.

Detection of PCR products of the ipaH gene from Shigella and enteroinvasive Escherichia coli by enzyme linked immunosorbent assay.

PCR techniques applied to diarrheal stools reliably diagnose Shigella and enteroinvasive Escherichia coli (EIEC) infections. Identification of PCR products using agarose gel electrophoresis (AGE) and hybridization with DNA probes has several shortcomings. Automated methods of identifying PCR products that process larger numbers of specimens can facilitate epidemiologic studies and standardize results. In this study, we used ELISA following PCR to detect ipaH gene sequences of Shigella and EIEC from 89 diarrheal stools. Results of ELISA were compared with AGE with and without DNA probe, and with culture. Two specimen preparation methods were compared as well: boiling/centrifugation, and purification with silicon dioxide (SiO(2)). Both PCR product-detection methods identified significantly more infections than did culture. PCR-ELISA detected significantly more infections than PCR-AGE when processed using SiO2 (P = 0.014). PCR-ELISA allows screening of larger numbers of specimens, automates test results, and avoids use of mutagenic reagents. PCR-ELISA is faster than PCR-AGE when testing large numbers of specimens, although not when testing small numbers of specimens.

Utility of a polymerase chain reaction diagnostic system in a study of the epidemiology of shigellosis among dysentery patients, family contacts, and well controls living in a shigellosis-endemic area.

Polymerase chain reaction (PCR) diagnostic methods have rarely been used in epidemiologic studies of Shigella and enteroinvasive Escherichia coli (EIEC) infections. In this study, amplification of the invasion plasmid antigen H (ipaH) gene by PCR and standard culture methods was used to identify Shigella species or EIEC among 154 patients with dysentery, 154 age-matched controls, and family contacts in Thailand. The ipaH PCR system increased the detection of Shigella species and EIEC from 58% to 79% among patients with dysentery and from 6% to 22% among 527 family contacts; 75% of infections in family members were asymptomatic. Detection of the ipaH gene was statistically associated with dysentery. Household contacts of patients with shigellosis diagnosed only by PCR had significantly higher rates of shigellosis than household contacts of patients who did not have Shigella or EIEC infections. Detection of the ipaH gene by PCR is far more sensitive than detection by standard culture and is highly correlated with evidence of Shigella transmission among family contacts.

A simple polymerase chain reaction technique to detect and differentiate Shigella and enteroinvasive Escherichia coli in human feces.

A simple polymerase chain reaction (PCR) procedure using IS630-specific primers was developed as a general diagnostic probe to detect Shigella and enteroinvasive Escherichia coli (EIEC). However, IS630 and the other two previously reported molecular probes, ipaH and ial, cannot be used to differentiate among Shigella serotypes and EIEC strains that cause dysentery. The sensitivity of PCR protocol was determined to be 100-200 shigellae for each PCR reaction. An enrichment incubation would allow the detection of shigellae in stool samples with low bacterial concentration; i.e., < 10(4) CFU/gram. Serotype-specific primers derived from the rfc genes of differentiate among Shigella serotypes in the laboratory, such as S. sonnei, S. flexneri, and S. dysenteriae 1. It was demonstrated further that the multiplex PCR system containing rfc-specific primers can efficiently identify the most prominent Shigella serotypes in raw stool samples of acute diarrheal patients.

Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria species.

A coccidian organism assigned to the genus Cyclospora has been increasingly recognized in association with prolonged diarrhea in humans throughout the world. Confusion surrounds the taxonomy of this fastidious organism, despite the availability of morphology and sporulation characteristics. The small subunit rRNA coding region from cyclosporan oocysts purified from a human fecal specimen was amplified and sequenced. The same sequence was present in specimens from 8 other patients with cyclosporan oocysts but absent in specimens from asymptomatic subjects and from cryptosporidiosis patients. Phylogenetic analysis of rDNA sequences reveals that the human-associated Cyclospora is closely related to members of the Eimeria genus. These results allow predictions concerning Cyclospora host specificity, life cycle, and epidemiology as well as the development of a specific polymerase chain reaction-based diagnostic assay.

Prevalence of Vibrio cholerae with heat-stable enterotoxin (NAG-ST) and cholera toxin genes; restriction fragment length polymorphisms of NAG-ST genes among V. cholerae O serogroups from a major shrimp production area in Thailand.

A total of 148 Vibrio cholerae isolates from a major shrimp production area in Southern Thailand were examined by colony hybridisation for genes encoding heat-stable enterotoxin (NAG-ST) and cholera toxin (CT). Only non-O1 V. cholerae strains were found to harbour NAG-ST (14 of 146) whereas no strains hybridised with the CT probe. NAG-ST-positive V. cholerae non-O1 strains were isolated from shrimp farms situated close to urban areas. Five different O serogroups were found among NAG-ST positive non-O1 strains. Southern blot and restriction endonuclease analysis of NAG-ST-positive strains revealed a high degree of genetic divergence. A total of seven classes of enterotoxin gene patterns were found with HindIII and EcoRI restriction endonucleases. Enterotoxin gene patterns correlated with O-antigen expression in 84% of isolates tested. In combination with other molecular techniques Southern blot analysis with an NAG-ST oligonucleotide probe could be useful for studying the molecular epidemiology of V. cholerae non-O1 strains.

Typing of human group A rotavirus with alkaline phosphatase-labeled oligonucleotide probes or a monoclonal enzyme immunoassay in unfrozen stools of children with diarrhea in Bangkok.

In developed countries, serotypes (or G types) have been identified in > 70% of group A rotavirus using monoclonal enzyme immunoassays (MEIAs); however, these assays have identified < 50% of rotavirus G types from developing countries presumably because the VP7 antigens were damaged by freezing and thawing during transportation of specimens. The VP7 (G) serotypes of rotavirus in unfrozen stool collected from children with acute diarrhea in Bangkok were determined using MEIA and compared to hybridization with alkaline phosphatase-labeled oligonucleotide probes. Reverse transcription of dsRNA coding for VP7 followed by polymerase chain reaction amplification of cDNA was used as an additional step prior to hybridization for 98 specimens that did not hybridize with the oligonucleotide probes. Of 251 rotavirus specimens, 208 (83%; 99% Cl = 76-89%) hybridized with G type specific oligonucleotides compared to 146 (58%; 99% Cl = 50-66%) that were typeable by MEIA. Forty-five (82%) of 55 stools containing G type 1, 80 of 84 (95%) containing G type 2, 0 of 3 containing G type 3, and 2 of 4 (50%) containing G type 4 as identified by MEIA hybridized with G type specific oligonucleotides. Differences in nucleotide sequences coding for VP7, in addition to destruction of the VP7 antigen by freezing and thawing of the specimen, may explain why not all rotavirus hybridized with G type specific probes.

Detection of Shigella and enteroinvasive Escherichia coli by PCR in the stools of patients with dysentery in Thailand.

The rate of detection of Shigella and enteroinvasive Escherichia coli (EIEC) using a PCR technique was compared with the rate detected by standard microbiological methods (bacteriology plus hybridization of E. coli colonies with a 17 kb EIEC probe) among patients with dysentery before and after antibiotic therapy. The PCR amplified DNA sequences encoding IpaH, a multiple copy sequence located on the chromosome and the invasion plasmid. Shigella or EIEC were detected using the IpaH PCR system among 72 (61%) of 119 patients with dysentery on the first day they were seen at hospital, compared to 50 (42%) using standard microbiological methods (p = 0.006). After three days of antibiotic therapy, IpaH sequences were detected in stools from 38 percent of patients, compared to 10 percent using standard microbiology (p < 0.001). After seven days of therapy, the rates were 26 percent vs. 8 percent respectively (p < 0.001). The IpaH PCR system appeared to be specific for Shigella or EIEC based on low rates of positive reactions among non-diarrhoea controls, and a strong correlation between persistently positive reactions and antibiotic resistance of bacterial isolates. IpaH sequences were detected in 10 (8%) of 119 drinking water samples from homes of patients with disease; none of these specimens were positive for Shigella or EIEC by standard microbiology. In conclusion, PCR amplification of IpaH sequences and detection of target DNA with a non-radioactive probe increased the rates of identification of Shigella and EIEC by 45% in initial clinical specimens and by nearly 300% in specimens obtained from patients receiving antibiotic therapy.

Modern diagnosis (with molecular tests) of acute infectious diarrhea.

There have been three significant technical developments in the molecular genetic diagnosis of infectious diarrhea. The first was the replacement of polynucleotide probes with more specific synthetic oligonucleotide probes. The second was the replacement of radiolabeled markers with nonradiolabeled markers, and the third was PCR amplification. In the PCR procedure, it is possible to increase the quantity of target nucleotide sequences to quantities easily detectable with nonradioactive oligonucleotide probes. It is now possible to amplify nucleotide sequences of more than one enteric pathogen with different primers simultaneously and to detect these amplified nucleotide sequences with nonradiolabeled oligonucleotide probes. With a scanning laser system, the results of enteric PCRs will be used to identify enteric pathogens on a routine basis in clinical and public health laboratories. Computers need to be used to analyze these results and transfer this information rapidly to clinicians and public health officials.

Epidemic of diarrhea caused by Vibrio cholerae non-O1 that produced heat-stable toxin among Khmers in a camp in Thailand.

An epidemic of a cholera-like disease occurred among Khmers in a camp in Aranyaprathet, Thailand, in May 1990. Of 215 patients with diarrhea, Vibrio cholerae O1 was isolated from 25 (12%) and V. cholerae non-O1 was isolated from 15 (7%). Five of 15 (33%) non-O1 V. cholerae isolates hybridized with two different oligonucleotide probes previously used to detect V. cholerae non-O1 that produces a heat-stable toxin. This is the first description of an epidemic of diarrhea caused by V. cholerae non-O1 that produces heat-stable toxin.

Detection of Shigellae and enteroinvasive Escherichia coli by amplification of the invasion plasmid antigen H DNA sequence in patients with dysentery.

Detection of Shigella organisms and enteroinvasive Escherichia coli (EIEC) by polymerase chain reaction (PCR) was evaluated in 20 patients with dysentery before and in 17 of the 20 after treatment with ciprofloxacin. DNA sequences coding for IpaH antigen, a multiple copy sequence found on the chromosome, and the invasion plasmid locus (ial) was detected after DNA amplification in 13 stools from patients from whom shigellae or EIEC were isolated but not in 21 nondysenteric stools containing other enteric bacteria. Although shigellae or EIEC were not isolated from any patient with dysentery after ciprofloxacin treatment, IpaH and ial sequences were found after PCR amplification in 7 patients after treatment with ciprofloxacin. IpaH sequences alone were detected in 4 patients; DNA augmentation of IpaH in stools in a specific way to identify Shigella or EIEC infection in persons from whom cultures cannot be obtained promptly after the onset of diarrhea or who have received antibiotics.

Typing of human group A rotavirus with alkaline phosphatase-labeled oligonucleotide probes.

Rotavirus (RV) in stools of children less than 1 year of age with diarrhea in Bangkok in 1989 were serotyped by monoclonal enzyme immunoassay (MEIA). RNA extracted from these specimens was tested for hybridization with alkaline phosphatase (AP) and 32P-labeled oligonucleotides constructed from the nucleotide sequences of VP7 of human G types 1 (HuG1Ac), 2 (HuG2Ac), 3 (HuG3Ac), and 4 (HuG4Ac). Of 148 specimens that contained RV, 72% (106/148) hybridized with RV G type specific AP-labeled oligonucleotides compared to 47% (70/148) that were serotyped by MEIA (P less than 0.001). Of 68 specimens that contained only one VP7 serotype (G-type), as identified by MEIA, 94% (16/17) of G1, 90% (27/30) of G2, 57% (4/7) of G3, and 36% (5/14) of G4 RV hybridized with the AP-labeled HuG1Ac, HuG2Ac, HuG3Ac, and HuG4Ac oligonucleotides, respectively. The probes for G1, 2, 3, and 4 RV were specific for each G type. The results of hybridizing specimens with 32P- and AP-labeled oligonucleotides were similar. After transcription and amplification of cDNA of gene 9, AP-labeled RV G type specific oligonucleotides hybridized with 90% (134/148) of RV specimens. The high sensitivity of these nonimmunological techniques could be of value in identifying G types of RV during vaccine trials.

Detection of Plasmodium falciparum by polymerase chain reaction in a field study.

Detection of Plasmodium falciparum by polymerase chain reaction (PCR) was evaluated in 33 P. falciparum-infected patients with two different amplification systems over 5-7 days of curative treatment. In the K1-14 system, a P. falciparum DNA fragment of 206 bp was detected, and in the circumsporozoite (CS) system, a fragment of 800 bp was detected. The K1-14 and CS systems identified 95% and 93%, respectively, of 103 microscopically identified specimens; both systems detected as few as 11 parasites/microliters among these specimens. Specimens from 20 smear- and history-negative controls were all negative by both PCR systems. The K1-14 and CS systems detected P. falciparum DNA in 53% and 20%, respectively, of blood films collected on the first day and 3% and 0 of the blood films collected on the fourth day after reversion to microscopic negative. The simultaneous use of two independent PCR systems to monitor patients during curative treatment of P. falciparum infections convincingly demonstrated that P. falciparum DNA was present transiently in the blood of infected patients at a time when the parasite could no longer be detected microscopically.

Shigella and enteroinvasive Escherichia coli infections in households of children with dysentery in Bangkok.

Shigellae and enteroinvasive Escherichia coli (EIEC) were identified in children with dysentery and their household contacts in Bangkok. Shigellae were isolated from 49% and EIEC from 6% of 306 children with dysentery seen at the outpatient department of Children's Hospital on weekdays during January through June 1989 and October 1989 through October 1990. The same serotype infecting the index child was isolated from 21 (4%) of 522 household contacts of 151 index children with Shigella infections and from none of 60 household contacts of 19 index children with EIEC infections. Amplification of DNA sequences coding for the invasion-associated locus (ial) by polymerase chain reaction increased the identification of Shigella and EIEC infections from 57% (111/193) to 68% (132/193). ial sequences were identified in 3 of 20 drinking water specimens from which shigellae or EIEC were not isolated. Amplification of ial sequences identified more shigellae and EIEC than did bacteriologic and colony hybridization methods in children with dysentery and in drinking water in Bangkok.

Microbiology and diagnosis of infections with Shigella and enteroinvasive Escherichia coli.

The etiology of dysentery in Thailand and the existing methods of diagnosing infections with Shigella and enteroinvasive Escherichia coli (EIEC) are reviewed. The four Shigella species (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) are classically identified by culture of fecal specimens on selective media and testing of isolates for agglutination in species-specific antisera. DNA probes have been used to identify both lactose-fermenting and non-lactose-fermenting EIEC as well as Shigella isolates that do not agglutinate in antisera. These DNA probes are not necessary for the identification of Shigella if a competent bacteriology laboratory with shigella antisera is available. In Thailand Shigella and EIEC are isolated more often from children greater than 2 years of age than from younger children. The clinical illness associated with EIEC infections is similar to shigellosis. Fewer children with EIEC infections than with shigellosis, however, have occult blood in stool (36% vs. 82%) and more than 10 fecal leukocytes per high-power field (36% vs. 67%). Standard bacteriologic methods and testing of E. coli isolates for hybridization with the shigella/EIEC probe are currently the most sensitive means of diagnosing infections caused by these enteric pathogens. A more rapid method of identifying Shigella and EIEC infections in a situation where a bacteriology laboratory is not available will probably involve immunologic assays.

Serotyping of human group A rotavirus with oligonucleotide probes.

Rotaviruses (RV) in stools of children with diarrhea in Thailand were serotyped by monoclonal enzyme immunoassay (MEIA), and RNA extracted from these specimens were tested for hybridization with oligonucleotides constructed from the nucleotide sequences of VP7 of human serotypes 1, 2, 3, and 4. Of 178 specimens that contained RV as identified with a monoclonal antibody to group A RV, 84% (149/178) hybridized with serotype-specific oligonucleotides, and 42% (74/178) were serotyped by MEIA (P less than .001). Of the 74 specimens that were serotyped by MEIA, 92% (35/38) of type 1, 97% (34/35) of type 2, and the one type 4 RV hybridized with the HuG1Ac, HuG2Ac, and HuG4Ac oligonucleotides, respectively. RV strains identified in children in Thailand in 1987 and 1988 to which a serotype could be assigned by either method were either type 1, type 2, or, less often, type 4. Testing RV for hybridization with oligonucleotides for genes encoding VP7 is an alternate method of determining RV serotypes.

Use of a synthetic oligonucleotide probe to detect strains of non-serovar O1 Vibrio cholerae carrying the gene for heat-stable enterotoxin (NAG-ST).

A synthetic oligonucleotide probe was developed to identify the gene for the heat-stable enterotoxin (NAG-ST) of non-serovar O1 Vibrio cholerae. Of 103 non-O1 V. cholerae isolates from Thailand, 31 isolates from Mexico, and 47 isolates from patients in the United States, only 7 (all from Thailand) hybridized with the probe. Probe-positive strains produced significantly higher fluid accumulations in infant mice than probe-negative strains.

Hybridization of Escherichia coli producing Shiga-like toxin I, Shiga-like toxin II, and a variant of Shiga-like toxin II with synthetic oligonucleotide probes.

Synthetic oligonucleotides, constructed from the nucleotide sequences of genes coding for the A subunit of Shiga-like toxin (SLT) I and the B subunit of SLT-II, were used as probes at different degrees of stringency to identify Escherichia coli producing different types of SLTs. At 45 degrees C, the A-I oligonucleotide probe hybridized with E. coli producing SLT-I, SLT-II, and variant of SLT-II (SLT-IIv). At 53 degrees C, only SLT-I-producing E. coli hybridized with this probe. At 45 degrees C, the B-II oligonucleotide probe hybridized with SLT-II- and SLT-IIv-producing E. coli. At 53 degrees C, this probe hybridized with only SLT-II-producing E. coli. The A-I and B-II oligonucleotide probes were subsequently tested for hybridization with 73 SLT-producing E. coli and 49 non-SLT-producing E. coli isolated in Asia and Canada. At 45 degrees C, the A-I oligomer had a sensitivity of 97% and a specificity of 100% in identifying SLT-producing E. coli. At 53 degrees C, the A-I oligonucleotide probe had a sensitivity of 92% and a specificity of 91% in identifying E. coli containing genes encoding SLT-I. At 45 degrees C, the B-II oligonucleotide had a 100% sensitivity and 97% specificity in identifying E. coli that hybridized with the SLT-II probe. Of 17 E. coli that hybridized only with the SLT-II probe, 10 did not hybridize with the B-II oligonucleotide at 53 degrees C. All 10 isolates were cytotoxic to Vero cells but not to HeLa cells, confirming that the B-II oligonucleotide probe used at 53 degrees C will differentiate isolates producing SLT-II and SLT-IIv.

A comparative field study of radiolabeled and enzyme-conjugated synthetic DNA probes for the diagnosis of falciparum malaria.

Synthetic, 21-base, DNA probes to the genome of Plasmodium falciparum were either 32P-labeled or enzyme-conjugated for comparative field studies. The sensitivity of both probes was compared with microscopy in the examination of blood samples from 97 Thai villagers, 47 Thai Rangers, and 19 malaria-free Bangkok residents. The probes were also used to monitor the therapeutic response of 18 of the Rangers during 7 days of treatment. The probes proved highly specific. Both probes had lower limits of detection of about 100 parasites per microliter blood. Thus, the low parasite densities in partially immune villagers from an endemic area were often missed, while higher parasite densities in the nonimmune Rangers were usually detected. As monitors of response to treatment, the probes paralleled microscopy in identifying reversion from positive to negative parasitemia. The enzymelabeled DNA probe as shown to perform similarly to the radiolabeled probe in populations with different malarial immune status and during curative treatment.

Clinical and microbiologic features of Shigella and enteroinvasive Escherichia coli infections detected by DNA hybridization.

To determine the clinical and microbiologic features of Shigella and enteroinvasive Escherichia coli (EIEC) infections, we investigated 410 children with diarrhea and 410 control children without diarrhea who were seen at Children's Hospital, Bangkok, Thailand, from January to June 1985. Shigella spp. were isolated from 96 (23%) and EIEC were isolated from 17 (4%) of 410 children with diarrhea and from 12 (3%) and 6 (1%) of 410 control children, respectively. The isolation rates of both pathogens increased with age and peaked in children 3 to 5 years old from whom Shigella spp. were isolated from 38% and EIEC were isolated from 9%. Shigella spp. were isolated from 52% and EIEC were isolated from 7% of 91 children with bloody diarrhea and from 15 and 3% of 319 children with nonbloody diarrhea. Fifteen (65%) of 23 EIEC were lactose positive, and all belonged to recognized EIEC serotypes. Among children with diarrhea, the stool blots of 76% of 17 children infected with EIEC, 45% of 96 children infected with Shigella spp., and 1% of 297 culture-negative children hybridized with the 17-kilobase EcoRI digestion fragment of pRM17, a recombinant plasmid containing DNA derived from the 140-megadalton Shigella flexneri plasmid. Although EIEC colonies can be reliably detected by DNA hybridization, detection by stool blot is less sensitive. Shigella spp. and EIEC are important causes of endemic diarrhea among children greater than 1 year old in Thailand.