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PubMed1997

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.

Centrifugation↗

PubMed2006

Comprehensive analysis of BCR-ABL transcript types in Korean CML patients using a newly developed multiplex RT-PCR.

Diagnosis of chronic myeloid leukemia (CML) is based on the detection of BCR-ABL gene or Philadelphia chromosome (Ph chromosome), and fusion proteins with different sizes are encoded depending on the breakpoint in the BCR gene. In general, 3 breakpoint cluster regions in the BCR gene have been described: major (M-bcr), minor (m-bcr), and micro (mu-bcr). This study was designed to determine the frequency of BCR-ABL transcripts using one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). Bone marrow (BM) or peripheral blood (PB) samples at diagnosis from 548 patients were obtained with a referring diagnosis of Ph-positive (Ph+) CML, and multistep RT-PCR and newly developed one-step multiplex RT-PCR were applied on each sample. Compared with the previous multistep RT-PCR, one-step multiplex RT-PCR with the primers is the more rapid and accurate method to identify the BCR-ABL breakpoints. Most patients (538/548, 98.18%) were found to have b3a2 or b2a2, and total frequency of occurrence of c3a2, e1a2, b2a3, b1a1, and e1a3 or coexpression of b2a2 and b3a2 was less than 2.00%. No differences were observed between women and men. As the multiplex RT-PCR technique distinguishes BCR-ABL transcripts in all samples with high sensitivity and specificity, it easily could be applied at early stages of diagnosis. The incidence of one or the other rearrangement in CML patients varies in different reported series, and the frequency in each type of BCR-ABL transcript in Korean CML patients seems to be different from those of Western countries.

Base Sequence↗

PubMed2005

Diagnosis of Pelizaeus-Merzbacher disease: detection of proteolipid protein gene copy number by real-time PCR.

Duplication of the proteolipid protein gene (PLP1) is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a severe X-linked myelination disorder. We developed an assay for the detection of the PLP1 gene dosage by real-time quantitative PCR using the ABI Prism 7700 Sequence Detection System and the TaqMan chemistry. Copy number of the PLP1 gene was determined by the standard curve method using GAPDH as the reference gene. The assay was tested both on 50 normal controls and on 20 subjects whose PLP1 gene copy number was previously determined by quantitative fluorescent multiplex PCR. The procedure confirmed the expected results both on the male and female normal controls as well as on the 20 subjects previously tested. Ratios corresponding to the presence of one, two or three PLP1 gene copies, distributed in three non-overlapping ranges, were obtained by real-time PCR analysis. Subsequently, 29 DNA samples of putative PMD patients and possible female carriers, with unknown PLP1 gene dosage, were analysed. Five affected males carrying the PLP1 gene duplication and four female heterozygotes carrying three PLP1 gene copies were identified among them. The method is suitable for the identification of affected male patients and female carriers. Specific ranges are widely spaced, ensuring a correct assignment of the PLP1 gene copy number.

Female↗

PubMed2006

Surveillance of respiratory virus infections in adult hospital admissions using rapid methods.

Both influenza and respiratory syncytial virus (RSV) cause epidemics of respiratory illness of variable severity during the winter season. Influenza in particular has been blamed for hospital winter bed pressures, although it is thought that RSV may also play a role. Human metapneumovirus (hMPV) is a new respiratory virus reported to be important in children; only a limited number of studies are available for adult populations. We aimed to determine initially the burden of virologically confirmed infections, i.e. influenza, RSV and hMPV using polymerase chain reaction (PCR) technology and, in addition, to assess the feasibility of this approach as a surveillance tool for these respiratory viruses. Adult patients admitted to hospital in the previous 24 hours with onset of acute respiratory symptoms in the last 14 days were asked to participate. Informed written consent was obtained and nose and throat swabs taken. Multiplex PCR for influenza A (H1N1 and H3N2), influenza B and RSV A and B were carried out together with a separate PCR for hMPV. A total of 219 patients in 2001-2002 and 216 in 2002-2003 were tested and the combined results for both seasons were: 8 positive for influenza A/H1N1, 14 for influenza A/H3N2, 2 for influenza B, 14 for RSV A and 6 for RSV B. Most patients (261/435) were >65 years and most positives (30/44) were found within this age group. A number of patients aged >65 years who were positive for influenza (12/15) reported having had vaccine. In total, 373 samples were tested for hMPV and 20 were found positive across all age groups except the 45-54 years age group. As influenza activity was low during the study period the impact of infection on admissions could not be assessed. Nevertheless the viruses studied accounted for 15% of hospital admissions for respiratory infection. Most patients were aged >65 years, as expected. In the two years studied RSV and hMPV were each responsible for as many hospitalized cases of respiratory infection as influenza. Influenza infection must be considered even in those who give a history of vaccination. The molecular methods used in this study showed that surveillance of these respiratory viruses can be conducted and may help in the management of patients.

Adult↗

PubMed2000

Application of a chromogenic medium and the PCR method for the rapid confirmation of L. monocytogenes in foodstuffs.

Detection of Listeria monocytogenes in foodstuffs by conventional cultivation methods carried out according to EN ISO guidelines is rather time-consuming. Therefore, two alternative methods were applied for rapid confirmation of L. monocytogenes in foodstuffs. Inoculum from liquid selective broth was plated on PALCAM and OXFORD agar and on chromogenic agar medium RAPID L. mono. Suspect colonies from PALCAM were confirmed according to EN ISO standards and by the multiplex PCR method. In total, 990 samples of foodstuffs were investigated and 63 strains of L. monocytogenes were isolated. The chromogenic medium RAPID L. mono provided results comparable to PCR, it is easier to handle and provides considerable financial savings.

Chromogenic Compounds↗

PubMed1998

PCR for detection of Shigella spp. in mayonnaise.

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.

Bacterial Proteins↗

PubMed2006

Molecular analysis of mitochondrial DNA point mutations by polymerase chain reaction.

Mitochondrial respiratory chain disorders are clinically and genetically heterogeneous. There are several mitochondrial DNA (mtDNA) point mutations responsible for common mitochondrial diseases such as mitochondrial encephalopathy, lactic acidosis, stroke-like events, myoclonic epilepsy and ragged red fibers, neuropathy, ataxia, retinitis pigmentosa, and Leber's hereditary optic neuropathy. As a result of the clinical overlap, it is usually necessary to analyze more than one mutation for a patient suspected of a mitochondrial disorder. Molecular diagnosis is often performed using polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis of the most likely point mutations. However, this method is time-consuming and often produces problems associated with incomplete restriction enzyme digestion. In addition, PCR/RFLP analysis may not be able to detect a low percentage of heteroplasmy. For a more effective method of diagnosing mtDNA disorders, we have developed a multiplex PCR/ allele-specific oligonucleotide (ASO) dot blot hybridization method to simultaneously analyze 11 point mutations. The PCR products from a DNA sample containing a homoplasmic wild-type or mutant mtDNA sequence will hybridize to either the wild-type or the mutant ASO probe. The PCR products of a heteroplasmic DNA sample will hybridize to both wild-type and mutant ASO probes. This PCR/ASO method allows the detection of low percentage mutant heteroplasmy.

Alleles↗

PubMed2000

Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA.

Reverse transcription polymerase chain reaction (RT-PCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol, RNAzol, FastTube reagent). RNA yield was slightly higher with RNAzol than with TRIzol as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol and TRIzol. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol, and 1 BCR-ABL-positive (specific for translocation t [9; 221) cell among 2x10(4) normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.

Base Sequence↗

PubMed2002

Assessment of the genetic diversity among arcobacters isolated from poultry products by using two PCR-based typing methods.

In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of Arcobacter isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as A. butzleri (n = 182) and A. cryaerophilus (n = 46). A total of 131 types (91 A. butzleri types and 40 A. cryaerophilus types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.

Arcobacter↗

PubMed2001

Emergence of rotavirus G4P8 strain among children suffering from watery diarrhea in Calcutta, India.

Rotavirus RNAs from the fecal samples were hybridized with cDNAs specific for G1 and G2 genotypes. 59 out of 138 samples (42.7%) did not hybridize with either probe. The cDNAs coding for VP7 and VP4 from one such sample, SC134, were synthesized by combined reverse transcriptase/polymerase chain reaction (PCR) using specific oligonucleotide primers and were used as probes to screen those local isolates which did not hybridize with either G1 or G2. 26/59 (44%) of fecal RNA hybridized with these cDNAs indicating a possible emergence of this strain in this part of India. The VP7 and VP4 genotype specificity of SC134 was found to be G4P8 by multiplex PCR. The VP7 gene of SC134 was cloned and characterized in detail by restriction enzyme digestion and DNA sequence analysis. Comparison of nucleotide and predicted amino acid sequence of the VP7 gene of SC134 with other serotypes revealed that the VP7 gene of SC134 was closely related to G4. However, amio acids within the VP7 sequence differed in seven positions with that of both the subtype 'A' and 'B' of the G4 serotype. To establish the relation of this emerging strain with the other reported G4 strains, a phylogenetic tree was constructed and SC134 was found to be more closely related to the ST3 strain isolated in England and included in the tetravalent vaccine formulation along with two Japanese strains, although all four were distinct and did not form any cluster as was evident by the horizontal distance separating them. The VP7 gene sequence of SC134 was submitted to EMBL and was assigned the accession number AJ278217.

Antigens, Viral↗

PubMed2006

MALDI-TOF mass spectrometry for multiplex genotyping of CYP2B6 single-nucleotide polymorphisms.

BACKGROUND: CYP2B6 is a highly variable and polymorphic cytochrome P450 (CYP) enzyme involved in the biotransformation of an increasing number of drugs, including cyclophosphamide, bupropion, and the nonnucleosidic reverse transcriptase inhibitor efavirenz. Several nonsynonymous and promoter single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with altered hepatic expression and function, which affect drug plasma concentrations. METHODS: We used multiplex PCR to amplify relevant gene fragments while avoiding amplification of the CYP2B7P1 pseudogene. Polymorphic sites were analyzed by allele-specific primer extension followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Method evaluation was performed on a panel of 287 genomic DNA samples previously genotyped by other methods. RESULTS: Five multiplex assays were developed, comprising the following 15 SNPs: -82T --> C (*22); 86G --> C (R29T, *17); 136A --> G (M46V, *11); 296G --> A (G99E, *12); 415A --> G (K139E, *8, *13); 419G --> A (R140Q, *14); 516G --> T (Q172H, *6, *7, *9, *13, *19, *20), 547G --> A (V183I); 769G --> A (D257N); 785A --> G (K262R, *4, *6, *7, *13, *16, *19, *20); 983T-->C (I328T, *16, *18); 1006C --> T (R336C, *19); 1172T --> A (I391N, *15); 1282C --> A (P428T, *21); 1459C --> T (R487C, *5, *7). In 9 DNA samples showing discrepant genotypes, correctness of the MALDI-TOF MS result was confirmed by direct sequencing. CONCLUSIONS: This genotyping method enabled sensitive, specific, accurate, and comprehensive determination of 15 relevant SNPs of CYP2B6. The assay design allows analysis of SNP subsets, incorporation of additional SNPs, and performance of high-throughput genotyping.

Aryl Hydrocarbon Hydroxylases↗

PubMed1999

Amplification of 16S ribosomal RNA gene sequences for the identification of streptococci of Lancefield group B.

The 16S r RNA gene of 49 streptococci of serological group B isolated from various origins was amplified by polymerase chain reaction (PCR) and subsequently digested with the restriction enzymes Rsa I and Msp I. The restriction profiles of all group B streptococci appeared to be identical indicating no intraspecies sequence variations of this gene. A fragment of the gene of two group B-streptococcal reference strains, including the hypervariable V2 region, could be amplified by PCR and sequenced. The sequence appeared to be identical and allowed the design of species-specific oligonucleotide primers. The primer pair used produced an amplicon with a size of 1250 bp and correctly identified all 49 group B-streptococci investigated but none of the control strains of various species and serogroups. This primer could be used in a multiplex PCR and allowed a rapid identification of bacteria of this species.

Base Sequence↗

PubMed2003

Differential expression of angiopoietins 1 and 2 and their receptor Tie-2 in human endometrium.

Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)-PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.

Angiopoietin-1↗

PubMed2006

Comparison of PCR, culture and microscopy of blood smears for the diagnosis of anthrax in sheep and cattle.

AIMS: To compare microscopy, culture and PCR for the diagnosis of anthrax in blood samples from sheep and cattle. METHODS AND RESULTS: Blood samples were stored at room temperature and at 37 degrees C after receipt, over a period of 15-17 days. Aliquots were plated onto blood agar and blood smears were prepared. Following microscopic examination, DNA was extracted from blood smears and subjected to a multiplex PCR assay targeting the Ba813, cap and lef markers. CONCLUSIONS: PCR provided the most reliable means for the detection of Bacillus anthracis in deteriorating blood samples (15-17 days) and was also successful in diagnosing anthrax in blood smears that had been stored for 6 years and a blood sample which had been stored for 18 months at -20 degrees C. While less successful than PCR, culture for B. anthracis on 7% sheep blood agar was typically more reliable (2-17 days) than the examination of blood smears (2-6 days) for encapsulated bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrated the superiority of PCR for the diagnosis of anthrax from blood smear scrapings, particularly when microscopy is unreliable.

Animals↗

PubMed2006

[The genetic polymorphisms of nine Y-STR loci with short fragment size alleles in southern Chinese Han population and its application in forensic science].

OBJECTIVE: To study the genetic polymorphisms of 9 Y-chromosome specific STR loci that the allele size is less than 180 bp in length in the southern Chinese Han population, and to utilize the studied result to forensic science. METHODS: Nine Y-STR loci were amplified by single multiplex PCR, and the PCR products were sequenced by using ABI Prism 3100 DNA Sequencer. The allele and haplotype frequencies at 9 Y-STR loci were determined in a total of 213 unrelated males from southern Chinese Han population. Eighty-four father/son pairs with demonstrated paternity and thirty-six non-paternity father/son pairs were detected by using our Y-STR multiplex system. RESULTS: Three Y-STR alleles for DYS426, five alleles for DYS393, DYS460, DYS391 and DYS389 I respectively, six alleles for DYS456, seven alleles for both H4 and DYS388, and eight alleles for DYS458 loci were detected in 213 unrelated male individuals. Except for the DYS426 locus with a low GD value of 0.1489, the GD values for other 8 Y-STR loci ranged from 0.5064 to 0.9133. A total of 178 haplotypes were found at 9 Y-STR loci, of which 154 haplotypes were observed only once, and the haplotype diversity was 0.9983. None of Y-STR allele mutation was observed in the 84 father/son pairs with demonstrated paternity. Among the 36 non-paternity father/son pairs, two cases could get the paternity exclusions at 2 Y-STR loci; and the paternity of 33 cases could be ruled out by 3 or more Y-STR loci; only one case was found no exclusion of paternity regardless of detecting 9 Y-STR loci. CONCLUSION: This result indicates that the 9 Y-STR loci with short fragment size alleles are highly polymorphic. The fluorescent multiplex amplification system that we developed is suitable for personal identification and paternity testing.

Alleles↗

PubMed2004

A novel PCR approach for prenatal detection of the common NEMO rearrangement in incontinentia pigmenti.

OBJECTIVES: Incontinentia pigmenti (IP) is a rare X-linked dominant genodermatosis that is usually lethal in males in the prenatal period. Largely 80% of cases are accounted for by a large-scale deletion encompassing exons 4 to 10 of the NEMO gene. The aim of this work was to facilitate prenatal diagnosis of IP by devising a novel test for detection of the prevalent NEMO deletion. METHODS: We devised a sensitive and reproducible multiplex PCR test enabling simultaneous amplification of the deleted and wild-type NEMO genes in IP female individuals. RESULTS: Combination of this DNA test, with Xq28 linkage analysis and X-inactivation pattern study enabled us to offer an IP prenatal diagnosis in 15 of the 16 couples at a 50% risk to have an affected offspring. CONCLUSION: A current approach to IP prenatal diagnosis is proposed on the basis of the previously mentioned molecular tools.

DNA Mutational Analysis↗

PubMed2001

Efficient primer design algorithms.

MOTIVATION: Primer design involves various parameters such as string-based alignment scores, melting temperature, primer length and GC content. This entails a design approach from multicriteria decision making. Values of some of the criteria are easy to compute while others require intense calculations. RESULTS: The reference point method was found to be tractable for trading-off between deviations from ideal values of all the criteria. Some criteria computations are based on dynamic programs with value iteration whose run time can be bounded by a low-degree polynomial. For designing standard PCR primers, the scheme offers in a relative gain in computing speed of up to 50: 1 over ad-hoc computational methods. Single PCR primer pairs have been used as model systems in order to simplify the quantization of the computational acceleration factors. The program has been structured so as to facilitate the analysis of large numbers of primer pairs with minor modifications. The scheme significantly increases primer design throughput which in turn facilitates the use of oligonucleotides in a wide range of applications including: multiplex PCR and other nucleic acid-based amplification systems, as well as in zip code targeting, oligonucleotide microarrays and nucleic acid-based nanoengineering.

Algorithms↗

PubMed2002

High prevalence of Neisseria gonorrhoeae and multiple sexually transmitted diseases among rural women in the Eastern Highlands Province of Papua New Guinea, detected by polymerase chain reaction.

BACKGROUND: In a previous community-based study among rural women in the Eastern Highlands Province (EHP) of Papua New Guinea we determined that the prevalences of Trichomonas vaginalis infection, Chlamydia trachomatis infection, and syphilis were 46%, 26%, and 4%, respectively. Surprisingly, however, the prevalence of Neisseria gonorrhoeae infection was only 1%, which we considered low in consideration of the high prevalence of other sexually transmitted diseases (STDs). The aim of the current study was to reexamine samples that were collected in that survey and retest them with use of polymerase chain reaction (PCR). STUDY DESIGN: Using a cluster-sampling method, we surveyed 201 women aged 15 to 45 years in a population of approximately 19,000 people. In addition, 243 other women living in the same area who wished to be screened for STDs were included in the study. METHODS: Endocervical samples that were stored frozen at -80 degrees C were retested with multiplex PCR (M-PCR) for the detection of both N gonorrhoeae and C trachomatis and with a separate PCR for the detection of T vaginalis. RESULTS: A total of 373 samples that were still available were analyzed. The prevalences of T vaginalis, C trachomatis, and N gonorrhoeae infections were 42.6%, 26.5%, and 18.2%, respectively; 59.8% of the women had at least one STD, while 21.7% had mixed infections, 5.9% of them with all three pathogens. CONCLUSIONS: STDs are very common among rural women in the EHP of Papua New Guinea and often present as multiple infections.

Adolescent↗