Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO.
Dengue viruses pose a considerable global public health problem with an estimated 100 million cases of illness every year. This illustrates the need for rapid and reliable diagnostic methods for proper patient management and disease control. Currently, laboratory diagnosis depends on serology or virus isolation, with both methods having certain drawbacks. Alternatively, reverse transcription and polymerase chain reaction (RT-PCR) offers the potential for the rapid, highly sensitive and specific detection of dengue viruses. Since we occasionally encounter the problem of insufficient amounts of patient serum for the direct detection of dengue viruses, a method was developed for the extraction of viral RNA after biological amplification in mosquito larvae. Using this method, 15 of 19 clinical samples tested were correctly identified using RT-PCR.
The potential of RT-PCR to rapidly diagnose dengue infections from both acute and convalescent phase patients' sera was evaluated. The RNA extraction method involved binding of the viral RNA to silica particles in the presence of high concentration of guanidine thiocyanate. The protocol that was established was sensitive enough to detect 40 plaque forming units per 100 microliter of serum and results could be obtained within one day. Results from this study indicate that clinical samples should be collected in the early acute phase of illness when anti-dengue antibodies were undetectable or of low titres to ensure a more reliable diagnosis.
Three species of Shorea (S. leprosula, S. acuminata and S. cursitii) were collected from a natural forest reserve of Malaysia and analyzed for genetic variation using the technique of random amplification of polymorphic DNA (RAPD) by the polymerase chain reaction (PCR). The average number of nucleotide substitutions was estimated. The nucleotide diversities within species were very similar and larger than those found in Drosophila melanogaster. The nucleotide divergences between these species are about 1.5 times the nucleotide diversities within the species, indicating that these species diverged from a common ancestor relatively recently.
Laboratories intending to adopt cycle sequencing of PCR products in their routine analysis often face a confusing range of methods and kits. Through the study of mitochondrial cytochrome b, we have shown that clean and highly reproducible sequences could be obtained by using a combination of existing simple and economical methods in the preparation of DNA templates, PCR, purification of PCR products and sequencing. Our protocol is useful in itself or as a standard in typing other PCR-amplified DNA at the population level.
A study was initiated to amplify by polymerase chain reaction (PCR), a short factor VIII gene fragment containing the Bcl I restriction site from hemophilia patients using published primer sequences. Preliminary findings indicated that the resulting fragment is 142 bp long. This fragment, when digested with Bcl I restriction enzyme produced two fragments, 99 bp and 43 bp in length. Polymorphism in the Bcl I region can be used to detect carrier state in the family members of the hemophiliacs.
A rapid diagnostic system for scrub typhus using nested polymerase chain reaction (PCR) was applied to clinical samples from Malaysian Aborigines. Whole blood from twenty-four patients suspected of scrub typhus infection were tested using nested polymerase chain reaction and sera were evaluated by the indirect immunoperoxidase test. Antibody responses towards Rickettsia tsutsugamushi were observed in seventeen patients with the majority having high titers of IgG antibodies. Seven patients were seronegative. The nested PCR amplified R. tsutsugamushi DNA from six patients, of which two were negative serologically and four had high titers of IgG antibodies. Second samples collected seven days after treatment were negative by PCR testing. Nested PCR is highly sensitive and specific and may be used to provide rapid confirmation of scrub typhus cases in endemic region.
A modified nested polymerase chain reaction (PCR) method for detection of Plasmodium falciparum, P. vivax and P. malariae was combined with a simple blood collection and deoxyribonucleic acid (DNA) extraction method and evaluated in Malaysia. Finger-prick blood samples from 46 hospital patients and 120 individuals living in malaria endemic areas were spotted on filter papers and dried. The simple Chelex method was used to prepare DNA templates for the nested PCR assay. Higher malaria prevalence rates for both clinical (78.2%) and field samples (30.8%) were obtained with the nested PCR method than by microscopy (76.1% and 27.5%, respectively). Nested PCR was more sensitive than microscopy in detecting mixed P. falciparum and P. vivax infections, detected 5 more malaria samples than microscopy on the first round of microscopical examination, and detected malaria in 3 microscopically negative samples. Nested PCR failed to detect parasite DNA in 2 microscopically positive samples, an overall sensitivity of 97.4% compared to microscopy. The nested PCR method, when coupled with simple dried blood spot sampling, is a useful tool for collecting accurate malaria epidemiological data, particularly in remote regions of the world.
The polymorphism of the human leucocyte antigen HLA-DR2 and the heterogeneity of HLA-DR2 class II-related haplotypes (HLA-DRB1-DRB5-DQA1-DQB1) were investigated in four populations of east and south-east Asia (SEA) and five Melanesian populations using TaqI restriction fragment length polymorphism (RFLP) analysis, and the polymerase chain reaction (PCR) amplification-based techniques PCR-RFLP and sequence-specific oligonucleotide (SSO) typing. The haplotype DRB1*1502-DRB5*0101-DQA1*0102-DQB1*0601 was common in Malaysians, Javanese, Thursday Islanders, Madang, Goroka and the Australian Aborigines, while DRB1*16021-DRB5*0101-DQA1*0102-DQB1*0502 was common in the Thai and Thursday Islanders. DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 was present at a high frequency in Northern Chinese, Goroka, Watut and Australian Aborigines. The study describes four rare or unusual haplotypes: HLA-DRB1*1501-DRB5*0101-DQA1*0101-DQB1*0601, DRB1*1502-DRB5*0101-DQA1*0101-DQB1*0502, DRB1*1502-DRB5*0102-DQA1* 0102-DQB1*0502 and DRB1*1501-DRB5*0101-DQA1*0101/2-DQB1*0503; the latter two were confirmed by segregation in two Javanese families. A new DR2 allele, initially detected by PCR-RFLP and confirmed by DNA sequencing as DRB1*16022 (previously designated DRB1*16Madang), was seen in a Madang individual. A new HLA-DR2 TaqI RFLP subtype, locally designated as DR15U, is also described. This RFLP subtype segregated in a Javanese family and correlated with a typically SEA haplotype, DRB1*1502-DRB5*0102-DQA1*0101-DQB1*0501. The allele HLA-DR16Thai, determined by TaqI DRB RFLP, was found by PCR-RFLP and SSO typing to correlate with a unique SEA haplotype, HLA-DRB1*16021-DRB5*0101-DQA1*0102-DQB1*0502, and was observed in the Thai, Malaysian, Thursday Islander, Javanese and Northern Chinese populations.
Hemophilia B is an X-linked recessive disorder of the hemostasis involving a defective clotting factor IX. Amplification of the regions containing restriction fragment length polymorphisms (RFLP) can be achieved by the use of polymerase chain reaction (PCR). This paper describes the analysis of 2 RFLPs involving the Dde1 and Taq1 restriction sites within the factor IX gene in a family with hemophilia B. Digestion of the PCR products with Taq1 revealed a 163bp fragment in all the family members. This finding suggests the absence of restriction site for Taq1 enzyme. However, the Dde1 digest results in bands 369bp and 319bp segregated amongst the family members. The pattern of inheritance of the 369bp fragment in this family suggested that both the patient's mother and aunt are not carriers and that the patient's factor IX gene could have undergone a de novo mutation producing a defective factor IX gene responsible for the hemophilia B. This is supported by the fact that no family history of hemophilia B is indicated in the other male members within the family.
One hundred and two Southern Thai-Muslims (STM) from Nakhon Si Thammarat province were studied for HLA class I and II by SSP ARMS-PCR and PCR-SSO, respectively. The allele frequencies, haplotype frequencies, delta value and linkage disequilibrium between alleles were expressed. The most frequent alleles for HLA-A, HLA-B and HLA-C were A*24(02,03), A*11 (01,02), A*02(01,03,05-07,11): B*15(01,04-07,12,19,20), B*07(02-05), B*51(01-05)/B*52 (011,012); and Cw*07(01-03), Cw*04(01,02), Cw*08(01-03), respectively. The HLA class II alleles frequently found were DRB1*1202, DRB1*15021, DRB1*0701; DRB3*0301; DRB5* 0101; DQA1*0101, DQA1*0103, DQA1*0601; DQB1*0301, DQB1*0501, DQB1*0201; and DPB1*1301, DPB1*2301 and DPB1*0501. Two common HLA class I and II haplotypes with significant linkage disequilibrium were A*24 (02,03)-Cw*08 (01-03)-B*15 (01,04-07,12,19,20) -DRB1*1202 and A*33 (01,02)-Cw*0302-B*5801-DQB1*0201. The absence of B*27 and DRB1 *1401, the presence of A*2301 and high frequency of A*68 were observed in STM.
The HLA-A*02 subtyping in Thais was conducted and included in the 12th International Histocompatibility Workshop (12WS). A total of 81 randomized individuals previously serologically or DNA typed as A2 were studied for A2 subtypings. The subjects consisted of 32 Southern Thai-Muslims (STM) and 49 Central Thais (CT). The 12WS HLA-A*02 subtyping DNA typing kit was employed. The most common A*02 subtypes in STM were A*0203,*0201 and *0207 while they were A*0203, *0207 and *0201 in CT. A*0202, *0204, *0208, *0209, *0212, *0213, *0214, *0215, *0216 and *0217 were not found in both STM and CT. The 12WS data indicated that A*0201 was also the most frequent allele of A*2 among North-East Asians. A2 subtype study in 32 STM revealed that 2 in 8 of A*0201 showed the absence of bands at 813 bp and 705 bp with primer mix number 03A and 517A and weak reaction band with primer mix number 33A. In addition, 3 subjects with A*0201 variations have one nucleotide difference in exon 2 by sequence base typing (by MGJ. Tilanus) which will be reported separately.
Direct microscopy is widely used for the diagnosis of parasitic infections although it often requires an experienced microscopist for accurate diagnosis, is labour intensive and not very sensitive. In order to overcome some of these shortcomings, molecular or nucleic acid-based diagnostic methods for parasitic infections have been developed over the past 12 years. The parasites which have been studied with these techniques include the human Plasmodia, Leishmania, the trypanosomes, Toxoplasma gondii, Entamoeba histolytica, Giardia, Trichomonas vaginalis, Cryptosporidium parvum, Taenia, Echinococcus, Brugia malayi, Wuchereria bancrofti, Loa loa and Onchocerca volvulus. Early methods, which involved hybridisation of specific probes (radiolabelled and non-radiolabelled) to target deoxyribonucleic acid (DNA), have been replaced by more sensitive polymerase chain reaction (PCR)-based assays. Other methods, such as PCR-hybridisation assays, PCR-restriction fragment length polymorphism (PCR-RFLP) assays and random amplified polymorphic DNA (RAPD) analysis have also proved valuable for epidemiological studies of parasites. The general principles and development of DNA-based methods for diagnosis and epidemiological studies will be described, with particular reference to malaria. These methods will probably not replace current methods for routine diagnosis of parasitic infections in developing countries where parasitic diseases are endemic, due to high costs. However, they will be extremely useful for genotyping parasite strains and vectors, and for accurate parasite detection in both humans and vectors during epidemiological studies.
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/microliter of whole blood by nested PCR without compromising the simplicity of specimen collection or DNA extraction.
A simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by < 90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.
Since the identification of the new human virus, GB virus C (GBV-C)/hepatitis G-virus (HGV), in 1995/1996, reverse transcription polymerase chain reaction remained the sole available diagnostic tool for GBV-C/HGV infection. Recently, a serologic test based on the detection of antibodies to the putative envelope protein 2 (anti-E2) has been introduced. We used this assay for a seroepidemiological survey including 3,314 healthy individuals from different parts of the world, 123 patients from Germany who were suspected to have an increased risk of acquiring GBV-C/HGV infection, 128 multiple organ donors, and 90 GBV-C/HGV RNA positive persons. In European countries, anti-E2 seropositivity ranged from 10.9% (Germany) to 15.3% (Austria). In South Africa (20.3%) and Brazil (19.5%), even higher anti-E2 prevalence rates were recorded. In Asian countries like Bhutan (3.9%), Malaysia (6.3%), and the Philippines (2.7%), anti-E2 positivity was significantly lower. GBV-C/HGV anti-E2 prevalence in potential "risk groups," i.e., patients on hemodialysis and renal transplant recipients, did not vary significantly from anti-E2 seroprevalence in German blood donors. Anti-E2 and GBV-C/HGV RNA were found to be mutually exclusive, confirming the notion that anti-E2 has to be considered as a marker of past infection.
Nine simple sequence repeat (SSR) markers were developed from Shorea curtisii using two different methods. One SSR locus was isolated by the commonly used method of screening by colony hybridization, and the other eight loci were isolated by a vectorette PCR method. Primer pairs were designed based on the sequences of all these SSR loci. Analysis of 40 individuals of S. curtisii from natural forest in Malaysia revealed that all SSR loci were polymorphic. Four SSR markers, Shc01, Shc04, Shc07 and Shc09, were highly polymorphic. We have also tested the applicability of these SSR printers to other species of Dipterocarpaceae using PCR amplification. Because the flanking region sequences of the S. curtisii SSRs were well conserved within this family, the SSR primers for S. curtisii can be applied to almost all species of Dipterocarpaceae.
A polymerase chain reaction assay based on the enzyme-linked immunosorbent assay (PCR-ELISA) has been developed to detect Brugia malayi infection in an area of low endemicity in Malaysia. Blood samples from 239 subjects were tested: 192 amicrofilaraemic individuals, 14 microfilaraemic persons and 3 chronic elephantiasis cases from endemic areas and 30 city-dwellers (non-endemic controls). PCR products were examined by ELISA and Southern hybridization. In the PCR-ELISA, digoxigenin-labelled PCR products were hybridized to a biotin-labelled probe. This was followed by incubation in streptavidin-coated microtitre wells and detection using anti-digoxigenin-peroxidase and ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)]. All microfilaraemic samples were positive by PCR-ELISA and Southern hybridization and all samples from non-endemic subjects and chronic elephantiasis patients were negative. The PCR-ELISA detected 12 times as many B. malayi infections as did thick blood film examination. Nineteen of the 194 samples from the endemic area gave positive results by both PCR-ELISA and Southern hybridization, and an additional 5 samples were positive by PCR-ELISA only. The PCR-ELISA was specific and sensitive, detected more infections, and was more reproducible than Southern hybridization.