Microfluidic PMMA-based microarray sensor chip with imaging analysis for ABO and RhD blood group typing.

BACKGROUND AND OBJECTIVES: Solid phase microarrays have been described for use in blood typing; red blood cells (RBCs) captured on immobilized antibodies were detected using surface plasmon resonance or fluorescence. We present antibody microarray on Poly (methylmethacrylate) (PMMA) surface coupled with microfluidic system for ABO and RhD blood typing. After immobilized by antigen-antibody interaction, the RBCs were detected by image recognition. MATERIALS AND METHODS: The sensor surface was produced from grafted aminopropyltriethoxysilane (APTES) on photochemical modified PMMA surface by UV irradiation and subsequently reacted with glutaraldehyde cross-linking. The amine group of monoclonal antibody of anti-A, anti-B and anti-D was reacted with an aldehyde group on the glutaraldehyde modified surface, forming an imine linkage. RBCs were captured by the coated antibody via antigen-antibody interaction, and blood grouping was determined by microarray image cell counting. RESULTS: Suitable condition for RBC detection was 10% RBC concentration at 10 µl/min flow rate. This setting eliminated non-specific RBC binding resulting in correct blood groups identification of all 136 samples tested. The platform showed good reproducibility with coefficient of variation of 2·17%, 3·62% and 2·51% for anti-A, anti-B and anti-D respectively. The antibody-coated surface can be stabilized by stabilizer coating and stored for long-term use. CONCLUSION: The PMMA array chip demonstrated its good accuracy and precision in rapid blood group testing. For its high throughput, the method has potential for use in large blood donation centre.

Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population.

OBJECTIVES: To develop reliable and convenient methods for Miltenberger (Mi(a) ) blood group typing. AIM: To apply real-time polymerase chain reaction (qPCR) melting curve analysis to Mi(a) blood group typing. BACKGROUND: The Mi(a) blood group is the collective set of glycophorin hybrids in the MNS blood group system. Mi(a+) blood is common among East Asians and is also found in the Thai population. Incompatible Mi(a) blood transfusions pose the risk of life-threatening haemolysis; therefore, Mi(a) blood group typing is necessary in ethnicities where the Mi(a) blood group is prevalent. METHODS/MATERIALS: One hundred and forty-three blood samples from Thai blood donors were used in the study. The samples included 50 Mi(a+) samples and 93 Mi(a-) samples, which were defined by serology. The samples were typed by Mi(a) typing qPCR, and 50 Mi(a+) samples were sequenced to identify the Mi(a) subtypes. Mi(a) subtyping qPCR was performed to define GP.Mur. Both Mi(a) typing and Mi(a) subtyping were tested on a conventional PCR platform. RESULTS: The results of Mi(a) typing qPCR were all concordant with serology. Sequencing of the 50 Mi(a+) samples revealed 47 GP.Mur samples and 3 GP.Hop or Bun samples. Mi(a) subtyping qPCR was the supplementary test used to further define GP.Mur from other Mi(a) subtypes. Both Mi(a) typing and Mi(a) subtyping performed well using a conventional PCR platform. CONCLUSION: Mi(a) typing qPCR correctly identified Mi(a) blood groups in a Thai population with the feasibility of Mi(a) subtype discrimination, and Mi(a) subtyping qPCR was able to further define GP.Mur from other Mi(a) subtypes.

Mycoplasma pneumoniae and Chlamydophila pneumoniae in children with community-acquired pneumonia in Thailand.

BACKGROUND: The prevalence of community-acquired pneumonia (CAP) caused by atypical pathogens in Thai children is unknown. OBJECTIVE: To examine the prevalence of Mycoplasma pneumoniae and Chlamydophila pneumoniae infections in paediatric patients (aged 2-15 years) with CAP in three academic hospitals using standardised laboratory techniques. The characteristics of atypical pneumonia were also compared with other causes of CAP. METHODS: Diagnosis of current infection was based on a four-fold or more rise in antibody serum samples or persistently high antibody titres together with the presence of mycoplasmal or chlamydial DNA in secretions. RESULTS: Of 245 patients with CAP, 17.5% of cases were caused by atypical pathogens (M. pneumoniae 14.3%, C. pneumoniae 2.8% and co-infection 0.4%). We also found atypical pathogens in young children aged 2-5 years. The clinical and laboratory findings did not distinguish atypical pneumonia from other CAPs. Segmental or lobar consolidation on chest X-rays was more common in atypical pneumonia, while dyspnoea was more prominent in other CAPs. CONCLUSION: Our data show a high prevalence of M. pneumoniae and C. pneumoniae in Thai children with CAP, including in children aged 2-5 years.

Comparison of three PCR primer sets for diagnosis of septicemic melioidosis.

Several sets of PCR primers have recently been developed for detection of Burkholderia pseudomallei. In this report, the performance of 16S rRNA gene primers (16S), rRNA spacer gene primers (spacer), and 'LPS' primers (LPS) were compared. All primer sets were tested by PCR amplification of the same DNA samples extracted from blood specimens of 46 patients from northeastern Thailand, of which 29 had melioidosis based on blood culture as a gold standard. The sensitivities were 41, 35.7, and 31% while the specificities were 47, 59, and 100% for the 16S, spacer, and LPS primers, respectively. The positive predictive values were 60, 59, and 100%, while negative predictive values were 35, 34, and 46%, for these primers. The low sensitivity of PCR was suspected to be because of small numbers of bacteria in the samples. In addition, one primer set could not detect all B. pseudomallei strains. To make PCR for melioidosis more practical, bacterial concentration steps must be added. Lastly, mixed infection of patients in endemic areas may be the cause of controversial false positive PCR results, and should be further investigated.

Overcoming the errors of in-house PCR used in the clinical laboratory for the diagnosis of extrapulmonary tuberculosis.

Our experiences from 1993 to 1997 in the development and use of IS6110 base PCR for the diagnosis of extrapulmonary tuberculosis in a routine clinical setting revealed that error-correcting processes can improve existing diagnostic methodology. The reamplification method initially used had a sensitivity of 90.91% and a specificity of 93.75%. The concern was focused on the false positive results of this method caused by product-carryover contamination. This method was changed to single round PCR with carryover prevention by uracil DNA glycosylase (UDG), resulting in a 100% specificity but only 63% sensitivity. Dot blot hybridization was added after the single round PCR, increasing the sensitivity to 87.50%. However, false positivity resulted from the nonspecific dot blot hybridization signal, reducing the specificity to 89.47%. The hybridization of PCR was changed to a Southern blot with a new oligonucleotide probe giving the sensitivity of 85.71% and raising the specificity to 99.52%. We conclude that the PCR protocol for routine clinical use should include UDG for carryover prevention and hybridization with specific probes to optimize diagnostic sensitivity and specificity in extrapulmonary tuberculosis testing.

Detection of HIV-1 window period infection in blood donors using borderline anti-HIV results, HIV-1 proviral DNA PCR, and HIV-1 antigen test.

Prevention of transmission of HIV-1 via blood transfusion has been carried out by the National Blood Center by screening donated blood with anti-HIV and HIV antigen tests. To increase the safety measure, detection of proviral DNA by PCR has been proposed; however, it was impractical to test all samples by PCR. From August 1994 to September 1995, there were 296,169 blood donors with 0.32 per cent prevalence of anti-HIV positive. From these donors, 153 samples of which the anti-HIV enzyme immunoassay optical density (OD) between cutoff and 80 per cent of cutoff value (borderline results) were selected for PCR testing. One out of 153 borderline cases showed positive by PCR test for HIV-1 proviral DNA. However, this case was also positive by HIV antigen test. Therefore, most of the samples with borderline anti-HIV results were true negative for HIV infection. On the other hand, there were 8 HIV antigen positive samples which had anti-HIV OD below the borderline value determined in this study. This finding confirmed the necessity of using both the anti-HIV and HIV antigen tests for screening of donated blood.

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization.

Serum ferritin levels in normal and HIV-1 infected pregnant women.

Between May, 1994 and May, 1995 serum ferritin concentrations were measured in 74 pregnant women who were HIV-1 positive (17 with CD4 cell counts below 200 cells/uL) and in 148 HIV-1 negative pregnant controls in first trimester of gestation to determine if a high level of serum ferritin is present in pregnant women with HIV-1 infection. Comparisons were made between groups stratified by CD4 cell counts. Pregnant women with HIV-1 infection had 92% higher mean serum ferritin levels (112.8 versus 58.8 ug/L, p < 0.005) compared to controls, whereas the mean maternal age, parity, gestational age, haemoglobin levels and body mass index at entry into the study did not differ significantly between the control and HIV-1 infection groups. The serum ferritin levels inversely correlated with the percentage of CD4 lymphocytes, CD4 cell counts and the CD4/CD8 ratio. This study suggests that serum ferritin levels can also be used as an immunological marker in HIV-1 infected pregnant women.

Vertical transmission of HIV-1 in mid-trimester gestation.

Between July, 1994 and March, 1995, 23 heart blood samples from fresh abortuses of HIV-1 seropositive pregnant women after elective termination of pregnancy between 18 and 25 weeks of gestation by prostaglandin E1 analogue vaginal administration were examined for polymerase chain reaction (PCR) of HIV-1 genome and p24 antigen to investigate the transplacental transfer of HIV-1 infection. All samples of fetal heart blood were positive for HIV-1 antibody (ELISA), but negative for PCR and HIV-1 p24 antigen assay. These negative results could be due to the lack of the virus in the peripheral blood or to a viral load low enough to be undetectable by PCR method at mid-trimester gestation and suggest that HIV-1 vertical transmission occurs mostly during the last trimester of pregnancy and/or at delivery.

Novel genotypes of hepatitis C virus in Thailand.

We examined 24 C-type hepatitis specimens from Thailand and detected hepatitis C virus (HCV) RNA in all of them by RT-nested PCR for a portion of the HCV 5' non-coding (5' NC) region and a portion of the HCV core region. However, we failed to detect HCV RNA in 11 specimens by RT-nested PCR for a portion in the non-structural protein 5 (NS5) region that has been used commonly for HCV genotyping. We designed a new primer set for a separate portion of the NS5 region. Using this primer set, we succeeded in amplifying this portion in all 24 specimens. Two novel HCV genotypes, tentatively designated HCV-VII and HCV-VIII, were identified by sequencing these amplified regions. Our newly designed primers for RT-nested PCR may be useful for diagnosing infection as well as for genotyping unidentified HCV genomes.

Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization.

Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially by antibody testing, which is not always sensitive or specific. We have developed two seminested PCR protocols combined with enzyme immunoassay (EIA) to detect the conserved ribosomal regulatory region of B. pseudomallei. Both PCRs used one biotinylated primer for capturing PCR products on EIA plates. One system, termed solution hybridization EIA (SHEIA), hybridized PCR products with a digoxigenin-labeled probe in solution. Another system, termed primer-labeled EIA (PLEIA), used a digoxigenin-labeled nested primer to generate products that were directly detected without hybridization. To prevent amplicon contamination, pre-PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was incorporated into each system. By a rapid method of blood sample preparation for PCR, these systems had sensitivities of 75 bacteria per ml for SHEIA and 300 bacteria per ml for PLEIA. No nonspecific amplification of other bacterial DNAs was detected. This seminested PCR coupled with SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic.

Routine voluntary antenatal anti-HIV screening in Bangkok, Thailand.

The following recommendations are made as a result of this study. 1. Routine voluntary screening for HIV infection in all pregnant women is feasible and worthwhile. 2. Every seropositive result should be repeated for confirmation before coming to a definitive conclusion to avoid a misdiagnosis. 3. Routine screening of seronegative pregnant women should be repeated during the third trimester to detect seroconversion since this offers a chance for AZT administration to the seroconverted pregnant women for reduction of perinatal transmission. 4. There should be available the appropriate back up services for seropositive pregnant women such as: (i) C--Choice. Having been appropriately counselled the pregnant women should be able to terminate or continue with the pregnancy. (ii) H--High-risk pregnancy concept. The pregnant women should be treated as high-risk cases. Throughout their pregnancy and delivery only experienced personnel should manage them. (iii) I--Integrated services. From our experience it would be reasonable to integrate the care of seropositive pregnant women with any other high-risk cases. Special or anonymous clinics may create an atmosphere of uneasy feelings among the women who could be made to feel alienated and discriminated against. (iv) P--Provision of care. Comprehensive services must be available. These include an experienced counselling team, adequate laboratory services, services for safe first and second trimester therapeutic abortions, appropriate facility in the delivery suite (including Caesarean section) for infected cases, and dedicated paediatricians.

PIP: During January 1991-December 1993 at Ramathibodi Hospital in Bangkok, Thailand, 91 of 24,856 (0.36%) pregnant women screened at their first prenatal visit for HIV tested positive for HIV antibodies. All were asymptomatic. AZT (Azidothymidine) was not administered. 8% of the HIV-seropositive women later admitted that they already knew their HIV status before coming for prenatal care from blood tests at other institutions. Eight women who tested HIV seronegative at the first screening tested HIV seropositive during the second routine screening at 28-32 weeks gestation, for a seroconversion rate of 0.03/100 seronegative tests at first screening. Researchers compared the 91 pregnant women testing HIV seropositive at the first screening with 182 HIV-seronegative pregnant women. After pretest counseling (a video presentation and information on voluntary testing), 100% of all pregnant women agreed to undergo HIV testing. The HIV seroprevalence rate increased from 0.13% to 0.47% during the study period. The leading risk factor for HIV infection was multiple sex partners (26.4% vs. 8.8%; p 0.05). The partners of 12% of the HIV-seropositive pregnant women tested HIV seronegative, resulting in considerable anxiety and difficulty in counseling the couples. After post-test counseling, 85.7% of the original 91 women opted for induced abortion. 13 of the original women and all of the seroconverted cases continued the pregnancy. The pregnant women who seroconverted made up 38% of the deliveries. The perinatal transmission rate was 19%. Infants born to the 8 mothers who seroconverted were more likely to test HIV positive than those born to mothers who tested positive at the first screening (37.5% vs. 7.7%; p 0.05). AZT administration to these women would have likely reduced the perinatal transmission rate. An infection led to the death of 1 infant in the seroconverted group at 5 months. Pregnancy outcomes did not differ between the HIV-seropositive group and the HIV-seronegative group.

Detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in breast milk and colostrum of seropositive mothers.

There is increasing evidence of vertical transmission of HIV-1 to infants through breast feeding of milk from HIV-1 infected mothers. It has been postulated that transmission occurs mainly via ingestion of infected cells in breast milk and colostrum. In this study, detection of HIV-1 proviral DNA was used to prove that cells from colostrum and milk do contain HIV. DNA were extracted from these cells of colostrum and milk of 18 seropositive mothers and amplified by nested PCR for HIV-1 gag and pol and 44 per cent were positive mostly by two primers. All ten negative control samples from seronegative mothers were negative. This study demonstrated the infectivity of breast milk and colostrum. Nevertheless, recommendation against breast-feeding should be weighed against poor alternatives in low socioeconomic families.

PIP: In Thailand clinicians gathered breast milk and colostrum samples (1 ml) at 1-10 days postpartum from 18 HIV-1 seropositive mothers at Ramathibodi Hospital and Maharaj Hospital and from 10 HIV-1 seronegative mothers at the same hospitals. Researchers used polymerase chain reaction to detect HIV-1 proviral DNA in cells in the breast milk and colostrum. Breast milk and colostrum samples from 44% of the HIV-1 seropositive women tested positive for HIV-1 DNA. The pol primers were superior to the gag primers. All of the colostrum samples of the HIV-1 seronegative women tested negative. These results suggest that HIV-1 seropositive lactating mothers can transmit HIV-1 via breast feeding after childbirth. The Obstetrics and Gynaecology Department at Ramathibodi Hospital in Bangkok advises HIV-1 infected mothers to not breast feed if there is a suitable alternative available. Health professionals should weigh breast feeding against poor alternatives in impoverished families.

Enzyme-linked immunosorbent assay for leptospirosis immunoglobulin M specific antibody using surface antigen from a pathogenic Leptospira: a comparison with indirect hemagglutination and microagglutination tests.

A search for a sensitive and specific test for human leptospirosis was made by enzyme-linked immunosorbent assay for immunoglobulin M specific antibody (IgM ELISA) using a surface antigen from L.interrogans serovar bataviae, L. interrogans serovar pyrogenes and L.interogans serovar icterohaemorrhagiae. The IgM ELISA tests using each of the three antigens were evaluated in 103 sera primarily positive by microagglutination test (MA). Optical density of these IgM ELISA tests showed good correlation. The IgM ELISA using antigen from serovar bataviae was compared with MA and indirect hemagglutination (IHA) in 20 sera primarily positive by IHA, and 103 sera primarily positive by MA. IgM ELISA and IHA using antigen prepared from serovar bataviae in 103 sera positive for MA had a sensitivity of 98.06 and 92.23 per cent respectively. In 20 sera primarily positive by IHA, IgM ELISA and MA showed sensitivity of 80 and 45 per cent respectively. The surface antigen used in IgM ELISA is broadly specific making IgM ELISA a sensitive and specific test for human leptospirosis. IHA agreed more with IgM ELISA in comparison to MA. As MA is not sensitive for early infection, IHA and IgM ELISA should be in routine use in general laboratories.

Laboratory diagnosis of congenital and maternal rubella infection: a review.

Physicians are aware of the congenital rubella syndrome. Serodiagnosis is usually used to detect rubella infection in pregnant women and their fetuses. Although being considered the cornerstone of serodiagnosis, the hemagglutination inhibition test is gradually being replaced by new more convenient methods. Tests to detect IgM eliminate the need for paired sera to diagnose acute rubella infection. However, because of the possibilities of false positive, IgM results should be interpreted with caution. Detection of IgM in cord blood and new genetic technology made the diagnosis of infection in utero possible. The evidence of reinfection in people considered to be immune is abundant; however, discovering new antigenic determinants correlating with immunity may solve the problem and a new vaccine and antibody test that is truly associated with immunity will be available in the future.

Antibodies to hepatitis C virus among patients with hepatocellular carcinoma and blood donors in Thailand.

HCC is the most cancer among Thai men. It is not known if HCV plays an oncogenic role in HCC in this country where HBV is endemic. Anti-HCV and HBsAg were assayed in 154 sera from HCC and 3,387 voluntary blood donors. The prevalence of anti-HCV in HCC (8.4%) was significantly higher than blood donors (1.38%). The prevalence of HBsAg in HCC (61%) was also significantly higher than blood donors (5.28%). The prevalence of anti-HCV in HCC was lower than that of Spain, Italy, Africa and Taiwan. Anti-HCV was found associated with a small portion of patients with HCC while HBV was found closely associated with the larger proportion of HCC. HCV in normal Thais was as common as those in southern Europe and HCV was found associated with HCC. However, HBV remains the major etiological factor of HCC in Thailand.

Gold blot for detection of immunoglobulin M (IgM)- and IgG-specific antibodies for rapid serodiagnosis of melioidosis.

Gold blot tests for rapid serodiagnosis of melioidosis were developed and evaluated with sera from 40 melioidosis patients and 159 normal controls. The sensitivity and specificity were 87.5 and 88%, respectively, for the immunoglobulin M (IgM) test and 100 and 91%, respectively, for the protein A test for IgG. Combination of the IgM gold blot and protein A gold blot yielded 97.5% sensitivity and 94.3% specificity. The tests were rapid and simple.

Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis.

Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis. However, in endemic areas, high background titers in normal populations and occasional low titers in patients with septicemic melioidosis prompted a search for a more sensitive and more specific method of serodiagnosis. An indirect fluorescent-antibody test for immunoglobulin M (IgM) specific antibody to Pseudomonas pseudomallei was more sensitive and more specific, but fluorescence microscopes are rarely available in the endemic areas. An enzyme-linked immunosorbent assay (ELISA) for IgM antibody is an attractive alternative. An indirect ELISA for IgM antibody (IgM ELISA) and an IgM antibody capture ELISA for melioidosis were developed. Both tests, together with IHA, were evaluated for 153 serum specimens from blood donors and 16 serum specimens from 16 melioidosis patients. It was found that IHA, the IgM ELISA, and the IgM antibody capture ELISA had sensitivities of 88, 88, and 75%, respectively, with specificities of 97.4, 92.2, and 91.5%, respectively. When IHA was combined with IgM ELISA, a sensitivity of 100% and a specificity of 95.4% were obtained. The IgM ELISA and IHA should be used in combination for serodiagnosis of melioidosis.