Evaluation of a rapid immunochromatographic test for detection of antibodies to human immunodeficiency virus.

A new immunochromatographic rapid test, Determine HIV-1/2, for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 in human whole blood, serum, and plasma was evaluated. Determine HIV-1/2 is a sandwich immunoassay and uses a nitrocellulose strip with a capture site for the patient's results and a procedural control site to confirm the validity of the assay. The results can be read visually, and a positive result is indicated by the formation of a red line within 15 min after sample application. The test showed 100% sensitivity for HIV-1 with 102 whole-blood, 152 serum, and 144 plasma samples obtained from Ramathibodi Hospital, Bangkok, Thailand. The sensitivity of the test for HIV-2 was 100% with 100 serum or plasma samples obtained from Ivory Coast. The sensitivity of the test with 4 anti-HIV-1 seroconversion panels from Boston Biomedica Inc. was equivalent to or better than those of another agglutination assay with serum or plasma and the enzyme immunoassay licensed by the U.S. Food and Drug Administration. The specificity was 100% with 367 sets of whole-blood, serum, and plasma samples from Ramathibodi Hospital. This method had an analytical sensitivity for the detection of HIV-1 equivalent to or better than that of another agglutination assay with serum or plasma. This test had an analytical sensitivity for the detection of HIV-1 better than that of another immunochromatographic test with whole blood. This evaluation demonstrated the excellent performance of this immunochromatographic test with EDTA-anticoagulated whole-blood, serum, and plasma samples. We conclude that this test is suitable for use in emerging countries and is an excellent alternative to HIV antibody testing at remote sites, as well as in traditional laboratories.

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.

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.

Epstein-Barr virus DNA in nasopharyngeal carcinoma in Thai patients at Ramathibodi Hospital, Bangkok.

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in Thailand. The association of Epstein-Barr virus (EBV) and NPC, especially undifferentiated type, has been documented. There is, however, conflicting evidence with regard to the squamous cell type. We have therefore investigated EBV-DNA in all the three types of NPC to assess the association of EBV and NPC in Thai patients. EBV-DNA was detected in formalin-fixed paraffin-embedded tissues from the patients of Ramathibodi Hospital, Bangkok using polymerase chain reaction. A primer pair that amplified EBV nuclear antigen gene was used in the reactions and the amplification products were hybridized with a specific EBV probe. EBV-DNA was identified in 24 of 28 of tissue samples from patients with undifferentiated NPC, 25 of 40 samples from patients with squamous cell NPC and 3 of 4 samples with nonkeratinizing NPC. None of 12 nasopharyngeal tissue samples without NPC contained detectable EBV-DNA. Our results indicated a strong association of EBV with undifferentiated as well as non-keratinizing NPC. EBV-DNA was demonstrated in most cases of squamous cell NPC but the association of EBV in this type of carcinoma was not as frequent as in the other two types of NPC.

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.

A new type of hepatitis C virus in patients in Thailand.

Partial nucleotide sequences in the tentative NS5 region of the hepatitis C viral genome obtained from patients with chronic hepatitis in Thailand were analyzed by reverse transcription followed by the polymerase chain reaction. Of ten samples studied, four showed low homologies to any known type of HCV: the homologies of the nucleotide sequences of these clones with HCV-J, -US, -K2a and -K2b were 66.5-69.1%, 66.5-68.2%, 61.2-64.1% and 64.4-66.2%, respectively, and the homologies of their deduced amino acids sequences were 71.7-75.2%, 71.7-75.2%, 69.0-72.6% and 69.9-73.5%, respectively. These four clones were classified a new distinct type of HCV, named HCV-T. Moreover, the nucleotide and amino acid sequence homologies of the four HCV-T clones showed that the HCV-T type could be classified into two genotypes, HCV-Ta and HCV-Tb.

Microscopic examination of Cryptosporidium oocysts in diarrhoeal stools.

Unconventional microscopic means for investigation of Cryptosporidium oocysts in patients' stools were explored in an attempt to obtain a more accurate diagnosis. The results showed that Nomarski interference contrast microscope provided clearer structures of oocysts in wet mount preparations than those under a normal light microscope and readily allowed distinction from yeast cells. Transmission electron microscopic study revealed that oocysts are thick walled and well sporulated. Their "untypical" appearance as seen by the light microscope resulted from sporozoites or the residuum that can be unfamiliar to some examiners. Electron microscopy provides definitive identification of Cryptosporidium spp. but Nomarski interference contrast microscopy was superior to bright field microscopy and may facilitate rapid diagnosis in routine fecal examination. The Ziehl-Neelsen modified acid fast technique was of value for differentiation and confirmation.

PIP: Histological studies were conducted with fecal specimens of Cryptosporidium oocysts, organisms that often cause fatal watery diarrhea in AIDS patients, to better distinguish them from yeasts. The specimens were from 3 patients with AIDS or suspected AIDS. The method used were bright field and Nomarski interference contrast microscopy of wet-mounted stools preserved in 10% formalin and stained by Giemsa and by the Neelsen modified acid-fast technique. Electron microscope sections were postfixed in osmium tetroxide. Ultra-thin sections were stained with and lead citrate before microscopic examination. Oocysts appeared under bright field microscopy as 3x4 mcm ellipsoidal bodies with a central large round granule, known as the residuum, and 1-4 granules. Interference contrast microscopy revealed banana-shaped sporozoites surrounding the residuum, clearly differentiating them from yeasts. Giemsa stains the sporozoites pale blue with purple dots, making it difficult to distinguish them from yeasts. Acid-fast stain turned the crescent-shaped sporozoites red and the residuum deep red, while yeasts stained blue. Cryptosporidium could easily by distinguished from yeast ultrastructurally by their double cell wall. Thus, interference microscopy and acid-fast staining are helpful to separate Cryptosporidium from similar sized yeasts, an alternative to intestinal biopsy which has formerly been required to diagnose this parasite in immune-compromised patients.

Evaluation of a locally developed direct immunofluorescence test for chlamydial infections.

The high cost of diagnostic tests for chlamydial infections limits their use which may result in under estimation of the incidence of chlamydial infections. This study was an attempt to reduce the cost of the test by developing an immunofluorescence test for C. trachomatis using monoclonal antibody to major outer membrane protein of C. trachomatis. Urethral swabs were obtained from patients with symptoms of urethritis. The developed immunofluorescence test was compared with culture method and a commercial immunofluorescence test kit (BioMerieux). Compared with the culture method, the sensitivity, specificity, predictive value of positive and predictive value of negative of the developed test were 79, 85, 61 and 93 per cent respectively. The results obtained from the comparison with commercial test kit showed an agreement of 88 per cent. The developed test was positive in 32 per cent of specimens while the commercial test was positive in 24 per cent. The commercial test kit showed excellent reactions and it contained monoclonal antibody to lipopolysaccharide of Chlamydiae in addition to monoclonal antibody to major outer membrane protein which can lead to stronger immunofluorescence staining. The locally developed test, however, costs much less without compromising the results.

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.

Intradermal hepatitis B virus immunization: immunogenicity and reactogenicity.

Mass immunization of hepatitis B virus (HBV) vaccine in adults is frequently demanded. However the high cost of conventional immunization is an obstacle to the provision of this vaccine. We investigated the serological response and adverse reactions following administration of a low-dose (1 or 2 micrograms of yeast-derived HBV vaccine (HB-VAX II, Merck, Sharp and Dohme) intradermally in young adults. Each 1 ml dose of the vaccine contained 10 micrograms of HBsAg protein. The study population included 58 female volunteers, aged 20-33 years, who were serologically-negative for HBV. They were alternately allocated to 1 microgram or 2 micrograms intradermal dose given by 2 experienced nurses as one or two 0.1 ml injections. Doses were given at 0, 1, and 6 months. Anti-HBs concentration was tested by enzyme-immunoassay on their sera obtained at 1, 6, and 7 months after the first dose. Positive seroconversion (anti-HBs greater than 10 IU/1) at 7 months was found in 90% (95% CL 79%, 100%) of the 1 microgram group and 96% (95% CL 89%, 100%) of the 2 micrograms group. Local reaction, a transient pigmented macule with an underlying nodule, was found in most volunteers but did not bother them. Intradermal HBV immunization could be an alternative strategy for mass immunization in young adults.

Prevalence of positive anti-HIV in pregnant women at Ramathibodi Hospital.

Anti-HIV screening has routinely been done at the antenatal clinic in Ramathibodi Hospital since January 1990. The prevalence of positive anti-HIV during the first and second half of 1990, and the first half of 1991 are 0.056, 0.2, 0.24 per cent respectively. Twenty positive cases are now under counselling. Age range is between 18-35 years. Seventeen cases are from rural areas. Twelve of seventeen cases were from the north-eastern part of Thailand. Fifteen cases (71.4%) had their pregnancies terminated. This study shows that the prevalence of positive anti-HIV is increasing. Vertical transmission has already been known to be one of the most important and serious transmissions to newborns and infants. The data indicated that screening test for anti-HIV in pregnant women, previously regarded as a low risk group, should be reviewed and routinely done.

PIP: In 1990-1991, health workers at the prenatal clinic at Ramathibodi Hospital in Bangkok, Thailand, screened 12,067 women in the first trimester of pregnancy for antibodies to HIV. Most of the pregnant women were from middle class areas of Bangkok. 21 of the women tested positive for HIV (0.2%). All HIV-positive women and their husbands underwent counseling. HIV prevalence between January and June 1990 was 0.056% and rose 3-fold by July-December 1990 (0.2%) and 4-fold by January-June 1991 (in reference to 1 year earlier, 0.244%). The highest number of cases/month occurred in December 1990. 85% of the HIV-positive women were 20-30 years old, but the age range was from 18-35 years. 95% were second gravida. 38% were housewives. 81% (17) were from rural areas. 90% were married. 66% of husbands of these HIV-positive women also tested positive for HIV. 71% of the HIV-positive women opted for termination of pregnancy (menstrual regulation in 57% and hypertonic saline solution in 14%). These findings motivated the hospital physicians to recommend obligatory HIV screening of all pregnant women to manage the HIV-positive cases and to prevent HIV from spreading to their offspring.

Gold immunoblot analysis of IgM-specific antibody in the diagnosis of human leptospirosis.

An immunoblot for the detection of leptospirosis was developed in our laboratory. Antigen prepared from Leptospira interrogans serovar bataviae was dotted onto nitrocellulose paper and blocked with skim milk. Test and control sera diluted 1:20 were applied to the dot, incubated, and washed. Anti-human IgM colloidal gold conjugate was added and the dots were washed. A positive reaction was shown by the development of a pink dot against a white background. The test was performed on 62 sera that tested positive for leptospirosis by a microagglutination (MA) test, on 40 sera that were positive by an indirect hemagglutination (IHA) test, and on sera from forty healthy blood donors. Four sera from the blood donors showed a faint pink dot, but the remainder showed a colorless reaction. All 62 sera that tested positive by MA were positive by this new test, while 95% of the 20 sera that tested positive by IHA were positive. Tests for IgG antibody were performed on 20 sera positive by MA using protein A-colloidal gold conjugate, and all showed weak reactivity. The results confirmed previous findings that most antibodies present in leptospirosis patients are of the IgM type. The ELISA takes three hours to perform, but the gold immunoblot can be completed in 30 min. In addition, the test blot can be kept as a permanent record, and is a significant improvement over existing tests.

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.

A comparison of passive haemagglutination test and immunofluorescent test for antibody to native deoxyribonucleic acid.

Antibody to double-stranded DNA is a specific marker for systemic lupus erythematosus. The recommended method for detection of this antibody is immunofluorescence. Haemagglutination was developed and the results of antibody detection were evaluated with those obtained by immunofluorescence. Human group O erythrocytes were treated with glutaraldehyde and coated with DNA from calf thymus. Testing in 169 active and inactive SLE sera, 59 sera were positive and 91 sera were negative by both methods. Five sera were negative by haemagglutination but positive by immunofluorescence. Fourteen sera with low haemagglutination titer were negative by immunofluorescence. The correlation between the results obtained by both methods were highly significance with contingency coefficient of 0.61 and correlation coefficient between the results of 78 sera positive by both or either method was 0.74 (p less than 0.001). Sixty-three sera from blood donors and seventy sera from pregnant women were negative by the two techniques. PHA is simpler, quicker and can be assayed in laboratories without the use of fluorescent microscope. It can be established as a very useful alternative test to immunofluorescence.

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.

Development of enzyme-linked immunosorbent assay for hepatitis B e antigen.

Hepatitis B e antigen (HBeAg) in sera indicates infectivity and when found during pregnancy, indicates a need for vaccination against hepatitis B virus. A sensitive test for HBeAg is needed in all hospitals but this test is expensive. Local development of enzyme-linked immunosorbent assay (ELISA) for HBeAg and its antibody (anti-HBe) was considered necessary and it was successfully conducted. The developed test was compared with ELISA test for HBeAg and anti-HBe manufactured by Organon Teknika (205 routine specimens and 103 sera positive for HBsAg) and Roche Diagnostic (160 routine specimens). The locally made and imported kits showed overall agreement of 97.5 to 98.1 per cent and the locally made test was always slightly more sensitive. The local test was also rapid, reproducible, and specific. The development lead to self reliance on ELISA test for HBeAg and anti-HBe.