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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Evaluation of two screening tests for human leptospirosis.

Leptospirosis is prevalent in Thailand but its diagnosis depends primarily on clinical awareness. Serodiagnosis is of great assistance in the diagnosis of leptospirosis but in Thailand microagglutination (MA) is the only serodiagnosis available. MA is not rapid and it is used mainly in the referent laboratory. In addition, its roles in early diagnosis are rarely available. Rapid screening serological test which is sensitive early in the infection is needed. Latex agglutination (LA), indirect hemagglutination (IHA) were developed and evaluated in 100 MA positive sera and 200 blood donors. Later on, IHA and LA were compared with MA in 30 patients with a clinical picture compatible with leptospirosis. IHA and LA had sensitivities of 94 and 98 per cent respectively in MA positive sera. The specificity of IHA and LA in 200 blood donors was 99 and 100 per cent respectively. The study in 30 patients showed that LA and IHA were definitely more sensitive than MA test in sera collected within two weeks after the onset of fever. LA is also one of the most rapid tests for leptospirosis. With either LA or IHA human leptospirosis will be diagnosed more readily and more accurately.

Evaluation of latex cryptococcal agglutination test in cryptococcal meningitis.

There is a growing demand for laboratory diagnosis of cryptococcal meningitis, which is partly due to the increasing incidence of AIDS in Thailand. Presently, latex cryptococcal agglutination test (LCAT) is the most sensitive and specific test for laboratory cryptococcal meningitis. However, the test is very expensive and not readily available. LCAT must be developed locally to meet the need in Thailand. Rabbit antibody to C. neoformans was raised and used to sensitize latex particles used in LCAT. The developed LCAT was compared with a reference LCAT. The locally made LCAT was almost identical to the reference LCAT in sensitivity and specificity. It was extensively compared with the culture and India ink examination, in 73 cerebrospinal fluid specimens from cryptococcal meningitis and 155 specimens from other diseases. LCAT was found specific and more sensitive than fungal culture and India ink examination. LCAT is now extensively used in Thailand and recommended by Thai experts for use in all general hospitals. It is a simple, sensitive, specific, rapid and inexpensive tool for both diagnosis, prognosis and follow-up of cryptococcal meningitis.

HBsAg carrier rate among institutionalised children from Phyathai Institute.

To identify the evidence supporting the horizontal transmission of hepatitis B virus, HBsAg carrier rate among institutionalised children is determined and compared to children of the same age-group from the well baby clinic, Ramathibodi hospital. The results of this study show that HBsAg is detected four times more frequently in institutionalised children than in children from a well baby clinic. The chance of becoming an HBsAg carrier increases with age and duration of stay in the institution. Repeated study one year later shows that the HBsAg carrier rate among the same group of institutionalised children had increased by 35 per cent. The evidence supports the hypothesis that hepatitis B virus can be transmitted nonparenterally. To prevent this mode of transmission in both institutions and the community, hepatitis B vaccine should be given to these children.

Alpha chain disease in the Thai man.

A 61-year-old Thai man presented with a history of chronic diarrhoea of 1-2 years duration. Stool examination revealed a heavy parasitic infection. Several anthelminthics were given without benefit, despite disappearance of the intestinal parasites. Serum protein studies revealed abnormal alpha heavy chain. Two courses of cyclophosphamide, vincristine and prednisolone were followed by a brief remission in his symptoms. A 21 day course of tetracycline was then given, resulting in an improvement in his condition. He is still symptom free nine months after confirmation of the diagnosis, without any further treatment. Serum protein studies should be included in laboratory investigations of a patient with chronic diarrhoea.