Relevance of antibody content and test format in HIV testing of pooled sera.

OBJECTIVE: To determine the effects of HIV-1 antibody level and test-format characteristics on testing pooled sera. DESIGN: This study was designed with a laboratory exercise followed by test observations on serosurveillance samples. METHODS: Sera with low, medium and high (n = 22, 12 and 20, respectively) antibody titers were pooled with HIV-1-negative sera and tested with two enzyme-linked immunosorbent assays (ELISA) and a particle agglutination test. The same kits were used to test single and pooled (batches of five, 10 and 20) samples collected from 3000 blood donors and sex workers. These samples were then seeded with 50 varying antibody-containing sera and similarly tested. Initial reactivities, sensitivities, and specificities for all test kits were calculated and compared. RESULTS: In the laboratory exercise, all reactive pools of five were detected. False-negative pools in batches of 10 and 20 with low antibody titers were noted with one or both ELISA, but not with the particle agglutination method. Testing 3000 samples revealed three confirmed reactive samples and 100% sensitivity/specificity for all kits, for both single and pooled sera testing. Increased initial reactivity (IR) was noted for the two ELISA. Examinations of pools of the seeded 3000 samples with the two ELISA showed false-negative reactivity with pools of 10 and 20 when pools contained low antibody sera (sensitivities and specificities of 92-97.9% and 98.1-100%, respectively). Again, increased IR was seen with the ELISA. False-negative pool and increased IR was not seen with the agglutination test (sensitivity/specificity 100%). CONCLUSIONS: We recommend the use of the particle agglutination assay for testing pooled sera of batches of 20 or less. Components of reactive pools should then be tested and reactive samples should undergo supplementary testing. Pooled samples tested by ELISA should not exceed five per batch. Retesting of reactive pools, testing of its components, and supplemental test(s) of reactive sera should then follow. The optimum pool size for most laboratories is five, with the best technical and economic performance seen with the particle agglutination assay.

Genetic subtypes of HIV-1 in the Philippines.

OBJECTIVES: To determine the genetic variability of HIV-1 amongst infected Filipinos and to analyze phylogenetic relationships, temporal introductions and transmission dynamics of identified variants. METHODS: Polymerase chain reaction amplification and direct sequencing of a 204 base-pair fragment of the env C2-V3 region from uncultured peripheral blood mononuclear cells obtained from 51 HIV-1-positive Filipinos infected from 1987 to mid-1996. Evolutionary distance and phylogenetic relationships among the DNA sequences were estimated. RESULTS: The 51 Philippine strains were classified into five env V3 subtypes, namely subtype B (n = 37), subtype E (n = 8), subtype A (n = 3), subtype C (n = 2) and subtype D (n = 1). The overall env nucleotide divergence ranged from 11.7 to 32.2%. The nucleotide variation appeared to be random and no temporal ordering was observed. The variation of the sequences at the tip of the V3 loop was very broad. Subtypes B and C isolates did not show close genetic relationship to other Asian variants. Only three of the subtype E strains had close affinity to known Asian sequences. The majority (94%) of the subjects acquired the infection by sexual transmission. About two-thirds were presumably infected outside the Philippines, whereas the remaining were infected indigenously. Information was limited to allow segregation of the identified subtypes by mode of transmission or risk groups. CONCLUSION: Our findings demonstrate the presence of multiple genetic subtypes of HIV-1 in the Philippines. The apparent geographic range of previously reported genotypes in South and South-east Asia was extended and has obvious implications for env-based antiviral interventions.

Coinfection is common in measles-associated pneumonia.

BACKGROUND: Measles continues to be a significant health problem in developing countries. OBJECTIVES: To describe the clinical features of measles-associated pneumonia (MAP) and to identify other pathogens involved. METHODS: Measles diagnosis was ascertained either by the typical symptom complex or by a sensitive enzyme immunoassay for antibody among children < 5 years of age admitted to the hospital with pneumonia. Other pathogens were identified by blood culture, virus isolation or antigen detection from nasopharyngeal aspirate and antibody determination from serum. RESULTS: Of 182 MAP cases 162 (89%) had clinically typical measles. Twenty patients had a diagnostic antibody finding with an atypical clinical presentation. Thirteen percent were younger than 9 months of age. The case fatality rate was 17%, with a significantly increased odds ratio (OR) for those with cyanosis [OR 4.6, 95% confidence interval (CI) 1.7 to 13], respiratory rate > or = 60/min (OR 3, 95% CI 1.3 to 7) or fulfilling criteria for very severe pneumonia (OR 5.3, 95% CI 2.3 to 12). Mixed infection was found in 53% of patients. Blood culture was positive in 10 patients, Streptococcus pneumoniae (N = 5) being the most common finding. Adenovirus (19%) and parainfluenza (25%) viruses were the most frequent other viruses. A dense infiltrate was seen significantly more often among measles patients with bacterial coinfection (87.5%) than those with other viruses (36%, P = 0.007) or no evidence of other infection (33%, P = 0.004). CONCLUSION: In MAP, coinfection with other microbes is common. Cyanosis and a respiratory rate of > or = 60/min predict a greater risk of dying.

A newly developed immunofluorescence assay for simultaneous detection of antibodies to human immunodeficiency virus type 1 and type 2.

Immunofluorescence assays (IFA) that simultaneously distinguish between antibodies against closely related human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) infections have not been readily available. Serum specimens from 95 HIV-1-infected, 26 HIV-2-infected and 3 HIV-1/HIV-2 dually infected individuals and 106 seronegative blood donors were evaluated for the ability to serologically discriminate HIV-1 and HIV-2 infections by means of IFA employing three types of cells whose morphology varied within one field of microscopy. Mixtures of HIV-1-infected, HIV-2-infected and uninfected cells were used in the present study. In consequence, all serum specimens from individuals infected with HIV were confirmed to contain antibodies to HIV-1 and/or HIV-2. None of the sera from the blood donors were positive. Serum specimens from HIV-1-infected or HIV-2-infected individuals were diagnosed as single infection with HIV-1 (85/95) and HIV-2 (22/26), respectively, by this new assay. Although another 14 (10/95 and 4/26) were shown to be seropositive for both HIV-1-infected and HIV-2-infected cells, these results suggest that this assay is potentially simple and useful for screening and confirming both HIV-1 and HIV-2 infections simultaneously.

Etiology of central nervous system infections in the Philippines and the role of serum C-reactive protein in excluding acute bacterial meningitis.

OBJECTIVES: The value of measurements of serum C-reactive protein (CRP) in differentiating central nervous system (CNS) infections of varying etiologies in the Philippines was investigated. METHODS: A wide array of bacteriologic and virologic methods as well as computed tomography, typical clinical presentation, and autopsy were used for etiologic diagnosis. RESULTS: Among 103 patients with CNS infection, etiology was identified in 60 (58%) cases. Bacteria were found in 19 (including 7 Streptococcus pneumoniae, 5 Haemophilus influenzae, 3 Neisseria meningitidis), tuberculosis in 4, viruses in 38 (including 20 coxsackievirus, 8 measles, 4 adenovirus, and 4 poliovirus infections), and brain abscess in 3 patients. C-reactive protein was elevated on admission in all 18 cases of bacterial meningitis tested, exceeding 50 mg/L in 17 (94%), and was not affected by prior antibacterial treatment. The mean CRP was significantly higher in the bacterial group than in the viral group (207 +/- 111 mg/L vs. 39 +/- 34 mg/L; P < 0.001). In the viral group one third had CRP above 50 mg/L. In patients with tuberculous meningitis, brain abscess, or cryptococcal meningitis, CRP was moderately to highly elevated. CONCLUSIONS: In the presence of a normal CRP concentration (below 10 mg/mL) acute bacterial meningitis is excluded even in a developing country setting and antimicrobial therapy is not warranted.

HIV-1 variants in South and South-East Asia.

HIV spread in South and South-East Asia is most alarming, and genetic variability of HIV-1 is an important consideration in vaccine development. In this study, we examined the third variable (V3) region of env gene of HIV-1 variants prevalent in Thailand, Malaysia, India, and the Philippines. By phylogenetic tree analyses, an HIV-1 variant from an injecting drug user (IDU) in Thailand belonged to subtype B, and HIV-1 variants from 2 IDUs in Malaysia were classified into 2 subtypes, B and E. One HIV-1 variant from a male homosexual in the Philippines belonged to subtype B. Out of 8 HIV-1 variants from sexually transmitted disease patients in India, 7 belonged to subtype C, and one to subtype A. Although the total number of individuals examined in this study was limited, 4 HIV-1 subtypes were found in South and South-East Asia and large international movements of HIV-1-infected individuals in this region could induce global dissemination of these HIV-1 variants.

Etiology of acute lower respiratory infection in Filipino children under five years.

This study conducted at the Research Institute for Tropical Medicine from April 1990 to December 1992, identified the etiology of acute lower respiratory tract infection (ALRI) in 119 (36.9%) of 317 hospitalized children < 5 years of age. A higher proportion of respiratory viruses (27.2%) than bacterial agents (10.7%) were identified. Viral agents (adenovirus, RSV, parainfluenza 3, influenza A and influenza B) and bacterial agents (mainly Haemophilus influenzae and Streptococcus pneumoniae) are the pathogenic agents involved in ALRI among Filipino children less than 5 years old.

Dengue surveillance in Metro Manila.

In a one-year surveillance of dengue infection in four hospitals in Metro Manila, Philippines, 143 patients were enrolled. Sixty seven were diagnosed by virus isolation and serology. Majority of patients had secondary antibody response. Only 5 patients developed dengue shock syndrome. Only dengue types 1 and type 2 were isolated. Highest incidence occurred among older children and young adults. Thrombocytopenia and hemoconcentration were observed in 37% and 77% of patients, respectively. There was no fatality. Several problems were identified in the course of the study which need to be corrected in order to have good quality surveillance data in future undertakings.

Development of a practicable method for isolation and identification of dengue viruses in developing countries.

We sought a practicable method for isolation and identification of dengue viruses in South-East Asia. We compared two mosquito cell lines, C6/36 and TRA-284-SFG, for virus isolation and two identification methods, immunofluorescent staining of infected cells with serotype-specific mouse monoclonal antibodies and a sandwich-type enzyme-linked immunosorbent assay with conventional mouse hyperimmune ascitic fluids. We found that the combination of TRA-284-SFG cells and ELISA is a useful and feasible method in developing countries.