Molecular surveillance of HIV-1 field strains in Nigeria in preparation for vaccine trials.

We conducted a national molecular epidemiologic survey of HIV-1 strains in Nigeria to determine the most prevalent subtype(s) for use in developing candidate vaccines. A total of 230 HIV-1-positive blood samples collected from 34 of the 36 Nigerian states were analyzed by our modified env gp41-based heteroduplex mobility assay (HMA) and/or gp41 sequencing and analysis. Overall, 103 (44.8%) were subtype A, 125 (54.3%) were subtype G, one (0.4%) was subtype C, and one (0.4%) was subtype J, and one (0.4%) was unclassifiable. To further characterize Nigerian viruses to aid in strain selection for candidate vaccines, one gp41 subtype G and five gp41 subtype A strains were selected for full envelope sequencing. The one subtype G sequence had consistent phylogenies throughout gp160, using programs to detect recombination. However, all five sequences that were primarily subtype A in gp41 were found to be recombinant viruses. Two of the five (40%) were A/G/J mosaics with common breakpoints. The remaining three gp160 recombinants all had their own unique break points: two A/? and one A/?/G, however, all five had the majority of their mosaic breakpoints occurring in gp41. None of the five were consistent with the circulating recombinant form (CRF)02_AG strain previously reported to be prevalent in West Africa. In conclusion, we showed a clear dominance and widespread distribution of gp41 subtypes A and G in fairly equal proportions, suggesting that vaccines designed for use in this geographic locale should incorporate the gene(s) of both subtypes. However, appreciating the magnitude of diversity of HIV-1 strains in Nigeria may require sequencing and analysis of longer gene regions for the identification of prevalent or emerging CRFs.

Substitution model of sequence evolution for the human immunodeficiency virus type 1 subtype B gp120 gene over the C2-V5 region.

Phylogenetic analyses frequently rely on models of sequence evolution that detail nucleotide substitution rates, nucleotide frequencies, and site-to-site rate heterogeneity. These models can influence hypothesis testing and can affect the accuracy of phylogenetic inferences. Maximum likelihood methods of simultaneously constructing phylogenetic tree topologies and estimating model parameters are computationally intensive, and are not feasible for sample sizes of 25 or greater using personal computers. Techniques that initially construct a tree topology and then use this non-maximized topology to estimate ML substitution rates, however, can quickly arrive at a model of sequence evolution. The accuracy of this two-step estimation technique was tested using simulated data sets with known model parameters. The results showed that for a star-like topology, as is often seen in human immunodeficiency virus type 1 (HIV-1) subtype B sequences, a random starting topology could produce nucleotide substitution rates that were not statistically different than the true rates. Samples were isolated from 100 HIV-1 subtype B infected individuals from the United States and a 620 nt region of the env gene was sequenced for each sample. The sequence data were used to obtain a substitution model of sequence evolution specific for HIV-1 subtype B env by estimating nucleotide substitution rates and the site-to-site heterogeneity in 100 individuals from the United States. The method of estimating the model should provide users of large data sets with a way to quickly compute a model of sequence evolution, while the nucleotide substitution model we identified should prove useful in the phylogenetic analysis of HIV-1 subtype B env sequences.

Development of an env gp41-based heteroduplex mobility assay for rapid human immunodeficiency virus type 1 subtyping.

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.

Failure of routine HIV-1 tests in a case involving transmission with preseroconversion blood components during the infectious window period.

CONTEXT: Current screening practices for blood donations have been successful in reducing human immunodeficiency virus (HIV) transmission through receipt of contaminated blood products. However, HIV-infected blood donations made prior to seroconversion and before high levels of viral replication occur could test negative using both serologic antigen and antibody tests. Testing based on nucleic acid amplification (NAT) is being implemented to screen for HIV-infected blood donated during this period, yet the issue of single vs minipool donation screening remains unresolved. OBJECTIVES: To determine HIV-1 genetic linkage between virus in 2 HIV-1-infected recipients of blood components and virus in the donor, who was HIV antigen and antibody negative at the time of donation; to screen the blood donor's plasma with HIV NAT assays, including those currently proposed for use in US blood donation screening. DESIGN AND SETTING: Case study conducted in October 1997 involving the Communicable Disease Centre, Singapore General Hospital, and the Singapore Blood Transfusion Service, Singapore. SUBJECTS: The blood donor and the 2 recipients of donor platelets and red blood cells. MAIN OUTCOME MEASURES: Genetic analysis of the HIV-1 p17 coding region of gag and the C2V5 region of env to determine the genetic relatedness of virus from the donor and recipients; reactivity in quantitative and qualitative assays, and reactivity in donor screening HIV NAT assays in single donation and minipool screening contexts. RESULTS: Direct DNA sequencing demonstrated identical HIV-1 subtype E viral sequences in the donor and recipients. Based on comparisons of a qualitative and quantitative assay for HIV-1 RNA levels, a low level of viremia (range, 5-39 copies/mL in plasma) was estimated to be in the donor's undiluted blood at the time of donation. Additional testing using donor-screening NAT assays showed consistent detection of HIV RNA in the undiluted donor plasma whereas detection was inconsistent at the 1:16 and 1:24 dilution levels currently used in minipool screening of blood donations in the United States. CONCLUSIONS: Transmission of HIV from a blood donor to a platelet recipient and a red blood cell recipient occurred in the preseroconversion infectious window period. The viral load in the implicated donation was estimated to be less than 40 copies/mL of plasma. Current US minipool HIV NAT screening protocols may not be sufficiently sensitive to detect all infectious window-period donations. JAMA. 2000;284:210-214

Genetic analysis of human immunodeficiency virus type 1 strains in Kenya: a comparison using phylogenetic analysis and a combinatorial melting assay.

We surveyed human immunodeficiency virus (HIV) subtype distribution from peripheral blood mononuclear cells (PBMCs) collected in 1995 from 24 HIV-1-infected Kenyan residents (specimens from predominantly male truck drivers and female sex workers near Mombasa and Nairobi). Processed lysates from the PBMC samples were used for env amplification, directly sequenced, and analyzed by phylogenetic analysis. Envelope amplification products were also used for analysis in a polymerase chain reaction (PCR)-based assay, called the combinatorial melting assay (COMA). Results of the two tests were compared for assignment of subtype for this Kenyan cohort. The COMA, a PCR capture technique with colorimetric signal detection, was used with HIV reference subtype strains as well as regional (East Africa) HIV strains for subtype identification. Performance of the COMA was at 100% concordance (24 of 24) as compared with DNA sequencing analysis. Phylogenetic analysis showed 17 isolates to be subtype A, 3 subtype D, and 4 subtype C viruses. This may represent an increase in subtype C presence in Kenya compared with previously documented reports. The COMA can offer advantages for rapid HIV-1 subtype screening of large populations, with the use of previously identified regional strains to enhance the identification of local strains. When more detailed genetic information is desired, DNA sequencing and analysis may be required.

Subtype G and multiple forms of A/G intersubtype recombinant human immunodeficiency virus type 1 in Nigeria.

Multiple human immunodeficiency virus type 1 (HIV-1) genetic subtypes, intersubtype recombinants, and group O have been found in west central Africa. In Nigeria, where HIV-1 prevalence is rising rapidly, characterization of HIV-1 strains has been limited. Each of three full-length genome sequences acquired to date shows evidence of recombination: two are largely subtype G with subtype A segments in the midgenome accessory region; the third, IbNG, is subtype G with the long terminal repeats and two segments of pol from subtype A. In this study, peripheral blood mononuclear cells obtained in 1994-1995 from 10 patients hospitalized in northeastern Nigeria were evaluated by sequencing of the complete envelope and, from 7 patients, a portion of gag. Four patients harbored subtype G viruses and six patients had recombinant viruses. Two had strains sharing the A/G recombinant structure of IbNG. Two had a previously undescribed recombinant, mostly subtype A, whose carboxyl-terminal gp41 could not be classified. An A/G recombinant different from IbNG but similar to CA1, a Cameroonian strain, was found in one patient. The remaining patient had a strain that was otherwise subtype G but shared an unclassified carboxyl-terminal gp41 segment with the CA1-like strains. Other subtypes and group O were not found.

HIV type 1 subtypes in Malaysia include B, C, and E.

PIP: Peripheral blood mononuclear cell specimens were collected from 13 HIV-1-infected IV drug users in Kuala Lumpur, Malaysia, as well as one HIV-infected baby, between 1992 and 1993. DNA was then amplified by nested polymerase chain reaction and a 345-bp fragment of the C2V3 region of the env gene was sequenced. 11 of the 14 Malaysian sequences clustered with the B' subtype, one different from the typical subtype B US strains HIVMN and HIVSF2. Two sequences grouped in the C subtype and had sister taxa closer to the Indian C subtype sequences than those from Zambia. The sequence from the infant was identified as a subtype E virus, grouped more closely with subtype E strains from Thailand than subtype E viruses from the Central African Republic.^ieng

Serodiagnosis of Lyme disease: accuracy of a two-step approach using a flagella-based ELISA and immunoblotting.

An ELISA containing a purified flagellar antigen from Borrelia burgdorferi (FLA-ELISA) was evaluated. The FLA-ELISA, detecting IgM and IgG together, did not have adequate specificity by itself. Good accuracy was obtained, however, when the FLA-ELISA was the first step in a two-step protocol that used immunoblotting as a conditional second test. Samples that scored positive or equivocal by the FLA-ELISA were evaluated with separate IgM and IgG immunoblots. The sensitivity of the two-step process for patients with erythema migrans or with later manifestations of Lyme disease was 64% and 100%, respectively. The specificity for health blood donors was 100% and was 90% for the aggregate of all persons with illness that may cause serologic cross-reactivity (98% if the samples from relapsing fever patients were excluded). Test precision was 96% overall, 99% for Lyme disease case serum samples, 100% for specimens from blood donors, and 88% for samples from persons with other illness.

Epidemiologic and diagnostic studies of patients with suspected early Lyme disease, Missouri, 1990-1993.

A retrospective case-control study investigated 45 Missouri outpatients with annular rashes meeting a surveillance case definition for erythema migrans and with onset in 1990-1991. Risk factors included being male, living near a body of water, and hunting. Twenty patients (44%) associated their rash with the bite of a tick; of these, 5 described an adult Amblyomma americanum. A typical rash was described as expanding over time and measuring 8 cm in diameter at 4 days after onset. Mild constitutional symptoms were common but fever was uncommon. Serologic tests failed to incriminate Borrelia burgdorferi or selected other arthropodborne pathogens. Skin specimens from suspected erythema migrans lesions of 23 Missouri patients sampled prospectively in 1991-1993 were culture-negative for B. burgdorferi. Thus, tick bite-associated annular rashes in Missouri remain idiopathic. Possible causes include infection with a novel A. americanum-transmitted pathogen and an atypical toxic or immunologic reaction to tick-associated proteins.

Detection of yellow fever virus by polymerase chain reaction.

BACKGROUND: Yellow fever virus continues to cause major epidemics. A sensitive rapid diagnostic test is required to identify cases and contacts in order to implement emergency immunization campaigns. OBJECTIVES: To identify YFV envelope protein gene fragments, construct a polymerase chain reaction (PCR) assay and test its utility in identifying viruses isolated from laboratory and clinical specimens. STUDY DESIGN: YFV RNA was transcribed with reverse transcriptase and the cDNA amplified by PCR using primers encoding a portion of the viral envelope protein gene. The identity of the 482 bp amplified product was confirmed by restriction enzyme analysis and by dot blot hybridization with a labelled oligonucleotide probe. The assay was tested for sensitivity and specificity on isolates from South America and Africa. Detection limits were determined using different probe labels. PCR inhibitory effects were analyzed with laboratory and clinical specimens. RESULTS: The assay was specific for YFV and did not detect any of 15 other flaviviruses. The amplified region was conserved among all 32 South American and African isolates tested. Four strains from Africa did not hybridize with the probe, indicating sequence divergence in the envelope protein gene. Samples containing 30 pfu of virus were detected by visual inspection of the ethidium bromide stained 482 bp DNA amplimer and 10 pfu were detected with a digoxigenin labelled probe. Inhibitory effects of human serum on the PCR were overcome by diluting samples 4-fold in buffer. Viral neutralizing antibody in experimental samples did not affect the sensitivity of detection. Yellow fever virus in serum from experimentally infected Cynomolgus monkeys (10(3.7)-10(7.0) pfu/0.1 ml) was detected with signal intensities corresponding to the amount of virus in the sample. When YFV was added to normal human serum and held at 27 degrees C and 80% humidity, the RNA could be detected for up to 3 weeks in samples that had no infectious virus. CONCLUSIONS: A PCR assay was constructed which detected YFV RNA in isolates from patients infected in South America and Africa. This assay is specific for YFV but some African strains were not detected. More clinical samples should be tested.

Evolutionary analysis of the 7.1-kb beta-lactamase-specifying R-plasmid of Neisseria gonorrhoeae by restriction endonucleases.

A physical map of restriction enzyme sites was made for the large beta-lactamase-specifying gonococcal R-plasmid (pMR0360) isolated in 1976. Single sites in the plasmid were mapped for 11 endonucleases, and multiple sites of cleavage were mapped for 17 other enzymes. Conclusions about the structure and origin of this plasmid are discussed.

Comparison of the histamine hypersensitivity and the Limulus amoebocyte lysate tests for endotoxin activity.

The histamine hypersensitivity test and the Limulus amoebocyte lysate test were compared for their effectiveness to quantitate endotoxin activity. The two tests compared favorably in all the trials, except with a sample of endotoxin from Brucella abortus that gave a positive Limulus amoebocyte lysate test at a concentration of 0.001 microgram, while failing to sensitize mice to histamine at a dose of 16 microgram per mouse. The Limulus amoebocyte lysate test was more sensitive than the histamine hypersensitivity test.