Evaluate performance of the Abbott chemiluminescent microparticle immunoassay assay for detection of syphilis infection in Chinese blood donors.

BACKGROUND AND OBJECTIVES: To prevent Treponema Pallidum (TP) transmission from blood transfusion, enzyme-linked immunosorbent assay (EIA) for anti-TP has been widely used in routine blood donation screening in China for many years. The aim of this study was to evaluate the performance of the Abbott CMIA assay for detection of anti-TP in Chinese blood donors. MATERIALS AND METHODS: A total of 2420 plasma samples, already routinely screened for anti-TP by two different EIAs, from four blood Centers were tested for anti-TP by Abbott CMIA. Subsequently, all samples with positive results by one or both EIAs and/or by Abbott CMIA were subjected to confirmatory testing (CT) using recombinant immunoblot assay (RIBA) or Treponema Pallidum particle agglutination assay (TPPA). TP infection was defined by a RIBA or TPPA positive. RESULTS: Compared with two EIAs strategy, Abbott CMIA showed a relatively best sensitivity as 98.80% (95% CI: 97.44%-100.16%) and a relatively best specificity as 99.58% (95% CI: 99.30%-99.85%), yielding the best consistency (99.49%) between anti-TP CT results with the highest κ value of .98. CONCLUSION: This is the first study to evaluate the performance of the Abbott CMIA assays for detection of syphilis in Chinese blood donors. Our results suggested that CMIA performed better than both EIAs, and implementation of CMIA replacing two different EIA reagents might help to further reduce the risk of transfusion-transmitted TP infection, decrease unnecessary blood waste and loss of blood donors.

International survey on the impact of parasitic infections: frequency of transmission and current mitigation strategies.

BACKGROUND AND OBJECTIVES: Globally, blood safety interventions have been successful in mitigating risk of the major transfusion-transmitted (TT) viruses. However, strategies that address risk from parasites are comparatively limited. TT parasites are often regional in nature, posing unique challenges; we sought to understand their impact on blood safety. MATERIALS AND METHODS: An electronic questionnaire was distributed to transfusion medicine leaders in 100 countries. The survey focused on specific questions pertaining to four parasitic diseases: babesiosis, Chagas, leishmaniasis and malaria. Respondents provided data on historical TT cases, local epidemiology, policies to mitigate risk and an assessment of public health perceptions for each aetiologic agent. RESULTS: Twenty-eight (28%) surveys were returned from countries in Europe (n = 13), the Americas (n = 6), Africa (n = 4), Asia (n = 3) and Oceana (n = 2). Historically, no cases of TT leishmaniasis were reported, TT babesiosis was exclusive to Canada and the USA, TT Chagas was limited to the Americas and Spain, while TT malaria was cosmopolitan. Mitigation efforts varied widely; malaria was the most frequently tested parasitic disease. The public's perception of risk for parasitic agents was low, while that of health authorities in endemic countries was higher. CONCLUSION: The global impact of parasitic infections on blood safety and related mitigation efforts varied widely by parasite epidemiology, test availability, public health priorities and socioeconomic constraints. While parasites continue to pose a risk to blood safety, the successful mitigation of viral risk has elevated the prominence of TT parasites in many locations, thereby requiring consideration of mitigation efforts.

Experimental transfusion-induced Babesia microti infection: dynamics of parasitemia and immune responses in a rhesus macaque model.

BACKGROUND: Babesiosis is an emerging tick-borne infection in humans. The increasing numbers of reported cases of transfusion-associated babesiosis (TAB), primarily caused by Babesia microti, represents a concern for the safety of the US blood supply. STUDY DESIGN AND METHODS: This study investigated kinetics of parasitemia and innate immune responses and dynamics of antibody responses during B. microti infection in rhesus macaques (RMs) using blood smears, quantitative polymerase chain reaction (qPCR), flow cytometry, and indirect fluorescent antibody testing. A total of six monkeys were transfused with either hamster or monkey-passaged B. microti-infected red blood cells (two and four monkeys, respectively) simulating TAB. RESULTS: The prepatent period in monkeys inoculated with hamster-passaged B. microti was 35 days compared with 4 days in monkeys transfused with monkey-passaged B. microti; the latter monkeys also had markedly higher parasitemia levels. The duration of the window period from the first detected parasitemia by qPCR analysis to the first detected antibody response ranged from 10 to 17 days. Antibody responses fluctuated during the course of the infection. Innate responses assessed by the frequencies of monocytes and activated B cells correlated with the kinetics and magnitude of parasitemia. On Day 14, additional activation peaks were noted for CD14+CD16+ and CD14-CD16+ monocytes and for CD11c+ myeloid dendritic cells, but only in animals transfused with monkey-passaged B. microti. Parasitemia persisted in these immunocompetent animals, similar to human infection. CONCLUSION: The results suggest that transfusion-associated transmission of B. microti leads to rapid onset of parasitemia (Day 4) in RMs, detectable antibody response 14 days later, and persistent parasitemia.

Confirmation of putative HIV-1 group P in Cameroon.

We report the second human immunodeficiency virus (HIV) belonging to the new HIV type 1 (HIV-1) group P lineage that is closely related to the simian immunodeficiency virus found in gorillas. This virus was identified in an HIV-seropositive male hospital patient in Cameroon, confirming that the group P virus is circulating in humans. Results from screening 1,736 HIV-seropositive specimens collected in Cameroon indicate that HIV-1 group P infections are rare, accounting for only 0.06% of HIV infections. Despite its rarity, group P shows evidence of adaptation to humans.

Infection, viral dissemination, and antibody responses of rhesus macaques exposed to the human gammaretrovirus XMRV.

Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.

Seroprevalence of xenotropic murine leukemia virus-related virus in normal and retrovirus-infected blood donors.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) has been reported in patients with prostate cancer and chronic fatigue syndrome. Although results have been conflicting, the potential of XMRV as an infectious human retrovirus has raised concerns about transfusion safety. To address this issue, normal and retrovirus-infected blood donors were screened for evidence of XMRV infection. STUDY DESIGN AND METHODS: Plasma from 1000 US, 100 human immunodeficiency virus Type 1-infected Cameroonian, and 642 human T-lymphotropic virus Type I (HTLV-I)-infected or uninfected Japanese blood donors as well as 311 sexually transmitted disease diagnostic specimens were screened for antibodies to XMRV gp70 and p15E using chemiluminescent immunoassays (CMIAs). CMIA-reactive samples were evaluated by p30 CMIA, Western blot, and real-time reverse transcriptase polymerase chain reaction. RESULTS: XMRV seroreactivity was low (0%-0.6%) with the exception of the HTLV-I-infected donors (4.9%). Antibody was detected against only a single XMRV protein (p15E or gp70); none of the seroreactive samples had detectable XMRV pol or env sequences. The elevated seroreactivity in HTLV-I-infected donors was due to an increased p15E seroreactive rate (4.1%). Inspection of XMRV and HTLV sequences revealed a high level of conservation within the immunodominant region (IDR) of the transmembrane protein. In some cases, HTLV IDR peptide competitively reduced the XMRV p15E signal. CONCLUSIONS: Based on the low prevalence of seroreactivity, detection of antibody to only a single XMRV protein and the absence of XMRV sequences, this study finds no compelling evidence of XMRV in normal or retrovirus-infected blood donors. The increased p15E seroreactivity observed in HTLV infection is likely due to cross-reactive antibodies.

Characterization of antibodies elicited by XMRV infection and development of immunoassays useful for epidemiologic studies.

BACKGROUND: Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a human gammaretrovirus recently identified in prostate cancer tissue and in lymphocytes of patients with chronic fatigue syndrome. To establish the etiologic role of XMRV infection in human disease requires large scale epidemiologic studies. Development of assays to detect XMRV-specific antibodies would greatly facilitate such studies. However, the nature and kinetics of the antibody response to XMRV infection have yet to be determined. RESULTS: Three rhesus macaques were infected with XMRV to determine the dynamics of the antibody responses elicited by infection with XMRV. All macaques developed antibodies to XMRV during the second week of infection, and the predominant responses were to the envelope protein gp70, transmembrane protein p15E, and capsid protein p30. In general, antibody responses to gp70 and p15E appeared early with higher titers than to p30, especially in the early period of seroconversion. Antibodies to gp70, p15E and p30 persisted to 158 days and were substantially boosted by re-infection, thus, were identified as useful serologic markers. Three high-throughput prototype assays were developed using recombinant proteins to detect antibodies to these viral proteins. Both gp70 and p15E prototype assays demonstrated 100% sensitivity by detecting all Western blot (WB) positive serial bleeds from the XMRV-infected macaques and good specificity (99.5-99.9%) with blood donors. Seroconversion sensitivity and specificity of the p30 prototype assay were 92% and 99.4% respectively. CONCLUSIONS: This study provides the first demonstration of seroconversion patterns elicited by XMRV infection. The nature and kinetics of antibody responses to XMRV in primates were fully characterized. Moreover, key serologic markers useful for detection of XMRV infection were identified. Three prototype immunoassays were developed to detect XMRV-specific antibodies. These assays demonstrated good sensitivity and specificity; thus, they will facilitate large scale epidemiologic studies of XMRV infection in humans.

Expanded evaluation of blood donors in the United States for human immunodeficiency virus type 1 non-B subtypes and antiretroviral drug-resistant strains: 2005 through 2007.

BACKGROUND: In a previous study of 66 human immunodeficiency virus (HIV)-infected US blood donors from 1999 to 2005, HIV-1 non-B and antiretroviral drug-resistant strains accounted for 4.7 and 6.5% of HIV infections, respectively. This study was expanded to include an additional 11 recently acquired infections and 197 established infections collected from January 2005 through December 2007. STUDY DESIGN AND METHODS: HIV-infected donors were detected using FDA-licensed assays. Drug resistance profiles for protease and reverse transcriptase (RT) genes were determined using a genotyping system (ViroSeq, Celera Diagnostics); genetic subtype was determined by phylogenetic analysis of these sequences. RESULTS: Drug resistance profiles were obtained for 203 of 208 specimens; 9.9% had mutations that confer drug resistance. Ten showed resistance to a single drug class: nine to nonnucleoside RT inhibitors (NNRTIs) and one to nucleoside RT inhibitors (NRTIs). Eight showed two drug class resistance: five NRTI plus NNRTI, two NRTI plus protease inhibitor (PI), and one NNRTI plus PI. Two showed three drug class resistance. Non-B strains were identified in 2.5% of donors and consisted of subtypes A1 and D, CRF02_AG, CRF43-02G, and URF_BF. CONCLUSIONS: Data from this and the previous study show that antiretroviral drug-resistant HIV-1 is present in 9.1% of HIV-infected donors from 1999 through 2007; 9.3% of established infections and 6.9% of recent infections. Diverse HIV-1 non-B strains presently account for 3.0% of HIV infections in US donors.

HIV testing in a high-incidence population: is antibody testing alone good enough?

BACKGROUND: The Centers for Disease Control and Prevention recently recommended the expansion of human immunodeficiency virus (HIV) antibody testing. However, antibody tests have longer "window periods" after HIV acquisition than do nucleic acid amplification tests (NAATs). METHODS: Public Health-Seattle & King County offered HIV antibody testing to men who have sex with men (MSM) using the OraQuick Advance Rapid HIV-1/2 Antibody Test (OraQuick; OraSure Technologies) on oral fluid or finger-stick blood specimens or using a first- or second-generation enzyme immunoassay. The enzyme immunoassay was also used to confirm reactive rapid test results and to screen specimens from OraQuick-negative MSM prior to pooling for HIV NAAT. Serum specimens obtained from subsets of HIV-infected persons were retrospectively evaluated by use of other HIV tests, including a fourth-generation antigen-antibody combination assay. RESULTS: From September 2003 through June 2008, a total of 328 (2.3%) of 14,005 specimens were HIV antibody positive, and 36 (0.3%) of 13,677 antibody-negative specimens were NAAT positive (indicating acute HIV infection). Among 6811 specimens obtained from MSM who were initially screened by rapid testing, OraQuick detected only 153 (91%) of 169 antibody-positive MSM and 80% of the 192 HIV-infected MSM detected by the HIV NAAT program. HIV was detected in serum samples obtained from 15 of 16 MSM with acute HIV infection that were retrospectively tested using the antigen-antibody combination assay. CONCLUSIONS: OraQuick may be less sensitive than enzyme immunoassays during early HIV infection. NAAT should be integrated into HIV testing programs that serve populations that undergo frequent testing and that have high rates of HIV acquisition, particularly if rapid HIV antibody testing is employed. Antigen-antibody combination assays may be a reasonably sensitive alternative to HIV NAAT.

Identification of human immunodeficiency virus type 1 non-B subtypes and antiretroviral drug-resistant strains in United States blood donors.

BACKGROUND: In this study, human immunodeficiency virus type 1 (HIV-1)-infected blood donors were evaluated for genetic subtype and drug resistance to determine the prevalence of divergent HIV strains in the US donor population. STUDY DESIGN AND METHODS: Subtype was determined by phylogenetic analysis of viral sequences amplified by reverse transcription-polymerase chain reaction. The drug resistance profile of the protease and reverse transcriptase (RT) genes was determined using an HIV-1 genotyping system (ViroSeq). RESULTS: From 1999 through 2005, 26 recently infected donors, defined as HIV-1 RNA-positive, antibody-negative (RNA+/Ab-), were identified (yield, 1:1.61 million). Over the same period, the frequency of anti-HIV-positive donors was 1:34,700. Twenty RNA+/Ab- specimens were evaluated; all were infected with HIV-1 subtype B. Drug resistance profiles obtained for 18 donors identified one strain with protease mutation L90M that confers resistance to nelfinavir and one with RT mutation Y188H that confers resistance to nevirapine. Genetic subtype was determined for 44 of 46 HIV antibody-reactive and confirmed-positive (Ab+) specimens. Three infections (6.8%) were due to circulating recombinant forms: 2 CRF01_AE and 1 CRF02_AG. In the Ab+ group, one strain was resistant to all nucleoside RT inhibitors and one had mutations that confer resistance to protease inhibitors. CONCLUSION: The data show that antiretroviral drug-resistant HIV strains are being transmitted in the United States. Overall 6.5 percent (4 of 62) of HIV-1-infected donors harbored drug-resistant strains. HIV-1 non-B strains accounted for 4.7 percent (3 of 64) of the infections in donors. HIV-1 subtype B is still the predominant strain in the United States; however, non-B strains are increasing.

Seroprevalence of HTLV-1/2 Among Voluntary Blood Donors in Vietnam.

Infection with human T lymphotropic virus (HTLV), although asymptomatic in most cases, can lead to severe illnesses, such as adult T cell leukemia/lymphoma or myelopathy/tropical spastic paraparesis. HTLV can be transmitted by whole-blood (WB) transfusion. The prevalence of HTLV among blood donor populations has not been characterized in Vietnam, although the screening has been partially implemented on voluntary basis since 2016. To determine the seroprevalence of HTLV-1/2 among blood donors, a total of 14,819 healthy blood donors in northern, central, and southern Vietnam and 1,003 samples from hepatitis B surface antigen (HbsAg), anti-hepatitis C (anti-HCV), or HIV Ag/Ab reactive blood donors were screened for anti-HTLV-1/2 antibodies by a chemiluminescence immunoassay using the Abbott ARCHITECT rHTLV-I/II assay. The anti-HTLV-1/2 repeat reactive (RR) samples were further tested by immunoblot (IB) method using MP Biomedicals HTLV Blot 2.4 for confirmation and differentiation of HTLV-1/2 infection. Proviral HTLV subgenomic amplification of the gag and tax regions was performed on the available WB RR samples (N = 11) by polymerase chain reaction (PCR). Among 14,819 blood donors, 34 samples (0.23%) were RR for anti-HTLV-1/2 antibodies, but only 1 case was confirmed HTLV-2 positive (0.0067%) and 5 cases were classified as indeterminate (0.034%) by IB. The RR rate was 0.39% among HBsAg/anti-HCV/HIV reactive sample groups, but none of them was confirmed by IB. Subgenomic PCR failed to amplify proviral DNA from WB samples of 11 RR samples. HTLV-1/2 prevalence was found to be low among blood donors in the study. Continued vigilance remains essential to maintain a low transfusion-transmitted risk in Vietnam.

HIV type 2 intergroup recombinant identified in Cameroon.

A unique HIV-2 intergroup recombinant strain was identified in Cameroon. The virus, CM-03-510-03, was amplified from blood collected from a 47-year-old female patient in Douala, Cameroon in 2003 who was seroreactive for HIV-2. A near full-length genome 9089 nucleotides in length was amplified from proviral DNA. The genome for CM-03-510-03 is composed of segments of HIV-2 groups A and B with four recombination break-points and has open reading frames for all the structural and regulatory genes. A comparison of CM-03-510-03 to the only previously reported HIV-2 intergroup recombinant shows that the two strains share one recombination breakpoint but are otherwise distinct from each other. Similar to HIV-1, HIV-2 intergroup recombination is an indication that coinfection with more than one strain has occurred in individuals and is a mechanism that increases strain genetic diversity.

Identification and characterization of HIV type 1 subtypes present in the Kingdom of Saudi Arabia: high level of genetic diversity found.

Saudi Arabia has a very low prevalence of HIV infections and nothing is known about HIV strains present in the population. Here specimens were collected from 62 HIV-1-infected patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Viral sequences were PCR amplified using primers for HIV-1 group M in gag p24, pol integrase, and env gp41 and genetic subtype was determined by phylogenetic analysis. HIV-1 viral sequences were amplified from 56 of the 62 specimens. Based on phylogenetic analysis of viral sequences, subtype C was the most common subtype present and accounted for 39.3% of the infections followed by subtype G (25%), subtype B (17.9%), subtype D (3.6%), and subtypes A and CRF02_AG (1.8% each). In addition, for six specimens subtype classifications were discordant between gag, pol, and/or env; these intersubtype recombinant viruses account for 10.7% of the infections and consisted of recombinants of subtypes A/CRF01, A/CRF02, A/G, B/G, and D/CRF02. The high HIV-1 strain diversity suggests that there have been multiple introductions of HIV-1 into Saudi Arabia from several sources. Within the study population, there were five husband/wife pairs. For each pair, the viral sequences obtained were closely related to each other showing that heterosexual transmission occurred.

HIV-1 strains identified in Brazilian blood donors: significant prevalence of B/F1 recombinants.

In the Brazilian HIV-1 epidemic subtypes B, C, and F1 are cocirculating in the high risk population groups, and there is a high prevalence of intersubtype recombinant forms. The dynamic nature of the HIV epidemic in Brazil led us to study HIV-1 subtypes present in HIV-infected blood donations collected from 2001 to 2003. Donations from 91 seropositive donors were evaluated. Genetic subtype was obtained for 88 specimens based on sequence analysis of gag p24, pol IN, and env gp41 IDR. HIV-1 subtype B was the predominant strain present in the donor population (73.9%). A significant prevalence of intersubtype recombinants of subtypes B and F1 was found (22.7%). Subtype C (1.1%) and F1 (2.3%) were rare. None of the B/F1 recombinants is CRF28_BF or CRF29_BF. The high level of unique B/F1 recombinant strains in this population demonstrates the dynamic and complex nature of the HIV epidemic in Brazil.

Analysis of HIV type 1 gp41 sequences in diverse HIV type 1 strains.

The HIV fusion inhibitor enfuvirtide (ENF/Fuzeon) targets the env gp41 transmembrane domain. Mutations in gp41 are associated with ENF resistance. We developed a prototype assay to genotype a 676-bp region spanning the heptad repeat domains (HR1 and HR2) of HIV-1 gp41. Plasma samples were collected from 126 HIV-1-infected blood donors in Cameroon, Brazil, Uganda, South Africa, Thailand, and Argentina. Based on analysis of gag p24, pol integrase, and env gp41 genes, the panel was composed of subtypes A/A2 (18), B (11), C (14), D (10), F/F2 (9), G (7), CRF01_AE (9), CRF02_AG (33), and recombinant strains (15). Genotyping was successful for 119 of the 126 samples (94.4%). Although numerous amino acid polymorphisms were detected in some samples, none had primary mutations associated with ENF resistance. The gp41 HIV-1 research reagents developed by Celera are useful tools for genotyping analysis of the gp41 region in diverse HIV-1 strains.

Near full-length genome characterization of three additional HIV type 1 CRF13_cpx strains from Cameroon.

Recombinant forms of HIV-1 contribute significantly to the ongoing epidemic. In the present study, we characterized the near full-length genomes of three candidate HIV-1 CRF13_cpx strains originating in Cameroon, 04CM-173-9, 04CM-632-28, and 02CM-A1394. Bootscanning, recombination breakpoint analysis, and phylogenetic trees confirmed similar genomic structures with identical breakpoint positions compared to the three available CRF13_cpx sequences. The candidate and reference sequences formed a distinct cluster well separated from other group M subtypes and had a mosaic structure derived from subtypes A1, G, J, and CRF01_AE. The similarity in genomic composition and position of recombination breakpoints suggest that these isolates share a common ancestor. The epidemiological significance of CRF13_cpx strains in Cameroon is unknown; however, the availability of three additional genomic sequences will improve our understanding of the overall genetic diversity within this recombinant form of HIV-1.

Partially double-stranded linear DNA probes: novel design for sensitive detection of genetically polymorphic targets.

Genetically polymorphic targets present a significant challenge to the reliability of detection and quantification by nucleic acid-based assays. A probe system with enhanced mismatch tolerance would be advantageous for such applications. The present study introduces a novel class of DNA probes, designated as partially double-stranded linear probes, composed of a long target-specific strand 5' labeled with a fluorophore and a markedly shorter quencher strand, complementary to the 5' end of the target-specific strand, that is 3' end-labeled with a quencher moiety. The utility of this probe system for sensitive detection of amplification products was demonstrated in a real-time PCR format. Comparison of multiple partially double-stranded linear probe combinations revealed that increased asymmetry in strand length was associated with improved mismatch tolerance. Notably, for a 45-mer/11-mer combination, the difference in threshold cycle values obtained for a perfectly matched target and one containing six mismatches was <1.5 cycles. The capacity for superior mismatch tolerance, ease of design, simplicity and flexibility of application are characteristics that make this new class of probes a desirable alternative for homogeneous detection of targets with a high level of genetic heterogeneity.

Evaluation of performance across the dynamic range of the Abbott RealTime HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses.

Performance of the Abbott m2000 instrument system and the Abbott RealTime HIV-1 assay was evaluated using a panel of 37 group M (subtypes A-D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTime HIV-1 detected an additional 31 samples at < 40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87% and 89% of values were within 0.5 log(10) copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by approximately 2 log(10) copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced "hands-on" time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.

Identification of HIV type 1 group N infections in a husband and wife in Cameroon: viral genome sequences provide evidence for horizontal transmission.

HIV-1 is classified into three groups, M (major), N (non-M non-O), and O (outlier); each group arose from a separate transmission of SIVcpz into humans. HIV-1 group N was recently discovered and infections with this virus are rare with only eight documented cases. All group N infections have been found in Cameroon and there is no evidence of direct linkage between the infected patients. We report here the identification of HIV-1 group N infections in a husband and wife. The group N infection in the husband, 1131-03, was identified first based on seroreactivity in peptide EIAs and confirmed by PCR amplification of group N viral sequences. Subsequently the wife, 1015-04, was evaluated and confirmed to also be infected with a group N virus. Near full-length viral genomes were amplified and sequenced from each patient's specimen. The low level of diversity between the two viral sequences provides evidence of horizontal transmission of group N from one spouse to the other. Patient 1131-03 was receiving antiviral therapy consisting of reverse transcriptase inhibitors; the treatment appears effective for suppression of group N viral replication based on apparently low viral load in plasma specimens collected from the patient and the absence of drug resistance mutations in RT sequences amplified from 1131-03. This report brings to 10 the number of group N infections identified and to 5 the number of group N genomes sequenced. Although group N infections continue to be rare, group N is a pathogenic virus and its prevalence needs to be monitored.

HIV-1 Group N: evidence of ongoing transmission in Cameroon.

An HIV-1 group N infection, 02CM-DJO0135, was identified among specimens collected in 2002 at the D'Joungolo Hospital, Yaoundé, Cameroon. Sequences were obtained from viral RNA extracted from plasma for regions of LTR-gag, pol-vif, and env. The virus amplified from the specimen is closely related to a previously reported group N virus, 02CM-DJO0131, that was also collected at this hospital in 2002. Although the viral sequences for the two isolates differ, their close relationship suggests that the two specimens are linked. No patient histories are available for 02CM-DJO0131 and 02CM-DJO0135; the specimens could have been drawn from a husband/wife, mother/child, or a single individual. However, differences in seroreactivity indicate that it is unlikely that the specimens were drawn from the same patient. This report documents the second case that suggests linkage between group N-infected individuals and indicates that there is ongoing transmission of HIV-1 group N in Cameroon.