Detection of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae merozoite surface protein 1-p19 antibodies in human malaria patients and experimentally infected nonhuman primates.

Approximately 3.2 billion people live in areas where malaria is endemic, and WHO estimates that 350 to 500 million malaria cases occur each year worldwide. This high prevalence, and the high frequency of international travel, creates significant risk for the exportation of malaria to countries where malaria is not endemic and for the introduction of malaria organisms into the blood supply. Since all four human infectious Plasmodium species have been transmitted by blood transfusion, we sought to develop an enzyme-linked immunosorbent assay (ELISA) capable of detecting antibodies elicited by infection with any of these species. The merozoite surface protein 1 (MSP1), a P. falciparum and P. vivax vaccine candidate with a well-characterized immune response, was selected for use in the assay. The MSP1 genes from P. ovale and P. malariae were cloned and sequenced (L. Birkenmeyer, A. S. Muerhoff, G. Dawson, and S. M. Desai, Am. J. Trop. Med. Hyg. 82:996-1003, 2010), and the carboxyl-terminal p19 regions of all four species were expressed in Escherichia coli. Performance results from individual p19 ELISAs were compared to those of a commercial test (Lab 21 Healthcare Malaria enzyme immunoassay [EIA]). The commercial ELISA detected all malaria patients with P. falciparum or P. vivax infections, as did the corresponding species-specific p19 ELISAs. However, the commercial ELISA detected antibodies in 0/2 and 5/8 individuals with P. malariae and P. ovale infections, respectively, while the p19 assays detected 100% of individuals with confirmed P. malariae or P. ovale infections. In experimentally infected nonhuman primates, the use of MSP1-p19 antigens from all four species resulted in the detection of antibodies within 2 to 10 weeks postinfection. Use of MSP1-p19 antigens from all four Plasmodium species in a single immunoassay would provide significantly improved efficacy compared to existing tests.

The prevalence of diverse HIV-1 strains was stable in Cameroonian blood donors from 1996 to 2004.

OBJECTIVE: The HIV epidemic in Cameroon is characterized by a high level of strain diversity despite a relatively low prevalence of infection. In this study, HIV strains infecting blood donors in Cameroon were characterized to determine the prevalence of subtypes and intersubtype recombinants and if strain prevalence was changing over time. METHODS: From 1996 through 2004, 676 HIV-infected blood donations were collected at blood banks in Douala and Yaoundé, Cameroon. A subset of the HIV-1 group M strains (n = 574) were classified based on phylogenetic analysis of viral sequences from the gag p24, pol integrase, and env gp41 regions. RESULTS: HIV-1 group M accounted for 97.3% (n = 658) of infections, whereas group O was present in 2.2% (n = 15) and HIV-2 in 0.4% (n = 3). Within the group M infections, 14 subtypes and circulating recombinant forms (CRFs) and unique recombinant forms (URFs) were identified. Overall, CRFO2_AG accounted for 58.2% of infections, URFs 14.8%, and levels of subtypes, A, B, C, D, F2, and G, and CRFs, 01, 06, 09, 11, 13, 22, and 37, varied from 0.2% to 6.1%. Evaluation of HIV strains present in the donor population over this 9-year period showed no substantial changes in the proportion of infections caused by each subtype and CRF, the percentage of intersubtype recombinants, or the strain composition of the URFs. CONCLUSIONS: HIV-1 strain diversity in Cameroon did not significantly change, suggesting a mature and relatively stable epidemic.

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.

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.

HIV global surveillance: foundation for retroviral discovery and assay development.

The high level of HIV genetic diversity has important implications for screening, diagnostic testing and patient monitoring. Continued diversification and global redistribution of HIV groups, subtypes and recombinants make it imperative that serological and molecular assays be designed and evaluated to ensure reliable performance on all HIV infections. Recognizing the importance of this issue, we initiated a comprehensive program to monitor global diversification of HIV, search for newly emerging variants, assemble large-volume panels of genetically and geographically diverse strains, and develop strategies to determine the impact of HIV diversity on assays used for detecting and monitoring HIV infection. Efforts to identify and characterize rare and emerging HIV strains have lead to the identification of HIV-1 group O, group N, and dual infections of groups M and O. A panel of plasma specimens was established that includes specimens collected from 12 countries in Africa, Asia, Europe, and South America; the panel comprises infections due to HIV-1 group M subtypes A, B, C, D, F, and G, as well as CRF01, CRF02, and unique recombinant forms, group N, and group O. Serological and molecular characterization of this unique panel has provided vital sequence data to support assay development and an invaluable source of well-defined specimens to evaluate and compare assay performance. The ability to address the challenge posed by ongoing evolution of HIV and the emergence of new variants requires continued surveillance of global HIV strain diversity, a sound scientific foundation for assay development, and suitable panels to evaluate and validate assay performance.

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 infections in northwestern Cameroon: identification of HIV type 1 group O and dual HIV type 1 group M and group O infections.

HIV-1 strain diversity was examined in a study population that consisted of hospital and clinic patients from seven cities and villages located in the northwestern regions of Cameroon. Specimens were screened using a serological algorithm designed to identify HIV-1 group M, N, and O, and SIVcpz-like infections followed by RT-PCR amplification to characterize the infecting virus. The results show that the HIV epidemic in northwest Cameroon is dominated by HIV-1 group M CRF02_AG infections (57%). Additional group M subtypes present include A, D, F2, G, and CRF01_AE. Based on discordant subtype classification between gag and env sequences, a high percentage (23%) of viral strains appear to be unique intersubtype recombinants with the majority (88%) involving recombination with CRF02_AG. Group O prevalence is low accounting for only 0.4% of HIV infections. However, group O strain diversity is high; isolates from clades I, IV, and V, as well as unclassified and recombinant strains, were found. Three dual infections by HIV-1 group M and group O were identified and characterized. In two specimens, both group M and O sequences were amplified in gag, pol, and env suggesting the presence of both viruses. Analysis of the third specimen shows the presence of a group O virus and an intergroup M/O recombinant virus. Finally, no infections due to HIV-1 group N or SIVcpz-like strains were found in the study population.

Identification and genomic sequence of an HIV type 1 group N isolate from Cameroon.

HIV-infected plasma specimens, collected in Cameroon between 1999 and 2002, were screened for HIV-1 group N and SIVcpz infections using a serological screening algorithm based on immunoassays with antigens derived from HIV-1 group M, N, and O, and SIVcpz strains. Specimens with reactivity to group N and SIVcpz antigens were characterized by RT-PCR and sequence analysis to identify the infecting virus. Although several specimens were serotyped as potential group N or SIVcpz infections, only one group N infection was confirmed. The specimen, 02CM-DJO0131, was collected in 2002 from a hospital patient at the D'Joungolo Hospital, Yaoundé. The virus genome was amplified as seven overlapping fragments comprising 8938 nucleotides. Phylogenetic analysis shows that 02CM-DJO0131 branches with group N sequences. With this study, three near full-length sequences are now available for group N. While we confirm the presence of group N in the Cameroonian population, group N infections continue to be rare and difficult to identify.