Judith N. Torimiro

Emergence of unique primate T-lymphotropic viruses among central African bushmeat hunters.

The human T-lymphotropic viruses (HTLVs) types 1 and 2 originated independently and are related to distinct lineages of simian T-lymphotropic viruses (STLV-1 and STLV-2, respectively). These facts, along with the finding that HTLV-1 diversity appears to have resulted from multiple cross-species transmissions of STLV-1, suggest that contact between humans and infected nonhuman primates (NHPs) may result in HTLV emergence. We investigated the diversity of HTLV among central Africans reporting contact with NHP blood and body fluids through hunting, butchering, and keeping primate pets. We show that this population is infected with a wide variety of HTLVs, including two previously unknown retroviruses: HTLV-4 is a member of a phylogenetic lineage that is distinct from all known HTLVs and STLVs; HTLV-3 falls within the phylogenetic diversity of STLV-3, a group not previously seen in humans. We also document human infection with multiple STLV-1-like viruses. These results demonstrate greater HTLV diversity than previously recognized and suggest that NHP exposure contributes to HTLV emergence. Our discovery of unique and divergent HTLVs has implications for HTLV diagnosis, blood screening, and potential disease development in infected persons. The findings also indicate that cross-species transmission is not the rate-limiting step in pandemic retrovirus emergence and suggest that it may be possible to predict and prevent disease emergence by surveillance of populations exposed to animal reservoirs and interventions to decrease risk factors, such as primate hunting.

Naturally acquired simian retrovirus infections in central African hunters

BACKGROUND Hunting and butchering of wild non-human primates infected with simian immunodeficiency virus (SIV) is thought to have sparked the HIV pandemic. Although SIV and other primate retroviruses infect laboratory workers and zoo workers, zoonotic retrovirus transmission has not been documented in natural settings. We investigated zoonotic infection in individuals living in central Africa. METHODS We obtained behavioural data, plasma samples, and peripheral blood lymphocytes from individuals living in rural villages in Cameroon. We did serological testing, PCR, and sequence analysis to obtain evidence of retrovirus infection. FINDINGS Zoonotic infections with simian foamy virus (SFV), a retrovirus endemic in most Old World primates, were identified in people living in central African forests who reported direct contact with blood and body fluids of wild non-human primates. Ten (1%) of 1099 individuals had antibodies to SFV. Sequence analysis from these individuals revealed three geographically-independent human SFV infections, each of which was acquired from a distinct non-human primate lineage: De Brazzas guenon (Cercopithecus neglectus), mandrill (Mandrillus sphinx), and gorilla (Gorilla gorilla), two of which (De Brazzas guenon and mandrill) are naturally infected with SIV. INTERPRETATION Our findings show that retroviruses are actively crossing into human populations, and demonstrate that people in central Africa are currently infected with SFV. Contact with non-human primates, such as happens during hunting and butchering, can play a part in the emergence of human retroviruses and the reduction of primate bushmeat hunting has the potential to decrease the frequency of disease emergence.

Presence of diverse human immunodeficiency virus type 1 viral variants in Cameroon.

Phylogenetic analysis of the gp41 region of 123 HIV-1-seropositive specimens from Cameroon showed that 89 were subtype A (71% of these sequences were IbNg-like), 12 (10%) were subtype D, 11 (9%) were subtype G, 5 (4%; closely related to subtype F2) were subtype F, 1 was subtype H, 2 (1.6%) remained unclassifiable, while 3 were group O. Further analysis of the two unclassifiable specimens in gag(p24), pol(prot), and env (C2V3 or gp41) showed that one (98CM19) was a complex mosaic between subtype A in p24 and subtype J prot, and unclassifiable in env (C2V3 or gp41). The second, 98CM63, clustered distinctly from all known subtypes in p24, prot, C2V3, or gp41. 98CM63 clustered with a specimen from Cyprus and these two geographically and epidemiologically unlinked specimens, with their distinct clustering pattern, may represent a new subcluster of subtype A. In conclusion, these findings confirm the high HIV-1 genetic variability and further suggest the continuous appearance of new viral strains in this population.

Human Immunodeficiency Virus Type 1 Group M Protease in Cameroon: Genetic Diversity and Protease Inhibitor Mutational Features

ABSTRACT To establish a baseline for monitoring resistance to protease inhibitors (PIs) and examining the efficacy of their use among persons in Cameroon infected with human immunodeficiency virus type 1 (HIV-1), we analyzed genetic variability and PI resistance-associated substitutions in PCR-amplified protease (PR) sequences in strains isolated from 110 HIV-1-infected, drug-naïve Cameroonians. Of the 110 strains, 85 were classified into six HIV-1 PR subtypes, A (n = 1), B (n = 1), F (n = 4), G (n = 7), H (n = 1), and J (n = 7), and a circulating recombinant form, CRF02-AG (n = 64). PR genes from the remaining 25 (23%) specimens were unclassifiable, whereas 2% (7 of 301) unclassifiable PR sequences were reported for a global collection. Two major PI resistance-associated mutations, 20M and 24I, were detected in strains from only two specimens, whereas secondary mutations were found in strains from all samples except one strain of subtype B and two strains of CRF02-AG. The secondary mutations showed the typical PI resistance-associated pattern for non-subtype B viruses in both classifiable and unclassifiable PR genes, with 36I being the predominant (99%) mutation, followed by 63P (18%), 20R (15%), 77I (13%), and 10I or 10V (11%). Of these mutations, dual and triple PI resistance-associated substitutions were found in 38% of all the Cameroonian strains. Compared with classifiable PR sequences, unclassifiable sequences had significantly more dual and triple substitutions (64% versus 30%; P = 0.004). Phenotypic and clinical evaluations are needed to estimate whether PI resistance during antiretroviral drug treatment occurs more rapidly in individuals infected with HIV-1 strains harboring multiple PI resistance-associated substitutions. This information may be important for determination of appropriate drug therapies for HIV-1-infected persons in Cameroon, where more than one-third of HIV-1 strains were found to carry dual and triple minor PI resistance-associated mutations.

Deforestation, hunting and the ecology of microbial emergence

Infectious agents represent a significant risk to humans, their domestic crops and animals, and the planet’s wildlife. The past century was punctuated by the emergence of a variety of infectious diseases. Perhaps most notable of the completely novel diseases emerging into the human population during this period was HIV-1, which will have a serious demographic impact on human populations. While recently emerged and reemerging pathogens has attracted substantial attention during the past decade, an understanding of the factors which influence microbial emergence remains elusive. Understanding the processes by which humans, or other organisms, acquire new diseases has practical implications for maintaining the health of communities as well as fundamental implications for understanding the functioning of ecosystems. Historically, approaches to the study of infectious agents have focused largely on the impact of these agents on morbidity and mortality, or on the development of medicines to treat them. These approaches have yielded significant progress in human and animal medicine. Nevertheless, these approaches have not generally placed infectious agents in the context of ecosystems or examined the general factors contributing to the emergence of such microbes. An ecological framework for examining emerging infectious diseases has a number of potential benefits. Such a framework will help to determine the impact of environmental factors on the changing prevalence of infectious diseases. It may also provide insights into the impact of human activities on the emergence of new disease. More generally, an ecological approach is likely to contribute to the well sought after goal of a predictive science of emerging infectious diseases. The problems of defining an ‘emerging infectious disease’ have been discussed considerably elsewhere. One set of emerging diseases are those that are completely novel, or thought never to have gained a foothold within a given species or population before. The goal of this paper is to examine factors which influence human acquisition of completely novel microbes. We approach this problem from three directions. The first examines the underlying diversity of potentially emerging microbes. The second explores the impact of human activities on contact with this microbial diversity. The final direction investigates the influence of environmental change on the frequency of human contact. To demonstrate how such phenomena might be approached empirically, we present a case study of hunting and deforestation in Cameroon. Among the conclusions drawn from this study is that certain synergies, for example between logging activities, hunting of nonhuman primates, and international travel are likely to increase the frequency at which novel microbes successfully enter into the global human population.

HIV-1 recombinants with multiple parental strains in low-prevalence, remote regions of Cameroon: Evolutionary relics?

BackgroundThe HIV pandemic disseminated globally from Central West Africa, beginning in the second half of the twentieth century. To elucidate the virologic origins of the pandemic, a cross-sectional study was conducted of the genetic diversity of HIV-1 strains in villagers in 14 remote locations in Cameroon and in hospitalized and STI patients. DNA extracted from PBMC was PCR amplified from HIV(+) subjects. Partial pol amplicons (N = 164) and nearly full virus genomes (N = 78) were sequenced. Among the 3956 rural villagers studied, the prevalence of HIV infection was 4.9%; among the hospitalized and clinic patients, it was 8.6%.ResultsVirus genotypes fell into two distinctive groups. A majority of the genotyped strains (109/164) were the circulating recombinant form (CRF) known to be endemic in West Africa and Central West Africa, CRF02_AG. The second most common genetic form (9/164) was the recently described CRF22_01A1, and the rest were a collection of 4 different subtypes (A2, D, F2, G) and 6 different CRFs (-01, -11, -13, -18, -25, -37). Remarkably, 10.4% of HIV-1 genomes detected (17/164) were heretofore undescribed unique recombinant forms (URF) present in only a single person. Nearly full genome sequencing was completed for 78 of the viruses of interest. HIV genetic diversity was commonplace in rural villages: 12 villages each had at least one newly detected URF, and 9 villages had two or more.ConclusionsThese results show that while CRF02_AG dominated the HIV strains in the rural villages, the remainder of the viruses had tremendous genetic diversity. Between the trans-species transmission of SIVcpz and the dispersal of pandemic HIV-1, there was a time when we hypothesize that nascent HIV-1 was spreading, but only to a limited extent, recombining with other local HIV-1, creating a large variety of recombinants. When one of those recombinants began to spread widely (i.e. became epidemic), it was recognized as a subtype. We hypothesize that the viruses in these remote Cameroon villages may represent that pre-epidemic stage of viral evolution.

Monitoring HIV Drug Resistance Early Warning Indicators in Cameroon: A Study Following the Revised World Health Organization Recommendations

Background The majority (>95%) of new HIV infection occurs in resource-limited settings, and Cameroon is still experiencing a generalized epidemic with ~122,638 patients receiving antiretroviral therapy (ART). A detrimental outcome in scaling-up ART is the emergence HIV drug resistance (HIVDR), suggesting the need for pragmatic approaches in sustaining a successful ART performance. Methods A survey was conducted in 15 ART sites of the Centre and Littoral regions of Cameroon in 2013 (10 urban versus 05 rural settings; 8 at tertiary/secondary versus 7 at primary healthcare levels), evaluating HIVDR-early warning indicators (EWIs) as-per the 2012 revised World Health Organization’s guidelines: EWI1 (on-time pill pick-up), EWI2 (retention in care), EWI3 (no pharmacy stock-outs), EWI4 (dispensing practices), EWI5 (virological suppression). Poor performance was interpreted as potential HIVDR. Results Only 33.3% (4/12) of sites reached the desirable performance for “on-time pill pick-up” (57.1% urban versus 0% rural; p<0.0001) besides 25% (3/12) with fair performance. 69.2% (9/13) reached the desirable performance for “retention in care” (77.8% urban versus 50% rural; p=0.01) beside 7.7% (1/13) with fair performance. Only 14.4% (2/13) reached the desirable performance of “no pharmacy stock-outs” (11.1% urban versus 25% rural; p=0.02). All 15 sites reached the desirable performance of 0% “dispensing mono- or dual-therapy”. Data were unavailable to evaluate “virological suppression” due to limited access to viral load testing (min-max: <1%-15%). Potential HIVDR was higher in rural (57.9%) compared to urban (27.8%) settings, p=0.02; and at primary (57.9%) compared to secondary/tertiary (33.3%) healthcare levels, p=0.09. Conclusions Delayed pill pick-up and pharmacy stock-outs are major factors favoring HIVDR emergence, with higher risks in rural settings and at primary healthcare. Retention in care appears acceptable in general while ART dispensing practices are standard. There is need to support patient-adherence to pharmacy appointments while reinforcing the national drug supply system.

Characterization of drug resistance mutations in naïve and ART-treated patients infected with HIV-1 in Yaounde, Cameroon.

Currently the prevalence of HIV‐1 infection in Cameroon is 5.1%, CRF02_AG subtype is responsible for about 50% of infections. Since an HIV‐1 drug resistance test is not yet available widely, accurate data on the prevalence of resistant viral strains are missing. The objective of this study was to determine HIV‐1 genetic diversity and to characterize HIV‐1 mutations conferring drug resistance among antiretroviral therapy (ART)‐naïve and ART‐treated patients. A cohort of 239 patients infected with HIV were followed‐up between January 2007 and July 2010 in Cameroon. Two hundred and sixteen plasma samples were sequenced for phylogenetic analysis and identification of drug resistance mutations in the HIV‐1 pol region. A significant genetic diversity was found: Seven pure subtypes (A1, A3, D, F1, F2, G, H), nine circulating recombinant forms (CRFs: 01_AE, 02_AG, 06cpx, 09cpx, 11cpx, 13cpx, 16cpx, 18cpx, 37cpx) and one new unique recombinant form (URF) (G/F2). The rate of transmitted drug resistance (TDR) in naïve patients was 8.2% (4/49). Around 80% of patients failing a first‐line ART harbored a virus with at least one resistance mutation to two antiretroviral (ARV) classes, and 36% of those failing a second‐line regimen carried a virus with at least one resistant mutation to three ARV classes. The high level of drug resistance observed in the cohort is alarming because this occurred as a result of only few years of treatment. Adherence to therapy, adequate education of physicians, and the appropriate use of genotypic resistance assay are critical points of intervention for the improvement of patient care. J. Med. Virol. 84:721–727, 2012.

Effects of Antioxidants on CD4 and Viral Load in HIV-Infected Women in Sub-Saharan Africa - Dietary Supplements vs. Local Diet

In sub-Sahara Africa, micronutrient deficiency, especially of antioxidant micronutrients including vitamins A, C, and E, beta-carotene, selenium, zinc, and polyphenols is very common in HIV-positive patients. Amongst adults, women are the most vulnerable. Antioxidants are known to play a vital role in the immune system, reducing oxidative stress. Oxidative stress is induced by excess production of reactive oxygen species (ROS), due to the HIV infection. Such damage may be prevented or moderated through adequate oral intake of antioxidants, scavenging ROS, as well as protecting cells and tissues against oxidative stress. Antioxidants can be provided to the body through locally available antioxidant rich-diets such as fruit-and-vegetable-based diets and/or dietary supplements. Provision of antioxidants through local diets or dietary supplements exercise beneficial effects on biological markers of the immune system (CD4 and viral load). However, while dietary supplements represent a costly and short-term strategy to limiting antioxidant deficiency, local diets, combined with adequate nutritional education, can provide a low-cost and long-term strategy to reduce oxidative stress, prevent micronutrient deficiency, and slow down HIV disease progression. The former can be applicable in countries around the West, Central, and South coast of Africa, which are rich in natural food resources. In contrast with significant evidence that dietary supplements confer benefits in HIV patients, fewer data are available relating to the benefits of local diets. Thus the need to do more research in this area arises. This review compares available data on effects of antioxidants on CD4 and viral load in HIV-positive women noneligible for antiretroviral therapy. Intake of antioxidants though dietary supplements and local diet, associated with nutritional education, is compared. Studies conducted in sub-Sahara Africa are considered.

Reference Values of Lymphocyte Subsets in Healthy, HIV-Negative Children in Cameroon

ABSTRACT Lymphocyte subset reference values used to monitor infectious diseases, including HIV/AIDS, tuberculosis, malaria, or other immunological disorders in healthy children in Cameroon, are lacking. Values for Caucasian cohorts are already being utilized for clinical decisions but could be inappropriate for African populations. We report here the immunological profile for children aged from birth through 6 years in Cameroon and also compare our values to data from other African and Caucasian populations. In a cohort of 352 healthy children, aged 0 to 6 years, the relative and absolute numbers of T-cell subsets, B cells, and NK lymphocytes were determined from peripheral blood collected in EDTA tubes. Samples were stained with BD Multitest reagents in Trucount tubes and analyzed by using CellQuest-Pro and FlowJo software. We evaluated about 23 different lymphocyte subsets in which the absolute number and percentage values differed significantly (P < 0.05) with age and peaked between 6 and 12 months. B-cell values were higher compared to reported values from developed countries. Differences in activated and differentiated T cells were observed in subjects between 1 and 6 years of age. The absolute CD8+ T-cell count and the CD4+/CD8+ ratio seem to depend on gender. Normal lymphocyte subsets values among children from Cameroon differ from reported values in Caucasian and some African populations. The differences observed could be due to genetic and environmental factors coupled with the methodology used. These values could be used as initial national reference guidelines as more data are assembled.