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.

Evaluation of hepatitis C virus antibody assay using dried blood spot samples.

Early diagnosis of hepatitis C virus (HCV) infection is essential for prompt initiation of treatment and prevention of transmission, yet several logistical barriers continue to limit access to HCV testing. Dried blood spot (DBS) technology involves a simple fingerstick that eliminates the need for trained personnel, and DBS can be stored and transported at room temperature. We evaluated the use of DBS whole blood samples in the modified Abbott ARCHITECT anti-HCV assay, comparing assay performance against the standard assay run using DBS and venous plasma samples. 144 HCV positive and 104 HCV negative matched venous plasma and whole blood specimens were selected from a retrospective study with convenience sampling in Cameroon. Results obtained using a modified volume DBS assay were highly correlated to the results of the standard assay run with plasma on clinical samples and dilution series (R2 = 0.71 and 0.99 respectively). The ARCHITECT Anti-HCV assay with input volume modification more accurately detects HCV antibodies in DBS whole blood samples with 100% sensitivity and specificity, while the standard assay had 90.97% sensitivity. The use of DBS has the potential to expand access to HCV testing to underserved or marginalized populations with limited access to direct HCV care.

A high prevalence of potential HIV elite controllers identified over 30 years in Democratic Republic of Congo.

BACKGROUND: In-depth analysis of the HIV pandemic at its epicenter in the Congo basin has been hampered by 40 years of political unrest and lack of functional public health infrastructure. In recent surveillance studies (2017-18), we found that the prevalence of HIV in Kinshasa, Democratic Republic of Congo (11%) far exceeded previous estimates. METHODS: 10,457 participants were screened in Kinshasa with rapid tests from 2017-2019. Individuals confirmed as reactive by the Abbott ARCHITECT HIV Ag/Ab Combo assay (n=1968) were measured by the Abbott RealTime HIV-1 viral load assay. Follow up characterization of samples was performed with alternate manufacturer viral load assays, qPCR for additional blood borne viruses, unbiased next generation sequencing, and HIV Western blotting. FINDINGS: Our data suggested the existence of a significant cohort (n=429) of HIV antibody positive/viral load negative individuals. We systematically eliminated collection site bias, sample integrity, and viral genetic diversity as alternative explanations for undetectable viral loads. Mass spectroscopy unexpectedly detected the presence of 3TC antiviral medication in approximately 60% of those tested (209/354), and negative Western blot results indicated false positive serology in 12% (49/404). From the remaining Western blot positives (n=53) and indeterminates (n=31) with reactive Combo and rapid test results, we estimate 2.7-4.3% of infections in DRC to be potential elite controllers. We also analyzed samples from the DRC collected in 1987 and 2001-03, when antiretroviral drugs were not available, and found similarly elevated trends. INTERPRETATION: Viral suppression to undetectable viral loads without therapy occurs infrequently in HIV-1 infected patients around the world. Mining of global data suggests a unique ability to control HIV infection arose early in central Africa and occurs in <1% of founder populations. Identification of this group of elite controllers presents a unique opportunity to study potentially novel genetic mechanisms of viral suppression. FUNDING: Abbott Laboratories funded surveillance in DRC and subsequent research efforts. Additional funding was received from a MIZZOU Award from the University of Missouri. Research was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH.

New Genomes from the Congo Basin Expand History of CRF01_AE Origin and Dissemination.

Although the first HIV circulating recombinant form (CRF01_AE) is the predominant strain in many Asian countries, it is uncommonly found in the Congo Basin from where it first originated. To fill the gap in the evolutionary history of this important strain, we sequenced near complete genomes from HIV samples with subgenomic CRF01_AE regions collected in Cameroon and the Democratic Republic of the Congo from 2001 to 2006. HIV genomes were generated from N = 13 plasma specimens by next-generation sequencing of metagenomic libraries prepared with spiked primers targeting HIV, followed by Sanger gap-filling. Genome sequences were aligned to reference strains, including Asian and African CRF01_AE sequences, and evaluated by phylogenetic and recombinant analysis to identify four CRF01_AE strains from Cameroon. We also identified two CRF02, one CRF27, and six unique recombinant form genomes (01|A1|G, 01|02|F|U, F|G|01, A1|D|01, F|G|01, and A1|G|01). Phylogenetic analysis, including the four new African CRF01_AE genomes, placed these samples as a bridge between basal Central African Republic CRF01_AE strains and all Asian, European, and American CRF01_AE strains. Molecular dating confirmed previous estimates indicating that the most recent common CRF01_AE ancestor emerged in the early 1970s (1968-1970) and spread beyond Africa around 1980 to Asia. The new sequences and analysis presented in this study expand the molecular history of the CRF01_AE clade, and are illustrated in an interactive Next Strain phylogenetic tree, map, and timeline at (https://nextstrain.org/community/EduanWilkinson/hiv-1_crf01).

Advanced molecular surveillance approaches for characterization of blood borne hepatitis viruses.

Defining genetic diversity of viral infections directly from patient specimens is the ultimate goal of surveillance. Simple tools that can provide full-length sequence information on blood borne viral hepatitis viruses: hepatitis C, hepatitis B and hepatitis D viruses (HCV, HBV and HDV) remain elusive. Here, an unbiased metagenomic next generation sequencing approach (mNGS) was used for molecular characterization of HCV infections (n = 99) from Israel which yielded full-length HCV sequences in 89% of samples, with 7 partial sequences sufficient for classification. HCV genotypes were primarily 1b (68%) and 1a (19%), with minor representation of genotypes 2c (1%) and 3a (8%). HBV/HDV coinfections were characterized by suppressed HBV viral loads, resulting in sparse mNGS coverage. A probe-based enrichment approach (xGen) aiming to increase HBV and HDV coverage was validated on a panel of diverse genotypes, geography and titers. The method extended HBV genome coverage a median 61% (range 8-84%) and provided orders of magnitude boosts in reads and sequence depth for both viruses. When HBV-xGen was applied to Israeli samples, coverage was improved by 28-73% in 4 samples and identified HBV genotype A1, A2, D1 specimens and a dual B/D infection. Abundant HDV reads in mNGS libraries yielded 18/26 (69%) full genomes and 8 partial sequences, with HDV-xGen only providing minimal extension (3-11%) of what were all genotype 1 genomes. Advanced molecular approaches coupled to virus-specific capture probes promise to enhance surveillance of viral infections and aid in monitoring the spread of local subtypes.

Author Correction: Development and performance of prototype serologic and molecular tests for hepatitis delta infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.

Molecular characterization of human immunodeficiency virus type 1 (HIV-1) and HIV-2 in Yaounde, Cameroon: evidence of major drug resistance mutations in newly diagnosed patients infected with subtypes other than subtype B.

Prior to current studies on the emergence of drug resistance with the introduction of antiretroviral therapy (ART) in Cameroon, we performed genotypic analysis on samples from drug-naïve, human immunodeficiency virus (HIV)-infected individuals in this country. Of the 79 HIV type 1 (HIV-1) pol sequences analyzed from Cameroonian samples, 3 (3.8%) were identified as HIV-1 group O, 1 (1.2%) was identified as an HIV-2 intergroup B/A recombinant, and the remaining 75 (95.0%) were identified as HIV-1 group M. Group M isolates were further classified as subtypes A1 (n = 4), D (n = 4), F2 (n = 6), G (n = 12), H (n = 2), and K (n = 1) and as circulating recombinant forms CRF02_AG (n = 41), CRF11_cpx (n = 1), and CRF13_cpx (n = 2). Two pol sequences were identified as unique recombinant forms of CRF02_AG/F2 (n = 2). M46L (n = 2), a major resistance mutation associated with resistance to protease inhibitors, was observed in 2/75 (2.6%) group M samples. Single mutations associated with resistance to nucleoside reverse transcriptase inhibitors (T215Y/F [n = 3]) and nonnucleoside reverse transcriptase inhibitors (V108I [n = 1], L100I [n = 1], and Y181C [n = 2]) were observed in 7 of 75 (9.3%) group M samples. None of the patients had any history of ART exposure. Population surveillance of transmitted HIV drug resistance is required and should be included to aid in the development of appropriate guidelines.

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.

ARCHITECT HIV Combo Ag/Ab and RealTime HIV-1 Assays Detect Diverse HIV Strains in Clinical Specimens.

Periodic evaluation of the impact of viral diversity on diagnostic tests is critical to ensure current technologies are keeping pace with viral evolution. To determine whether HIV diversity impacts the ARCHITECT HIV Combo Ag/Ab (HIV Combo) or RealTime HIV-1 (RT) assays, a set of N = 199 HIV clinical specimens from Cameroon, Senegal, Saudi Arabia, and Thailand were sequenced and tested in both assays. The panel included historical groups N and P specimens and a newly identified group N specimen. These and specimens classified as H, U (unclassified)/URF (unique recombinant form), CRF (circulating recombinant form) 01, 02, 06, 09, 11, 13, 18, 22, 37, and 43 were detected by both the RT assay (1.75-6.84 log copies/ml) and the HIV Combo assay (3.26-1121.96 sample to cutoff ratios). Sequence alignment identified 3 or fewer mismatches to the RT assay oligos in 82.4% of samples. Altogether, these data demonstrate the HIV Combo and RT assays detect diverse strains of HIV in clinical specimens.

Universal Target Capture of HIV Sequences From NGS Libraries.

Background: Global surveillance of viral sequence diversity is needed to keep pace with the constant evolution of HIV. Recent next generation sequencing (NGS) methods have realized the goal of sequencing circulating virus directly from patient specimens. Yet, a simple, universal approach that maximizes sensitivity and sequencing capacity remains elusive. Here we present a novel HIV enrichment strategy to yield near complete genomes from low viral load specimens. Methodology: A non-redundant biotin-labeled probe set (HIV-xGen; n = 652) was synthesized to tile all HIV-1 (groups M, N, O, and P) and HIV-2 (A and B) strains. Illumina Nextera barcoded libraries of either gene-specific or randomly primed cDNA derived from infected plasma were hybridized to probes in a single pool and unbound sequences were washed away. Captured viral cDNA was amplified by Illumina adaptor primers, sequenced on a MiSeq, and NGS reads were demultiplexed for alignment with CLC Bio software. Results: HIV-xGen probes selectively captured and amplified reads spanning the entirety of the HIV phylogenetic tree. HIV sequences clearly present in unenriched libraries of specimens but previously not observed due to high host background levels, insufficient sequencing depth or the extent of multiplexing, were now enriched by >1,000-fold. Thus, xGen selection not only substantially increased the depth of existing sequence, but also extended overall genome coverage by an average of 40%. We characterized 50 new, diverse HIV strains from clinical specimens and demonstrated a viral load cutoff of approximately log 3.5 copies/ml for full length coverage. Genome coverage was <20% for 5/10 samples with viral loads 90% for 35/40 samples with higher viral loads. Conclusions: Characterization of >20 complete genomes at a time is now possible from a single probe hybridization and MiSeq run. With the versatility to capture all HIV strains and the sensitivity to detect low titer specimens, HIV-xGen will serve as an important tool for monitoring HIV sequence diversity.

High prevalence of hepatitis delta virus in Cameroon.

Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), infects an estimated 15-20 million people worldwide and confers a greater risk for accelerated progression to liver disease. However, limited HDV surveillance data are available in sub-Saharan Africa where HDV diversity is high. To determine the prevalence and diversity of HDV in Cameroon, serological and molecular characterization was performed on 1928 HBsAg positive specimens selected from retrospective viral surveillance studies conducted in Cameroon from 2010-2016. Samples were screened for HDV antibodies on the Abbott ARCHITECT instrument and for HDV RNA on the Abbott m2000 instrument by research assays. HDV positive specimens with sufficient viral load were selected for genomic sequencing. The seroprevalence of HDV in HBsAg positive samples from Cameroon was 46.73% [95% CI; 44.51-48.96%], with prevalence of active HDV infection being 34.2% [95% CI; 32.09-36.41%]. HDV genotypes 1, 6, 7 and 8 were identified amongst N = 211 sequences, including N = 145 genomes. HDV prevalence is high within the study cohort, indicating that a large portion of HBV infected individuals in Cameroon are at elevated risk for severe hepatitis and death. Collectively, these results emphasize the need for HBV vaccination and HDV testing in HBsAg positive patients in Cameroon.

Development and performance of prototype serologic and molecular tests for hepatitis delta infection.

Worldwide, an estimated 5% of hepatitis B virus (HBV) infected people are coinfected with hepatitis delta virus (HDV). HDV infection leads to increased mortality over HBV mono-infection, yet HDV diagnostics are not widely available. Prototype molecular (RNA) and serologic (IgG) assays were developed for high-throughput testing on the Abbott m2000 and ARCHITECT systems, respectively. RNA detection was achieved through amplification of a ribozyme region target, with a limit of detection of 5 IU/ml. The prototype serology assay (IgG) was developed using peptides derived from HDV large antigen (HDAg), and linear epitopes were further identified by peptide scan. Specificity of an HBV negative population was 100% for both assays. A panel of 145 HBsAg positive samples from Cameroon with unknown HDV status was tested using both assays: 16 (11.0%) had detectable HDV RNA, and 23 (15.7%) were sero-positive including the 16 HDV RNA positive samples. Additionally, an archival serial bleed panel from an HDV superinfected chimpanzee was tested with both prototypes; data was consistent with historic testing data using a commercial total anti-Delta test. Overall, the two prototype assays provide sensitive and specific methods for HDV detection using high throughput automated platforms, allowing opportunity for improved diagnosis of HDV infected patients.

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.

Monitoring antiretroviral therapy in HIV/AIDS patients in resource-limited settings: CD4 counts or total lymphocyte counts?

OBJECTIVE: In order to improve the monitoring of disease progression and therapeutic effectiveness in the management of HIV/AIDS in a resource-limited setting, this study was carried out to establish a correlation between total lymphocyte counts (TLC) and CD4 lymphocyte counts in HIV-1 infected/AIDS adults in Yaoundé, Cameroon. METHODS: Full blood counts, differential white, and CD4 counts were measured in 149 patients using standard methods. The correlation coefficient established correlation between values. Sensitivity, specificity, and positive predictive values were calculated as required. RESULTS: The mean TLC, CD4 count, and CD4% as well as CD4/CD8 ratios were 1.932+/-0.895 x 10(9)/L, 268+/-183 cells/mm(3), 14.51+/-15.9%, and 0.34+/-0.25, respectively. Only a weak correlation was observed between TLC and CD4 counts (r=0.41, p=0.05). As a predictor of CD4 count, TLC cut-offs <2.0 and <1.0 x 10(9)/L were unable to predict these values reliably, but showed that at TLC cut-offs of <1.0 x 10(9)/L there was a high chance of CD4 counts being under 200 cells/mm(3). CONCLUSIONS: These data suggest that TLC are of limited value in predicting CD4 counts and should not be substituted for CD4 counts whenever possible. However, TLC may be reliably used in designing algorithms and programs for initiating patient management and follow-up in this setting.

Assessing the prevalence of urogenital schistosomaisis and transmission risk factors amongst school-aged children around Mapé dam ecological suburbs in Malantouen district, Cameroon.

BACKGROUND: Urogenital schistosomiasis is a parasitic infection of public health importance that affects over 112 million people worldwide. The study aimed at assessing the urogenital schistosomiasis prevalence and risk factors of transmission around Mape dam suburds in Malantouen district, West, Cameroon. METHODS: The study was conducted using semi-structured pretested questionnaires to collect socio-demographic and ecological data. Urine samples were also collected and used to confirm the prevalence of schistosomiasis in consented school-aged children in four primary schools between March - July 2014. Snails' samples around the dam surburbs were also collected for taxonomy characterization and species identification. Data were compiled and quality control assessed and analysed using SPSS version 17 and Epiinfo data 3.1. P < 0.05 was considered statistical significance. RESULTS: Questionnaires were administered to 229 pupils, with gender ratio of 1.04 (m/f). The prevalence of schistosomiasis haematobium was 16.6%. Mambonko school site, which is the closest to the dam suburbs, registered the greatest prevalence rate of 40%. The age group beween 10-13 years was the most infected (18.3%) and boys were more infested than girls (21.0% vs. 15.5%). Haematuria, urination pain, school absentiesm and poor performance were the major recorded complications in 39.5 and 26.3% males to female respectively. Infection rate gender disparity documented is still poorly understood and Bulinus truncatus collected from Mambonko suburb as potential snail intermediate host requires further studies. CONCLUSIONS: Authors advocated that schools and dam suburds sustained and innovative community-based surveillance and response targeted interventions implementation are needed to inform and support decision-making policy, but also in improving effective contextual behavioural communication changes and MDA improved uptake measures on national schistosomiasis control and elimination in Cameroon.

Functional domains of the human immunodeficiency virus type 1 Nef protein are conserved among different clades in Cameroon.

The Nef protein of human immunodeficiency virus type 1 (HIV-1) has multiple functional domains, is immunogenic, and contains several cytotoxic T lymphocyte (CTL)-targeted epitopes. Several defined subfunctions of Nef are important for the pathogenesis of HIV-1 infection. In this study, we present the genetic diversity of the nef gene of 55 newly derived HIV-1 sequences obtained from Cameroonian patients. Four genetic subtypes and three circulating recombinant forms (CRFs) were identified: subtypes A (11%), G (7.3%), D (5.4%), F1 (1.8%), F2 (5.4%), CRF01_AE (5.4%), CRF02_AG (58.2%), and CRF11_cpx (1.8%). Two isolates clustered distinctly from the known HIV-1 genetic subtypes in nef and were designated as unclassified. Interestingly, the majority of all functional domains including the myristoylation signal, CD4 binding motif, beta turn motif, and the phosphorylation sites were well conserved in our cohort. Putative CTL-epitopic domains of the central portion of Nef were also well conserved, whereas those at the C-term were not. Our study demonstrated that despite high genetic diversity observed in the nef gene, most described functional domains and CTL epitopes were well conserved among Cameroonian HIV-1 subtypes. These findings could be used for the development of antiretroviral-acting therapeutics and anti-HIV-1 vaccines.

HIV genetic diversity in Cameroon: possible public health importance.

To monitor the evolving molecular epidemiology and genetic diversity of HIV in a country where many distinct strains cocirculate, we performed genetic analyses on sequences from 75 HIV-1-infected Cameroonians: 74 were group M and 1 was group O. Of the group M sequences, 74 were classified into the following env gp41 subtypes or recombinant forms: CRF02 (n = 54), CRF09 (n = 2), CRF13 (n = 2), A (n = 5), CRF11 (n = 4), CRF06 (n = 1), G (n = 2), F2 (n = 2), and E (n = 1, CRF01), and 1 was a JG recombinant. Comparison of phylogenies for 70 matched gp41 and protease sequences showed inconsistent classifications for 18 (26%) strains. Our data show that recombination is rampant in Cameroon with recombinant viruses continuing to recombine, adding to the complexity of circulating HIV strains. This expanding genetic diversity raises public health concerns for the ability of diagnostic assays to detect these unique HIV mosaic variants and for the development of broadly effective HIV vaccines.

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.