Purifying selection decreases the potential for Bangui orthobunyavirus outbreaks in humans.

Pathogens carried by insects, such as bunyaviruses, are frequently transmitted into human populations and cause diseases. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling the detection of any pathogen from clinical specimens. mNGS was performed on blood samples to identify potential viral coinfections in human immunodeficiency virus (HIV)-positive individuals from Kinshasa, the Democratic Republic of the Congo (DRC), participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from a hepatitis B virus-positive patient. A quantitative polymerase chain reaction assay was designed and used to screen >2,500 plasma samples from Cameroon, the DRC, and Uganda, failing to identify any additional cases. The recent sequencing of a US Center for Disease Control Arbovirus Reference Collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest that this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS.

HPLC-MS identification and expression of Candida drug-resistance proteins from African HIV-infected patients.

The objective of this study was to elucidate the proteomic mechanisms of drug resistance in HIV-infected African patients. Cell membrane fractions from forty oral Candida isolates isolated from African HIV-positive patients were analysed using HPLC-MS with the aim of identifying proteins associated with their pathogenicity and drug resistance. Heat shock proteins that mediate the fungicidal activity of salivary peptides were found in all tested Candida fractions, with pH-responsive proteins associated with increased pathogenicity only being present in the three most commonly isolated species. ABC multidrug transporter efflux pumps and estrogen binding proteins were only found in C. albicans fractions, while ergosterol biosynthesis proteins were identified in four species. The combination of various adherence, invasion, upregulation and efflux pump mechanisms appear to be instrumental for the Candida host colonization and drug resistance emergence in HIV-infected individuals.

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.

Increased HIV in Greater Kinshasa Urban Health Zones: Democratic Republic of Congo (2017-2018).

BACKGROUND: Diagnosis of people living with HIV (PLHIV) is the first step toward achieving the new Fast Track Strategy to end AIDS by 2030: 95-95-95. However, reaching PLHIV is especially difficult in resource-limited settings such as the Democratic Republic of Congo (DRC), where reliable prevalence data is lacking. This study evaluated the prevalence of HIV in patients in the urban Kinshasa area. METHODS: Individuals seeking healthcare were tested for HIV between February 2017 and July 2018 at existing Kinshasa urban clinics. The study was conducted in two phases. Case finding was optimized in a pilot study phase using a modified cell phone-based Open\Data Kit (ODK) collection system. HIV prevalence was then determined from data obtained between March-July of 2018 from 8320 individuals over the age of 18 years receiving care at one of 47 clinics in Kinshasa. RESULTS: The prevalence of HIV in our study was 11.0% (95% CI 10.3-11.6%) overall and 8.14% in the subset of N = 1240 participants who were healthy mothers seeking prenatal care. These results are in sharp contrast to President's Emergency Plan for AIDS Relief (PEPFAR) estimates of 2.86%, but are consistent with data from surrounding countries. CONCLUSION: While this data is sub-national and reflects an urban healthcare setting, given the large population of Kinshasa and rapidly changing age demographics, the results suggest that HIV prevalence in the DRC is substantially higher than previously reported.

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).

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.

Proteomic Profiling of Exosomes Derived from Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers.

Abuse of alcohol and tobacco could exacerbate HIV pathogenesis by transferring materials through exosomes (small nanovesicles). Exosomes present a stable and accessible source of information concerning the health and/or disease status of patients, which can provide diagnostic and prognostic biomarkers for myriad conditions. Therefore, we aimed to study the specific exosomal proteins that are altered in both HIV-infected subjects and alcohol/tobacco users. Exosomes were isolated from plasma of the following subjects: a) HIV-negative subjects (healthy), b) HIV-positive subjects (HIV), c) HIV-negative alcohol drinkers (drinkers), d) HIV-negative tobacco smokers (smokers), e) HIV-positive drinkers (HIV + drinkers), and f) HIV-positive smokers (HIV + smokers). Quantitative proteomic profiling was then performed from these exosomes. Sixteen proteins were significantly altered in the HIV group, ten in drinkers, four in HIV + drinkers, and fifteen in smokers compared to healthy subjects. Only one protein, fibulin-1 (FBLN1), was significantly altered in HIV + smokers. Interestingly, hemopexin was not significantly altered in drinkers or HIV patients but was significantly altered in HIV + drinkers. Further, our study is the first to show properdin expression in plasma exosomes, which was decreased in HIV + smokers and HIV + drinkers compared to HIV patients. The present findings suggest that hemopexin and properdin show potential as markers for physiological effects that may arise in HIV-infected individuals who abuse alcohol and tobacco. Graphical abstract This study presents a proteomic analysis of plasma-derived exosomes from HIV-infected alcohol drinkers and smokers. Among the proteins altered due to drug-abuse, hemopexin and properdin were of highest significance. These proteins can be potential biomarkers for co-morbid conditions associated with drug abuse in HIV-patients.

Brief Report: Complete Genome Sequence of CG-0018a-01 Establishes HIV-1 Subtype L.

BACKGROUND: The full spectrum of HIV-1 diversity can be found in Central Africa, including 2 divergent HIV-1 strains collected in 1983 and 1990 in Democratic Republic of Congo (DRC) that were preliminarily classified as group M subtype L. However, a third epidemiologically distinct subtype L genome must be identified to designate L as a true subtype. METHODS: Specimen CG-0018a-01 was collected in 2001 in DRC as part of an HIV diversity study. Previous subgenomic HIV-1 sequences from this specimen branched closely with proposed subtype L references. Metagenomic next-generation sequencing (mNGS) and HIV-specific target-enriched (HIV-xGen) libraries were combined for NGS to extend genome coverage. mNGS reads were analyzed for the presence of other coinfections with the sequence-based ultrarapid pathogen identification bioinformatics pipeline. RESULTS: A complete HIV-1 genome was generated with an average coverage depth of 47,783×. After bioinformatic analysis also identified hepatitis B virus reads, a complete hepatitis B virus genotype A genome was assembled with an average coverage depth of 73,830×. The CG-0018a-01 HIV-1 genome branched basal to the 2 previous putative subtype L strains with strong bootstrap support of 100. With no evidence of recombination present, the strain was classified as subtype L. CONCLUSIONS: The CG-0018a-01 HIV-1 genome establishes subtype L and confirms ongoing transmission in DRC as recently as 2001. Since CG-0018a-01 is more closely related to an ancestral strain than to isolates from 1983 to 1990, additional strains are likely circulating in DRC and possibly elsewhere.

HIV Capsid Inhibitors Beyond PF74.

Human immunodeficiency virus (HIV) capsid plays important roles at multiple stages of viral replication. At the initial stages, controlled uncoating (disassembly) of the capsid ensures efficient reverse transcription of the single-stranded RNA genome, into the double-stranded DNA. Whereas at later stages, a proper assembly of capsid ensures the formation of a mature infectious virus particle. Hence, the inhibition of capsid assembly and/or disassembly has been recognized as a potential therapeutic strategy, and several capsid inhibitors have been reported. Of these, PF-3450074 (PF74) has been extensively studied. Recently reported GS-CA inhibitors (GS-CA1 and GS-6207), have shown a strong potential and appear to contain a PF74 scaffold. The location of resistance mutations and the results of structural studies further suggest that GS-CA compounds and PF74 share the same binding pocket, which is located between capsid monomers. Additionally, phenylalanine derivatives containing the PF74 scaffold show slightly enhanced capsid inhibiting activity. A comparison of capsid structures in complex with host factors and PF74, reveals the presence of common chemical entities at topologically equivalent positions. Here we present the status of capsid inhibitors that contain PF74 scaffolds and propose that the PF74 scaffold may be used to develop strong and safe capsid inhibitors.

Contribution of HIV Infection, AIDS, and Antiretroviral Therapy to Exocrine Pathogenesis in Salivary and Lacrimal Glands.

The structure and function of exocrine glands are negatively affected by human immunodeficiency virus (HIV) infection and its co-morbidities, including innate and adaptive immune responses. At the same time, exocrine function may also be influenced by pharmacotherapies directed at the infectious agents. Here, we briefly review the role of the salivary glands and lacrimal glands in normal physiology and exocrine pathogenesis within the context of HIV infection and acquired immune deficiency syndrome (AIDS), including the contribution of antiretroviral therapies on both. Subsequently, we discuss the impact of HIV infection and the types of antiretroviral therapy on disease management and therapy development efforts.

Cytokine profiling of exosomes derived from the plasma of HIV-infected alcohol drinkers and cigarette smokers.

Cytokines and chemokines circulate in plasma and may be transferred to distant sites, via exosomes. HIV infection is associated with dysregulation of cytokines and chemokines, which subsequently contribute to the pathogenesis of HIV. Alcohol and tobacco exposure, which are prevalent in HIV-infected individuals, may induce changes in the expression of cytokines and chemokines. Therefore, our aim in this study was to quantify plasma exosomal cytokines and chemokines that we expect to exacerbate toxicity or disease progression in HIV-positive drug abusers. We measured the levels of cytokines and chemokines in the plasma and plasma exosomes of 39 patients comprising six groups: HIV-negative and HIV-positive non drug abusers, HIV-negative and HIV-positive alcohol users, and HIV-negative and HIV positive tobacco smokers. We measured six cytokines (TNF-α, IL-1ß, IL-8, IL-6, IL-1ra, IL-10) and two chemokines (MCP-1 and RANTES). All were present in exosomes of healthy subjects, but their levels varied between different study groups. HIV-positive alcohol drinkers had higher levels of plasma IL-8 compared to those of HIV-positive non-drinkers. The IL-1ra level was significantly higher in exosomes of non-HIV-infected alcohol drinkers compared to those of HIV-positive alcohol drinkers. Interestingly, the IL-10 level was higher in exosomes compared with their respective plasma levels in all study groups except HIV-positive non-alcohol drinkers. IL-10 was completely packaged in exosomes of HIV-positive smokers. HIV-positive smokers had significantly higher levels of plasma IL-8 compared with HIV-positive non-smokers and significantly higher exosomal IL-6 levels compared with HIV-negative subjects. HIV-positive smokers had significantly increased plasma levels of IL-1ra compared to HIV-positive non-smokers. The MCP-1 levels in the plasma of HIV-positive smokers was significantly higher than in either HIV-positive non-drug abusers or HIV-negative smokers. Overall, the findings suggest that plasma cytokines and chemokines are packaged in exosomes at varying degrees in different study groups. Exosomal cytokines and chemokines are likely to have a significant biological role at distant sites including cells in the brain.

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.

Malaria over-diagnosis in Cameroon: diagnostic accuracy of Fluorescence and Staining Technologies (FAST) Malaria Stain and LED microscopy versus Giemsa and bright field microscopy validated by polymerase chain reaction.

BACKGROUND: Malaria is a major world health issue and its continued burden is due, in part, to difficulties in the diagnosis of the illness. The World Health Organization recommends confirmatory testing using microscopy-based techniques or rapid diagnostic tests (RDT) for all cases of suspected malaria. In regions where Plasmodium species are indigenous, there are multiple etiologies of fever leading to misdiagnoses, especially in populations where HIV is prevalent and children. To determine the frequency of malaria infection in febrile patients over an 8-month period at the Regional Hospital in Bamenda, Cameroon, we evaluated the clinical efficacy of the Flourescence and Staining Technology (FAST) Malaria stain and ParaLens AdvanceTM microscopy system (FM) and compared it with conventional bright field microscopy and Giemsa stain (GS). METHODS: Peripheral blood samples from 522 patients with a clinical diagnosis of "suspected malaria" were evaluated using GS and FM methods. A nested PCR assay was the gold standard to compare the two methods. PCR positivity, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined. RESULTS: Four hundred ninety nine samples were included in the final analysis. Of these, 30 were positive via PCR (6.01%) with a mean PPV of 19.62% and 27.99% for GS and FM, respectively. The mean NPV was 95.01% and 95.28% for GS and FM, respectively. Sensitivity was 26.67% in both groups and specificity was 92.78% and 96.21% for GS and FM, respectively. An increased level of diagnostic discrepancy was observed between technicians based upon skill level using GS, which was not seen with FM. CONCLUSIONS: The frequency of malarial infections confirmed via PCR among patients presenting with fever and other symptoms of malaria was dramatically lower than that anticipated based upon physicians' clinical suspicions. A correlation between technician skill and accuracy of malaria diagnosis using GS was observed that was less pronounced using FM. Additionally, FM increased the specificity and improved the PPV, suggesting this relatively low cost approach could be useful in resource-limited environments. Anecdotally, physicians were reluctant to not treat all patients symptomatically before results were known and in spite of a negative microscopic diagnosis, highlighting the need for further physician education to avoid this practice of overtreatment. A larger study in an area with a known high prevalence is being planned to compare the two microscopy methods against available RDTs.

Sensitive Next-Generation Sequencing Method Reveals Deep Genetic Diversity of HIV-1 in the Democratic Republic of the Congo.

As the epidemiological epicenter of the human immunodeficiency virus (HIV) pandemic, the Democratic Republic of the Congo (DRC) is a reservoir of circulating HIV strains exhibiting high levels of diversity and recombination. In this study, we characterized HIV specimens collected in two rural areas of the DRC between 2001 and 2003 to identify rare strains of HIV. The env gp41 region was sequenced and characterized for 172 HIV-positive specimens. The env sequences were predominantly subtype A (43.02%), but 7 other subtypes (33.14%), 20 circulating recombinant forms (CRFs; 11.63%), and 20 unclassified (11.63%) sequences were also found. Of the rare and unclassified subtypes, 18 specimens were selected for next-generation sequencing (NGS) by a modified HIV-switching mechanism at the 5' end of the RNA template (SMART) method to obtain full-genome sequences. NGS produced 14 new complete genomes, which included pure subtype C (n = 2), D (n = 1), F1 (n = 1), H (n = 3), and J (n = 1) genomes. The two subtype C genomes and one of the subtype H genomes branched basal to their respective subtype branches but had no evidence of recombination. The remaining 6 genomes were complex recombinants of 2 or more subtypes, including subtypes A1, F, G, H, J, and K and unclassified fragments, including one subtype CRF25 isolate, which branched basal to all CRF25 references. Notably, all recombinant subtype H fragments branched basal to the H clade. Spatial-geographical analysis indicated that the diverse sequences identified here did not expand globally. The full-genome and subgenomic sequences identified in our study population significantly increase the documented diversity of the strains involved in the continually evolving HIV-1 pandemic.IMPORTANCE Very little is known about the ancestral HIV-1 strains that founded the global pandemic, and very few complete genome sequences are available from patients in the Congo Basin, where HIV-1 expanded early in the global pandemic. By sequencing a subgenomic fragment of the HIV-1 envelope from study participants in the DRC, we identified rare variants for complete genome sequencing. The basal branching of some of the complete genome sequences that we recovered suggests that these strains are more closely related to ancestral HIV-1 strains than to previously reported strains and is evidence that the local diversification of HIV in the DRC continues to outpace the diversity of global strains decades after the emergence of the pandemic.

Enhanced oxidative stress by alcohol use in HIV+ patients: possible involvement of cytochrome P450 2E1 and antioxidant enzymes.

BACKGROUND: Alcohol consumption is prevalent amongst HIV positive population. Importantly, chronic alcohol use is reported to exacerbate HIV pathogenesis. Although alcohol is known to increase oxidative stress, especially in the liver, there is no clinical evidence that alcohol increases oxidative stress in HIV positive patients. The mechanism by which alcohol increases oxidative stress in HIV positive patients is also unknown. METHODS: To examine the effects of alcohol use on oxidative stress we recruited HIV+ patients who reported mild-to-moderate alcohol use. Strict inclusion and exclusion criteria were applied to reduce the effect of other therapeutic drugs metabolized via the hepatic system as well as the effect of co-morbidities such as active tuberculosis on the interaction between alcohol and HIV infection, respectively. Blood samples were collected from HIV-negative alcohol-users and HIV positive alcohol-users followed by collection of plasma and isolation and fractionation of monocytes from peripheral blood. We then determined oxidative DNA damage, glutathione level, alcohol level, transcriptional level of cytochrome P450 2E1 (CYP2E1) and several antioxidant enzymes, and plasma level of cytokines. RESULTS: Compared to HIV-negative alcohol users, HIV-positive alcohol users demonstrated an increase in oxidative DNA damage in both plasma and CD14+ monocytes, as well as, a relative increase in oxidized/reduced glutathione (GSSG/GSH) in plasma samples. These results suggest an increase in oxidative stress in HIV-positive alcohol users compared with HIV-negative alcohol users. We also examined whether alcohol metabolism, perhaps by CYP2E1, and antioxidant enzymes are involved in alcohol-mediated increased oxidative stress in HIV-positive patients. The results showed a lower plasma alcohol level, which was associated with an increased level of CYP2E1 mRNA in monocytes, in HIV-positive alcohol users compared with HIV-negative alcohol users. Furthermore, the transcription of major antioxidants enzymes (catalase, SOD1, SOD2, GSTK1), and their transcription factor, Nrf2, were reduced in monocytes obtained from HIV positive alcohol users compared to the HIV-negative alcohol user group. However, no significant change in levels of five major cytokines/chemokines were observed between the two groups. CONCLUSIONS: The data suggests that alcohol increases oxidative stress in HIV+ patients, perhaps through CYP2E1- and antioxidant enzymes-mediated pathways. The enhanced oxidative stress is accompanied by a failure of cellular antioxidant mechanisms to maintain redox homeostasis. Overall, the enhanced oxidative stress in monocytes may exacerbate HIV pathogenesis in HIV positive alcohol users.

Effect of mild-to-moderate smoking on viral load, cytokines, oxidative stress, and cytochrome P450 enzymes in HIV-infected individuals.

Mild-to-moderate tobacco smoking is highly prevalent in HIV-infected individuals, and is known to exacerbate HIV pathogenesis. The objective of this study was to determine the specific effects of mild-to-moderate smoking on viral load, cytokine production, and oxidative stress and cytochrome P450 (CYP) pathways in HIV-infected individuals who have not yet received antiretroviral therapy (ART). Thirty-two human subjects were recruited and assigned to four different cohorts as follows: a) HIV negative non-smokers, b) HIV positive non-smokers, c) HIV negative mild-to-moderate smokers, and d) HIV positive mild-to-moderate smokers. Patients were recruited in Cameroon, Africa using strict selection criteria to exclude patients not yet eligible for ART and not receiving conventional or traditional medications. Those with active tuberculosis, hepatitis B or with a history of substance abuse were also excluded. Our results showed an increase in the viral load in the plasma of HIV positive patients who were mild-to-moderate smokers compared to individuals who did not smoke. Furthermore, although we did not observe significant changes in the levels of most pro-inflammatory cytokines, the cytokine IL-8 and MCP-1 showed a significant decrease in the plasma of HIV-infected patients and smokers compared with HIV negative non-smokers. Importantly, HIV-infected individuals and smokers showed a significant increase in oxidative stress compared with HIV negative non-smoker subjects in both plasma and monocytes. To examine the possible pathways involved in increased oxidative stress and viral load, we determined the mRNA levels of several antioxidant and cytochrome P450 enzymes in monocytes. The results showed that the levels of most antioxidants are unaltered, suggesting their inability to counter oxidative stress. While CYP2A6 was induced in smokers, CYP3A4 was induced in HIV and HIV positive smokers compared with HIV negative non-smokers. Overall, the findings suggest a possible association of oxidative stress and perhaps CYP pathway with smoking-mediated increased viral load in HIV positive individuals.

Drug resistance in non-B subtype HIV-1: impact of HIV-1 reverse transcriptase inhibitors.

Human immunodeficiency virus (HIV) causes approximately 2.5 million new infections every year, and nearly 1.6 million patients succumb to HIV each year. Several factors, including cross-species transmission and error-prone replication have resulted in extraordinary genetic diversity of HIV groups. One of these groups, known as group M (main) contains nine subtypes (A-D, F-H and J-K) and causes ~95% of all HIV infections. Most reported data on susceptibility and resistance to anti-HIV therapies are from subtype B HIV infections, which are prevalent in developed countries but account for only ~12% of all global HIV infections, whereas non-B subtype HIV infections that account for ~88% of all HIV infections are prevalent primarily in low and middle-income countries. Although the treatments for subtype B infections are generally effective against non-B subtype infections, there are differences in response to therapies. Here, we review how polymorphisms, transmission efficiency of drug-resistant strains, and differences in genetic barrier for drug resistance can differentially alter the response to reverse transcriptase-targeting therapies in various subtypes.

Multi-drug resistant oral Candida species isolated from HIV-positive patients in South Africa and Cameroon.

Candida species are a common cause of infection in immune-compromised HIV-positive individuals, who are usually treated with the antifungal drug, fluconazole, in public hospitals in Africa. However, information about the prevalence of drug resistance to fluconazole and other antifungal agents on Candida species is very limited. This study examined 128 Candida isolates from South Africa and 126 Cameroonian Candida isolates for determination of species prevalence and antifungal drug susceptibility. The isolates were characterized by growth on chromogenic and selective media and by their susceptibility to 9 antifungal drugs tested using the TREK™ YeastOne9 drug panel (Thermo Scientific, USA). Eighty-three percent (82.8%) of South African isolates were Candida albicans (106 isolates), 9.4% were Candida glabrata (12 isolates), and 7.8% were Candida dubliniensis (10 isolates). Of the Cameroonian isolates, 73.02% were C. albicans (92 isolates); 19.05% C. glabrata (24 isolates); 3.2% Candida tropicalis (4 isolates); 2.4% Candida krusei (3 isolates); 1.59% either Candida kefyr, Candida parapsilopsis, or Candida lusitaneae (2 isolates); and 0.79% C. dubliniensis (1 isolate). Widespread C. albicans resistance to azoles was detected phenotypically in both populations. Differences in drug resistance were seen within C. glabrata found in both populations. Echinocandin drugs were more effective on isolates obtained from the Cameroon than in South Africa. A multiple-drug resistant C. dubliniensis strain isolated from the South African samples was inhibited only by 5-flucytosine in vitro on the YO9 panel. Drug resistance among oral Candida species is common among African HIV patients in these 2 countries. Regional surveillance of Candida species drug susceptibility should be undertaken to ensure effective treatment for HIV-positive patients.

Enhanced nicotine metabolism in HIV-1-positive smokers compared with HIV-negative smokers: simultaneous determination of nicotine and its four metabolites in their plasma using a simple and sensitive electrospray ionization liquid chromatography-tandem mass spectrometry technique.

Smoking is approximately three times more prevalent in HIV-1-positive than HIV-negative individuals in the United States. Nicotine, which is the major constituent of tobacco, is rapidly metabolized mainly by cytochrome P450 (CYP2A6) to many metabolites. In this study, we developed a simple, fast, and sensitive electrospray ionization liquid chromatography-tandem mass spectrometry method using a strong cation solid phase extraction, and determined the concentration of nicotine and its four major metabolites (cotinine, nornicotine, norcotinine, and trans-3'-hydroxycotinine) in the plasma of HIV-1-positive and HIV-negative smokers. The multiple reaction monitoring transitions for nicotine, cotinine, trans-3'-hydroxycotinine, nornicotine, norcotinine, nicotine-d4, and cotinine-d3 were selected at mass-to-charge ratios of 163.3/117.1, 177.5/80.3, 193.2/80.1, 149.5/132.3, 163.4/80.3, 167.3/121.4, and 180.3/101.2, respectively. The lower limit of quantitation for nicotine and its metabolites was 0.53 ng/ml, which is relatively more sensitive than those previously reported. The concentration of nicotine was detected 5-fold lower in HIV-1-positive smokers (7.17 ± 3.8 ng/ml) than that observed in HIV-negative smokers (33.29 ± 15.4 ng/ml), whereas the concentration of the metabolite nornicotine was 3-fold higher in HIV-1-positive smokers (6.8 ± 2.9 ng/ml) than in HIV-negative smokers (2.3 ± 1.2 ng/ml). Although it was statistically nonsignificant, the concentration of the metabolite cotinine was also higher in HIV-1-positive smokers (85.6 ± 60.5 ng/ml) than in HIV-negative smokers (74.9 ± 40.5 ng/ml). In conclusion, a decrease in the concentration of nicotine and an increase in the concentration of its metabolites in HIV-1-positive smokers compared with HIV-negative smokers support the hypothesis that nicotine metabolism is enhanced in HIV-1-positive smokers compared with HIV-negative smokers.