Biochemical Detection of Capsid in the Nucleus During HIV-1 Infection.

Recent evidence has shown that uncoating and reverse transcription precede nuclear import. These recent breakthroughs have been made possible through the development of innovative biochemical and imaging techniques. This method outlines the biochemical assay used for detecting the presence of the HIV-1 core in the nuclear compartment. In this procedure, human cells are infected with HIV-1NL4-3, with or without the inclusion of PF74, a small molecule that inhibits core entry into the nuclear compartment. Subsequently, cells are separated into cytosolic and nuclear fractions. To assess whether the capsid protein has reached the nuclear compartment, cytosolic and nuclear fractions are subjected to Western blot analysis, utilizing antibodies specific to the HIV-1 capsid protein p24. To validate the true origin of these fractions, Western blot analysis employing antibodies against cytosolic and nuclear markers are also performed. In summary, this assay provides a reliable and efficient means to detect the presence of the HIV-1 capsid protein in the nucleus during infection under various conditions.

An Ultrasensitive p24 Assay to Measure HIV-1 in Diverse Biological Matrixes.

Assays to study HIV persistence are crucial to evaluate therapeutic strategies aimed toward an HIV cure. Several assays have been developed to date that rely on the measurement of nucleic acids. In recent years, the advancement of ultrasensitive technologies for the detection of proteins has improved our understanding of the role of translation-competent reservoirs in HIV persistence. In this chapter, we describe the development of an ultrasensitive p24 ELISA that uses planar array technology. This assay allows for the detection of HIV-1 p24 in the low fg/ml range in different biological matrixes, including cell lysates. This assay can be used to investigate the efficacy of latency reversing agents to reactivate HIV or to evaluate the persistence of translation-competent reservoirs in people living with HIV (PWH) in cells or diverse biological fluids.

In-house ELISA protocols for capsid p24 detection of diverse HIV isolates.

BACKGROUND: The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates. METHODS: Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes. RESULTS: Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype. CONCLUSIONS: These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits.

Point-of-care p24 antigen detection for early infant diagnosis of HIV infection: cross-sectional and longitudinal studies in Zambia.

BACKGROUND: Early infant diagnosis of HIV infection is challenging in sub-Saharan Africa, particularly in rural areas, leading to delays in diagnosis and treatment. Use of a point-of-care test would overcome many challenges. This study evaluated the validity of a novel point-of-care p24 antigen detection test (LYNX) in rural and urban settings in southern Zambia. METHODS: Two studies were conducted: a cross-sectional study from 2014 to 2015 at Macha Hospital (LYNX Hospital study) and a longitudinal study from 2016 to 2018 at 12 health facilities in Southern Province, Zambia (NSEBA study). In both studies, children attending the facilities for early infant diagnosis were enrolled and a blood sample was collected for routine testing at the central lab and immediate on-site testing with the LYNX test. The performance of the LYNX test was measured in comparison to nucleic acid-based testing at the central lab. RESULTS: In the LYNX Hospital study, 210 tests were performed at a median age of 23.5 weeks (IQR: 8.9, 29.0). The sensitivity and specificity of the test were 70.0 and 100.0%, respectively. In the NSEBA study, 2608 tests were performed, including 1305 at birth and 1222 on children ≥4 weeks of age. For samples tested at birth, sensitivity was 13.6% (95% CI: 2.9, 34.9) and specificity was 99.6% (95% CI: 99.1, 99.9). While specificity was high for all ages, sensitivity increased with age and was higher for participants tested at ≥4 weeks of age (80.6%; 95% CI: 67.4, 93.7). Children with positive nucleic acid tests were more likely to be negative by the LYNX test if their mother received antiretroviral therapy during pregnancy (60.7% vs. 24.2%; p = 004). CONCLUSIONS: Considering the high specificity and moderate sensitivity that increased with age, the LYNX test could be of value for early infant diagnosis for infants ≥4 weeks of age, particularly in rural areas where centralized testing leads to long delays. Point-of-care tests with moderate sensitivity and high specificity that are affordable, easy-to-use, and easily implemented and maintained should be developed to expand access to testing and deliver same-day results to infants in areas where it is not feasible to implement nucleic acid-based point-of-care assays.

Controlled copper in situ growth-amplified lateral flow sensors for sensitive, reliable, and field-deployable infectious disease diagnostics.

A polyethyleneimine (PEI)-assisted copper in-situ growth (CISG) strategy was proposed as a controlled signal amplification strategy to enhance the sensitivity of gold nanoparticle-based lateral flow sensors (AuNP-LFS). The controlled signal amplification is achieved by introducing PEI as a structure-directing agent to regulate the thermodynamics of anisotropic Cu nanoshell growth on the AuNP surface, thus controlling shape and size of the resultant AuNP@Cu core-shell nanostructures and confining free reduction and self-nucleation of Cu2+ for improved reproducibility and decreased false positives. The PEI-CISG-enhanced AuNP-LFS showed ultrahigh sensitivities with the detection limits of 50 fg mL-1 for HIV-1 capsid p24 antigen and 6 CFU mL-1 for Escherichia coli O157:H7. We further demonstrated its clinical diagnostic efficacy by configuring PEI-CISG into a commercial AuNP-LFS detection kit for SARS-CoV-2 antibody detection. Altogether, this work provides a reliable signal amplification platform to dramatically enhance the sensitivity of AuNP-LFS for rapid and accurate diagnostics of various infectious diseases.

Ultra-low HIV-1 p24 detection limits with a bioelectronic sensor.

Early diagnosis of the infection caused by human immunodeficiency virus type-1 (HIV-1) is vital to achieve efficient therapeutic treatment and limit the disease spreading when the viremia is at its highest level. To this end, a point-of-care HIV-1 detection carried out with label-free, low-cost, and ultra-sensitive screening technologies would be of great relevance. Herein, a label-free single molecule detection of HIV-1 p24 capsid protein with a large (wide-field) single-molecule transistor (SiMoT) sensor is proposed. The system is based on an electrolyte-gated field-effect transistor whose gate is bio-functionalized with the antibody against the HIV-1 p24 capsid protein. The device exhibits a limit of detection of a single protein and a limit of quantification in the 10 molecule range. This study paves the way for a low-cost technology that can quantify, with single-molecule precision, the transition of a biological organism from being "healthy" to being "diseased" by tracking a target biomarker. This can open to the possibility of performing the earliest possible diagnosis.

Activate capture and digital counting (AC + DC) assay for protein biomarker detection integrated with a self-powered microfluidic cartridge.

We demonstrate a rapid, 2-step, and ultrasensitive assay approach for quantification of target protein molecules from a single droplet test sample. The assay is comprised of antibody-conjugated gold nanoparticles (AuNPs) that are "activated" when they are mixed with the test sample and bind their targets. The resulting liquid is passed through a microfluidic channel with a photonic crystal (PC) biosensor that is functionalized with secondary antibodies to the target biomarker, so that only activated AuNPs are captured. Utilizing recently demonstrated hybrid optical coupling between the plasmon resonance of the AuNP and the resonance of the PC, each captured AuNP efficiently quenches the resonant reflection of the PC, thus enabling the captured AuNPs to be digitally counted with high signal-to-noise. To achieve a 2-step assay process that is performed on a single droplet test sample without washing steps or active pump elements, controlled single-pass flow rate is obtained with an absorbing paper pad waste reservoir embedded in a microfluidic cartridge. We use the activate capture and digital counting (AC + DC) approach to demonstrate HIV-1 capsid antigen p24 detection from a 40 µL spiked-in human serum sample at a one thousand-fold dynamic range (1-103 pg mL-1) with only a 35-minute process that is compatible with point-of-care (POC) analysis. The AC + DC approach allows for ultrasensitive and ultrafast biomolecule detection, with potential applications in infectious disease diagnostics and early stage disease monitoring.

Smartphone assisted immunodetection of HIV p24 antigen using reusable, centrifugal microchannel array chip.

An integrated immunodetection platform employing a simple, reusable, centrifugal microchannel array chip and a smartphone as detection unit was developed. The applicability of the platform to the detection of HIV p24 antigen was demonstrated. The microchip was made of polycarbonate and contained 4 × 8 zigzag microchannels. After the monoclonal antibody of HIV p24 was adsorbed onto the channel surfaces, HIV p24 was introduced into the microchannel to react with the antibody. A biotin linked polyclonal antibody was then brought in to react with HIV p24, and SP80 (containing streptavidin and horseradish peroxidase) was introduced to react with the biotin. Finally, a solution containing 3,3',5,5'-tetramethylbenzidine and other reagents was passed through the above channels, horseradish peroxidase catalyzed the oxidation of tetramethylbenzidine (to 3,3',5,5'- tetramethylbenzidine diamine) forming a dark color. The color intensity, indicating HIV p24 antigen quantity, was then photographed via a smartphone, and the color of each microchannel was processed via a computer to determine the HIV p24 antigen concentration. Under the optimized conditions, limits of detection (LODs) of 0.17 ng/ml and 0.11 ng/ml were obtained for p24 antigen in a buffer solution and human serum, respectively. Channel washing/rinsing was implemented via a centrifugal force. An economic portable centrifugal device that could accommodate up to 4 microchips was assembled, and multi-step solution loading and rinsing involved in this sandwich immunoassay were performed conveniently. The microchip could be reused after a simple regeneration process. The low-cost polycarbonate microchip and centrifugal device together with the simple but efficient operation make the method a promising tool for HIV screening in resource limited areas.

Cross-Subtype Detection of HIV-1 Capsid p24 Antigen Using a Sensitive Europium Nanoparticle Assay.

Accurate and early detection of diverse HIV-1 subtypes using currently available p24 antigen assays have been a major challenge. We report the development of a sensitive time resolved fluorescence (TRF) europium nanoparticle immuno assay for cross subtype detection of p24 antigen using broadly cross-reactive antibodies. Several antibodies were tested for optimal reactivity with antigens of diverse HIV-1 subtypes and circulating recombinant forms. We tested HIV strains using this assay for sensitivity and quantification ability at the pico-gram per millilter level. We identified two broadly cross-reactive HIV-1 p24 antibodies C65690M and ANT-152, which detected all strains of HIV tested. These two antibodies also yielded a better signal to cutoff ratio for the same amount of antigen tested in comparison to a commercial assay. Using an appropriate combination of C65690M and ANT-152 p24 antibodies capable of detecting all HIV types and highly sensitive TRF-based europium nano particle assay platform, we developed a sensitive p24 antigen assay that can detect HIV infection of all HIV subtypes and may be useful in early detection.

Identification, Localization, and Quantification of HIV Reservoirs Using Microscopy.

The major barrier to eradicating human immunodeficiency virus-1 (HIV) infection is the generation and extended survival of HIV reservoirs. In order to eradicate HIV infection, it is essential to detect, quantify, and characterize circulating and tissue-associated viral reservoirs in infected individuals. Currently, PCR-based technologies and Quantitative Viral Outgrowth Assays (Q-VOA) are the gold standards to detect viral reservoirs. However, these methods are limited to detecting circulating viral reservoirs, and it has been shown that they misrepresent the size of the reservoirs, largely because they detect only one component of the HIV life cycle and are unable to detect viral reservoirs in tissues. Here, we described the use of multiple detection systems to identify integrated HIV DNA or viral mRNA and several HIV proteins in circulating and tissue reservoirs using improved staining and microscopy techniques. We believe that this imaging-based approach for detecting HIV reservoirs will lead to breakthroughs necessary to eradicate these reservoirs. © 2018 by John Wiley & Sons, Inc.

Self-Assembly of Antigen Proteins into Nanowires Greatly Enhances the Binding Affinity for High-Efficiency Target Capture.

High-efficiency target capture is an essential prerequisite for sensitive immunoassays. However, the current available immunoassay approaches are subject to deficient binding affinities between predator-prey molecules that greatly restrict the target capture efficiency and immunoassay sensitivity. Herein, we present a new strategy through the self-assembly of antigen proteins into nanowires to enhance the binding affinity between an antigen and antibody. Through the genetic fusion of antigen proteins (e.g., HIV p24) with the yeast amyloid protein Sup35 self-assembly domain, specific antigen nanowires (Ag nanowires) were constructed and demonstrated a remarkable enhancement in binding affinity compared with that of the monomeric antigen molecule. The Ag nanowires were further combined with magnetic beads to form a 3D magnetic probe based on a seed-induced self-assembly strategy. Taking advantage of both the strong binding affinity and the rapid magnetic separation and enrichment capacity, the specific 3D magnetic probe achieved a 100-fold improvement in detection sensitivity within a significantly shorter period of 20 min over that of the conventional enzyme-linked immunosorbent assay method.

Different kinetics of viral replication and DNA integration in the main HIV-1 cellular reservoirs in the presence and absence of integrase inhibitors.

To compare the kinetics of integration, p24 production and equilibrium of the different HIV-DNA forms in human primary cells in the presence/absence of integrase-inhibitors (INIs) in vitro. Monocyte-derived-macrophages (MDMs), CD4+ T-cells and peripheral blood mononuclear cells (PBMCs) were infected with HIV-1 in the presence/absence of raltegravir and dolutegravir. HIV-DNA levels and p24 production were measured by qPCR and ELISA assays, respectively. In the absence of INIs, levels of HIV-DNA forms were initially very low, with an increase in the integration process starting at 3 dpi. HIV-DNA increased more slowly in MDMs than it did in CD4+ T-cells and PMBCs peaking at 21 dpi with a mean of 1580 (±890) and 615 (±37) copies/103 cells for proviral and unintegrated HIV-DNA, and 455,972 (±213,255) pg/mL of p24 at the same time point. In CD4+ T-cells the proviral HIV-DNA increased together with unintegrated HIV-DNA peaking at 7 dpi (583 ±â€¯261 and 338 ±â€¯254 copies/103 cells) when the p24 was 218,000 (±75,600) pg/mL. A similar trend was observed in PBMCs (494 ±â€¯361 and 350 ±â€¯123 copies/103 cells for proviral and unintegrated HIV-DNA, and p24 production of 149,400 ±â€¯131,800 pg/mL). Both INIs inhibited viral replication and integration in all the cell types that were tested, especially starting at 3 dpi. However, a small but measurable amount of HIV-DNA (<5 copies/103 cells) was still observed in treated-MDMs up to 30 dpi. In conclusion, our study showed differences in HIV-DNA kinetic integration between CD4+ T-cells and MDMs, which could explain the divergent kinetics of viral-replication. Both INIs inhibited HIV-1 integration and replication with no difference found between CD4+ T-cells and MDMs. However, residual HIV-DNA remained detectable up to 30 dpi in INI-treated MDMs although complete inhibition of HIV replication was achieved. The clinical significance of this minor DNA persistence deserves further investigation considering the role of macrophages as reservoirs.

Interleukin 21 (IL-21)/microRNA-29 (miR-29) axis is associated with natural resistance to HIV-1 infection.

BACKGROUND: Interleukin-21 (IL-21) modulates HIV-1 infection through the elicitation of different antiviral mechanisms, including Th17 lineage commitment and induction of microRNA (miR)-29, a miRNA endowed with anti-HIV activity. As miR-29 expression is significantly increased in HIV-1-exposed seronegative individuals (HESN), we investigated the role of miR-29/IL21 axis in the natural control of HIV-1 infection. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from 15 Italian sexually exposed HESN and 15 HIV-unexposed healthy controls were in-vitro infected with an R5-tropic HIV-1Ba-L strain. Seven days post HIV-1 infection we evaluated: 1) p24 production (ELISA); 2) CD4/IL-21 and CD4/IL-17 T lymphocytes (FACS); 3) IL-17 concentration in supernatants (ELISA); and 4) IL-6, IL-17, IL-21, and miR-29a,b,c expression by CD4 T lymphocytes as well as perforin and granzyme by peripheral blood mononuclear cells (qPCR). The same analyses were performed on the 15 HIV-positive partners. RESULTS: At baseline IL-6 expression alone was increased in HESN compared to healthy controls. Seven days after in-vitro HIV-1 infection, nevertheless, differences emerged. Thus, CD4/IL21 and CD4/IL17 T lymphocytes, as well as IL-21 and IL-17 expression and production were significantly augmented in HESN compared to healthy controls. Interestingly, IL-21 upregulation correlated with a significantly increased expression of miR-29a,b,c and a reduced susceptibility to in-vitro HIV-1 infection in HESN alone. No differences were observed in perforin and granzyme expression. CONCLUSION: The IL-21/miR-29 axis is upregulated by HIV-1 infection in HESN suggesting its involvement in the natural resistance to HIV-1 infection in HESN. Approaches that exogenously increase IL-21 production or prompt preexisting cellular IL-21 reservoir could confine the magnitude of the initial HIV-1 infection.

Expanding the Scope of Protein-Detecting Electrochemical DNA "Scaffold" Sensors.

The ability to measure the levels of diagnostically relevant proteins, such as antibodies, directly at the point of care could significantly impact healthcare. Thus motivated, we explore here the E-DNA "scaffold" sensing platform, a rapid, convenient, single-step means to this end. These sensors comprise a rigid nucleic acid "scaffold" attached via a flexible linker to an electrode and modified on its distal end with a redox reporter and a protein binding "recognition element". The binding of a targeted protein reduces the efficiency with which the redox reporter approaches the electrode, resulting in an easily measured signal change when the sensor is interrogated voltammetrically. Previously we have demonstrated scaffold sensors employing a range of low molecular weight haptens and linear peptides as their recognition elements. Expanding on this here we have characterized sensors employing much larger recognition elements (up to and including full length proteins) in order to (1) define the range of recognition elements suitable for use in the platform; (2) better characterize the platform's signaling mechanism to aid its design and optimization; and (3) demonstrate the analytical performance of sensors employing full-length proteins as recognition elements. In doing so we have enlarged the range of molecular targets amenable to this rapid and convenient sensing platform.

Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultrabroad Dynamic Range.

Paper-based lateral flow immunoassays (LFIAs) are one of the most widely used point-of-care (PoC) devices; however, their application in early disease diagnostics is often limited due to insufficient sensitivity for the requisite sample sizes and the short time frames of PoC testing. To address this, we developed a serum-stable, nanoparticle catalyst-labeled LFIA with a sensitivity surpassing that of both current commercial and published sensitivities for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV. We report the synthesis and characterization of porous platinum core-shell nanocatalysts (PtNCs), which show high catalytic activity when exposed to complex human blood serum samples. We explored the application of antibody-functionalized PtNCs with strategically and orthogonally modified nanobodies with high affinity and specificity toward p24 and established the key larger nanoparticle size regimes needed for efficient amplification and performance in LFIA. Harnessing the catalytic amplification of PtNCs enabled naked-eye detection of p24 spiked into sera in the low femtomolar range (ca. 0.8 pg·mL-1) and the detection of acute-phase HIV in clinical human plasma samples in under 20 min. This provides a versatile absorbance-based and rapid LFIA with sensitivity capable of significantly reducing the HIV acute phase detection window. This diagnostic may be readily adapted for detection of other biomolecules as an ultrasensitive screening tool for infectious and noncommunicable diseases and can be capitalized upon in PoC settings for early disease detection.

Short Communication: Tenofovir Disoproxil Fumarate/Emtricitabine Fits for All as Appropriate HIV-1 Pre-Exposure Prophylaxis?

Pre-exposure prophylaxis (PrEP) with TDF/FTC significantly reduces the risk for HIV-1 transmission, but, to date, nobody knows if PrEP can still be useful in case of viruses harboring resistances to TDF/FTC or if alternative antiretroviral drugs might be rather proposed in selected cases. We promote the debate by describing a case of an acute infection with multiresistant HIV that occurred in a young girl who started a relationship with an HIV-infected partner who had a known multiresistant HIV.

Heterogeneity of HIV-1 Replication in Ectocervical and Vaginal Tissue Ex Vivo.

In clinical trials evaluating HIV-1 prevention products, ex vivo exposure of mucosal tissue to HIV-1 is performed to inform drug levels needed to suppress viral infection. Understanding assay and participant variables that influence HIV-1 replication will help with assay implementation. Demographic and behavioral data were obtained from 61 healthy women aged 21-45. Paired cervical tissue (CT) and vaginal tissue (VT) biopsies were collected and treated with HIV-1BaL or HIV-1JR-CSF, washed, and cultured. On days 3, 7, and/or 11, culture supernatant was collected, and viral replication was monitored by p24 ELISA. Tissue was extracted at study end, and HIV-1 relative RNA copies were determined by polymerase chain reaction. Cumulative p24 and RNA were log-transformed and analyzed using a linear mixed model, t-test, and an intraclass correlation coefficient (ICC). HIV replication was similar between CT and VT for each virus, but HIV-1BaL had 1.5 log10 and 0.9 log10 higher levels of p24 than HIV-1JR-CSF in CT and VT, respectively (p < .001), which correlated with HIV-1 relative RNA copies. Cumulative p24 and RNA copies in both tissues demonstrated low intraperson correlation for both viruses (ICC ≤0.513 HIV-1BaL; ICC ≤0.419 HIV-1JR-CSF). Enrollment into previous clinical studies in which genital biopsies were collected modestly decreased the HIV-1BaL cumulative p24 for CT, but not for VT. To improve the ex vivo challenge assay, viruses should be evaluated for replication in mucosal tissue before study implementation, baseline mucosal tissue is not needed if a placebo/no treatment group is included within the clinical trial, and previous biopsy sites should be avoided.

HIV-associated benign lymphoepithelial cysts of the parotid glands confirmed by HIV-1 p24 antigen immunostaining.

Approximately 1%-10% of patients with HIV infection have been reported to have salivary gland enlargement. Parotid swelling in patients with HIV is often associated with salivary gland disease, including benign lymphoepithelial cysts (BLECs). The presence of BLEC can serve as an indicator of HIV infection, and the diagnosis of HIV-associated BLEC is usually based on clinical course, HIV confirmatory blood testing, such as western blot or viral detection, and imaging studies, but not on biopsies or immunostaining. To exclude other diseases such as tuberculosis and malignant lymphoma and to further improve the diagnostic accuracy of BLEC, the detection of the HIV-1 p24 antigen by immunohistochemistry is a useful diagnostic method. We report a case of a 65-year-old Japanese man with swelling of the parotid glands and HIV-associated BLEC confirmed via HIV-1 p24 immunohistochemical staining.

Regulatory T Cells Contribute to HIV-1 Reservoir Persistence in CD4+ T Cells Through Cyclic Adenosine Monophosphate-Dependent Mechanisms in Humanized Mice In Vivo.

Background: Regulatory T cells (Tregs) suppress T-cell immune activation and human immunodeficiency virus type 1 (HIV-1) replication, but the role of Tregs in HIV-1 reservoir persistence is poorly defined. Methods: Tregs were depleted by denileukin diftitox in humanized mice with chronic HIV-1 infection. Viral replication in lineage cells was determined by p24 expression. Levels of HIV-1 RNA and DNA in human cells, as well as replication-competent-virus-producing cells, were measured to quantified viral replication and reservoirs. Results: Treg depletion resulted in a blip of HIV-1 replication in T cells but not in myeloid cells. The major activated reservoir cells were memory CD4+ T cells in vivo. Interestingly, the transient activation of viral replication led to HIV-1 reservoir reduction after viremia resuppression, as indicated by the quantity of HIV-1 DNA and replication-competent-virus-producing cells. Furthermore, we demonstrated that Tregs use cyclic adenosine monophosphate (cAMP)-dependent protein kinase A pathway to inhibit HIV-1 activation and replication in resting conventional T cells in vitro. Conclusion: Tregs suppress HIV-1 replication in T cells and contribute to HIV-1 reservoir persistence. cAMP produced in Tregs is involved in their suppression of viral gene activation and expression. Treg depletion combined with combination antiretroviral therapy provides a novel strategy for HIV-1 cure.

Human immunodeficiency virus-associated cystic lymphoid hyperplasia: An immunohistochemical description.

BACKGROUND: Cystic lymphoid hyperplasia (CLH) describes benign salivary lymphoepithelial cysts with a strong link to human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). The pathogenesis is related to ductal dilatation of entrapped salivary elements due to intranodal HIV-associated atypical lymphoid hyperplasia. Very little is known about the immunophenotypic profile of this entity. AIM: This study aims to describe the immunopathological features of a series of CLH cases in HIV-positive patients to clarify the etiopathogenesis. MATERIALS AND METHODS: Paraffin-embedded tissue from 25 cases of parotid CLH in HIV seropositive patients was immunohistochemically analyzed with CD3, CD20, CD4, CD8, and p24 using standard procedures. STATISTICAL ANALYSIS: The data are mostly descriptive and were analyzed using EpiInfo (3.5.1) (CDC, Atlanta, USA); significant differences were analyzed using the Student's t-test and the Chi-square test with a statistical significance level of P < 0.05 being used. RESULTS: Immunostaining showed a CD8:CD4 of ~1:1 except in selected cases with decreased CD4 and increased CD8 expression in the interfollicular (IF) areas. p24 staining revealed 100% specificity in HIV-associated CLH. CONCLUSION: The immunohistochemical description of CD20, CD3, CD4, and CD8 provides an understanding of CLH pathogenesis. CLH of parotid lymph nodes in confirmed HIV-positive patients with 100% specificity with HIV p24 antibody validates the strong association of CLH with HIV and AIDS. The CD4:CD8 ratio was ~1:1; however, increased CD8 expression within IF areas may indicate possible HIV-related CLH as compared to other cystic parotid lesions.