Multiparameter immunohistochemistry analysis of HIV DNA, RNA and immune checkpoints in lymph node tissue.

The main barrier to a cure for HIV is the persistence of long-lived and proliferating latently infected CD4+ T-cells despite antiretroviral therapy (ART). Latency is well characterized in multiple CD4+ T-cell subsets, however, the contribution of regulatory T-cells (Tregs) expressing FoxP3 as well as immune checkpoints (ICs) PD-1 and CTLA-4 as targets for productive and latent HIV infection in people living with HIV on suppressive ART is less well defined. We used multiplex detection of HIV DNA and RNA with immunohistochemistry (mIHC) on formalin-fixed paraffin embedded (FFPE) cells to simultaneously detect HIV RNA and DNA and cellular markers. HIV DNA and RNA were detected by in situ hybridization (ISH) (RNA/DNAscope) and IHC was used to detect cellular markers (CD4, PD-1, FoxP3, and CTLA-4) by incorporating the tyramide system amplification (TSA) system. We evaluated latently infected cell lines, a primary cell model of HIV latency and excisional lymph node (LN) biopsies collected from people living with HIV (PLWH) on and off ART. We clearly detected infected cells that coexpressed HIV RNA and DNA (active replication) and DNA only (latently infected cells) in combination with IHC markers in the in vitro infection model as well as LN tissue from PLWH both on and off ART. Combining ISH targeting HIV RNA and DNA with IHC provides a platform to detect and quantify HIV persistence within cells identified by multiple markers in tissue samples from PLWH on ART or to study HIV latency.

Herpes Simplex Virus Keratitis Reactivation after SARS-CoV-2 BNT162b2 mRNA Vaccination: A Report of Two Cases.

PURPOSE: To report two cases of herpes simplex virus keratitis reactivation following Pfizer-BioNTech COVID-19 (BNT162b2) mRNA vaccination. METHODS: Two patients (one male, age 42 years, and one female, age 29 years) who are known to have herpetic keratitis presented to our emergency room in a time frame between 4 days and 4 weeks of receiving the vaccine. One patient presented with necrotizing stromal keratitis; the other presented with endotheliitis and epithelial keratitis. PCR for herpes simplex virus (HSV) was obtained from the two patients, and all cases received systemic acyclovir. RESULTS: PCR for HSV came positive in both cases. Patients responded well to the provided treatment. CONCLUSION: Ocular herpetic infection may be activated by COVID-19 (BNT162b2) mRNA vaccine. Treating physician should be alert to such associations, and patients should be followed closely. No direct causality has been proven, but further reporting and investigating similar conditions is recommended.

HIV and Chagas Disease: An Evaluation of the Use of Real-Time Quantitative Polymerase Chain Reaction to Measure Levels of Trypanosoma cruzi Parasitemia in HIV Patients in Cochabamba, Bolivia.

This cross-sectional study evaluated epidemiologic characteristics of persons living with HIV (PWH) coinfected with Trypanosoma cruzi in Cochabamba, Bolivia, and estimated T. cruzi parasitemia by real-time quantitative polymerase chain reaction (qPCR) in patients with and without evidence of reactivation by direct microscopy. Thirty-two of the 116 HIV patients evaluated had positive serology for T. cruzi indicative of chronic Chagas disease (27.6%). Sixteen of the 32 (50%) patients with positive serology were positive by quantitative polymerase chain reaction (qPCR), and four of the 32 (12.5%) were positive by direct microscopy. The median parasite load by qPCR in those with CD4+ < 200 was 168 parasites/mL (73-9951) compared with 28.5 parasites/mL (15-1,528) in those with CD4+ ≥ 200 (P = 0.89). There was a significant inverse relationship between the degree of parasitemia estimated by qPCR from blood clot and CD4+ count on the logarithmic scale (rsBC= -0.70, P = 0.007). The correlation between T. cruzi estimated by qPCR+ blood clot and HIV viral load was statistically significant with rsBC = 0.61, P = 0.047. Given the significant mortality of PWH and Chagas reactivation and that 57% of our patients with CD4+ counts < 200 cells/mm3 showed evidence of reactivation, we propose that screening for chronic Chagas disease be considered in PWH in regions endemic for Chagas disease and in the immigrant populations in nonendemic regions. Additionally, our study showed that PWH with advancing immunosuppression have higher levels of estimated parasitemia measured by qPCR and suggests a role for active surveillance for Chagas reactivation with consideration of treatment with antitrypanosomal therapy until immune reconstitution can be achieved.

Long-Term Safety of Rituximab (Risks of Viral and Opportunistic Infections).

PURPOSE OF REVIEW: We reviewed the current data on infections associated with rituximab use published over the last 5 years. RECENT FINDINGS: New literature was available on rates of serious infections, Hepatitis B reactivation and screening, and infection with Severe Acute Respiratory Syndrome Coronavirus 2. Rates of infection varied by study and population, however, higher risk of infection in patients with underlying rheumatologic diseases was seen in those who required a therapy switch, had a smoking history, and those undergoing retreatment who had a serious infection with their first course of therapy. With regards to HBV, the proportion of patients screened continues to be inadequate. Despite the upfront cost, HBV screening and prophylaxis were found to be cost effective. There is still limited data regarding COVID-19 severity in the setting of rituximab, however, rituximab, especially in combination with steroids, may lead to more severe disease and higher mortality.

ELISPOT assay of interferon-γ secretion for evaluating human cytomegalovirus reactivation risk in allo-HSCT recipients.

Human cytomegalovirus (HCMV) is a common cause of significant morbidity and mortality in transplant recipients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We evaluated interferon-γ (IFN-γ) secretion by HCMV NLV-specific CD8+ T cells in HCMV-reactivated allo-HSCT recipients using an enzyme-linked immunospot (ELISPOT) assay at 3 months post-transplantation. Blood samples from 47 recipients were tested for HCMV DNAemia, HCMV pp65 antigenemia, and anti-HCMV immunoglobulins (IgG/IgM) over 3 months post-transplantation. Of the 47 transplant recipients, 26 were HLA-A*02 positive and 21 were HLA-A*02 negative. The results were essentially consistent between the 47 transplant recipients and the HLA-A*02-positive recipients. HCMV DNAemia was not linearly correlated with IFN-γ spot-forming cells (SFCs) counts; IFN-γ SFCs counts did not differ significantly between the HCMV DNAemia-positive and -negative groups, whereas the HCMV-DNA virus loads were inversely correlated with the IFN-γ SFCs counts. HCMV pp65 antigenemia was not linearly correlated with IFN-γ SFCs counts; IFN-γ SFCs counts in the HCMV pp65 antigenemia-positive and -negative groups were similar. More IFN-γ SFCs counts were detected in transplant recipients with high anti-HCMV-IgG antibody titers than in those with low anti-HCMV-IgG titers pre-transplantation in the 47 recipients. Anti-HCMV-IgG antibody titers were positively linearly correlated with IFN-γ SFCs counts in HLA-A*02-positive recipients. The HCMV infection indicators used to monitor HCMV reactivation had different values in transplant recipients. The use of the IFN-γ SFCs counts measured by ELISPOT to evaluate the risk of HCMV reactivation needs further study.

Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies.

The number of new cases of leprosy reported worldwide has remained essentially unchanged for the last decade despite continued global use of free multidrug therapy (MDT) provided to any diagnosed leprosy patient. In order to more effectively interrupt the chain of transmission, new strategies will be required to detect those with latent disease who contribute to furthering transmission. To improve the ability to diagnose leprosy earlier in asymptomatic infected individuals, we examined the combined use of two well-known biomarkers of M. leprae infection, namely the presence of M. leprae DNA by PCR from earlobe slit skin smears (SSS) and positive antibody titers to the M. leprae-specific antigen, Phenolic Glycolipid I (anti-PGL-I) from leprosy patients and household contacts living in seven hyperendemic cities in the northern state of Pará, Brazilian Amazon. Combining both tests increased sensitivity, specificity and accuracy over either test alone. A total of 466 individuals were evaluated, including 87 newly diagnosed leprosy patients, 52 post-treated patients, 296 household contacts and 31 healthy endemic controls. The highest frequency of double positives (PGL-I+/RLEP+) were detected in the new case group (40/87, 46%) with lower numbers for treated (12/52, 23.1%), household contacts (46/296, 15.5%) and healthy endemic controls (0/31, 0%). The frequencies in these groups were reversed for double negatives (PGL-I-/RLEP-) for new cases (6/87, 6.9%), treated leprosy cases (15/52, 28.8%) and the highest in household contacts (108/296, 36.5%) and healthy endemic controls (24/31, 77.4%). The data strongly suggest that household contacts that are double positive have latent disease, are likely contributing to shedding and transmission of disease to their close contacts and are at the highest risk of progressing to clinical disease. Proposed strategies to reduce leprosy transmission in highly endemic areas may include chemoprophylactic treatment of this group of individuals to stop the spread of bacilli to eventually lower new case detection rates in these areas.

Clinical efficacy of a novel, high-sensitivity HBcrAg assay in the management of chronic hepatitis B and HBV reactivation.

BACKGROUND & AIMS: A fully automated, novel high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developed. We demonstrate the clinical utility of iTACT-HBcrAg for monitoring chronic hepatitis B (CHB) and for the early detection of HBV reactivation. METHODS: After fundamental assessments, the clinical performance of iTACT-HBcrAg was compared with other HBV markers. i) Serial sera, available from 161 HBeAg-negative patients with CHB and persistently undetectable HBV DNA, were measured by iTACT-HBcrAg and a conventional HBcrAg assay (G-HBcrAg). ii) Serial sera from 13 HBV-reactivated patients were measured by iTACT-HBcrAg and an ultra-high-sensitivity HBsAg immune complex transfer-chemiluminescent enzyme immunoassay (lower limit of detection; 0.0005 IU/ml, ICT-CLEIA) to compare HBV DNA detection. iii) To elucidate the various HBcrAg components detected by iTACT-HBcrAg, OptiPrep density gradient centrifugation analysis was performed on sera obtained before and after HBV reactivation. RESULTS: The analytical performance of iTACT-HBcrAg was satisfactory. The sensitivity of iTACT-HBcrAg (2.1 Log U/ml) was approximately 10-fold greater than that of G-HBcrAg (2.8 Log U/ml). i) HBcrAg was detectable in the sera of 97.5% (157/161) of patients with CHB by iTACT-HBcrAg, of whom 75.2% (121/161) had ≥2.8 Log U/ml HBcrAg and 22.4% (36/161) had 2.1-2.8 Log U/ml HBcrAg, which was undetectable by G-HBcrAg. ii) 9 and 2 of 13 HBV-reactivated patients were HBcrAg-positive by iTACT-HBcrAg before and at HBV DNA positivity, respectively; 7 and 4 were HBcrAg-positive by iTACT-HBcrAg before and at HBsAg-positivity by ICT-CLEIA, respectively. iii) The HBcrAg detected by iTACT-HBcrAg before HBV reactivation was contained in empty particles (22 KDa precore protein). CONCLUSIONS: iTACT-HBcrAg could be used to better monitor responses to anti-HBV treatments in HBeAg-negative patients and for the early detection of HBV reactivation. LAY SUMMARY: A fully automated, novel high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developed. iTACT-HBcrAg can be used to monitor HBeAg-negative patients with chronic hepatitis B, as well as for the early detection of HBV reactivation. iTACT-HBcrAg could be used as a general marker of disease progression and treatment response.

Zoster meningitis in an immunocompetent host with headache and atypical rash.

Headache is a frequent emergency department (ED) complaint. Secondary headache, due to infectious causes, must be carefully evaluated as a differential diagnosis. Red flag signs and classic physical examination findings are available to aid the diagnosis and evaluation of secondary headache. These findings, however, are limited by poor sensitivity and predictive value. We present a case of Herpes zoster (HZ) meningitis in a young healthy male adult with major presenting symptom of headache and new-onset rash to underscore the variation in atypical presentations of aseptic meningitis. HZ-associated aseptic meningitis often presents with characteristic, but at times atypical rash. We recommend skin lesions be thoroughly evaluated, along with classic signs of fever and nuchal rigidity, to assist in the diagnosis of meningitis.

Presumed Herpes Zoster Ophthalmicus Reactivation Following Recombinant Zoster Vaccination.

PURPOSE: To report a case of herpes zoster ophthalmicus (HZO) reactivation after recombinant zoster vaccination. METHODS: A 78-year-old woman, with a history of HZO 20 years ago, was referred for progressive corneal thinning in her left eye that started 1 week after her second dose of recombinant zoster vaccination. RESULTS: At presentation, visual acuity was counting fingers. Corneal sensation was markedly decreased. Slit lamp examination revealed a temporal paracentral epithelial defect 1.5 × 2.0 mm in size with 40% thinning and surrounding stromal inflammation suggestive of stromal keratitis with ulceration. The patient was started on oral valacyclovir, topical erythromycin ointment, and hourly topical lubrication. A bandage contact lens was placed and was replaced 1 week later with self-retained cryopreserved amniotic membrane ring. The ring was removed in the following week when the thinned area was epithelialized with no further evidence of stromal lysis. CONCLUSIONS: HZO reactivation after recombinant zoster vaccination is uncommon but possible. Ophthalmologists should remain aware of potential risks of zoster vaccination and take special precautions in patients with HZO history.

Strongyloides infection manifested during immunosuppressive therapy for SARS-CoV-2 pneumonia.

BACKGROUND: SARS-CoV-2 pandemic has posed formidable public health and clinical challenges. The use of immunosuppressive agents, such as high dose corticosteroids and cytokine inhibitors (e.g., Tocilizumab) has been suggested to contrast the hyperinflammatory process involved in the pathogenesis of the severe disease, with conflicting evidence. Among the drawbacks of immunosuppressive therapy, the risk of reactivation of latent infections, including parasitic infestations, is to be considered. CASE PRESENTATION: We report a case of a 59-year-old Italian patient treated with high dose intravenous dexamethasone and two intravenous doses of Tocilizumab for interstitial bilateral pneumonia associated with SARS-CoV-2 infection who developed itching, abdominal pain, and an increased eosinophil count. Stool examination confirmed the presence of S. stercoralis larvae. The patient was treated with a 4-day course of Ivermectin with full recovery. DISCUSSION: We report the first case of S. stercoralis infection following an 11-day treatment with high-dose steroids and Tocilizumab for severe COVID-19. Clinicians should be aware of the risk of strongyloidiasis as a complication of the treatment for severe COVID-19.

Pathogenic load and frailty in older adults: Singapore longitudinal ageing study.

Human evidence for the role of continuous antigenic stimulation from persistent latent infections in frailty is limited. We conducted a nested case-control study (99 deceased and 43 survivors) of participants aged 55 and above in a longitudinal ageing cohort followed up from 2003 to 2017. Using blood samples and baseline data collected in 2003-2004, we examined the association of pathogenic load (PL) count of seropositivity to 10 microbes (viruses, bacteria and mycoplasma) with cumulated deficit-frailty index (CD-FI) and the physical frailty (PF) phenotype, and mortality. Controlling for age, sex, education, smoking and alcohol histories, high PL (7-9) versus low PL (3-6) was associated with an estimated increase of 0.035 points in the CD-FI (Cohen's D=0.035 / 0.086, or 0.41). High PL was associated with 8.5 times odds of being physically frail (p=0.001), 2.8 times odds of being weak (p=0.010), 3.4 times odds of being slow (p=0.024), and mortality hazard ratio of 1.53 (p=0.046). There were no significant associations for specific pathogens, except marginal associations for Epstein-Barr virus and Chikungunya. Conclusion: A high pathogenic load of latent infections was associated with increased risks of frailty and mortality.

Risk Factors for Cytomegalovirus Reactivation in Lung Transplant Recipients.

PURPOSE: We examined risk factors that may have contributed to Cytomegalovirus (CMV) reactivation among patients who underwent lung transplantation (LTx). METHODS: We reviewed medical records of patients who underwent LTx at a tertiary healthcare hospital in South Korea between January 2013 and May 2017. We excluded patients who died within the first year after LTx and those lost to follow-up. CMV reactivation was defined as the detection of CMV titers above 3000 copies/ml regardless of specific symptoms after prophylaxis cessation. RESULTS: Of 89 patients included, 39 (43.8%) developed CMV reactivation. Of those 39 patients, 16 (41.0%) experienced additional CMV reactivation. Multivariate analysis identified lymphocyte counts below 1.0 × 103/µl (hazard ratio [HR] 49.33, p < 0.001) and use of steroids at more than twice the standard dose (HR 8.07, p < 0.001) as risk factors for CMV reactivation. The multivariate model also identified chronic kidney disease (CKD; HR 5.19, p = 0.016) and pneumonia (HR 17.22, p = 0.013) as risk factors for repetitive CMV reactivation. CONCLUSION: This study suggests that lymphopenia and high doses of steroids may be important risk factors for CMV reactivation in LTx patients. Our results also suggest that repetitive CMV reactivation may be associated with CKD and pneumonia.

Characteristics of imported Plasmodium ovale spp. and Plasmodium malariae in Hubei Province, China, 2014-2018.

BACKGROUND: There have been an increasing number of imported cases of malaria in Hubei Province in recent years. In particular, the number of cases of Plasmodium ovale spp. and Plasmodium malariae significantly increased, which resulted in increased risks during the malaria elimination phase. The purpose of this study was to acquire a better understanding of the epidemiological characteristics of P. ovale spp. and P. malariae imported to Hubei Province, China, so as to improve case management. METHODS: Data on all malaria cases from January 2014 to December 2018 in Hubei Province were extracted from the China national diseases surveillance information system (CNDSIS). This descriptive study was conducted to analyse the prevalence trends, latency periods, interval from onset of illness to diagnosis, and misdiagnosis of cases of P. ovale spp. and P. malariae malaria. RESULTS: During this period, 634 imported malaria cases were reported, of which 87 P. ovale spp. (61 P. ovale curtisi and 26 P. ovale wallikeri) and 18 P. malariae cases were confirmed. The latency periods of P. ovale spp., P. malariae, Plasmodium vivax, and Plasmodium falciparum differed significantly, whereas those of P. ovale curtisi and P. ovale wallikeri were no significant difference. The proportion of correct diagnosis of P. ovale spp. and P. malariae malaria cases were 48.3% and 44.4%, respectively, in the hospital or lower-level Centers for Disease Control and Prevention (CDC). In the Provincial Reference Laboratory, the sensitivity of microscopy and rapid diagnostic tests was 94.3% and 70.1%, respectively, for detecting P. ovale spp., and 88.9% and 38.9%, respectively, for detecting P. malariae. Overall, 97.7% (85/87) of P. ovale spp. cases and 94.4% (17/18) of P. malariae cases originated from Africa. CONCLUSION: The increase in the number of imported P. ovale spp. and P. malariae cases, long latency periods, and misdiagnosis pose a challenge to this region. Therefore, more attention should be paid to surveillance of imported cases of P. ovale spp. and P. malariae infection to reduce the burden of public health and potential risk of malaria.

Development and application of a combined molecular and tissue culture-based approach to detect latent infectious laryngotracheitis virus (ILTV) in chickens.

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in chickens. ILTV can establish latency and reactivate later in life, but there have been few investigations of ILTV latency. This study aimed to contribute to the methodologies available to detect latent ILTV. A nested PCR was developed which was more sensitive than three other molecular methods investigated in this study. This nested PCR was then used in conjunction with in vitro reactivation culture methods that were optimized and applied to trigeminal ganglia (TG) and tracheal samples from ILTV-vaccinated commercial layer birds (n = 30). ILTV DNA was detected by nested PCR in the upper respiratory tract (URT) or eye of 22 birds. Of the remaining 8 birds, ILTV could be detected by co-culture in TG of 5 birds, with reactivated virus mostly detected 6 days post-explant (dpe). ILTV was also detected in tracheal cultures by 6 dpe. In the ILTV-positive URT samples, the virus could be characterised as vaccine strains SA2 (n = 9) or A20 (n = 5). This study provides evidence for reactivation and shedding of vaccine ILTV in commercial layer birds. Moreover, this study produced a molecular and in-vitro culture method to detect latent viral infection.