Pre-pandemic antibodies screening against SARS-CoV-2 and virus detection among children diagnosed with eruptive fevers.

OBJECTIVES: This study aims to assess the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies against the spike (S) and nucleocapsid (NP) proteins, as well as neutralizing antibodies against the receptor-binding domain (RBD). Additionally, it aims to detect viral RNA of SARS-CoV-2 in pre-pandemic archival pediatric specimens collected before the announcement of the COVID-19 pandemic spread on March 20th, 2020, in Morocco. The objective is to investigate the existence of pre-pandemic immunity to SARS-CoV-2. METHODS: We conducted a cross-sectional study, to analyze IgG antibody levels in a cohort of 106 pre-pandemic pediatric participants. Using an indirect enzyme-linked immunosorbent assay (ELISA), we measured the IgG levels against the S and NP proteins of SARS-CoV-2. Additionally, we staged a competitive ELISA assay to evaluate the neutralizing capability of these antibodies. We used reverse transcription polymerase chain reaction (rRT-PCR) to detect viral NP and ORF1ab genes of SARS-CoV-2 in oropharyngeal swabs. Moreover, we conducted on the same specimens a multiplexed RT-PCR to detect RNA of the most common 27 pathogens involved in lower respiratory tract infections. RESULTS: Among the 106 serum samples, 13% (nn = =14) tested positive for SARS-CoV-2 IgG antibodies using ELISA. Temporal analysis indicated varying IgG positivity levels across 2019. Neutralizing antibodies were found in 21% of the 28 samples analyzed, including two with high inhibition rates (93%). The SARS-CoV-2 RNA was detected using rRT-PCR in 14 samples. None of the samples tested positive for the other 27 pathogens associated with lower respiratory tract infections, using multiplexed RT-PCR. CONCLUSION: Our study addresses the possibility, that COVID-19 infections occurred in Morocco before the recognized outbreak. On the other hand, some of the cases might reflect cross-reactivity with other coronaviruses or be influenced by previous viral exposures or vaccinations. Understanding these factors is crucial to comprehending pediatric immune responses to newly emerging infectious diseases.

Prevalence of HBsAg among Moroccan HIV-1 infected patients and APOBEC3G variant frequencies in HIV-1/HBV co-infection.

INTRODUCTION: Human immunodeficiency virus (HIV) / hepatitis B virus (HBV) causes higher rates of liver disease compared to infection with just one virus. Co-infection can accelerate the progression to liver fibrosis or hepatocellular carcinoma and disturb the treatment response. APOBEC3G is a host defense factor which interferes with HIV-1 and HBV. We aimed to determine the prevalence of hepatitis B surface antigen (HBsAg) among HIV-infected patients and seronegative controls, and screen the HIV/HBV population for APOBEC3G variants rs8177832, rs35228531 and rs2294367, previously associated with HIV-1 infection susceptibility in Morocco. METHODOLOGY: A case control study was conducted on 404 individuals (204 HIV-infected and 200 eligible blood donors) from April to November 2021. HBsAg was measured on the Roche Cobas e411 automatic analyzer (Roche Diagnostics, Basel, Switzerland) and APOBEC3G polymorphisms were identified using the TaqMan genotyping allelic discrimination method. Fisher Exact test, odds ratio (OR) with 95% confidence interval (CI), and haplotype frequencies were calculated. RESULTS: Of the 204 HIV-1 seropositive patients and 200 controls, 4.9% (95%CI: 2.38-8.83) and 2.50% (95% CI: 0.82-5.74) were HBsAg-positive respectively. There was a significant association between increasing age (> 40 years) and HBV infection among controls (p = 0.04). The distribution of genotypes and alleles frequencies of APOBEC3G variants was heterogenous and five different haplotypes with frequencies ≥ 5% were obtained, of which ACC (rs8177832, rs35228531, rs2294367) was the most prevalent. CONCLUSIONS: HBV co-infection is common among HIV-1 infected individuals in Morocco. Efforts should be made to prevent, treat and control HBV transmission in this population.

Assessment of two POC technologies for CD4 count in Morocco.

BACKGROUND: In the era of "test and treat strategy", CD4 testing remains an important tool for monitoring HIV-infected individuals. Since conventional methods of CD4 count measurement are costly and cumbersome, POC CD4 counting technique are more affordable and practical for countries with limited resources. Before introducing such methods in Morocco, we decided to assess their reliability. METHODS: In this study 92 blood samples from HIV-infected patients, were tested by PIMA and FACSPresto to derive CD4 count. Flow cytometry using FacsCalibur, was used as reference method for CD4 count comparison. Linear regression, Bland-Altman analysis were performed to assess correlation and agreement between these POC methods and the reference method. In addition, sensitivity and specificity, positive predictive value (PPV), negative predictive value (NPV) and misclassification percentage at 350 and 200 CD4 count thresholds; were also determined. Finally, because FACSPresto can also measure hemoglobin (Hb) concentration, 52 samples were used to compare FACSPresto against an automated hematology analyzer. RESULTS: The coefficient of determination R2 was 0.93 for both methods. Bland-Altman analysis displayed a mean bias of - 32.3 and - 8.1 cells/µl for PIMA and FACSPresto, respectively. Moreover, with a threshold of 350 CD4 count, PIMA displayed a sensitivity, specificity, PPV, NPV, were 88.57%, 94.12%, 91.18%, 92.31%; respectively. FACSPresto showed 88.23%, 96.23%, 93.75% and 92.73%; respectively. Furthermore, the upward misclassification percentage was 8.57 and 5.88%, for PIMA and FACSPresto, respectively; whereas the downward misclassification percentage was 7.84% and 7.54%; respectively. With 200 cells/µl threshold, PIMA had a sensitivity, specificity, PPV and NPV of 83.33%, 98.53%, 93.75% and 95.71%, respectively. Regarding FACSPresto, sensitivity, specificity, PPV and NPV was 82.35%, 98.57%, 88.57% and 95.83%; respectively. Upward misclassification percentage was 5.56% and 5.88%, for PIMA and FACSPresto, respectively; whereas downward misclassification percentage was 4.41% and 4.29%; respectively. Finally, the hemoglobin measurement evaluation displayed an R2 of 0.80 and a mean bias of - 0.12 with a LOA between - 1.75 and 1.51. CONCLUSION: When compared to the reference method, PIMA and FACSPresto have shown good performance, for CD4 counting. The introduction of such POC technology will speed up the uptake of patients in the continuum of HIV care, in our country.

Seroprevalence of measles vaccine antibody response in vertically HIV-infected children, in Morocco.

BACKGROUND: The widespread use of an effective and safe vaccine to measles has substantially decreased morbidity and mortality from this epidemic. Nevertheless, HIV-infected children vaccinated against measles may develop an impaired vaccine response and remain susceptible to this disease. In Morocco, infants are routinely vaccinated against measles, regardless of their HIV serostatus. An evaluation of the immunization of these children may be of paramount importance to implement timely measures aimed at preventing measles transmission. METHODS: In this study, we have enrolled 114 children vaccinated against measles, 50 children prenatally infected with HIV and 64 HIV-uninfected children. For all children, blood samples were taken to measure anti-measles IgG by EIA and CD4 count by flow cytometry. Additionally, HIV viral load was determined by automated real time PCR, for HIV-infected children. RESULTS: The seroprotective rate of IgG anti-measles antibodies was significantly lower among HIV-infected children (26%) compared with HIV-uninfected children (73%) (p < 0.001). Within HIV-infected children group, the comparison of variables between children without seroprotective seroconversion to measles and those with seroprotective immunity, displayed that sex and age were not statistically different, p > 0.999 and p = 0.730, respectively. However, CD4 count was lower among children with negative serostatus to measles (23% versus 32%, p < 0.001). Furthermore, viral load was higher, with 2.91 log10 ± 2.24 versus 1.7 log10 ± 1.5 (p = 0.042). Finally, 62% of children with a negative vaccine response to measles were under HAART therapy, versus 92% (p = 0.008). CONCLUSION: The majority of HIV-infected children vaccinated against measles develop a suboptimal seroprotective titer, and therefore remain at risk for this highly infectious disease. These data in combination with international recommendations, including recent WHO guidance on vaccination of HIV-infected children, suggest there is a need for national measures to prevent these children from measles.

Assessment of the HBV vaccine response in a group of HIV-infected children in Morocco.

BACKGROUND: Since its development in the early 1980s, Hepatitis B virus (HBV) vaccine has been proven to be highly protective. However, its immunogenicity may be ineffective among HIV-infected children. In Morocco, HBV vaccine was introduced in 1999, and since then all infants, including vertically HIV-infected infants, have been following the vaccination schedule, implemented by the Moroccan ministry of health. An assessment of the immunization of these children is important to optimize efforts aimed at tackling Hepatitis B coinfection, within the country. METHODS: Forty-nine HIV-infected children (HIV group) and 112 HIV uninfected children (control group) were enrolled in this study. Samples were tested by Elisa (Monolisa Anti-HBs, Biorad) to quantify the anti-HBs antibodies. The % of lymphocyte subsets i.e. CD4+ T cells, CD8+ T cells, B cells, and NK, was determined by flow cytometry, using CellQuest Pro software (Becton-Dickinson), and for HIV group, HIV viral load was measured by real time PCR assay (Abbott). All variables were statistically compared in the two groups. RESULTS: The median age was 51 ± 35 months for the HIV group and 50 ± 36 months (p > 0.05) for the control group. Female represented 63% and 41% (p = 0.01), among the HIV group and the control group, respectively. Among HIV-infected children, 71.4% (35/49) were under HAART therapy at the enrollment in the study. Seroprotection titer i.e. anti-HBs ≥10mUI/ml among control group was 76% (85/112), and only 29% (14/49) among the perinatally HIV-infected children (p < 0.0001). Lower % of CD4 + T cells was observed in HIV-infected children with a poor anti-HBs response. CONCLUSION: In this studied group, we have shown that despite the vaccination of HIV-children with HBV vaccine, 71% did not show any seroprotective response. These findings support the need for monitoring HBV vaccine response among HIV-infected children in Morocco, in order to revaccinate non-immunized children.

HIV-1 Subtype distribution in morocco based on national sentinel surveillance data 2004-2005.

BACKGROUND: Little is known about HIV-1 subtype distribution in Morocco. Some data suggest an emergence of new HIV subtypes. We conducted phylogenetic analysis on a nationally representative sample of 60 HIV-1 viral specimens collected during 2004-2005 through the Morocco national HIV sentinel surveillance survey. RESULTS: While subtype B is still the most prevalent, 23.3% of samples represented non-B subtypes, the majority of which were classified as CRF02_AG (15%). Molecular clock analysis confirmed that the initial introduction of HIV-1B in Morocco probably came from Europe in the early 1980s. In contrast, the CRF02_AG strain appeared to be introduced from sub-Saharan Africa in two separate events in the 1990s. CONCLUSIONS: Subtype CRF02_AG has been emerging in Morocco since the 1990s. More information about the factors introducing HIV subtype-specific transmission will inform the prevention strategy in the region.

Evaluation of a dry format reagent alternative for CD4 T-cell enumeration for the FACSCount system: a report on a Moroccan-Canadian study.

BACKGROUND: Efforts to improve alternative CD4 T-cell counting methods are critical to accelerate the implementation of HIV antiretroviral therapy in resources limited regions. Substituting liquid format reagents to eliminate cold-chain transportation and refrigerated storage with dry format reagents contributes to higher efficiency supply management solution especially for laboratories at remote locations. ReaMetrix has developed dry format reagent kits compatible with the FACSCount system, a dedicated flow cytometer for T-cell subset enumeration widely used in resource limited settings. A dual site collaborative study was designed to compare T-cell subsets using both the new dry format ReaMetrix reagent and the original BD Biosciences liquid reagents. METHOD: A total of 167 HIV positive samples prepared with Rea T Count (ReaMetrix) and FACSCount (BD Biosciences) reagents were analyzed using FACSCount Systems. To compare both methods, Bland-Altman, Pollock, Scott % similarity and correlation coefficient statistical analysis was applied. Immuno-Trol served as an assay processing control and quality indicator of interlaboratory and intralaboratory variation. RESULTS: The mean bias and limits of agreement for CD4 T-cell measurements between Rea T Count and FACSCount reagents were -16 cells/microl (-4.6%) and -74 to +43, respectively. The correlation obtained was 0.988 with a similarity of 97.9%. Between laboratory variation data was very good with %CV below 10%. CONCLUSION: The introduction of dry reagents permits the elimination of cold-chain transportation and the on-site refrigerated storage without compromise to assay quality. The substitution of dry reagents facilitates easier supply management practice that will assure wider access to quality HIV treatment.