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Influenza viruses know no boundaries, representing an example of rapid virus evolution combined with pressure exerted by the host's immune system. Seasonal influenza causes 4-50 million symptomatic cases in the EU/EEA each year, with a global death toll reaching 650,000 deaths. That being the case, in 2014 North Macedonia introduced the sentinel surveillance in addition to the existing influenza surveillance in order to obtain more precise data on the burden of disease, circulating viruses and to implement timely preventive measures. The aims of this study were to give a comprehensive virological and epidemiological overview of four influenza seasons (2016-2020), assess the frequency and distribution of influenza circulating in North Macedonia and to carry out molecular and phylogenetic analyses of the hemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09, A(H3N2) from ILI and SARI patients. Our results showed that out of 1,632 tested samples, 46.4% were influenza positive, with influenza A(H1N1)pdm09 accounting for the majority of cases (44%), followed by influenza B (32%) and A(H3N2) (17%). By comparing the sentinel surveillance system to the routine surveillance system, we showed that the newly applied system works efficiently and gives great results in the selection of cases. Statistically significant differences (p = < 0.0000001) were observed when comparing the number of reported ILI cases among patients aged 0-4, 5-14, 15-29, and 30-64 years to the reference age group. The phylogenetic analysis of the HA sequences unveiled the resemblance of mutations circulating seasonally worldwide, with a vast majority of circulating viruses belonging to subclade 6B.1A. The PROVEAN analysis showed that the D187A substitution in the receptor binding site (RBS) of the A(H1N1)pdm09 HA has a deleterious effect on the its function. The A(H3N2) viruses fell into the 3C.2a and 3C.3a throughout the analyzed seasons. Molecular characterization revealed that various substitutions in the A(H3N2) viruses gradually replaced the parental variant in subsequent seasons before becoming the dominant variant. With the introduction of sentinel surveillance, accompanied by the advances made in whole-genome sequencing and vaccine therapeutics, public health officials can now modify their approach in disease management and intervene effectively and in a timely manner to prevent major morbidity and mortality from influenza.

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BACKGROUND: The quantitative fluorescent polymerase chain reaction (QF-PCR) has proven to be a reliable method for detection of common fetal chromosomal aneuploidies. However, there are some technical shortcomings, such as uncertainty of aneuploidy determination when the short tandem repeats (STR) height ratio is unusual due to a large size difference between alleles or failure due to the presence of maternal cell contamination (MCC). The aim of our study is to facilitate the implementation of the QF-PCR as a rapid diagnostic test for common fetal aneuploidies. METHODS: Here, we describe an in-house one-tube multiplex QF-PCR method including 20 PCR markers (15 STR markers and 5 fixed size) for rapid prenatal diagnosis of chromosome 13, 18, 21, X and Y aneuploidies. In order to improve the aneuploidy classification of a given diallelic STR marker, we have employed a multilevel logistic regression analysis using "height-ratio" and "allele-size-difference" as fixed effects and "marker" as a random effect. We employed two regression models, one for the 2:1 height ratio (n = 48 genotypes) and another for the 1:2 height ratio (n = 41 genotypes) of the trisomic diallelic markers while using the same 9015 genotypes with normal 1:1 height ratio in both models. Furthermore, we have described a simple procedure for the treatment of the MCC, prior DNA isolation and QF-PCR analysis. RESULTS: For both models, we have achieved 100% specificity for the marker aneuploidy classification as compared to 98.60% (2:1 ratio) and 98.04% (1:2 ratio) specificity when using only the height ratio for classification. Treatment of the MCC enables a successful diagnosis rate of 76% among truly contaminated amniotic fluids. CONCLUSIONS: Adjustment for the allele size difference and marker type improves the STR aneuploidy classification, which, complemented with appropriate treatment of contaminated amniotic fluids, eliminates sample re-testing and reinforces the robustness of the QF-PCR method for prenatal testing.

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AIM: To assess the association between azoospermia factor c microrearrangements and semen quality, and between Y-chromosome background with distinct azoospermia factor c microrearrangements and semen quality impairment. METHODS: This retrospective study, carried out in the Research Center for Genetic Engineering and Biotechnology "Georgi D. Efremov," involved 486 men from different ethnic backgrounds referred for couple infertility from 2002-2017: 338 were azoospermic/oligozoospermic and 148 were normozoospermic. The azoospermia factor c microrearrangements were analyzed with sequence tagged site and sequence family variant markers, quantitative fluorescent polymerase chain reaction, and multiplex ligation probe amplification analysis. The Y-haplogroups of all participants were determined with direct single nucleotide polymorphism typing and indirect prediction with short tandem repeat markers. RESULTS: Our participants had two types of microdeletions: gr/gr and b2/b3; three microduplications: b2/b4, gr/gr, and b2/b3; and one complex rearrangement gr/gr deletion + b2/b4 duplication. Impaired semen quality was not associated with microrearrangements, but b2/b4 and gr/gr duplications were significantly associated with haplogroup R1a (P<0.001 and P=0.003, respectively) and b2/b3 deletions with haplogroup E (P=0.005). There were significantly more b2/b4 duplication carriers in Albanians than in Macedonians with haplogroup R1a (P=0.031). CONCLUSION: Even though azoospermia factor c partial deletions/duplications and Y-haplogroups were not associated with impaired semen quality, specific deletions/duplications were significantly associated with distinct haplogroups, implying that the Y chromosome background may confer susceptibility to azoospermia factor c microrearrangements.

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Previous molecular analyses of α-thalassemia (α-thal) in the Republic of Macedonia have identified the following genetic defects: -α3.7 (rightward), -(α)20.5 and - -MED I deletions and Hb Icaria [α142, Term→Lys (α2), HBA2: c.427T>A] and polyadenylation signal (polyA) [AATAAA>AATGAA (α2), HBA2: c.*92A>G] point mutations. Here, we report two unrelated patients from the Romani population in the Republic of Macedonia, homozygotes for the α2-globin gene variant Hb Agrinio [α29(B10)Leu→Pro; HBA2: c.89T>C]. To date, Hb Agrinio has been described only in individuals of Greek, Cypriot and Spanish origin. Both of our patients had early presentation of the disease (3.5 years and 2 months, respectively) with frequent blood transfusions from early infancy. They have a severe intermediate phenotype of thalassemia (Hb H disease) with hemoglobin (Hb) levels of 7.8 and 7.7 g/dL, respectively. Although the HBA2: c.89T>C mutation results in an α+ allele, the severe phenotype of the homozygotes is due to the production of hyperunstable α chains that undergo post translational precipitation. This leads to a greater degree of red cell damage and hemolytic anemia. The detection of Hb Agrinio in two unrelated families of Romani ethnic origin, may suggest it is a founder mutation in this population living in the Republic of Macedonia. Considering the severity of the clinical presentation of the homozygotes or compound heterozygotes for this rare Hb variant, a targeted molecular screening for Hb Agrinio mutation carriers should be considered in all patients of Romani ethnic origin with manifested microcytosis.

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BACKGROUND: Although age-related loss of chromosome Y (LOY) in normal hematopoietic cells is a well-known phenomenon, the phenotypic consequences of LOY have been elusive. However, LOY has been found in association with smoking, shorter survival and higher risk of cancer. It was suggested that LOY in blood cells could become a predictive biomarker of male carcinogenesis. AIMS, METHODS & FINDINGS: To investigate the association of LOY in blood cells with the risk for development of colorectal (CC) and prostate cancers (PC), we have analyzed DNA samples from peripheral blood of 101 CC male patients (mean age 60.5±11.9 yrs), 70 PC patients (mean age 68.8±8.0 yrs) and 93 healthy control males (mean age 65.8±16.6 yrs). The methodology included co-amplification of homologous sequences on chromosome Y and other chromosomes using multiplex quantitative fluorescent (QF) PCR followed by automatic detection and analysis on ABI 3500 Genetic Analyzer. The mean Y/X ratio was significantly lower in the whole group of cancer patients (0.907±0.12; p = 1.17x10-9) in comparison to the controls (1.015±0.15), as well as in CC (0.884±0.15; p = 3.76x10-9) and PC patients (0.941±0.06; p = 0.00012), when analyzed separately. Multivariate logistic regression analysis adjusting for LOY and age showed that LOY is a more significant predictor of cancer presence than age, and that age probably does not contribute to the increased number of subjects with detectable LOY in cancer patients cohort. CONCLUSION: In conclusion, our results support the recent findings of association of LOY in blood cells with carcinogenesis in males.

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