[Epidemiological characteristics and clinical features of black African subject's resistant hypertension]. / Caractéristiques épidémio-cliniques de l'hypertension artérielle résistante du sujet noir africain.

INTRODUCTION: Few studies in sub-Saharan Africa were interested in resistant hypertension. The objectives of this study were to determine the frequency of resistant hypertension in hypertensive black African population, and to describe its clinical and therapeutic features. PATIENTS AND METHODS: From May 1, 2010 to May 31, 2012, we included consecutively hypertensive followed in two hospitals in the city of Ouagadougou, under antihypertensive treatment at optimum dose and observant. Patients whose blood pressure was uncontrolled despite a triple antihypertensive therapy at the optimal dose including a diuretic associated with dietary measures have received ambulatory blood pressure monitoring. Following this examination, patients whose blood pressure was ≥135/85mmHg during the day and/or ≥120/70mmHg at night were considered resistant hypertension. We investigated the cardiovascular risk factors as well as target organ damages. We combined spironolactone 50mg in treatment when absence of contra-indication appreciated the evolution of blood pressure under this treatment. The measurement of plasma renin activity was not performed. Statistical analysis was performed using SPSS Version 17 for Windows. RESULTS: We included 692 patients with 14.6% of resistant hypertension. The average age of patients was 54.8±11.1years in the general population, 56.5±11.8years in the subgroup of non-resistant hypertension and 64.2±5.4years in the subgroup of resistant hypertension. The symptoms were represented by headache (11.9%), dizziness (9.9%) and chest pain (8.9%). Modifiable cardiovascular risk factors were dominated by dyslipidemia, diabetes and obesity/overweight. These risk factors were significantly more frequent in the subgroup of resistant hypertension. The global cardiovascular risk was high in 24.9% of cases in the general population, 22.5% in the subgroup of non-resistant hypertension and 38.6% in the subgroup of resistant hypertension. The target organ damages were significantly more frequent in the same subgroup of resistant hypertension. After addition of spironolactone, 21.8% of resistant hypertensive patients were controlled. CONCLUSION: This study shows that resistant hypertension is common in black Africans. It is mostly subjects of the sixth decade, with limited economic income and living in rural areas. In the absence of contra-indication, spironolactone contributed to decrease the morbidity of this pathology.

[Infective endocarditis in sub-Saharan african children, cross-sectional study about 19 cases in Ouagadougou at Burkina Faso]. / Les endocardites infectieuses chez l'enfant africain subsaharien, étude transversale à propos de 19 cas à Ouagadougou au Burkina Faso.

INTRODUCTION: Infective endocarditis is a transplant of a microorganism on a most often injured endocardium. It is rare in children. This work aimed to determine the frequency of endocarditis of the child, to describe clinical presentation, data from echocardiography, microbiological profile and clinical course. PATIENTS AND METHODS: From May 1 2010 to April 30 2011, we consecutively included children received for infective endocarditis in two medical centers in the city of Ouagadougou: Saint-Camille medical center and teaching hospital Yalgado-Ouedraogo. We investigated the functional and general signs and treatment already received. The physical examination looking for an infectious syndrome, pneumonia, heart failure and entrance doors. Blood cultures, blood count, creatinine, blood chemistry, HIV status, electrocardiogram, chest radiography and cardiac Doppler ultrasound were systematic. The diagnosis of the disease was based on Duke criteria. RESULTS: Nineteen endocarditis in children were reported, that is 1.7% of admissions. The average age was 4.7 ± 2.6 years (extremes: 1 and 14). The sex ratio was 1.7 for girls. The clinical presentation was a common infectious syndrome. Impaired general condition and congestive heart failure were present on admission in six cases, respectively. The front door was dental in nine cases (47.4%), skin in four cases (21%) and ENT in three cases (15.8%). A peripheral vein was implicated in one case. In the two other cases, no front door had been found. HIV serology was positive in four cases. As for the blood cultures, they were positive in 13 cases. The germs found were Streptococcus in 10 cases and staphylococcus in three cases. Echocardiography had revealed vegetations in 18 cases. These vegetations were localized on the mitral in nine cases. Multiple locations were found in four cases. Underlying heart disease was dominated by rheumatic valve disease (68.4%), healthy heart forms were found in two cases. Treatment consisted of antibiotics, antipyretic treatment and that of heart failure as appropriate. The evolution was marked by five deaths (26.3%) in an array of septic shock. Death was more important in congenital heart disease. CONCLUSION: Infective endocarditis of the child is common in our practice. The clinical syndrome is common infectious. Streptococcus and Staphylococcus are the two germs found. The main door is dental. Hence, dental care should be promoted for better prevention of infective endocarditis in our context.

[Prevalence of intestinal parasites at Saint-Camille medical center in Ouagadougou (Burkina Faso), 1991 to 2010]. / Prévalence des parasites intestinaux de 1991 à 2010 au centre médical Saint-Camille de Ouagadougou (Burkina Faso).

BACKGROUND: Intestinal parasitoses continue to be a major public health problem in developing countries. OBJECTIVES: This study sought to evaluate the prevalence of intestinal parasites and their coinfection rates in patients attending the Saint-Camille medical center in Ouagadougou. Methodology. This retrospective study covers the period from 1991 through 2010. RESULTS: In all, 292,148 stool samples were analyzed, and 177,672 contained at least one parasite, for a prevalence rate of 60.82%. Protozoans accounted for 90.53% of the parasites identified (160,838) and helminthes 9.47% (16,834). The most common protozoans were Entamœba histolytica/dispar (39.88%), Trichomonas intestinalis (25.78%) and Giardia intestinalis (24.83%). The helminthes encountered most frequently were Hymenolepis nana (3.99%) and Ancylostoma spp (3.65%). Globally, the prevalence of parasites decreased over the 20-year study period. The prevalence of E. histolytica/dispar decreased while that of Giardia intestinalis became more frequent. The most common parasitic associations were E. histolytica/dispar-G. intestinalis (26.24%) and G. intestinalis-T. intestinalis (20.09%). CONCLUSION: Our results indicate that Burkina Faso is a zone at high prevalence of intestinal parasitosis, even though this prevalence appears to be decreasing. Appropriate strategies should be developed to accelerate the reduction in the incidence of these parasites.

[Epidemiology, characterization of genotypes of human papillomavirus in a population of women in Ouagadougou]. / Épidémiologie et caractérisation des génotypes de papillomavirus humain dans une population de femmes à Ouagadougou.

OBJECTIVES: This work was carried out in order to determine the prevalence of different HPV genotypes in a population of women attending gynecological consultation. MATERIAL AND METHODS: From May to June 2010, cervical samples were obtained from 300 women attending gynecological consultation in two health centers in Ouagadougou. The strains of HPV genotyping was done using the technique of polymerase chain reaction (PCR) followed by reverse hybridization on nitrocellulose strips. RESULTS: Among the 73 women(24.3%) infected with HPV, only 27.4% (20/73) of them were infected with a HPV low risk (BR), the 72.6% (53/73). Other women were infected with at least one high risk HPV (HR). By combining the HPV genotypes found without taking into account the number of infected women, we found a total of 84 HPV among whom we have high-risk HPV : HPV-50'S(26/84 or 31.0%), HPV-18 (12/84 or 14.3%), HPV-16 (9/84 or 10.7%), HPV-30'S (5/84 or 5.9%), HPV-HR (5/84 or 5.9%) and HPV-45 (3/84 or 3.6%) and low-risk HPV: HPV-6 (15/84 or 17.9%) and HPV-BR (9/84 or 10.7%). We have found no HPV-11. DISCUSSION AND CONCLUSION: The prevalence of HPV found in our series is comparable to that found in the world. To complete this study, it would be necessary to investigate the prevalence of HPV found in cervical lesions in Burkina Faso.

Prevalence and genotype characterization of human papillomaviruses among HIV-seropositive in Ouagadougou, Burkina Faso.

BACKGROUND: Approximately, 15-20 of 40 HPVs that infect the female genital tract confer a high-risk of invasive cancer, thus HPVs account for 95% of cervix cancers. The objectives of this study were to: (i) estimate the prevalence of HPV infection in women infected with HIV in Ouagadougou, (ii) identify potential carcinogenic HPV strains and (iii) determine whether existing HPV vaccines match the isolated strains. METHODS: From May 2009 to April 2010, 250 HIV-infected women were included in this study. Each woman was screened for the presence of HPV and for HPV genotype using PCR/hybridization technique. RESULTS: Of the 250 HIV-infected women, 59.6% were infected with at least one type of HPV. High-risk HPVs were identified with the following prevalence: HPV-18 (25.0%); HPV-50'S (25.5%); HPV-30'S (20.8%); HPV-16 (4.7%); HPV-45 (3.7%). Low-risk HPVs were represented by HPV-6 (5.7%) and HPV-11 (0.9%). CONCLUSION: The issue of the study showed that the existing vaccines: Gardasil and Cervarix may be used in the country although they match only HPV-16, HPV-18, HPV-6 and HPV-11. Further investigations should be continued for the establishment of vaccine that matches all genotypes circulating in the country.

Knowledge, attitudes and practice (KAP) regarding newborn feeding modalities in HIV-infected and HIV-uninfected pregnant women in sub-Saharan Africa: a multicentre study.

Exclusive formula feeding, exclusive breastfeeding (EBF) with early weaning or the administration of antiretroviral therapy to lactating mothers and/or to breastfed newborns may lower postnatal HIV transmission. The aim of this study was to assess mothers' knowledge, attitudes and practice (KAP) on lactation in various real-life settings in sub-Saharan Africa. A questionnaire survey investigating KAP with regard to breastfeeding in pregnant women of unknown status (Questionnaire A, 16 items) or HIV-infected women (Questionnaire B, 37 items) was administered. Associations between newborn feeding KAP and demographic, socioeconomic, cultural and obstetric variables were investigated. From January 2007 to January 2008, 2112 pregnant women answered Questionnaire A in Burkina Faso, Cameroon, Chad, Tanzania, Uganda and Zambia. Most women (53.0%) declared EBF as the preferred feeding modality. The practice of strictly defined EBF in previous pregnancies was only 11.4%, which was inversely correlated with education and parity. Questionnaire B was answered by 225 HIV-infected pregnant women in Burkina Faso, Tanzania and Uganda. Knowledge about the lactation-associated risk was associated with previous dead children. Significant variability was observed among collaborating sites. The introduction of fluids other than maternal milk within 6 months of age is common practice in sub-Saharan Africa, requiring intensive health education efforts if strictly defined EBF is to be adopted to decrease HIV postnatal transmission. Significant variation in newborn feeding determinants was observed.

[Evolution of antibiotic-resistance Staphylococcus aureus in Saint Camille Medical Centre in Ouagadougou]. / Evolution de la résistance de Staphylococcus aureus aux antibiotiques au Centre Médical Saint Camille de Ouagadougou.

BACKGROUND: Monitoring the antibiotic resistance of microorganisms in a specific geographic area can be useful in developing new approaches to first-intention antibiotherapy. OBJECTIVE: The purpose of this study was to describe the evolution of resistance of Staphylococcus aureus to antibiotics routinely used at Saint Camille Medical Centre in Ouagadougou, Burkina Faso from 1996 to 2006. METHOD: Strains of S. aureus, isolated from various pathologic sources were tested to determine their susceptibility to antibiotics. Sensitivity tests were performed in accordance with the guidelines of the Antibiogram Committee of the French Society for Microbiology (version 2007). RESULTS: During the study period, 1160 staphylococci strains were isolated including 73.45% identified as S. aureus. Susceptibility tests demonstrated a significant increase in resistance to beta-lactam antibiotics. The proportion of strains showing resistance to ampicillin reached 58.29% in 2000. Resistance to these antibiotics regressed significantly from 2000 to 2006. Resistance to pristinamycin and erythromycin showed a tendency to increase while resistance to gentamicin and oleandomycin showed no statistically significant change. CONCLUSION: This study demonstrated that S. aureus was the most common Staphylococcus genus present at the center and that it was resistant to several antibiotics. Reducing use of beta-lactam probably accounted for the significant decline in resistance to this type of antibiotic. Care should also be given to the use of other antibiotics such as pristinamycin and erythromycin since resistance appears to be increasing.

Co-infection of Toxoplasma gondii with HBV in HIV-infected and uninfected pregnant women in Burkina Faso.

Toxoplasma gondii infections can induce serious complications in HIV-infected pregnant women, leading to miscarriage; favour the mother-to-child transmission of HBV and HIV and birth defects. The purposes of this study were: (1) to quantify IgM and IgG antibodies to Toxoplasma gondii in HIV-seropositive and seronegative pregnant women, (2) to identify hepatitis B antigens (HBsAg) in pregnant women and (3) to determine T. gondii and HBV co-infections among these patients. The study was conducted at Centre Medical Saint Camille, in Burkina Faso from January to June 2009. A total of 276 HIV-infected and uninfected pregnant women were included. All women had less than 32 weeks of amenorrhoea and were aged from 19 to 42 years. Toxoplasma gondii antibodies and HBsAg were detected using ELISA method. In addition, women freely agreed to answer a questionnaire. The results of our investigations revealed that, among these pregnant women, 38.8% were illiterates, 50.4% were housewives and only 5.4% were civil servants. Positive T. gondii-specific IgM (4.7%) and IgG (27.2%) were detected. In this study, we found that HIV-seropositive status seem to be associated with great prevalence rates of both T. gondii (31.9 vs. 22.5%) and HBV (13.0 vs. 5.8%). The elevated co-infection rate in HIV-positive women suggested that they are exposed to T. gondii and HBV infections prevalently because of their immune depression. Therefore, to reduce the prevalence of T. gondii and HBV among HIV-seropositive pregnant women, lamivudine could be included in their HEART and women should follow healthy lifestyle formation.

Mother-to-child HIV and HHV-8 transmission in neonates at Saint Camille Medical Centre in Burkina Faso.

In Sub-Saharan Africa, many HIV infected people are co-infected with Human Herpes Virus 8 (HHV-8). Therefore, the present study aimed to: (1) identify the pregnant women co-infected by HIV and HHV-8 at Saint Camille Medical Centre; (2) use three molecules (Zidovudine, Nevirapine and Lamivudine) to interrupt the vertical transmission of HIV and (3) use the PCR technique to diagnose children, who were infected by these viruses, in order to offer them an early medical assistance. A total of 107 pregnant women, aged from 19 to 42 years were diagnosed to be HIV positive at Saint Camille Centre; among them 13 were co-infected with HHV-8. All included women received the HAART. Two to six months after childbirth their babies underwent PCR diagnosis for HIV and HHV-8. The results revealed that, among these mothers, 68.2% were housewives, 34.6% were illiterates and 60.7% did not have university degree. The prevalence of HHV-8 among these pregnant women was 12.15% and the rate of vertical transmission of both HIV and HHV-8, was 0.0%. The issue of this study revealed that the antiretroviral therapy increased the mother CD4 T-cells, prevented the transcription of the mRNA of HHV-8 and blocked HIV vertical transmission.

Aetiology of acute gastro-enteritis in children at Saint Camille Medical Centre, Ouagadougou, Burkina Faso.

UNLABELLED: The present study aims at identifying the infectious agents responsible for child Acute GastroEnteritis (AGE) in Ouagadougou. From May 5 2006 to June 22 2008, 648 children aged from 2 to 41 months, with at least an average of 3 loose stools per day have been enrolled for coproculture, parasitology and virology test. Among them, 34 (5.25%) were HIV seropositive. A single sample of faeces from each child was used to identify enteropathogens. An infectious aetiology was identified in 41.20% of cases. The pathogenic agents detected as responsible for the AGE are: Rotavirus 21.1%; Adenovirus 1.9%; Giardia 7.6% Entamoeba; 1.08%; entero-pathogenic E. coli 41.7%; Salmonella 3.40%; Shigella 1.85% and Yersinia 1.70%. CONCLUSION: Therefore, these AGE etiologic agents constitute a problem of public health in Burkina Faso. Their control for the child would require: (1) a regular paediatric and clinical follow up; (2) health education of the population for food hygiene and (3) in case of absence of HIV infection in the mother, a promotion of exclusive breast-feeding up to the age of 4 months.

Effective program against mother-to-child transmission of HIV at Saint Camille Medical Centre in Burkina Faso.

The present research was aimed to prevent mother-to-child transmission of HIV; to use RT-PCR in order to detect, 6 months after birth, infected children; and to test the antiretroviral resistance of both children and mothers in order to offer them a suitable therapy. At the Saint Camille Medical Centre, 3,127 pregnant women (aged 15-44 years) accepted to be enrolled in the mother-to-child transmission prevention protocol that envisages: (i) Voluntary Counselling and Testing for all the pregnant women; (ii) Antiretroviral therapy for HIV positive pregnant women and for their newborns; (iii) either powdered milk feeding or short breast-feeding and RT-PCR test for their children; (iv) finally, pol gene sequencing and antiretroviral resistance identifications among HIV positive mothers and children. Among the patients, 227/3,127 HIV seropositive women were found: 221/227 HIV-1, 4/227 HIV-2, and 2/227 mixed HIV infections. The RT-PCR test allowed the detection of 3/213 (1.4%) HIV infected children: 0/109 (0%) from mothers under ARV therapy and 3/104 (2.8%) from mothers treated with Nevirapine. All children had recombinant HIV-1 strain (CRF06_CPX) with: minor PR mutations (M36I, K20I) and RT mutations (R211K). Among them, two twins had Non-Nucleoside Reverse Transcriptase Inhibitor mutation (Y18CY). Both mothers acquired a major PR mutation (V8IV), investigated 6 months after a single-dose of Nevirapine. Prevention by single-dose of Nevirapine reduced significantly mother-to-child transmission of HIV, but caused many mutations and resistance to antiretroviral drugs. Based on present study the antiretroviral therapy protocol, together with the artificial-feeding, might represent the ideal strategy to avoid transmission of HIV from mother-to-child.

Epidemiology of neonatal infections.

The World Health Organization estimates that more than 4 million neonates die each year and 98% of these deaths occur in developing countries. Infections are among the main causes of neonatal mortality. The neonatal health is intrinsically linked to the mother's health and the care she receives before, during and immediately after delivery. If in resource-rich countries improvements in perinatal conditions, prevention and management of fetal-neonatal infections have reduced the burden of neonatal morbidity and mortality, in resource-limited areas they have only just improved and many barriers remain still to be overcome. Prematurity is often due to fetal infections and represents a significant risk factor for nosocomial infections.

Characterization of drug-resistance mutations in HIV-1 isolates from non-HAART and HAART treated patients in Burkina Faso.

Non-B HIV subtypes have been estimated to account for 88% of HIV infections in the world. These subtypes are particularly relevant in view of the availability of antiretroviral (ARV) drugs, since subtype-specific mutations are associated with drug-resistance in developing countries. Therefore, the pol gene sequences in HIV-1 isolates were examined from the three distinct groups of 39 infected patients from Ouagadougou in Burkina Faso: 17 patients who had not received any antiretroviral therapy (ART); 16 patients received ART, and 6 HIV-infected children, from infected mothers, received a single Nevirapine dose prophylaxis during birth. HIV-1 pol sequencing was successful for 29 samples. As expected, all patients presented the common (non-B subtype) M36I polymorphism and 26/29 (90%) the K20I mutation. Phylogenetic studies showed high predominance of recombinant HIV-1 strains: CRF06_cpx 16/29 (55.17%), CRF02_AG 9/29 (31.03%), A1 2/29 (6.89%), G 1/29 (3.44%), and CRF09_cpx 1/29 (3.44%). Two twins showed, 6 months after birth, a NNRTI-mutation (Y181C/Y). During the same period, the twin mother presented a different NNRTI-mutation (V106I), thus suggesting that the different blood drug concentration may determine a different drug-resistance pathway. Among 17 non-highly active antiretroviral therapy (HAART) patients, 3/17 (17.64%) presented virus with reverse transcriptase (RT) mutations [V118I: 1/17 patients (5.88%), V179E: 2/17 patients (11.76%)]. 10/17 (58.82%) presented virus with minor protease (PR) mutations [L63P: 5/17 patients (29.41%), V77I: 3/17 patients (17.64%), L10I: 2/17 patients (11.76%)]. 4/17 patients did not show any PR and RT mutations (23.52%). Among six HAART-treated patients, 6/6 and 3/6 had M36I and L63LP protease minor subtypes, respectively; and only two (33.33%) presented virus with K103N mutation. The low prevalence of drug-resistant associated mutations in Burkina Faso is encouraging. However, further studies with a larger cohort with a high non-B subtype prevalence are necessary to optimize ART in developing countries.

Reduction of mother-to-child transmission of HIV at Saint Camille Medical Centre in Burkina Faso.

One thousand three hundred and twenty-eight pregnant women with less than 32 weeks of amenorrhea received voluntary counseling and testing at Saint Camille Medical Center from May 1, 2002 to December 30, 2004. Following informed consent and pre-test counseling, HIV screening was performed in 1,202 women. According to the prevention protocol, HIV-positive women received a single dose of Nevirapine (200 mg) during their labor, while their newborn received a single dose of Nevirapine (2 mg/kg) within 72 hr from birth. HIV seroprevalence (11.2%) was higher than in the overall population. One hundred and ninety-three children were born at the end of December 2004; 53 children (27.5%) followed a short breastfeeding protocol for 4 months, while 140 (72.5%) were fed artificially. All the children underwent RT-PCR test for HIV 5-6 months after their birth: 173 (89.6%) were HIV negative whilst 20 children (10.4%) were HIV positive. Out of the 20 positive children 5/53 (9.4%) had received breast milk for 4 months, while the remaining 15/140 (10.7%) had been fed artificially (P = 0.814). Artificially fed babies (3/140 (2.1%)) and 1/53 (1.9%) of those breast fed for 4 months deceased according to mortality rate of HIV-positive children. This shows that there is no statistically significant difference (P = 0.648) between the mortality of artificially fed (3/140 or 2.1%) and breast-fed (1/53 or 1.9%) children. Artificially fed children (20/140 (14.3%)) and 5/53 (9.4%) of breast-fed children died within 6-10 months. This figure indicates that there is no significant difference between the mortality rate of artificially and that of breast-fed children (P = 0.427). Although the HIV prevention program reduced significantly the vertical transmission of HIV at Saint Camille Medical Center, the mortality of artificially fed children was still high due to gastrointestinal diseases. The HIV diagnosis by RT-PCR technique was of great help in the early identification of HIV-infected children.

Linkage of human cytomegalovirus glycoprotein gO variant groups identified from worldwide clinical isolates with gN genotypes, implications for disease associations and evidence for N-terminal sites of positive selection.

Previously, we identified the glycoprotein gO gene, UL74, as a hypervariable locus in the human cytomegalovirus (HCMV) genome [Virology 293 (2002) 281]. Here, we analyze gO from 50 isolates from congenitally infected newborns, transplant recipients, and HIV/AIDS patients from Italy, Australia, and UK. These are compared to four gO groups described from USA transplantation patients [J. Virol. 76 (2002) 10841]. Phylogenetic analyses identified seven genotypes. Divergence between genotypes was up to 55% and within 3%. Discrete linkage was shown between seven hypervariable gO and gN genotypes, but not with gB. This suggests interactions, while gN and gO are known to form complexes with distinct conserved glycoproteins gM, gH/gL, respectively, both are involved in fusogenic entry and exit. Codon-based maximum likelihood models showed evidence for sites of positive selection. Further analyses of disease relationships should take into account these newly defined gO/gN groups.

Antibiotic resistance in Burkina Faso.

Burkina Faso is one of the Subsaharan African nations. No national services for monitoring of antibiotic resistance are available, so the number of reports of resistance patterns among hospital pathogens are inconsistent. In order to evaluate antibiotic resistance, a total of 1998 valuable microrganisms were analysed during 2000 at the Medical Centre St. Camille of Ouagadougou, Burkina Faso's capital. They were isolated as follows: 1012 from urine-culture, 503 from tonsil swabs, 398 from pus, 53 from sputum and 32 from blood-cultures. Escherichia coli was the most isolated microrganism from urine (44%); Enterococcus faecalis from tonsil swabs (96.4%), Staphylococcus aureus from pus (17%) and K. pneumoniae (70%) from sputum. In general, resistance to the old antibiotics, such as aminopenicillins and cotrimoxazole was shown. The most active antibiotic was norfloxacin, a rarely used antibiotic in this country. In conclusion, our study shows that it is necessary to create antibiotic-resistance surveillance centers in the developing countries to adopt an accurate therapy to avoid exporting of antibiotic resistance to the developed countries linked to increased emigration.

Human cytomegalovirus glycoprotein N (gpUL73-gN) genomic variants: identification of a novel subgroup, geographical distribution and evidence of positive selective pressure.

Human cytomegalvirus (HCMV) ORF UL73 is a polymorphic locus, encoding the viral glycoprotein gpUL73-gN, a component of the gC-II envelope complex. The previously identified gN genomic variants, denoted gN-1, gN-2, gN-3 and gN-4, were further investigated in this work by analysing a large panel of HCMV clinical isolates collected from all over the world (223 samples). Sequencing and phylogenetic analysis confirmed the existence of the four gN genotypes, but also allowed the identification of a novel subgroup belonging to the gN-3 genotype, which was designated gN-3b. The number of non-synonymous (d(N)) and synonymous (d(S)) nucleotide substitutions and their ratio (d(N)/d(S)) were estimated among the gN genotypes to evaluate the possibility of positive selection. Results showed that the four variants evolved by neutral (random) selection, but that the gN-3 and gN-4 genotypes are maintained by positive selective pressure. The 223 HCMV clinical isolates were subdivided according to their geographical origin, and four main regions of gN prevalence were identified: Europe, China, Australia and Northern America. The gN variants were found to be widespread and represented within the regions analysed without any significant difference, and no new genotype was detected. Finally, for clinical and epidemiological purposes, a rapid and low-cost method for genetic grouping of the HCMV clinical isolates was developed based on the RFLP revealed by SacI, ScaI and SalI digestion of the PCR-amplified UL73 sequence. This technique enabled us to distinguish all four gN genomic variants and also their subtypes.

Immunoelectron microscopy analysis of HCMV gpUL73 (gN) localization.

Human Cytomegalovirus (HCMV) UL73 encodes for a polymorphic structural glycoprotein, gpUL73(gN), conserved among herpesviruses. This study analyzed the intracellular and intraviral localization of gpUL73 by immunoelectron-microscopy comparing the reactivity of two different antibodies. We found that gN is an envelope component of the mature viral particle with at least a portion exposed at the virus surface and another at the internal side of the envelope. Furthermore, gpUL73 is also present in the matrix of dense bodies and "black holes". These results, as well as immunoblotting analysis, suggest that the two antibodies recognize different forms, fully processed or unprocessed, of gpUL73-gN.

Analysis of intracellular and intraviral localization of the human cytomegalovirus UL53 protein.

Human cytomegalovirus (HCMV) UL53 belongs to a family of conserved herpesvirus genes. In this work, the expression and localization of the UL53 gene product was analysed. Results obtained showed that pUL53 is a new structural protein. In infected human fibroblasts, pUL53 localizes in cytoplasmic perinuclear granular formations together with other structural viral proteins. In the nucleus, pUL53 forms patches at the nuclear periphery and co-localizes with lamin B at the internal nuclear membrane level. Immunoelectron microscopy studies have disclosed that nuclear pseudo-inclusions are labelled, whereas nucleocapsid formations within the intranuclear skein are negative. Furthermore, the mature virus particle maintains pUL53 at its tegumental level. These data suggest that pUL53 could be involved either in nucleocapsid maturation or in the egress of nucleocapsids from the nucleus to the cytoplasm through the nuclear membrane, a role compatible with the function hypothesized for UL31, its positional homologue in herpes simplex virus type 1.