Isolation,genotyping and pathogenicity of a Toxoplasma gondii strain isolated from a Serval (Leptailurus serval) in China

Toxoplasma gondii typically causes lifelong chronic infection and has been identified in a variety of intermediate and definitive hosts. Felids are capable of serving as both intermediate and definite hosts for T. gondii infection. However, there is no direct evidence to prove that servals are the intermediate host of T. gondii. In this study, T. gondii antibodies were detected in a serval by a modified agglutination test (titer, 1:200). Viable T. gondii was isolated from the striated muscles of the serval. This strain was further propagated in cell culture and designated as TgServalCHn1. Genetic characterization of DNA derived from cell culture was performed by RFLP‐PCR of 10 markers, as well as polymorphic ROP5 and ROP18 genes. Results showed that this strain of T. gondii belonged to the genotype ToxoDB#20. The ROP5 allele 4 and ROP18 allele 3 suggested that this strain was avirulent, which was further supported by infection in mice. Encephalitis, immune organ necrosis and focal mononuclear cell infiltration in multiple organs were the main pathology characteristics observed in BALB/C mice infected with the TgServalCHn1 strain. To our knowledge, the present study is the first to report the isolation of T. gondii from a serval, which gives direct evidence for servals serving as an intermediate host of T. gondii. The genotyping results revealed the presence of genotype ToxoDB#20 in central China, enriching the scope of the distribution of T. gondii genotypes in Asia.     

Evolutionary puzzle of Toxoplasma gondii with suicidal ideation and suicide attempts: An updated systematic review and meta‐analysis

The World Health Organization has reported an annual global suicide rate of 14.5 per 100,000 people. On the other hand, it is estimated that approximately one‐third of the global population are infected with Toxoplasma gondii (T. gondii) parasite. It is widely assumed that microbial pathogens, such as T. gondii, are probably associated with affective and behavioural modulation. The present article aimed to assess the proposed role of toxoplasmosis in raising the risk of suicidal ideation (SI) and suicide attempts (SA) using the available epidemiological data. Seven major electronic databases and the Internet search engine Google were searched for all the studies published between the 1st of January 1950 and 31st of October 2019. The heterogeneity and the risk of bias within and across studies were assessed. Following data extraction, pooled odds ratios (ORs) with 95% confidence interval (CI) across studies were calculated using the random‐effects models. A total number of 9,696 articles were screened and 27 studies were regarded as eligible in our systematic review (SI with five papers and 22 papers on SA). A significant association was detected between antibodies against T. gondii with TA (ORs = 1.57; 95% confidence interval [CI] 1.23–2.00, p = .000). Exploration of the association between T. gondii and SA yielded a positive effect of seropositivity for IgG antibodies but not IgM. Despite the limited number of studies, a statistical association was detected between suicidal behaviours and infection with latent T. gondii.     

Seroepidemiology of ovine toxoplasmosis and neosporosis in breeding rams from Rio Grande do Sul,Brazil

This study determined the prevalence of ovine toxoplasmosis and neosporosis and the risk factors associated with the development of these diseases in breeding rams from the State of Rio Grande do Sul (RS), Southern Brazil. Serum samples (n = 1,800) from breeding rams maintained on 705 sheep farms from seven mesoregions were evaluated serologically to detect anti‐IgG Toxoplasma gondii by indirect ELISA and anti‐IgG Neospora caninum by the indirect immunofluorescence assay (IFA). The prevalence of T. gondii was 33.05% (595/1,800); seropositivity to N. caninum was 18.44% (332/1,800). Additionally, there was simultaneous seropositivity (8.94%;161/1,800) to N. caninum and T. gondii. The variables size of the property (<500 ha) (Prevalence Ratio, PR = 1.36); breeding system (semi‐intensive/intensive) (PR = 1.23); and natural mounting without control (PR = 1.50) were considered as risk factors for the occurrence of T. gondii. Size of the property (<500 ha) (PR = 1.58) and natural mounting without control (PR = 2.32) were risk factors associated with the prevalence of N. caninum in rams. Additionally, separation of ewes prior to parturition was considered as a protective factor for the occurrence of T. gondii (PR = 0.82) and N. caninum (PR = 0.74). These results demonstrated that these two parasitic disease agents are endemic in rams throughout all regions of RS.     

Multilocus genotyping of Theileria parva isolates associated with a live vaccination trial in Kenya provides evidence for transmission of immunizing parasites into local tick and cattle populations

The live infection and treatment (ITM) vaccination procedure using the trivalent Muguga cocktail is increasingly being used to control East Coast fever, with potential implications for Theileria parva population genetic structure in the field. Transmission of the Kiambu V T. parva component to unvaccinated cattle has previously been described in Uganda. We monitored the T. parva carrier state in vaccinated and control animals on a farm in West Kenya where an ITM stabilate derived from the Kenyan T. parva Marikebuni stock was evaluated for field efficacy. A nested PCR‐based Marikebuni‐specific marker identified a carrier state in nine of ten vaccinated animals, detectable for a period of two years. We used 22 variable number tandem repeat (VNTR) markers to determine multilocus genotypes (MLGs) of 19 T. parva schizont‐infected lymphocyte isolates derived from cattle and field ticks. Two isolates from unimmunized cattle were identical to the Marikebuni vaccination stock. Two cattle isolates were identical to a Muguga cocktail component Kiambu V. Seven isolates from ticks exhibited MLGs that were identical to the Serengeti/Muguga vaccine stocks. Six cattle and two tick‐derived stocks exhibited unique MLGs. The data strongly suggest transmission of immunizing genotypes, from Marikebuni vaccine‐induced carrier cattle to unimmunized cattle. It is possible that genotypes similar to those in the Muguga cocktail are present in the field in Western Kenya. An alternative hypothesis is that these parasites may have originated from vaccine trial sites in Eastern Uganda. If correct, this suggests that T. parva stocks used for immunization can potentially be disseminated 125 km beyond the immediate vaccination site. Regardless of their origin, the data provide evidence that genotypes similar to those in the Muguga cocktail are circulating in the field in East Africa, alleviating concerns about dissemination of ‘alien’ T. parva germplasm through live vaccination.     

Antigen gene and variable number tandem repeat (VNTR) diversity in Theileria parva parasites from Ankole cattle in south‐western Uganda: Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci

Theileria parva is a tick‐transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti‐transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south‐western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen‐encoding genes that are targets of CD8+T‐cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south‐western Uganda. Cattle‐derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved.     

Genetic diversity of Bartonella spp. in vampire bats from Brazil

Recently, an increasing number of Bartonella species have been emerged to cause human diseases. Among animal reservoirs for Bartonella spp., bats stand out due to their high mobility, wide distribution, social behaviour and long‐life span. Although studies on the role of vampire bats in the epidemiology of rabies have been extensively investigated in Latin America, information on the circulation and genetic diversity of Bartonella species in these bat species is scarce. In the present work, 208 vampire bats, namely Desmodus rotundus (the common vampire bat; n = 167), Diphylla ecaudata (the hairy‐legged vampire bat; n = 32) and Diaemus youngii (the white‐winged vampire bat; n = 9) from 15 different states in Brazil were sampled. DNA was extracted from liver tissue samples and submitted to real‐time PCR (qPCR) and conventional PCR (cPCR) assays for Bartonella spp. targeting five genetic loci, followed by phylogenetic and genotype network analyses. Fifty‐one out of 208 liver samples (24.51%) were positive for Bartonella DNA in the ITS real‐time PCR assay [40 (78.43%) of them were from D. rotundus from 11 states, and 11 (21.57%) samples from D. ecaudata from three states. Eleven genotypes were found for each gltA and rpoB genes. Several ITS sequences detected in the present study clustered within the lineage that includes B. bacilliformis and B. ancachensis. The Bayesian phylogenetic inference based on the gltA gene positioned the obtained sequences in six different clades, closely related to Bartonella genotypes previously detected in D. rotundus and associated ectoparasites sampled in Latin America. On the other hand, the Bartonella rpoB genotypes clustered together with the ruminant species, B. schoenbuchensis and B. chomelii. The present study describes for the first time the molecular detection of Bartonella spp. in D. ecaudata bats. It also indicates that Bartonella spp. of vampire bats are genetically diverse and geographically widespread in Brazil.     

First Molecular Identification and Genetic Characterization of Theileria lestoquardi in Sheep of the Maghreb Region

Theileria lestoquardi is the most prominent Theileria species in small ruminants that causes malignant theileriosis of sheep in Africa and Asia. In the present survey, blood samples and ticks were collected in Kebili (southern Tunisia) from 166 Queue Fine de l'Ouest sheep. Giemsa‐stained blood smears, immunofluorescent antibody test (IFAT) and PCR were performed. The DNA was extracted from blood and analysed by PCR targeting 18S rRNA gene of Theileria spp. and then sequenced. A total number of 140 ticks were collected from a total number of 166 sheep during the four seasons. The ticks belonged to two genera and 4 species; the most frequent tick was Hyalomma excavatum 84.3% (118/140) and then Rhipicephalus spp. 15.7% (22/140). Only two animals had positive Giemsa‐stained blood smears, and they were also positive by IFAT. The amplicons had 99.3 and 99.6% homology with the BLAST published T. lestoquardi amplicons. To our knowledge, this is the first report of T. lestoquardi in small ruminants within the Maghreb region.     

High syphilis seropositivity in European brown hares (Lepus europaeus), Lower Saxony,Germany

The lagomorph‐infecting Treponema paraluisleporidarum is a close relative of the human syphilis‐bacterium Treponema pallidum. There is a paucity of information on the epidemiology of hare syphilis and its relationship to the rabbit‐ and human‐infecting treponemes that cause syphilis. In our study, we tested 734 serum samples from European brown hares (Lepus europaeus) collected between 2007 and 2019 in the federal state of Lower Saxony, Germany, for the presence of antibodies against T. paraluisleporidarum. Since T. paraluisleporidarum cross‐reacts with T. pallidum antigen, we used a commercially available T. pallidum‐particle agglutination (TP‐PA) assay to test for the presence of antibodies. A high seropositivity (n = 405/734) was detected. An additional 233 serum samples were retested using a fluorescent treponemal antibody absorption test to confirm the results of the TP‐PA assay. Our results show that infection is widespread in Lower Saxony and suggest a horizontal (sexual) transmission mode since adult hares show significantly higher seropositivity than subadults (odds ratio: 0.03 [95% CI 0.02–0.05], p < .0001). No difference was detected based on gender (odds ratio: 0.79 [95% Cl 0.58–1.07], p = .1283). Further studies are warranted to genetically characterize the T. paraluisleporidarum strains that infect wild hares.     

Investigation of the molecular epizootiological characteristics and tracking of the geographical origins of Brucella canis strains in China

Brucellosis is a global pandemic infectious zoonosis. Brucella canis is a rare source of human brucellosis in China, and its public health significance remains under debate. Moreover, data pertaining to the epizootiological characteristics and geographical origin of B. canis on a nationwide scale are limited, and the risk to public safety posed by B. canis infections is unknown. The MLVA (multilocus variable‐number tandem repeat analysis) assay can be helpful to analyse epidemiological correlations among Brucella isolates and to track their geographic origins. To accomplish this task, MLVA‐16 was used to analyse the epidemiological links of 63 isolates obtained from dogs and humans. Sixty‐three B. canis strains were sorted into three large clusters (A, B and C) and 50 different genotypes (GT1–50), and 43 unique genotypes were represented by single isolates, suggesting that these strains had no obvious epidemiological links and that canine brucellosis is predominantly sporadic in China. The other seven shared genotypes (among a total of 20 isolates) were each represented by two to eight isolates, indicating that strains from each shared genotype were epidemiologically correlated. Five of the shared genotypes were from 16 strains obtained from Beijing, indicating that canine brucellosis in Beijing originates from multipoint outbreaks with multiple sources of infection. Based on comprehensive case analysis of clinical B. canis infection, we preliminarily suggest that human B. canis infections are associated with Mycoplasma pneumoniae infection that results in decreased patient immunity. B. canis may have limited epidemiological significance for the healthy population, but it remains a significant threat to the canine breeding industry and to humans who come into close contact with dogs. Based on MLVA‐11 data, B. canis strains were clustered into 16 genotypes and divided into five evolutionary branches; these data confirm that this population covers an extensive geographic area and exhibits characteristics of the origin and evolution of co‐existing introduced and locally native lineages. We believe this study will contribute to strengthening efforts to prevent and control canine brucellosis and to improve public understanding of the health risks posed by B. canis.     

Sequencing and phylogenetic characterization of Brucella canis isolates,Ohio, 2016

Brucella canis is one of zoonotic pathogens causing infection in human. In this study, we isolated and sequenced 38 B. canis strains from 11 cases. Core genome multilocus sequence typing analysis classified all B. canis isolates into two genogroups, GI and GII . All 38 isolates cluster together forming a 2016 Ohio cluster, in which they form five subclusters reflecting their geographical differences. Unlike GI , the isolates of the GII are from diverse geographical locations including Asia, America, Africa, and Europe and form Asia and South America clusters. Overall, our findings could be useful to investigate and track B. canis of future outbreaks.     

Serosurvey on Schmallenberg Virus and Selected Ovine Reproductive Pathogens in Culled Ewes From Southern Spain

After the first case of Schmallenberg virus (SBV) was reported in southern Spain (March 2012), a retrospective serological study was carried out in extensive sheep flocks from nearby areas to assess the history of exposure to SBV and other selected ovine reproductive pathogens (Chlamydophila abortus, Coxiella burnetii, Border Disease virus ‘BDV’, Toxoplasma gondii and Neospora caninum). Secondly, the presence of antibodies was investigated in meat juice samples against selected pathogens to validate their use in serosurveys in sheep. A total of 209 Merina and cross‐bred culled ewes belonging to 12 outdoor flocks managed in extensive breeding systems were sampled. Serum and meat juice samples were collected at the slaughterhouse and analysed using commercial ELISA kits. Chlamydophila abortus (62.68%, CI₉₅ 56.13–69.23) and Toxoplasma gondii (57.42%, CI₉₅ 50.72–64.12) were the most prevalent pathogens. The seroprevalence of BDV (16.27%, CI₉₅ 11.27–21.27) and Coxiella burnetii (13.88%, CI₉₅ 9.2–18.56) was moderate, and only 4 of 209 animals (1.91%, CI₉₅ 1.82–2.96) presented specific antibodies against Neospora caninum or SBV. All the examined ovine flocks were seropositive to three or more pathogens. The highest percentage of seropositive animals was detected for T. gondii‐C. abortus coseropositive (25.36%) culled ewes. The concordance between serum and meat juice samples was moderate for T. gondii (κ = 0.419) and BDV (κ = 0.568), and fair for C. abortus (κ = 0.311). Our results show evidence of circulation of SBV from summer 2011 in southern Spain. Furthermore, C. abortus and T. gondii were the most prevalent pathogens associated with sheep in outdoor rearing systems. Finally, these preliminary results point to meat juice samples as a potential biological sample for serosurveys studies on sheep.     

HIV knowledge trends during an era of rapid antiretroviral therapy scale‐up: an analysis of 33 sub‐Saharan African countries

Introduction

Population‐level improvements in knowledge about HIV may reduce the stigma attached to HIV and ensure maximal uptake of HIV prevention initiatives. The extent to which levels of HIV knowledge in the general population of sub‐Saharan Africa have changed in the current era of antiretroviral therapy (ART) scale‐up remains unknown.

Methods

Data on HIV knowledge in the general population were drawn from the 2003 to 2015 Demographic and Health Surveys (DHS) and AIDS Indicator Surveys (AIS) of 33 countries in sub‐Saharan Africa. The DHS/AIS contain five questions on HIV prevention and transmission that have been used by the Joint United Nations Programme on HIV/AIDS (UNAIDS) as a core indicator of HIV knowledge. We created a composite HIV knowledge variable equal to the number of correct responses to these five questions; a participant was considered to have comprehensive knowledge of HIV (yes/no) if he/she answered all five questions correctly. We fitted negative binomial regression models with cluster‐correlated robust standard errors and country fixed effects, adjusted for socio‐demographic variables, specifying HIV knowledge as the dependent variable and year as the explanatory variable. As an alternative parameterization, we also fitted a multivariable linear probability model with cluster‐correlated robust standard errors and country fixed effects specifying comprehensive knowledge of HIV as the dependent variable.

Results

A total of 791,186 women and 395,891 men participating in 75 DHS/AIS were included in the analyses. The mean HIV knowledge score was 3.7 among women and 3.9 among men (p < 0.001). Only 35% of women and 41% of men (p < 0.001) had a comprehensive knowledge of HIV. We estimated a modest but statistically significant positive association between year of DHS/AIS and HIV knowledge (adjusted b = 0.005; 95% confidence interval (CI), 0.001 to 0.009). Similarly, we estimated a statistically significant positive association between year of DHS/AIS and comprehensive knowledge of HIV (adjusted b = 0.011; 95% CI, 0.005 to 0.017), suggesting an approximately 1% relative increase per year in the percentage of the general population who possess a comprehensive knowledge of HIV.

Conclusions

There have been minimal improvements over time in HIV knowledge across sub‐Saharan Africa.

     

Association between two single nucleotide polymorphisms of interleukin‐27 gene and increased cryptorchidism risk

Growing evidences have suggested the association between interleukin‐27 and cryptorchidism. We aimed to investigate the relationship between IL‐27 polymorphisms and cryptorchidism susceptibility. A total of 519 males were enrolled in a case–control study (150 cases and 369 normal subjects). The variants were discriminated using polymerase chain reaction–restriction fragment length polymorphism methods. The proportions of the major allele for rs153109 and rs17855750 were A and T with frequencies of 0.56 and 0.85 in cases and 0.51 and 0.91 in controls respectively (P values = 0.002, P value = 0.002). The heterozygous genotype of rs153109 and 17855750 was A/G and T/G with frequencies of 0.62 and 0.25 in cases and 0.39 and 0.17 in controls respectively (P values <0.001, P values <0.001). The A allele and A/G genotype of rs153109 polymorphisms contribute to increase cryptorchidism susceptibility, and G allele and T/G genotype of rs17855750 also contribute to increase cryptorchidism susceptibility, which implies that these allele and genotypes may be risk factors for the development of cryptorchidism.     

Detection of Toxoplasma gondii in the milk of naturally infected goats in the Northeast of Brazil

The aim of the present study was to detect the genomic DNA of Toxoplasma gondii in milk samples from naturally infected goats in the state of Pernambuco, (Brazil). In total, 248 blood serum samples were collected and processed from lactating goats and then submitted to a search for antibodies to T. gondii through the indirect immunofluorescence reaction. Samples with a score of 64 or more were considered positive. In total, 248 milk samples were collected and processed from the same group of goats in order to study the DNA of T. gondii using the polymerase chain reaction (PCR) technique. In the serum samples, 56/248 (22.58%) of the animals were positive, whereas the DNA of the parasite was detected in 15/248 (6.05%) of the milk samples. Five of these 15 samples were animals who were also positive in the serology. This study reports the first occurrence of the elimination of T. gondii from the milk of naturally infected goats in the north‐east of Brazil. It is suggested that the consumption of in natura goat milk may constitute a potential risk to the health of milk consumers in this region.     

Serum‐free in vitro cultivation of Theileria annulata and Theileria parva schizont‐infected lymphocytes

Theileriosis is a tick‐borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata‐infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%–20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch‐to‐batch variation, safety and ethical concerns. In this study, the suitability of serum‐free media for the cultivation of Theileria‐transformed cell lines was examined. Three commercial serum‐free media (HL‐1, ISF‐1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS‐containing RPMI‐1640 medium. ISF‐1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF‐1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF‐1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin‐free, serum‐free, protein‐free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF‐1 medium. The use of serum‐free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case‐by‐case basis. The relevance of Theileria cultivation in serum‐free media for applications such as vaccine development requires further examination.     

Effect of Typha capensis (Rohrb.)N.E.Br. rhizome extract F1 fraction on cell viability,apoptosis induction and testosterone production in TM3‐Leydig cells

Typha capensis (Rohrb.)N.E.Br. (bulrush) is used by traditional healers in Southern Africa to treat male reproductive problems. This study aimed at investigating the effects of T. capensis on TM3‐Leydig cells. T. capensis rhizome crude extract obtained from autumn, winter, spring and summer harvest was fractionated using HPLC into four fractions, and TM3‐Leydig cells were incubated with different concentrations of the F1 fraction (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) for 24, 48 and 96 hr respectively. The following parameters were evaluated: cell morphology, viability (MTT assay), testosterone production (testosterone ELISA test), apoptosis (Annexin V‐Cy3 binding) and DNA fragmentation (TUNEL assay). Results revealed that the summer harvest obtained the highest amount of extract. The F1 fraction of all harvests was the most effective. This fraction significantly enhanced testosterone production in TM3 cells in a dose‐dependent manner with maximum effect at 0.1 μg/ml. At higher concentrations, lower testosterone production was observed. Cell viability including apoptosis was not affected at concentrations used by the traditional healers to treat patients. This study shows that T. capensis enhanced testosterone production and might be useful to treat male infertility and ageing male problems.     

Comprehensiveness of HIV care provided at global HIV treatment sites in the IeDEA consortium: 2009 and 2014

Introduction: An important determinant of the effectiveness of HIV treatment programs is the capacity of sites to implement recommended services and identify systematic changes needed to ensure that invested resources translate into improved patient outcomes. We conducted a survey in 2014 of HIV care and treatment sites in the seven regions of the International epidemiologic Database to Evaluate AIDS (IeDEA) Consortium to evaluate facility characteristics, HIV prevention, care and treatment services provided, laboratory capacity, and trends in the comprehensiveness of care compared to data obtained in the 2009 baseline survey. Methods: Clinical staff from 262 treatment sites in 45 countries in IeDEA completed a site survey from September 2014 to January 2015, including Asia‐Pacific with Australia (n = 50), Latin America and the Caribbean (n = 11), North America (n = 45), Central Africa (n = 17), East Africa (n = 36), Southern Africa (n = 87), and West Africa (n = 16). For the 55 sites with complete data from both the 2009 and 2014 survey, we evaluated change in comprehensiveness of care. Results: The majority of the 262 sites (61%) offered seven essential services (ART adherence, nutritional support, PMTCT, CD4+ cell count testing, tuberculosis screening, HIV prevention, and outreach). Sites that were publicly funded (64%), cared for adults and children (68%), low or middle Human Development Index (HDI) rank (68%, 68%), and received PEPFAR support (71%) were most often fully comprehensive. CD4+ cell count testing was universally available (98%) but only 62% of clinics offered it onsite. Approximately two‐thirds (69%) of sites reported routine viral load testing (44–100%), with 39% having it onsite. Laboratory capacity to monitor antiretroviral‐related toxicity and diagnose opportunistic infections varied widely by testing modality and region. In the subgroup of 55 sites with two surveys, comprehensiveness of services provided significantly increased across all regions from 2009 to 2014 (5.7 to 6.5, p < 0.001). Conclusions: The availability of viral load monitoring remains suboptimal and should be a focus for site capacity, particularly in East and Southern Africa, where the majority of those initiating on ART reside. However, the comprehensiveness of care provided increased over the past 5 years and was related to type of funding received (publicly funded and PEPFAR supported).     

Analysis of Three Functional Polymorphisms in Relation to Osteoporosis Phenotypes: Replication in a Spanish Cohort

Osteoporosis is a complex disease involving many putative genetic factors. Association analysis of functional SNPs in candidate genes is an important tool for their identification. However, this approach is affected by limited power, population stratification, and other drawbacks that lead to discordant results. Replication in independent cohorts is essential. We performed association analyses of three functional polymorphisms previously associated with bone phenotypes—namely, Ala222Val in MTHFR, Ile1062Val in LRP6, and −13910C>T in LCT—in a cohort of 944 postmenopausal Spanish women, all of them with lumbar spine (LS) bone mineral density (BMD) data and most with femoral neck (FN) BMD and fracture data. We found significant differences between genotypes only for the MTHFR polymorphism and vertebral factures, with an OR of 2.27 (95% CI 1.17–4.38) for the TT vs. CC/CT genotypes, P = 0.018. We present genotype and allele frequency data for LCT −13910C>T for a Spanish population, where the T allele (conferring lactase persistence) has a frequency of 38.6%. Genotype frequencies were consistent with observed clines in Europe and with the prevalence of lactase nonpersistence. The LCT −13910C>T polymorphism was significantly associated with height and weight, such that T allele carriers were 0.88 cm taller (95% CI 0.08–1.59 cm, P = 0.032, adjusted by age) than CC individuals and TT homozygotes were 1.91 kg heavier than CC/CT individuals (95% CI 0.11–3.71 kg, P = 0.038, adjusted by age). In conclusion, no significant association was observed between the studied polymorphisms and LS BMD or FN BMD in postmenopausal Spanish women, and only MTHFR Ala222Val was associated with vertebral fractures.