Correction: Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants.

[This corrects the article DOI: 10.1371/journal.ppat.1006402.].

Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants.

The most immediate and evident effect of mucosal exposure to semen in vivo is a local release of proinflammatory mediators accompanied by an influx of leukocytes into the female genital mucosa (FGM). The implication of such response in HIV-1 transmission has never been addressed due to limitations of currently available experimental models. Using human tissue explants from the uterine cervix, we developed a system of mucosal exposure to seminal plasma (SP) that supports HIV-1 replication. Treatment of ectocervical explants with SP resulted in the upregulation of inflammatory and growth factors, including IL-6, TNF, CCL5, CCL20, CXCL1, and CXCL8, and IL1A, CSF2, IL7, PTGS2, as evaluated by measuring protein levels in explant conditioned medium (ECM) and gene expression in tissue. SP treatment was also associated with increased recruitment of monocytes and neutrophils, as observed upon incubation of peripheral blood leukocytes with ECM in a transwell system. To evaluate the impact of the SP-mediated response on local susceptibility to HIV-1, we infected ectocervical explants with the CCR5-tropic variant HIV-1BaL either in the presence of SP, or after explant pre-incubation with SP. In both experimental settings SP enhanced virus replication as evaluated by HIV-1 p24gag released in explant culture medium over time, as well as by HIV-1 DNA quantification in explants infected in the presence of SP. These results suggest that a sustained inflammatory response elicited by SP soon after coitus may promote HIV-1 transmission to the FGM. Nevertheless, ectocervical tissue explants did not support the replication of transmitted/founder HIV-1 molecular clones, regardless of SP treatment. Our system offers experimental and analytical advantages over traditional models of HIV-1 transmission for the study of SP immunoregulatory effect on the FGM, and may provide a useful platform to ultimately identify new determinants of HIV-1 infection at this site.

Antibody acquisition models: A new tool for serological surveillance of malaria transmission intensity.

Serology has become an increasingly important tool for the surveillance of a wide range of infectious diseases. It has been particularly useful to monitor malaria transmission in elimination settings where existing metrics such as parasite prevalence and incidence of clinical cases are less sensitive. Seroconversion rates, based on antibody prevalence to Plasmodium falciparum asexual blood-stage antigens, provide estimates of transmission intensity that correlate with entomological inoculation rates but lack precision in settings where seroprevalence is still high. Here we present a new and widely applicable method, based on cross-sectional data on individual antibody levels. We evaluate its use as a sero-surveillance tool in a Tanzanian setting with declining malaria prevalence. We find that the newly developed mathematical models produce more precise estimates of transmission patterns, are robust in high transmission settings and when sample sizes are small, and provide a powerful tool for serological evaluation of malaria transmission intensity.

Submicroscopic infections with Plasmodium falciparum during pregnancy and their association with circulating cytokine, chemokine, and cellular profiles.

The immunological consequences of pregnancy-associated malaria (PAM) due to Plasmodium falciparum have been extensively investigated in cross-sectional studies conducted at delivery, but there have been very few longitudinal studies of changes due to PAM during pregnancy. We conducted a prospective study in Benin to investigate the changes associated with PAM in groups of 131 and 111 women at inclusion in the second trimester and at delivery, respectively. Infected women were identified by standard microscopic examinations of blood smears and by quantitative PCR (qPCR) assays and were matched to uninfected control women by age, gestational age, and gravidity. We quantified plasma levels of a panel of soluble immunological mediators and other mediators, as well as the frequencies of peripheral blood mononuclear cell types. Comparisons of these variables in infected and uninfected women used multivariate analyses, and we also assessed the predictive value of variables measured at inclusion for pregnancy outcomes at delivery. In multivariate analyses, peripheral plasma interleukin 10 (IL-10) and gamma interferon-inducible protein 10 (IP-10) levels were associated with PAM at inclusion and at delivery, while higher IL-10 levels distinguished qPCR-detectable submicroscopic infections at inclusion but not at delivery. Maternal anemia at delivery was associated with markers of proinflammatory (increased frequency of monocytes) and anti-inflammatory (increased IL-10 levels and increased activation of regulatory T cells) activity measured at inclusion. Elevated concentrations of IL-10 are associated with the majority of P. falciparum infections during pregnancy, but this marker alone does not identify all submicroscopic infections. Reliably identifying such occult infections will require more sensitive and specific methods.

Haptoglobin phenotype prevalence and cytokine profiles during Plasmodium falciparum infection in Dogon and Fulani ethnic groups living in Mali.

BACKGROUND: The Fulani are known to have a lower parasitaemia and less clinical episodes of malaria as compared to the Dogon sympatric ethnic group, living in Mali. Higher circulating malaria-specific antibody titers and increased pro-inflammatory cytokine levels have been shown in Fulani individuals. Several studies have tried to link haptoglobin (Hp) phenotypes with susceptibility to malaria, but without consensus. This study investigated the role of Hp phenotypes and cytokine levels in Dogon and Fulani during asymptomatic Plasmodium falciparum infection. METHODS: Two different cohorts were combined in this study: a 2008 cohort with 77 children aged between two and ten years and a 2001 cohort, with 82 children and adults, aged between 11 and 68 years. Hp phenotypes in plasma were measured by Western Blot. Circulating levels of sCD163, IL-6, IL-10, IFN-γ and TNF were measured by ELISA. Multiple regression analysis was performed to associate Hp phenotypes with cytokine profiles. In addition, in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with Hp:Hb complexes was performed and cytokine release in corresponding supernatants were measured using cytometric bead array. RESULTS: The results revealed a higher Hp2-2 phenotype prevalence in the Fulani. The Hp2-2 phenotype was associated with a higher susceptibility to P. falciparum infection in Dogon, but not in Fulani. In concordance with previous studies, Fulani showed increased inflammatory mediators (IL-6, IFN-γ) and additionally also increased sCD163 levels compared to Dogon, irrespective of infection. Furthermore, infected individuals showed elevated sCD163 levels compared to uninfected individuals, in both Fulani and Dogon. Multiple regression analysis revealed that the Hp1-1 phenotype was associated with higher levels of TNF and IFN-γ, as compared to the Hp2-2 phenotype. In vitro stimulation of PBMCs with Hb:Hp1-1 complexes resulted in a pro-inflammatory cytokine profile, whilst stimulation with Hb:Hp2-2 complexes showed a more balanced profile. CONCLUSIONS: Ethnicity might be an important confounder on the Hp phenotype-dependent susceptibility to malaria and future studies could consider taking this into account when designing new immunological studies. Although, the relatively small sample size used in this study warrens for precautions in the interpretation of the data and these findings should ideally be validated in a bigger cohort.

Plasmodium falciparum mutant haplotype infection during pregnancy associated with reduced birthweight, Tanzania.

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is a key strategy in the control of pregnancy-associated malaria. However, this strategy is compromised by widespread drug resistance from single-nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. During September 2008-October 2010, we monitored a cohort of 924 pregnant women in an area of Tanzania with declining malaria transmission. P. falciparum parasites were genotyped, and the effect of infecting haplotypes on birthweight was assessed. Of the genotyped parasites, 9.3%, 46.3%, and 44.4% had quadruple or less, quintuple, and sextuple mutated haplotypes, respectively. Mutant haplotypes were unrelated to SP doses. Compared with infections with the less-mutated haplotypes, infections with the sextuple haplotype mutation were associated with lower (359 g) birthweights. Continued use of the suboptimal IPTp-SP regimen should be reevaluated, and alternative strategies (e.g., intermittent screening and treatment or intermittent treatment with safe and effective alternative drugs) should be evaluated.

Malaria and fetal growth alterations in the 3(rd) trimester of pregnancy: a longitudinal ultrasound study.

BACKGROUND: Pregnancy associated malaria is associated with decreased birth weight, but in-utero evaluation of fetal growth alterations is rarely performed. The objective of this study was to investigate malaria induced changes in fetal growth during the 3(rd) trimester using trans-abdominal ultrasound. METHODS: An observational study of 876 pregnant women (398 primi- and secundigravidae and 478 multigravidae) was conducted in Tanzania. Fetal growth was monitored with ultrasound and screening for malaria was performed regularly. Birth weight and fetal weight were converted to z-scores, and fetal growth evaluated as fetal weight gain from the 26th week of pregnancy. RESULTS: Malaria infection only affected birth weight and fetal growth among primi- and secundigravid women. Forty-eight of the 398 primi- and secundigravid women had malaria during pregnancy causing a reduction in the newborns z-score of -0.50 (95% CI: -0.86, -0.13, P = 0.008, multiple linear regression). Fifty-eight percent (28/48) of the primi- and secundigravidae had malaria in the first half of pregnancy, but an effect on fetal growth was observed in the 3(rd) trimester with an OR of 4.89 for the fetal growth rate belonging to the lowest 25% in the population (95%CI: 2.03-11.79, P<0.001, multiple logistic regression). At an individual level, among the primi- and secundigravidae, 27% experienced alterations of fetal growth immediately after exposure but only for a short interval, 27% only late in pregnancy, 16.2% persistently from exposure until the end of pregnancy, and 29.7% had no alterations of fetal growth. CONCLUSIONS: The effect of malaria infections was observed during the 3(rd) trimester, despite infections occurring much earlier in pregnancy, and different mechanisms might operate leading to different patterns of growth alterations. This study highlights the need for protection against malaria throughout pregnancy and the recognition that observed changes in fetal growth might be a consequence of an infection much earlier in pregnancy.

Biomarkers of Plasmodium falciparum infection during pregnancy in women living in northeastern Tanzania.

In pregnant women, Plasmodium falciparum infections are an important cause of maternal morbidity as well as fetal and neonatal mortality. Erythrocytes infected by these malaria-causing parasites accumulate through adhesive interactions in placental intervillous spaces, thus evading detection in peripheral blood smears. Sequestered infected erythrocytes induce inflammation, offering the possibility of detecting inflammatory mediators in peripheral blood that could act as biomarkers of placental infection. In a longitudinal, prospective study in Tanzania, we quantified a range of different cytokines, chemokines and angiogenic factors in peripheral plasma samples, taken on multiple sequential occasions during pregnancy up to and including delivery, from P. falciparum-infected women and matched uninfected controls. The results show that during healthy, uninfected pregnancies the levels of most of the panel of molecules we measured were largely unchanged except at delivery. In women with P. falciparum, however, both comparative and longitudinal assessments consistently showed that the levels of IL-10 and IP-10 increased significantly whilst that of RANTES decreased significantly, regardless of gestational age at the time the infection was detected. ROC curve analysis indicated that a combination of increased IL-10 and IP-10 levels and decreased RANTES levels might be predictive of P. falciparum infections. In conclusion, our data suggest that host biomarkers in peripheral blood may represent useful diagnostic markers of P. falciparum infection during pregnancy, but placental histology results would need to be included to verify these findings.

Factors associated with and causes of perinatal mortality in northeastern Tanzania.

OBJECTIVE: To identify factors associated with perinatal mortality in northeastern Tanzania. DESIGN: Prospective cohort study. SETTING: Northeastern Tanzania. Population. 872 mothers and their newborns. METHODS: Pregnant women were screened for factors possibly associated with perinatal mortality, including preeclampsia, small-for-gestational age, preterm delivery, anemia, and health-seeking behavior. Fetal growth was monitored using ultrasound. Finally, the specific causes of the perinatal deaths were evaluated. MAIN OUTCOME MEASURE: Perinatal mortality. RESULTS: Forty-six deaths occurred. Key factors associated with perinatal mortality were preterm delivery (adjusted odds ratio (OR) 14.47, 95% confidence interval (CI) 3.23-64.86, p < 0.001), small-for-gestational age (adjusted OR 3.54, 95%CI 1.18-10.61, p = 0.02), and maternal anemia (adjusted OR 10.34, 95%CI 1.89-56.52, p = 0.007). Adherence to the antenatal care program (adjusted OR 0.027, 95%CI 0.003-0.26, p = 0.002) protected against perinatal mortality. The cause of death in 43% of cases was attributed to complications related to labor and specifically to intrapartum asphyxia (30%) and neonatal infection (13%). Among the remaining deaths, 27% (7/26) were attributed to preeclampsia and 23% (6/26) to small-for-gestational age. Of these, 54% (14/26) were preterm. CONCLUSIONS: Preeclampsia, small-for-gestational age and preterm delivery were key risk factors and causes of perinatal mortality in this area of Tanzania. Maternal anemia was also strongly associated with perinatal mortality. Furthermore, asphyxia accounted for a large proportion of the perinatal deaths. Interventions should target the prevention and handling of these conditions in order to reduce perinatal mortality.

Reliability of rapid diagnostic tests in diagnosing pregnancy-associated malaria in north-eastern Tanzania.

BACKGROUND: Accurate diagnosis and prompt treatment of pregnancy-associated malaria (PAM) are key aspects in averting adverse pregnancy outcomes. Microscopy is the gold standard in malaria diagnosis, but it has limited detection and availability. When used appropriately, rapid diagnostic tests (RDTs) could be an ideal diagnostic complement to microscopy, due to their ease of use and adequate sensitivity in detecting even sub-microscopic infections. Polymerase chain reaction (PCR) is even more sensitive, but it is mainly used for research purposes. The accuracy and reliability of RDTs in diagnosing PAM was evaluated using microscopy and PCR. METHODS: A cohort of pregnant women in north-eastern Tanzania was followed throughout pregnancy for detection of plasmodial infection using venous and placental blood samples evaluated by histidine rich protein 2 (HRP-2) and parasite lactate dehydrogenase (pLDH) based RDTs (Parascreen™) or HRP-2 only (Paracheck Pf® and ParaHIT®f), microscopy and nested Plasmodium species diagnostic PCR. RESULTS: From a cohort of 924 pregnant women who completed the follow up, complete RDT and microscopy data was available for 5,555 blood samples and of these 442 samples were analysed by PCR. Of the 5,555 blood samples, 49 ((proportion and 95% confidence interval) 0.9% [0.7 -1.1]) samples were positive by microscopy and 91 (1.6% [1.3-2.0]) by RDT. Forty-six (50.5% [40.5 - 60.6]) and 45 (49.5% [39.4 - 59.5]) of the RDT positive samples were positive and negative by microscopy, respectively, whereas nineteen (42.2% [29.0 - 56.7]) of the microscopy negative, but RDT positive, samples were positive by PCR. Three (0.05% [0.02 - 0.2]) samples were positive by microscopy but negative by RDT. 351 of the 5,461 samples negative by both RDT and microscopy were tested by PCR and found negative. There was no statistically significant difference between the performances of the different RDTs. CONCLUSIONS: Microscopy underestimated the real burden of malaria during pregnancy and RDTs performed better than microscopy in diagnosing PAM. In areas where intermittent preventive treatment during pregnancy may be abandoned due to low and decreasing malaria risk and instead replaced with active case management, screening with RDT is likely to identify most infections in pregnant women and out-performs microscopy as a diagnostic tool.

Changes in the levels of cytokines, chemokines and malaria-specific antibodies in response to Plasmodium falciparum infection in children living in sympatry in Mali.

BACKGROUND: The Fulani are known to be less susceptible to Plasmodium falciparum malaria as reflected by lower parasitaemia and fewer clinical symptoms than other sympatric ethnic groups. So far most studies in these groups have been performed on adults, which is why little is known about these responses in children. This study was designed to provide more information on this gap. METHODS: Circulating inflammatory factors and antibody levels in children from the Fulani and Dogon ethnic groups were measured. The inflammatory cytokines; interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor (TNF) and the chemokines; regulated on activation normal T cell expressed and secreted (RANTES), monokine-induced by IFN-gamma (MIG), monocyte chemotactic protein (MCP)-1 and IFN-gamma-inducible protein (IP)-10 were measured by cytometric bead arrays. The levels of interferon (IFN)-alpha, IFN-gamma and malaria-specific antibodies; immunoglobulin (Ig) G, IgM and IgG subclasses (IgG1-IgG4) were measured by ELISA. RESULTS: The results revealed that the Fulani children had higher levels of all tested cytokines compared to the Dogon, in particular IFN-gamma, a cytokine known to be involved in parasite clearance. Out of all the tested chemokines, only MCP-1 was increased in the Fulani compared to the Dogon. When dividing the children into infected and uninfected individuals, infected Dogon had significantly lower levels of RANTES compared to their uninfected peers, and significantly higher levels of MIG and IP-10 as well as MCP-1, although the latter did not reach statistical significance. In contrast, such patterns were not seen in the infected Fulani children and their chemokine levels remained unchanged upon infection compared to uninfected counterparts. Furthermore, the Fulani also had higher titres of malaria-specific IgG and IgM as well as IgG1-3 subclasses compared to the Dogon. CONCLUSIONS: Taken together, this study demonstrates, in accordance with previous work, that Fulani children mount a stronger inflammatory and antibody response against P. falciparum parasites compared to the Dogon and that these differences are evident already at an early age. The inflammatory responses in the Fulani were not influenced by an active infection which could explain why less clinical symptoms are seen in this group.

Interethnic differences in antigen-presenting cell activation and TLR responses in Malian children during Plasmodium falciparum malaria.

The Fulani ethnic group from West Africa is relatively better protected against Plasmodium falciparum malaria as compared to other sympatric ethnic groups, such as the Dogon. However, the mechanisms behind this lower susceptibility to malaria are largely unknown, particularly those concerning innate immunity. Antigen-presenting cells (APCs), and in particular dendritic cells (DCs) are important components of the innate and adaptive immune systems. Therefore, in this study we investigated whether APCs obtained from Fulani and Dogon children exhibited differences in terms of activation status and toll-like receptor (TLR) responses during malaria infection. Lower frequency and increased activation was observed in circulating plasmacytoid DCs and BDCA-3+ myeloid DCs of infected Fulani as compared to their uninfected counterparts. Conversely, a higher frequency and reduced activation was observed in the same DC subsets obtained from peripheral blood of P. falciparum-infected Dogon children as compared to their uninfected peers. Moreover, infected individuals of both ethnic groups exhibited higher percentages of both classical and inflammatory monocytes that were less activated as compared to their non-infected counterparts. In line with APC impairment during malaria infection, TLR4, TLR7 and TLR9 responses were strongly inhibited by P. falciparum infection in Dogon children, while no such TLR inhibition was observed in the Fulani children. Strikingly, the TLR-induced IFN-γ release was completely abolished in the Dogon undergoing infection while no difference was seen within infected and non-infected Fulani. Thus, P. falciparum infection is associated with altered activation status of important APC subsets and strongly inhibited TLR responses in peripheral blood of Dogon children. In contrast, P. falciparum induces DC activation and does not affect the innate response to specific TLR ligands in Fulani children. These findings suggest that DCs and TLR signalling may be of importance for the protective immunity against malaria observed in the Fulani.