Drug-Resistant Polymorphisms and Copy Numbers in Plasmodium falciparum, Mozambique, 2015.

One of the fundamental steps toward malaria control is the use of antimalarial drugs. The success of antimalarial treatment can be affected by the presence of drug-resistant populations of Plasmodium falciparum. To assess resistance, we used molecular methods to examine 351 P. falciparum isolates collected from 4 sentinel sites in Mozambique for K13, pfmdr1, pfcrt, and pfdhps polymorphisms and for plasmepsin2 (pfpm2) and pfmdr1 copy numbers. We found multiple copies of pfpm2 in 1.1% of isolates. All isolates carried K13 wild-type alleles (3D7-like), except 4 novel polymorphisms (Leu619Leu, Phe656Ile, Val666Val, Gly690Gly). Prevalence of isolates with pfcrt mutant (K76T) allele was low (2.3%). Prevalence of isolates with pfdhps mutant alleles (A437G and K540E) was >80%, indicating persistence of sulfadoxine/pyrimethamine resistance; however, markers of artemisinin were absent, and markers of piperaquine resistance were low. Piperaquine resistance isolates may spread in Mozambique as dihydroartemisinin/piperaquine drug pressure increases.

A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants.

BACKGROUND: The candidate malaria vaccine RTS,S/AS01 reduced episodes of both clinical and severe malaria in children 5 to 17 months of age by approximately 50% in an ongoing phase 3 trial. We studied infants 6 to 12 weeks of age recruited for the same trial. METHODS: We administered RTS,S/AS01 or a comparator vaccine to 6537 infants who were 6 to 12 weeks of age at the time of the first vaccination in conjunction with Expanded Program on Immunization (EPI) vaccines in a three-dose monthly schedule. Vaccine efficacy against the first or only episode of clinical malaria during the 12 months after vaccination, a coprimary end point, was analyzed with the use of Cox regression. Vaccine efficacy against all malaria episodes, vaccine efficacy against severe malaria, safety, and immunogenicity were also assessed. RESULTS: The incidence of the first or only episode of clinical malaria in the intention-to-treat population during the 14 months after the first dose of vaccine was 0.31 per person-year in the RTS,S/AS01 group and 0.40 per person-year in the control group, for a vaccine efficacy of 30.1% (95% confidence interval [CI], 23.6 to 36.1). Vaccine efficacy in the per-protocol population was 31.3% (97.5% CI, 23.6 to 38.3). Vaccine efficacy against severe malaria was 26.0% (95% CI, -7.4 to 48.6) in the intention-to-treat population and 36.6% (95% CI, 4.6 to 57.7) in the per-protocol population. Serious adverse events occurred with a similar frequency in the two study groups. One month after administration of the third dose of RTS,S/AS01, 99.7% of children were positive for anti-circumsporozoite antibodies, with a geometric mean titer of 209 EU per milliliter (95% CI, 197 to 222). CONCLUSIONS: The RTS,S/AS01 vaccine coadministered with EPI vaccines provided modest protection against both clinical and severe malaria in young infants. (Funded by GlaxoSmithKline Biologicals and the PATH Malaria Vaccine Initiative; RTS,S ClinicalTrials.gov number, NCT00866619.).

Long-lasting insecticidal nets no longer effectively kill the highly resistant Anopheles funestus of southern Mozambique.

BACKGROUND: Chemical insecticides are crucial to malaria control and elimination programmes. The frontline vector control interventions depend mainly on pyrethroids; all long-lasting insecticidal nets (LLINs) and more than 80% of indoor residual spraying (IRS) campaigns use chemicals from this class. This extensive use of pyrethroids imposes a strong selection pressure for resistance in mosquito populations, and so continuous resistance monitoring and evaluation are important. As pyrethroids have also been used for many years in the Manhiça District, an area in southern Mozambique with perennial malaria transmission, an assessment of their efficacy against the local malaria vectors was conducted. METHODS: Female offspring of wild-caught Anopheles funestus s.s. females were exposed to deltamethrin, lambda-cyhalothrin and permethrin using the World Health Organization (WHO) insecticide-resistance monitoring protocols. The 3-min WHO cone bioassay was used to evaluate the effectiveness of the bed nets distributed or available for purchase in the area (Olyset, permethrin LLIN; PermaNet 2.0, deltamethrin LLIN) against An. funestus. Mosquitoes were also exposed to PermaNet 2.0 for up to 8 h in time-exposure assays. RESULTS: Resistance to pyrethroids in An. funestus s.s. was extremely high, much higher than reported in 2002 and 2009. No exposure killed more than 25.8% of the mosquitoes tested (average mortality, deltamethrin: 6.4%; lambda-cyhalothrin: 5.1%; permethrin: 19.1%). There was no significant difference in the mortality generated by 3-min exposure to any net (Olyset: 9.3% mortality, PermaNet 2.0: 6.0%, untreated: 2.0%; p = 0.2). Six hours of exposure were required to kill 50% of the An. funestus s.s. on PermaNet 2.0. CONCLUSIONS: Anopheles funestus s.s. in Manhiça is extremely resistant to pyrethroids, and this area is clearly a pyrethroid-resistance hotspot. This could severely undermine vector control in this district if no appropriate countermeasures are undertaken. The National Malaria Control Programme (NMCP) of Mozambique is currently improving its resistance monitoring programme, to design and scale up new management strategies. These actions are urgently needed, as the goal of the NMCP and its partners is to reach elimination in southern Mozambique by 2020.

Intermittent preventive treatment of malaria in pregnancy with mefloquine in HIV-infected women receiving cotrimoxazole prophylaxis: a multicenter randomized placebo-controlled trial.

BACKGROUND: Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended for malaria prevention in HIV-negative pregnant women, but it is contraindicated in HIV-infected women taking daily cotrimoxazole prophylaxis (CTXp) because of potential added risk of adverse effects associated with taking two antifolate drugs simultaneously. We studied the safety and efficacy of mefloquine (MQ) in women receiving CTXp and long-lasting insecticide treated nets (LLITNs). METHODS AND FINDINGS: A total of 1,071 HIV-infected women from Kenya, Mozambique, and Tanzania were randomized to receive either three doses of IPTp-MQ (15 mg/kg) or placebo given at least one month apart; all received CTXp and a LLITN. IPTp-MQ was associated with reduced rates of maternal parasitemia (risk ratio [RR], 0.47 [95% CI 0.27-0.82]; p=0.008), placental malaria (RR, 0.52 [95% CI 0.29-0.90]; p=0.021), and reduced incidence of non-obstetric hospital admissions (RR, 0.59 [95% CI 0.37-0.95]; p=0.031) in the intention to treat (ITT) analysis. There were no differences in the prevalence of adverse pregnancy outcomes between groups. Drug tolerability was poorer in the MQ group compared to the control group (29.6% referred dizziness and 23.9% vomiting after the first IPTp-MQ administration). HIV viral load at delivery was higher in the MQ group compared to the control group (p=0.048) in the ATP analysis. The frequency of perinatal mother to child transmission of HIV was increased in women who received MQ (RR, 1.95 [95% CI 1.14-3.33]; p=0.015). The main limitation of the latter finding relates to the exploratory nature of this part of the analysis. CONCLUSIONS: An effective antimalarial added to CTXp and LLITNs in HIV-infected pregnant women can improve malaria prevention, as well as maternal health through reduction in hospital admissions. However, MQ was not well tolerated, limiting its potential for IPTp and indicating the need to find alternatives with better tolerability to reduce malaria in this particularly vulnerable group. MQ was associated with an increased risk of mother to child transmission of HIV, which warrants a better understanding of the pharmacological interactions between antimalarials and antiretroviral drugs. TRIAL REGISTRATION: ClinicalTrials.gov NCT 00811421; Pan African Clinical Trials Registry PACTR 2010020001813440 Please see later in the article for the Editors' Summary.

Evaluation of Omnigene-Sputum for Preservation of Sputum Samples for Diagnosis of Mycobacterium tuberculosis.

In several low-income countries, the transport of sputa could take up to one week to reach the laboratories, resulting in increased contamination rates and a loss of growth. The aim of this study was to evaluate the effect of the OMNIgene-SPUTUM in preserving Mycobacterium tuberculosis on sputum samples simulating three hypothetical scenarios for conservation and/or decontamination: (1) sputum was mixed with OMN and conserved at room temperature for five days and then processed for culture (OMN); (2) sputum cultures followed the routine standing operating procedure at day 0 (STD); and (3) sputum samples were kept at room temperature for five days and mixed with the standard decontamination reagent (SDT5) and then processed for culture. The positivity rate based on smear microscopy was 36.4%, 29.1%, and 27.3% for STD, STD5, and OMN, respectively. The proportion of positive results by liquid culture (MGIT) was 39.1% (43/110) for STD, 26.4% (29/110) for STD5, and 20.0% for OMN (22/110). The overall concordance of liquid culture results was 51.8% (57/110): 37.3% (41/110) for negative results, 11.8% (13/110) for MTBC growth, and 2.7% (3/110) for contaminated results. The OMN arm showed better performance in solid culture than in liquid culture, with a notable reduction in contaminated results.

Direct genotyping of Mycobacterium tuberculosis from Xpert® MTB/RIF remnants.

Genotyping of Mycobacterium tuberculosis (MTB) isolates has markedly improved our knowledge of its transmission dynamics. MIRU-VNTR is considered the reference molecular tool for MTB fingerprinting. However, the dependence of this technique on cultured isolates means that we lack molecular epidemiology data from many settings where culture facilities have not been implemented. Efforts have been made to adapt the MIRU-VNTR procedure to direct analysis of clinical specimens, although implementation of these efforts has not proven successful. The large-scale roll-out of Xpert MTB/RIF (Xpert) technology, which is now in almost every TB-endemic country, including many where MTB is not cultured, provides us with a new opportunity to explore whether MTB genotyping could be performed from the remnants of the Xpert cartridge. We ran a pilot study in Mozambique in which the remnants of 24 positive Xpert assays for detection of MTB were used as template material for the 15-locus or the more discriminatory 24-locus MIRU-VNTR technique. MTB fingerprinting was possible in specimens with a high bacterial burden, according to the Xpert load categories, and within the first week after Xpert was performed. Given the wide availability, simple processing, and rapid reporting of results with Xpert, our findings suggest that MIRU-VNTR-based fingerprinting from remnants of Xpert could play a major role in extending MTB molecular epidemiology studies to settings where information on the transmission dynamics of this pathogen is lacking.

Correlation of Xpert MTB/RIF with measures to assess Mycobacterium tuberculosis bacillary burden in high HIV burden areas of Southern Africa.

Traditionally, smear microscopy has been used as a point-of-care measure of bacillary burden in tuberculosis patients to inform infection control and contact tracing. Xpert MTB/RIF has the potential to replace smear. However, data to support the use of its quantitative output [cycle threshold (CT)] as an alternate point-of-care measure of bacillary burden are limited. This study assessed the correlation (Spearman's) between CT, smear, culture time-to-positivity (TTP), and clinical factors in patients with Xpert-positive sputum from Mozambique (n = 238) and South Africa (n = 462). Mean CT and smear grade correlated well (ρ0.72); compared to TTP and smear (ρ0.61); and mean CT and TTP (ρ0.50). In multivariate analyses, lower CT (higher bacillary load) was associated with negative HIV serostatus and low BMI. A smear positivity rule-out (95% sensitivity) CT cut-off of 28.0 was identified, with 54.1% specificity, 2.07 positive likelihood ratio, 0.09 negative likelihood ratio and 79.0% correctly classified. Cut-offs were higher for HIV positive compared to HIV negative individuals for any set sensitivity level. This study suggests Xpert CT values correlate well with smear, both in HIV positive and negative individuals, and that CT cut-offs might be broadly applicable to multiple settings. Studies to directly assess the association of CT with infectiousness are needed.

High Rates of Non-Tuberculous Mycobacteria Isolation in Mozambican Children with Presumptive Tuberculosis.

INTRODUCTION: Non-tuberculous mycobacteria (NTM) can cause disease which can be clinically and radiologically undistinguishable from tuberculosis (TB), posing a diagnostic and therapeutic challenge in high TB settings. We aim to describe the prevalence of NTM isolation and its clinical characteristics in children from rural Mozambique. METHODS: This study was part of a community TB incidence study in children <3 years of age. Gastric aspirate and induced sputum sampling were performed in all presumptive TB cases and processed for smear testing using fluorochrome staining and LED Microscopy, liquid and solid culture, and molecular identification by GenoType® Mycobacterium CM/AS assays. RESULTS: NTM were isolated in 26.3% (204/775) of children. The most prevalent NTM species was M. intracellulare (N = 128), followed by M. scrofulaceum (N = 35) and M. fortuitum (N = 9). Children with NTM were significantly less symptomatic and less likely to present with an abnormal chest radiograph than those with M. tuberculosis. NTM were present in 21.6% of follow-up samples and 25 children had the same species isolated from ≥2 separate samples. All were considered clinically insignificant and none received specific treatment. Children with NTM isolates had equal all cause mortality and likelihood of TB treatment as those with negative culture although they were less likely to have TB ruled out. CONCLUSIONS: NTM isolation is frequent in presumptive TB cases but was not clinically significant in this patient cohort. However, it can contribute to TB misdiagnosis. Further studies are needed to understand the epidemiology and the clinical significance of NTM in children.

Effects of HIV infection on maternal and neonatal health in southern Mozambique: A prospective cohort study after a decade of antiretroviral drugs roll out.

INTRODUCTION: The HIV epidemic is concentrated in sub-Saharan Africa. However, limited information exists on its impact on women and infant's health since the introduction of antiretroviral drugs in this region, where health resources are often scarce. METHODS: The effect of HIV infection on maternal health, birth outcomes and infant health was analysed in two contemporary cohorts of HIV-uninfected and HIV-infected pregnant women from southern Mozambique. Pregnant women attending the first antenatal care visit were followed until one month after delivery. Antiretroviral therapy was administered based on CD4+T cell count and clinical stage. Maternal and neonatal morbidity and mortality, as well as pregnancy outcomes were assessed by mother's HIV status. RESULTS: A total of 1183 HIV-uninfected and 561 HIV-infected pregnant women were enrolled. HIV-infected women were more likely to have anaemia both at the first antenatal care visit and at delivery than HIV-uninfected women (71.5% versus 54.8% and 49.4% versus 40.6%, respectively, p<0.001). Incidence of hospital admissions during pregnancy was increased among HIV-infected women (RR, 2.04, [95%CI, 1.45; 2.86]; p<0.001). At delivery, 21% of HIV-infected women reported being on antiretroviral therapy, and 70% having received antiretroviral drugs for prevention of mother to child transmission of HIV. The risk of stillbirths was doubled in HIV-infected women (RR, 2.16 [95%CI 1.17; 3.96], p = 0.013). Foetal anaemia was also increased among infants born to HIV-infected women (10.6% versus 7.3%, p = 0.022). No differences were found in mean birth weight, malaria, prematurity and maternal and neonatal deaths between groups. CONCLUSIONS: HIV infection continues to be associated with significant maternal morbidity and poor neonatal health outcomes. Efforts should urgently be made to identify the barriers that impede improvements on the devastating effects of HIV in African women and their infants. TRIAL REGISTRATION: ClinicalTrials.gov NCT 00811421.

Correction: High Rates of Non-Tuberculous Mycobacteria Isolation in Mozambican Children with Presumptive Tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0169757.].

IP-10 Kinetics in the First Week of Therapy are Strongly Associated with Bacteriological Confirmation of Tuberculosis Diagnosis in HIV-Infected Patients.

Simple effective tools to monitor the long treatment of tuberculosis (TB) are lacking. Easily measured host derived biomarkers have been identified but need to be validated in larger studies and different population groups. Here we investigate the early response in IP-10 levels (between day 0 and day 7 of TB therapy) to identify bacteriological status at diagnosis among 127 HIV-infected patients starting TB treatment. All participants were then classified as responding or not responding to treatment blindly using a previously described IP-10 kinetic algorithm. There were 77 bacteriologically confirmed cases and 41 Xpert MTB/RIF® and culture negative cases. Most participants had a measurable decline in IP-10 during the first 7 days of therapy. Bacteriologically confirmed cases were more likely to have high IP-10 levels at D0 and had a steeper decline than clinically diagnosed cases (mean decline difference 2231 pg/dl, 95% CI: 897-3566, p = 0.0013). Bacteriologically confirmed cases were more likely to have a measurable decline in IP-10 at day 7 than clinically diagnosed cases (48/77 (62.3%) vs 13/41 (31.7%), p < 0.001). This study confirms the association between a decrease in IP-10 levels during the first week of treatment and a bacteriological confirmation at diagnosis in a large cohort of HIV positive patients.

Do Xpert MTB/RIF Cycle Threshold Values Provide Information about Patient Delays for Tuberculosis Diagnosis?

INTRODUCTION: Early diagnosis and initiation to appropriate treatment is vital for tuberculosis (TB) control. The XpertMTB/RIF (Xpert) assay offers rapid TB diagnosis and quantitative estimation of bacterial burden through Cycle threshold (Ct) values. We assessed whether the Xpert Ct value is associated with delayed TB diagnosis as a potential monitoring tool for TB control programme performance. MATERIALS AND METHODS: This analysis was nested in a prospective study under the routine TB surveillance procedures of the National TB Control Program in Manhiça district, Maputo province, Mozambique. Presumptive TB patients were tested using smear microscopy and Xpert. We explored the association between Xpert Ct values and self-reported delay of Xpert-positive TB patients as recorded at the time of diagnosis enrolment. Patients with >60 days of TB symptoms were considered to have long delays. RESULTS: Of 1,483 presumptive TB cases, 580 were diagnosed as TB of whom 505 (87.0%) were due to pulmonary TB and 302 (94.1%) were Xpert positive. Ct values (range, 9.7-46.4) showed a multimodal distribution. The median (IQR) delay was 30 (30-45) days. Ct values showed no correlation with delay (R2 = 0.001, p = 0.621), nor any association with long delays: adjusted odds ratios (AOR) (95% confidence interval [CI]) comparing to >28 cycles 0.99 (0.50-1.96; p = 0.987) for 23-28 cycles, 0.93 (0.50-1.74; p = 0.828) for 16-22 cycles; and 1.05 (0.47-2.36; p = 0.897) for <16 cycles. Being HIV-negative (AOR [95% CI]), 2.05 (1.19-3.51, p = 0.009) and rural residence 1.74 (1.08-2.81, p = 0.023), were independent predictors of long delays. CONCLUSION: Xpert Ct values were not associated with patient delay for TB diagnosis and cannot be used as an indicator of TB control program performance.

Incidence of Tuberculosis Among Young Children in Rural Mozambique.

BACKGROUND: Tuberculosis (TB) contributes significantly to child morbidity and mortality. This study aimed to estimate the minimum community-based incidence rate of TB among children <3 years of age in Southern Mozambique. METHODS: Between October 2011 and October 2012, in the Manhiça District Health and Demographic Surveillance System, we enrolled prospectively all presumptive TB cases younger than 3 years of age through passive and active case finding. Participants included all children who were either symptomatic or were close contacts of a notified adult smear-positive pulmonary TB. Children were clinically evaluated at baseline and follow-up visits. Investigation for TB disease included chest radiography, HIV and tuberculin skin testing as well as gastric aspirate and induced sputum sampling, which were processed for smear, culture and mycobacterial molecular identification. RESULTS: During the study period, 13,764 children <3 years contributed to a total of 9575 person-year. Out of the 789 presumptive TB cases enrolled, 13 had TB culture confirmation and 32 were probable TB cases. The minimum community-based incidence rate of TB (confirmed plus probable cases) was 470 of 100,000 person-year (95% confidence interval: 343-629 of 100,000). HIV co-infection was present in 44% of the TB cases. CONCLUSION: These data highlight the huge burden of pediatric TB. This study provides one of the first prospective population-based incidence data of childhood tuberculosis and adds valuable information to the global effort of producing better estimates, a critical step to inform public health policy.

Drug-resistant polymorphisms and copy numbers in plasmodium falciparum, mozambique, 2015

One of the fundamental steps toward malaria control is the use of antimalarial drugs. The success of antimalarial treatment can be affected by the presence of drug-resistant populations of Plasmodium falciparum. To assess resistance, we used molecular methods to examine 351 P. falciparum isolates collected from 4 sentinel sites in Mozambique for K13, pfmdr1, pfcrt, and pfdhps polymorphisms and for plasmepsin2 (pfpm2) and pfmdr1 copy numbers. We found multiple copies of pfpm2 in 1.1% of isolates. All isolates carried K13 wild-type alleles (3D7-like), except 4 novel polymorphisms (Leu619Leu, Phe656Ile, Val666Val, Gly690Gly). Prevalence of isolates with pfcrt mutant (K76T) allele was low (2.3%). Prevalence of isolates with pfdhps mutant alleles (A437G and K540E) was >80%, indicating persistence of sulfadoxine/pyrimethamine resistance; however, markers of artemisinin were absent, and markers of piperaquine resistance were low. Piperaquine resistance isolates may spread in Mozambique as dihydroartemisinin/piperaquine drug pressure increases.

Tuberculosis on the rise in southern Mozambique (1997-2012)

Tuberculosis (TB) remains an important public health concern and a leading cause of disease and death worldwide. Mozambique is one of the few high-burden countries where incidence rates have not improved in recent years. The estimated TB incidence rate in 2014 was 552 cases per 100 000 population and the estimated case detection rate is very low at just around 39% [1, 2]. The National Tuberculosis Control Programme (NTP) in Mozambique was launched in 1977 and expanded nationwide in 1985 [3]. The TB patient registration system began in 1984 and short-course therapy followed in the late eighties [3, 4], despite a civil war which resulted in a shortage of qualified medical staff and supplies, and fragile deployment systems. By the year 2000, the Directly Observed Therapy, Short-Course (DOTS) strategy had complete coverage in all districts of Mozambique

Effects of HIV infection on maternal and neonatal health in southern Mozambique: A prospective cohort study after a decade of antiretroviral drugs roll out

The HIV epidemic is concentrated in sub-Saharan Africa. However, limited information exists on its impact on women and infant's health since the introduction of antiretroviral drugs in this region, where health resources are often scarce. Methods: The effect of HIV infection on maternal health, birth outcomes and infant health was analysed in two contemporary cohorts of HIV-uninfected and HIV-infected pregnant women from southern Mozambique. Pregnant women attending the first antenatal care visit were followed until one month after delivery. Antiretroviral therapy was administered based on CD4+T cell count and clinical stage. Maternal and neonatal morbidity and mortality, as well as pregnancy outcomes were assessed by mother's HIV status. Results: A total of 1183 HIV-uninfected and 561 HIV-infected pregnant women were enrolled. HIV-infected women were more likely to have anaemia both at the first antenatal care visit and at delivery than HIV-uninfected women (71.5% versus 54.8% and 49.4% versus 40.6%, respectively, p<0.001). Incidence of hospital admissions during pregnancy was increased among HIV-infected women (RR, 2.04, [95%CI, 1.45; 2.86]; p<0.001). At delivery, 21% of HIV-infected women reported being on antiretroviral therapy, and 70% having received antiretroviral drugs for prevention of mother to child transmission of HIV. The risk of stillbirths was doubled in HIV-infected women (RR, 2.16 [95%CI 1.17; 3.96], p = 0.013). Foetal anaemia was also increased among infants born to HIV-infected women (10.6% versus 7.3%, p = 0.022). No differences were found in mean birth weight, malaria, prematurity and maternal and neonatal deaths between groups. Conclusions: HIV infection continues to be associated with significant maternal morbidity and poor neonatal health outcomes. Efforts should urgently be made to identify the barriers that impede improvements on the devastating effects of HIV in African women and their infants.