Intermittent screening and treatment with artemether-lumefantrine versus intermittent preventive treatment with sulfadoxine-pyrimethamine for malaria in pregnancy: a facility-based, open-label, non-inferiority trial in Nigeria.

BACKGROUND: The spread of SP resistance may compromise the effectiveness of intermittent preventive treatment of malaria in pregnancy (MiP) with sulfadoxine-pyrimethamine (IPTp-SP) across Africa. However, there is no recommended alternative medicine for IPTp or alternative strategy for prevention of MiP. This poses problems for the prevention of MiP. This study investigated, whether screening with a rapid diagnostic test for malaria at routine antenatal clinic attendances and treatment of only those who are positive (intermittent screening and treatment) with artemether-lumefantrine is as effective and safe as IPTp-SP in pregnant women. METHODS: During antenatal clinic sessions at the General Hospital Calabar, Nigeria, held between October 2013 and November 2014, 459 pregnant women were randomized into either the current standard IPTp-SP or intermittent screening and treatment with artemether-lumefantrine (ISTp-AL). All women received a long-lasting insecticide-treated net at enrolment. Study women had a maximum of four scheduled visits following enrolment. Haemoglobin concentration and peripheral parasitaemia were assessed in the third trimester (36-40 weeks of gestation). Birth weight was documented at delivery or within a week for babies delivered at home. RESULTS: In the third trimester, the overall prevalence of severe anaemia (Hb < 8 g/dl) and moderate (8-10.9 g/dl) anaemia was 0.8 and 27.7%, respectively, and was similar in both treatment groups (p = 0.204). The risk of third-trimester severe anaemia did not differ significantly between both treatment arms (risk difference - 1.75% [95% CI - 4.16 to 0.66]) although the sample was underpowered for this outcome due to several participants being unavailable to give a blood sample. The risk of third-trimester maternal parasitaemia was significantly lower in the ISTp-AL arm (RD - 3.96% [95% CI - 7.76 to - 0.16]). The risk of low birthweight was significantly lower in the ISTp-AL arm after controlling for maternal age, gravidity and baseline parasitaemia (risk difference - 1.53% [95% CI - 1.54 to - 1.15]). Women in the ISTp-AL arm complained of fever more frequently compared to women in the IPTp-SP arm (p = 0.022). CONCLUSIONS: The trial results suggest that in an area of high malaria transmission with moderate sulfadoxine-pyrimethamine resistance, ISTp with artemether-lumefantrine may be an effective strategy for controlling malaria in pregnancy. Trial registration PACTR, PACTR201308000543272. Registered 29 April 2013, http://www.pactr.org/ATMWeb/appmanager/atm/atmregistry?dar=true&tNo=PACTR201308000543272.

Active case finding: comparison of the acceptability, feasibility and effectiveness of targeted versus blanket provider-initiated-testing and counseling of HIV among children and adolescents in Cameroon.

BACKGROUND: Children and adolescents still lag behind adults in accessing antiretroviral therapy (ART), which is largely due to their limited access to HIV testing services. This study compares the acceptability, feasibility and effectiveness of targeted versus blanket provider-initiated testing and counseling (PITC) among children and adolescents in Cameroon. METHODS: During a 6-month period in three hospitals in Cameroon, we invited HIV-positive parents to have their biological children (6 weeks-19 years) tested for HIV (targeted PITC). During that same period and in the same hospitals, we also systematically offered HIV testing to all children evaluated at the outpatient department (blanket PITC). Children of consenting parents were tested for HIV, and positive cases were enrolled on ART. We compared the acceptability, feasibility and effectiveness of targeted and blanket PITC using Chi-square test at 5% significant level. RESULTS: We enrolled 1240 and 2459 eligible parents in the targeted PITC (tPITC) and blanket PITC (bPITC) group, and 99.7% and 98.8% of these parents accepted the offer to have their children tested for HIV, respectively. Out of the 1990 and 2729 children enrolled in the tPITC and bPITC group, 56.7% and 90.3% were tested for HIV (p < 0.0001), respectively. The HIV positivity rate was 3.5% (CI:2.4-4.5) and 1.6% (CI:1.1-2.1) in the tPITC and bPITC (p = 0.0008), respectively. This finding suggests that the case detection was two times higher in tPITC compared to bPITC, or alternatively, 29 and 63 children have to be tested to identify one HIV case with the implementation of tPITC and bPITC, respectively. The majority (84.8%) of HIV-positive children in the tPITC group were diagnosed earlier at WHO stage 1, and cases were mostly diagnosed at WHO stage 3 (39.1%) (p < 0.0001) in the bPITC group. Among the children who tested HIV-positive, 85.0% and 52.5% from the tPITC and bPITC group respectively, were enrolled on ART (p = 0.0018). CONCLUSIONS: The tPITC and bPITC strategies demonstrated notable high HIV testing acceptance. tPITC was superior to bPITC in terms of case detection, case detection earliness and linkage to care. These findings indicate that tPITC is effective in case detection and linkage of children and adolescents to ART. TRIAL REGISTRATION: Trial registration Number: NCT03024762 . Name of Registry: ClinicalTrial.gov. Date registration: January 19, 2017 ('retrospectively registered'). Date of enrolment first patient: 15/07/2015.

Prevalence of the Pfdhfr and Pfdhps mutations among asymptomatic pregnant women in Southeast Nigeria.

Sulfadoxine-pyrimethamine (SP) is the recommended drug for intermittent preventive treatment of malaria in pregnancy in most of sub-Saharan Africa. Resistance to SP is related to mutations in the dhfr and dhps gene of Plasmodium falciparum. This study determined the prevalence of Pfdhfr and Pfdhps polymorphisms found in asymptomatic pregnant women attending antenatal care in Calabar, Nigeria. From October 2013 to November 2014, asymptomatic pregnant women attending antenatal care clinics were enrolled after obtaining informed consent. Malaria diagnosis testing was done using thick and thin smears. Dried blood spot filter papers were collected. Parasite DNA was extracted from the filter papers using a chelex extraction. Extraction was followed by nested PCR and restriction enzyme digestion. P. falciparum infection was detected by microscopy in 7% (32/459) participants. Twenty-eight P. falciparum isolates were successfully genotyped. In the Pfdhfr gene, the triple mutation was almost fixed; S108N mutation was (100%), N51I (93%) and C59R mutations (93%), whereas the I164L mutation was absent. The prevalence of Pfdhps S436A, A437G, A581G and A613S mutations was 82.1% (23/28), 96.4% (27/28), 71.4% (20/28) and 71.4% (20/28) respectively. The K540E mutation was absent. The prevalence of the Pfdhfr triple mutation IRNI was 92.9% (26/28). The efficacy of SP as IPTp in Southeast Nigeria may be severely threatened. The continuous monitoring of SP molecular markers of resistance is required to assess thresholds. The evaluation of alternative preventive treatment strategies and drug options for preventing malaria in pregnancy may be necessary.

Cytokine response to selected MTB antigens in Ghanaian TB patients, before and at 2 weeks of anti-TB therapy is characterized by high expression of IFN-γ and Granzyme B and inter- individual variation.

BACKGROUND: There has been a long held belief that patients with drug-susceptible TB are non-infectious after two weeks of therapy. Recent microbiological and epidemiological evidence has challenged this dogma, however, the nature of the Mtb-specific cellular immune response during this period has not been adequately investigated. This knowledge could be exploited in the development of immunological biomarkers of early treatment response. METHODS: Cellular response to four Mtb infection phase-dependent antigens, ESAT-6/CFP-10 fusion protein and three DosR encoded proteins (Rv1733c, Rv2029c, Rv2628) were evaluated in a Ghanaian TB cohort (n=20) before and after 2 weeks of anti TB therapy. After 6-days in vitro stimulation, Peripheral blood mononuclear cell (PBMC) culture supernatant was harvested and the concentration of IFN-γ, Granzyme B, IL-10, IL-17, sIL2Rα and TNF-α were determined in a 6-plex Luminex assay. Frequencies of IFN-γ + CD4 and CD8 T cells were also determined in an intracellular cytokine assay. RESULTS: All antigens induced higher levels of IFN-γ, followed by Granzyme B, TNF-α and IL-17 and low levels of IL-10 and sIL-2R-α in PBMC before treatment and after 2 weeks of treatment. Median cytokine levels of IFN-γ, Granzyme B, IL-17 and sIL-2R-α increased during week two, but it was significant for only Rv1733-specific production of Granzyme B (P = 0. 013). The median frequency of antigen specific IFN-γ + CD4 T cells increased at week two; however, only the increase in the ESAT-6/CFP-10-specific response was significant (P = 0. 0008). In contrast, the median frequency of ESAT-6/CFP-10- specific IFN-γ + CD8 T cell responses declined during week two (P = 0. 0024). Additionally, wide inter-individual variation with three distinct patterns were observed; increase in all cytokine levels, decrease in all cytokine levels and fluctuating cytokine levels after 2 weeks of treatment. CONCLUSION: The second week of effective chemotherapy was characterized by a general increase in cytokine response to Mtb-specific antigens suggestive of an improvement in cellular response with therapy. However, the wide inter-individual variation observed would limit the utility of cytokine biomarkers during this period.

Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions.

BACKGROUND: Real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) is a sensitive method for detection of sub-microscopic gametocytaemia by measuring gametocyte-specific mRNA. Performing analysis on fresh whole blood samples is often not feasible in remote and resource-poor areas. Convenient methods for sample storage and transport are urgently needed. METHODS: Real-time QT-NASBA was performed on whole blood spiked with a dilution series of purified in-vitro cultivated gametocytes. The blood was either freshly processed or spotted on filter papers. Gametocyte detection sensitivity for QT-NASBA was determined and controlled by microscopy. Dried blood spot (DBS) samples were subjected to five different storage conditions and the loss of sensitivity over time was investigated. A formula to approximate the loss of Pfs25-mRNA due to different storage conditions and time was developed. RESULTS: Pfs25-mRNA was measured in time to positivity (TTP) and correlated well with the microscopic counts and the theoretical concentrations of the dilution series. TTP results constantly indicated higher amounts of RNA in filter paper samples extracted after 24 hours than in immediately extracted fresh blood. Among investigated storage conditions freezing at -20°C performed best with 98.7% of the Pfs25-mRNA still detectable at day 28 compared to fresh blood samples. After 92 days, the RNA detection rate was only slightly decreased to 92.9%. Samples stored at 37°C showed most decay with only 64.5% of Pfs25-mRNA detectable after one month. The calculated theoretical detection limit for 24 h-old DBS filter paper samples was 0.0095 (95% CI: 0.0025 to 0.0380) per µl. CONCLUSIONS: The results suggest that the application of DBS filter papers for quantification of Plasmodium falciparum gametocytes with real-time QT-NASBA is practical and recommendable. This method proved sensitive enough for detection of sub-microscopic densities even after prolonged storage. Decay rates can be predicted for different storage conditions as well as durations.

Parental and child-level predictors of HIV testing uptake, seropositivity and treatment initiation among children and adolescents in Cameroon.

BACKGROUND: There is a growing body of evidence positioning targeted provider-initiated testing and counselling (tPITC, also known as index case testing) as a promising HIV case-finding and linkage strategy among children and adolescents. However, the effectiveness and efficiency of this strategy is limited by low HIV testing uptake and case detection rates. Despite this fact, there is very little literature on factors associated with HIV testing uptake, HIV seropositivity and ART-enrolment in tPITC implementation among African children. This study aims to bridge this information gap and contribute in improving the effectiveness and efficiency of tPITC among children and adolescents in Cameroon and beyond. METHODS: In three ART clinics where tPITC was previously inexistent, we introduced the routine implementation of this strategy by inviting parents living with HIV/AIDS in care to have their biological children (6 weeks-19 years) HIV-tested. Children of consenting parents were HIV-tested; those testing positive were enrolled on ART. Parental and child-level characteristics associated with HIV testing uptake, seropositivity and ART-enrollment were assessed using bivariate and multivariate regression analysis at 5% significance level. RESULTS: We enrolled 1,236 parents, through whom 1,990 children/adolescents were recruited for HIV testing. Among enrolled parents, 46.2% (571/1,236) had at least one child tested, and 6.8% (39/571) of these parents had at least one HIV-positive child. Among enrolled children/adolescents, 56.7% (1,129/1,990) tested for HIV and 3.5% (40/1129) tested HIV-positive. Parental predictors of HIV testing uptake among children/adolescents were sex, occupation and duration on ART: female [aOR = 1.6 (1.1-2.5)], office workers/students [aOR = 2.0 (1.2-3.3)], and parents with ART duration > 5 years [aOR = 2.0 (1.3-2.9)] had significantly higher odds to test a child than male, farmers/traders, and parents with ART duration < 5 years respectively. The only child-level predictor of testing uptake was age: children < 18 months [aOR = 5(2-10)] had significantly higher odds to test for HIV than adolescents > 15 years. Parents of children identified as HIV-positive were more likely to be female, aged 40-60 years, farmers/traders, widows/divorcees and not on ART. Children found HIV-positive and who were ART-enrolled were more likely to be female and aged 5-9 years. However, none of the above-mentioned associations was statistically significant. CONCLUSIONS: Parents who were male, farmers/traders, and on ART for ≤ 5 years were less likely to test their children for HIV. Also, adolescents 10-19 years old were less likely to be tested. Therefore, these groups should be targeted with intensive counseling and follow-up to facilitate optimal testing uptake. No association was found between parental or child-level characteristics and HIV seropositivity among tested children. This finding prompts for further research to investigate approaches to better identify and target HIV testing to children/adolescents with the highest likelihood of HIV seropositivity. CLINICAL TRIAL REGISTRATION: Reg: CinicalTrials.gov # NCT03024762.

Dry reagent-based polymerase chain reaction compared with other laboratory methods available for the diagnosis of Buruli ulcer disease.

BACKGROUND: Because of the multifaceted clinical presentation of Buruli ulcer disease, misclassification of clinically diagnosed cases may occur frequently. Laboratory tests for the confirmation of suspected cases include microscopic examination, culture, polymerase chain reaction (PCR), and histopathologic examination. However, microscopic examination, the only test usually available in areas of endemicity, has a low sensitivity. METHODS: To make a highly sensitive diagnostic method locally available, dry reagent-based PCR (DRB-PCR), which is well adapted to tropical conditions, was pilot-tested in Ghana. Subsequently, the assay was used for the routine diagnosis of Buruli ulcer disease over a period of 2 years. The method was compared with other diagnostic tests to evaluate its performance under field conditions. RESULTS: The interassay agreement rate between DRB-PCR and standard PCR was 91.7% for swab specimens and 95% for tissue specimens. Among all of the locally available tests, DRB-PCR revealed the highest overall positivity ratio. Sixty percent of patients with clinical diagnoses of Buruli ulcer disease had the diagnoses confirmed by DRB-PCR of swab or tissue specimens, compared with 30%-40% of patients who had diagnoses confirmed by microscopic examination of swab or tissue specimens. The positivity ratio of DRB-PCR varied considerably when analyzed per treatment center. Standardization of specimen collection resulted in a 30% increase in the positivity ratio of the assay, compared with that in the pilot-testing phase. CONCLUSIONS: DRB-PCR is a reliable tool for the diagnosis of Buruli ulcer disease. However, PCR assays are suitable for detection only during early stages of the disease, when samples still contain bacilli. The quality of clinical diagnosis and the quality of diagnostic specimens strongly influence the positivity ratio.