Viruses associated with measles-like illnesses in Uganda.

OBJECTIVES: In this study, we investigated the causes of measles-like illnesses (MLI) in the Uganda national surveillance program in order to inform diagnostic assay selection and vaccination strategies. METHODS: We used metagenomic next-generation sequencing (M-NGS) on the Illumina platform to identify viruses associated with MLI (defined as fever and rash in the presence of either cough, coryza or conjunctivitis) in patient samples that had tested IgM negative for measles between 2010 and 2019. RESULTS: Viral genomes were identified in 87/271 (32%) of samples, of which 44/271 (16%) contained 12 known viral pathogens. Expected viruses included rubella, human parvovirus B19, Epstein Barr virus, human herpesvirus 6B, human cytomegalovirus, varicella zoster virus and measles virus (detected within the seronegative window-period of infection) and the blood-borne hepatitis B virus. We also detected Saffold virus, human parvovirus type 4, the human adenovirus C2 and vaccine-associated poliovirus type 1. CONCLUSIONS: The study highlights the presence of undiagnosed viruses causing MLI in Uganda, including vaccine-preventable illnesses. NGS can be used to monitor common viral infections at a population level, especially in regions where such infections are prevalent, including low and middle income countries to guide vaccination policy and optimize diagnostic assays.

East Coast Fever Carrier Status and Theileria parva Breakthrough Strains in Recently ITM Vaccinated and Non-Vaccinated Cattle in Iganga District, Eastern Uganda.

East Coast fever (ECF) is a tick-borne disease of cattle that hinders the development of the livestock industry in eastern, central and southern Africa. The 'Muguga cocktail' live vaccine, delivered by an infection and treatment method (ITM), remains the only immunisation strategy of controlling ECF. However, there are challenges of the live vaccine inducing ECF carrier status in immunised animals and the possibility of lack of protection from parasite strains that are antigenically different from the vaccine strains. In Uganda, there are insufficient data regarding the ECF carrier status and T. parva genetic diversity in vaccinated and associated non-vaccinated cattle to assess the effectiveness of ITM vaccination. Blood was collected from recently ECF vaccinated (98) and non-vaccinated (73) cattle from Iganga district in Eastern Uganda at 120 days post-vaccination. The p104 gene nested PCR was used to screen for T. parva DNA, 11 minisatellite and 3 microsatellite markers (SSR) were used for genotyping. Two minisatellite markers (MS7 and MS19) were used to determine whether ECF carrier status was due to the T. parva vaccine or local strains. The prevalence of T. parva based on p104 nPCR was 61.2% (60/98) (RR 2.234, 95% CI 1.49-3.35, p-value < 0.001) among recently vaccinated cattle and 27.4% (20/73) (RR 1.00) among associated non-vaccinated cattle. The Muguga cocktail vaccine strains were responsible for carrier status in 10 (58.8%) by MS7 and 11 (64.7%) by MS19 in vaccinated cattle. Genotypes of T. parva with different-sized alleles to the vaccine strains that could be potential 'breakthroughs' were detected in 2 (11.8%)) and 4 (23.5%) isolates from vaccinated cattle based on MS7 and MS19 minisatellite markers, respectively. Using 14 SSR markers, T. parva diversity was higher in vaccinated (Na = 2.214, Ne = 1.978, He = 0.465) than associated non-vaccinated (Na = 1.071, Ne = 1.048, He = 0.259) cattle. The principal component analysis (PCA) showed isolates from vaccinated cattle were closely related to those from non-vaccinated cattle. The analysis of molecular variance (AMOVA) revealed high genetic variation (96%) within T. parva isolates from vaccinated and non-vaccinated cattle but low variation (4%) between vaccinated and non-vaccinated cattle. This study reveals the role of ITM in inducing the carrier status and higher T. parva genetic diversity in vaccinated cattle. The low genetic variation between T. parva isolates in both vaccinated and non-vaccinated cattle may be suggestive of the protective role of vaccine strains against genetically related local strains in the study area.

Assessment of the Impact of Early Diagnosis and Early Treatment in the Integrated Control of East Coast Fever (ECF) Involving Acquired Immunity Induced by Natural Infection in Ankole Cattle.

The integrated control of East Coast fever (ECF) by early diagnosis and treatment involving acquired immunity induced by natural infection in Ankole cattle was assessed. A longitudinal study was carried out in Kiruhura district, southwestern Uganda for six months on 244 Ankole breed of cattle from 18 herds under natural tick challenge and relaxed tick control measures. Calves aged three to six months old were recruited and monitored daily by farmers for detection of ECF clinical symptoms. The reported sick animals were treated using Buparvaquone and treatment outcome determined. Monthly follow-ups and blood collections were done to monitor ECF status. Blood was analyzed for Theileria parva parasites by microscopy, DNA by polymerase chain reaction (PCR) and antibodies by enzyme-linked immunosorbent assay (ELISA). The overall prevalence of ECF clinical disease within six months period was 30.3% (74). The major symptoms of early clinical ECF disease were fever and enlarged parotid or prescapular lymph nodes. Clinical cases were categorized as mild, 24% (18) or moderate, 76% (56). There was an overall recovery rate of 100% (74) of the ECF cases whereby 94.6% (70) recovered promptly and 5.4% (4) recovered slowly. Based on blood analysis, prevalence of ECF at baseline was 3.7% (9) by microscopy, 31.1% (76) by PCR and 38.1% (93) by ELISA. A significant increase (p < 0.05) was shown by the increased number of calves with T. parva specific antibodies in the sera from 38.1% at baseline to 68.8% after six months. High antibody levels (positive percentage ≥ 50%) were detected in all ECF-treated and recovered calves at the end of six months. The acquired immunity to ECF was high in treated and recovered cattle, indicating that natural exposure to infection, accurate early diagnosis and effective treatment enhance development of immune-protection in indigenous cattle in an endemic area. The prominent early clinical symptoms for ECF could be exploited in the development of decision support tools for chemotherapy and other integrated control measures.

The prevalence and genetic characterisation of Cryptosporidium isolates from cattle in Kiruhura district, South Western Uganda.

Cryptosporidium is an emerging opportunistic zoonotic pathogen that causes diarrheal illness in a wide range of hosts including livestock and humans. This study set out to establish the prevalence of Cryptosporidium as well as the circulating genotypes in order to elucidate the potential role of cattle in the spread of human cryptosporidiosis. Rectal coprological samples from 363 cattle in 11 households in Kiruhura district, Southwestern Uganda were collected and screened for the presence of Cryptosporidium oocysts using the phenol auramine staining method followed by fluorescent microscopy. DNA was extracted from the microscopy positive samples and the COWP gene amplified using PCR. PCR products were sequenced and subjected to phylogenetic analysis. Additionally a multiplex realtime PCR was used to identify the Cryptosporidium spp. Multivariable mixed effect logistic regression models were used to identify potential risk factors for Cryptosporidium infection. The overall prevalence of Cryptosporidium was 7.7% (95% CI 5.1-10.9), and herd level prevalence was 33.3% (95% CI 18.5-52.2). We found a statistically significant difference (OR = 30.78, 95% CI 4.31-219.95, p = 0.001) between infection in bulls as compared to cows. There was no significant difference in the prevalence among the different cattle breeds sampled. All the sequenced COWP gene DNA amplicons were confirmed to be C. hominis, with 93%-100% identity to sequences in the GenBank. The amplification of the small subunit rRNA by multiplex realtime PCR further established that the isolates in this study are C. hominis. This study represents the first time naturally occurring C. hominis has been detected from cattle in Uganda.

Performance evaluation of a prototype rapid diagnostic test for combined detection of gambiense human African trypanosomiasis and malaria.

BACKGROUND: Malaria is endemic in all regions where gambiense or rhodesiense human African trypanosomiasis (HAT) is reported, and both diseases have similarities in their symptomatology. A combined test could be useful for both diseases and would facilitate integration of the screening for gambiense HAT (gHAT) and malaria diagnosis. This study aimed to evaluate a combined prototype rapid diagnostic test (RDT) for gHAT and malaria. METHODS: Blood samples were collected in the Democratic Republic of the Congo and in Uganda to evaluate the performance of a prototype HAT/Malaria Combined RDT in comparison to an individual malaria RDT based on Plasmodium falciparum (P.f.) Histidine Rich Protein II (HRP-II or HRP2) antigen (SD BIOLINE Malaria Ag P.f. RDT) for malaria detection and an individual gHAT RDT based on recombinant antigens, the SD BIOLINE HAT 2.0 RDT for HAT screening. Due to the current low prevalence of gHAT in endemic regions, the set of blood samples that were collected was used to evaluate the specificity of the RDTs for gHAT, and additional archived plasma samples were used to complete the evaluation of the HAT/Malaria Combined RDT in comparison to the HAT 2.0 RDT. RESULTS: Frozen whole blood samples from a total of 486 malaria cases and 239 non-malaria controls, as well as archived plasma samples from 246 gHAT positive and 246 gHAT negative individuals were tested. For malaria, the sensitivity and specificity of the malaria band in the HAT/Malaria Combined RDT were 96.9% (95% CI: 95.0-98.3) and 97.1% (95% CI: 94.1-98.8) respectively. The sensitivity and specificity of the SD BIOLINE malaria Ag P.f. RDT were 97.3% (95% CI: 95.5-98.6) and 97.1% (95% CI: 94.1-98.8) respectively. For gHAT, using archived plasma samples, the sensitivity and specificity were respectively 89% (95% CI: 84.4-92.6) and 93.5% (95% CI: 89.7-96.2) with the HAT/Malaria Combined RDT, and 88.2% (95% CI: 83.5-92) and 94.7% (95% CI: 91.1-97.2) with the HAT 2.0 RDT. Using the whole blood samples that were collected during the study, the specificity of the HAT/Malaria Combined RDT for gHAT was 95.8% (95% CI: 94.3-97.0). CONCLUSION: The HAT/Malaria Combined prototype RDT was as accurate as the individual malaria or gHAT RDTs. The HAT/Malaria Combined prototype RDT is therefore suitable for both malaria diagnosis and gHAT screening. However, there is a need to assess its accuracy using fresh samples in prospective clinical trials.

Towards Point-of-Care Diagnostic and Staging Tools for Human African Trypanosomiaisis.

Human African trypanosomiasis is a debilitating disease prevalent in rural sub-Saharan Africa. Control of this disease almost exclusively relies on chemotherapy that should be driven by accurate diagnosis, given the unacceptable toxicity of the few available drugs. Unfortunately, the available diagnostics are characterised by low sensitivities due to the inherent low parasitaemia in natural infections. Demonstration of the trypanosomes in body fluids, which is a prerequisite before treatment, often follows complex algorithms. In this paper, we review the available diagnostics and explore recent advances towards development of novel point-of-care diagnostic tests.

Drug resistance in human African trypanosomiasis.

Human African trypanosomiasis or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.

The diamidine diminazene aceturate is a substrate for the high-affinity pentamidine transporter: implications for the development of high resistance levels in trypanosomes.

African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(-/-), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The K(m) for diminazene transport in bloodstream tbat1(-/-) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(-/-), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes.

Genotypic status of the TbAT1/P2 adenosine transporter of Trypanosoma brucei gambiense isolates from Northwestern Uganda following melarsoprol withdrawal.

BACKGROUND: The development of arsenical and diamidine resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 purine transporter as a result of alterations in the corresponding T. brucei adenosine transporter 1 gene (TbAT1). Previously, specific TbAT1 mutant type alleles linked to melarsoprol treatment failure were significantly more prevalent in T. b. gambiense from relapse patients at Omugo health centre in Arua district. Relapse rates of up to 30% prompted a shift from melarsoprol to eflornithine (alpha-difluoromethylornithine, DFMO) as first-line treatment at this centre. The aim of this study was to determine the status of TbAT1 in recent isolates collected from T. b. gambiense sleeping sickness patients from Arua and Moyo districts in Northwestern Uganda after this shift in first-line drug choice. METHODOLOGY AND RESULTS: Blood and cerebrospinal fluids of consenting patients were collected for DNA preparation and subsequent amplification. All of the 105 isolates from Omugo that we successfully analysed by PCR-RFLP possessed the TbAT1 wild type allele. In addition, PCR/RFLP analysis was performed for 74 samples from Moyo, where melarsoprol is still the first line drug; 61 samples displayed the wild genotype while six were mutant and seven had a mixed pattern of both mutant and wild-type TbAT1. The melarsoprol treatment failure rate at Moyo over the same period was nine out of 101 stage II cases that were followed up at least once. Five of the relapse cases harboured mutant TbAT1, one had the wild type, while no amplification was achieved from the remaining three samples. CONCLUSIONS/SIGNIFICANCE: The apparent disappearance of mutant alleles at Omugo may correlate with melarsoprol withdrawal as first-line treatment. Our results suggest that melarsoprol could successfully be reintroduced following a time lag subsequent to its replacement. A field-applicable test to predict melarsoprol treatment outcome and identify patients for whom the drug can still be beneficial is clearly required. This will facilitate cost-effective management of HAT in rural resource-poor settings, given that eflornithine has a much higher logistical requirement for its application.

Two mutations in dihydrofolate reductase combined with one in the dihydropteroate synthase gene predict sulphadoxine-pyrimethamine parasitological failure in Ugandan children with uncomplicated falciparum malaria.

The point mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) and the dihydropteroate synthase (dhps) genes that are linked to sulphadoxine-pyrimethamine (SP) resistance in vitro have been well characterised. To determine whether a few of these mutations could predict SP treatment failure in vivo, two mutations (Asn-108 and Arg-59) in the dhfr gene and one (Glu-540) in the dhps gene were analysed according to the risk of SP parasitological failure (RI-RIII) at day 28 in pre-treatment isolates in 79 Ugandan children aged 6-59 (mean = 18.4, S.D. = 8.8) months with uncomplicated falciparum malaria. Neither the dhfr-108 (P = 0.3) nor the dhps-540 (P = 0.6) or dhfr-108 + dhps-540 (P = 0.04) mutations were significantly associated with SP parasitological failure. However, the dhfr-108 + dhfr-59 (P = 0.04), the dhfr-59 + dhps-540 (P = 0.04) and the dhfr-108 + dhfr-59 + dhps-540 (P = 0.02) mutations significantly increased the risk for SP parasitological failure. Our findings confirm an earlier report that the dhfr-59 and the dhps-540 mutations could be useful genetic markers for rapid screening of populations at high risk of SP resistance.

Efficacy of sulphadoxine-pyrimethamine alone or combined with amodiaquine or chloroquine for the treatment of uncomplicated falciparum malaria in Ugandan children.

The rapid development of falciparum resistance to sulphadoxine-pyrimethamine (SP) in East and Central Africa has raised concerns as to the efficacy of combining it with another drug. In 2002, we assessed the efficacy of SP alone and combined with amodiaquine (AQ/SP) or chloroquine (CQ/SP) in Ugandan children with uncomplicated falciparum malaria. At day 14, adequate clinical response was 100% (84/84) for AQ/SP, 93% (92/101) for CQ/SP and 91% (73/80) for SP. At day 28, parasitological failure (RI-RIII) occurred in 16% (13/80) of children treated with AQ/SP, in 48% (48/100) of those treated with CQ/SP and in 61% (48/79) of those treated with SP alone. Compared with the AQ/SP arm, the odds for parasitological failure at day 28 were five times higher (95% CI, 2-10) in the CQ/SP group and sevenfold higher (95% CI, 3-17) in that of SP alone. CQ/SP does not offer any significant added benefit over SP alone while AQ/SP is an efficacious low-cost combination. These findings have important policy implications for Uganda and other resource-constrained African countries faced with the problematic choice of a new first-line antimalarial treatment in a context of high CQ resistance.