We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes. Lack of amplification of Pfhrp2, Pfhrp3 and their flanking genes in two P. falciparum field isolates of ...India. Row 1: Lanes 1 and 7=100bp marker, Lanes 6 and 12=negative control (distilled water), Lanes 2–5=Pfhrp2 gene, Lanes 8–11=Pfhrp3 gene. Row 2: Lanes 1 and 7=100bp marker, Lanes 6 and 12=negative control (distilled water), Lanes 2–5=upstream Pfhrp2 gene, Lanes 8–11=downstream Pfhrp2 gene. Display omitted
► Failure to express Pfhrp2 and Pfhrp3 due to gene deletion may contribute to variable performance of malaria RDTs. ► We report two Indian P. falciparum isolates lacking these genes. ► Genetic deletion of Pfhrp2 and Pfhrp3 may lead to false negative results in RDTs.
Genetic polymorphisms in diagnostic antigens are important factors responsible for variable performance of rapid diagnostic tests. Additionally, the failure of antigen expression due to gene deletion may also contribute to variable performance. We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes leading to false negative results of rapid diagnostic tests. The study highlights need to determine the prevalence of P. falciparum isolates lacking these genes in larger field populations in India.
Malaria due to Plasmodium falciparum (Pf) remains a major public threat in India. Artemisinin-based combination therapy (ACT) has been the country's first-line drug for uncomplicated Pf malaria. In ...2013-2014, Artesunate plus sulfadoxine (AS+SP) was replaced by Artemether Lumefantrine (AL) as the first- line antimalarial in North East (NE) states of the country which are endemic for Pf malaria. Regular monitoring of antimalarial drugs is of utmost importance to achieve the goal of elimination. This study aimed to assess the efficacy and safety of ACT for treating uncomplicated Pf malaria in the NE states of India.
A prospective study of 28-day follow-up was conducted to monitor the efficacy and safety of AL from 2018-2019 in four districts, Udalgiri, Meghalaya, Lawngtlai, and Dhalai of NE, India. The clinical and parasitological response and the polymorphism analysis of the Pfdhps, P/dhfr, and Pfkelch 13 gene were evaluated.
A total of 234 patients were enrolled in the study out of 216 patients who completed the follow-up to 28 days. One-hundred percent adequate clinical and parasitological responses (ACPR) were observed with polymerase chain reaction (PCR) correction. The genotype results suggest no recrudescence in the treatment-failure patients. The classical single nucleotide polymorphisms (SNP) in the Pfdhfr gene was S108N (94.9%), followed by C59R (91.5%), whereas, in the Pfdhps gene, the common SNP was A437G (79.6%), followed by S3436A. No associated or validated mutations were found in the propeller region of the PfKelch13 gene.
AL was efficacious and safe in uncomplicated P. falciparum malaria in North East India. In contrast, mutations in the genes responsible for sulfadoxine and pyrimethamine resistance have been fixed in northeast India's population.
•Co-circulation of all four Dengue Virus serotypes in Delhi.•Coinfection of dengue with malaria and chikungunya.•Occurrence of dengue and malaria inversely related.•Post monsoon peak in dengue cases ...and high vector indices
To study the epidemiology of dengue with reference to serological, demographic profile, spatio-temporal distribution, vectors, circulating serotypes and coinfections.
Demographic data and presenting symptoms of fever cases reporting to the clinic were recorded. Suspected patients were tested for dengue, chikungunya and malaria. Dengue specific RT-PCR was performed to detect circulating DENV serotypes. Vector surveys were carried out to detect Aedes breeding.
Of the 5536 fever patients tested during 2012 to 2015, 1536 (27.7%) had confirmed dengue. The peak in dengue positivity was observed during September and October. Of the 60 samples analysed, 10 (16.7%) had concurrent infection with multiple dengue serotypes; one of them had all the four serotypes. Coinfection of dengue with malaria and chikungunya was also observed. The occurrence of dengue and malaria was inversely related. Seven percent of the dengue patients required hospitalization. Vector surveys in the draining area revealed Aedes breeding with a high house index.
Delhi being hyperendemic, the occurrence of concurrent infections with multiple DENV serotypes has become a frequent finding. The study emphasizes the need of epidemiological and entomological surveillance to monitor trends in dengue distribution, seasonal patterns and circulating serotypes to guide dengue control activities.
Parenteral artesunate is the treatment of choice for severe malaria. It is safe, efficacious and well tolerated anti-malarial. However, delayed haemolysis has been reported in travellers, non-immune ...individuals and in African children.
A prospective, observational study was carried out in admitted severe malaria patients receiving parenteral artesunate. The patients were followed up until day 28 for monitoring clinical as well as laboratory parameters for haemolytic anaemia.
Twenty-four patients with severe malaria receiving injection artesunate were enrolled in the study. Post-artesunate delayed haemolysis following parenteral artesunate therapy was observed in three of 24 patients (12.5%, 95% confidence interval 4.5-31.2%). Haemolysis was observed in two more patients possibly due to other reasons. The haemoglobin fall ranged from 13.6 to 38.3% from day 7 to day 28 in these patients.
The possibility of delayed haemolysis should be considered while treating the severe malaria patients with parenteral artesunate. The study highlights the need for further studies in different epidemiological settings.
Rapid diagnostic tests (RDTs) have revolutionized the diagnosis of malaria. Among the various factors affecting RDTs sensitivity is genetic variation of the antigen used. The genetic variation in ...PfHRP2 and PfHRP3 proteins was studied among the Indian Plasmodium falciparum isolates.
One hundred and forty isolates of P. falciparum were collected from six geographical regions of India. Target genes encoding PfHRP2 and PfHRP3 antigens were sequenced to study genetic polymorphism. Minimum detection limit giving a positive rapid diagnostic test was also determined.
Extensive variations were observed in amino acid repeat types of PfHRP2 and PfHRP3. PfHRP2 exhibited more polymorphism than PfHRP3. Significant relation was observed between type 2 and type 7 repeats and RDT detection rate as higher number of these repeats showed better sensitivity with RDTs.
The results provide insights into the genetic diversity of Pfhrp2 and Pfhrp3 genes among Indian P. falciparum population and its relation to RDT sensitivity.
A young male returned from the Democratic Republic of the Congo (DRC) to India after four months during his official work. Within a week of his arrival, he developed a high-grade fever with nausea ...and was hospitalized in a private hospital in New Delhi. He was diagnosed with malaria, treated with an artesunate injection as antimalarial, and discharged on day 5th from the hospital. A week later, he was diagnosed with malaria and dengue positive at ICMR-National Institute of Malaria Research, New Delhi. Artesunate with sulphadoxine and pyrimethamine (AS+SP) was administered following India's malaria treatment policy. However, high-grade fever, along with the asexual stage of the P. falciparum parasite, was observed within 28 days of treatment with AS+SP, signifying late treatment failure (LTF). Further, the molecular analysis from both the days of episodes was analyzed using genomic DNA from dried blood spots, revealing resistance to sulphadoxine-pyrimethamine with mutations at codons pfdhfr 51I, pfdhfr 59 R, pfdhfr 108 N, pfdhps 437 A, pfdhps 581 G. No functional mutation associated was found in pfKelch13, but interestingly the sensitive codons to chloroquine (CQ) (wild type pfcrtK76 and pfmdrN86) revealed the probably reversible CQ sensitivity in the sample from DRC.
In India, the recommended first-line treatment for malaria in the second and third trimester of pregnancy is artesunate + sulfadoxine-pyrimethamine (AS+SP). However, data on safety and efficacy of ...artemisinin-based combination therapy (ACT) in pregnancy is limited. This study assessed the safety and efficacy of AS+SP and artesunate + mefloquine (AS+MQ) for treatment of Plasmodium falciparum in pregnancy in India.
This open-label, randomized clinical trial was conducted from October 2010 to December 2013 at three sites in India (Ranchi and Jamshedpur in Jharkhand state, and Rourkela in Odisha state). Pregnant women in the second or third trimester who had P. falciparum mono-infection of any parasite density with or without fever were randomized to receive AS+SP or AS+MQ. Blood slides and filter paper samples for Polymerase Chain Reaction (PCR) were collected on days 0, 1, 2, 3, 14, 21, 28, 42 and 63 post treatment. Women were followed up at delivery and at day 42 postpartum.
Two hundred and forty-eight women of 7064 pregnant women (3.5%) who were screened at monthly antenatal clinics had a P. falciparum mono-infection and were randomized to receive AS+SP (125) or AS+MQ (123) and all of these women were included in the intention to treat (ITT) analysis. The primary endpoint of an adequate clinical and parasite response (ACPR) on day 63 was not available for 9 women who were counted as treatment failure in the ITT analysis. In the ITT population, the ACPR was 121/125 (96.8%; 95% Confidence interval (CI) 92.0-99.1%) in the AS+SP group and 117/123 (95.1%; 95% CI 89.7-98.2) in the AS+MQ group. Among the 239 women (121 from the AS+SP arm and 118 from the AS+MQ arm) who completed the day 63 follow up (per protocol analysis) the ACPR was 100% in the AS+SP group and 99.2% (117/118) in the AS+MQ group. There were five serious adverse events (SAE) among pregnant women (4 in the AS+SP group and 1 in the AS+MQ group) and 13 fetal/neonatal SAEs (7 in the AS+SP group and 6 in the AS+MQ) but none of them were related to the study drugs. A higher proportion of women in the AS+MQ arm reported vomiting within 7 days post-treatment than did women in the AS+SP arm (6.9 vs. 1.6%; p = 0.001).
Both AS+SP and AS+MQ are safe and effective for treatment of uncomplicated falciparum malaria in pregnancy in India. Trial registration CTRI This study is registered with Clinical Trial Registry India (CTRI), number CTRI/2009/091/001055. Date of Registration 11 January 2010, http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=1185&EncHid=&userName=anvikar.
India's target of malaria elimination by 2030 may not be achieved solely by detecting Plasmodium falciparum and P. vivax, the two common Plasmodium species causing infections in humans. Sporadic ...reports have been documented on other Plasmodium species in the country, associated mostly with travel history. A febrile patient of Indian origin (Non-resident Indian (NRI)) was diagnosed with an infection of Plasmodium ovale curtisi malaria on his arrival from Sudan. A case report from Kerala was published in December 2020 and this is second report. Due to the inaccessibility of molecular techniques for routine diagnosis, this neglected non-falciparum malaria goes undetected. For an accurate diagnosis, suspected malaria cases should be tested using PCR and other advanced methods.
Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low ...parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.
Available DNA isolation methods for
involve numerous processing steps, adding to the cost and conferring risk of contamination. Here we devise a simple and cost-effective method for direct extraction ...of
DNA from dried filter paper spot (DBS), appropriate for resource-limited setups.
The protocol involves simple freezing and thawing of DBS, neither involves any purification step nor any chemical reagent. The method was assessed in terms of DNA quantity, PCR detection sensitivity, time requirement, cost effectiveness, labor intensiveness and degree of shearing. The reliability of this method was confirmed by comparing it with other in use methods for
DNA isolation.
Pure DNA was obtained with this method, as exemplified by the absorbance ratio (260nm /280nm) of 1.2. The protocol produced digestible, PCR-grade genomic DNA, also found to be suitable for sequencing. DNA isolated remained stable and retained its integrity after storage for one month at 4 °C.
Our process substantiated as efficient, reproducible, simple, fast, and inexpensive. Development of this optimized freeze-thaw based DNA extraction method for malaria parasite may provide a valuable tool for molecular analysis in resource-limited setups. This is the first report of DNA extraction from DBS of
utilizing freeze-thaw.
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