Primaquine Administration after Falciparum Malaria Treatment in Malaria Hypoendemic Areas with High Incidence of Falciparum and Vivax Mixed Infection: Pros and Cons

Mixed infections of Plasmodium falciparum and Plasmodium vivax is high (~30%) in some malaria hypoendemic areas where the patients present with P. falciparum malaria diagnosed by microscopy. Conventional treatment of P. falciparum with concurrent chloroquine and 14 days of primaquine for all falciparum malaria patients may be useful in areas where mixed falciparum and vivax infections are high and common and also with mild or moderate G6PD deficiency in the population even with or without subpatent vivax mixed infection. It will be possibly cost-effective to reduce subsequent vivax illness if the patients have mixed vivax infection. Further study to prove this hypothesis may be warranted.     

Malaria in pregnant women living in areas of low transmission on the southeast Brazilian Coast: molecular diagnosis and humoural immunity profile

Studies on autochthonous malaria in low-transmission areas in Brazil have acquired epidemiological relevance because they suggest continued transmission in what remains of the Atlantic Forest. In the southeastern portion of the state of São Paulo, outbreaks in the municipality of Juquitiba have been the focus of studies on the prevalence of Plasmodium, including asymptomatic cases. Data on the occurrence of the disease or the presence of antiplasmodial antibodies in pregnant women from this region have not previously been described. Although Plasmodium falciparum in pregnant women has been widely addressed in the literature, the interaction of Plasmodium vivax and Plasmodium malariae with this cohort has been poorly explored to date. We monitored the circulation of Plasmodium in pregnant women in health facilities located in Juquitiba using thick blood film and molecular protocols, as well as immunological assays, to evaluate humoural immune parameters. Through real-time and nested polymerase chain reaction, P. vivax and P. malariae were detected for the first time in pregnant women, with a positivity of 5.6%. Immunoassays revealed the presence of IgG antibodies: 44% for ELISA-Pv, 38.4% for SD-Bioline-Pv and 18.4% for indirect immunofluorescence assay-Pm. The high prevalence of antibodies showed significant exposure of this population to Plasmodium. In regions with similar profiles, testing for a malaria diagnosis might be indicated in prenatal care.     

Mass blood survey for malaria: pooling and realtime PCR combined with expert microscopy in north-west Thailand

ABSTRACT: BACKGROUND: Asymptomatic carriage of Plasmodium falciparum and Plasmodium vivax is common in both low-and high-transmission settings and represents an important reservoir of infection that needs to be targeted if malaria elimination is to succeed. METHODS: Mass blood examinations (475 individuals) were conducted in two villages in Mae Hong Son, an area of endemic but low-transmission malaria in the north-west of Thailand. The microscopist at the local malaria clinic did not detect any infections. Pools of four samples were screened by real-time PCR; individual members of all of the positive pools were then re-examined by expert microscopy and by a second species-specific PCR reaction. RESULTS: Eight subjects were found to be positive by both PCR and expert microscopy and one was found to be positive by PCR alone. The slides contained asexual stage parasites of P. vivax, P. falciparum and Plasmodium malariae, but no gametocytes. The local clinic was notified within two to eight days of the survey. CONCLUSION: A combination of pooling, real-time PCR and expert microscopy provides a feasible approach to identifying and treating asymptomatic malaria infections in a timely manner.     

A research agenda for malaria eradication: diagnoses and diagnostics

Many of malaria's signs and symptoms are indistinguishable from those of other febrile diseases. Detection of the presence of Plasmodium parasites is essential, therefore, to guide case management. Improved diagnostic tools are required to enable targeted treatment of infected individuals. In addition, field-ready diagnostic tools for mass screening and surveillance that can detect asymptomatic infections of very low parasite densities are needed to monitor transmission reduction and ensure elimination. Antibody-based tests for infection and novel methods based on biomarkers need further development and validation, as do methods for the detection and treatment of Plasmodium vivax. Current rapid diagnostic tests targeting P. vivax are generally less effective than those targeting Plasmodium falciparum. Moreover, because current drugs for radical cure may cause serious side effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency, more information is needed on the distribution of G6PD-deficiency variants as well as tests to identify at-risk individuals. Finally, in an environment of very low or absent malaria transmission, sustaining interest in elimination and maintaining resources will become increasingly important. Thus, research is required into the context in which malaria diagnostic tests are used, into diagnostics for other febrile diseases, and into the integration of these tests into health systems.     

A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

Formerly known as a hypoendemic malaria country, the Republic of Djibouti declared the goal of pre-eliminating malaria in 2006. The aim of the present study was to evaluate the prevalence of Plasmodium falciparum, Plasmodium vivax and mixed infections in the Djiboutian population by using serological tools and to identify potential determinants of the disease and hotspots of malaria transmission within the country. The prevalence of P. falciparum and P. vivax within the districts of the capital city and the rest of the Republic of Djibouti were assessed using 13 and 2 serological markers, respectively. The relationship between the immune humeral response to P. falciparum and P. vivax and variables such as age, gender, wealth status, urbanism, educational level, distance to rivers/lakes, living area, having fever in the last month, and staying in a malaria-endemic country more than one year was estimated and analysed by questionnaires administered to 1910 Djiboutians. Multivariate ordinal logistic regression models of the immune humeral response were obtained for P. falciparum and P. vivax. The P. falciparum and P. vivax seroprevalence rates were 31.5%, CI95% [29.4-33.7] and 17.5%, CI95% [15.8-19.3], respectively. Protective effects against P. falciparum and P. vivax were female gender, educational level, and never having visited a malaria-endemic area for more than one year. For P. falciparum only, a protective effect was observed for not having a fever in the last month, living more than 1.5 km away from lakes and rivers, and younger ages. This is the first study that assessed the seroprevalence of P. vivax in the Republic of Djibouti. It is necessary to improve knowledge of this pathogen in order to create an effective elimination programme. As supported by recent observations on the subject, the Republic of Djibouti has probably demonstrated a real decrease in the transmission of P. falciparum in the past seven years, which should encourage authorities to improve efforts toward elimination.     

Correlates of HIV and malaria co-infection in Southern India

ABSTRACT: BACKGROUND: Malaria and HIV co-infection adversely impact the outcome of both diseases and previous studies have mostly focused on falciparum malaria. Plasmodium vivax contributes to almost half of the malaria cases in India, but the disease burden of HIV and P. vivax co-infection is unclear. METHODS: HIV-infected subjects (n=460) were randomly selected from the 4,611 individuals seen at a Voluntary Counseling and Testing Center in Chennai, India between Jan 2 to Dec 31 2008. Malaria testing was performed on stored plasma samples by both nested PCR using both genus-specific and species-specific primers and immunochromatography-based rapid diagnostic test for detecting antibodies against both Plasmodium falciparum and P. vivax. RESULTS: Recent malaria co-infection, defined by the presence of antibodies, was detected in 9.8% (45/460) participants. Plasmodium vivax accounted for majority of the infections (60%) followed by P. falciparum (27%) and mixed infections (13%). Individuals with HIV and malaria co-infection were more likely to be men (p=0.01). Between those with and without malaria, there was no difference in age (p=0.14), CD4+ T-cell counts (p=0.19) or proportion CD4+ T-cell below 200/mL (p=0.51). CONCLUSIONS: Retrospective testing of stored plasma samples for malaria antibodies can facilitate identification of populations with high rates of co-infection, and in this southern India HIVinfected cohort there was a considerable burden of malaria co-infection, predominantly due to P. vivax. However, the rate of P. falciparum infection was more than 6-fold higher among HIV-infected individuals than what would be expected in the general population in the region. Interestingly, individuals co-infected with malaria and HIV were not more likely to be immunosuppressed than individuals with HIV infection alone.     

Epidemiological study of malaria in north Sulawesi, Indonesia by fluorescence and Giemsa staining.

An epidemiological study of malaria infection was conducted in the Likupang District, Minahasa Regency, North Sulawesi Province, Indonesia, during August 2-15, 1991. In this study, 510 people of six villages, representing ages between 1 month to 84 years cooperated voluntarily. Blood smears stained with Giemsa and acridine orange (AO), revealed 33 and 83 malaria parasite positives respectively. This significant difference was due particularly to the fact that AO staining examined under either a daylight- or halogen-illuminated microscope equipped with interference filters was sensitive to detect low-density parasitemia in many subjects previously diagnosed negative by Giemsa staining in the field. The low malaria prevalence obtained by Giemsa staining may have been attributable to the lack of standard-quality diagnostic tools in the field or inadequate observation of the slides. In both staining methods, Plasmodium falciparum was found to be the predominant species, while the remainings were P. vivax or a mixture of both. Subjects infected with P. vivax revealed higher density of parasitemia and gametocytemia than those with P. falciparum.     

Glucose-6-phosphate metabolism in Plasmodium falciparum

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.     

Validation of G6PD Point-of-Care Tests among Healthy Volunteers in Yangon,Myanmar

Primaquine and other 8-amnoquinoline based anti-malarials can cause haemolysis in subjects with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Correct diagnosis of G6PD status in patients is crucial for safe treatment of both relapsing stages of Plasmodium vivax and transmitting forms of Plasmodium falciparum. Lack of suitable point-of-care tests has hampered a much needed wide use of primaquine for malaria elimination. In this study we have assessed the performances of two qualitative tests, the fluorescent spot test (FST) and the G6PD CareStart test (CST), against the gold standard quantitative spectrophotometric assay in a population of 1000 random adult healthy volunteers living in Yangon, Myanmar. The prevalence of G6PD deficiency in the Bamar, Karen and in the whole sample set was 6.6% (10.1% in males), 9.2% (21.0% in males) and 6.8% (11.1% in males) respectively. The FST and CST showed comparable performances with sensitivity over 95% and specificity over 90%, however for cases with severe G6PD activity the FTS had improved performance. If used with a conservative interpretation of the signal, the CareStart test has the potential to be used in the field and, by allowing a wider use of primaquine, to help malaria elimination.     

First Evaluation of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Vivax Malaria Endemic Regions in the Republic of Korea

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK).

Methods

Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK.

Results

Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants.

Conclusions

No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.     

Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase

Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.     

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.     

Safety and tolerability of elubaquine (bulaquine, CDRI 80/53) for treatment of Plasmidium vivax malaria in Thailand

We conducted a study to compare the safety and tolerability of anti-relapse drugs elubaquine and primaquine against Plasmodium vivax malaria. After standard therapy with chloroquine, 30 mg/kg given over 3 days, 141 patients with P. vivax infection were randomized to receive primaquine or elubaquine. The 2 treatment regimens were primaquine 30 mg once daily for 7 days (group A, n = 71), and elubaquine 25 mg once daily for 7 days (group B, n = 70). All patients cleared parasitemia within 7 days after chloroquine treatment. Among patients treated with primaquine, one patient relapsed on day 26; no relapse occurred with elubaquine treatement. Both drugs were well tolerated. Adverse effects occurred only in patients with G6PD deficiency who were treated with primaquine (group A, n = 4), whose mean hematocrit fell significantly on days 7, 8 and 9 (P = 0.015, 0.027, and 0.048, respectively). No significant change in hematocrit was observed in patients with G6PD deficiency who were treated with elubaquine (group B, n = 3) or in patients with normal G6PD. In conclusion, elubaquine, as anti-relapse therapy for P. vivax malaria, was as safe and well tolerated as primaquine and did not cause clinically significant hemolysis.     

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.     

ITS1, 5.8S and A-type ITS2 rDNA sequences from Plasmoidum vivax and development of a method for retrospective PCR diagnosis of malaria by stained thick blood smears]

Stages life cycle of the malaria parasite differ in the rate of replication and the structural properties of functionally active A-, S-, and O-type ribosomes. Regions of A-type rDNA including ITS1, 5.8S, and ITS2 from two strains of Plasmodium vivax with different incubation periods were amplified and sequenced. No substantial differences in the sequences of two strains were revealed. Phylogenetic analysis of the obtained and homologous sequences of ITS1 rDNA of A, S, and O types of P. vivax; A and S types of P. falciparum; and Cryptosporidium parvum, Eimeria maxima, Toxoplasma gondii as outgroup, by the maximum parsimony method using PAUP 4.0 revealed that divergence of ITS1 might have occurred after speciation and at different rates in individual lineages of the Plasmodium genus. Basing on the results of the analysis of orthologous sequences of P. vivax and P. falciparum, we developed genus- and species-specific primers for PCR diagnostics of malaria, as well as a one-step effective method of DNA isolation from Giemsa-Romanovsky-stained thick blood smears. It was demonstrated that stained preparations could be a reliable source of plasmodial DNA, and the quality of preparations and storage time (10-20 years) did not interfere with the results of PCR analysis.     

Review of pyronaridine anti-malarial properties and product characteristics

ABSTRACT: Pyronaridine was synthesized in 1970 at the Institute of Chinese Parasitic Disease and has been used in China for over 30 years for the treatment of malaria. Pyronaridine has high potency against Plasmodium falciparum, including chloroquine-resistant strains. Studies in various animal models have shown pyronaridine to be effective against strains resistant to other anti-malarials, including chloroquine. Resistance to pyronaridine appears to emerge slowly and is further retarded when pyronaridine is used in combination with other antimalarials, in particular, artesunate. Pyronaridine toxicity is generally less than that of chloroquine, though evidence of embryotoxicity in rodents suggests use with caution in pregnancy. Clinical pharmacokinetic data for pyronaridine indicates an elimination T1/2 of 13.2 and 9.6 days, respectively, in adults and children with acute uncomplicated falciparum and vivax malaria in artemisinin-combination therapy. Clinical data for mono or combined pyronaridine therapy show excellent anti-malarial effects against P. falciparum and studies of combination therapy also show promise against Plasmodium vivax. Pyronaridine has been developed as a fixed dose combination therapy, in a 3:1 ratio, with artesunate for the treatment of acute uncomplicated P. falciparum malaria and blood stage P. vivax malaria with the name of Pyramax(R) and has received Positive Opinion by European Medicines Agency under the Article 58 procedure.     

Severe disease in children hospitalized with a diagnosis of Plasmodium vivax in south-eastern Pakistan

ABSTRACT: BACKGROUND: Infection by Plasmodium vivax has been considered rarely threatening to life, but recent studies challenge this notion. This study documented the frequency and character of severe illness in paediatric patients admitted to a hospital in south-eastern Pakistan with a laboratory-confirmed diagnosis of vivax malaria. METHODS: An observational study of all 180 paediatric patients admitted with any diagnosis of malaria during 2010 was conducted: 128 P. vivax; 48 Plasmodium falciparum; and four mixed infections of these species. Patients were classified as having severe illness with any of the following indicators: Glascow coma scale <11; >2 convulsions; haemoglobin <5g/dL; thrombocytes <50,000/mL; blood glucose <45mg%; >70 breaths/min; or intravenous antimalarial therapy. Additionally, 64 patients with a diagnosis of vivax malaria were treated during 2009, and the 21 of these having severe illness were included in analyses of the frequency and character of severe illness with that diagnosis. RESULTS: During 2010, 39 (31%) or 37 (77%) patients with a diagnosis of P. vivax or P. falciparum were classified as having severe disease. Including the 2009 records of 64 patients having vivax malaria, a total of 60 (31%) patients with severe illness and a diagnosis of P. vivax were available. Altered mental status (Glascow coma scale score <11; or >2 convulsions) dominated at 54% of the 83 indicators of severe illness manifest among the patients with vivax malaria, as was true among the 37 children with a diagnosis of falciparum malaria and being severely ill; 58% of the 72 indicators of severe disease documented among them. No statistically significant difference appeared in frequencies of any other severe disease indicators between patients diagnosed with vivax or falciparum malaria. Despite such similarities, a diagnosis of falciparum malaria nonetheless came with 3.8-fold (95% CI = 1.8- 8.1) higher risk of presenting with severe illness, and 8.0-fold (95% CI = 2.1-31) greater likelihood of presenting with three or more severe disease indicators. Two patients did not survive hospitalization, one each with a diagnosis of falciparum or vivax malaria. CONCLUSIONS: Vivax malaria caused a substantial burden of potentially life-threatening morbidity on a paediatric ward in a hospital in south-eastern Pakistan.     

Real-life implementation of a G6PD deficiency screening qualitative test into routine vivax malaria diagnostic units in the Brazilian Amazon (SAFEPRIM study)

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency greatly hinders Plasmodium vivax malaria radical cure and further elimination due to 8-aminoquinolines-associated hemolysis. Although the deleterious health effects of primaquine in G6PD deficient individuals have been known for over 50 years, G6PD testing is not routinely performed before primaquine treatment in most P. vivax endemic areas.Method/Principal findingsThe qualitative CareStart G6PD screening test was implemented in 12 malaria treatment units (MTUs) in the municipality of Rio Preto da Eva, Western Brazilian Amazon, a malaria endemic area, between February 2019 and early January 2020. Training materials were developed and validated; evaluations were conducted on the effectiveness of training health care professionals (HCPs) to perform the test, the interpretation and reliability of routine testing performed by HCPs, and perceptions of HCPs and patients. Most HCPs were unaware of G6PD deficiency and primaquine-related adverse effects. Most of 110 HCPs trained (86/110, 78%) were able to correctly perform the G6PD test after a single 4-hour training session. The test performed by HCPs during implementation showed 100.0% (4/4) sensitivity and 68.1% (62/91) specificity in identifying G6PD deficient patients as compared to a point-of-care quantitative test (Standard G6PD).Conclusions/SignificanceG6PD screening using the qualitative CareStart G6PD test performed by HCPs in MTUs of an endemic area showed high sensitivity and concerning low specificity. The amount of false G6PD deficiency detected led to substantial loss of opportunities for radical cure.