• View in gallery

    Giemsa-stained thin blood films (A–D). A, Early trophozoite with single chromatin dot in a crenated red cell. B, Two late trophozoites. C, Trophozoite, band form. D, Schizont. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Detection of P. knowlesi DNA by nested PCR. The expected PCR product is 153 bp in size. Negative controls consisting of water and positive controls from DNA extracted from a previously identified P. knowlesi infection in human and PCR product of DNA extracted from whole blood of our patient were carried out in duplicate. Molecular size markers in base pairs are in the rightmost lane.

  • 1

    Johnson BW, Russell BJ, Lanciotti RS, 2005. Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J Clin Microbiol 43 :4977–4983.

    • Search Google Scholar
    • Export Citation
  • 2

    Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, Haton K, 2004. Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species specific real-time PCR assays. J Clin Microbiol 42 :5636–5643.

    • Search Google Scholar
    • Export Citation
  • 3

    Somchai J, Putaporntip C, Iwasaki T, Sata T, Kanbara H, 2004. Naturally acquired Plasmodium knowlesi malaria in human, Thailand. Emerg Infect Dis 10 :2211–2213.

    • Search Google Scholar
    • Export Citation
  • 4

    Chin W, Contacos PG, Coatney GR, 1965. A naturally acquired quotidian-type malaria in man transferable to monkeys. Science 149 :865.

  • 5

    Singh B, Sung LK, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, Thomas A, Conway DJ, 2004. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 363 :1017–1024.

    • Search Google Scholar
    • Export Citation
  • 6

    Ng OT, Ooi EE, Lee CC, Lee PJ, Ng LC, Pei SW, Tu TM, Loh JP, Leo YS, 2008. Naturally acquired human Plasmodium knowlesi infection, Singapore. Emerg Infect Dis 14 :814–816.

    • Search Google Scholar
    • Export Citation
  • 7

    De Monbrison F, Gérome P, Chaulet JF, Wallon M, Picot S, Peyron F, 2004. Comparative diagnostic performance of two commercial rapid tests for malaria in a non-endemic area. Eur J Clin Microbiol Infect Dis 23 :784–786.

    • Search Google Scholar
    • Export Citation
  • 8

    BinaxNow© Malaria Test Kit, 2007. Inverness Medical Professional Diagnostics, Scarborough, Maine. Rev. 2. 19 July 2007.

  • 9

    McCutchan TF, Piper RC, Makler MT, 2008. Use of malaria rapid diagnostic test to identify Plasmodium knowlesi infection. Emerg Infect Dis 14 :1750–1752.

    • Search Google Scholar
    • Export Citation
  • 10

    Cox-Singh J, Davis TM, Lee KS, Shamsul SS, Matusop A, Ratnam S, Rahman HA, Conway DJ, Singh B, 2008. Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clin Infect Dis 46 :165–171.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Monkey Malaria in Humans: A Diagnostic Dilemma with Conflicting Laboratory Data

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  • 1 National University Health System, Singapore; Defence Medical and Environmental Research Institute, Singapore

Plasmodium knowlesi has recently been recognized as the fifth Plasmodium species causing malaria in humans. Diagnosis is difficult morphologically, and currently, available rapid tests have not been comprehensively evaluated with this pathogen. We report a case of P. knowlesi malaria that was confirmed after the initial clue of discordant microscopy and immunochromatographic results, highlighting the importance of molecular diagnostics in cases with the relevant clinical and epidemiologic history.

INTRODUCTION

Plasmodium knowlesi is a simian malaria parasite and is recently recognized as the fifth malaria parasite infecting humans. As this is a newly recognized infection, rapid diagnostic tests are not yet established, and blood film microscopy is difficult as the parasite is often incorrectly identified. Polymerase chain reaction remains the gold standard for diagnosis but requires time. We report such a case of P. knowlesi infection with discordant microscopy and immunochromatographic results, emphasizing the need to be vigilant especially in regions which are endemic and in close proximity to habitats of macaques.

CASE REPORT

A previously healthy 33-year-old Singaporean was admitted in October 2007 with a 3-day history of fever, malaise, nausea, and vomiting. He had just completed military reserve training in a forested area in northwestern Singapore a week before admission. His last overseas travel was a month before to Phuket, Thailand.

He had a fever of 40°C for 3 days with no chills or rigors and mild jaundice but no hepatosplenomegaly. His white cell count was 4.44 × 109/L, hematocrit was 41.6%, and platelet was 44 × 109/L. Serum bilirubin was 48 Umol/L (N < 30), aspartate transaminase (AST) was 294 U/L, and alanine transaminase (ALT) was 282 U/L. Renal function was normal. Dengue fever was ruled out with negative serology and reverse transcription-polymerase chain reaction (RT-PCR).1

His thin blood film for malaria parasites showed possible mixed Plasmodium falciparum with P. malariae infection with a parasite load of 0.2% (equivalent to 7,700 parasites/mmol/L blood) as shown in Figure 1.

The OptiMAL rapid malaria test (DiaMed AG, Morat, Switzerland) was positive for P. falciparum. The BinaxNow Malaria test (Inverness Medical, Binax) had both bands positive, signifying either a mixed infection of P. falciparum with another species or P. falciparum alone. The conflicting microscopic and immunochromatographic results with no history of exposure to P. falciparum–endemic areas led to a molecular investigation.

The patient’s sample was positive for malaria DNA based on the real-time PCR for small subunit ribosomal RNA (SSUrRNA) of Rougemont and others.2 The sample was negative for P. falciparum, P. vivax, P. ovale, and P. malariae. Using a nested PCR that specifically targeted another site on the SSUrRNA but was previously shown to be specific for P. knowlesi3 showed an expected 153-bp product (Figure 2). Sequence analysis of the SSU rRNA PCR product using Pmk8 and Pmkr9 primers with a direct sequencing approach showed highest (97%) homology with P. knowlesi (U83876).

The patient was treated with chloroquine and then primaquine with a rapid response. He was well on follow-up a month later without recurrent parasitemia on malaria blood film examination.

DISCUSSION

Plasmodium knowlesi is a well-known cause of simian malaria, predominantly affecting long-tailed macaques, which was first detected in a human > 40 years ago.4 Human infections have been reported in East Malaysia,5 Thailand,3 and recently in Singapore,6 suggesting a wider distribution across Southeast Asia. Diagnosis is a challenge. Indeed, the first human case of P. knowlesi malaria was initially thought to be P. falciparum, and then P. malariae the next day, only to be confirmed as P. knowlesi after inoculation of the infected human blood into rhesus monkeys.4 This common misidentification occurs as early trophozoites of P. knowlesi appear as ring forms, similar to P. falciparum. Late trophozoites of P. knowlesi appearing as “band forms,” schizonts and gametocytes resemble P. malariae.5 Similar findings were also detected in our patient (Figure 1).

The OptiMAL rapid malaria test detects parasite-specific lactate dehydrogenase, whereas the BinaxNow Malaria test detects histidine-rich protein II (HRP-2) specific to P. falciparum and a pan-malarial antigen common to all four human malaria species. Both tests have their limitations,7 and the unusual result in our patient indicating either mixed infection or P. falciparum alone led to more specific molecular testing. The specific reason for the observed cross-reaction of BinaxNow Malaria test with P. knowlesi antigens was not determined in our study. However, it is likely that the antigen capture test was developed, and its specificity was determined by comparing the reactivity of the antibodies used in the assay against P. falciparum, P. vivax, P. ovale, and P malariae, but not P knowlesi.8 Our observation underscores the need to include P. knowlesi in the development and validation of malaria diagnostics, as well as in proficiency panels for quality assurance testing of laboratories offering malaria diagnostic services. The antigen-capture test recently developed based on monoclonal antibodies (mAbs) to Plasmodium lactate dehydrogenase (pLDH) that was able to rapidly diagnose P. knowlesi9 similarly needs to be validated for its specificity and sensitivity.

Plasmodium knowlesi infection has been shown to be transmitted between humans and non-human primates.4 Although most human infections are mild, severe infections have occurred. 10 The treatment is typically with chloroquine and primaquine. Quinine has also been shown to be effective.5 Relapse after treatment and drug resistance have not been reported, and the optimal type of malaria prophylaxis is unknown.

In conclusion, P. knowlesi is a fifth Plasmodium species causing human malaria. Large prospective studies are needed to determine its true prevalence in humans in the region and beyond. Clinicians should suspect this diagnosis in individuals with the appropriate travel or contact history (in areas with macaques) and the degree of suspicion should be enhanced by ambiguous microscopic and rapid diagnostic tests.

Figure 1.
Figure 1.

Giemsa-stained thin blood films (A–D). A, Early trophozoite with single chromatin dot in a crenated red cell. B, Two late trophozoites. C, Trophozoite, band form. D, Schizont. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 6; 10.4269/ajtmh.2009.80.927

Figure 2.
Figure 2.

Detection of P. knowlesi DNA by nested PCR. The expected PCR product is 153 bp in size. Negative controls consisting of water and positive controls from DNA extracted from a previously identified P. knowlesi infection in human and PCR product of DNA extracted from whole blood of our patient were carried out in duplicate. Molecular size markers in base pairs are in the rightmost lane.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 6; 10.4269/ajtmh.2009.80.927

*

Address correspondence to Catherine W. M. Ong, Division of Infectious Disease, Department of Medicine, National University Health System, 5 Lower Kent Ridge Road, Singapore 119074. E-mail: catongwm@yahoo.com

Authors’ addresses: Catherine W. M. Ong and Paul A. Tambyah, Division of Infectious Disease, Department of Medicine, National University Health System, 5 Lower Kent Ridge Road, Singapore 119074, Tel: 65-6772-4362/4380, Fax: 65-6779-4361. Shir Ying Lee, Department of Haematology, National University Health System, 5 Lower Kent Ridge Road, Singapore 119074, Tel: 65-6772-4362, Fax: 65-6779-4361. Wee Hong Koh and Eng-Eong Ooi, Defence Medical and Environmental Research Institute, 27 Medical Drive, Singapore 11750, Tel: 65-6776-2255, Fax: 65-6775-9011.

Acknowledgments: The authors thank Seok Wei Chew for helping with the definitive testing by molecular methods for this case. KWH and OEE conducted the molecular analysis of the specimen. LSY made the figures. CWMO, PAT, and OEE all contributed in writing the paper.

Disclosure: PAT has received research support from Baxter, Pfizer, Merck, Wyeth, and Interimmune.

REFERENCES

  • 1

    Johnson BW, Russell BJ, Lanciotti RS, 2005. Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J Clin Microbiol 43 :4977–4983.

    • Search Google Scholar
    • Export Citation
  • 2

    Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, Haton K, 2004. Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species specific real-time PCR assays. J Clin Microbiol 42 :5636–5643.

    • Search Google Scholar
    • Export Citation
  • 3

    Somchai J, Putaporntip C, Iwasaki T, Sata T, Kanbara H, 2004. Naturally acquired Plasmodium knowlesi malaria in human, Thailand. Emerg Infect Dis 10 :2211–2213.

    • Search Google Scholar
    • Export Citation
  • 4

    Chin W, Contacos PG, Coatney GR, 1965. A naturally acquired quotidian-type malaria in man transferable to monkeys. Science 149 :865.

  • 5

    Singh B, Sung LK, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, Thomas A, Conway DJ, 2004. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 363 :1017–1024.

    • Search Google Scholar
    • Export Citation
  • 6

    Ng OT, Ooi EE, Lee CC, Lee PJ, Ng LC, Pei SW, Tu TM, Loh JP, Leo YS, 2008. Naturally acquired human Plasmodium knowlesi infection, Singapore. Emerg Infect Dis 14 :814–816.

    • Search Google Scholar
    • Export Citation
  • 7

    De Monbrison F, Gérome P, Chaulet JF, Wallon M, Picot S, Peyron F, 2004. Comparative diagnostic performance of two commercial rapid tests for malaria in a non-endemic area. Eur J Clin Microbiol Infect Dis 23 :784–786.

    • Search Google Scholar
    • Export Citation
  • 8

    BinaxNow© Malaria Test Kit, 2007. Inverness Medical Professional Diagnostics, Scarborough, Maine. Rev. 2. 19 July 2007.

  • 9

    McCutchan TF, Piper RC, Makler MT, 2008. Use of malaria rapid diagnostic test to identify Plasmodium knowlesi infection. Emerg Infect Dis 14 :1750–1752.

    • Search Google Scholar
    • Export Citation
  • 10

    Cox-Singh J, Davis TM, Lee KS, Shamsul SS, Matusop A, Ratnam S, Rahman HA, Conway DJ, Singh B, 2008. Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clin Infect Dis 46 :165–171.

    • Search Google Scholar
    • Export Citation
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