BackgroundBoth Plasmodium vivax and Plasmodium falciparum are prevalent in Pakistan, yet up-to-date data on the epidemiology of malaria in Pakistan are not available. This study was undertaken to determine the current prevalence and distribution of Plasmodium species across the country.MethodsA malariometric population survey was conducted in 2011 using blood samples collected from 801 febrile patients of all ages in four provinces and the capital city of Islamabad. Microscopically confirmed Plasmodium-positive blood samples were reconfirmed by polymerase chain reaction (PCR). Confirmed parasite-positive samples were subjected to species-specific PCR capable of detecting four species of human malaria.ResultsOf the 707 PCR-positive samples, 128 (18%) were P. falciparum, 536 (76%) were P. vivax, and 43 (6%) were mixed P. falciparum and P. vivax. Ninety-four microscopy-positive samples were PCR-negative, and Plasmodium malariae and Plasmodium ovale were not detected. Prevalence of P. vivax ranged from 2.4% in Punjab Province to 10.8% in Sindh Province and prevalence of P. falciparum ranged from 0.1% in Islamabad to 3.8% in Balochistan.ConclusionsPlasmodium infections in Pakistan are largely attributed to P. vivax but P. falciparum and mixed species infections are also prevalent. In addition, regional variation in the prevalence and species composition of malaria is high.

BackgroundHighly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans.MethodsA reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR.ResultsLimits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80 % humidity, the LoD was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR.ConclusionsThis ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomatic P. falciparum and P. vivax parasitaemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1038-z) contains supplementary material, which is available to authorized users.

The Mandatory Guidelines for Federal Workplace Drug Testing Programs provide criteria for specimen validity testing, including urine pH cut-offs, to report a urine specimen as adulterated or invalid. Since the urine pH criteria for invalid classifications, > or = 3 and < 4.5 or > or = 9 and < 11, became effective in November 2004, a number of specimens with results within the upper invalid limits, typically in the range of 9.1 to 9.3, have been reported with no evidence of adulteration. This study evaluated the hypothesis that these pH findings were the result of exposure to increased environmental temperatures during specimen standing and transport. Indeed, increased storage temperatures were associated with increased urine pH, with the magnitude of the change related to both storage time and temperature. The pH values of specimens stored at -20 degrees C are relatively stable, whereas pH results > 9 are achieved at storage temperatures of room temperature or higher. It is noteworthy that no condition(s) produced a specimen with a pH > 9.5. Degradation of nitrogenous urine analytes is most likely responsible for the noted increases in pH. These findings are intended to supplement information used by the Medical Review Officers who are responsible for interpreting such marginally invalid pH results.